电针通过调节AMPK/mTOR/ULK1信号通路对衰老小鼠肝脏自噬与氧化应激的影响

目的:观察电针对衰老小鼠体力及肝脏AMP活化的蛋白激酶(AMPK)、哺乳动物雷帕霉素靶蛋白(mTOR)、unc-51样自噬激活激酶1(ULK1)、Atg5、Atg7、Atg13和Beclin1表达的影响,探索其通过激活AMPK/mTOR/ULK1通路延缓衰老的机制.方法:将30周龄雄性SAMP8小鼠随机分为模型组、雷帕...     

肝豆汤对Wilson病模型高铜诱导的SH-SY5Y细胞自噬效应的影响及其作用机制

目的:研究肝豆汤对高铜诱导的人神经母细胞瘤(SH-SY5Y)细胞自噬效应的影响及其作用机制,为中医药防治脑型Wilson病(Wilson disease,WD)提供新的治疗靶点和研究思路。方法:噻唑蓝(MTT)比色法筛选硫酸铜(CuSO_4)造模浓度(0,100,200,400,800,1 600μmol·L-1)及时间; MTT比色法筛选含药血清浓度(5%,10%,15%,20%)及时间;乳酸脱氢酶(LDH)释放实验检测细胞LDH漏出率;流式细胞法检测细胞内活性氧(ROS)的含量;荧光染料JC-1检测细胞线粒体膜电位;流式细胞仪对自噬进行定量分析。蛋白免疫印迹法(Western blot)检测肝激酶B1(LKB1),腺苷酸活化蛋白激酶(AMPK),自噬微管相关蛋白轻链3A/B(LC3A/B),哺乳动物雷帕霉素靶蛋白(mTOR),unc-51样激酶1(ULK1),磷酸化ULK(p-ULK),磷酸化AMPK(p-AMPK)蛋白的表达。结果:MTT结果显示,CuSO_4对细胞的损伤呈现一定的量效和时效关系(P 0. 01),随着CuSO_4作用浓度及时间的增加,细胞存活率呈现下降趋势; 10%含肝豆汤兔血清可显著抑制CuSO_4诱导的细胞死亡(P 0. 01)。LDH释放实验显示,与正常组比较,CuSO_4作用细胞后LDH漏出率显著增加(P 0. 01),与模型组比较,含肝豆汤兔血清明显降低CuSO_4损伤细胞的LDH漏出率(P 0. 05)。DCFH-DA荧光染色显示,与正常组比较,CuSO_4可显著增加细胞内ROS生成(P 0. 01),与模型组比较,含肝豆汤兔血清可显著抑制CuSO_4诱导的细胞内ROS产生(P 0. 01)。JC-1染色结果显示,与正常组比较,CuSO_4诱导细胞线粒体膜电位Δψm显著降低(P 0. 01),与模型组比较,含肝豆汤兔血清明显抑制CuSO_4诱导的线粒体膜电位降低Δψm(P 0. 05)。Western blot结果显示,与正常组比较,模型组细胞内LKB1,AMPK,LC3A/B,ULK1及p-AMPK蛋白的表达显著增加,mTOR及p-ULK蛋白的表达显著降低(P 0. 01)。与模型组比较,含肝豆汤兔血清组LKB1,AMPK,LC3A/B,ULK1及p-AMPK蛋白表达显著降低,mTOR及p-ULK蛋白表达显著增加(P 0. 01)。结论:高铜可通过诱导细胞内线粒体氧化应激,上调自噬相关蛋白LKB1,p-AMPK,AMPK,LC3A/B及ULK1的表达,下调自噬相关蛋白mTOR及p-ULK的表达,导致细胞发生自噬性死亡,而肝豆汤可通过调控LKB1/AMPK信号通路,下调自噬相关蛋白LKB1,p-AMPK,LC3A/B,ULK及AMPK的表达,上调自噬相关蛋白及基因mTOR及p-ULK的表达,抑制自噬的发生,阻断高铜诱导的神经元损伤,从而发挥神经保护作用。     

Ginkgo biloba extract protects against diabetic cardiomyopathy by restoring autophagy via adenosine monophosphate-activated protein kinase/mammalian target of the rapamycin pathway modulation

