Silvestrol alleviates glioblastoma progression through ERK pathway modulation and MANBA and NRG-1 expression

Background:Glioblastoma,a notably malignant tumor within the central nervous system,is distinguished by its aggressive behavior.Silvestrol,a robust inhibitor of the RNA helicase eukaryotic initiation factor 4A(eIF4A),has shown significant potential as an anticancer compound.Yet,the impact of silvestrol on glioblastoma,especially its molecular mechanisms,has not been fully elucidated.Methods:This investigation employed a variety of in vitro assays,such as cell counting kit-8(CCK-8),clonogenic,5-ethynyl-2′-deoxyuridine(EDU),wound healing,and flow cytometry,to evaluate cell cycle progression,apoptosis,cell viability,and migration.Western blot analysis was also performed to study the apoptosis and extracellular regulated kinase(ERK)pathways.After the ERK pathway was inhibited,differentially expressed genes(DEGs)in U87 cells were identified,followed by an analysis of target genes using the gene expression profiling interactive analysis(GEPIA)database.Results:Silvestrol significantly suppressed the proliferation,migration,and colony formation of glioma cells.It caused cell cycle arrest and enhanced apoptosis in these cells.Additionally,silvestrol stimulated the ERK pathway,with these effects being reversible by an ERK phosphorylation inhibitor.Transcriptome combined with GEPIA,GSCA,UALCAN,TIMER database screened 4 potential drug targets of silvestrol:chromosome 1 open reading frame 226(C1ORF226),mannosidase beta A(MANBA),IQ motif and Sec7 domain 2(IQSEC2),neuregulin 1(NRG-1).Among them,C1ORF226 was lower risk gene while MANBA,IQSEC2,and NRG-1 were high-risk genes.Furthermore,silvestrol notably reduced MANBA mRNA levels,which could be reversed by inhibiting ERK phosphorylation.Furthermore,silvestrol markedly decreased NRG-1 protein levels,with an additional reduction observed when the ERK pathway was blocked.Conclusion:Silvestrol’s anti-glioma effects are primarily due to the suppression of MANBA expression via the ERK pathway and possibly by hindering the translation of NRG-1 protein,thus reducing its expression.The downregulation of MANBA and NRG-1 proteins may be crucial in hindering glioma development and progression.These results highlight the intricate relationship between the ERK pathway and gene expression regulation in silvestrol’s therapeutic effectiveness against glioma.

Discovering differential protein expression caused by CagA-induced ERK pathway activation in AGS cells using the SELDI-ProteinChip platform

AIM： To identify the protein expression differences related to the CagA-induced ERK pathway activation in AGS cells. METHODS： Human AGS cells transfected with cagA and blank vector were treated with specific mitogenactivated protein kinase kinase （MEK） inhibitor. Total cell proteins were combined by strong anion exchange （SAX2） and weak cation exchange （CM10） ProteinChip arrays and analyzed using surface-enhanced laser desorption/ ionization time-of-flight mass spectrometry （SELDI-TOF- MS） proteomics technology. Protein expression profiles were compared with those of inhibitor-untreated cagA transfectants. SwissProt/TrEMBL database searching for differentially expressed proteins was carried out using the TagIdent tool with the pI and mass information. RESULTS： When a total of 16 proteins that showed expression differences in inhibitor-untreated cagA transfectants were compared with vector transfectants, three proteins with m/z 4229, 8162 and 9084 were found to have no expression differences after treatment with MEK inhibitor, while the other 13 maintained the same expression differences after inhibitor treatment. Seven pieces of meaningful matching information for the three proteins were obtained from database searching. CONCLUSION： Biomarkers with m/z 4229, 8162 and 9084 are ERKI/2 phosphorylation dependent, andtherefore are the downstream molecules of ERK1/2 in the ERK/MAPK signaling pathway. The three biomarkers may be important cancer-associated proteins according to SwissProt/TrEMBL database information.

