BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effect...BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.展开更多
AIMTo investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase (ERK) signaling pathway in vitro.METHODSThe expression levels of phosphorylate...AIMTo investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase (ERK) signaling pathway in vitro.METHODSThe expression levels of phosphorylated ERK (P-ERK), keratin family members including K19 and K10 and the ocular master control gene Pax-6 were measured in 16 surgically excised pterygium tissues and 12 eye bank conjunctiva. In colony-forming cell assays, the differences in clone morphology and in K10, K19, P-ERK and Pax-6 expression between the head and body were investigated. When cocultured with the ERK signaling pathway inhibitor PD98059, the changes in clone morphology, colony-forming efficiency, differentiated marker K10, K19 and Pax-6 expression and P-ERK protein expression level were examined by immunoreactivity and Western blot analysis.RESULTSThe expression of K19 and Pax-6 decreased in the pterygium, especially in the head. No staining of K10 was found in the normal conjunctiva epithelium, but it was found to be expressed in the superficial cells in the head of the pterygium. Characteristic upregulation of P-ERK was observed by immunohistochemistry. The clone from the head with more differentiated cells in the center expressed more K10, and the clone from the body expressed more K19. The P-ERK protein level increased in the pterygium epithelium compared with conjunctiva and decreased when cocultured with PD98059. The same medium with the ERK inhibitor PD98059 was more effective in promoting clonal growth than conventional medium with 3T3 murine feeder layers. It was observed that the epithelium clone co-cultured with the inhibitor had decreased K10 expression and increased K19 and Pax-6 expression.CONCLUSIONWe suggest ERK signaling pathway activation might play a role in the pterygium epithelium abnormal differentiation.展开更多
BACKGROUND Immunological dysfunction-induced low-grade inflammation is regarded as one of the predominant pathogenetic mechanisms in post-infectious irritable bowel syndrome(PI-IBS).γδT cells play a crucial role in ...BACKGROUND Immunological dysfunction-induced low-grade inflammation is regarded as one of the predominant pathogenetic mechanisms in post-infectious irritable bowel syndrome(PI-IBS).γδT cells play a crucial role in innate and adaptive immunity.Adenosine receptors expressed on the surface ofγδT cells participate in intestinal inflammation and immunity regulation.AIM To investigate the role ofγδT cell regulated by adenosine 2A receptor(A2AR)in PI-IBS.METHODS The PI-IBS mouse model has been established with Trichinella spiralis(T.spiralis)infection.The intestinal A2AR and A2AR inγδT cells were detected by immunohistochemistry,and the inflammatory cytokines were measured by western blot.The role of A2AR on the isolatedγδT cells,including proliferation,apoptosis,and cytokine production,were evaluated in vitro.Their A2AR expression was measured by western blot and reverse transcription polymerase chain reaction(RT-PCR).The animals were administered with A2AR agonist,or A2AR antagonist.Besides,γδT cells were also injected back into the animals,and the parameters described above were examined,as well as the clinical features.Furthermore,the A2AR-associated signaling pathway molecules were assessed by western blot and RT-PCR.RESULTS PI-IBS mice exhibited elevated ATP content and A2AR expression(P<0.05),and suppression of A2AR enhanced PI-IBS clinical characteristics,indicated by the abdominal withdrawal reflex and colon transportation test.PI-IBS was associated with an increase in intestinal T cells,and cytokine levels of interleukin-1(IL-1),IL-6,IL-17A,and interferon-α(IFN-α).Also,γδT cells expressed A2AR in vitro and generated IL-1,IL-6,IL-17A,and IFN-α,which can be controlled by A2AR agonist and antagonist.Mechanistic studies demonstrated that the A2AR antagonist improved the function ofγδT cells through the PKA/CREB/NF-κB signaling pathway.CONCLUSION Our results revealed that A2AR contributes to the facilitation of PI-IBS by regulating the function ofγδT cells via the PKA/CREB/NF-κB signaling pathway.展开更多
Dietary flavonoids are abundant in natural plants and possess multiple pharmacological and nutritional activities.In this study,apigenin,luteolin,and baicalein were chosen to evaluate their anti-diabetic effect in hig...Dietary flavonoids are abundant in natural plants and possess multiple pharmacological and nutritional activities.In this study,apigenin,luteolin,and baicalein were chosen to evaluate their anti-diabetic effect in high-glucose and dexamethasone induced insulin-resistant(IR)HepG2 cells.All flavonoids improves the glucose consumption and glycogen synthesis abilities in IR-HepG2 cells via activating glucose transporter protein 4(GLUT4)and phosphor-glycogen synthase kinase(GSK-3β).These fl avonoids signifi cantly inhibited the production of reactive oxygen species(ROS)and advanced glycation end-products(AGEs),which were closely related to the suppression of the phosphorylation form of NF-κB and P65.The expression levels of insulin receptor substrate-1(IRS-1),insulin receptor substrate-2(IRS-2)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway in IR-HepG2 cells were all partially activated by the fl avonoids,with variable effects.Furthermore,the intracellular metabolic conditions of the fl avonoids were also evaluated.展开更多
An increasing number of studies report that the Ras/Raf/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway has a death-promoting apoptotic function in neural cells. We hypothesized that the Ras/Raf...An increasing number of studies report that the Ras/Raf/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway has a death-promoting apoptotic function in neural cells. We hypothesized that the Ras/Raf/ERK1/2 signaling pathway may be abnormally regulated in rat injured spinal cord models. The weight drop method was used to establish rat spinal cord injury at T9. Western blot analysis and immunohistochemical staining revealed Ras expression was dramatically elevated, and the phosphorylations of A-Raf, B-Raf and C-Raf were all upregulated in the injured spinal cord. Both mitogen-activated protein kinase kinase 1/2 and ERK1/2, which belong to the Ras/Raf signaling kinases, were upregulated. These results indicate that Ras/Raf/ ERK1/2 signaling may be upregulated in injured spinal cord and are involved in recovery after spinal cord injury.展开更多
Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell diff...Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively, mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.展开更多
BACKGROUND Intestinal inflammation is a common digestive tract disease, which is usually treated with hormone medicines. Hormone medicines are effective to some extent, but long-term use of them may bring about many c...BACKGROUND Intestinal inflammation is a common digestive tract disease, which is usually treated with hormone medicines. Hormone medicines are effective to some extent, but long-term use of them may bring about many complications.AIM To explore the protective effects of panax notoginseng saponin(PNS) against dextran sulfate sodium(DSS)-induced intestinal inflammatory injury through phosphoinositide-3-kinase protein kinase B(PI3K/AKT) signaling pathway inhibition in rats.METHODS Colitis rat models were generated via DSS induction, and rats were divided into control(no modeling), DSS, DSS + PNS 50 mg/k, and DSS + PNS 100 mg/kg groups. Then, the intestinal injury, oxidative stress parameters, inflammatory indices, tight junction proteins, apoptosis, macrophage polarization, and TLR4/AKT signaling pathway in colon tissues from rats in each of the groups were detected. The PI3 K/AKT signaling pathway in the colon tissue of rats was blocked using the PI3K/AKT signaling pathway inhibitor, LY294002.RESULTS Compared with rats in the control group, rats in the DSS group showed significantly shortened colon lengths, and significantly increased disease activity indices, oxidative stress reactions and inflammatory indices, as well as significantly