Studies demonstrated that Ginkgo biloba extract (GBE) played a cardioprotective role in diabetic conditions. Impaired autophagy is one of the mechanisms underlying diabetic cardiomyopathy (DCM). The effect of GBE on autophagy has been observed in several diseases; however, whether GBE can ameliorate DCM by regulating autophagy remains unclear. Here, we investigated the effect of GBE on DCM and the potential mechanisms regarding autophagy using a streptozotocin (STZ)-induced diabetic rat model and a high-glucose (HG)-stimulated H9C2 cell model. We demonstrated that GBE attenuated metabolic disturbances, improved cardiac function, and reduced myocardial pathological changes in diabetic rats. Impaired autophagy as well as dysregulation of the adenosine monophosphate-activated protein kinase/ mammalian target of the rapamycin (AMPK/mTOR) signaling pathway were observed in diabetic hearts, as evidenced by the reduced conversion of LC3B-I to LC3B-II along with excessive p62 accumulation, decreased AMPK phosphorylation, and increased mTOR phosphorylation, which could be reversed by GBE treatment. In vitro, GBE reduced the apoptosis induced by HG in H9C2 cells by activating AMPK and inhibiting mTOR to restore autophagy. However, this effect was inhibited by the AMPK inhibitor Compound C. In conclusion, the ameliorative effect of GBE on DCM might be dependent on the restoration of autophagy through modulation of the AMPK/mTOR pathway.     

Qili Qiangxin,a compound herbal medicine formula,alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro

ObjectiveQili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model.MethodsThe main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia–reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.ResultsApoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX.ConclusionQLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.     

基于LKB1/AMPK/PGC-1α的泽泻汤改善非酒精性脂肪肝作用机制研究

以棕榈酸(palmitic acid,PA)诱导的脂质堆积细胞模型和高脂诱导的非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)动物模型,探讨泽泻汤(Zexie Decoction)体内外改善NAFLD的药效,以LKB1/AMPK/PGC-1α通路为切入点探讨其可能作用机制。MTT结果显示泽泻汤对HepG2细胞活力无影响,泽泻汤剂量依赖性下调PA诱导的肝细胞培养基谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)的水平,降低高脂饮食(high-fat diet,HFD)小鼠血浆ALT、AST及肝脏总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)水平。尼罗红染色观察PA诱导的细胞内脂质沉积,PA诱导的肝细胞脂质蓄积显著增加,体外细胞脂质堆积模型诱导成功,泽泻汤有效改善PA诱导的肝细胞脂质堆积;小鼠肝脏油红染色结果同样表明,泽泻汤剂量依赖性减少HFD小鼠肝脏脂质堆积。线粒体膜电位染色显示泽泻汤能够逆转PA诱导肝细胞损伤引起的线粒体膜电位的下降;同时泽泻汤激活PGC-1α上调其靶基因ACADS、CPT-1α、CPT-1β、UCP-1、ACSL-1、NRF-1等的表达;Western blot及免疫组化结果显示泽泻汤可体内外上调LKB1、p-AMPK、p-ACC、PGC-1α蛋白表达水平。综上所述,泽泻汤能够有效改善NAFLD,其机制可能与调控LKB1/AMPK/PGC-1α通路相关。     

