Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

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.

Loss of monopolar spindle-binding protein 3B expression promotes colorectal cancer malignant behaviors by activation of target of rapamycin kinase/autophagy signaling

BACKGROUND Monopolar spindle-binding protein 3B(MOB3B)functions as a signal transducer and altered MOB3B expression is associated with the development of human cancers.AIM To investigate the role of MOB3B in colorectal cancer(CRC).METHODS This study collected 102 CRC tissue samples for immunohistochemical detection of MOB3B expression for association with CRC prognosis.After overexpression and knockdown of MOB3B expression were induced in CRC cell lines,changes in cell viability,migration,invasion,and gene expression were assayed.Tumor cell autophagy was detected using transmission electron microscopy,while nude mouse xenograft experiments were performed to confirm the in-vitro results.RESULTS MOB3B expression was reduced in CRC vs normal tissues and loss of MOB3B expression was associated with poor CRC prognosis.Overexpression of MOB3B protein in vitro attenuated the cell viability as well as the migration and invasion capacities of CRC cells,whereas knockdown of MOB3B expression had the opposite effects in CRC cells.At the molecular level,microtubule-associated protein light chain 3 II/I expression was elevated,whereas the expression of matrix metalloproteinase(MMP)2,MMP9,sequestosome 1,and phosphorylated mechanistic target of rapamycin kinase(mTOR)was downregulated in MOB3B-overexpressing RKO cells.In contrast,the opposite results were observed in tumor cells with MOB3B knockdown.The nude mouse data confirmed these in-vitro findings,i.e.,MOB3B expression suppressed CRC cell xenograft growth,whereas knockdown of MOB3B expression promoted the growth of CRC cell xenografts.CONCLUSION Loss of MOB3B expression promotes CRC development and malignant behaviors,suggesting a potential tumor suppressive role of MOB3B in CRC by inhibition of mTOR/autophagy signaling.

TopoisomeraseⅡalpha promotes gallbladder cancer proliferation and metastasis through activating phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.

Micro RNA-21 promotes phosphatase gene and protein kinase B/phosphatidylinositol 3-kinase expression in colorectal cancer

AIM: To explore the regulatory mechanism of the target gene of micro RNA-21(mi R-21), phosphatase gene(p TEN), and its downstream proteins, protein kinase B(AKT) and phosphatidylinositol 3-kinase(p I3K), in colorectal cancer(CRC) cells. METHODS: Quantitative real-time p CR(q RT-p CR) and Western blot were used to detect the expression levels of mi R-21 and p TEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of p TEN m RNA and its downstream proteins AKT and p I3 K in HCT116 cells after downregulating mi R-21 were investigated. RESULTS: Comparing the mi R-21 expression in CRC cells, the expression levels of mi R-21 were highest in HCT116 cells, and the expression levels of mi R-21 were lowest in SW480 cells. In comparing mi R-21 and p TEN expression in CRC cells, we found that the protein expression levels of mi R-21 and p TEN were inversely correlated(p < 0.05); when mi R-21 expression was reduced, m RNA expression levels of p TEN did not significantly change(p > 0.05), but the expression levels of its protein significantly increased(p < 0.05). In comparing the levels of p TEN protein and downstream AKT and p I3 K in HCT116 cells after downregulation of mi R-21 expression, the levels of AKT and p I3 K protein expression significantly decreased(p < 0.05). CONCLUSION: p TEN is one of the direct target genesof mi R-21. Thus, phosphatase gene and its downstream AKT and p I3 K expression levels can be regulated by regulating the expression levels of mi R-21, which in turn regulates the development of CRC.

Cytotoxicity of nonylphenol on spermatogonial stem cells via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway

BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stable degradation product widely used in daily life and production and has been proven to affect male fertility.However,the underlying mechanisms therein are unclear.Thus,it is necessary to study the effect and mechanism of NP on spermatogonial stem cells(SSCs).AIM To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway.METHODS SSCs were treated with NP at 0,10,20 or 30μmol.MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs.Flow cytometry was conducted to measure SSC apoptosis.The expression of Bad,Bcl-2,cytochrome-c,pro-Caspase 9,SOX-2,OCT-4,Nanog,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot,and the mRNA expression of SOX-2,OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction.RESULTS Compared with untreated cells(0μmol NP),SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2,Nanog,OCT-4,SOX-2,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,and PLZF(P<0.05),whereas the expression of Bad,cytochrome-c,and pro-Caspase 9 increased significantly(P<0.05).We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K,AKT,mTORC1,and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs(P<0.05).NP exerted the greatest effect at 30μmol among all NP concentrations.CONCLUSION NP attenuated the proliferation,differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress.The associated mechanism may be related to the PI3K/AKT/mTOR pathway.

Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway

Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU. In this study, we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process. Western blot analysis demonstrated that telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid, while they were expressed in PAJU cells transfected with a telencephalin expression plasmid. After treatment with 1.0 nM amyloid beta protein 42, expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished, while levels of phosphorylated ezrin/radixin/moesin increased. In addition, the high levels of telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

Mechanism of total glucosides from Chishao(Radix Paeoniae Rubra)on proliferation and apoptosis of hepatocellular carcinoma cells via phosphatase and tensin homolog deleted on chromosome ten/phosphatidylinositol 3-kinase/protein kinase B signaling pathway

OBJECTIVE:To investigate the possible molecular mechanism of total glycosides of Chishao(Radix Paeoniae Rubra)(TG-RPR)on proliferation and apoptosis of hepatocellular carcinoma cells.METHODS:The proliferation of TG-RPR on Hep G2 cells was detected using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.The apoptosis of Hep G2 cells was measured by annexin V-FITC/double staining.The phosphatase and tensin homolog deleted on chromosome ten(PTEN)/phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(Akt)signaling pathway was evaluated by Western Blot and reverse transcription-polymerase chain reaction(RT-PCR).RESULTS:TG-RPR can up-regulation the expression of pro-apoptotic factors such as PTEN and BCL2-Associated X(Bax),down-regulation the expression of anti-apoptotic factors including B-cell lymphoma-2(Bcl-2),PI3 K,and Akt.CONCLUSION:TG-RPR significantly inhibits the proliferation of Hep G2 cells in a dose-dependent manner and promotes apoptosis.These results demonstrated TG-RPR has significant inhibitory effect on Hep G2 cells.These results identify a critical role of TG-RPR in proliferation and apoptosis of Hep G2 cells via modulating PTEN/PI3 K/Akt signaling pathway.TG-RPR may offer a promise as a potential pharmaceutical therapy for hepatocellular carcinoma.

Response of Subcutaneous Xenografts of Endometrial Cancer in Nude Mice to Inhibitors of Phosphatidylinositol 3-Kinase/Akt and Mitogen-Activated Protein Kinase (MAPK) Pathways: An Effective Therapeutic Strategy for Endometrial Cancer

Objective: This study was designed to explore whether inhibition of the extracellular-regulated kinase (ERK) and phosphatidylinositol-3-kinase (PI3K) signaling pathways can inhibit the growth of xenografts of endometrial cancer cell lines with different estrogen receptors (ER) profiles in vivo and to provide preliminary laboratory basis for the probability of endometrial adenocarcinoma treatment with blockage of the two pathways, especially to endometrial cancer with low ER status. Methods: Human endometrial cancer Ishikawa bearing ER and HEC-1Awith low ER status cells were subcutaneously injected into BALB/c nude mice to establish endometrial cancer xenograft tumor models. The effects of PI3K/Akt inhibitor LY294002, MAPK/ERK1/2 inhibitor PD-98059 and their combinations on the growth of the xenograft tumors and apoptotic state of Ishikawa and HEC-1Acells were tested in vivo using the inhibitory rate, the terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, H/E-stain. Western blot analysis was used to detect the alterations of activated ERK (P-ERK) and AKT (P-AKT) during this process. Results: LY294002, a PI3K/Akt pathway inhibitor, induced significant suppression in the growth of both Ishikawa and HEC-1Acell xenograft tumors, concomitant with increased apoptosis in xenografts as evidenced by TUNEL. A similar effect was also observed when the MAPK/ERK1/2 signaling pathway was inhibited by PD98059. Concurrent inhibition of the PI3K/Akt and MAPK/ERK1/2 pathways showed enhanced anti-tumor effects in vivo as indicated by increased apoptosis. At the same time, the levels of P-ERK and P-AKT in both xenograft tumors decreased, and their levels in combination group was the lowest. Conclusions: PD98059, LY294002 and their combinations showed remarkable inhibitory effects on xenograft tumors of endometrial carcinoma cell lines with different expression status of ER in vivo through blockage of PI3K/Akt and MAPK/ERK1/2 signaling pathways. This suggests that targeting these pathways may be an effective therapeutic strategy against endometrial carcinomas, especially for ER-negative cancers which show poor response to endocrinal therapy.

