虎杖苷调节Sirt1-FoxO1信号通路对高糖诱导的心肌细胞焦亡的影响

目的 基于沉默信息调节因子1(silence information regulator1,Sirt1)-叉头状转录因子O1(forkhead transcription factor O1,FoxO1)信号通路研究虎杖苷(polydatin,PD)抑制高糖诱导的心肌细胞焦亡的作用机制。方法 以心肌细胞H9c2为研究对象,使用不同浓度PD处理筛选PD浓度。将细胞分为模型组、PD低、中、高剂量组(PD-L、M、H,20、40、80μmol/L)、空白对照(control)组、Sirt1抑制剂[PD-H+sirtinol (10μmol/L)]组。细胞计数试剂盒(cell counting Kit-8,CCK8)法检测细胞活性;酶联免疫吸附试验(enzyme-linked immunosorbent assay,Elisa)法检测细胞内乳酸脱氢酶(lactate dehydrogenase,LDH)释放量、白细胞介素1β(Interleukin-1β,IL-1β)水平;二氯荧光素双醋酸盐(dichlorodihydrofluorescein diacetate,DCFH-DA)荧光探针检测细胞活性氧(reactive oxygen species,ROS)水平;流式细胞术检测细胞焦亡情况;实时荧光定量PCR检测细胞中Nod样受体蛋白3(Nod-like receptor protein 3,NLRP3) mRNA、消皮素D(gasdermin D,GSDMD) mRNA表达水平;Western blot检测蛋白表达。结果 与control组比较,模型组细胞活性下降(P<0.01),经过PD不同浓度处理后,细胞活性逐渐上升(P<0.01);与control组比较,模型组LDH释放量、IL-1β水平、ROS水平、细胞焦亡率、NLRP3 mRNA、GSDMD mRNA表达、焦亡蛋白NLRP3、裂解的半胱氨酸天冬氨酸酶(cleaved-caspase-1,c-caspase-1)、GSDMD、GSDMDN表达均上升,Sirt1-Foxo1信号通路蛋白表达下降(P<0.01);与模型组比较,PD各组及PD-H+sirtinol组LDH释放量、IL-1β水平、ROS水平、细胞焦亡率、NLRP3 mRNA、GSDMD mRNA表达、焦亡蛋白NLRP3、c-caspase-1、GSDMD、GSDMD-N表达均下降,Sirt1-FoxO1信号通路蛋白表达上升(P<0.05)。sirtinol可以逆转PD对于高糖诱导的心肌细胞焦亡的保护作用。结论 PD可以通过上调Sirt1-FoxO1信号通路蛋白表达,改善细胞炎症损伤,抑制高糖诱导的心肌细胞焦亡。

Corilagin alleviates podocyte injury in diabetic nephropathy by regulating autophagy via the SIRT1-AMPK pathway

BACKGROUND Diabetic nephropathy(DN)is the most frequent chronic microvascular consequence of diabetes,and podocyte injury and malfunction are closely related to the development of DN.Studies have shown that corilagin(Cor)has hepatoprotective,anti-inflammatory,antibacterial,antioxidant,anti-hypertensive,antidiabetic,and anti-tumor activities.AIM To explore the protective effect of Cor against podocyte injury in DN mice and the underlying mechanisms.METHODS Streptozotocin and a high-fat diet were combined to generate DN mice models,which were then divided into either a Cor group or a DN group(n=8 in each group).Mice in the Cor group were intraperitoneally injected with Cor(30 mg/kg/d)for 12 wk,and mice in the DN group were treated with saline.Biochemical analysis was used to measure the blood lipid profiles.Hematoxylin and eosin staining was used to detect pathological changes in kidney tissue.Immunohistochemistry and Western blotting were used to assess the protein expression of nephrin and podocin.Mouse podocyte cells(MPC5)were cultured and treated with glucose(5 mmol/L),Cor(50μM),high glucose(HG)(30 mmol/L),and HG(30 mmol/L)plus Cor(50μM).Real-time quantitative PCR and Western blotting RESULTS Compared with the control group,the DN mice models had increased fasting blood glucose,glycosylated hemoglobin,triglycerides,and total cholesterol,decreased nephrin and podocin expression,increased apoptosis rate,elevated inflammatory cytokines,and enhanced oxidative stress.All of the conditions mentioned above were alleviated after intervention with Cor.In addition,Cor therapy improved SIRT1 and AMPK expression(P<0.001),inhibited reactive oxygen species and oxidative stress,and elevated autophagy in HG-induced podocytes(P<0.01).CONCLUSION Cor alleviates podocyte injury by regulating autophagy via the SIRT1-AMPK pathway,thereby exerting its protective impact on renal function in DN mice.