迷迭香酸通过AMPK/mTOR通路减轻新生大鼠缺血缺氧性脑损伤研究

目的 探讨迷迭香酸对新生大鼠缺血缺氧脑损伤（hypoxic-ischemic encephalopathy,HIE）的影响,及其对单磷酸腺苷活化蛋白激酶（adenosine monophosphate activated protein kinase,AMPK）/雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）通路的调控作用,初步探讨其脑保护机制。方法 取7 d龄SD新生大鼠,随机分为对照组、模型组、迷迭香酸（300 mg/kg）组、AMPK/mTOR激动剂MT6378（10 mg/kg）组、AMPK抑制剂GSK-690693（30 mg/kg）组和迷迭香酸（300 mg/kg）+MT6378（10 mg/kg）组,每组20只。建立HIE模型,给予相应药物进行干预,采用TTC染色法检测大鼠脑梗死情况;透射电镜（TEM）观察大鼠海马神经元结构损伤及自噬状况;免疫荧光法检测大鼠海马神经元自噬标记物微管相关蛋白1轻链3B（microtubule-associated protein 1 light chain 3B,LC3B）阳性表达;TUNEL法检测大鼠海马神经元凋亡率;免疫组化法检测大鼠海马神经元磷酸化AMPK（p-AMPK）阳性表达;Western blotting检测大鼠海马组织活化的半胱氨酸蛋白酶3（cleaved Caspase-3）、mTOR及其磷酸化蛋白（p-mTOR）、Unc-51样自噬激活激酶1（uncoordinated-51 like autophagy activating kinase 1,Ulk1）及其磷酸化蛋白（p-Ulk1）、LC3B表达。结果 与对照组相比,模型组大鼠脑梗死严重,海马神经元结构损伤及自噬空泡形成较多,细胞自噬及凋亡水平升高,AMPK/mTOR通路活化（P<0.05）。与模型组相比,迷迭香酸组及GSK-690693组大鼠脑梗死、海马神经元结构损伤、凋亡及自噬减弱,AMPK/mTOR通路被抑制（P<0.05）;MT6378组海马组织AMPK/mTOR通路进一步激活,大鼠脑梗死、海马神经元结构损伤、凋亡及自噬进一步加重（P<0.05）;MT6378可逆转迷迭香酸的上述作用（P<0.05）。结论 迷迭香酸可能通过抑制AMPK/mTOR通路激活,降低海马神经元自噬及凋亡进程,发挥抗HIE脑损伤作用。     

Quercetin improves nonalcoholic fatty liver by ameliorating inflammation,oxidative stress,and lipid metabolism in db/db mice

Multiphase pathological processes involve in Type 2 diabetes (T2DM)‐induced nonalcoholic fatty liver disease (NAFLD). However, the therapies are quite limited. In the present study, the hepatoprotective effects and underlying mechanisms of quercetin in T2DM‐induced NAFLD were investigated. T2DM‐induced NAFLD and quercetin treatment models were established in vivo and in vitro. The results revealed that quercetin alleviated serum transaminase levels and markedly reduced T2DM‐induced histological alterations of livers. Additionally, quercetin restored superoxide dismutase, catalase, and glutathione content in livers. Not only that, quercetin markedly attenuated T2DM‐induced production of interleukin 1 beta, interleukin 6, and TNF‐α. Accompanied by the restoration of the increased serum total bile acid (p = .0001) and the decreased liver total bile acid (p = .0005), quercetin could reduce lipid accumulation in the liver of db/db mice. Further mechanism studies showed that farnesoid X receptor 1/Takeda G‐protein‐coupled receptor 5 signaling pathways was involved in quercetin regulation of lipid metabolism in T2DM‐induced NAFLD. In high D‐glucose and free fatty acid cocultured HepG2 cells model, quercetin eliminated lipid droplets and restored the upregulated total cholesterol and triglyceride levels. Similar to the findings in mice, quercetin could also activate farnesoid X receptor 1/Takeda G‐protein‐coupled receptor 5 signaling pathway. These findings suggested that quercetin might be a potentially effective drug for the treatment of T2DM‐induced NAFLD.     

当归多糖对糖尿病肾病KK-Ay小鼠肾脏AMPK信号通路及线粒体自噬的影响

目的 研究当归多糖对糖尿病肾病（diabetic nephropathy,DN）KK-Ay小鼠肾脏磷酸腺苷激活的蛋白激酶（AMPactivated protein kinase,AMPK）信号通路及线粒体自噬的影响。方法 SPF级雄性KK-Ay小鼠用高糖高脂饲料喂养,随机分为模型组、厄贝沙坦（25 mg/kg）组和当归多糖高、中、低剂量（400、200、100 mg/kg）组,每组10只;将10只雄性C57BL/6J小鼠作为对照组。给予药物干预4周,观察小鼠一般情况,每周称定体质量并检测血糖;末次给药后,心脏取血并处死小鼠,分离血清检测尿微量白蛋白（urine microalbuminuria,U-ALB）、肌酐（creatinine,SCr）、尿素氮（urea nitrogen,BUN）;采用苏木素-伊红（HE）染色观察肾组织病理变化;采用Western blotting检测肾组织线粒体自噬相关蛋白[微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、p62、Nix]和线粒体裂变蛋白[线粒体动力相关蛋白1(dy...     