Acupuncture at Back-Shu point improves insomnia by reducing inflammation and inhibiting the ERK/NF-κB signaling pathway

BACKGROUND Insomnia is a disease where individuals cannot maintain a steady and stable sleep state or fail to fall asleep.Western medicine mainly uses sedatives and hypnotic drugs to treat insomnia,and long-term use is prone to drug resistance and other adverse reactions.Acupuncture has a good curative effect and unique advantages in the treatment of insomnia.AIM To explore the molecular mechanism of acupuncture at Back-Shu point for the treatment of insomnia.METHODS We first prepared a rat model of insomnia,and then carried out acupuncture for 7 consecutive days.After treatment,the sleep time and general behavior of the rats were determined.The Morris water maze test was used to assess the learning ability and spatial memory ability of the rats.The expression levels of inflammatory cytokines in serum and the hippocampus were detected by ELISA.qRTPCR was used to detect the mRNA expression changes in the ERK/NF-κB signaling pathway.Western blot and immunohistochemistry were carried out to evaluate the protein expression levels of RAF-1,MEK-2,ERK1/2 and NF-κB.RESULTS Acupuncture can prolong sleep duration,and improve mental state,activity,diet volume,learning ability and spatial memory.In addition,acupuncture increased the release of 1L-1β,1L-6 and TNF-αin serum and the hippocampus and inhibited the mRNA and protein expression of the ERK/NF-κB signaling pathway.CONCLUSION These findings suggest that acupuncture at Back-Shu point can inhibit the ERK/NF-κB signaling pathway and treat insomnia by increasing the release of inflammatory cytokines in the hippocampus.

Timosaponin AⅢ induces drug-metabolizing enzymes by activating constitutive androstane receptor (CAR) via dephosphorylation of the EGFR signaling pathway

The current study aimed to assess the effect of timosaponin AⅢ(T-AⅢ)on drug-metabolizing enzymes during anticancer therapy.The in vivo experiments were conducted on nude and ICR mice.Following a 24-day administration of T-AⅢ,the nude mice exhibited an induction of CYP2B10,MDR1,and CYP3A11 expression in the liver tissues.In the ICR mice,the expression levels of CYP2B10 and MDR1 increased after a three-day T-AⅢ administration.The in vitro assessments with HepG2 cells revealed that T-AⅢ induced the expression of CYP2B6,MDR1,and CYP3A4,along with constitutive androstane receptor(CAR)activation.Treatment with CAR siRNA reversed the T-AⅢ-induced increases in CYP2B6 and CYP3A4 expression.Furthermore,other CAR target genes also showed a significant increase in the expression.The up-regulation of murine CAR was observed in the liver tissues of both nude and ICR mice.Subsequent findings demonstrated that T-AⅢ activated CAR by inhibiting ERK1/2 phosphorylation,with this effect being partially reversed by the ERK activator t-BHQ.Inhibition of the ERK1/2 signaling pathway was also observed in vivo.Additionally,T-AⅢ inhibited the phosphorylation of EGFR at Tyr1173 and Tyr845,and suppressed EGF-induced phosphorylation of EGFR,ERK,and CAR.In the nude mice,T-AⅢ also inhibited EGFR phosphorylation.These results collectively indicate that T-AⅢ is a novel CAR activator through inhibition of the EGFR pathway.

Blocking the JAK2/STAT3 and ERK pathways suppresses the proliferation of gastrointestinal cancers by inducing apoptosis

Dysregulated crosstalk between different signaling pathways contributes to tumor development,including resistance to cancer therapy.In the present study,we found that the mitogen-activated extracellular signal-regulated kinase(MEK)inhibitor trametinib failed to suppress the proliferation of PANC-1 and MGC803 cells by activating the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway,while the JAK2 inhibitor fedratinib failed to inhibit the growth of the PANC-1 cells upon stimulation of extracellular signal-regulated kinase(ERK)signaling.In particular,the most prominent enhancement of the anti-proliferative effect resulted from the concurrent blockage of the JAK2/STAT3 and ERK signaling pathways.Furthermore,the combination of the two inhibitors resulted in a reduced tumor burden in mice.Our evidence suggests novel crosstalk between JAK2/STAT3 and ERK signaling in gastric cancer(GC)and pancreatic ductal adenocarcinoma(PDAC)cells and provides a therapeutic strategy to overcome potential resistance in gastrointestinal cancer.

