Electroacupuncture preconditioning attenuates ischemic brain injury by activation of the adenosine monophosphate-activated protein kinase signaling pathway

Electroacupuncture has therapeutic effects on ischemic brain injury, but its mechanism is still poorly understood. In this study, mice were stimulated by electroacupuncture at the Baihui（GV20） acupoint for 30 minutes at 1 m A and 2/15 Hz for 5 consecutive days. A cerebral ischemia model was established by ligating the bilateral common carotid artery for 15 minutes. At 72 hours after injury, neuronal injury in the mouse hippocampus had lessened, and the number of terminal deoxynucleotide transferase-mediated d UTP nick-end labeling-positive cells reduced after electroacupuncture treatment. Moreover, expression of adenosine monophosphate-activated protein kinase α（AMPKα） and phosphorylated AMPKα was up-regulated. Intraperitoneal injection of the AMPK antagonist, compound C, suppressed this phenomenon. Our findings suggest that electroacupuncture preconditioning alleviates ischemic brain injury via AMPK activation.

Adenosine monophosphate-activated protein kinase activation enhances embryonic neural stem cell apoptosis in a mouse model of amyotrophic lateral sclerosis

Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dis- mutase 1 mutant （SOD1G93A） and wild-type （SOD1wv） mouse models were exposed to H202. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by flow cytometry. Moreover, we evaluated the expression of the adenos- ine monophosphate-activated protein kinase （AMPK） ct-subunit, paired box 3 （Pax3） protein, and p53 in western blot analyses. Compared with SOD1wr cells, SOD1~93A embryonic neural stem cells were more likely to undergo H202-induced apoptosis. Phosphorylation of AMPKct in SOD1G93A cells was higher than that in SOD1wr cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKct. p53 protein levels were also correlated with AMPKct phosphorylation levels. Compound C, an inhibitor of AMPKa, attenuated the effects of H20~. These results suggest that embryonic neural stem cells from SOD1C93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKa pathway.

AB015.Metabolic stress in glaucoma engages early activation of the energy biosensor adenosine monophosphate-activated protein kinase leading to neuronal dysfunction

Background:Metabolic stress has been proposed to contribute to neuronal damage in glaucoma,but the mechanism driving this response is not understood.The adenosine monophosphate-activated protein kinase(AMPK)is a master regulator of energy homeostasis that becomes active at the onset of energy stress.AMPK is a potent inhibitor of the mammalian target of rapamycin complex 1(mTORC1),which we showed is essential for the maintenance of retinal ganglion cell(RGC)dendrites,synapses,and survival.Here,we tested the hypothesis that AMPK is an early mediator of metabolic stress in glaucoma.Methods:Unilateral elevation of intraocular pressure was induced by injection of magnetic microbeads into the anterior chamber of mice expressing yellow fluorescent protein in RGCs.Inhibition of AMPK was achieved by administration of siRNA or compound C.RGC dendritic trees were 3D-reconstructed and analyzed with Imaris(Bitplane),and survival was assessed by counting Brn3a or RBPMS-labeled soma and axons in the optic nerve.RGC function was examined by quantification of anterograde axonal transport after intraocular administration of cholera toxinβ-subunit.Retinas from glaucoma patients were analyzed for expression of active AMPK.Results:Ocular hypertension triggered rapid upregulation of AMPK activity in RGCs concomitant with loss of mTORC1 function.AMPK inhibition with compound C or siRNA effectively restored mTORC1 activity and promoted an increase in total dendritic length,surface and complexity relative to control retinas.Attenuation of AMPK activity led to robust RGC soma and axon survival.For example,95%of RGCs(2,983±258 RGCs/mm2,mean±S.E.M.)survived with compound C compared to 77%in vehicle-treated eyes(2,430±233 RGCs/mm2)(ANOVA,P<0.001)at three weeks after glaucoma induction(n=8-10/group).Importantly,blockade of AMPK activity effectively restored anterograde axonal transport.Lastly,RGC-specific upregulation of AMPK activity was detected in human glaucomatous retinas relative to age-matched controls(n=10/group).Conclusions:Metabolic stress in glaucoma involves AMPK activation and mTORC1 inhibition promoting early RGC dendritic pathology,dysfunction and neurodegeneration.

Metformin attenuates motility,contraction,and fibrogenic response of hepatic stellate cells in vivo and in vitro by activating AMP-activated protein kinase

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.