Protective effects of panax notoginseng saponin on dextran sulfate sodium-induced colitis in rats through phosphoinositide-3-kinase protein kinase B signaling pathway inhibition

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.

Alisol B acetate induces apoptosis of SGC7901 cells via mitochondrial and phosphatidylinositol 3-kinases/Akt signaling pathways

AIM: To examine the effect of alisol B acetate on the growth of human gastric cancer cell line SGC7901 and its possible mechanism of action. METHODS: The cytotoxic effect of alisol B acetate on SGC7901 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Phase-contrast and electron microscopy were used to observe the morphological changes. Cell cycle and mitochondrial transmembrane potential (△Ψm) were determined by flow cytometry. Western blotting was used to detect the expression of apoptosis-regulated gene Bcl-2, Bax, Apaf-1, caspase-3, caspase-9, Akt, P-Akt and phosphatidylinositol 3-kinases (PI3K). RESULTS: Alisol B acetate inhibited the proliferation of SGC7901 cell line in a time-and dose-dependent manner. PI staining showed that alisol B acetate can change the cell cycle distribution of SGC7901, increase the proportion of cells in G0-G1 phase and decrease the proportion of S phase cells and G2-M phase cells. Alisol B acetate at a concentration of 30 μmol/L induced apoptosis after 24, 48 and 72 h incubation, with occurrence rates of apoptotic cells of 4.36%, 14.42% and 21.16%, respectively. Phase-contrast and electron microscopy revealed that the nuclear fragmentation and chromosomal condensed, cells shrank and attachment loss appeared in the SGC7901 treated with alisol B acetate. Apoptosis of SGC7901cells was associated with cell cycle arrest, caspase-3 and caspase-9 activation, loss of mitochondrial membrane potential and up-regulation of the ratio of Bax/Bcl-2 and inhibition of the PI3K/Akt. CONCLUSION: Alisol B acetate exhibits an antiproliferative effect in SGC7901 cells by inducing apoptosis. Apoptosis of SGC7901 cells involves mitochondria-caspase and PI3K/Akt dependent pathways.

Anti-silencing function 1B knockdown suppresses the malignant phenotype of colorectal cancer by inactivating the phosphatidylinositol 3-kinase/AKT pathway

BACKGROUND Mounting studies have highlighted the pivotal influence of anti-silencing function 1B(ASF1B)on the malignancy of cancers.AIM To explore the influence and mechanism of ASF1B in colorectal cancer(CRC).METHODS Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect mRNA expression of ASF1B.Immunohistochemical staining was performed to detect protein expression of ASF1B and Ki67 in tumor tissues.Western blot analysis was used to determine levels of ASF1B and proliferation/epithelial mesenchymal transition(EMT)/stemness-related proteins.In addition,the proliferation of CRC cells was assessed using Cell Counting Kit-8 and 5-Ethynyl-2’-Deoxyuridine assays.The migration and invasion of CRC cells were evaluated using transwell assays.Stemness of CRC cells was tested using the sphere formation assay.To construct a xenograft tumor model,HCT116 cells were introduced into mouse flanks via subcutaneous injection.RESULTS ASF1B expression was markedly increased in CRC tissues and cells,and it was inversely correlated with overall survival of CRC patients and was positively associated with the tumor node metastasis(TNM)stage of CRC patients.Silencing of ASF1B suppressed proliferation,migration,invasion,stemness and EMT of CRC cells as well as tumorigenesis of xenograft mice.Furthermore,protein levels of Pphosphatidylinositol 3-kinase(p-PI3K)and p-AKT were decreased after silencing of ASF1B in CRC cells.The inhibitory effects of ASF1B knockdown on cell proliferation,stemness and EMT were partly abolished by PI3K activator in CRC cells.CONCLUSION Silencing of ASF1B inactivated the PI3K/AKT pathway to suppress CRC malignancy in vitro.