Baicalein Triggers Autophagy and Inhibits the Protein Kinase B/Mammalian Target of Rapamycin Pathway in Hepatocellular Carcinoma HepG2 Cells

Baicalein (BA), isolated from the Chinese medicinal herb Scutellariae radix (Huangqin in Chinese), is a flavonoid with various pharmacological effects. Herein, we found that BA only slightly reduced the cell viability on HepG2 cells after 24‐h treatment as determined by 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyl tetrazolium bromide (MTT) assay. However, BA (50 μM) effectively blocked the colony formation. Meanwhile, BA remarkably induced the formation of autophagosomes after 24‐h treatment as determined by immunofluorescence with monodansylcadaverine staining as well as transmission electron microscopy, respectively. Moreover, BA obviously up‐regulated the expression of microtubule‐associated protein 1A/1B‐light chain 3‐II in concentration‐dependent and time‐dependent manners in HepG2 cells. When combined with the autophagy inhibitor chloroquine and BA, the cell viability and colony formation were significantly decreased, indicating that BA triggered protective autophagy, which prevented cell death. Further study showed that BA concentration‐dependently and time‐dependently decreased the expression of p‐AKT (S473), p‐ULK1 (S757) and p‐4EBP1 (T37 and S65), suggesting the involvement of protein kinase B (AKT)/mammalian target of rapamycin (mTOR) in BA‐triggered autophagy. Copyright © 2015 John Wiley & Sons, Ltd.     

Cassiae Semen improves non-alcoholic fatty liver disease through autophagy-related pathway

Objective Cassiae Semen (CS, Juemingzi in Chinese) has been used for thousands of years in ancient Chinese history for relieving constipation, improving liver function as well as preventing myopia. Here we aimed to elucidate the anti-steatosis effect and underlying mechanism of CS against non-alcoholic fatty liver disease (NAFLD). Methods High-performance liquid chromatography (HPLC) was used to identify the major components of CS water extract. Mice were fed with a high-fat and sugar-water (HFSW) diet to induce hepatic steatosis and then treated with CS. The anti-NAFLD effect was determined by measuring serum biomarkers and histopathology staining. Additionally, the effects of CS on cell viability and lipid metabolism in oleic acid and palmitic acid (OAPA)-treated HepG2 cells were measured. The expression of essential genes and proteins involved in lipid metabolism and autophagy signalings were measured to uncover the underlying mechanism. Results Five compounds, including aurantio-obtusin, rubrofusarin gentiobioside, cassiaside C, emodin and rhein were simultaneously identified in CS extract. CS not only improved the diet-induced hepatic steatosis in vivo, as indicated by decreased number and size of lipid droplets, hepatic and serum triglycerides (TG) levels, but also markedly attenuated the OAPA-induced lipid accumulation in hepatocytes. These lipid-lowering effects induced by CS were largely dependent on the inhibition of fatty acid synthase (FASN) and the activation of autophagy-related signaling, including AMP-activated protein kinase (AMPK), light chain 3-II (LC3-II)/ LC3-1 and autophagy-related gene5 (ATG5). Conclusion Our study suggested that CS effectively protected liver steatosis via decreasing FASN-related fatty acid synthesis and activating AMPK-mediated autophagy, which might become a promising therapeutic strategy for relieving NAFLD.     

Gastrodin Ameliorates Oxidative Stress and Proinflammatory Response in Nonalcoholic Fatty Liver Disease through the AMPK/Nrf2 Pathway

This study was designed to investigate the antioxidative, antiinflammatory and metabolism‐regulating effects of gastrodin (GSTD) in the treatment of nonalcoholic fatty liver disease (NAFLD). Oleic acid (OA) was used to induce steatosis in HL‐7702 cells; a high‐fat or high‐fat and high‐cholesterol diet was used to induce NAFLD in mice and rats. Our results showed that GSTD significantly increased hepatic superoxide dismutase (SOD) but decreased reactive oxygen species (ROS)/malondialdehyde (MDA) and reduced the mRNA levels of proinflammatory cytokines both in vitro and in vivo. GSTD promoted the phosphorylation of nuclear factor erythroid‐2‐related factor‐2 (Nrf2) at serine (Ser) 40, stimulated its nuclear translocation and increased hepatic expression of heme oxygenase‐1 (HO‐1). GSTD activated AMP‐activated protein kinase (AMPK), suppressed hepatic steatosis, lowered serum triglyceride (TG)/glucose and decreased body weight gain in animals with NAFLD. The stimulating effects of GSTD on the Nrf2 pathway as well as its antioxidative/antiinflammatory activities were abolished by compound C in OA‐treated HL‐7702 cells. In summary, our results demonstrate that GSTD activates the AMPK/Nrf2 pathway, ameliorates oxidative stress/proinflammatory response and improves lipid metabolism in NAFLD. Our findings may support the future clinical application of GSTD for the treatment of NAFLD to reduce hepatic steatosis, oxidative stress and proinflammatory response. Copyright © 2015 John Wiley & Sons, Ltd.     