Experimental study on the effect of cryoablation on lung cancer mice based on MAPK/ERK signaling pathway

Objective:To study the regulatory effect of cryoablation on MAPK/ERK signaling pathway in mice with lung adenocarcinoma.Methods:Lewis mouse lung adenocarcinoma cell line was used to establish subcutaneous transplanted tumor model in C57BL/6 mice.Ten mice were randomly divided into two groups:sham operation group and cryoablation group,with 5 mice in each group.The cryoablation group was treated with double circulation-rewarming ablation,and the sham operation group was treated with incision and suture at the transplanted tumor.The tumor tissues were taken 14 days after operation.Detect the effect of cryoablation on MAPK/ERK pathway related proteins by Western blot,such as KRAS,RAF1,MEK1,ERK1/2,P-RAF1,P-MEK1,P-ERK1/2.The expression of KRAS gene was further verified by qRt-PCR.Results:Compared with the sham operation group,the phosphorylated proteins P-RAF1,P-MEK1 and P-ERK1/2 in tumor tissue after cryoablation were decreased(P<0.05),and the key molecule KRAS in MAPK/ERK pathway was decreased in protein and gene expression(P<0.05).Conclusion:Cryoablation can negatively regulate MAPK/ERK signaling pathway by down-regulating KRas expression.

Longan Aril Reverses H2O2 Cytotoxicity in PC12 Cells via RAS/MEK/ERK Signaling Pathway

[Objectives]To explore the neuroprotective effects and mechanism of Longan Aril(LA)effective parts on PC12 cells injured by H2O2.[Methods]The neuroprotective effects of LA were evaluated by the cell viability,SOD and MDA content,apoptosis assay and relative protein expression of Aβand p-Tau.The neuroprotective mechanism of LA was studied by using metabolomics and network pharmacology,and the expressions of RAS/MEK/ERK signaling pathway-related proteins were detected by western blotting.[Results]LA could improve the cell survival rate and SOD content,and reduce apoptosis and expression of Aβand p-tau.Inhibition of RAS/MEK/ERK signaling pathway is a possible mechanism of LA neuroprotective effects.[Conclusions]LA has a neuroprotective effects in vitro and be likely to inhibit the process of AD by inhibition of RAS/MEK/ERK signalling pathway.

Vitamin C induces periodontal ligament progenitor cell differentiation via activation of ERK pathway mediated by PELP1

The differentiation of periodontal ligament(PDL)progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone.Vitamin C(VC),a water-soluble nutrient that cannot be biosynthesized by humans,is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling.Therefore,the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level.We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents.During the process,VC preferentially activated ERK1/2 but did not affect JNK or p38.Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2.ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation.PELP1,a nuclear receptor co-regulator,was up-regulated under VC treatment.PELP1 knockdown inhibited ERK phosphorylation.The overexpression of PELP1 had a positive relationship with Runx2 expression.Taken together,we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis.Our fi nding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.

ERK signaling pathway may induce gemcitabine chemoresistance in pancreatic cancer cell line SW1990 by regulating the expression of mdr-1 and RRM1 gene

Objective： To investigate the relationship between extracellular signal-regulated kinase （ERK） pathway, multidrug resistance gene （mdr-1）, ribonucleotide recluctase M1 （RRM1） gene and their roles in gemcitabine （GEM） chemoresistance in pancreatic cancer cell line SW1990. Methods： The GEM-resistance cell model was constructed by a stepwise method. Immunohistochemistry was used to measure the expression of ERK protein （ERK1/2） in the established cell strains in a semiquantitative way. The mRNA expression of mdr-1 and RRM1 were detected by RT-PCR. MTT assay was performed to determine the IC50 value. Results： The established GEM-resistant cell strains were able to gain stable growth and passage ability in the medium contained different concentration levels of GEM （0, 30, 60, 100, 150 and 200 nmol/L）. The expression of ERK protein, mdr-1 and RRM1 gene were elevated accompanied by the increase of GEM concentration. There was a highly positive correlation between mdr-1, RRM1 expression and GEM-resistanca level （r = 0.960, P = 0.002 and r = 0.966, P = 0.002）. The expression of ERK protein also correlated with the mdr-1 and RRM1 level （r = -0.943, P = 0.005 and r = -0.883, P = 0.02）. At the GEM-resistance level of 200 nmol/L, the grey scale value of ERK1/2 was 164.22 ±13.17, mdr-1/β-actin and RRM1/β-actin were 1.41 ±0.04 and 1.45 ± 0.18, respectively; after treated with ERK pathway inhibitor U0126, these values synchronously decreased to 186.85 ± 13.14, 0.2 3± 0.02 and 0.21 ± 0.03, respectively; inversely, the ERK1/2 grey scale value was 106.55 ± 16.45, mdr-l/β-actin and RRMl/β-actin were 1.50± 0.07 and 1.52 ± 0.12, respectively, which presented a tendency of synchronously increase after treated with ERK pathway activator EGF. The IC50 values in GEM-resistant cells of 0 nmol/L and 200 nmol/L levels were 4.104 and 10.20, respectively. After treated with U0126, these values decreased to 3.26 and 4.50, respectively; while treated with EGF, the IC50 values increased to 8.89 and 17.17, respectively. Conclusion： The ERK pathway may induce the GEM-chemoresistance in pancreatic cell line SW1990 through the participation in the regulation of the mdr-1 and RRM1 gene expression.

