痰湿型多囊卵巢综合征大鼠颗粒细胞AMPK/UCP2途径与线粒体功能变化的研究

目的观察痰湿型多囊卵巢综合征(PCOS)大鼠颗粒细胞腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)/解偶联蛋白2(uncoupling protein 2,UCP2)(AMPK/UCP2)途径与线粒体功能的变化,探讨“痰湿不孕”的机制。方法选取动情周期规律SD大鼠22只。运用高脂+来曲唑灌胃法复制痰湿型PCOS大鼠,随机分为对照组和模型组,造模成功后,取卵巢组织提取原代颗粒细胞,比色法检测三磷酸腺苷(ATP)含量;化学发光法检测活性氧(ROS)水平;Western blot法检测AMPKα、p-AMPKα及UCP2蛋白表达。结果与对照组比较,模型组大鼠颗粒细胞线粒体数量减少,出现肿胀,嵴断裂及空泡化改变;ATP降低、ROS增高(P<0.01);p-AMPKα/AMPKα比值降低(P<0.05);UCP2蛋白表达升高(P>0.05)。结论“痰湿不孕”可能与AMPK/UCP2途径所致的线粒体受损相关。

基于APN/AdipoR1/AMPK信号通路探讨滋肾丸对老年糖尿病大鼠骨骼肌胰岛素抵抗的改善作用

目的研究滋肾丸对D-半乳糖联合链脲佐菌素(Streptozotocin,STZ)诱导的老年糖尿病(Diabetes mellitus,DM)大鼠骨骼肌胰岛素抵抗(Insulin resistance,IR)的改善作用。方法取D-半乳糖联合STZ构建成功的30只老年DM大鼠,随机分为3组(每组10只):模型组、二甲双胍组(0.25 g·kg-1)和滋肾丸组(相当于28.0 g生药·kg-1),另取同批10只大鼠为对照组。对照组与模型组予等体积生理盐水灌胃,余各组予对应药物剂量灌胃,连续干预7周。末次给药后,测量计算腓肠肌指数;按对应试剂盒的说明流程分别检测大鼠空腹血糖(Fasting blood-glucose,FBG)、空腹胰岛素(Fasting insulin,FINS)以及血清三酰甘油(Triglyceride,TG)、总胆固醇(Total cholesterol,TC)、低密度脂蛋白胆固醇(Low density lipoprotein cholesterol,LDL-C)、高密度脂蛋白胆固醇(High-density lipoprotein cholesterol,HDL-C)、谷氨酸丙酮酸转氨酶(Glutamic pyruvic transaminase,ALT)、谷氨酸草酰乙酸转氨酶(Glutamic oxaloacetic transaminase,AST)、脂联素(Adiponectin,APN)、瘦素(Leptin,LP)、肿瘤坏死因子(Tumor necrosis factor-alpha,TNF-α)水平和骨骼肌丙二醛(Malondialdehyde,MDA)、超氧化物歧化酶(Superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH)、单磷酸腺苷(Adenosine monophosphate,AMP)和腺嘌呤核苷三磷酸(Adenosine triphosphate,ATP)活性,并计算胰岛素抵抗指数(Homeostasis Model Assessment-Insulin Resistance,HOMA-IR)和AMP/ATP比率;利用苏木精-伊红(Hematoxylin-eosin,HE)、过碘酸-希夫(Periodic acid-schiff,PAS)染色法和透射电镜(Transmission electron microscope,TEM)观察骨骼肌组织病理学和超微结构变化;蛋白免疫印迹法(Western blot)检测骨骼肌组织中胰岛素信号通路、腺苷酸活化蛋白激酶(Adenosine monophosphate activated protein kinase,AMPK)信号通路相关蛋白如胰岛素受体(Insulin receptor,IR)、胰岛素受体底物-1(Insulin receptor substrate-1,IRS-1)、磷酸化IRS-1(p-IRS-1)、磷脂酰肌醇3激酶(Phosphoinositide 3-kinase,PI3K)、总蛋白激酶B(Protein kinase B,AKT)、磷酸化AKT(p-AKT)、葡萄糖转运蛋白4(Glucose transporter-4,GLUT4),脂联素受体1(Adiponectin receptor 1,AdipoR1)、AdipoR2、AMPK、磷酸化AMPK(p-AMPK)、过氧化物酶体增殖物激活受体γ辅助激活因子-1α(Peroxisome proliferators-activated receptorγcoactivator alpha,PGC-1α)的蛋白表达;实时荧光定量聚合酶链式反应法(Real-time PCR)法检测它们相关基因的表达。结果与对照组相比,模型组的大鼠腓肠肌指数、FINS和血清LDL-C、APN以及骨骼肌SOD、GSH、AMP、ATP、AMP/ATP显著降低(P<0.01或P<0.001),FBG、HOMA-IR和血清TG、TC、HDL-C、ALT、AST、LP、TNF-α及骨骼肌MDA显著升高(均P<0.001);PAS染色结果显示模型组的大鼠骨骼肌大量肌纤维染色明显变浅、Ⅰ型和Ⅱ型肌纤维着色分辨深浅难辨;TEM结果显示模型组的大鼠骨骼肌细胞胞内局部轻度水肿,肌纤维排列局部紊乱、断裂,线粒体部分水肿,嵴减少,膜内基质变淡。模型组的大鼠骨骼肌组织p-IRS-1、PI3K、p-AKT、GLUT4、AdipoR1、p-AMPK、PGC-1α蛋白和IR、IRS-1、PI3K、AKT、GLUT4、APN、AdipoR1、AMPK、PGC-1αmRNA表达均显著下调(均P<0.001),且p-AKT/AKT和p-AMPK/AMPK蛋白表达比率也显著和明显降低(P<0.001和P<0.05)。经二甲双胍和滋肾丸治疗后,与模型组相比,二甲双胍组以及滋肾丸组腓肠肌指数、FINS和血清LDL-C、APN以及骨骼肌组织AMP、ATP、AMP/ATP显著升高(P<0.01或P<0.001),FBG、HOMA-IR和血清TG、TC、HDL-C、ALT、AST、LP、TNF-α及骨骼肌MDA明显或显著降低(P<0.05或P<0.01或P<0.001),PAS染色结果显示二甲双胍组和滋肾丸组的大鼠骨骼肌Ⅱ型肌纤维均有所增多且Ⅰ型和Ⅱ型肌纤维差别明显;TEM结果显示二甲双胍组和滋肾丸组的大鼠骨骼肌胞内和线粒体水肿等均有不同程度的改善。二甲双胍组以及滋肾丸组p-IRS-1、PI3K、p-AKT、GLUT4、AdipoR1、PGC-1α蛋白和IR、IRS-1、PI3K、AKT、GLUT4、APN、AdipoR1、AMPK、PGC-1αmRNA表达均显著上调(P<0.05或P<0.01或P<0.001),p-AKT/AKT蛋白表达比率显著升高(P<0.001)且滋肾丸组p-AMPK/AMPK蛋白表达比率明显升高(P<0.05)。结论滋肾丸能够改善糖脂代谢紊乱老年DM大鼠骨骼肌IR,其机制可能与增强骨骼肌PI3K/AKT信号通路、增加机体循环APN浓度和上调骨骼肌AdipoR1的表达并激活其下游AMPK/PGC1-α信号通路有关。