Dan-gua Fang (丹瓜方) Improves Glycolipid Metabolic Disorders by Promoting Hepatic Adenosine 5'-monophosphate Activated Protein Kinase Expression in Diabetic Goto-kakizaki Rats

Objective：To investigate the effect of Dan-gua Fang（丹瓜方） on adenosine 5’-monophosphate（AMP） activated protein kinase（AMPK） α expression in liver and subsequent improvement of glucose and lipid metabolism.Methods：Forty 13-week-old diabetic Goto-Kakizaki（GK） rats were randomly divided into model,Dan-gua Fang,metformin and simvastatin groups（n=10 for each）,and fed high-fat diet ad libitum.Ten Wistar rats were used as normal group and fed normal diet.After 24 weeks,liver expression of AMPK α mRNA was assessed by real-time PCR.AMPK α and phospho-AMPK α protein expression in liver was evaluated by Western blot.Liver histomorphology was carried out after hematoxylin-eosin staining,and blood glucose（BG）,glycosylated hemoglobin A1c（HbA1c）,food intake and body weight recorded.Results：Similar AMPK α mRNA levels were found in the Dan-gua Fang group and normal group,slightly higher than the values obtained for the remaining groups（P〈0.05）.AMPK α protein expression in the Dan-gua Fang group animals was similar to other diabetic rats,whereas phospho-AMPK α（Thr-172） protein levels were markedly higher than in the metformin group and simvastatin group（P〈0.05）,respectively.However,phosphor-AMPKa/AMPK α ratios were similar in all groups.Dan-gua Fang reduced fasting blood glucose with similar strength to metformin,and was superior in reducing cholesterol,triglycerides,high-density lipoprotein cholesterol as well as improving low-density lipoprotein cholesterol in comparison with simvastatin and metformin.Dan-gua Fang decreases plasma alanine aminotransferase（ALT） significantly.Conclusion：Dan-gua Fang,while treating phlegm-stasis,could decrease BG and lipid in type 2 diabetic GK rats fed with high-fat diet,and effectively protect liver histomorphology and function.This may be partly explained by increased AMPK expression in liver.Therefore,Dan-gua Fang might be an ideal drug for comprehensive Intervention for glucose and lipid metabolism disorders in type 2 diabetes mellitus.

Amino acids regulate energy utilization through mammalian target of rapamycin complex 1 and adenosine monophosphate activated protein kinase pathway in porcine enterocytes

As major fuels for the small intestinal mucosa,dietary amino acids(AA)are catabolized in the mitochondria and serve as sources of energy production.The present study was conducted to investigate AA metabolism that supply cell energy and the underlying signaling pathways in porcine enterocytes.Intestinal porcine epithelial cells(IPEC-J2)were treated with different concentrations of AA,inhibitor,or agonist of mammalian target of rapamycin complex 1(mTORCl)and adenosine monophosphate activated protein kinase(AMPK),and mitochondrial respiration was monitored.The results showed that AA treatments resulted in enhanced mitochondrial respiration,increased intracellular content of pyruvic acid and lactic acid,and increased hormone-sensitive lipase mRNA expression.Meanwhile,decreased citrate synthase,isocitrate dehydrogenase alpha,and carnitine palmitoyltransferase 1 mRNA expression were also observed.We found that AA treatments increased the protein levels of phosphorylated mammalian target of rapamycin(p-mTOR),phosphorylated-p70 ribosomal protein S6 kinase,and phosphorylated-4 E-binding protein 1.What is more,the protein levels of phosphorylated AMPKα(pAMPKa)and nicotinamide adenine dinucleotide(NAD)-dependent protein deacetylase sirtuin-1(SIRT1)were decreased by AA treatments in a time depending manner.Mitochondrial bioenergetics and the production of tricarboxylic acid cycle intermediates were decreased upon inhibition of mTORCl or AMPK.Moreover,AMPK activation could up-regulate the mRNA expressions of inhibitor of nuclear factor kappa-B kinase subunit beta(Ikbk(3),integrin-linked protein kinase(ILK),unconventional myosin-Ic(Myolc),ribosomal protein S6 kinase beta-2(RPS6 Kβ2),and vascular endothelial growth factor(VEGF)-β,which are downstream effectors of mammalian target of rapamycin(mTOR).The mRNA expressions of phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform(PIK3 CD)and5’-AMP-activated protein kinase subunit gamma-1(PRKAG1),which are upstream regulators of mTOR,were also up-regulated by AMPK activation.On the other hand,AMPK activation also down-regulated FK506-binding protein 1 A(FKBP1 A),serine/threonine-protein phosphatase 2 A 55 kDa regulatory subunit B beta isoform,phosphatase and tensin homolog(PTEN),and unc-51 like autophagy activating kinase 1(Ulkl),which are up-stream regulators of mTORCl.Taken together,these data indicated that AA regulated cellular energy metabolism through mTOR and AMPK pathway in porcine enterocytes.These results demonstrated interactions of AMPK and mTORCl pathways in AA catabolism and energy metabolism in intestinal mucosa cells of piglets,and also provided reference for using AA to remedy human intestinal diseases.