Alpinetin alleviates osteoporosis by promoting osteogenic differentiation in BMSCs by triggering autophagy via PKA/mTOR/ULK1 signaling

Osteoporosis, a systemic bone disease that is characterized by a reduction in bone mass and destruction of bone microstructure, is becoming a serious problem worldwide. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into bone-forming osteoblasts, and play an important role in maintaining homeostasis of bone metabolism, thus being a potential therapeutic target for osteoporosis. Although the phytochemical alpinetin (APT) has been reported to possess a variety of pharmacological activities, it is still unclear whether APT can influence the osteogenic differentiation of on BMSCs and if it can improve osteoporosis. In this study, we found that APT treatment was able to enhance osteogenic differentiation levels of human BMSCs in vitro and mouse ones in vivo as revealed by multiple osteogenic markers including increased alkaline phosphatase activity and osteocalcin expression. Mechanistically, the protein kinase A (PKA)/mTOR/ULK1 signaling was involved in the action of APT to enhance the osteogenic differentiation of BMSCs. In addition, oral administration of APT significantly mitigated the bone loss in a dexamethasone-induced mouse model of osteoporosis through strengthening PKA signaling and autophagy. Altogether, these data demonstrate that APT promotes osteogenic differentiation in BMSCs by augmenting the PKA/mTOR/ULK1 autophagy signaling, highlighting its potential therapeutic application for treating osteoporotic diseases.     

Hypolipogenic effects of Icariside E4 via phosphorylation of AMPK and inhibition of MID1IP1 in HepG2 cells

Though icariside E4 (IE4) is known to have anti-noceptive, anti-oxidant, anti-Alzheimer and anti-inflammatory effects, there was no evidence on the effect of IE4 on lipid metabolism so far. Hence, the hypolipogenic mechanism of IE4 was investigated in HepG2 hepatocellular carcinoma cells (HCCs) in association with MID1 Interacting Protein 1(MID1IP1) and AMPK signaling. Here, IE4 did not show any toxicity in HepG2 cells, but reduced lipid accumulation in HepG2 cells by Oil Red O staining. MID1IP1 depletion decreased the expression of SREBP-1c and fatty acid synthase (FASN) and induced phosphorylation of ACC in HepG2 cells. Indeed, IE4 activated phosphorylation of AMPK and ACC and inhibited the expression of MID1IP1 in HepG2 cells. Furthermore, IE4 suppressed the expression of SREBP-1c, liver X receptor-α (LXR), and FASN for de novo lipogenesis in HepG2 cells. Interestingly, AMPK inhibitor compound C reversed the ability of IE4 to reduce MID1IP1, SREBP-1c, and FASN and activate phosphorylation of AMPK/ACC in HepG2 cells, indicating the important role of AMPK/ACC signaling in IE4-induced hypolipogenic effect. Taken together, these findings suggest that IE4 has hypolipogenic potential in HepG2 cells via activation of AMPK and inhibition of MID1IP1 as a potent candidate for treatment of fatty liver disease.     