卡格列净通过SIRT1-FOXO3α信号通路改善小鼠肾小球系膜细胞自噬功能

目的:探讨高糖条件下卡格列净对小鼠肾小球系膜细胞系SV40 Mes13自噬功能的影响及可能机制。方法:将SV40 Mes13细胞分别置于以下3组条件中:正常浓度葡萄糖(NG, 5.6 mmol/L)、高浓度葡萄糖(HG,30 mmol/L)和高浓度葡萄糖加卡格列净(100 nmol/L),干预时间为48 h。采用Western blot法检测观察沉默信息调节蛋白1(SIRT1)、叉头框蛋白O3α(FOXO3α)、p62、LC3B、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原α1(COL1A1)和Ⅲ型胶原α1(COL3A1)的表达水平,采用RT-qPCR检测FOXO3α和BNIP3及纤维化指标的表达水平。用携带SIRT1 shRNA的慢病毒转染细胞,构建SIRT1敲减的SV40 Mes13细胞株,置于上述3组条件下干预48 h,采用Western blot和RT-qPCR法再次检测上述指标的表达情况,并通过自噬双标腺病毒(mRFP-GFP-LC3)标记观察细胞内自噬流的变化。结果:(1)Western blot结果显示,卡格列净可以改善SV40 Mes13细胞自噬功能,且该作用依赖于SIRT1:HG干预后,与NG组相比,SV40 Mes13细胞LC3B-Ⅱ/LC3B-Ⅰ水平降低,而p62蛋白水平升高(P<0.05);与HG组相比,HG与卡格列净共干预后,LC3B-Ⅱ/LC3B-Ⅰ水平上升,p62水平降低(P<0.05)。当SIRT1被敲减后,卡格列净对LC3B-Ⅱ/LC3B-Ⅰ的上调作用和对p62蛋白水平的下调作用消失(P>0.05)。(2)卡格列净上调SIRT1-FOXO3α通路:空载病毒对照组中,与NG组相比,HG干预后,SV40 Mes13细胞SIRT1蛋白水平、FOXO3α蛋白水平及核转位和BNIP3 mRNA水平降低(P<0.05);HG与卡格列净共干预后,与HG组相比,SIRT1、核内FOXO3α和BNIP3水平上升(P<0.05)。当SIRT1被敲减后,卡格列净上述作用消失(P>0.05)。(3)卡格列净改善SV40 Mes13细胞纤维化:HG干预后,与NG组相比,SV40 Mes13细胞α-SMA、COL1A1和COL3A1水平上升(P<0.05);HG和卡格列净共干预后,与HG组相比,上述纤维化指标水平下降(P<0.05)。当SIRT1被敲减后,卡格列净上述作用消失(P>0.05)。结论:在小鼠肾小球系膜细胞SV40 Mes13中:(1)卡格列净通过上调SIRT1-FOXO3α信号通路,改善高糖环境下小鼠SV40Mes13细胞自噬功能;(2)上调SIRT1-FOXO3α信号通路从而改善自噬可能是卡格列净改善高糖状态下小鼠SV40Mes13细胞纤维化的机制之一。

矢志方激活Sirt1-Foxo1通路调控内质网应激相关蛋白改善高尿酸血症小鼠肾损伤

[目的]基于沉默信息调节因子1(Sirt1)-叉头转录因子1(Foxo1)通路观察矢志方对高尿酸血症(HUA)小鼠肾组织内质网应激(ERS)及下游分子增强子结合蛋白同源蛋白(Chop)、天冬氨酸特异性半胱氨酸蛋白酶(Caspase)12表达的影响,探讨其作用机制。[方法] 32只SPF级雄性BALB/c小鼠随机分为正常组、模型组、非布司他组和矢志方组,每组8只。正常组给予生理盐水灌胃,其余各组予氧嗪酸钾250 mg/kg灌胃制备HUA小鼠模型。非布司他组和矢志方组分别按6 mg/kg、562.5 mg/kg灌胃相应药物,正常组和模型组予相同体积生理盐水灌胃,连续2周。体外实验培养人肾小管上皮细胞(HK2),分为正常组、模型组、矢志方组,饥饿24 h后予200μg/mL尿酸和300μg/mL矢志方干预24 h。苏木精-伊红(HE)染色和Masson染色观察小鼠肾脏组织病理变化,蛋白免疫印迹法(Western blot)和免疫组化染色检测小鼠肾组织Sirt1、内质网跨膜蛋白肌醇酶1α(IRE1α)、Foxo1、Caspase 12、Chop表达,Western blot和免疫荧光染色检测HK2细胞Sirt1、IRE1α、Foxo1、Caspase 12、Chop表达。[结果] HE染色和Masson染色结果显示:与正常组比较,模型组小鼠肾小管管壁变薄,管腔扩张,炎性细胞浸润,大量蓝色胶原纤维沉积。与模型组比较,矢志方组小鼠肾小管管壁变薄、管腔扩张、蓝色胶原纤维集聚程度减少。Western blot结果显示:模型组较正常组相比肾组织和HK2细胞IRE1α、Foxo1、Caspase 12、Chop表达显著升高(P<0.01,P<0.001),Sirt1蛋白表达显著降低(P<0.001),用药组较模型组相比肾组织和HK2细胞IRE1α、Foxo1、Caspase 12、Chop蛋白表达显著降低(P<0.05,P<0.001),Sirt1蛋白表达显著升高(P<0.001)。免疫组化染色和免疫荧光染色结果与Western blot结果一致。[结论]矢志方减轻肾脏病理损伤的机制可能与抑制HUA小鼠肾组织ERS、激活Sirt1-Foxo1通路表达有关。

基于AMPK/SIRT1-FoxO1信号通路探讨葛根芩连汤对db/db糖尿病小鼠肝脏氧化应激的影响

目的观察葛根芩连汤对db/db小鼠肝脏氧化应激的影响,探讨其改善2型糖尿病的作用机制。方法将75只自发性2型糖尿病db/db小鼠随机分为模型组、二甲双胍组和葛根芩连汤高、中、低剂量组,每组15只,另取15只db/m小鼠作为空白组,分别予相应药物灌胃12周。检测小鼠体质量、空腹血糖(FPG)及糖化血红蛋白(HbA1c)含量,HE染色观察肝脏组织病理改变,化学发光免疫分析法检测肝组织丙二醛(MDA)含量和谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)活性,Western blot检测肝组织腺苷酸活化蛋白激酶(AMPK)、p-AMPK、沉默信息调节因子1(SIRT1)、叉头框蛋白1(FoxO1)、p-FoxO1蛋白表达,实时荧光定量PCR检测肝组织AMPK、SIRT1、FoxO1 mRNA表达。结果与空白组比较,模型组小鼠体质量、FPG及HbA1c含量显著增加(P<0.01);肝细胞脂肪变性、点状坏死,肝窦淤血;肝组织MDA含量显著增加,GSH-Px、CAT活性显著降低(P<0.01),AMPK、p-AMPK和SIRT1蛋白表达显著降低,FoxO1和p-FoxO1蛋白表达显著升高(P<0.01),AMPK和SIRT1 mRNA表达显著降低,FoxO1 mRNA表达显著升高(P<0.01)。与模型组比较,各给药组小鼠体质量、FPG及HbA1c含量显著减少(P<0.05,P<0.01);肝组织病理改变有所减轻;肝组织MDA含量显著减少,GSH-Px、CAT活性显著升高(P<0.05,P<0.01),p-AMPK、AMPK和SIRT1蛋白表达显著升高,FoxO1和p-FoxO1蛋白表达显著降低(P<0.05,P<0.01),AMPK和SIRT1 mRNA表达显著升高,FoxO1 mRNA表达显著降低(P<0.05)。与二甲双胍组比较,葛根芩连汤高剂量组各指标变化更显著(P<0.05)。结论葛根芩连汤可能通过调节AMPK/SIRT1-FoxO1信号通路降低db/db小鼠肝脏氧化应激水平,改善2型糖尿病。