Beneficial effects of metformin on primary cardiomyocytes via activation of adenosine monophosphate-activated protein kinase

Background Metformin has become a cornerstone in the treatment of patients with type-2 diabetes. Accumulated evidence suggests that metformin supports direct cardiovascular effects. The present study aimed to investigate if metformin has beneficial effects on primary cardiomyocytes damaged by H2O2, and reveal the potential mechanism of action of metformin. Methods Cardiomyocytes were incubated in the presence of 100μmol/L H2O2 for 12 hours. Cardiomyocytes were pretreated with metformin at different concentrations and time and with aminoimidazole carboxamide ribonucleotide （AICAR） （500μmol/L）, an adenosine monophophate （AMP）-activated protein kinase （AMPK） agonist for 60 minutes before the addition of H2O2. Other cells were preincubated with compound C （an AMPK antagonist, 20μmol/L） for 4 hours. The viability and apoptosis of cells were analyzed. AMPK, endothelial nitric oxide synthase （eNOS）, and transforming growth factor （TGF）-β1 were analyzed using immunblotting. Results Metformin had antagonistic effects on the influences of H2O2 on cell viability and attenuated oxidative stress-induced apoptosis. Metformin also increased phosphorylation of AMPK and eNOS, and reduced the expression of TGF-β1, basic fibroblast growth factor （bFGF）, and tumor necrosis factor （TNF）-α. Conclusions Metformin has beneficial effects on cardiomyocytes, and this effect involves activation of the AMPK-eNOS pathway. Metformin may be potentially beneficial for the treatment of heart disease.

Metformin inhibits nuclear factor-κB activation and inflammatory cytokines expression induced by high glucose via adenosine monophosphate-activated protein kinase activation in rat glomerular mesangial cells in vitro

Background The renoprotective mechanisms of adenosine monophosphate （AMP）-activated protein kinase （AMPK） agonist-metformin have not been stated clearly.We hypothesized that metformin may ameliorate inflammation via AMPK interaction with critical inflammatory cytokines The aim of this study was to observe the effects of metformin on expression of nuclear factor-κB （NF-κB）,monocyte chemoattractant protein-1 （MCP-1）,intercellular adhesion molecule-1 （ICAM-1） and transforming growth factor-beta 1 （TGF-β1） induced by high glucose （HG） in cultured rat glomerular mesangial cells （MCs）.Methods MCs were cultured in the medium with normal concentration glucose （group NG,5.6 mmol/L）,high concentration glucose （group HG,25 mmol/L） and different concentrations of metformin （group M1,M2,M3）.After 48-hour exposure,the supernatants and MCs were collected.The expression of NF-κB,MCP-1,ICAM-1,and TGF-β1 mRNA was analyzed by real time polymerase chain reaction.Westem blotting was used to detect the expression of AMPK,phospho-Thr-172 AMPK （p-AMPK）,NF-κB p65,MCP-1,ICAM-1,and TGF-β1 protein.Results After stimulated by HG,the expression of NF-κB,MCP-1,ICAM-1,TGF-β1 mRNA and protein of MCs in group HG increased significantly compared with group NG （P ＜0.05）.Both genes and protein expression of NF-κB,MCP-1,ICAM-1,TGF-β1 of MCs induced by high glucose were markedly reduced after metformin treatment in a dose-dependent manner （P ＜0.05）.The expression of p-AMPK increased with the rising of metformin concentration,presenting the opposite trend,while the level of total-AMPK protein was unchanged with exposure to HG or metformin.Conlusion Metformin can suppress the expression of NF-κB,MCP-1,ICAM-1 and TGF-β1 of glomerular MCs induced by high glucose via AMPK activation,which may partlv contribute to its reno-protection.

金合欢素调节Sirt1/AMPK/Nrf2信号通路对糖尿病白内障大鼠氧化应激损伤的影响

目的探讨金合欢素对糖尿病白内障(DC)大鼠氧化应激损伤的影响及其对沉默调节蛋白1(Sirt1)/腺苷酸活化蛋白激酶(AMPK)/核因子E2相关因子2(Nrf2)信号通路的调控作用。方法60只SD大鼠随机分为对照组、模型组、金合欢素低剂量组、金合欢素高剂量组、金合欢素+Sirt1抑制剂(EX527)组,除对照组以外均构建DC大鼠模型,其中,金合欢素低剂量组、金合欢素高剂量组大鼠分别经颈部皮下注射10 mg·kg^(-1)、20 mg·kg^(-1)的金合欢素,金合欢素+EX527组大鼠经颈部皮下注射20 mg·kg^(-1)金合欢素,均为每天2次,同时金合欢素+EX527组大鼠经皮下埋入渗透微型泵每天泵入3.5 mg·kg^(-1)EX527,其余组别均泵入等量生理盐水,给药持续4周。给药结束后,测量血压和空腹血糖(FBG),裂隙灯照射法观察大鼠晶状体混浊状况,HE染色观察晶状体组织病理学变化,ELISA测定血清丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、白细胞介素(IL)-6、IL-1β的含量,Western blot检测Sirt1、p-AMPK、AMPK、Nrf2蛋白表达水平。结果与对照组相比,模型组大鼠晶状体上皮细胞呈片状、条索状,发生迁移性聚集,收缩压、FBG、晶状体混浊评分、MDA、IL-6、IL-1β水平均升高,SOD、GSH-Px含量及Sirt1、p-AMPK/AMPK、Nrf2蛋白表达水平均降低(均为P<0.05);与模型组比较,金合欢素低、高剂量组大鼠晶状体上皮细胞迁移性聚集现象改善,收缩压、FBG、晶状体混浊评分、MDA、IL-6、IL-1β水平均降低,SOD、GSH-Px含量及Sirt1、p-AMPK/AMPK、Nrf2蛋白表达水平均升高(均为P<0.05);与金合欢素高剂量组比较,金合欢素+EX527组晶状体上皮细胞形态改变和聚集现象加重,收缩压、FBG、晶状体混浊评分、MDA、IL-6、IL-1β水平均升高,SOD、GSH-Px含量及Sirt1、p-AMPK/AMPK、Nrf2蛋白表达水平均降低(均为P<0.05)。结论金合欢素可能通过激活Sirt1/AMPK/Nrf2通路保护DC大鼠免受氧化应激损伤。

