Objective To investigate the effect of emodin on high glucose(HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to induction of adenosine-monophosphate-activated pr...Objective To investigate the effect of emodin on high glucose(HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to induction of adenosine-monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)-mediated autophagy in podocytes(MPC5 cells)in vitro.Methods MPC5 cells were treated with different concentrations of HG(2.5,5,10,20,40,80 and 160 mmol/L),emodin(2,4,8µmol/L),or HG(40 mmol/L)and emodin(4µmol/L)with or without rapamycin(Rap,100 nmol/L)and compound C(10µmol/L).The viability and apoptosis of MPC5 cells were detected using cell counting kit-8(CCK-8)assay and flow cytometry analysis,respectively.The expression levels of cleaved caspase-3,autophagy marker light chain 3(LC3)Ⅰ/Ⅱ,and AMPK/mTOR signaling pathway-related proteins were determined by Western blot.The changes of morphology and RFP-LC3 fluorescence were observed under microscopy.Results HG at 20,40,80 and 160 mmol/L dose-dependently induced cell apoptosis in MPC5 cells,whereas emodin(4µmol/L)significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage(P<0.01).Emodin(4µmol/L)significantly increased LC3-Ⅱ protein expression levels and induced RFP-LC3-containing punctate structures in MPC5 cells(P<0.01).Furthermore,the protective effects of emodin were mimicked by rapamycin(100 nmol/L).Moreover,emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR.The AMPK inhibitor compound C(10µmol/L)reversed emodin-induced autophagy activation.Conclusion Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway,which might provide a potential therapeutic option for diabetic nephropathy.展开更多
AIM To investigate the effect of metformin on activated hepatic stellate cells(HSCs) and the possible signaling pathways involved. METHODS A fibrotic mouse model was generated by intraperitoneal injection of carbon te...AIM To investigate the effect of metformin on activated hepatic stellate cells(HSCs) and the possible signaling pathways involved. METHODS A fibrotic mouse model was generated by intraperitoneal injection of carbon tetrachloride(CCl_4) and subsequent treatment with or without metformin. The level of fibrosis was detected by hematoxylin-eosin staining, Sirius Red staining, and immunohistochemistry. The HSC cell line LX-2 was used for in vitro studies. The effect of metformin on cell proliferation(CCK8 assay),motility(scratch test and Transwell assay), contraction(collagen gel contraction assay), extracellular matrix(ECM) secretion(Western blot), and angiogenesis(ELISA and tube formation assay) was investigated. We also analyzed the possible signaling pathways involved by Western blot analysis.RESULTS Mice developed marked liver fibrosis after intraperitoneal injection with CCl_4 for 6 wk. Metformin decreased the activation of HSCs, reduced the deposition of ECM, and inhibited angiogenesis in CCl_4-treated mice. Platelet-derived growth factor(PDGF) promoted the fibrogenic response of HSCs in vitro, while metformin inhibited the activation, proliferation, migration, and contraction of HSCs, and reduced the secretion of ECM. Metformin decreased the expression of vascular endothelial growth factor(VEGF) in HSCs through inhibition of hypoxia inducible factor(HIF)-1α in both PDGF-BB treatment and hypoxic conditions, and it down-regulated VEGF secretion by HSCs and inhibited HSC-based angiogenesis in hypoxic conditions in vitro. The inhibitory effects of metformin on activated HSCs were mediated by inhibiting the Akt/mammalian target of rapamycin(m TOR) and extracellular signal-regulated kinase(ERK) pathways via the activation of adenosine monophosphate-activated protein kinase(AMPK).CONCLUSION Metformin attenuates the fibrogenic response of HSCs in vivo and in vitro, and may therefore be useful for the treatment of chronic liver diseases.展开更多