Huangqin decoction alleviates lipid metabolism disorders and insulin resistance in nonalcoholic fatty liver disease by triggering Sirt1/NF-κB pathway

BACKGROUND Nonalcoholic fatty liver disease(NAFLD)is a clinicopathological entity characterized by intrahepatic ectopic steatosis.As a consequence of increased consumption of high-calorie diet and adoption of a sedentary lifestyle,the incidence of NAFLD has surpassed that of viral hepatitis,making it the most common cause of chronic liver disease globally.Huangqin decoction(HQD),a Chinese medicinal formulation that has been used clinically for thousands of years,has beneficial outcomes in patients with liver diseases,including NAFLD.However,the role and mechanism of action of HQD in lipid metabolism disorders and insulin resistance in NAFLD remain poorly understood.AIM To evaluate the ameliorative effects of HQD in NAFLD,with a focus on lipid metabolism and insulin resistance,and to elucidate the underlying mechanism of action.METHODS High-fat diet-induced NAFLD rats and palmitic acid(PA)-stimulated HepG2 cells were used to investigate the effects of HQD and identify its potential mechanism of action.Phytochemicals in HQD were analyzed by highperformance liquid chromatography(HPLC)to identify the key components.RESULTS Ten primary chemical components of HQD were identified by HPLC analysis.In vivo,HQD effectively prevented rats from gaining body and liver weight,improved the liver index,ameliorated hepatic histological aberrations,decreased transaminase and lipid profile disorders,and reduced the levels of pro-inflammatory factors and insulin resistance.In vitro studies revealed that HQD effectively alleviated PA-induced lipid accumulation,inflammation,and insulin resistance in HepG2 cells.In-depth investigation revealed that HQD triggers Sirt1/NF-κB pathwaymodulated lipogenesis and inflammation,contributing to its beneficial actions,which was further corroborated by the addition of the Sirt1 antagonist EX-527 that compromised the favorable effects of HQD.CONCLUSION In summary,our study confirmed that HQD mitigates lipid metabolism disorders and insulin resistance in NAFLD by triggering the Sirt1/NF-κB pathway.

Effect and Mechanism of Dicliptera chinensis Polysaccharide on miR-141/AMPK/SIRT1 Signaling Pathway in Rats with NAFLD

[Objectives]Non-alcoholic fatty liver disease(NAFLD)rat model was established by feeding high-fat and high-sugar fodder to rats,and the protective effect of Dicliptera chinensis polysaccharide(DCP)on NAFLD rats was studied to explore its potential mechanism.[Methods]45 SD rats were randomly divided into 4 groups:normal control group,model control group and DCP treatment groups(100 and 300 mg/kg).The rats in the normal control group were fed with ordinary fodder,and the rats in other groups were fed with high-fat and high-sugar diet for 14 weeks to establish NAFLD model.From the 9^(th)week,the rats in the DCP treatment groups were given different doses of DCP by intragastric administration(5 mL/kg)for 6 weeks.After the last intragastric administration,the rats fasted for 16 h,and the serum and liver of rats were collected for detection.Hematoxylin-eosin(HE)staining was conducted to observe the histopathological changes of rat liver,and alanine aminotransferase(ALT),aspartate aminotransferase(AST),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),malondialdehyde(MDA),triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),and high density lipoprotein cholesterol(HDL-C)were detected by biochemical method.Interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor(TNF-α)and micrornA-141(micro RNA-141)were detected by reverse transcription-polymerase chain reaction(RT-PCR).The expression of SIRT1 and adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)in rat liver was detected by western blot.[Results]Compared with the model control group,the inflammatory damage and steatodegeneration of rats in the DCP groups were relieved to varying degrees,and the number of lipid vacuoles significantly reduced.The ALT,AST,TC,TG and LDL-C content in the serum and MDA content in the liver tissue decreased to varying degrees,while the HDL-C,SOD and GSH-Px content increased.The expression of SIRT1 and AMPK increased,while the expression of miR-141,TNF-α,IL-6 and IL-1βdeclined,and the DCP 300 mg/kg treatment group had better improvement effect.[Conclusions]DCP had a certain protective effect on NAFLD rats,which may be related to the regulation of miR-141/AMPK/SIRT1 signaling pathway.