基于“肾脑相济”理论探讨艾灸对阿尔茨海默病大鼠海马AMPK/mTOR信号通路的影响

目的观察艾灸对阿尔茨海默病(Alzheimer’s disease,AD)大鼠腺苷酸活化蛋白激酶(adenosine 5-monophosphate activated protein kinase,AMPK)/雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路相关递质表达的影响,探讨艾灸治疗AD的作用机制。方法将SD大鼠按照随机数字表法分为正常组8只、模型组32只,采取侧脑室注射β淀粉样蛋白(amyloidβ-protein,Aβ)_(25-35)的方法建立大鼠AD模型。将模型复制成功的大鼠随机分为模型组、药物组、艾灸组,每组8只。艾灸组大鼠用艾条灸“百会”“肾俞”“三阴交”,每次15 min,同时按3 mg/kg灌胃蒸馏水;药物组大鼠按3 mg/kg灌胃盐酸多奈哌齐;对照组及模型组大鼠按3 mg/kg灌胃蒸馏水。采用Morris水迷宫法检测大鼠行为学表现,苏木精—伊红染色法观察大鼠海马病理组织改变,Western blot法检测大鼠海马磷酸化雷帕霉素靶蛋白(phosphorylated mammalian target of rapamycin,p-mTOR)、核糖体蛋白S6激酶(ribosomal protein S6 kinase p70,P70S6K)、自噬相关基因5(autophagy-related gene 5,ATG5)、磷酸化腺苷酸活化蛋白激酶(phosphorylated adenosine 5-monophosphate activated protein kinase,p-AMPK)、微管相关蛋白1轻链3B(microtubule associated protein light chain 3B,LC3B)-Ⅱ/LC3B-Ⅰ的表达水平。结果苏木精—伊红染色结果表明,模型组海马神经元萎缩明显,与模型组比较,药物组和艾灸组海马神经元形态及分化程度均有明显改善。与正常组比较,模型组大鼠的逃避潜伏期显著延长(P<0.05),p-mTOR及P70S6K表达水平均显著升高(P<0.05),ATG5、LC3B-Ⅱ/LC3B-Ⅰ、p-AMPK表达水平均显著降低(P<0.05)。与模型组比较,药物组和艾灸组大鼠的逃避潜伏期均显著缩短(P<0.05),p-mTOR及P70S6K表达水平均显著下降(P<0.05),ATG5、LC3B-Ⅱ/LC3B-Ⅰ、p-AMPK表达水平均显著上升(P<0.05)。与药物组比较,艾灸组大鼠逃避潜伏期显著缩短(P<0.05);p-mTOR及P70S6K表达水平显著降低(P<0.05),ATG5、LC3B-Ⅱ/LC3B-Ⅰ、p-AMPK表达水平均显著上升(P<0.05)。结论艾灸能够调控AMPK/mTOR信号通路,诱导细胞自噬,阻断脑内Aβ表达,从而改善认知功能。

Effects of Ginsenoside Rb1 on Skeletal Muscle Insulin Resistance and Adenosine Monophosphate?activated Protein Kinase Signaling Pathway in Obese Mice

