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.

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.

Endogenous hydrogen sulfide and ERK1/2-STAT3 signaling pathway may participate in the association between homocysteine and hypertension

Background Homocysteine(Hcy)is a risk factor for hypertension,although the mechanisms are poorly understood.Methods We first explored the relationship between Hcy levels and blood pressure(BP)by analyzing the clinical data of primary hypertensive patients admitted to our hospital.Secondly,we explored a rat model to study the effect of Hcy on blood pressure and the role of H2S.An hyperhomocysteinemia(HHcy)rat model was induced to explore the effect of Hcy on blood pressure and the possible mechanism.We carried out tissue histology,extraction and examination of RNA and protein.Finally,we conducted cell experiments to determine a likely mechanism through renin-angiotensin-aldosterone system(RAAS)and extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway.Results In primary hypertensive inpatients with HHcy,blood pressure was significantly higher as compared with inpatient counterparts lacking HHcy.In the rat model,blood pressure of the Wistar rats was significantly increased with increases in serum Hcy levels and decreased after folate treatment.Angiotensin converting enzyme 1(ACE1)expression in the Wistar Hcy group was enhanced comparing to controls,but was decreased in the Wistar folate group.Angiotensin II receptor type 1(AGTR1)levels in the kidney tissue increased in the Wistar folate group.Both serum H2S and kidney cystathionineγ-lyase decreased with elevated levels of serum Hcy.In vitro,increased concentrations and treatment times for Hcy were associated with increased expression of collagen type 1 and AGTR1.This dose and time dependent response was also observed for p-STAT3 and p-ERK1/2 expression.Conclusion Endogenous H2S might mediate the process of altered blood pressure in response to changes in serum Hcy levels,in a process that is partly dependent on activated RAAS and ERK1/2-STAT3 signaling pathway.

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.

Estrogen up-regulates MMP2/9 expression in endometrial epithelial cell via VEGF-ERK1/2 pathway

Objective:To study the effect of estrogen on anovulatory dysfunctional uterine bleeding(ADUB).Methods:Primary endometrial epithelial cells of Hainan Lizu female was cultured and hydrolylic activity of gelalinase was determined by gelatin zymography analysis.Cellular mRNA and protein synthesis was blocked respectively to determine whether the increased expression of MMP-2/9 was induced by estrogen.The expression of VEGF was blocked by siRNA.After treatment with various factors.MMP-9,VEGF,total Erk and phosphorylated Erk expression in primary uterine epithelial cells was detected by Western blotting analysis.Cell MMP-2/9mRNA levels was measured by real-time RT-PCR.Results:The activity and expression of MMP2/9 was inereased in the endometrium of patients with ADUB.Estrogen could up-regulate the expression of VEGF and activate Erk 1/2-Elk1 signal path.After interference by siRNA,ERK1/2 pathway was blocked in cells,and the expression of MMP-2/9 was down-regulated.ERK1/2 specific blocker U0126 blocked ERK phosphorylation,and it could down-regulate the expression of MMP-2/9.Conclusions:The results showed that the estrogen can increase the expression of VEGF,and thus activate ERK1/2 pathway to induce MMP-2/9 expression.

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.

Cytokine receptor-like factor 1(CRLF1)promotes cardiac fibrosis via ERK1/2 signaling pathway

Cardiac fibrosis is a cause of morbidity and mortality in people with heart disease.Anti-fibrosis treatment is a significant therapy for heart disease,but there is still no thorough understanding of fibrotic mechanisms.This study was carried out to ascertain the functions of cytokine receptor-like factor 1(CRLF1)in cardiac fibrosis and clarify its regulatory mechanisms.We found that CRLF1 was expressed predominantly in cardiac fibroblasts.Its expression was up-regulated not only in a mouse heart fibrotic model induced by myocardial infarction,but also in mouse and human cardiac fibroblasts provoked by transforming growth factor-β1(TGF-β1).Gain-and loss-of-function experiments of CRLF1 were carried out in neonatal mice cardiac fibroblasts(NMCFs)with or without TGF-β1 stimulation.CRLF1 overexpression increased cell viability,collagen production,cell proliferation capacity,and myofibroblast transformation of NMCFs with or without TGF-β1 stimulation,while silencing of CRLF1 had the opposite effects.An inhibitor of the extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway and different inhibitors of TGF-β1 signaling cascades,comprising mothers against decapentaplegic homolog(SMAD)-dependent and SMAD-independent pathways,were applied to investigate the mechanisms involved.CRLF1 exerted its functions by activating the ERK1/2 signaling pathway.Furthermore,the SMAD-dependent pathway,not the SMAD-independent pathway,was responsible for CRLF1 up-regulation in NMCFs treated with TGF-β1.In summary,activation of the TGF-β1/SMAD signaling pathway in cardiac fibrosis increased CRLF1 expression.CRLF1 then aggravated cardiac fibrosis by activating the ERK1/2 signaling pathway.CRLF1 could become a novel potential target for intervention and remedy of cardiac fibrosis.