薯蓣皂苷通过调控SIRT1-FoxO1-自噬通路减轻糖尿病大鼠胰岛素抵抗

目的:探究薯蓣皂苷是否通过调控沉默信息调节因子1(sirtuin 1,SIRT1)-叉头框蛋白O1(forkhead box protein O1,FoxO1)-自噬通路减轻糖尿病大鼠胰岛素抵抗。方法:将60只SPF级SD大鼠随机分为对照组、模型组、低剂量(5 mg/kg)薯蓣皂苷组、中剂量(10 mg/kg)薯蓣皂苷组、高剂量(20 mg/kg)薯蓣皂苷组和薯蓣皂苷(20 mg/kg)+EX-527(SIRT1抑制剂)组,每组10只。高脂饲料喂养4周后腹腔注射链脲佐菌素以构建2型糖尿病(type 2 diabetes mellitus,T2DM)大鼠模型,低、中、高剂量薯蓣皂苷组和薯蓣皂苷+EX-527组大鼠分别灌胃相应剂量药物,对照组和模型组大鼠灌胃等量生理盐水,每天1次,为期4周。全自动生化分析仪检测血清中空腹血糖(fasting blood glucose,FBG)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)、甘油三酯(triglyceride,TG)和总胆固醇(total cholesterol,TC)水平,酶联免疫吸附实验检测血清空腹胰岛素(fasting insulin,FINS)水平,计算胰岛素抵抗指数(homeostasis model assessment-insulin resistance,HOMA-IR)和胰岛素敏感指数(insulin sensitivity index,ISI),行口服葡萄糖耐量实验(oral glucose tolerance test,OGTT),计算OGTT曲线下区域面积(area under curve,AUC);HE染色观察大鼠胰腺组织损伤情况;使用Western blot检测胰腺组织中beclin-1、LC3及SIRT1-FoxO1自噬通路相关蛋白的表达。结果:与对照组相比,模型组FBG、AUC、FINS、HOMA-IR、体重、TG、TC和LDL-C水平、胰腺组织损伤程度及FoxO1水平显著增加,ISI、beclin-1、LC3-II/LC3-I和SIRT1水平显著降低(P<0.05);与模型组相比,低、中、高剂量组FBG、AUC、FINS、HOMA-IR、体重、TG、TC和LDL-C水平、胰腺组织损伤程度及FoxO1水平以薯蓣皂苷剂量依赖性的方式显著降低,ISI、beclin-1、LC3-II/LC3-I和SIRT1水平以薯蓣皂苷剂量依赖性的方式显著增加(P<0.05);与高剂量薯蓣皂苷组相比,薯蓣皂苷+EX-527组FBG、AUC、FINS、HOMA-IR、体重、TG、TC和LDL-C水平、胰腺组织损伤程度及FoxO1水平显著增加,ISI、beclin-1、LC3-II/LC3-I和SIRT1水平显著降低(P<0.05)。结论:薯蓣皂苷可能通过激活SIRT1-FoxO1-自噬通路减轻T2DM大鼠胰岛素抵抗。

毓麟珠对卵巢早衰大鼠SIRT1-FoxO1-自噬通路的调控作用

目的:探讨毓麟珠对卵巢早衰(POF)大鼠自噬的影响,并分析其防治POF的作用机制。方法:雌性SD大鼠采用随机数字表法选取16只为正常组,其余大鼠通过腹腔注射环磷酰胺建立POF模型。造模成功大鼠分为POF组、戊酸雌二醇(0.09 mg/kg)组(简称雌二醇组)、低剂量(7.56 g/kg)毓麟珠组、高剂量(15.12 g/kg)毓麟珠组和毓麟珠+沉默信息调节因子1(SIRT1)抑制剂EX527组(简称毓麟珠+EX527组),每组16只。低、高剂量毓麟珠组大鼠分别灌胃相应剂量的毓麟珠药液,雌二醇组大鼠给予戊酸雌二醇溶液灌胃,毓麟珠+EX527组大鼠在给予15.12 g/kg毓麟珠药液灌胃的同时腹腔注射10 mg/kg EX527,正常组和POF组大鼠给予等体积的生理盐水干预,每天1次,连续给药3周。给药结束后,记录各组大鼠的双侧卵巢重量和体重,计算卵巢指数;ELISA检测各组大鼠血清雌二醇(E2)、促卵泡激素(FSH)和抗缪勒管激素(AMH)水平;检测各组大鼠卵巢组织丙二醛(MDA)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)水平;苏木精-伊红(HE)染色和TUNEL染色观察各组大鼠卵巢组织病理变化,计数各级卵泡数,并分析窦卵泡中颗粒细胞凋亡水平;免疫组织化学法检测各组大鼠卵巢组织微管相关蛋白1轻链3B(LC3B)蛋白表达;RT-qPCR检测各组大鼠卵巢组织LC3B、p62、beclin-1、Atg5和Atg7的mRNA表达;Western blot检测各组大鼠卵巢组织LC3A/B、p62、beclin-1、Atg5、Atg7、SIRT1、叉头框蛋白O1(FoxO1)和乙酰化FoxO1(Ac-FoxO1)的蛋白水平;评估POF大鼠的生育能力。结果:与正常组相比,POF组大鼠卵巢指数,血清E2和AMH水平,卵巢组织SOD和CAT活性,卵巢原始卵泡、初级卵泡、次级卵泡和窦卵泡数量,p62 mRNA和蛋白水平,SIRT1和FoxO1蛋白水平,以及受孕率和胚胎数均显著降低,而血清FSH水平,卵巢组织MDA水平,闭锁卵泡数量,颗粒细胞凋亡水平,卵巢LC3B蛋白阳性表达,LC3B、beclin-1、Atg5和Atg7 mRNA水平,beclin-1、Atg5、Atg7和Ac-FoxO1蛋白水平,以及LC3-II/LC3-I比值均显著升高(P<0.05);与POF组相比,高剂量毓麟珠组和雌二醇组大鼠卵巢指数,血清E2和AMH水平,卵巢组织SOD和CAT活性,卵巢原始卵泡、初级卵泡、次级卵泡和窦卵泡数量,p62 mRNA和蛋白水平,SIRT1和FoxO1蛋白水平,以及受孕率和胚胎数均显著升高,而血清FSH水平,卵巢组织MDA水平,闭锁卵泡数量,颗粒细胞凋亡水平,卵巢LC3B蛋白阳性表达,LC3B、beclin-1、Atg5和Atg7 mRNA水平,beclin-1、Atg5、Atg7和Ac-FoxO1蛋白水平,以及LC3-II/LC3-I比值均显著降低(P<0.05);EX527可显著减弱毓麟珠对POF大鼠卵巢功能的保护作用。结论:毓麟珠可能通过激活SIRT1-FoxO1通路抑制氧化应激及细胞自噬,从而改善POF大鼠卵巢功能。