Objectives: The objective of the study is to observe the effects of ginsenoside Rb1 on indexes of body weight, body composition, blood lipid, skeletal muscle endurance, and insulin sensitivity in obese mice, probe into its pharmacological action, and further explore its effects on adenosine monophosphate-activated protein kinase(AMPK) signaling pathway in skeletal muscle. Materials and Methods: Eight-week-old C57 BL/6 J mice were fed with high-fat diet for 12 weeks to establish obese mouse model. The model-establishment obese mice were randomly divided into three groups including model control group, metformin group, and ginsenoside Rb1 group. In the normal control group, normal diet was administered. The intervention period was 8 weeks. Body weight and food intake of the mice were measured regularly every week. The treadmill test was performed at weeks 3 and 7, and the oral glucose tolerance test was carried out at weeks 4 and 8. Body composition of the mice was detected by applying NMR Animal Body Composition Analyzer at week 8. Four parameters of blood lipids and free fatty acid(FFA)levels were detected. The m RNA expression of AMPKα and proliferator-activated receptor gamma coactivator-1α(PGC-1α) in skeletal muscle was examined by real-time fluorescence quantitative polymerase chain reaction, and the influence of ginsenoside Rb1 on protein expression of AMPKα, p-AMPKα, and PGC-1α was observed by western blotting. Results: The body weight(since the 5 th week of drug administration)and food intake of the mice in the ginsenoside Rb1 group were significantly lower than those in the model control group(P < 0.05) in a time-dependent manner. Ginsenoside Rb1 could significantly reduce the levels of triglyceride and low-density lipoprotein cholesterol, while increase the high-density lipoprotein cholesterol level(P < 0.05). In addition, ginsenoside Rb1 could reduce the serum FFA level(P < 0.05).After the administration of ginsenoside Rb1 for 8 weeks, the body fat mass of obese mice decreased and the lean mass increased(P < 0.05).The skeletal muscle endurance and the oral glucose tolerance of the obese mice improved using ginsenoside Rb1. At the molecular level,ginsenoside Rb1 could up-regulate the mRNA and protein expression of AMPKα in skeletal muscle, and increase the content of p-AMPK protein significantly(P < 0.01). At the same time, the mRNA and protein level of PGC-1α was also un-regulated, correspondingly(P < 0.01).Conclusion: Ginsenoside Rb1 exerts effects on reducing body weight, decreasing blood lipid levels, enhancing the skeletal muscle endurance,and increasing the insulin sensitivity in obese mice by activating the related proteins in AMPK signaling pathway in skeletal muscle.

阿江榄仁酸由AMPK/mTOR/HO-1信号通路调控自噬对糖尿病视网膜病变影响

目的探讨阿江榄仁酸(arjunolic acid,AA)对糖尿病视网膜病变(diabetic retinopathy,DR)大鼠视网膜细胞自噬及AMPK/mTOR/HO-1信号通路的影响。方法以健康SD大鼠为研究对象,构建链脲佐菌素(STZ)诱导的糖尿病大鼠模型,随机分为对照组(Con)组、模型(STZ)组、AA低剂量(AAL,10 mg/kg)组和AA高剂量(AAH,10 mg/kg)组。连续给药10周后,HE染色检测视网膜组织病理结构;qRT-PCR检测视网膜组织白介素(IL)-1β、IL-6和线粒体丙酮酸转运载体(MPC)-1的mRNA表达;二氢乙锭(DHE)染色评估视网膜组织ROS产生;Western blot检测自噬和AMPK/mTOR/HO-1信号通路相关蛋白表达。结果与Con组比较,STZ组大鼠视网膜出现肿胀和空泡样变化等病理变化,中央视网膜ONL层厚度和细胞核计数明显降低(P<0.01);IL-1β、IL-6和MCP-1的mRNA水平显著增高(P<0.05);视网膜外核层(ONL)、内核层(INL)和神经节细胞层(GCL)中ROS产生增加(P<0.01);LC3II/I比率、HO-1和p-AMPK/AMPK蛋白表达显著降低,p62和p-mTOR/mTOR表达升高(P<0.01)。与STZ组比较,AAL和AAH组大鼠视网膜ONL厚度和细胞核计数逐渐升高,结构相对规整(P<0.05);AAH组IL-1β、IL-6和MCP-1的mRNA表达明显降低(P<0.05);视网膜ONL、INL和GCL中ROS产生逐渐降低(P<0.01);LC3II/I比率、p-AMPK/AMPK和HO-1表达逐渐升高,p62和p-mTOR/mTOR表达逐渐降低(P<0.01)。结论阿江榄仁酸可能是治疗DR的候选药物,可能机制为通过AMPK/mTOR/HO-1调节的自噬途径保护视网膜细胞免受STZ诱导的氧化应激和炎症损伤。

基于AMPK/mTOR信号通路探讨人参-黄芪对人胃癌细胞自噬的影响

目的:探究益气扶正药人参-黄芪对人胃癌细胞自噬的影响,并探讨腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路在其中的作用机制。方法:将人胃癌细胞株(SGC-7901)随机分为对照组(人参-黄芪0 mg/ml)和人参-黄芪组(1、2、4、8、16、32、64 mg/ml),经药物处理后,CCK-8法观察人胃癌细胞增殖情况;筛选最佳浓度并将其作为后续实验中的实验组;MDC免疫荧光染色观察细胞自噬情况;Western blot法检测人胃癌细胞自噬相关蛋白(Beclin-1、LC3B)及AMPK/mTOR信号通路相关蛋白(AMPK、p-AMPK、mTOR及p-mTOR)的表达情况。结果:CCK-8法结果显示,人参-黄芪组人胃癌细胞增殖作用降低(P<0.01),且呈浓度依赖性;MDC染色荧光检测显示实验组的荧光强度显著增强(P<0.01),表明人参-黄芪可以诱导人胃癌细胞产生自噬;Western blot结果显示,实验组的Beclin-1、LC3B、p-AMPK/AMPK蛋白表达水平上升(P<0.01),p-mTOR/mTOR蛋白表达水平下降(P<0.01)。结论:人参-黄芪配伍能够抑制人胃癌细胞的增殖、促进人胃癌细胞自噬,其机制可能与激活AMPK/mTOR信号通路相关。