黄芪阳和汤调控PI3K/AKT/NF-κB信号通路促进糖尿病足溃疡大鼠创面愈合

目的基于磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/核因子-κB(NF-κB)信号通路探究黄芪阳和汤对糖尿病足溃疡(DFU)大鼠创面愈合的影响。方法构建DFU大鼠模型,将建模成功的48只大鼠随机分为模型组,黄芪阳和汤低(8.5 g/kg)、高(17 g/kg)剂量组,黄芪阳和汤高剂量(17 g/kg)+LY294002(PI3K/AKT通路抑制剂,0.3 mg/kg)组;每组12只;另取12只大鼠为对照组。各组大鼠给予对应药物干预,连续4周。第14、28天给药后,观察大鼠一般状态及创面变化,计算创面愈合率,检测大鼠空腹血糖(FBG)水平和大鼠创面周围组织经皮氧分压(TcpO2);酶联免疫吸附试验检测大鼠血清血管内皮生长因子(VEGF)、缺氧诱导因子-1α(HIF-1α)、C反应蛋白(CRP)、白细胞介素(IL)-6水平;苏木素-伊红染色观察大鼠创面组织病理学变化;免疫组织化学染色测定大鼠创面组织微血管密度;蛋白免疫印迹法检测大鼠创面组织中PI3K、磷酸化PI3K(p-PI3K)、AKT、磷酸化AKT(p-AKT)、NF-κB p65、磷酸化NF-κB p65(p-NF-κB p65)、NF-κB抑制蛋白α(IκB-α)蛋白表达。结果对照组大鼠毛色光滑,饮食、饮水、排泄均正常,较活跃,创面愈合快,创面组织炎症反应较轻,新生血管较多,肉芽组织中成纤维细胞及胶原基质丰富;模型组大鼠毛色暗淡无光泽,活动减少,且出现多饮、多食、多尿症状,创面颜色较深,且周围组织出现水肿、溃疡,创面组织可见大量炎性细胞浸润,伴组织坏死、渗出,新生血管及成纤维细胞较少,创面愈合率、创面周围组织TcpO2、血清VEGF、HIF-1α、创面组织微血管密度、p-PI3K、p-AKT、IκB-α蛋白表达水平降低,FBG、血清CRP、IL-6、创面组织p-NF-κB p65蛋白表达升高(P<0.05);与模型组相比,黄芪阳和汤低、高剂量组大鼠状态逐渐改善,创面组织病变程度依次减轻,创面愈合率、创面周围组织TcpO2、血清VEGF、HIF-1α、创面组织微血管密度、p-PI3K、p-AKT、IκB-α蛋白表达水平依次升高,FBG、血清CRP、IL-6、创面组织p-NF-κB p65蛋白表达依次降低(P<0.05);LY294002能部分逆转高剂量黄芪阳和汤对DFU大鼠的治疗作用(P<0.05)。结论黄芪阳和汤能调控PI3K/AKT/NF-κB信号通路,抑制DFU大鼠炎症反应,促进血管新生,从而促进创面愈合。

Fenofibrate Pre-treatment Suppressed Inflammation by Activating Phosphoinositide 3 Kinase/Protein Kinase B(PI3K/Akt) Signaling in Renal Ischemia-Reperfusion Injury

The aim of this study was to investigate the possible beneficial effects of Fenofibrate on renal ischemia-reperfusion injury（IRI） in mice and its potential mechanism. IRI was induced by bilateral renal ischemia for 60 min followed by reperfusion for 24 h. Eighteen male C57BL/6 mice were randomly divided into three groups： sham-operated group（sham）, IRI＋saline group（IRI group）, IRI＋Fenofibrate（FEN） group. Normal saline or Fenofibrate（3 mg/kg） was intravenously injected 60 min before renal ischemia in IRI group and FEN group, respectively. Blood samples and renal tissues were collected at the end of reperfusion. The renal function, histopathologic changes, and the expression levels of pro-inflammatory cytokines [interleukin-8（IL-8）, tumor necrosis factor alpha（TNF-α） and IL-6] in serum and renal tissue homogenate were assessed. Moreover, the effects of Fenofibrate on activating phosphoinositide 3 kinase/protein kinase B（PI3K/Akt） signaling and peroxisome proliferator-activated receptor-α（PPAR-α） were also measured in renal IRI. The results showed that plasma levels of blood urea nitrogen and creatinine, histopathologic scores and the expression levels of TNF-α, IL-8 and IL-6 were significantly lower in FEN group than in IRI group. Moreover, Fenofibrate pretreatment could further induce PI3K/Akt signal pathway and PPAR-α activation following renal IRI. These findings indicated PPAR-α activation by Fenofibrate exerts protective effects on renal IRI in mice by suppressing inflammation via PI3K/Akt activation. Thus, Fenofibrate could be a novel therapeutic alternative in renal IRI.