栀子苷改善大鼠非酒精性脂肪性肝病游离脂肪酸代谢的机制研究

观察栀子苷改善非酒精性脂肪性肝病的效果,从游离脂肪酸探讨栀子苷调节非酒精性脂肪性肝病的作用机制。健康雄性Wistar大鼠40只,随机分为4组:正常组、模型组、栀子苷组和血脂康组,每组大鼠10只,正常组大鼠给予正常大鼠饲料喂养,其余3组大鼠均采用高脂饲料喂养以诱导非酒精性脂肪性肝病,造模时间为8周,从第5周起至第8周末,栀子苷组和血脂康组分别灌服相应的药物。记录大鼠体重、肝湿重、脂肪质量;用相应方法检测肝组织TG,FFA,FAS,AMPK,ACCase及Malonyl-Co A含量,血清CHO,LDL-C的含量,血清AST,ALT的活性;观察肝脏组织肉眼及病理变化(HE染色法)。结果显示,与正常组相比,模型组大鼠的体重;肝湿重;脂肪重;血清CHO,LDL-C,ALT,AST;肝组织TG,FFA,FAS,ACCase及Malonyl-Co A含量皆显著升高(P0.01);肝组织AMPK活性显著降低(P0.01),肝组织肉眼外观及病理切片脂肪变性明显,并出现炎症损伤;与模型组相比,栀子苷组大鼠体重、脂肪质量、肝组织FFA含量、血清ALT,AST活性均显著降低(P0.01),肝湿重,肝组织TG,FAS,ACCase及Malonyl-Co A含量明显降低(P0.05),肝组织AMPK活性明显增多(P0.05),肝脏肉眼外观及病理学表现均有所改善;与模型组相比,血脂康组大鼠的肝湿重、脂肪质量、肝组织TG,FFA和血清LDL-C水平明显降低(P0.05);与血脂康组相比,栀子苷组大鼠的体重、脂肪质量、肝组织FFA含量均显著降低(P0.01),其他方面无明显差异。结果表明,栀子苷具有显著的改善高脂饮食诱导的大鼠非酒精性脂肪性肝病的药理效应;其改善大鼠非酒精性脂肪性肝病的游离脂肪酸代谢是通过调节"AMPK-ACCase-Malonyl-Co A-FFA"轴来实现的。     

养心康片通过Akt/AMPK-mTOR通路对心肌梗死后心力衰竭模型兔心肌细胞自噬的影响

目的观察养心康片通过Akt/AMPK-mTOR通路对心肌梗死后心力衰竭模型兔心肌细胞自噬的影响。方法对实验兔应用结扎冠脉的方法建立心肌梗死后心力衰竭兔模型,随机分为模型组、养心康组、AMPK抑制剂组、Akt抑制剂组和mTOR抑制剂组。并另取不造模的兔设立空白对照组。每组5只,共30只。造模后第3天开始分别给予相应的处理措施,共4周。观察养心康片对模型兔心肌beclin 1、LC3蛋白表达,心肌beclin 1和Atg5 mRNA的表达的影响,电镜检测各组心肌细胞超微结构。结果养心康组的beclin 1 mRNA的表达量、beclin 1蛋白表达量较模型组、mTOR抑制剂组降低(P<0.05或P<0.01),较空白对照组、AMPK抑制剂组升高(P<0.05或P<0.01);养心康组的LC3-Ⅱ/LC3-I比值较模型组、mTOR抑制剂组降低(P<0.05),较空白对照组、AMPK抑制剂组升高(P<0.05)。养心康组的Atg5 mRNA表达量较模型组降低(P<0.05),较空白对照组、AMPK抑制剂组升高(P<0.05或P<0.01)。电镜结果显示养心康组的自噬体、自噬体溶酶体数量较模型组、Akt抑制剂组和mTOR抑制剂组降低减少,较AMPK抑制剂组增多。结论养心康片可通过干预Akt/AMPK-mTOR通路调控心肌细胞自噬水平,改善心肌梗死后心衰模型的心肌细胞超微结构。     

Preventive and curative effects of Berberis aristata Fruit extract on paracetamol- and CCl4-induced hepatotoxicity

The effect of an aqueous-methanol extract of Berberis aristata fruits (Berberidaceae) was investigated against paracetamol- and CCl₄-induced hepatic damage. Paracetamol produced 100% mortality at a dose of 1 g/kg in mice while pre-treatment of animals with crude extract (500 mg/kg) reduced the death rate to 10%. Pre-treatment of rats with fruit extract (500 mg/kg, orally twice daily for 2 days) prevented (p<0.05) the paracetamol (640 mg/kg) as well as CCl₄ (1.5 mL/kg)-induced rise in serum transaminases (GOT and GPT). Post-treatment with three successive doses of extract (500 mg/kg, 6h) restricted the hepatic damage induced by acetaminophen (p<0.01) but CCl₄-induced hepatotoxicity was not altered (p>0.05). Plant extract (500 mg/kg) caused significant prolongation (p<0.01) in pentobarbital (75 mg/kg)-induced sleep as well as increased strychnine-induced lethality in mice suggestive of inhibitory effect on microsomal drug metabolizing enzymes (MDME). These results indicate that the crude extract of Berberis aristata fruits exhibits hepatoprotective action partly through MDME inhibitory action.     