肺心汤通过调控AMPK/mTOR信号通路抑制自噬对低氧性肺动脉高压大鼠的保护作用

目的探究肺心汤通过调控腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路对低氧性肺动脉高压(hypoxic pulmonary hypertension,HPH)大鼠的治疗作用及机制。方法36只SD雄性大鼠随机分为正常组、模型组、西地那非组以及肺心汤低、中、高剂量组。除正常组外,将其余5组置于氧气浓度为10.0%±0.5%的低氧舱内造模28 d,每天8 h,造模同时灌胃给药。4周后检测大鼠心肺血流动力学指标[右心室收缩压(right ventricular systolic pressure,RVSP),右心室肥厚指数(right ventricular hypertrophy index,RVHI),肺动脉加速时间/肺动脉射血时间(pulmonary acceleration time/pulmonary ejection time,PAT/PET)、三尖瓣环收缩期位移(tricuspid annulus plane systolic excursion,TAPSE)、右心室前壁厚度(right ventricular anterior wall thickness,RVAWT)];HE染色法检测肺动脉重塑;免疫荧光法检测α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白共定位表达;透射电镜观察自噬溶酶体的数量;免疫组织化学法检测肺组织中p-AMPK、p-mTOR、微管相关蛋白1轻链3B(microtubule-associated protein light chain 3B,LC3B)、Beclin1和p62蛋白阳性表达。结果与正常组相比,模型组大鼠RVSP、RVHI及RVAWT显著升高(P<0.01),PAT/PET和TAPSE显著降低(P<0.01);肺小动脉壁明显增厚,管腔狭窄,肺小动脉管壁厚度占血管直径的百分比(the percentage of wall thickness of pulmonary arterioles to vascular diameter,WT%)显著升高(P<0.01);α-SMA和PCNA共定位表达显著增加;自噬溶酶体数量增加;肺组织中p-AMPK、LC3B和Beclin1蛋白阳性表达上调(P<0.01),p-mTOR及p62蛋白阳性表达下调(P<0.01)。与模型组相比,各给药组大鼠RVSP、RVHI及RVAWT显著下降(P<0.01),PAT/PET、TAPSE显著升高(P<0.05,P<0.01);肺小动脉壁增厚程度减轻,WT%显著降低(P<0.01);α-SMA和PCNA共定位表达减少;自噬溶酶体数量减少;肺组织中p-AMPK、LC3B和Beclin1蛋白阳性表达显著下降(P<0.05,P<0.01),p-mTOR及p62蛋白阳性表达显著上调(P<0.01)。与西地那非组相比,肺心汤低、中剂量组TAPSE降低(P<0.01);肺心汤低、中剂量组肺动脉中膜增厚程度增加,WT%增加(P<0.01);肺心汤低、中剂量组p-AMPK蛋白阳性表达升高(P<0.01),肺心汤高剂量组p-AMPK蛋白阳性表达降低(P<0.05);肺心汤低剂量组p-mTOR蛋白阳性表达降低(P<0.01)。结论肺心汤可能通过下调AMPK/mTOR信号通路抑制自噬,发挥改善HPH的作用。

Adenosine triphosphate promotes locomotor recovery after spinal cord injury by activating mammalian target of rapamycin pathway in rats

The mammalian target of rapamycin （mTOR） pathway plays an important role in neuronal growth, proliferation and differentiation. To better understand the role of mTOR pathway involved in the induction of spinal cord injury, rat models of spinal cord injury were established by modified Allen＇s stall method and interfered for 7 days by intraperitoneal administration of mTOR activator adenosine triphosphate and mTOR kinase inhibitor rapamycin. At 1-4 weeks after spinal cord injury induction, the Basso, Beattie and Bresnahan locomotor rating scale was used to evaluate rat locomotor function, and immunohistochemical staining and western blot analysis were used to detect the expression of nestin （neural stem cell marker）, neuronal nuclei （neuronal marker）, neuron specific enolase, neurofilament protein 200 （axonal marker）, glial fibrillary acidic protein （astrocyte marker）, Akt, mTOR and signal transduction and activator of transcription 3 （STAT3）. Results showed that adenosine triphosphate-mediated Akt/mTOR/STAT3 pathway increased endogenous neural stem cells, induced neurogenesis and axonal growth, inhibited excessive astrogliosis and improved the locomotor function of rats with spinal cord injury.