Expression of phosphatidylinositol-3 kinase and effects of inhibitor Wortmannin on expression of tumor necrosis factor-α in severe acute pancreatitis associated with acute lung injury

BACKGROUND: Acute lung injury(ALI) is a common and serious complication of severe acute pancreatitis(SAP). The study aimed to investigate the protective effect and mechanism of phosphatidylinositol-3 kinase(PI3K) inhibitor Wortmannin in SAP associated with ALI.METHODS: Ninety rats were randomly divided into three groups: sham operation(SO) group(n=30), SAP group(n=30), and SAP+Wortmannin(SAP+W) group(n=30). SAP model was induced by retrograde injection of 4% sodium taurocholate into the biliopancreatic duct of rats. The rate of lung water content, myeloperoxidase(MPO), matrix metalloproteinase 9(MMP-9), protein kinase B(PKB), abdphosphorylation of protein kinase B(P-PKB) activity in the lung tissue were evaluated.RESULTS: In the SAP group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours(P<0.05); the rate of lung water content, MPO and TNF-α activity were also gradually increased, and the degree of lung lesion gradually increased(P<0.05). In the SAP+Wortmannin group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours; it was higher than that in the SO group(P<0.05), but significantly lower than that in the SAP group(P<0.05). The rest indicators in the SAP+Wortmannin group were also significantly decreased as compared with the SAP group(P<0.05).CONCLUSIONS: The expression of phosphatidylinositol-3 kinase/protein kinase B was elevated in severe pancreatitis rats with lung injury. This suggested that PI3 K signal transduction pathway is involved in the control and release of proinfl ammatory cytokines TNF-α, which may play an important role in the pathogenesis of severe acute pancreatitis associated with lung injury. This finding indicated that Wortmannin can block the PI3 K signal transduction pathway, and inhibit the release of infl ammatory factor TNF-α.

白芍总苷调节磷脂酰肌醇3-激酶/蛋白激酶B信号通路对大鼠原发性痛经的改善作用实验研究

目的:探讨白芍总苷(TGP)调节磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路对大鼠原发性痛经(PDM)的改善作用。方法:将雌性SD大鼠采用随机数字表法分为正常对照组(Nor组)、PDM模型组(PDM组)、低剂量TGP组(TGP-L组)、高剂量TGP组(TGP-H组)和高剂量TGP+PI3K激活剂740Y-P组(TGP-H+740Y-P组),每组12只。除Nor组外,其余各组大鼠采用苯甲酸雌二醇联合缩宫素制备大鼠PDM模型,并分别用50、100 mg/kg TGP干预TGP-L组、TGP-H组大鼠,TGP-H+740Y-P组大鼠另需10 mg/kg 740Y-P干预。末次给药后观察大鼠的扭体反应,记录扭体次数和潜伏期,行扭体反应评分。HE染色观察大鼠子宫组织的病理学变化,行病理学评分。ELISA法检测大鼠子宫组织前列腺素F2α(PGF2α)、前列腺素E2(PGE2)水平以及血清β-内啡肽(β-EP)水平。免疫印迹法测量子宫组织PI3K、Akt表达及其磷酸化水平。结果:与Nor组比较,PDM组子宫病理损伤严重,子宫组织病理学评分、PGF2α以及p-PI3K、p-Akt水平升高,子宫组织PGE2及血清β-EP水平降低(均P<0.05)。与PDM组比较,TGP-L组、TGP-H组大鼠扭体反应潜伏期延长,扭体反应次数减少,扭体反应评分降低,子宫组织病理损伤减轻,子宫组织病理学评分、PGF2α以及p-PI3K、p-Akt水平降低,子宫组织PGE2及血清β-EP水平升高(均P<0.05)。高剂量TGP对大鼠PDM的改善作用更显著,而740Y-P减弱了TGP对大鼠PDM的改善作用。结论:TGP可能通过抑制PI3K/Akt信号通路改善大鼠PDM。