decreased expression of tight junction-associated proteins. In addition, the DSS group showed significantly increased apoptotic cell numbers,and showed significantly increased M1 macrophages in spleen and colon tissues.They also showed significantly decreased M2 macrophages in colon tissues, as well as activation of the PI3K/AKT signaling pathway(all P < 0.05). Compared with rats in the DSS group, rats in the DSS + PNS group showed significantly lengthened colon lengths, decreased disease activity indices, and significantly alleviated oxidative stress reactions and inflammatory responses. In addition, this group showed significantly increased expression of tight junction-associated proteins, significantly decreased apoptotic cell numbers, and significantly decreased M1 macrophages in spleen and colon tissues. This group further showed significantly increased M2 macrophages in colon tissues, and significantly suppressed activation of the PI3K/AKT signaling pathway, as well as a dose dependency(all P < 0.05). When the PI3K/AKT signaling pathway was inhibited, the apoptosis rate of colon tissue cells in the DSS + LY294002 group was significantly lower than that of the DSS group(P < 0.05).CONCLUSION PNS can protect rats against DSS-induced intestinal inflammatory injury by inhibiting the PI3K/AKT signaling pathway, and therefore may be potentially used in the future as a drug for colitis.展开更多
BACKGROUND: Studies have shown that electro-acupuncture at the Ren meridian could improve proliferation of subventricular zone neural stem cells in cerebral-ischemic rats. However, there are few reports on the influe...BACKGROUND: Studies have shown that electro-acupuncture at the Ren meridian could improve proliferation of subventricular zone neural stem cells in cerebral-ischemic rats. However, there are few reports on the influence of electro-acupuncture at the Du meridian on neural stem cell proliferation. OBJECTIVE: To observe the influence of electro-acupuncture at Ren and Du meridians on neural stem cell proliferation in the subventricular zone and altered signal transduction in cerebral ischemia rats. DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Laboratory of Human Anatomy, Medical College of Sun Yat-sen University from May 2006 to February 2008. MATERIALS: Mouse anti-rat bromodeoxyuridine (BrdU) monoclonal antibody was provided by Sigma, USA; mouse anti-rat nestin monoclonal antibody and extracellular signal-regulated protein kinase (ERK) specific inhibitor PD98059 were provided by Calbiochem, Germany; acupuncture needle was provided by Suzhou Acupuncture Supplies, China. METHODS: A total of 126 rats were randomly assigned to four groups: model (n = 36), Du meridian (n = 36), Ren/Du meridian (n = 36), and Ren/Du meridian + PD98059 (n = 18). Rats in the Ren/Du meridian + PD98059 group were observed on days 7 (n = 6) and 14 (n = 12) after cerebral ischemia injury. Rats in the model, Du meridian, and Ren/Du meridian groups were observed on days 7, 14, and 28 after cerebral ischemia injury, with 12 rats per group at each time point. Thread occlusion was used to establish middle cerebral artery occlusion models. Electro-acupuncture was performed at Renzhong (DU 26) and Baihui (DU 20) acupoints in the Du meridian group, as well as Chengjiang (RN 24), Guanyuan (RN 4), Renzhong, and Baihuiacupoints in the Ren/Du meridian and Ren/Du meridian + PD98059 groups 2 days after model establishment. In addition, electro-acupuncture stimulation with disperse-dense waves was performed, with 30 Hz disperse wave, 100 Hz dense wave, and 5 V intensity for 20 minutes. Rats in the Ren/Du meridian + PD98059 group were treated with 0.2 pg PD98059 injection into the subventricular zone, 2 pL per rat. Rats in the model group were not treated with electro-acupuncture. MAIN OUTCOME MEASURES: BrdU/nestin immunofluorescent staining was used to detect proliferating neural stem cells in the subventricular zone of cerebral ischemia rats; Western blot was used to determine phosphorylated ERK1 and 2 (pERK1/2) expression in the subventricular zone. RESULTS: On days 14 and 28 after cerebral ischemia, there were significantly more BrdU-positive and BrdU/nestin-positive cells in the Ren/Du meridian group compared with the Du meridian group (P 〈 0.05). PD98059 decreased the number of BrdU-positive and BrdU/nestin-positive cells induced by electro-acupuncture at the/:ten and Du meridians (P 〈 0.05). On days 7, 14, and 28 after treatment, pERK1/2 expression was significantly greater in the Du meridian and Ren/Du meridian groups compared with the model group (P 〈 0.05). The promoting effect of electro-acupuncture at Ren and Du meridians on ERK1/2 phosphorylation was superior to electro-acupuncture at the Du meridian alone on day 14 after model induction (P 〈 0.05). However, PD98059 completely abolished the promoting effect of electro-acupuncture at Ren/Du meridians on pERK1/2 expression (P 〈 0.05). CONCLUSION: Electro-acupuncture at Ren and Du meridians increased proliferation of subventricular zone neural stem cells, which was related to activation of the ERK pathway in a rat model of cerebral ischemia injury.展开更多
Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-reg...Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-regulated kinase(ERK)pathway is thought to increase cell proliferation and to protect cells from apoptosis.The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.Methods:Four human colon cancer cell lines,HCT1116 and Lovo(wildtype p53),DLD1 and SW1116(mutant p53),were used.Lovo stable transfectants with XAF1 sense and antisense were established.The effects of dominant-negative MEK1(DN-MEK1)and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined.The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay.Cell proliferation was measured by MTT assay.Apoptosis was determined by Hoechst 33258 staining.Results:U0126 increased the expression of XAF1 in a time-and dose-dependent manner.A similar result was obtained in cells transfected with DN-MEK1 treatment.Conversely,the expression of XIAP was down-regulated.Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection.rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression.Overexpression of XAF1 was more sensitive to U0126-induced apoptosis,whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.Conclusions:XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation,which required de novo protein synthesis.The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.展开更多
AIM:To explore the role and mechanisms of extracellular signal-regulated protein kinase-mitogen-activated protein kinase(ERK-MAPK) signaling in pentagastrinregulated growth of large intestinal carcinoma.METHODS:HT-29 ...AIM:To explore the role and mechanisms of extracellular signal-regulated protein kinase-mitogen-activated protein kinase(ERK-MAPK) signaling in pentagastrinregulated growth of large intestinal carcinoma.METHODS:HT-29 cells were incubated in different media and divided into the control group,pentagastrin group,proglumide group,and pentagastrin + proglumide group.No reagent was added to the control group,and other groups were incubated with reagent at different concentrations.Changes in proliferation of HT-29 cells were detected by MTT assay,and the optimal concentrations of pentagastrin and proglumide were determined.The changes in proliferation index(PI) and apoptosis rate(AR) of HT-29 cells were detected by Annexin V-fluorescein isothiocyanate flow cytometry.mRNA expression of pentagastrin receptor/cholecystokinin-B receptor(CCK-BR),ERK1/2 and K-ras were detected by reverse transcriptase polymerase chain reaction.The protein and phosphorylation level of ERK1/2 and K-ras were detected by western blotting.All data were analyzed by analysis of variance and SNK-q test.RESULTS:The proliferation of HT-29 cells was stimulated by pentagastrin at a concentration of 6.25-100 mg/L,and the optimal concentration of pentagastrin was 25.0 mg/L(F = 31.36,P < 0.05).Proglumide had no obvious effect on the proliferation of HT-29 cells,while it significantly inhibited the proliferation of HT-29 cells stimulated by pentagastrin when the concentration of proglumide was 8.0-128.0 mg/L,and the optimal concentration was 32.0 mg/L(F = 24.31,P < 0.05).The PI of the pentagastrin(25.0 mg/L) group was 37.5% ± 5.2%,which was significantly higher than 27.7% ± 5.0% of the control group and 27.3% ± 5.8% of the pentagastrin(25.0 mg/L) + proglumide(32.0 mg/L) group(Q = 4.56-4.75,P < 0.05).The AR of the pentagastrin(25.0 mg/L) group was 1.9% ± 0.4%,which was significantly lower than 2.5% ± 0.4% of the control group and 2.4% ± 0.3% of the pentagastrin(25.0 mg/L) + proglumide(32.0 mg/L) group(Q = 4.23-4.06,P < 0.05).mRNA expression of CCK-BR was detected in HT-29 cells.The phosphorylation levels of ERK1/2 protein and phosphorylated K-ras protein of the pentagastrin group were 0.43% ± 0.04% and 0.45% ± 0.06%,which were significantly higher than 0.32% ± 0.02% and 0.31% ± 0.05% of the control group(Q = 7.78-4.95,P < 0.05),and 0.36% ± 0.01% and 0.35% ± 0.04% of the pentagastrin + proglumide group(Q = 5.72-4.08,P < 0.05).There were no significant differences in the mRNA and protein expression of ERK1/2 and K-ras among the control,pentagastrin,proglumide and pentagastrin + proglumide groups(F = 0.52,0.72,0.78,0.28;P > 0.05).CONCLUSION:Gastrin stimulates proliferation of HT-29 cells and inhibits apoptosis by upregulating phosphorylation of ERK and K-ras through the Ras-Raf-MEK1/2-ERK1/2 pathway,and this is restrained by proglumide.展开更多
Aim: To determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth. Methods: The cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were...Aim: To determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth. Methods: The cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were determined by proliferation assay and fluorescence-activated cell sorter. Western blot analysis was carried out to evaluate the effects of dexamethasone on phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and expression of cyclin D1 in DU145 cells with or without glucocorticoid receptor (GR) antagonist RU486. Reverse transcription- polymerase chain reaction verified the expression of GR mRNA in DU145 cells. Results: Dexamethasone significantly inhibited DU 145 cell proliferation at the G0/G1 phase. Westem blot analysis showed a dramatic reduction of ERK1/2 activity and cyclin D1 expression in dexamethasone-treated cells. The decreased phosphorylation of ERK1/2 in dexamethasone-treated cells was attenuated by GR blockade. Additionally, the effects of dexamethasone in inhibiting cyclin D1 expression were altered by GR blockade. Conclusion: Dexamethasone suppresses DU145 cell proliferation and cell cycle, and the underlying mechanisms are through the inhibition of phosphorylation of ERK1/2 and cyclin D1 expression. The inhibition of ERK1/2 phosphorylation and cyclin D1 expression is attenuated by GR blockade, suggesting that GR regulates ERK1/2 and cyclin D1 pathways. These observations suggest that dexamethasone has a potential clinical application in prostate cancer therapy.展开更多
The RON receptor tyrosine kinase, a member of the MET proto-oncogene family, is a pathogenic factor im- plicated in tumor malignancy. Specifically, aberrations in RON signaling result in increased cancer cell growth, ...The RON receptor tyrosine kinase, a member of the MET proto-oncogene family, is a pathogenic factor im- plicated in tumor malignancy. Specifically, aberrations in RON signaling result in increased cancer cell growth, survival, invasion, angiogenesis, and drug resistance. Biochemical events such as ligand binding, receptor over- expression, generation of structure-defected variants, and point mutations in the kinase domain contribute to RON signaling activation. Recently, functional crosstalk between RON and signaling proteins such as MET and EFGR has emerged as an additional mechanism for RON activation, which is critical for tumorigenic develop- ment. The RON signaling crosstalk acts either as a regulatory feedback loop that strengthens or enhances tumor- igenic phenotype of cancer cells or serves as a signaling compensatory pathway providing a growth/survival ad- vantage for cancer cells to escape targeted therapy. Moreover, viral oncoproteins derived from Friend leukemia or Epstein-Barr viruses interact with RON to drive viral oncogenesis. In cancer cells, RON signaling is integrated into cellular signaling network essential for cancer cell growth and survival. These activities provide the mo- lecular basis of targeting RON for cancer treatment. In this review, we will discuss recent data that uncover the mechanisms of RON activation in cancer cells, review evidence of RON signaling crosstalk relevant to cancer malignancy, and emphasize the significance of the RON signaling addiction by cancer cells for tumor therapy. Understanding aberrant RON signaling will not only provide insight into the mechanisms of tumor pathogenesis, but also lead to the development of novel strategies for molecularly targeted cancer treatment.展开更多
Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2)induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protei...Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2)induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protein expressions were measured by Western blot.MnSOD2,Drp1 and PGC-1αmRNA expressions were detected by real time PCR.Results:Results showed that high glucose significantly up-regulated the protein expressions of MYPT1,pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells;while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose.Importantly,fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-la in HK-2 cell=s induced by high glucose.Conclusions:Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α.Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy.展开更多
It has been established that cancer can be promoted and exacerbated by inflammation.Hepatocellular carcinoma(HCC) is the fifth most common cancer worldwide,and its long-term prognosis remains poor.Although HCC is a co...It has been established that cancer can be promoted and exacerbated by inflammation.Hepatocellular carcinoma(HCC) is the fifth most common cancer worldwide,and its long-term prognosis remains poor.Although HCC is a complex and heterogeneous tumor with several genomic mutations,it usually develops in the context of chronic liver damage and inflammation,suggesting that understanding the mechanism(s) of inflammation-mediated hepatocarcinogenesis is essential for the treatment and prevention of HCC.Chronic liver damage induces a persistent cycle of necroinflammation and hepatocyte regeneration,resulting in genetic mutations in hepatocytes and expansion of initiated cells,eventually leading to HCC development.Recently,several inflammation-and stress-related signaling pathways have been identified as key players in these processes,which include the nuclear factor B,signal transducer and activator of transcription,and stress-activated mitogen-activated protein kinase pathways.Although these pathways may suggest potential therapeutic targets,they have a wide range of functions and complex crosstalk occurs among them.This review focuses on recent advances in our understanding of the roles of these signaling pathways in hepatocarcinogenesis.展开更多
AIM: To investigate whether extracellular signal-regu- lated kinase 1 (ERK1) is activated and associated with hepatic stellate cell (HSC) proliferation in fibrotic rat liver tissue. METHODS: Rat hepatic fibrosis was i...AIM: To investigate whether extracellular signal-regu- lated kinase 1 (ERK1) is activated and associated with hepatic stellate cell (HSC) proliferation in fibrotic rat liver tissue. METHODS: Rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were eval- uated by hematoxylin and eosin staining, and Masson’ s trichrome method. ERK1 mRNA in rat liver tissue was determined by reverse transcription-polymerase chain reaction, while the distribution of ERK1 was assessed by immunohistochemistry. ERK1 protein was detected by Western blotting analysis. The number of activated HSCs was quantified after alpha smooth muscle actin (α-SMA) staining. RESULTS: With the development of hepatic fibrosis, the positive staining cells of α-SMA increased obviously, and mainly resided in the portal ducts. Fiber septa and peri- sinuses were accompanied with proliferating bile ducts. The positive staining areas of the rat livers in model groups 1-4 wk after ligation of common bile duct (12.88% ± 2.63%, 22.65% ± 2.16%, 27.45% ± 1.86%, 35.25% ± 2.34%, respectively) were significantly larger than those in the control group (5.88% ± 1.46%, P < 0.01). With the development of hepatic fibrosis, the positive cells of ERK1 increased a lot, and were mainly distributed in portal ducts, fiber septa around the bile ducts, vas- cular endothelial cells and perisinusoidal cells. Western blotting analysis displayed that the expression of ERK1 and ERK2 protein was up-regulated during the model course, and its level was the highest 4 wk after opera- tion, being 3.9-fold and 7.2-fold higher in fibrotic rat liver than in controls. ERK1 mRNA was expressed in normal rat livers as well, which was up-regulated two days af- ter BDL and reached the highest 4 wk after BDL. The expression of ERK1 was positively correlated with α-SMAexpression (r = 0.958,P < 0.05).展开更多