目的：观察气血并治方有效组分对缺氧/复氧（H/R）损伤心肌细胞一磷酸腺苷酸活化蛋白激酶（AMPK）相关糖脂代谢通路的作用机制。方法：分离、提取、培养出生1~2 d SD乳鼠原代心肌细胞,于常规倒置相差显微镜下观察原代心肌细胞形态及生...目的：观察气血并治方有效组分对缺氧/复氧（H/R）损伤心肌细胞一磷酸腺苷酸活化蛋白激酶（AMPK）相关糖脂代谢通路的作用机制。方法：分离、提取、培养出生1~2 d SD乳鼠原代心肌细胞,于常规倒置相差显微镜下观察原代心肌细胞形态及生长状态,经α-横纹肌辅肌动蛋白（α-actinin）免疫荧光染色鉴定为心肌细胞后,进行缺氧3 h复氧2 h处理制作H/R损伤模型,随机分为正常组（正常氧）,模型组（缺氧/复氧）,曲美他嗪组（缺氧/复氧＋100μmol·L-1盐酸曲美他嗪,TMZ）,气血并治方有效组分组（缺氧/复氧＋1 mmol·L-1气血并治方有效组分,CWQB）。采用实时荧光定量聚合酶链反应（Real-time PCR）和蛋白免疫印迹法（Western blot）测定AMPK代表性亚基心肌一磷酸腺苷酸活化蛋白激酶α（AMPKα）,及其糖代谢通路中葡萄糖转运体4（GLUT4）,磷酸果糖激酶2（PFK2）,脂肪酸代谢通路中乙酰辅酶A羧化酶（ACC2）,脂肪酸移位酶（FAT/CD36）的基因及蛋白表达情况。结果：与正常组比较,模型组,TMZ组,CWQB组的AMPKα,GLUT4,PFK2基因和蛋白表达上调,ACC2,FAT/CD36基因和蛋白表达下调（P〈0.05）;与模型组比较,TMZ组,CWQB组AMPKα,GLUT4,PFK2,ACC2,FAT/CD36基因和蛋白表达均上调（P〈0.05）,其中TMZ组上调AMPKα,FAT/CD36基因和蛋白,上调GLUT4,PFK2基因表达的效果更为显著（P〈0.05）。结论：气血并治方有效组分可以激活H/R损伤心肌细胞的AMPK信号通路,增强GLUT4介导的葡萄糖转送,PFK2参与的糖酵解,同时促进FAT/CD36调控的脂肪酸转运,上调ACC2抑制脂肪酸氧化过程,进而提高缺氧/复氧条件下心肌细胞对葡萄糖、脂肪酸等产能底物的利用能力,改善H/R损伤心肌细胞的能量代谢。展开更多
基金Supported by the Chinese Medicine Research Project of Hubei Provincial Health Commission(No.ZY2019Q024)Scientific Research Project of Wuhan Municipal Health Commission(No.WX20B11)Scientific Research Project of Wuhan Municipal Health Commission(No.WZ20C01)。
文摘Objective To investigate the effect of emodin on high glucose(HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to induction of adenosine-monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)-mediated autophagy in podocytes(MPC5 cells)in vitro.Methods MPC5 cells were treated with different concentrations of HG(2.5,5,10,20,40,80 and 160 mmol/L),emodin(2,4,8µmol/L),or HG(40 mmol/L)and emodin(4µmol/L)with or without rapamycin(Rap,100 nmol/L)and compound C(10µmol/L).The viability and apoptosis of MPC5 cells were detected using cell counting kit-8(CCK-8)assay and flow cytometry analysis,respectively.The expression levels of cleaved caspase-3,autophagy marker light chain 3(LC3)Ⅰ/Ⅱ,and AMPK/mTOR signaling pathway-related proteins were determined by Western blot.The changes of morphology and RFP-LC3 fluorescence were observed under microscopy.Results HG at 20,40,80 and 160 mmol/L dose-dependently induced cell apoptosis in MPC5 cells,whereas emodin(4µmol/L)significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage(P<0.01).Emodin(4µmol/L)significantly increased LC3-Ⅱ protein expression levels and induced RFP-LC3-containing punctate structures in MPC5 cells(P<0.01).Furthermore,the protective effects of emodin were mimicked by rapamycin(100 nmol/L).Moreover,emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR.The AMPK inhibitor compound C(10µmol/L)reversed emodin-induced autophagy activation.Conclusion Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway,which might provide a potential therapeutic option for diabetic nephropathy.
基金Supported by National Natural Science Foundation of China,No.81370590
文摘AIM To investigate the effect of metformin on activated hepatic stellate cells(HSCs) and the possible signaling pathways involved. METHODS A fibrotic mouse model was generated by intraperitoneal injection of carbon tetrachloride(CCl_4) and subsequent treatment with or without metformin. The level of fibrosis was detected by hematoxylin-eosin staining, Sirius Red staining, and immunohistochemistry. The HSC cell line LX-2 was used for in vitro studies. The effect of metformin on cell proliferation(CCK8 assay),motility(scratch test and Transwell assay), contraction(collagen gel contraction assay), extracellular matrix(ECM) secretion(Western blot), and angiogenesis(ELISA and tube formation assay) was investigated. We also analyzed the possible signaling pathways involved by Western blot analysis.RESULTS Mice developed marked liver fibrosis after intraperitoneal injection with CCl_4 for 6 wk. Metformin decreased the activation of HSCs, reduced the deposition of ECM, and inhibited angiogenesis in CCl_4-treated mice. Platelet-derived growth factor(PDGF) promoted the fibrogenic response of HSCs in vitro, while metformin inhibited the activation, proliferation, migration, and contraction of HSCs, and reduced the secretion of ECM. Metformin decreased the expression of vascular endothelial growth factor(VEGF) in HSCs through inhibition of hypoxia inducible factor(HIF)-1α in both PDGF-BB treatment and hypoxic conditions, and it down-regulated VEGF secretion by HSCs and inhibited HSC-based angiogenesis in hypoxic conditions in vitro. The inhibitory effects of metformin on activated HSCs were mediated by inhibiting the Akt/mammalian target of rapamycin(m TOR) and extracellular signal-regulated kinase(ERK) pathways via the activation of adenosine monophosphate-activated protein kinase(AMPK).CONCLUSION Metformin attenuates the fibrogenic response of HSCs in vivo and in vitro, and may therefore be useful for the treatment of chronic liver diseases.