基于UBA2/PTEN/PI3K/Akt通路探讨蔓荆子黄素对结直肠癌细胞增殖、迁移和侵袭的影响

目的 基于泛素样修饰激活酶2(UBA2)/磷酸酶及张力蛋白同源物(PTEN)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路探究蔓荆子黄素对结直肠癌SW480细胞增殖、迁移和侵袭的影响。方法 取对数生长期的SW480细胞,对照组细胞常规培养,蔓荆子黄素组细胞加入10μmol/L蔓荆子黄素培养,UBA2抑制剂组细胞加入0.5μmol/L UBA2抑制剂培养,蔓荆子黄素+UBA2抑制剂组细胞加入10μmol/L蔓荆子黄素和0.5μmol/L UBA2抑制剂共培养。CCK-8实验检测细胞增殖情况,克隆形成实验观察细胞的单克隆形成能力,划痕实验观察细胞的迁移能力,Transwell实验观察细胞的侵袭能力,Western blot法检测细胞中UBA2/PTEN/PI3K/Akt通路相关蛋白表达情况。结果 CCK-8实验和克隆形成实验显示,UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组培养72 h后的细胞增殖吸光度OD值明显低于蔓荆子黄素组(P均<0.05),细胞克隆形成数量均明显少于蔓荆子黄素组(P均<0.05),UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组培养不同时间的细胞增殖吸光度OD值和细胞克隆形成数量比较差异均无统计学意义(P均>0.05)。划痕实验和Transwell实验显示,UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组划痕间距均明显宽于蔓荆子黄素组(P均<0.05),穿膜细胞数量均明显少于蔓荆子黄素组(P均<0.05),UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组比较差异均无统计学意义(P均>0.05)。蔓荆子黄素组、UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组细胞中PTEN蛋白相对表达量均明显高于对照组(P均<0.05),UBA2、p-PI3K、p-Akt蛋白相对表达量均明显低于对照组(P均<0.05);UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组细胞中PTEN蛋白相对表达量均明显高于蔓荆子黄素组(P均<0.05),UBA2、p-PI3K、p-Akt蛋白相对表达量均明显低于蔓荆子黄素组(P均<0.05),UBA2抑制剂组和蔓荆子黄素+UBA2抑制剂组UBA2、PTEN、p-PI3K、p-Akt蛋白相对表达量比较差异均无统计学意义(P均>0.05)。结论 蔓荆子黄素可能通过抑制UBA2/PTEN/PI3K/Akt信号通路发挥抗结直肠癌SW480细胞增殖、迁移和侵袭的能力。

薯蓣皂苷经PI3K/AKT通路对肝癌Bel-7402细胞增殖和凋亡的影响

目的分析薯蓣皂苷对肝癌Bel-7402细胞增殖和凋亡的影响并探讨其机制。方法肝癌Bel-7402细胞分为空白组和薯蓣皂苷低、中、高剂量组(给予1、2、8μmol/L的薯蓣皂苷)及薯蓣皂苷+抑制剂组(给予8μmol/L的薯蓣皂苷+10μmol/L的磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路抑制剂LY294002),于处理后12、24、36、48及72 h时采用四甲基偶氮唑盐(MTT)比色法测定细胞活力,于24 h时采用流式细胞术检测细胞凋亡情况、采用蛋白免疫印迹法(Western blot)检测p-PI3K和p-AKT的表达。结果与空白组相比较,薯蓣皂苷低、中、高剂量组细胞活力及p-PI3K、p-AKT表达均下降,凋亡率升高(P<0.05),且各剂量组间两两比较,上述指标水平差异均有统计学意义(P<0.05);与薯蓣皂苷高剂量组比较,薯蓣皂苷+抑制剂组细胞活力及p-PI3K、p-AKT表达下降,凋亡率升高(P<0.05)。结论薯蓣皂苷可能通过抑制PI3K/AKT通路抑制肝癌Bel-7402细胞增殖,诱导Bel-7402细胞凋亡。