肾缺血再灌注损伤与线粒体自噬SIRT1-FOXO3-PINK1-Parkin调节轴的研究进展

肾缺血再灌注损伤(IRI)是导致急性排斥反应、移植肾功能延迟恢复和移植肾间质纤维化的重要原因,已成为制约移植受者和肾长期存活的瓶颈,如何减轻肾IRI是改善肾移植远期预后的关键问题。自噬与肾IRI有着密切关系,线粒体自噬在肾IRI中的作用越来越受到关注。阐明肾IRI与线粒体自噬SIRT1-FOXO3-PINK1-Parkin调节轴的调控关系,对探讨肾IRI后肾小管上皮细胞损伤修复的新机制具有重要意义。

益气养阴活血通络法对急性心肌梗死大鼠Sirt1-FoxO1-FoxO3a信号通路的影响

目的探讨益气养阴、活血通络法对急性心肌梗死大鼠Sirt1-FoxO1-FoxO3a信号通路的影响。方法随机将60只大鼠分组,共6组,依次为空白组,模型组,中药双参活血颗粒高、中、低剂量组和西药(盐酸曲美他嗪)组,将大鼠冠状动脉左前降支结扎后制成急性心肌梗死模型,模型制备成功后连续给药2周,随后处死,采用Western Blot、RT-PCR检测模型大鼠缺血心肌组织中Sirt1、FoxO1、FoxO3a各个蛋白含量与其mRNA的表达。结果模型组中的Sirt1、FoxO1、FoxO3a蛋白含量、mRNA表达与空白组比均下降(P<0.05),中药各剂量组与西药组Sirt1、FoxO1、FoxO3a蛋白含量、mR-NA表达与模型组比均上升(P<0.05),中药组上升幅度具有药物浓度依赖性,西药组与中药高剂量组比较差异无统计学意义(P>0.05)。结论具有益气养阴、活血通络功效的中药双参活血颗粒可以影响Sirt1-FoxO1-FoxO3a信号通路而减轻大鼠急性心肌梗死的损伤。

Quercetin ameliorates oxidative stress-induced senescence in rat nucleus pulposus-derived mesenchymal stem cells via the miR-34a-5p/SIRT1 axis

BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchymal stem cells(NPMSCs)and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs(IVDs).Quercetin(Que)has been demonstrated to reduce oxidative stress in diverse degenerative diseases.AIM To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism.METHODS In vitro,NPMSCs were isolated from rat tails.Senescence-associatedβ-galactosidase(SA-β-Gal)staining,cell cycle,reactive oxygen species(ROS),realtime quantitative polymerase chain reaction(RT-qPCR),immunofluorescence,and western blot analyses were used to evaluated the protective effects of Que.Meanwhile the relationship between miR-34a-5p and Sirtuins 1(SIRT1)was evaluated by dual-luciferase reporter assay.To explore whether Que modulates tert-butyl hydroperoxide(TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway,we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression.In vivo,a puncture-induced rat IDD model was constructed,and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo.RESULTS We found that TBHP can cause NPMSCs senescence changes,such as reduced cell proliferation ability,increased SA-β-Gal activity,cell cycle arrest,the accumulation of ROS,and increased expression of senescence-related proteins.While abovementioned senescence indicators were significantly alleviated by Que treatment.Que decreased the expression levels of senescence-related proteins(p16,p21,and p53)and senescence-associated secreted phenotype(SASP),including IL-1β,IL-6,and MMP-13,and it increased the expression of SIRT1.In addition,the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown.In vivo,X-ray,and histological analyses indicated that Que alleviated IDD in a punctureinduced rat model.CONCLUSION In summary,the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway,suggesting that Que may be a potential agent for the treatment of IDD.

积雪草苷调控SIRT1-FOXO3-PINK1-Parkin通路介导的线粒体自噬保护肾缺血再灌注损伤的机制研究

目的探讨积雪草苷(AC)调控沉默信息调节因子1(SIRT1)-叉头盒转录因子O3(FOXO3)-PTEN诱导性激酶蛋白1(PINK1)-E3泛素连接酶(Parkin)通路介导线粒体自噬对肾缺血再灌注损伤(RIRI)的保护作用及机制。方法采用随机数字表法将50只雄性SD大鼠分为假手术组(Sham组)、模型组(Model组)、AC组、AC+SIRT1抑制剂(EX-527)组、EX-527组,每组10只。构建RIRI模型;AC组造模前予以AC混悬液80 mg/(kg·d)连续灌胃4周;AC+EX-527组造模前3 d予以含EX-527(5 mg/kg)的1%DMSO溶液腹腔注射,术中再灌注前20 min腹腔注射1次,余处理同AC组;EX-527组仅予以等量含EX-527的1%DMSO溶液腹腔注射。造模24 h后取材,检测血肌酐(Scr)、尿素氮(BUN)水平,HE染色观察肾组织病理改变及评分,Western blot检测组织SIRT1、FOXO3、PINK1、Parkin通路蛋白和自噬相关蛋白Beclin1、微管相关蛋白轻链3(LC3)A/B-Ⅰ、LC3A/B-Ⅱ表达水平,并计算(LC3A/B-Ⅱ)/(LC3A/B-Ⅰ);紫外分光光度法检测组织ATP含量;JC染色法检测线粒体膜电位变化。结果与Sham组比较,Model组Scr、BUN水平升高,肾组织发生病理损伤,通路及自噬相关蛋白表达量出现不同程度升高,ATP含量减少,线粒体膜电位水平下降(P<0.05);相比Model组,AC组Scr、BUN水平明显降低,肾组织病理损伤减轻,通路及自噬相关蛋白表达水平升高,ATP含量增高,线粒体膜电位升高(P<0.05);与AC组比较,经EX-527干预的AC+EX-527、EX-527组Scr、BUN水平出现不同程度升高,组织病理损伤加重现象,通路及自噬相关蛋白表达量均减少,ATP含量减少,线粒体膜电位水平下降(P<0.05),EX-527组程度较为明显。结论AC通过上调SIRT1-FOXO3-PINK1-Parkin信号通路蛋白表达,促进线粒体自噬来改善肾组织细胞线粒体功能,抑制细胞凋亡,对RIRI起到保护作用。