Portal hypertension(PHT) is an important consequence of liver cirrhosis, which can lead to complications that adversely affect a patient's quality of life and survival, such as upper gastrointestinal bleeding, asc...Portal hypertension(PHT) is an important consequence of liver cirrhosis, which can lead to complications that adversely affect a patient's quality of life and survival, such as upper gastrointestinal bleeding, ascites, and portosystemic encephalopathy. In recent years, advances in molecular biology have led to major discoveries in the pathological processes of PHT, including the signaling pathways that may be involved: PI3 K-AKT-mTOR, RhoA/Rho-kinase, JAK2/STAT3, and farnesoid X receptor. However, the pathogenesis of PHT is complex and there are numerous pathways involved. Therefore, the targeting of signaling pathways for medical management is lagging. This article summarizes the progress that has been made in understanding the signaling pathways in PHT, and provides ideas for treatment of the disorder.展开更多
Tongluojiunao (TLJN) is an herbal medicine consisting of two main components, geniposide and ginsenoside Rg1. TLJN has been shown to protect primary cultured hippocampal neurons. How-ever, its mechanism of action re...Tongluojiunao (TLJN) is an herbal medicine consisting of two main components, geniposide and ginsenoside Rg1. TLJN has been shown to protect primary cultured hippocampal neurons. How-ever, its mechanism of action remains unclear. In the present study, primary cultured hippocampal neurons treated with Aβ1-42 (10 μmol/L) signiifcantly increased the release of lactate dehydroge-nase, which was markedly reduced by TLJN (2 μL/mL), speciifcally by the component geniposide (26 μmol/L), but not ginsenoside Rg1 (2.5 μmol/L). hTe estrogen receptor inhibitor, ICI182780 (1 μmol/L), did not block TLJN-or geniposide-mediated decrease of lactate dehydrogenase under Aβ1-42-exposed conditions. However, the phosphatidyl inositol 3-kinase or mitogen-activated protein kinase pathway inhibitor, LY294002 (50 μmol/L) or U0126 (10 μmol/L), respectively blo cked the decrease of lactate dehydrogenase mediated by TLJN or geniposide. hTerefore, these results suggest that the non-classical estrogen pathway (i.e., phosphatidyl inositol 3-kinase or mitogen-activated protein kinase) is involved in the neuroprotective effect of TLJN, speciifcally its component, geniposide, against Aβ1-42-mediated cell death in primary cultured hippocampal neurons.展开更多
Objective: To study the influence of glycyrrhetinic acid(GA) on bronchial asthma(BA)smooth muscle proliferation and apoptosis as well as inflammatory factor expression and its molecular mechanism.Methods: Male SD guin...Objective: To study the influence of glycyrrhetinic acid(GA) on bronchial asthma(BA)smooth muscle proliferation and apoptosis as well as inflammatory factor expression and its molecular mechanism.Methods: Male SD guinea pigs were selected and made into asthma models, bronchial asthma smooth muscle cells were cultured and divided into BA group, GA group and GA + LM group that were treated with serum-free RPMI1640 culture medium, serumfree RPMI1640 culture medium containing 50 ng/mL glycyrrhetinic acid, serum-free RPMI1640 culture medium containing 50 ng/mL glycyrrhetinic acid and 100 ng/mL LM22B-10 respectively; normal guinea pigs were collected and bronchial smooth muscle cells were cultured as control group. The cell proliferation activity as well as the expression of proliferation and apoptosis genes, inflammatory factors and p-ERK1/2 was determined.Results: Proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6,YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in BA group were significantly higher than those of control group while m RNA expression levels of Bax,caspase-9 as well as caspase-3 were significantly lower than that of control group(P < 0.05); proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in GA group were significantly lower than those of BA group(P < 0.05) while the m RNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly higher than those of BA group(P < 0.05); proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40 of airway smooth muscle cell in GA + LM group were significantly higher than those of GA group(P < 0.05) while m RNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly lower that of GA group(P < 0.05).Conclusion: GA can inhibit the proliferation of bronchial smooth muscle cells and reduce the expression of inflammatory factors by inhibiting the phosphorylation of ERK1/2.展开更多
BACKGROUND: The pathogenesis of hepatic fibrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentia...BACKGROUND: The pathogenesis of hepatic fibrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentiation. The aim of this study was to investigate the effects of PD98059, a specific inhibitor of ERK, on the cell cycle, cell proliferation, secretion of type I collagen and expression of cyclin D1 mRNA, CDK4 mRNA and transforming growth factor-beta 1 (TGF-beta 1) mRNA in rat hepatic stellate cells (HSCs) stimulated by acetaldehyde. METHODS: Rat HSCs stimulated by acetaldehyde were incubated with PD98059 at different concentrations. The cell cycle was analysed by flow cytometry. Cell proliferation was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of cyclin D1, CDK4 and TGF-beta 1 was examined using the reverse transcriptase-polymerase chain reaction. Type I collagen in the culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: 20, 50 and 100 mu mol/L PD98059 significantly inhibited the proliferation and provoked a G0/G1-phase arrest of acetaldehyde-induced HSCs in a dose-dependent manner. The secretion of type I collagen and the expression of cyclin D1, CDK4 and TGF-beta 1 mRNA in acetaldehyde-induced HSCs were markedly inhibited by 50 and 100 mu mol/L PD98059, respectively. CONCLUSIONS: The ERK pathway regulates the cell proliferation, secretion of type I collagen and the expression of TGF-beta 1 mRNA in rat HSCs stimulated by acetaldehyde, which is likely related to its regulative effect on the cell cycle.展开更多
Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from inju...Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from injury in a diabetic model after cerebral ischemia/reperfusion. In this study, transient whole-brain ischemia was induced by four-vessel occlusion in normal and diabetic rats, and extracellular signal-regulated kinase 1/2 inhibitor (U0126) was administered into diabetic rats 30 minutes before ischemia as a pretreatment. Results showed that the number of surviving neurons in the hippocampal CA1 region was reduced, extracellular signal-regulated kinase 1/2 phosphorylation and KuT0 activity were decreased, and pro-apoptotic Bax expression was upregulated after intervention using U0126. These findings demonstrate that inhibition of extracellular signal-regulated kinase 1/2 activity aggravated neuronal loss in the hippocampus in a diabetic rat after cerebral ischemia/reperfusion, further decreased DNA repairing ability and ac- celerated apoptosis in hippocampal neurons. Extracellular signal-regulated kinase 1/2 activation plays a neuroprotective role in hippocampal neurons in a diabetic rat after cerebral ischemia/ reperfusion.展开更多
文摘BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.