白花蛇舌草提取物通过AMPK/ATG5信号通路对急性胰腺炎大鼠肺损伤的保护作用机制研究

目的:通过5’-单磷酸腺苷活化蛋白激酶(AMPK)/自噬相关蛋白5(ATG5)信号通路,探讨白花蛇舌草提取物减轻急性胰腺炎(AP)大鼠肺损伤的机制。方法:建立AP肺损伤大鼠模型,随机分为模型组、提取物(白花蛇舌草提取物)组、3-MA(自噬抑制剂3-甲基腺嘌呤)组、AICAR(AMPK激活剂)组、提取物+AICAR组,每组15只,另取15只大鼠作为假手术组;ELISA检测血清淀粉酶(AMY)、IL-1β、IL-6、IL-18水平;检测大鼠腹水量及肺湿/干重比(W/D);HE观察胰腺及肺组织病理损伤;Western blot检测溶酶体相关膜蛋白2(LAMP2)、自噬底物p62、IL-1β前体蛋白(pro-IL-1β)、胰蛋白酶原活化肽(TAP)、AMPK、p-AMPK、ATG5、自噬标志物LC3-Ⅱ/Ⅰ、泛素特异性蛋白酶10(UPS10)表达。结果:与假手术组相比,模型组大鼠腹水量、肺W/D、胰腺和肺组织病理损伤评分、血清AMY、IL-1β、IL-6、IL-18水平、胰腺组织p62、TAP、pro-IL-1β表达、肺组织pAMPK/AMPK、ATG5、LC3-Ⅱ/Ⅰ、UPS10、pro-IL-1β表达均升高(P<0.05),胰腺和肺组织LAMP2蛋白表达降低(P<0.05);模型大鼠经白花蛇舌草提取物或自噬抑制剂3-MA干预后,上述指标均得到显著改善(P<0.05),且白花蛇舌草提取物的改善效果优于3-MA(P<0.05);而AMPK激活剂AICAR可削弱白花蛇舌草提取物对AP大鼠肺损伤的改善作用(P<0.05)。结论:白花蛇舌草提取物可通过抑制AMPK/ATG5信号通路减轻炎症反应、降低自噬水平改善AP大鼠肺损伤。

右美托咪定通过激活AMPK减轻TNF-α诱导人成神经细胞瘤细胞系SH-SY5Y损伤

目的 研究右美托咪定(DEX)对TNF-α诱导的人成神经细胞瘤细胞系的作用及其机制。方法 采用CCK8检测细胞活性,确定最佳DEX和TNF-α剂量,细胞分为:对照组(control组)、模型组(model组)、DEX干预模型组(DEX组)、DEX联合compound C干预模型组(DEX+CC组);Western blot检测细胞中p-AMPK、SNHP、KIF5B、Drp1、OPA1蛋白表达,ELISA检测IL-1β、IL-6水平,相应试剂盒测定线粒体膜电位(Δψm)、呼吸链复合酶活性(complexⅠ-Ⅳ)、ATP、MDA、SOD、GSH、ROS。结果 模型组较对照组p-AMPK活性、OPA1及SNPH水平显著降低,但Drp1和KIF5B水平显著增高(P<0.01),DEX组较model组complexⅠ~Ⅳ及Δψm、ATP、GSH、SOD水平显著增高,而MDA、IL-1β、IL-6水平显著降低(P<0.01);DEX+CC组较DEX组complexⅠ~Ⅳ及Δψm、ATP、GSH、SOD水平显著降低,而MDA、IL-1β、IL-6水平显著增高(P<0.01)。结论 DEX通过依赖AMPK的方式改善线粒体功能、减少氧化应激及炎性反应,减轻TNF-α诱导的细胞损伤。