The role of ERK1/2 signaling pathway in coronary microembolization-induced rat myocardial inflammation and injury

Objectives In this work,we explore the effect of atorvastatin on myocardial apoptosis and caspase-8 acti- vation after coronary microembolization(CME) in rats. Methods Fifty rats were randomly divided into five groups; the coronary microembolization(CME) group,the sham-operated (sham) control group,the gastric lavage control group, the atorvastatin lavage group,and the caspasse-8 inhibitor (N-acetyl-Ile-Glu-Thr-Asp-CHO,abbreviated as CHO) group,with 10 rats for each group.A microembolization ball was injected through the left ventricle for constructing the CME model.Animals in the sham control group were given an injection of physiological saline instead of the microembolization ball.Seven days before the operation,the atorvastatin group underwent gastric lavage with 20 mg/kg of atorvastatin once a day.Gastric lavage control animals underwent gastric lavage with an equivalent dose of physiological saline instead of the atorvastatin.Animals in the CHO group were given an intraperitoneal injection of 10 mg/kg of CHO 30 min before the operation.Six hours after the operation,cardiac ultrasonic detection was conducted on each group to measure the cardiac function indexes.TUNEL(Terminal-deoxynucleoitidyl transferase mediated dUTP nick end labeling) assays were used to measure myocardial apoptosis,and western blots were used to quantify the expression levels of activated caspase-3 and -8.Results(1) The echocardiographic parameters showed that,compared to the sham control animals,the left ventricular ejection fraction(LVEF) of the CME group was significantly decreased(P【0.05).In addition, cardiac sonography revealed a decrease in the left ventricular shortening fraction(FS) and cardiac output(CO), but an increase in the left ventricular end-diastolic dimension (LVEDd).Compared to the CME group,the atorvastatin and CHO groups exhibited significantly improved cardiac function (P【0.05).(2) When compared with the sham control,the myocardical apoptotic rate of the CME group,as well as the levels of activated caspase-3 and-8,increased significantly (P【0.05).The myocardial apoptotic rate,as well as the levels of activated caspase-3 and caspase-8 in the atorvastatin and CHO groups,decreased significandy(P【0.05) in comparison to the CME group.Conclusions The atorvastatin pretreatment clearly suppressed post-CME myocardial apoptosis and improved cardiac function.The most likely mechanism for these effects is the blockade of the myocardial death receptor -mediated apoptosis pathway.

Therapeutic effect and mechanism of Shenqi Zhilong Decoction on mice with membranous nephropathy through ERK/cPLA2 signaling pathway

Objective: To explore the therapeutic effect and underlying mechanism of Shenqi Zhilong Decoction on mice with membranous nephropathy (MN). Methods:Mice with MN was established by injecting cationic bovine serum albumin (c-BSA) into tail vein for several times. model mice were randomly divided into MN group (equal amount of distilled water), Shenqi Zhilong Decoction low dose group (12 g crude drug/kg), Shenqi Zhilong Decoction high dose group (24 g crude drug/kg), and Tripterygium wilfordii polyglycoside tablet group (14 mg/ kg). Another 10 un-treatment mice were taken as control group (equal amount of distilled water). The drug was administered orally once a day for 4 weeks. After the last administration, 24 hours urine was collected to determine the urinary protein content;blood from inner canthus was collected to measure the changes of kidney function, liver function, blood lipid and levels of IL-6, IL-4 and TNF-α in serum in each group;HE staining was used to observe the pathological changes of kidney. Immunohistochemical staining was used to observe the expression of IgG in kidney. The protein expression of ERK1/2 and cPLA2 in renal tissues was determined by Western-blot method. The gene expression of Neph1, Nephrin and Podocin mRNA in kidney tissues were detected by RT-PCR. Results: Compared with model group, Shenqi Zhilong decoction at low-dose and high-dose could significantly reduce the value of urine protein in MN mice;Decreased TC and TG levels (P<0.05 or P<0.01);Increased the levels of ALB and TP in liver function (P<0.05 or P<0.01);has no significant effects on the levels of CRE, UREA and UA in renal function (P>0.05). Decreased the contents of IL-6, IL-4 and TNF-α in serum (P<0.05 or P<0.01);Significantly down-regulated the protein expression levels of p-ERK1/2 and p-cPLA2 in kidney tissues of MN mice (P<0.05 or P<0.01);Significantly increased the expression levels of NephP1, Nephrin and Podocin mRNA in renal tissues (P<0.01). Conclusion: Shenqi Zhulong Decoction has a good therapeutic effect on MN mice, and the mechanism of action is related to regulate the expression of related genes of Nephrin-Podocin-Neph1 receptor complex for protecting the glomerular filtration barrier, and inhibite the activation of ERK/cPLA2 pathway for relieving damage of GEC and reduceing secretion of pro-inflammatory cytokines.