基金Supported by National Natural Science Foundation of China (No.81100649)
文摘AIMTo investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase (ERK) signaling pathway in vitro.METHODSThe expression levels of phosphorylated ERK (P-ERK), keratin family members including K19 and K10 and the ocular master control gene Pax-6 were measured in 16 surgically excised pterygium tissues and 12 eye bank conjunctiva. In colony-forming cell assays, the differences in clone morphology and in K10, K19, P-ERK and Pax-6 expression between the head and body were investigated. When cocultured with the ERK signaling pathway inhibitor PD98059, the changes in clone morphology, colony-forming efficiency, differentiated marker K10, K19 and Pax-6 expression and P-ERK protein expression level were examined by immunoreactivity and Western blot analysis.RESULTSThe expression of K19 and Pax-6 decreased in the pterygium, especially in the head. No staining of K10 was found in the normal conjunctiva epithelium, but it was found to be expressed in the superficial cells in the head of the pterygium. Characteristic upregulation of P-ERK was observed by immunohistochemistry. The clone from the head with more differentiated cells in the center expressed more K10, and the clone from the body expressed more K19. The P-ERK protein level increased in the pterygium epithelium compared with conjunctiva and decreased when cocultured with PD98059. The same medium with the ERK inhibitor PD98059 was more effective in promoting clonal growth than conventional medium with 3T3 murine feeder layers. It was observed that the epithelium clone co-cultured with the inhibitor had decreased K10 expression and increased K19 and Pax-6 expression.CONCLUSIONWe suggest ERK signaling pathway activation might play a role in the pterygium epithelium abnormal differentiation.
基金Supported by National Natural Science Foundation of China,No.81160057,No.81860102,and No.82060102Natural Science Foundation of Hainan Province,High-level Personnel Program,No.821RC1116+1 种基金Research Project of Health Industry in Hainan Province,No.20A200066Hainan Provincial Clinical Medical Center.
文摘BACKGROUND Immunological dysfunction-induced low-grade inflammation is regarded as one of the predominant pathogenetic mechanisms in post-infectious irritable bowel syndrome(PI-IBS).γδT cells play a crucial role in innate and adaptive immunity.Adenosine receptors expressed on the surface ofγδT cells participate in intestinal inflammation and immunity regulation.AIM To investigate the role ofγδT cell regulated by adenosine 2A receptor(A2AR)in PI-IBS.METHODS The PI-IBS mouse model has been established with Trichinella spiralis(T.spiralis)infection.The intestinal A2AR and A2AR inγδT cells were detected by immunohistochemistry,and the inflammatory cytokines were measured by western blot.The role of A2AR on the isolatedγδT cells,including proliferation,apoptosis,and cytokine production,were evaluated in vitro.Their A2AR expression was measured by western blot and reverse transcription polymerase chain reaction(RT-PCR).The animals were administered with A2AR agonist,or A2AR antagonist.Besides,γδT cells were also injected back into the animals,and the parameters described above were examined,as well as the clinical features.Furthermore,the A2AR-associated signaling pathway molecules were assessed by western blot and RT-PCR.RESULTS PI-IBS mice exhibited elevated ATP content and A2AR expression(P<0.05),and suppression of A2AR enhanced PI-IBS clinical characteristics,indicated by the abdominal withdrawal reflex and colon transportation test.PI-IBS was associated with an increase in intestinal T cells,and cytokine levels of interleukin-1(IL-1),IL-6,IL-17A,and interferon-α(IFN-α).Also,γδT cells expressed A2AR in vitro and generated IL-1,IL-6,IL-17A,and IFN-α,which can be controlled by A2AR agonist and antagonist.Mechanistic studies demonstrated that the A2AR antagonist improved the function ofγδT cells through the PKA/CREB/NF-κB signaling pathway.CONCLUSION Our results revealed that A2AR contributes to the facilitation of PI-IBS by regulating the function ofγδT cells via the PKA/CREB/NF-κB signaling pathway.
基金supported by National Natural Science Foundation of China(32072212)Multi-Year Research Grant of University of Macao(MYRG2018-00169-ICMS)+5 种基金Science and Technology Development Fund of Macao(FDCT)(0098/2020/A)MICINN supporting the Ramón y Cajal grant for M.A.Prieto(RYC-201722891)Jianbo Xiao(RYC2020-030365-I)Xunta de Galicia supporting the Axudas Conecta Peme,the IN852A 2018/58 Neuro Food Project,the program EXCELENCIA-ED431F 2020/12the pre-doctoral grants of P.García-Oliveira(ED481A-2019/295)to Ibero-American Program on Science and Technology(CYTED-AQUA-CIBUS,P317RT0003).
文摘Dietary flavonoids are abundant in natural plants and possess multiple pharmacological and nutritional activities.In this study,apigenin,luteolin,and baicalein were chosen to evaluate their anti-diabetic effect in high-glucose and dexamethasone induced insulin-resistant(IR)HepG2 cells.All flavonoids improves the glucose consumption and glycogen synthesis abilities in IR-HepG2 cells via activating glucose transporter protein 4(GLUT4)and phosphor-glycogen synthase kinase(GSK-3β).These fl avonoids signifi cantly inhibited the production of reactive oxygen species(ROS)and advanced glycation end-products(AGEs),which were closely related to the suppression of the phosphorylation form of NF-κB and P65.The expression levels of insulin receptor substrate-1(IRS-1),insulin receptor substrate-2(IRS-2)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway in IR-HepG2 cells were all partially activated by the fl avonoids,with variable effects.Furthermore,the intracellular metabolic conditions of the fl avonoids were also evaluated.
基金funded by the National Natural Science Foundation for Young Scholars of China,No.81101362 and 81401784the Key Project of National Natural Science Foundation of China,No.81330042
文摘An increasing number of studies report that the Ras/Raf/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway has a death-promoting apoptotic function in neural cells. We hypothesized that the Ras/Raf/ERK1/2 signaling pathway may be abnormally regulated in rat injured spinal cord models. The weight drop method was used to establish rat spinal cord injury at T9. Western blot analysis and immunohistochemical staining revealed Ras expression was dramatically elevated, and the phosphorylations of A-Raf, B-Raf and C-Raf were all upregulated in the injured spinal cord. Both mitogen-activated protein kinase kinase 1/2 and ERK1/2, which belong to the Ras/Raf signaling kinases, were upregulated. These results indicate that Ras/Raf/ ERK1/2 signaling may be upregulated in injured spinal cord and are involved in recovery after spinal cord injury.
基金sponsored by the National Natural Science Foundation of China,No.81102595the Natural Science Foundation of Guangxi,No.2012GXNSFAA053113
文摘Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively, mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.
