目的探讨血管软化丸能否通过调控腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)/NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)通路抑制细胞焦亡改善载脂蛋白E基因...目的探讨血管软化丸能否通过调控腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)/NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)通路抑制细胞焦亡改善载脂蛋白E基因敲除(apolipoprotein E knockout,APOE^(-/-))小鼠动脉粥样硬化(atherosclerosis,AS)。方法10只C57BL/6J小鼠作为空白对照组,60只APOE^(-/-)小鼠随机分为模型组、血管软化丸低剂量组、血管软化丸高剂量组、阿托伐他汀组、血管软化丸高剂量和AMPK抑制剂联合组、AMPK抑制剂组,给予高脂饮食饲养18周建造动脉粥样硬化小鼠模型,并于第13周给予不同方法干预,干预6周后取材,分别检测各组小鼠甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,TC)、低密度脂蛋白-胆固醇(low density lipoprotein-eholesterol,LDL-C)、高密度脂蛋白-胆固醇(high density lipoprotein-eholesterol,HDL-C)水平,HE和油红O染色观察小鼠主动脉组织病理形态学变化,Elisa测定主动脉组织白细胞介素(interleukin,IL)-1β和IL-18水平,Western Blot检测AMPK、P-AMPK、NLRP3、裂解的半胱氨酸天冬酶1(Cleaved-Caspase-1)、Gasdermin D(GSDMD)-N蛋白表达,免疫荧光检测P-AMPK、NLRP3蛋白在主动脉根部的分布情况,透射电子显微镜观察主动脉内皮超微结构改变情况。结果与对照组相比,模型组大鼠血清TG、TC、LDL-C水平显著升高,HDL-C水平显著降低(P<0.01),主动脉根部细胞排列紊乱,且脂肪空泡明显,主动脉有明显粥样斑块沉积,P-AMPK蛋白表达水平显著降低,NLRP3、Cleaved-Caspase 1、GSDMD-N蛋白表达水平及IL-1β和IL-18水平显著升高(P<0.05或P<0.01);与模型组比较,血管软化丸高低剂量组及阿托伐他汀组血脂紊乱得到改善,主动脉组织及细胞病变程度减轻,P-AMPK蛋白表达水平显著升高,NLRP3、Cleaved-Caspase 1、GSDMD-N蛋白表达水平显著降低(P<0.05或P<0.01),IL-1β和IL-18水平显著降低(P<0.05或P<0.01);与血管软化丸高剂量+AMPK抑制剂组比较,血管软化丸高剂量组焦亡相关蛋白水平显著降低(P<0.05或P<0.01),主动脉内皮细胞损伤程度减轻,而AMPK抑制剂组与之相反。结论血管软化丸可能通过调控AMPK/NLRP3通路减轻细胞焦亡发挥对动脉粥样硬化小鼠的保护作用。展开更多
OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compoun...OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compound derived from traditional Chinese medicine,used in Asian countries for the prevention and treatment of hepatic disease.Therefore,the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs.METHODS The murine HSC(tHSC/Cl-6)cell line were incubated in 96-well plates and treated with TNF-αand CuE at various concentrations and indicated times.Cell viability was assessed with MTT assay.Another,t-HSC/Cl-6 were incubated in 6-well plates and also treated with TNF-α,CuE,AICAR or metformin for the indicated time and concentration.Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blotting and RT-PCR.RESULTS CuE inhibited cell proliferation of activated HSC/T-6cells in concentration-and time-dependent manner.CuE triggered the activation of caspase-3,cleaved the PARP,ration of bcl-2/bax,and cytochrom c protein in a time-and concentration-dependent manner.CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK.CuE inhibited the protein and mRNA expressions ofα-SMA,TIMP-1 and collagenⅠ in activated HSC-T6.CuE broadly blocked p-PI3 K,p-Akt,p-mTOR and p-p70S6 Kexpressions.CuE significantly increased phosphorylated AMPK expression as well as AICAR and metoformin.And metformin showed significantly higher activation of AMPK than AICAR.Ability of CuE on activation of AMPK was between AICAR and metformin.It′s also found that CuE significantly decreased p-mTOR as well as AICAR and metformin.CONCLUSION CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment.The findings demonstrate that CuE induced HSC apoptosis via ERK/MAPK and PI3K/Akt-AMPK-mTOR signaling.展开更多
OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were t...OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were treated with SRT1720(10μmol·L^(-1))and AICAR(500μmol·L^(-1))prior to ethanol(50 mmol·L^(-1)) for 24 and 48 h.Cell viability was analyzed by methyl thiazolyl tetrazolium assay.SIRT1,AMPK and p-AMPK m RNA levels for 24 h and 48 h were analyzed by RT-PCR,SIRT1,AMPK and p-AMPK protein expressions in the supernatant at 24 and 48 h was detected by Western blot.RESULTS SRT1720 and AICAR effectively decreased LX-2 cell viabilities and exhibited scarcely little toxicity in human Chang liver cells.SRT1720 and AICAR attenuated collagen-I,α-smooth muscle actin(α-SMA)levels,activated liver kinase B-1(LKB1)and AMPK phosphorylation in ethanol treated LX-2 cells.Meanwhile,SRT1720 and AICAR enhanced SIRT1 expression mediated by ethanol both in Chang liver cells and LX-2 cells.Furthermore,SRT1720 and AICAR suppressed the expression of sterol regulatory element-binding protein-1(SREBP-1)to regulate fatty acid synthesis.CONCLUSION SIRT1 agonist and AMPK agonist blocked the crosstalk between hepatocytes and HSCs via SIRT1/AMPK signaling pathway to modulate hepatocytes accumulation of lipid and HSCs activation.展开更多