紫檀芪对宫颈癌细胞自噬及SIRT1-FoxO信号通路的影响

目的:探讨紫檀芪(PTE)对宫颈癌细胞自噬及SIRT1-FoxO信号通路的影响。方法:以人宫颈癌HeLa细胞作为研究对象。采用CCK-8法和流式细胞术分别测定不同浓度的PTE对HeLa细胞活力和凋亡率的影响;透射电镜观察细胞中自噬体数目;qPCR测定细胞中SIRT1和FoxO的mRNA表达;Western blot测定细胞中SIRT1、FoxO、LC3-Ⅰ、LC3-Ⅱ、p62、Bax和Bcl-2水平。结果:PTE作用于HeLa细胞24和48 h后,细胞活力抑制率随浓度增加而升高。与0μmol/L PTE相比,15、30和60μmol/L PTE处理后,HeLa细胞凋亡率、细胞中自噬体数目及Bax、LC3-Ⅱ/LC3-Ⅰ、SIRT1和FoxO水平显著升高(P<0.05),Bcl-2和p62水平显著降低(P<0.05),且呈浓度依赖性。结论:PTE可能通过激活SIRT1-FoxO信号通路诱导HeLa细胞自噬和凋亡,从而抑制HeLa细胞活力。

七氟醚可能通过调控SIRT1-FOXO1介导的自噬对大鼠脑缺血再灌注损伤的影响

目的:本研究探讨七氟醚对大鼠脑缺血再灌注损伤的影响以及机制研究。方法:将SD大鼠随机分为假手术组(Sham)、缺血再灌注损伤组(I/R)、缺血再灌注损伤组+七氟醚组(I/R+Sev)、缺血再灌注损伤组+七氟醚组+EX527组(I/R+Sev+EX527)。使用Morris水迷宫实验和TTC染色观察脑缺血再灌注后损伤情况;TUNEL染色检测神经细胞凋亡情况;透射电镜检测自噬情况;Western blotting检测自噬和凋亡相关蛋白以及SIRT1-FOXO1通路蛋白的表达。结果:与Sham组比较,I/R组大鼠逃避潜伏期延长,穿越平台次数减少,脑梗死体积显著增加,神经细胞凋亡率显著升高,自噬空泡数量明显增加,Bad、cleaved-caspase-3、LC3-Ⅱ/Ⅰ蛋白表达显著升高,Bcl-2、p62、SIRT1、FOXO1蛋白表达显著降低,差异均有统计学意义(P<0.05)。与I/R组比较,I/R+Sev组大鼠逃避潜伏期减少,穿越平台次数增多,脑梗死体积显著减小,神经细胞凋亡率显著降低,自噬空泡数量明显减少,Bad、cleaved-caspase-3、LC3-Ⅱ/Ⅰ蛋白表达显著降低,Bcl-2、p62、SIRT1、FOXO1蛋白表达显著升高,差异均有统计学意义(P<0.05)。与I/R+Sev组比较,I/R+Sev+EX527组大鼠逃避潜伏期延长,穿越平台次数减少,脑梗死体积增加,神经细胞凋亡率升高,自噬空泡数量增加,Bad、cleaved-caspase-3、LC3-Ⅱ/Ⅰ蛋白表达升高,Bcl-2、p62、SIRT1、FOXO1蛋白表达降低,差异均有统计学意义(P<0.05)。结论:七氟醚处理后可能通过抑制自噬和凋亡保护大鼠脑缺血再灌注损伤,其机制与SIRT1-FOXO1信号通路有关。