基金National Natural Science Foundation of China,No.81704059Scientific Research Project of Hebei Province Traditional Chinese Medicine Administration,No.2017130。
文摘BACKGROUND Intestinal inflammation is a common digestive tract disease, which is usually treated with hormone medicines. Hormone medicines are effective to some extent, but long-term use of them may bring about many complications.AIM To explore the protective effects of panax notoginseng saponin(PNS) against dextran sulfate sodium(DSS)-induced intestinal inflammatory injury through phosphoinositide-3-kinase protein kinase B(PI3K/AKT) signaling pathway inhibition in rats.METHODS Colitis rat models were generated via DSS induction, and rats were divided into control(no modeling), DSS, DSS + PNS 50 mg/k, and DSS + PNS 100 mg/kg groups. Then, the intestinal injury, oxidative stress parameters, inflammatory indices, tight junction proteins, apoptosis, macrophage polarization, and TLR4/AKT signaling pathway in colon tissues from rats in each of the groups were detected. The PI3 K/AKT signaling pathway in the colon tissue of rats was blocked using the PI3K/AKT signaling pathway inhibitor, LY294002.RESULTS Compared with rats in the control group, rats in the DSS group showed significantly shortened colon lengths, and significantly increased disease activity indices, oxidative stress reactions and inflammatory indices, as well as significantly decreased expression of tight junction-associated proteins. In addition, the DSS group showed significantly increased apoptotic cell numbers,and showed significantly increased M1 macrophages in spleen and colon tissues.They also showed significantly decreased M2 macrophages in colon tissues, as well as activation of the PI3K/AKT signaling pathway(all P < 0.05). Compared with rats in the DSS group, rats in the DSS + PNS group showed significantly lengthened colon lengths, decreased disease activity indices, and significantly alleviated oxidative stress reactions and inflammatory responses. In addition, this group showed significantly increased expression of tight junction-associated proteins, significantly decreased apoptotic cell numbers, and significantly decreased M1 macrophages in spleen and colon tissues. This group further showed significantly increased M2 macrophages in colon tissues, and significantly suppressed activation of the PI3K/AKT signaling pathway, as well as a dose dependency(all P < 0.05). When the PI3K/AKT signaling pathway was inhibited, the apoptosis rate of colon tissue cells in the DSS + LY294002 group was significantly lower than that of the DSS group(P < 0.05).CONCLUSION PNS can protect rats against DSS-induced intestinal inflammatory injury by inhibiting the PI3K/AKT signaling pathway, and therefore may be potentially used in the future as a drug for colitis.
基金the National Natural Science Foundation of China,No.30371808the Natural Science Foundation of Guangdong Province,No.5009688
文摘BACKGROUND: Studies have shown that electro-acupuncture at the Ren meridian could improve proliferation of subventricular zone neural stem cells in cerebral-ischemic rats. However, there are few reports on the influence of electro-acupuncture at the Du meridian on neural stem cell proliferation. OBJECTIVE: To observe the influence of electro-acupuncture at Ren and Du meridians on neural stem cell proliferation in the subventricular zone and altered signal transduction in cerebral ischemia rats. DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Laboratory of Human Anatomy, Medical College of Sun Yat-sen University from May 2006 to February 2008. MATERIALS: Mouse anti-rat bromodeoxyuridine (BrdU) monoclonal antibody was provided by Sigma, USA; mouse anti-rat nestin monoclonal antibody and extracellular signal-regulated protein kinase (ERK) specific inhibitor PD98059 were provided by Calbiochem, Germany; acupuncture needle was provided by Suzhou Acupuncture Supplies, China. METHODS: A total of 126 rats were randomly assigned to four groups: model (n = 36), Du meridian (n = 36), Ren/Du meridian (n = 36), and Ren/Du meridian + PD98059 (n = 18). Rats in the Ren/Du meridian + PD98059 group were observed on days 7 (n = 6) and 14 (n = 12) after cerebral ischemia injury. Rats in the model, Du meridian, and Ren/Du meridian groups were observed on days 7, 14, and 28 after cerebral ischemia injury, with 12 rats per group at each time point. Thread occlusion was used to establish middle cerebral artery occlusion models. Electro-acupuncture was performed at Renzhong (DU 26) and Baihui (DU 20) acupoints in the Du meridian group, as well as Chengjiang (RN 24), Guanyuan (RN 4), Renzhong, and Baihuiacupoints in the Ren/Du meridian and Ren/Du meridian + PD98059 groups 2 days after model establishment. In addition, electro-acupuncture stimulation with disperse-dense waves was performed, with 30 Hz disperse wave, 100 Hz dense wave, and 5 V intensity for 20 minutes. Rats in the Ren/Du meridian + PD98059 group were treated with 0.2 pg PD98059 injection into the subventricular zone, 2 pL per rat. Rats in the model group were not treated with electro-acupuncture. MAIN OUTCOME MEASURES: BrdU/nestin immunofluorescent staining was used to detect proliferating neural stem cells in the subventricular zone of cerebral ischemia rats; Western blot was used to determine phosphorylated ERK1 and 2 (pERK1/2) expression in the subventricular zone. RESULTS: On days 14 and 28 after cerebral ischemia, there were significantly more BrdU-positive and BrdU/nestin-positive cells in the Ren/Du meridian group compared with the Du meridian group (P 〈 0.05). PD98059 decreased the number of BrdU-positive and BrdU/nestin-positive cells induced by electro-acupuncture at the/:ten and Du meridians (P 〈 0.05). On days 7, 14, and 28 after treatment, pERK1/2 expression was significantly greater in the Du meridian and Ren/Du meridian groups compared with the model group (P 〈 0.05). The promoting effect of electro-acupuncture at Ren and Du meridians on ERK1/2 phosphorylation was superior to electro-acupuncture at the Du meridian alone on day 14 after model induction (P 〈 0.05). However, PD98059 completely abolished the promoting effect of electro-acupuncture at Ren/Du meridians on pERK1/2 expression (P 〈 0.05). CONCLUSION: Electro-acupuncture at Ren and Du meridians increased proliferation of subventricular zone neural stem cells, which was related to activation of the ERK pathway in a rat model of cerebral ischemia injury.
基金Shanghai Medical Key Discipline Construction Foundation(05-Ⅲ-005-017).
文摘Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-regulated kinase(ERK)pathway is thought to increase cell proliferation and to protect cells from apoptosis.The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.Methods:Four human colon cancer cell lines,HCT1116 and Lovo(wildtype p53),DLD1 and SW1116(mutant p53),were used.Lovo stable transfectants with XAF1 sense and antisense were established.The effects of dominant-negative MEK1(DN-MEK1)and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined.The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay.Cell proliferation was measured by MTT assay.Apoptosis was determined by Hoechst 33258 staining.Results:U0126 increased the expression of XAF1 in a time-and dose-dependent manner.A similar result was obtained in cells transfected with DN-MEK1 treatment.Conversely,the expression of XIAP was down-regulated.Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection.rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression.Overexpression of XAF1 was more sensitive to U0126-induced apoptosis,whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.Conclusions:XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation,which required de novo protein synthesis.The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.