Protective effects of Yishen Sanjie Huayu compound on the renal artery disease in rats with IgA nephropathy through ERK/NF-κB pathway

Objective:To observe the effect of Yishen Sanjie Huayu compound prescription on ERK/NF-κB signaling pathway in IgA nephropathy(IgAN)rats,and explore its effect on preventing and treating IgA nephropathy intrarenal arteriole disease.Methods:Fifty-five male SD rats were randomly divided into blank group,model group,ShenfukangⅡcapsule group and Losartan potassium tablet group.Bovine serum albumin(BSA)was used for intragastric administration and carbon tetrachloride(CCl4).IgAN rat model was established by subcutaneous injection and lipopolysaccharide(LPS)tail vein injection.ShenfukangⅡcapsule group and Losartan potassium tablet group were given each drug suspension 2ml/head/d one week after modeling Gavage was started.The blank group and the model group were given an equal volume of normal saline.The 24h urine protein(UTP)of the rats was measured at 4,8,and 12 weeks after the administration,and the blood creatinine(SCr)was measured after 12 weeks.,urea nitrogen(BUN),aldosterone(ADS),angiotensinⅡ(AngⅡ),immunohistochemical Envi-sion System two-step method to detect vascular endothelial growth factor(VEGF)and human matrix in the whole rat kidney and small artery area The expression of metalloproteinase-9(MMP-9),proliferating cell nuclear antigen(PCNA),extracellular regulatory protein kinase(ERK)1/2,nuclear transcription factor-κB(NF-κB),and the small arteries of rat kidney tissue The intima,media,vessel wall/vascular outer diameter value.Results:Compared with the model group,the expressions of VEGF,MMP-9,PCNA,ERK1/2 and NF-κB in kidney tissues of the ShenfukangⅡcapsule group and the Losartan potassium tablet group decreased(P<0.05),24hUTP and SCr,BUN level decreased(P<0.05),kidney tissue damage was alleviated;intima and vessel wall/vascular outer diameter values were significantly reduced(P<0.01),there was no significant difference in ADS between the groups.The AngⅡof the Tanpotassium tablets group was lower than that of the model group(P<0.05).Conclusion:Yishen Sanjie Huayu compound can inhibit the ERK/NF-κB signaling pathway in rats with IgA nephropathy,reduce the levels of VEGF,MMP-9,PCNA,ERK1/2,NF-κB,and inhibit intrarenal arteriole vascular endothelial cells Proliferate and reduce kidney damage.

Circ_0053943 complexed with IGF2BP3 drives uveal melanoma progression via regulating N6-methyladenosine modification of Epidermal growth factor receptor

Numerous studies have characterized the critical role of circular RNAs(circRNAs)as regulatory factors in the progression of multiple cancers.However,the biological functions of circRNAs and their underlying molecular mechanisms in the progression of uveal melanoma(UM)remain enigmatic.In this study,we identified a novel circRNA,circ_0053943,through re-analysis of UM microarray data and quantitative RT-PCR.Circ_0053943 was found to be upregulated in UM and to promote the proliferation and metastatic ability of UM cells in both in vitro and in vivo settings.Mechanistically,circ_0053943 was observed to bind to the KH1 and KH2 domains of insulin-like growth factor 2 mRNA-binding protein 3(IGF2BP3),thereby enhancing the function of IGF2BP3 by stabilizing its target mRNA.RNA sequencing assays identified epidermal growth factor receptor(EGFR)as a target gene of circ_0053943 and IGF2BP3 at the transcriptional level.Rescue assays demonstrated that circ_0053943 exerts its biological function by stabilizing EGFR mRNA and regulating the downstream mitogen-activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK)signaling pathway.Collectively,circ_0053943 may promote UM progression by stabilizing EGFR mRNA and activating the MAPK/ERK signaling pathway through the formation of a circ_0053943/IGF2BP3/EGFR RNA-protein ternary complex,thus providing a potential biomarker and therapeutic target for UM.