丁酸钠经AMPK/Nrf2/HO-1信号通路调节脂多糖诱导肺泡巨噬细胞极化的作用机制

目的探讨丁酸钠(sodium butyrate,SB)对脂多糖(lipopolysaccharide,LPS)诱导肺泡巨噬细胞极化的影响及其作用机制。方法小鼠肺泡巨噬细胞(MH-S细胞)随机分为对照(Control)组、LPS组、SB组、LPS+SB(LB)组、LPS+SB+腺苷酸活化蛋白激酶(AMPK)抑制剂(Compound C)(LC)组、LPS+SB+核因子E2相关因子2(Nrf2)抑制剂(ML385)(LM)组。通过CCK8检测MH-S细胞活力,筛选出最佳的1000 ng/mL LPS、1 mmol/L SB、10μmol/L Compound C、5μmol/L ML385药物浓度进行后续实验;实时荧光定量(qRT-PCR)检测MH-S细胞白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-10(IL-10)、白细胞分化抗原86(CD86)、巨噬细胞甘露糖受体(CD206)、AMPK、Nrf2和血红素加氧酶1(HO-1)的mRNA表达水平;酶联免疫吸附试验(ELISA)检测培养基上清IL-6、TNF-α、IL-1β和IL-10蛋白含量;流式细胞术测定M1和M2型巨噬细胞相关标记物CD86和CD206的表达。各组数据通过单因素方差分析和Tukey法进行检验。结果通过CCK8选取了1000 ng/mL LPS、1 mmol/L SB、10μmol/L Compound C和5μmol/L ML385进行造模和干预。qRT-PCR和ELISA结果一致显示,与LPS组比较,LB组M1型巨噬细胞相关促炎细胞因子IL-6、TNF-α、IL-1β显著降低(均P<0.01),但M2型巨噬细胞相关抑炎细胞因子IL-10显著升高(均P<0.01)。qRT-PCR和流式细胞术结果一致显示,与Control组比较,LPS组CD86水平显著升高(均P<0.01),SB组差异无统计学意义;与LPS组比较,LB组CD86表达水平显著降低(均P<0.01),但M2型巨噬细胞标记物CD206的变化趋势与CD86相反。qRT-PCR结果显示,与LPS组比较,LB组促进AMPK/Nrf2/HO-1的表达(均P<0.05);与LB组比较,LC组降低了AMPK/Nrf2/HO-1的表达(均P<0.05),LM组降低了Nrf2/HO-1的表达(均P<0.05)。流式细胞术结果显示,与LB组比较,LC组和LM组逆转了SB对CD86水平的抑制作用(均P<0.01);M2型巨噬细胞标记物CD206的表达趋势与M1型巨噬细胞标记物CD86相反。结论SB通过激活AMPK/Nrf2/HO-1信号通路,抑制LPS诱导的M1型、促进M2型肺泡巨噬细胞极化,改善了炎症反应。

AMPK及mTOR与多囊卵巢综合征的关系及中药干预研究进展

多囊卵巢综合征(Polycystic ovary syndrome,PCOS)是一组生殖内分泌代谢紊乱的综合征,临床以稀发排卵、高雄激素体征、胰岛素抵抗为主要特征,其中育龄期发病率高,对女性生育力造成严重不良影响。PCOS的发生发展涉及多种信号通路,腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)及哺乳动物雷帕霉素靶蛋白(Mammalian target of rapamycin,mTOR)作为细胞能量感受器是其中两个关键靶点。二者在PCOS各个发病部位包括下丘脑-垂体-卵巢轴、子宫内膜、脂肪与骨骼肌中发挥重要的调节作用,通过影响细胞自噬、氧化应激、炎症、线粒体功能、葡萄糖摄取等,促进卵泡的发育和成熟,改善胰岛素抵抗。近年来,中医药因其成分多样、靶点众多等优势广泛应用于临床,研究人员已对PCOS的发病以及中药治疗及改善PCOS的机制进行了大量研究,结果提示AMPK与mTOR相关通路在其中发挥关键作用。通过总结中药干预AMPK与mTOR及其相关通路治疗PCOS的研究结果,为临床治疗及基础研究提供参考。

黄芪多糖调控AMPK⁃mTORC1通路对牙周膜成纤维细胞增殖、凋亡、自噬的影响

目的探究黄芪多糖(Astragalus polysaccharide,APS)对牙周膜成纤维细胞(human periodontal ligament fibroblasts,HPLF)增殖、凋亡、自噬及磷酸腺苷蛋白激酶(AMPK)/雷帕霉素靶蛋白(mTORC1)通路的影响。方法原代培养HPLF细胞;免疫组织化学法鉴定HPLF细胞;设置对照组、0.1 mg·mL^(-1)组(0.1 mg·mL^(-1) APS)、0.2 mg·mL^(-1)组(0.2 mg·mL^(-1) APS)、0.4 mg·mL^(-1)组(0.4 mg·mL^(-1) APS),细胞增殖及毒性(CCK-8)法检测各组细胞增殖抑制率;用流式细胞仪检测各组细胞凋亡率;透射电子显微镜观察各组HPLF细胞自噬情况;Western blot检测各组细胞自噬、凋亡、通路相关蛋白表达情况。结果与对照组相比,细胞增殖抑制率、细胞凋亡率在0.1 mg·mL^(-1)组、0.2 mg·mL^(-1)组显著降低,而0.4 mg·mL^(-1)组与0.2 mg·mL^(-1)组相比细胞增殖抑制率与凋亡率则明显增多。与对照组相比,0.1 mg·mL^(-1)和0.2 mg·mL^(-1) APS显著抑制细胞自噬,而0.4 mg·mL^(-1) APS则促进细胞自噬;与对照组相比,Beclin⁃1、LC3⁃Ⅱ/LC3⁃Ⅰ、半胱氨酸蛋白酶⁃3(Caspase⁃3)、bcl⁃2相关X蛋白(Bax)、p⁃AMPK/AMPK在0.1 mg·mL^(-1)组、0.2 mg·mL^(-1)组显著降低,而0.4 mg·mL^(-1)组与0.2 mg·mL^(-1)组相比表达水平则显著增加;p62、survivin、Bcl⁃2、p⁃mTORC1/mTORC1表达水平与其呈现相反趋势。结论APS具有促进HPLF细胞增殖、抑制细胞凋亡的作用,并可能通过调节AMPK⁃mTORC1通路抑制细胞自噬的发生。