Study on the Relationship between AMPK/UCP2 Pathway and Mitochondrial Dysfunction in Granulosa Cells of Polycystic Ovay Syndrome

Objective:To investigate the changes of the AMP-activated protein kinase(AMPK)/uncoupling protein 2(UCP2)(AMPK/UCP2)pathway in ovarian granulosa cells with PCOS and its relationship with mitochondrial dysfunction.Methods:PCOS mouse models and normally fed mice,ovarian granulosa cells from the two mice were extracted,and the protein expression levels of AMPKα,p-AMPKαand UCP2 were detected by western blotting.The ROS and ATP content of granulosa cells were determined by colorimetric and chemiluminescence immunoassays to assess mitochondrial function.Pearson correlation analysis was used to determine the correlation between AMPK/UCP2 pathway-related proteins,ROS and ATP.Results:P-AMPKα/GAPDH(0.12±0.09),AMPKα/GAPDH(0.35±0.40),P-AMPKα/AMPKα(0.56±0.33)and ATP(0.36±0.04)pmol/mg in PCOS model mice were lower than those in non-POCS groups,while UCP2/GAPDH(1.18±0.28)and ROS(48810.92±4498.08)were lower than those in non-POCS groups.The fluorescence intensity of DCF was higher than that of the non-POCS group(P<0.05).AMPK was positively correlated with ATP and negatively correlated with ROS.UCP2 was positively correlated with ROS and negatively correlated with ATP.Conclusion:There are abnormal changes such as decreased AMPK expression and increased UCP2 expression in ovarian granulosa cells of PCOS,and AMPK is positively and negatively correlated with mitochondrial function indexes ATP and ROS,while UCP2 is the opposite,suggesting that the imbalance in the expression and activity of AMPK/UCP2 pathway in PCOS may be one of the molecular mechanisms leading to mitochondrial dysfunction.Regulation of AMPK/UCP2 pathway activity may be a potential therapeutic target to ameliorate PCOS-related mitochondrial dysfunction.