丹参提取物对脑外伤模型大鼠脑神经元SIRT1-FoxO1-自噬通路的影响

目的研究丹参提取物(SME)对脑外伤大鼠神经元沉默信息调节因子2相关酶1(SIRT1)-叉头框蛋白O1(FoxO1)-自噬通路的影响。方法采用改良Feeney法构建大鼠脑外伤模型,将78只SD大鼠按照随机数字表法分成假手术组、模型组、SME低、中、高剂量组和抑制剂组,每组13只。假手术组和模型组大鼠腹腔注射生理盐水2mL,SME低、中、高剂量组大鼠分别按0.75、1.50、3.00mg/kg给予SME,抑制剂组予SME中剂量组剂量给药同时按照5mg/kg注射SIRT1抑制剂(EX-527)。通过尼氏染色观察大鼠右侧脑组织神经元损伤情况;免疫组织化学染色、逆转录聚合酶链式反应(RT-PCR)、蛋白免疫印迹法(Western blot)分别测定SIRT1和FoxO1的细胞阳性率、转录(mRNA)水平和蛋白表达水平。结果与假手术组比较,模型组SIRT1和FoxO1的阳性细胞比例、mRNA和蛋白表达量均显著升高[(12.29±2.42)%比(4.36±1.21)%,(13.15±1.67)%比(3.31±1.50)%,(0.87±0.06)比(0.62±0.05),(0.89±0.03)比(0.69±0.04),(0.72±0.04)比(0.43±0.04),(0.76±0.08)比(0.31±0.09),P<0.05];与模型组比较,抑制剂组SIRT1阳性细胞比例、mRNA和蛋白表达量均显著降低[(6.88±2.12)%比(12.29±2.42)%,(0.71±0.09)比(0.87±0.06),(0.54±0.08)比(0.72±0.04),P<0.05],SME低、中剂量两组SIRT1蛋白水平升高[(0.98±0.05)、(1.12±0.08)比(0.72±0.04),P<0.05]及SME高剂量组SIRT1、FoxO1阳性细胞比例和mRNA和蛋白表达量均显著升高[(19.04±1.81)%比(12.29±2.42)%,(18.09±1.34)%比(13.15±1.67)%,(1.21±0.10)比(0.87±0.06),(1.19±0.03)比(0.89±0.03),(1.15±0.13)比(0.72±0.04),(0.95±0.09)比(0.76±0.08),P<0.05];与抑制剂组比较,SME中、高剂量两组FoxO1阳性细胞比例升高[(14.63±2.22)%、(18.09±1.34)%比(9.85±1.31)%,P<0.05],SME低、中、高剂量三组SIRT1阳性细胞比例、SIRT1、FoxO1的mRNA和SIRT1蛋白水平都显著升高[(12.52±2.01)%、(14.82±2.11)%、(19.04±1.81)%比(6.88±2.12)%,(0.86±0.08)、(0.92±0.05)、(1.21±0.10)比(0.71±0.09),(0.89±0.06)、(0.98±0.07)、(1.19±0.03)比(0.82±0.05),(0.98±0.05)、(1.12±0.08)、(1.15±0.13)比(0.54±0.08),P<0.05];与SME低剂量组比较,SME中剂量组FoxO1 mRNA和SIRT1蛋白表达量[(0.98±0.07)比(0.89±0.06),(1.12±0.08)比(0.98±0.05),P<0.05]及SME高剂量组SIRT1阳性细胞比例、SIRT1和FoxO1的mRNA和蛋白表达量均显著升高[(19.04±1.81)%比(12.52±2.01)%,(1.21±0.10)比(0.86±0.08),(1.19±0.03)比(0.89±0.06),(1.15±0.13)比(0.98±0.05),(0.95±0.09)比(0.79±0.07),P<0.05];与SME中剂量组比较,SME高剂量组SIRT1和FoxO1 mRNA表达显著升高[(1.21±0.10)比(0.92±0.05),(1.19±0.03)比(0.98±0.07),P<0.05]。结论SME能够明显促进脑外伤模型大鼠脑神经元SIRT1表达,增强SIRT1的去乙酰化活性,正向调节SIRT1-FoxO1-自噬通路发挥神经保护作用。

CHANGES IN NEUROPEPTIDES AFTER MUSIC EXPOSURE 429Cardioprotective effect of ivabradine via the AMPK/SIRT1/PGC-1αsignaling pathway in myocardial ischemia/reperfusion injuryinduced in H9c2 cell

Post-resuscitation myocardial dysfunction(PRMD)is the most severe myocardial ischemia-reperfusion injury(MIRI)and is characterized by difficult treatment and poor prognosis.Research has shown the protective effects of the rational use of ivabradine(IVA)against PRMD,however,the molecular mechanisms of IVA remain unknown.In this study,an ischemia-reperfusion injury(IRI)model was established using hypoxic chambers.The results demonstrated that pretreatment with IVA reduced IRI-induced cytotoxicity and apoptosis.IVA attenuated mitochondrial damage,eliminated excess reactive oxygen species(ROS),suppressed IRI-induced ATP and NAD+,and increased the AMP/ATP ratio.We further found that IVA increased the mRNA levels of sirtuin 1(SIRT1)and peroxisome proliferator-activated receptor-γcoactivator 1α(PGC-1α)and upregulated the expression levels of phosphorylated AMP-activated protein kinase(p-AMPK)/AMPK,SIRT1,and PGC-1αproteins.Interestingly,no change in AMPK mRNA levels was observed.Cardiomyocyte energy metabolism significantly changed after IRI.The aim of this study was to demonstrate the cardioprotective effect of Ivabradine via the AMPK/SIRT1/PGC-1αsignaling pathway in myocardial ischemia/reperfusion injury-induced in H9c2 cell.

Mechanism of Resveratrol on autophagy mediated by Mst1/Sirt3 signaling pathway in diabetic cardiomyopathy

Objective:To observe the effects of resveratrol on myocardial cell injury and Mst1/Sirt3 signaling pathway mediated autophagy in type 2 diabetic mice. Methods:C57 BL/KSJ db/db mice were allocated to the normal control group,the model group,and the resveratrol group;C57 BL/KSJ db/m mice served as the melbine group,with 10 mice each. The resveratrol group and the melbine group were treated with resveratrol and metformin by gavage,respectively. The normal control group and the model group were treated with equal volume of normal saline by gavage,for 8 consecutive weeks. H & E staining,transmission electron microscopy and immunofluorescence were used to observe the pathological morphology,ultrastructure and apoptosis levels of myocardial tissues,respectively. RT-qPCR method was used to detect the expression levels of apoptosis genes Bax and Bcl-2 in myocardial tissues,and Western-blot method was used to detect the expression levels of autophagy proteins(LC3 and p62),Mst1 and Sirt3 proteins in myocardial tissue. Results:Compared with the model group,resveratrol can significantly reduce the body weight,blood glucose level and serum CK and LDH levels of db/db mice,and the differences were statistically significant(P<0.05;P<0.01). Meanwhile,after resveratrol treatment,myocardial inflammation score,apoptosis rate,Bax mRNA expression level and Bax/Bcl-2 ratio in myocardial tissue were significantly reduced,and Bcl-2 mRNA expression level was significantly increased,and the differences were statistically significant(P<0.01). In addition,compared with the model group,the expression level of p62 and p-Mst1 protein in the myocardial tissue of the resveratrol group was significantly reduced,and the expression level of Sirt3 protein and the ratio of LC3Ⅱ/LC3Ⅰ were significantly increased,and the differences were statistically significant(P<0.01). Conclusion:Resveratrol promotes the autophagy level of cardiomyocytes by activating the Mst1/Sirt3 signaling pathway and inhibits cardiomyocyte apoptosis to play a protective role in diabetic cardiomyopathy.