Oxidative stress is one of the main causes of neurodegenerative diseases such as Alzheimer disease(AD).Our previous studies have shown that artemisinin,a anti-malaria Chinese medicine,with neuroprotective effect,howev...Oxidative stress is one of the main causes of neurodegenerative diseases such as Alzheimer disease(AD).Our previous studies have shown that artemisinin,a anti-malaria Chinese medicine,with neuroprotective effect,however,the antioxidative effect of artemisinin and its potential mechanism remain to be elucidated.In the present study,the protective effect and the underlying mechanism of artemisinin against injury of hydrogen peroxide(H_2O_2) in SH-SY5Y and hippocampal neurons were studied.Our results show that artemisinin protected SH-SY5Y and hippocampal neuronal cells from H_2O_2-induced cell death at clinically relevant concentrations in a concentration-dependent manner.Further studies showed that artemisinin significantly reduced cell death caused by H_2O_2 by restoring nuclear morphology,abnormal changes in intracellular ROS,activation of caspase 3,lactate dehydrogenase release and mitochondrial membrane potential.Hoechst staining and flow cytometry showed that artemisinin significantly reduced the apoptosis of SH-SY5Y cells exposed to H_2O_2.Western blotting analysis showed that artemisinin stimulated the phosphorylation and activation of AMP-activated protein kinase(AMPK) in SH-SY5Y cells in a time and concentration-dependent manner,whereas the application of AMPK inhibitor Compound C or decrease in expression of AMPKα with shRNA specific for AMPKα blocked the protective effect of artemisinin.Similar results were obtained in primary cultured hippocampal neurons.Taken together,these results indicate that artemisinin can protect neuronal cells from oxidative damage,at least in part through the activation of AMPK.Because artemisinin is relatively inexpensive and has few side effects,our findings support the role of artemisinin as a potential therapeutic agent for neurodegenerative diseases.展开更多
文摘目的探讨血管软化丸能否通过调控腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)/NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)通路抑制细胞焦亡改善载脂蛋白E基因敲除(apolipoprotein E knockout,APOE^(-/-))小鼠动脉粥样硬化(atherosclerosis,AS)。方法10只C57BL/6J小鼠作为空白对照组,60只APOE^(-/-)小鼠随机分为模型组、血管软化丸低剂量组、血管软化丸高剂量组、阿托伐他汀组、血管软化丸高剂量和AMPK抑制剂联合组、AMPK抑制剂组,给予高脂饮食饲养18周建造动脉粥样硬化小鼠模型,并于第13周给予不同方法干预,干预6周后取材,分别检测各组小鼠甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,TC)、低密度脂蛋白-胆固醇(low density lipoprotein-eholesterol,LDL-C)、高密度脂蛋白-胆固醇(high density lipoprotein-eholesterol,HDL-C)水平,HE和油红O染色观察小鼠主动脉组织病理形态学变化,Elisa测定主动脉组织白细胞介素(interleukin,IL)-1β和IL-18水平,Western Blot检测AMPK、P-AMPK、NLRP3、裂解的半胱氨酸天冬酶1(Cleaved-Caspase-1)、Gasdermin D(GSDMD)-N蛋白表达,免疫荧光检测P-AMPK、NLRP3蛋白在主动脉根部的分布情况,透射电子显微镜观察主动脉内皮超微结构改变情况。结果与对照组相比,模型组大鼠血清TG、TC、LDL-C水平显著升高,HDL-C水平显著降低(P<0.01),主动脉根部细胞排列紊乱,且脂肪空泡明显,主动脉有明显粥样斑块沉积,P-AMPK蛋白表达水平显著降低,NLRP3、Cleaved-Caspase 1、GSDMD-N蛋白表达水平及IL-1β和IL-18水平显著升高(P<0.05或P<0.01);与模型组比较,血管软化丸高低剂量组及阿托伐他汀组血脂紊乱得到改善,主动脉组织及细胞病变程度减轻,P-AMPK蛋白表达水平显著升高,NLRP3、Cleaved-Caspase 1、GSDMD-N蛋白表达水平显著降低(P<0.05或P<0.01),IL-1β和IL-18水平显著降低(P<0.05或P<0.01);与血管软化丸高剂量+AMPK抑制剂组比较,血管软化丸高剂量组焦亡相关蛋白水平显著降低(P<0.05或P<0.01),主动脉内皮细胞损伤程度减轻,而AMPK抑制剂组与之相反。结论血管软化丸可能通过调控AMPK/NLRP3通路减轻细胞焦亡发挥对动脉粥样硬化小鼠的保护作用。
基金The project supported by National Natural Science Foundation of China(81260497,81460564)Science and Technology Department of Jilin Province Youth Scientific Research Fund Project(201201075)
文摘OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compound derived from traditional Chinese medicine,used in Asian countries for the prevention and treatment of hepatic disease.Therefore,the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs.METHODS The murine HSC(tHSC/Cl-6)cell line were incubated in 96-well plates and treated with TNF-αand CuE at various concentrations and indicated times.Cell viability was assessed with MTT assay.Another,t-HSC/Cl-6 were incubated in 6-well plates and also treated with TNF-α,CuE,AICAR or metformin for the indicated time and concentration.Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blotting and RT-PCR.RESULTS CuE inhibited cell proliferation of activated HSC/T-6cells in concentration-and time-dependent manner.CuE triggered the activation of caspase-3,cleaved the PARP,ration of bcl-2/bax,and cytochrom c protein in a time-and concentration-dependent manner.CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK.CuE inhibited the protein and mRNA expressions ofα-SMA,TIMP-1 and collagenⅠ in activated HSC-T6.CuE broadly blocked p-PI3 K,p-Akt,p-mTOR and p-p70S6 Kexpressions.CuE significantly increased phosphorylated AMPK expression as well as AICAR and metoformin.And metformin showed significantly higher activation of AMPK than AICAR.Ability of CuE on activation of AMPK was between AICAR and metformin.It′s also found that CuE significantly decreased p-mTOR as well as AICAR and metformin.CONCLUSION CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment.The findings demonstrate that CuE induced HSC apoptosis via ERK/MAPK and PI3K/Akt-AMPK-mTOR signaling.
基金supported by National Natural Science Foundation of China(81700523)
文摘OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were treated with SRT1720(10μmol·L^(-1))and AICAR(500μmol·L^(-1))prior to ethanol(50 mmol·L^(-1)) for 24 and 48 h.Cell viability was analyzed by methyl thiazolyl tetrazolium assay.SIRT1,AMPK and p-AMPK m RNA levels for 24 h and 48 h were analyzed by RT-PCR,SIRT1,AMPK and p-AMPK protein expressions in the supernatant at 24 and 48 h was detected by Western blot.RESULTS SRT1720 and AICAR effectively decreased LX-2 cell viabilities and exhibited scarcely little toxicity in human Chang liver cells.SRT1720 and AICAR attenuated collagen-I,α-smooth muscle actin(α-SMA)levels,activated liver kinase B-1(LKB1)and AMPK phosphorylation in ethanol treated LX-2 cells.Meanwhile,SRT1720 and AICAR enhanced SIRT1 expression mediated by ethanol both in Chang liver cells and LX-2 cells.Furthermore,SRT1720 and AICAR suppressed the expression of sterol regulatory element-binding protein-1(SREBP-1)to regulate fatty acid synthesis.CONCLUSION SIRT1 agonist and AMPK agonist blocked the crosstalk between hepatocytes and HSCs via SIRT1/AMPK signaling pathway to modulate hepatocytes accumulation of lipid and HSCs activation.
基金National Natural Science Foundation of China(31771128)the University of Macao (MYRG2016-00052-FHS+2 种基金MYRG2018-00134-FHS)Science and Technology Development Fund (FDCT)of Macao (FDCT 021/2015/A1016/2016/A1).
文摘Oxidative stress is one of the main causes of neurodegenerative diseases such as Alzheimer disease(AD).Our previous studies have shown that artemisinin,a anti-malaria Chinese medicine,with neuroprotective effect,however,the antioxidative effect of artemisinin and its potential mechanism remain to be elucidated.In the present study,the protective effect and the underlying mechanism of artemisinin against injury of hydrogen peroxide(H_2O_2) in SH-SY5Y and hippocampal neurons were studied.Our results show that artemisinin protected SH-SY5Y and hippocampal neuronal cells from H_2O_2-induced cell death at clinically relevant concentrations in a concentration-dependent manner.Further studies showed that artemisinin significantly reduced cell death caused by H_2O_2 by restoring nuclear morphology,abnormal changes in intracellular ROS,activation of caspase 3,lactate dehydrogenase release and mitochondrial membrane potential.Hoechst staining and flow cytometry showed that artemisinin significantly reduced the apoptosis of SH-SY5Y cells exposed to H_2O_2.Western blotting analysis showed that artemisinin stimulated the phosphorylation and activation of AMP-activated protein kinase(AMPK) in SH-SY5Y cells in a time and concentration-dependent manner,whereas the application of AMPK inhibitor Compound C or decrease in expression of AMPKα with shRNA specific for AMPKα blocked the protective effect of artemisinin.Similar results were obtained in primary cultured hippocampal neurons.Taken together,these results indicate that artemisinin can protect neuronal cells from oxidative damage,at least in part through the activation of AMPK.Because artemisinin is relatively inexpensive and has few side effects,our findings support the role of artemisinin as a potential therapeutic agent for neurodegenerative diseases.