基金Supported by Natural Science Foundation of Anhui Province,No.1408085MH148Natural Science Fund of Education Bureau of Anhui Province,No.kj2010b242+1 种基金Natural Science Fund of Wannan Medical College,No.wk2012zf02the key science and technology project of Wuhu City,No.health-2-4
文摘AIM:To explore the role and mechanisms of extracellular signal-regulated protein kinase-mitogen-activated protein kinase(ERK-MAPK) signaling in pentagastrinregulated growth of large intestinal carcinoma.METHODS:HT-29 cells were incubated in different media and divided into the control group,pentagastrin group,proglumide group,and pentagastrin + proglumide group.No reagent was added to the control group,and other groups were incubated with reagent at different concentrations.Changes in proliferation of HT-29 cells were detected by MTT assay,and the optimal concentrations of pentagastrin and proglumide were determined.The changes in proliferation index(PI) and apoptosis rate(AR) of HT-29 cells were detected by Annexin V-fluorescein isothiocyanate flow cytometry.mRNA expression of pentagastrin receptor/cholecystokinin-B receptor(CCK-BR),ERK1/2 and K-ras were detected by reverse transcriptase polymerase chain reaction.The protein and phosphorylation level of ERK1/2 and K-ras were detected by western blotting.All data were analyzed by analysis of variance and SNK-q test.RESULTS:The proliferation of HT-29 cells was stimulated by pentagastrin at a concentration of 6.25-100 mg/L,and the optimal concentration of pentagastrin was 25.0 mg/L(F = 31.36,P < 0.05).Proglumide had no obvious effect on the proliferation of HT-29 cells,while it significantly inhibited the proliferation of HT-29 cells stimulated by pentagastrin when the concentration of proglumide was 8.0-128.0 mg/L,and the optimal concentration was 32.0 mg/L(F = 24.31,P < 0.05).The PI of the pentagastrin(25.0 mg/L) group was 37.5% ± 5.2%,which was significantly higher than 27.7% ± 5.0% of the control group and 27.3% ± 5.8% of the pentagastrin(25.0 mg/L) + proglumide(32.0 mg/L) group(Q = 4.56-4.75,P < 0.05).The AR of the pentagastrin(25.0 mg/L) group was 1.9% ± 0.4%,which was significantly lower than 2.5% ± 0.4% of the control group and 2.4% ± 0.3% of the pentagastrin(25.0 mg/L) + proglumide(32.0 mg/L) group(Q = 4.23-4.06,P < 0.05).mRNA expression of CCK-BR was detected in HT-29 cells.The phosphorylation levels of ERK1/2 protein and phosphorylated K-ras protein of the pentagastrin group were 0.43% ± 0.04% and 0.45% ± 0.06%,which were significantly higher than 0.32% ± 0.02% and 0.31% ± 0.05% of the control group(Q = 7.78-4.95,P < 0.05),and 0.36% ± 0.01% and 0.35% ± 0.04% of the pentagastrin + proglumide group(Q = 5.72-4.08,P < 0.05).There were no significant differences in the mRNA and protein expression of ERK1/2 and K-ras among the control,pentagastrin,proglumide and pentagastrin + proglumide groups(F = 0.52,0.72,0.78,0.28;P > 0.05).CONCLUSION:Gastrin stimulates proliferation of HT-29 cells and inhibits apoptosis by upregulating phosphorylation of ERK and K-ras through the Ras-Raf-MEK1/2-ERK1/2 pathway,and this is restrained by proglumide.
文摘Aim: To determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth. Methods: The cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were determined by proliferation assay and fluorescence-activated cell sorter. Western blot analysis was carried out to evaluate the effects of dexamethasone on phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and expression of cyclin D1 in DU145 cells with or without glucocorticoid receptor (GR) antagonist RU486. Reverse transcription- polymerase chain reaction verified the expression of GR mRNA in DU145 cells. Results: Dexamethasone significantly inhibited DU 145 cell proliferation at the G0/G1 phase. Westem blot analysis showed a dramatic reduction of ERK1/2 activity and cyclin D1 expression in dexamethasone-treated cells. The decreased phosphorylation of ERK1/2 in dexamethasone-treated cells was attenuated by GR blockade. Additionally, the effects of dexamethasone in inhibiting cyclin D1 expression were altered by GR blockade. Conclusion: Dexamethasone suppresses DU145 cell proliferation and cell cycle, and the underlying mechanisms are through the inhibition of phosphorylation of ERK1/2 and cyclin D1 expression. The inhibition of ERK1/2 phosphorylation and cyclin D1 expression is attenuated by GR blockade, suggesting that GR regulates ERK1/2 and cyclin D1 pathways. These observations suggest that dexamethasone has a potential clinical application in prostate cancer therapy.
基金supported in part by National Institutes of Health grantR01 CA91980 (MHW)a grant from the Amarillo Area Foundation(MHW)supported by NIH grants R01 CA112029 and CA121211
文摘The RON receptor tyrosine kinase, a member of the MET proto-oncogene family, is a pathogenic factor im- plicated in tumor malignancy. Specifically, aberrations in RON signaling result in increased cancer cell growth, survival, invasion, angiogenesis, and drug resistance. Biochemical events such as ligand binding, receptor over- expression, generation of structure-defected variants, and point mutations in the kinase domain contribute to RON signaling activation. Recently, functional crosstalk between RON and signaling proteins such as MET and EFGR has emerged as an additional mechanism for RON activation, which is critical for tumorigenic develop- ment. The RON signaling crosstalk acts either as a regulatory feedback loop that strengthens or enhances tumor- igenic phenotype of cancer cells or serves as a signaling compensatory pathway providing a growth/survival ad- vantage for cancer cells to escape targeted therapy. Moreover, viral oncoproteins derived from Friend leukemia or Epstein-Barr viruses interact with RON to drive viral oncogenesis. In cancer cells, RON signaling is integrated into cellular signaling network essential for cancer cell growth and survival. These activities provide the mo- lecular basis of targeting RON for cancer treatment. In this review, we will discuss recent data that uncover the mechanisms of RON activation in cancer cells, review evidence of RON signaling crosstalk relevant to cancer malignancy, and emphasize the significance of the RON signaling addiction by cancer cells for tumor therapy. Understanding aberrant RON signaling will not only provide insight into the mechanisms of tumor pathogenesis, but also lead to the development of novel strategies for molecularly targeted cancer treatment.
基金supported by National Natural Science Foundation of China(No.81560124)Hainan Key Research and Development Projects(ZDYF2018131,ZDYF2017113,ZDYF2017114)+1 种基金Hainan Science and Technology Planned Project of Youth Outstanding Ability of Innovation(201704)Hainan Health Family Planning Industry Project(13A210277)
文摘Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2)induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protein expressions were measured by Western blot.MnSOD2,Drp1 and PGC-1αmRNA expressions were detected by real time PCR.Results:Results showed that high glucose significantly up-regulated the protein expressions of MYPT1,pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells;while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose.Importantly,fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-la in HK-2 cell=s induced by high glucose.Conclusions:Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α.Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy.
基金Supported by A fellowship from the Daiichi Sankyo Foundation of Life Science,to Nakagawa H
文摘It has been established that cancer can be promoted and exacerbated by inflammation.Hepatocellular carcinoma(HCC) is the fifth most common cancer worldwide,and its long-term prognosis remains poor.Although HCC is a complex and heterogeneous tumor with several genomic mutations,it usually develops in the context of chronic liver damage and inflammation,suggesting that understanding the mechanism(s) of inflammation-mediated hepatocarcinogenesis is essential for the treatment and prevention of HCC.Chronic liver damage induces a persistent cycle of necroinflammation and hepatocyte regeneration,resulting in genetic mutations in hepatocytes and expansion of initiated cells,eventually leading to HCC development.Recently,several inflammation-and stress-related signaling pathways have been identified as key players in these processes,which include the nuclear factor B,signal transducer and activator of transcription,and stress-activated mitogen-activated protein kinase pathways.Although these pathways may suggest potential therapeutic targets,they have a wide range of functions and complex crosstalk occurs among them.This review focuses on recent advances in our understanding of the roles of these signaling pathways in hepatocarcinogenesis.