Modulation effects of pressing manipulation on local inflammatory responses and ERK/NF-κB pathway in trigger point model rats

Objective:To investigate the mechanism of trigger point deactivation induced by pressing manipulation in a rat model and to explore its potential regulation of the inflammatory response through the extracellular signal-regulated kinase(ERK)/nuclear factor-κB(NF-κB)pathway.Methods:Fifty male Sprague-Dawley rats were randomly divided into a blank group,a model group,a pressing manipulation group,an ERK agonist group,and a pressing manipulation+ERK agonist group,with 10 rats in each group.Except for the blank group,rats in other groups were used to establish the trigger point rat model using the blunt blow combined with the eccentric exercise method.The pressing manipulation group underwent pressing manipulation intervention at the trigger points.The ERK agonist group received an injection of recombinant human epidermal growth factor via the tail vein.The pressing manipulation+ERK agonist group received interventions from both the pressing manipulation and ERK agonist groups.The pressure pain threshold(PPT)was measured by a mechanical pain threshold detector before and after the intervention.The histological changes were evaluated by hematoxylin-eosin staining after the intervention;the expression levels of ERK,phosphorylated ERK(p-ERK),NF-κB p65(p65),phosphorylated NF-κB p65(p-p65),and phosphorylated NF-κB inhibitor(p-IκB)were detected by Western blotting;the levels of interleukin(IL)-1β,IL-6,and tumor necrosis factor(TNF)-αwere detected by enzyme-linked immunosorbent assay.Results:The PPT increased(P<0.05);the inflammatory cells disappeared;the ratios of p-ERK/ERK,p-p65/p65,and p-IκB/β-actin,also the levels of IL-1β,IL-6,and TNF-αall decreased in the pressing manipulation group after the intervention compared with the model group(P<0.05).The PPT decreased significantly(P<0.05),the inflammatory cell presence increased,and the ratios of p-ERK/ERK and p-p65/p65 were elevated(P<0.05);additionally,the levels of IL-6 and TNF-αwere significantly higher in the pressing manipulation+ERK agonist group compared with the pressing manipulation group(P<0.05).The PPT was significantly lower(P<0.05),the inflammatory cell count was higher,the ratios of p-ERK/ERK and p-IκB/β-actin and the levels of IL-1βand TNF-αwere significantly higher in the ERK agonist group compared with the pressing manipulation+ERK agonist group(P<0.05).Conclusion:Pressing manipulation can effectively alleviate inflammation and pain in trigger point model rats,potentially by inhibiting the ERK/NF-κB signaling pathway.

Hypoxia-inducible factor-1α–mediated upregulation of CD99 promotes the proliferation of placental mesenchymal stem cells by regulating ERK1/2

BACKGROUND As human placenta-derived mesenchymal stem cells(hP-MSCs)exist in a physiologically hypoxic microenvironment,various studies have focused on the influence of hypoxia.However,the underlying mechanisms remain to be further explored.AIM The aim was to reveal the possible mechanisms by which hypoxia enhances the proliferation of hP-MSCs.METHODS A hypoxic cell incubator(2.5%O2)was used to mimic a hypoxic microenvironment.Cell counting kit-8 and 5-ethynyl-20-deoxyuridine incorporation assays were used to assay the proliferation of hP-MSCs.The cell cycle was profiled by flow cytometry.Transcriptome profiling of hP-MSCs under hypoxia was performed by RNA sequencing.CD99 mRNA expression was assayed by reverse transcription-polymerase chain reaction.Small interfering RNA-mediated hypoxia-inducible factor 1α(HIF-1α)or CD99 knockdown of hP-MSCs,luciferase reporter assays,and the ERK1/2 signaling inhibitor PD98059 were used in the mechanistic analysis.Protein expression was assayed by western blotting;immunofluorescence assays were conducted to evaluate changes in expression levels.RESULTS Hypoxia enhanced hP-MSC proliferation,increased the expression of cyclin E1,cyclin-dependent kinase 2,and cyclin A2,and decreased the expression of p21.Under hypoxia,CD99 expression was increased by HIF-1α.CD99-specific small interfering RNA or the ERK1/2 signaling inhibitor PD98059 abrogated the hypoxia-induced increase in cell proliferation.CONCLUSION Hypoxia promoted hP-MSCs proliferation in a manner dependent on CD99 regulation of the MAPK/ERK signaling pathway in vitro.