TTF1调控ERK信号转导通路的作用

[目的]探讨TTF1调控ERK信号转导通路的作用.[方法]建立人肝癌HepG2裸鼠移植瘤模型,取40只随机分为阴性对照组、紫杉醇组、TTF1小剂量组、TTF1中剂量组及TTF1大剂量组,每组各为8只,药物干预21 d后处死裸鼠,采用Western blot技术检测肿瘤组织ERK1/2和p-ERK1/2蛋白表达.[结果]TTF1对人肝癌HepG2裸鼠移植瘤的生长有抑制作用;ERK1/2蛋白表达未见明显变化,p-ERK1/2蛋白表达随着TTF1剂量的升高而减少.[结论]TTF1对肿瘤生长有抑制作用,其机制可能与通过调节ERK信号转导通路有关.

Up-regulation of Ras/Raf/ERK1/2 signaling in the spinal cord impairs neural cell migration, neurogenesis, synapse formation, and dendritic spine development

Background The Ras/Raf/ERK1/2 signaling pathway controls many cellular responses such as cell proliferation, migration, differentiation, and death. In the nervous system, emerging evidence also points to a death-promoting role for ERK1/2 in both in vitro and in vivo models of neuronal death. To further investigate how Ras/Raf/ERK1/2 up-regulation may lead to the development of spinal cord injury, we developed a cellular model of Raf/ERK up-regulation by over- expressing c-Raf in cultured spinal cord neurons （SCNs） and dorsal root ganglions （DRGs）.

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.

The effects of claudin 14 during early Wallerian degeneration after sciatic nerve injury

Claudin 14 has been shown to promote nerve repair and regeneration in the early stages of Wallerian degeneration （0-4 days） in rats with sciatic nerve injury, but the mechanism underlying this process remains poorly understood. This study reported the effects of claudin 14 on nerve degeneration and regeneration during early Wallerian degeneration. Claudin 14 expression was up-regulated in sciatic nerve 4 days after Wallerian degeneration. The altered expression of claudin 14 in Schwann cells resulted in expression changes of cytokines in vitro. Expression of claudin 14 affected c-Jun, but not Akt anal ERK1/2 patl^ways, l^urther studies reve^ed that enhanced expression of claudin 14 could promote Schwann cell proliferation and migration. Silencing of claudin 14 expression resulted in Schwann cell apoptosis and reduction in Schwann cell proliferation. Our data revealed the role of claudin 14 in early Wallerian degeneration, which may provide new insights into the molecular mechanisms of Wallerian degeneration.

Apoptosis in glioma-bearing rats after neural stem cell transplantation

Abnormal activation of the Ras/Raf/Mek/Erk signaling cascade plays an important role in glioma. Inhibition of this aberrant activity could effectively hinder glioma cell proliferation and promote cell apoptosis. To investigate the mechanism of gJioblastoma treatment by neural stem ceiJ trans- plantation with respect to the Ras/Raf/Mek/Erk pathway, C6 glioma cells were prepared in sus- pension and then infused into the rat brain to establish a glioblastoma model. Neural stem cells isolated from fetal rats were then injected into the brain of this glioblastoma model. Results showed that Raf-1, Erk and Bcl-2 protein expression significantly increased, while Caspase-3 protein expression decreased. After transplantation of neural stem cells, Raf-1, Erk and Bcl-2 protein expression significantly decreased, while Caspase-3 protein expression significantly in-creased. Our findings indicate that transplantation of neural stem cells may promote apoptosis of glioma cells by inhibiting Ras/Raf/Mek/Erk signaling, and thus may represent a novel treatment approach for glioblastoma.