文摘AIM: To investigate whether extracellular signal-regu- lated kinase 1 (ERK1) is activated and associated with hepatic stellate cell (HSC) proliferation in fibrotic rat liver tissue. METHODS: Rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were eval- uated by hematoxylin and eosin staining, and Masson’ s trichrome method. ERK1 mRNA in rat liver tissue was determined by reverse transcription-polymerase chain reaction, while the distribution of ERK1 was assessed by immunohistochemistry. ERK1 protein was detected by Western blotting analysis. The number of activated HSCs was quantified after alpha smooth muscle actin (α-SMA) staining. RESULTS: With the development of hepatic fibrosis, the positive staining cells of α-SMA increased obviously, and mainly resided in the portal ducts. Fiber septa and peri- sinuses were accompanied with proliferating bile ducts. The positive staining areas of the rat livers in model groups 1-4 wk after ligation of common bile duct (12.88% ± 2.63%, 22.65% ± 2.16%, 27.45% ± 1.86%, 35.25% ± 2.34%, respectively) were significantly larger than those in the control group (5.88% ± 1.46%, P < 0.01). With the development of hepatic fibrosis, the positive cells of ERK1 increased a lot, and were mainly distributed in portal ducts, fiber septa around the bile ducts, vas- cular endothelial cells and perisinusoidal cells. Western blotting analysis displayed that the expression of ERK1 and ERK2 protein was up-regulated during the model course, and its level was the highest 4 wk after opera- tion, being 3.9-fold and 7.2-fold higher in fibrotic rat liver than in controls. ERK1 mRNA was expressed in normal rat livers as well, which was up-regulated two days af- ter BDL and reached the highest 4 wk after BDL. The expression of ERK1 was positively correlated with α-SMAexpression (r = 0.958,P < 0.05).
基金Supported by the National Natural Science Foundation of China,No.81573948
文摘Portal hypertension(PHT) is an important consequence of liver cirrhosis, which can lead to complications that adversely affect a patient's quality of life and survival, such as upper gastrointestinal bleeding, ascites, and portosystemic encephalopathy. In recent years, advances in molecular biology have led to major discoveries in the pathological processes of PHT, including the signaling pathways that may be involved: PI3 K-AKT-mTOR, RhoA/Rho-kinase, JAK2/STAT3, and farnesoid X receptor. However, the pathogenesis of PHT is complex and there are numerous pathways involved. Therefore, the targeting of signaling pathways for medical management is lagging. This article summarizes the progress that has been made in understanding the signaling pathways in PHT, and provides ideas for treatment of the disorder.
基金supported by the National Natural Science Foundation of China No.81072901the New Teacher Fund for Doctor Station,Ministry of Education,No.20120013110013+1 种基金grants from the Nautical Traditional Chinese Medicine Discipline,No.522/0100604054grants from the Nautical Traditional Chinese Medicine Collaborative Innovation Center,No.522/0100604299
文摘Tongluojiunao (TLJN) is an herbal medicine consisting of two main components, geniposide and ginsenoside Rg1. TLJN has been shown to protect primary cultured hippocampal neurons. How-ever, its mechanism of action remains unclear. In the present study, primary cultured hippocampal neurons treated with Aβ1-42 (10 μmol/L) signiifcantly increased the release of lactate dehydroge-nase, which was markedly reduced by TLJN (2 μL/mL), speciifcally by the component geniposide (26 μmol/L), but not ginsenoside Rg1 (2.5 μmol/L). hTe estrogen receptor inhibitor, ICI182780 (1 μmol/L), did not block TLJN-or geniposide-mediated decrease of lactate dehydrogenase under Aβ1-42-exposed conditions. However, the phosphatidyl inositol 3-kinase or mitogen-activated protein kinase pathway inhibitor, LY294002 (50 μmol/L) or U0126 (10 μmol/L), respectively blo cked the decrease of lactate dehydrogenase mediated by TLJN or geniposide. hTerefore, these results suggest that the non-classical estrogen pathway (i.e., phosphatidyl inositol 3-kinase or mitogen-activated protein kinase) is involved in the neuroprotective effect of TLJN, speciifcally its component, geniposide, against Aβ1-42-mediated cell death in primary cultured hippocampal neurons.
基金supported by Guangdong Medical Science and Technology Research Fund Project(No:A2017331)
文摘Objective: To study the influence of glycyrrhetinic acid(GA) on bronchial asthma(BA)smooth muscle proliferation and apoptosis as well as inflammatory factor expression and its molecular mechanism.Methods: Male SD guinea pigs were selected and made into asthma models, bronchial asthma smooth muscle cells were cultured and divided into BA group, GA group and GA + LM group that were treated with serum-free RPMI1640 culture medium, serumfree RPMI1640 culture medium containing 50 ng/mL glycyrrhetinic acid, serum-free RPMI1640 culture medium containing 50 ng/mL glycyrrhetinic acid and 100 ng/mL LM22B-10 respectively; normal guinea pigs were collected and bronchial smooth muscle cells were cultured as control group. The cell proliferation activity as well as the expression of proliferation and apoptosis genes, inflammatory factors and p-ERK1/2 was determined.Results: Proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6,YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in BA group were significantly higher than those of control group while m RNA expression levels of Bax,caspase-9 as well as caspase-3 were significantly lower than that of control group(P < 0.05); proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in GA group were significantly lower than those of BA group(P < 0.05) while the m RNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly higher than those of BA group(P < 0.05); proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40 of airway smooth muscle cell in GA + LM group were significantly higher than those of GA group(P < 0.05) while m RNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly lower that of GA group(P < 0.05).Conclusion: GA can inhibit the proliferation of bronchial smooth muscle cells and reduce the expression of inflammatory factors by inhibiting the phosphorylation of ERK1/2.
文摘BACKGROUND: The pathogenesis of hepatic fibrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentiation. The aim of this study was to investigate the effects of PD98059, a specific inhibitor of ERK, on the cell cycle, cell proliferation, secretion of type I collagen and expression of cyclin D1 mRNA, CDK4 mRNA and transforming growth factor-beta 1 (TGF-beta 1) mRNA in rat hepatic stellate cells (HSCs) stimulated by acetaldehyde. METHODS: Rat HSCs stimulated by acetaldehyde were incubated with PD98059 at different concentrations. The cell cycle was analysed by flow cytometry. Cell proliferation was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of cyclin D1, CDK4 and TGF-beta 1 was examined using the reverse transcriptase-polymerase chain reaction. Type I collagen in the culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: 20, 50 and 100 mu mol/L PD98059 significantly inhibited the proliferation and provoked a G0/G1-phase arrest of acetaldehyde-induced HSCs in a dose-dependent manner. The secretion of type I collagen and the expression of cyclin D1, CDK4 and TGF-beta 1 mRNA in acetaldehyde-induced HSCs were markedly inhibited by 50 and 100 mu mol/L PD98059, respectively. CONCLUSIONS: The ERK pathway regulates the cell proliferation, secretion of type I collagen and the expression of TGF-beta 1 mRNA in rat HSCs stimulated by acetaldehyde, which is likely related to its regulative effect on the cell cycle.
基金supported by the Hebei Province Natural Science Program,No.H2012401007a grant from the foundation Key Project of Hebei Province Education Ministry,No.ZD2010106
文摘Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from injury in a diabetic model after cerebral ischemia/reperfusion. In this study, transient whole-brain ischemia was induced by four-vessel occlusion in normal and diabetic rats, and extracellular signal-regulated kinase 1/2 inhibitor (U0126) was administered into diabetic rats 30 minutes before ischemia as a pretreatment. Results showed that the number of surviving neurons in the hippocampal CA1 region was reduced, extracellular signal-regulated kinase 1/2 phosphorylation and KuT0 activity were decreased, and pro-apoptotic Bax expression was upregulated after intervention using U0126. These findings demonstrate that inhibition of extracellular signal-regulated kinase 1/2 activity aggravated neuronal loss in the hippocampus in a diabetic rat after cerebral ischemia/reperfusion, further decreased DNA repairing ability and ac- celerated apoptosis in hippocampal neurons. Extracellular signal-regulated kinase 1/2 activation plays a neuroprotective role in hippocampal neurons in a diabetic rat after cerebral ischemia/ reperfusion.