四氢姜黄素固体分散体通过调节肝脏AMPK/UCP2表达改善代谢综合征大鼠糖脂代谢紊乱

目的:观察四氢姜黄素固体分散体对高脂诱导的代谢综合征大鼠肝脏脂质,抗氧化系统,AMP-活化蛋白激酶(AMPK)磷酸化以及解偶联蛋白2(UCP2)表达的影响。方法:除空白组外,其余SD大鼠给予高脂喂养4 w后,按照糖耐量异常水平,动物随机分为模型组、二甲双胍150 mg/kg组、四氢姜黄素固体分散体20 mg/kg、40 mg/kg、80 mg/kg组。给药8 w后测定动物空腹血糖(FBG)、血清胰岛素(FIns)、C反应蛋白(CRP)、口服葡萄糖耐量(OGTT)、计算胰岛素抵抗指数(HOMAIR);测定肝脏中胆固醇(TC)、甘油三酯(TG)水平及氧化应激相关指标;油红O染色检测肝脏脂肪病变;免疫印迹法检测肝脏中AMPK磷酸化及UCP2的表达水平。结果:四氢姜黄素固体分散体40、80 mg/kg组能显著降低高脂饲养大鼠的FBG、FIns、CRP、HOMAIR,降低OGTT实验中AUC值,显著降低肝脏组织中TC、TG、丙二醛(MDA)含量,提高过氧化氢酶(CAT)、超氧化物岐化酶(SOD)活力,升高谷胱甘肽(GSH)含量,增加肝脏AMPK磷酸化及UCP2的表达。四氢姜黄素固体分散体20 mg/kg组能显著降低该模型大鼠血清CRP值、AUC值,显著降低肝脏组织中TC、TG、MDA含量,升高GSH含量,增加肝脏AMPK磷酸化。结论:四氢姜黄素固体分散体能显著改善代谢综合征大鼠的糖脂代谢紊乱同时改善肝脏抗氧化系统功能,该作用可能与激活肝脏中AMPK信号以及提高UCP2的表达有关。

Matrine attenuates oxidative stress and cardiomyocyte apoptosis in doxorubicin-induced cardiotoxicity via maintaining AMPKα/UCP2 pathway

Oxidative stress and cardiomyocyte apoptosis are involved in the pathogenesis of doxorubicin(DOX)-induced cardiotoxicity. Matrine is well-known for its powerful anti-oxidant and anti-apoptotic capacities. Our present study aimed to investigate the effect of matrine on DOX-induced cardiotoxicity and try to unearth the underlying mechanisms. Mice were exposed with DOX to generate DOX-induced cardiotoxicity or normal saline as control. H9 C2 cells were used to verify the effect of matrine in vitro.DOX injection triggered increased generation of reactive oxygen species(ROS) and excessive cardiomyocyte apoptosis, which were significantly mitigated by matrine. Mechanistically, we found that matrine ameliorated DOX-induced uncoupling protein 2(UCP2) downregulation, and UCP2 inhibition bygenipin could blunt the protective effect of matrine on DOX-induced oxidative stress and cardiomyocyte apoptosis. Besides, 50-AMP-activated protein kinase α2(Ampkα2) deficiency inhibited matrine-mediated UCP2 preservation and abolished the beneficial effect of matrine in mice. Besides, we observed that matrine incubation alleviated DOX-induced H9 C2 cells apoptosis and oxidative stress level via activating AMPKα/UCP2, which were blunted by either AMPKα or UCP2 inhibition with genetic or pharmacological methods. Matrine attenuated oxidative stress and cardiomyocyte apoptosis in DOX-induced cardiotoxicity via maintaining AMPKα/UCP2 pathway, and it might be a promising therapeutic agent for the treatment of DOX-induced cardiotoxicity.