黄连温胆汤对胰岛素抵抗大鼠肝脏脂质蓄积的影响

目的观察黄连温胆汤对胰岛素抵抗(IR)大鼠肝脏脂质蓄积的影响,从自噬角度探讨其作用机制。方法50只雄性SD大鼠随机分为空白组10只和造模组40只,造模组采用高脂高糖饮食诱导IR大鼠模型,将成模大鼠随机分为模型组、黄连温胆汤组和二甲双胍组,分别予蒸馏水、黄连温胆汤药液和二甲双胍溶液灌胃,连续8周。透射电镜观察大鼠肝组织自噬小体形成情况;油红O染色观察肝脏脂质蓄积;ELISA检测大鼠空腹血糖(FPG)、空腹胰岛素(FINS),计算胰岛素抵抗指数(HOMA-IR);GPO-PAP法检测大鼠肝组织三酰甘油(TG)、总胆固醇(TC)含量;Western blot检测肝组织LC3、p62、哺乳动物雷帕霉素靶蛋白(mTOR)和p-mTOR蛋白表达;RT-PCR检测肝组织mTOR mRNA表达。结果与空白组比较,模型组大鼠肝组织红色脂滴明显增多,并有少量自噬小体形成,FPG、FINS和HOMA-IR明显升高(P<0.01),肝组织TC、TG含量明显增加(P<0.05,P<0.01),LC3Ⅱ/LC3Ⅰ比值升高,p62蛋白表达降低,p-mTOR/mTOR比值降低(P<0.01);与模型组比较,黄连温胆汤组和二甲双胍组大鼠肝组织红色脂滴数量减少,未见明显自噬小体,FPG、FINS和HOMA-IR明显降低(P<0.05,P<0.01),肝组织TC、TG含量减少(P<0.05,P<0.01),LC3Ⅱ/LC3Ⅰ比值降低,p62蛋白表达升高,p-mTOR/mTOR比值升高(P<0.05,P<0.01)。各组大鼠肝组织mTOR mRNA表达差异无统计学意义(P>0.05)。结论黄连温胆汤可改善IR大鼠肝脏脂质蓄积,其机制与上调大鼠肝组织p-mTOR蛋白表达,抑制肝脏自噬有关。     

九味肝泰胶囊对高脂饮食诱导大鼠非酒精性脂肪肝的治疗作用

目的研究九味肝泰胶囊对高脂饮食诱导的大鼠非酒精性脂肪性肝病(NAFLD)的治疗作用和可能机制。方法采用高脂饮食诱导大鼠NAFLD动物模型。设对照组,模型组,水飞蓟素阳性对照组(0.05 g/kg),九味肝泰胶囊高、中、低剂量(1.80、0.90、0.45g/kg)组。除对照组给予普通饲料饲养外,其他各组以高脂饲料喂养,饲养10周。从第5周开始,4个给药组分别ig给予相应药物,共给药6周。观察和检测大鼠体质量、肝湿质量、肝脏指数和肝脏形态学变化;检测血清中的高密度脂蛋白-胆固醇(HDL-C)、游离脂肪酸(FFA)、天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)水平;检测肝脏组织中总胆固醇(TC)、三酰甘油(TG)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平。结果与模型组比较,九味肝泰胶囊高、中剂量组能显著降低NAFLD大鼠血清中的FFA、AST、ALT水平(P0.05),升高HDL-C水平(P0.05);显著降低NAFLD大鼠肝脏组织中的TC、TG、MDA水平(P0.05),升高SOD的活性(P0.05);NAFLD大鼠的肝组织病理学形态均得到不同程度改善,大鼠肝组织脂肪变程度减轻。结论九味肝泰胶囊对NAFLD大鼠具有调节脂质和保护肝脏的作用,其作用机制可能与减少脂质在肝脏的沉积、减轻过氧化损伤有关。