肠病药方调控PI3K/AKT/NF-κB信号通路改善溃疡性结肠炎的研究

目的探讨肠病药方调控磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/核因子kappa B(NF-κB)信号通路对抗溃疡性结肠炎(UC)的作用机制。方法42只C57BL/6小鼠随机分为正常对照组(n=6,Control组,自由饮去离子水)、模型组(n=12,DSS组)、美沙拉嗪组(n=12,MES组)及肠病药方组(n=12,CBD组),后3组小鼠自由饮用3%葡聚糖硫酸钠(DSS)溶液7 d诱导UC模型,后两组同期灌胃美沙拉嗪或肠病药方,前两组灌胃等体积去离子水;观察实验期间小鼠体质量改变,评测小鼠疾病活动指数(DAI);造模结束后麻醉处死小鼠,测量结肠长度、观察结肠内容物及黏膜,采用苏木素-伊红(HE)染色观察各组小鼠结肠病理组织学变化,酶联免疫吸附试验(ELISA)检测各组小鼠结肠组织匀浆白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平,蛋白免疫印迹法(WB)检测各组小鼠结肠组织中磷酸化PI3K(p-PI3K)-p85、磷酸化AKT1(p-AKT1)、磷酸化NF-κB(p-NF-κB)-p65蛋白表达,实时荧光定量PCR技术(qRT-PCT)检测各组小鼠结肠组织PI3K、AKT、NF-κB信使RNA(mRNA)表达水平。结果与DSS组比较,CBD组治疗后,有效缓解UC小鼠症状,粪便性状及便血情况改善,结肠黏膜炎症浸润减少,体质量下降有所缓解,DAI评分降低及结肠长度接近正常,结肠组织匀浆中IL-6、TNF-α表达降低(P<0.05),p-PI3K-p85、p-AKT1、p-NF-κB-p65蛋白及mRNA表达明显减少(P<0.05)。结论肠病药方可缓解UC小鼠的症状、减轻结肠黏膜损伤,其机制可能与调解结肠组织中PI3K/AKT/NF-κB信号通路中相关蛋白质及mRNA表达、降低结肠组织炎症因子表达有关。

黄芩素调节PI3K/Akt/NF-κB信号通路及对细菌性脑膜炎大鼠血脑屏障的影响

目的:探讨黄芩素调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/核因子κB(NF-κB)信号通路对细菌性脑膜炎(BM)大鼠血脑屏障的影响。方法:制备大鼠BM模型,将大鼠分为对照组、模型组、黄芩素组(腹腔注射30 mg/kg黄芩素)、黄芩素+抑制剂组(腹腔注射30 mg/kg黄芩素+7.5 mg/kg LY294002),对大鼠进行Loeffler神经行为评分,H-E染色观察大鼠脑组织病理学变化,伊文思蓝染色检测血脑屏障通透性,分别检测脑脊液IL-1β、IL-6水平及白细胞计数(WBC)、脑组织含水量,水通道蛋白4(AQP4)、闭锁蛋白-5(claudin-5)、PI3K/Akt/NF-κB通路相关蛋白表达。结果:对照组大鼠脑组织结构正常,细胞排列整齐,模型组大鼠脑组织细胞排列紊乱,大量炎症细胞浸润。与对照组比较,模型组Loeffler评分及脑组织AQP4、claudin-5、p-PI3K、p-Akt表达水平显著降低,脑脊液IL-1β、IL-6水平及WBC、脑组织EB含量及含水量、p-NF-κB p65表达水平显著升高;黄芩素组较模型组病理损伤减轻,炎症细胞减少。与模型组比较,黄芩素组Loeffler评分及脑组织AQP4、claudin-5、p-PI3K、p-Akt表达水平显著升高,脑脊液IL-1β、IL-6水平及WBC、脑组织EB含量及含水量、p-NF-κB p65表达水平显著降低;LY294002可部分逆转黄芩素对BM大鼠血脑屏障通透性的改善作用。结论:黄芩素可改善BM大鼠脑水肿及血脑屏障通透性,可能与调控PI3K/Akt/NF-κB信号通路有关。

Tumor-related factor complement Clq/TNF-related protein 6 affects the development of digestive system tumors through the phosphatidylinositol 3-kinase pathway

In this editorial,we review the work of Razali et al published in World J Gas-troenterology,with a particular focus on the effect of rs10889677 variation in the phosphatidylinositol 3-kinase(PI3K)pathway and buparlisib on colitis-associated cancer.The role of PI3K in promoting cancer progression has been widely recognized,as it is involved in regulating the survival,differentiation,and prolif-eration of cancer cells.The complement Clq/TNF-related protein 6(CTRP6)is a newer tumor-associated factor.Recent studies have revealed the pro-tumor effect of CTRP6 in gastric cancer,hepatocellular carcinoma,colorectal cancer,and other gastrointestinal tumors through the PI3K pathway.This article attempts to reveal the mechanism through which the CTRP6 affects the development of digestive system tumors through the PI3K pathway by summarizing recent research.