Inhibitory effects of oxyresveratrol on ERK and Smad1/2 phosphorylation and HSC activation in preventing carbon tetrachloride-induced rat liver fibrosis

Oxyresveratrol(ORes,trans-2,4,3′,5′-tetrahydroxy stilbene)naturally exists in mulberry,grapes,peanuts and other plants.It belongs to stilbene polyphenolic family and has an extra hydroxyl group at 2-position comparing with resveratrol(Res).Hence,ORes has stronger antioxidant activity than resveratrol.In present study,we employed a rat hepatic fibrosis model induced by carbon tetrachloride(CCl_(4))and administrated ORes via gavage feeding to study the protective effects and potential mechanisms of ORes against hepatic fibrosis.We demonstrated that rat liver oxidative damage induced by CCl_(4)was significantly alleviated after ORes feeding.Furthermore,the mRNA transcription levels ofα-smooth muscle actinn(˛-SMA),desmin,and two MMPs(MMP2 and MMP9)were reduced and the expression levels of transforming growth factorβ1(TGF-β1),p-small mother against decapen-taplegic protein(Smad)1/2 and p-extracellular signal-regulated kinases(ERK)1/2 in the liver tissue down-regulated dramatically.In a parallel study with Res,ORes showed more efficacious protective effect than Res against rat liver fibrosis,which is attributed to extended conjugation system due to the extra hydroxyl group at 2-position on ORes making it more electron-rich and susceptible to oxidation than Res.Therefore,dietary consumption of mulberry and other fruits containing ORes may be beneficial in the prevention of liver fibrosis.

Different Concentrations of Notoginsenoside Rg1 Attenuate Hypoxic and Hypercapnia Pulmonary Hypertension by Reducing the Expression of ERK in Rat PASMCs

Pulmonary arterial hypertension (PAH) is a serious disease which is characterized by increased vascular resistance and pressure. We have previously hypothesized that panax notoginseng saponins (PNS) might attenuate pulmonary vasoconstriction under hypoxia and hypercapnia condition. This study aims to investigate the effect of notoginsenoside Rg1, a main ingredient of PNS, with various concentrations (8, 40, 100 mg/L, respectively) on extracellular signal regulated kinase (ERK1/2) signaling pathway in pulmonary arterial smooth muscle cells (PASMCs). In addition, PASMCs were randomly divided into six groups: SD rat under normoxic condition as control group (N group), hypoxia hypercapnia group (H group), DMSO control group (HD group), Rg1-treatment groups (RgLRgM and RgH group). Western-blot and RT-PCR were used to test the expression of p-ERK protein and the expression of ERK1 mRNA and ERK2 mRNA. This study provided the evidence that the expression of p-ERK protein and the expression of ERK1 mRNA and ERK2 mRNA in HD group and H group were obviously higher than that in N group (P < 0.01), Whereas the level of ERK1/2 mRNA in Rg1-treatment groups was significantly lower than that in HD group and H group (P < 0.01), and the proper concentration of Rg1 is 40 mg/L. These results suggested that notoginsenoside Rg1 can attenuate pulmonary vasoconstriction which may lead to HHPV through reducing the expression of ERK1/2.

C-X-C chemokine receptor type 7 antibody enhances neural plasticity after ischemic stroke

Stromal cell-derived factor-1 and its receptor C-X-C chemokine receptor 4(CXCR4) have been shown to regulate neural regeneration after stroke.Howeve r,whether stromal cell-derived factor-1 receptor CXCR7,which is widely distributed in the develo ping and adult central nervous system,participates in neural regeneration remains poorly unde rstood.In this study,we established rat models of focal cerebral ischemia by injecting endothelin-1 into the cerebral co rtex and striatum.Starting on day 7 after injury,CXCR7-neutralizing antibody was injected into the lateral ventricle using a micro drug delivery system for 6 consecutive days.Our results showed that CXCR7-neutralizing antibody increased the total length and number of sprouting co rticospinal tra ct fibers in rats with cerebral ischemia,increased the expression of vesicular glutamate transporter 1 and growth-related protein 43,marke rs of the denervated spinal cord synapses,and promoted the differentiation and maturation of oligodendrocyte progenitor cells in the striatum.In addition,CXCR7 antibody increased the expression of CXCR4 in the striatum,increased the protein expression of RAS and ERK1/2 associated with the RAS/ERK signaling pathway,and im proved rat motor function.These findings suggest that CXCR7 improved neural functional recovery after ischemic stroke by promoting axonal regeneration,synaptogenesis,and myelin regeneration,which may be achieved by activation of CXCR4 and the RAS/ERK1/2 signaling pathway.

MAPK/ERK regulation of P53 in human epidermoid carcinoma cell line A431

Objective:To observe the impact of activation and inhibition of mitogen activated protein kinases(MAPK)/extracellular signalregulated protein kinase(ERK)signaling pathway on the proliferation and apoptosis of cutaneous squamous cell carcinoma(SCC).cells and investigate the interaction mechanism between MAPK/ERK signaling pathway and tumor suppressor gene P53 in SCC.Methods:Human A431 cells were cultured and divided into MAPK/ERK inhibition groups with low-,medium-and highconcentration of inhibitors(PD98059+DMSO),MAPK/ERK activation groups with low-,medium-and high-concentration of stimuli(IGF+PBS)and blank control group(DMSO).The cell proliferation in vitro was detected by MTT assay,with the cell apoptosis detected by flow cytometry(FCM)and the protein expression of P-ERK and P53 detected by western blot in each group.Results:The A431 cell proliferation was inhibited by different concentrations of PD98059 with a clear concentration-effect and time-effect relationship(p<.05);and the cell proliferation was promoted by the different concentrations of IGF with a clear concentration-effect and time-effect relationship(p<.05).The FCM results showed a significant increase in the apoptosis rate of A431 cells which were treated with PD98059,with a clear concentration-effect relationship(p<.05);while the apoptosis rate was decreased significantly after A431 cells were treated with IGF,also with a concentration-effect relationship(p<.05).The western blot results showed that the expression of P-ERK protein was decreased but the expression of P53 was increased after A431 cells were treated with PD98059.With the concentration of PD98059 going up,the decrease in P-ERK and the increase in P53 were more significant(p<.05);while the expression of P-ERK protein was increased but the expression of P53 was decreased after A431 cells were treated with IGF.With the concentration of IGF going up,the increase in P-ERK and the decrease in P53 were more significant(p<.05).According to Pearson correlation analysis,the expression of P53 was negatively correlated to that of P-ERK(p<.05).Conclusions:After MAPK/ERK signaling pathway was activated by IGF in A431 cells,the expression of pro-apoptotic factor P53 was decreased with the ability of cell proliferation enhanced and the ability of apoptosis reduced.However,after the inhibition of MAPK/ERK signaling pathway,the expression of pro-apoptotic factor P53 was increased with the ability of cell proliferation reduced and the ability of apoptosis increased.

Apolipoprotein A1 suppresses the hypoxia-induced angiogenesis of human retinal endothelial cells by targeting PlGF

AIM:To investigate the anti-angiogenic effect of apolipoprotein A1(apoA1)on primary human retinal vascular endothelial cells(HRECs)and explore the possible mechanism.METHODS:The primary HRECs were transfected with apoA1-GFP recombinant lentiviral and were compared with cells undergoing transfection with empty lentiviral vectors.Hypoxia chambers were used to simulate the anoxic environment of cells under pathological condition.The concentrations of secreted vascular endothelial growth factor(VEGF)and placental growth factor(PlGF)were measured by enzyme-linked immunosorbent assay(ELISA).Cell migration ability was detected by wound healing assay.The sprouting of HRECs was determined by tube formation assay.The protein levels of extracellular signal regulated kinase 1/2(ERK1/2)and phosphor ylated ERK1/2(p-ERK1/2)were measured by Western blot.RESULTS:Overexpressed apoA1 in hypoxia-induced HRECs significantly suppressed PlGF(0.67±0.10 folds,P=0.007).Overexpressed apoA1 also attenuated hypoxiainduced cell migration(0.32±0.11 folds,P<0.0001),tube formation(0.66±0.01 folds,P<0.0001)and the phosphorylation levels of ERK(0.6±0.11 folds,P=0.025).Pretreatment of mitogen-activated protein kinase kinase(MEK)inhibitor(U0126)further reduced the PlGF and angiogenesis in hypoxia-induced HRECs.CONCLUSION:ApoA 1 inhibits the angiogenesis at least in part by inactivating ERK1/2 in hypoxia-induced HRECs.Moreover,apoA1 suppresses the PlGF expression,which selectively associated with pathological angiogenesis.