Mechanism of hesperidin improving myocardial ischemia/reperfusion injury in type 2 diabetic rats through SIRT1/Nrf2/HO-1 signaling pathway

Objective:To observe the protective effect of hesperidin on myocardial ischemia/reperfusion injury in type 2 diabetes mellitus and its effect on SIRT1/Nrf2/HO-1 signaling pathway.Methods:50 Sprague-Dawley(SD)rats were randomly assigned to the normal control group(NC),model group,ischemia-reperfusion group(IR),hesperidin group,SIRT1 inhibitor group and hesperidin plus SIRT1 inhibitor group.In addition to NC,the rats in the remaining groups were replicated by intraperitoneal of high-fat diet combined with injection of streptozotocin for type 2 diabetic rats.After then,the myocardial ischemia/reperfusion injury(MIRI)rat model was established by LAd for 30 minutes with 2 hours reperfusion.He staining was used to observe the pathological changes of myocardial tissue,and the levels of serum LDH,CK-MB and SOD,GSH and MDA in myocardial tissue were detected by kit methods,and the expression abundance of related proteins in 4-HNE and SIRT1/Nrf2/HO-1 signal pathway were detected by immunohistochemistry and Western blot;Results:Hesperidin could significantly inhibit cardiomyocyte necrosis and inflammatory cell infiltration,reduce LDH activity,CK-MB and MDA level,and increase SOD activity,GSH and 4-HNE level,the differences were statistically significant when compared with IR group(P<0.01).In addition,compared with the ischemia-reperfusion group,the expressions of SIRT1,Nrf2 and HO-1 proteins in hesperidin group were significantly up-regulated,the differences were statistically significant(P<0.01);Conclusion:Hesperidin inhibits oxidative stress by activating SIRT1/Nrf2/HO-1 signaling pathway,and play a protective effect of myocardial ischemia reperfusion injury in diabetic rats.

Yiqi Yangyin and Huatan Quyu granule can improve skeletal muscle energy metabolism in a type 2 diabetic rat model by promoting the AMPK/SIRT/PGC-1α signalling pathway

Objective:To investigate how Yiqi Yangyin and Huatan Quyu granule (YYHO) improves skeletal muscle insulin resistance in a type 2 diabetic rat model and to discover whether the molecular mechanism is related to the promotion of the AMPK/SIRT/PGC-1α signalling pathway.Methods:Rats were randomly divided into 4 groups:the normal group,the model group,the YYHQ granule group,and the pioglitazone group.The type 2 diabetic rat model was established by feeding a high-fat diet for 5 weeks along with a single intraperitoneal injection of 30 mg/kg streptozotocin (STZ).After modelling successfully,the appropriate drug was intragastrically administered to diabetic rats for 2 weeks,once per day.The YYHQ granule group was given a dose of 4.8 g/kg body weight per day,the pioglitazone group was given a dose of 1.35 mg/kg body weight per day.The doses for both groups were equivalent to the clinical equivalent dose based on a previous study.Other groups were gavaged with the same amount of saline water.Body weight,food intake,water intake,urine volume and grip strength were recorded weekly.The fasting blood glucose(FBG) was determined weekly using blood glucose test strips.The related glucose and lipid metabolism indexes,e.g.,fasting insulin (Fins),glycated haemoglobin (GHb),HOMA-IR,ISI,triglycerides (TG),total cholesterol (TC),high-density lipoprotein cholesterol (HDL-C),low-density lipoprotein cholesterol (LDL-C) and free fatty acid (FFA),were determined using biochemical method.The mRNA expression levels of adenosine monophosphate-activated protein kinase (AMPK),peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α),carnitine palmitoyl transterase-1 (CPT-1),Sirtuin 1 (SIRT1),and Sirtuin 3 (SIRT3) were assessed using quantitative real-time PCR (qRT-PCR).The protein expression levels of creatine kinase (CK),Ca2+ ATPase,α-Actin,AMPK,PGC-1α and CPT-1 were determined using enzyme-linked immunosorbent assay method (ELISA).Results:Body weight decreased significantly (P <.01),food intake,water intake and urine volume increased significantly (P <.01),and grip strength decreased significantly (P <.01) in the model group compared with the normal group.The levels of FBG,Fins,GHb and HOMA-IR increased significantly (P <.01),and the ISI decreased significantly (P <.01) in the model group.The levels of TG,TC,LDL-C and FFA increased significantly (P <.05 or P <.01),and the level of HDL-C decreased significantly (P <.05) in the model group.These changes were reversed after treatment with YYHQ granule or pioglitazone.Compared with the model group,the YYHQ granule and pioglitazone groups significantly improve body weight,water intake and urine volume (P <.05 or P <.01),however,both treatments had no significant effect on food intake (P >.05).The levels of FBG,Fins,GHb,HOMA-IR and ISI were improved significantly (P <.01) and the levels of TG,TC and LDL-C were improved significantly (P <.05 or P <.01),however,both treatments had no significant effect on the levels of HDL-C and FFA (P >.05).Further results indicated that YYHQ granule significantly decreased the mRNA expression of AMPK,PGC-1α,CPT-1,SIRT1 and SIRT3 in skeletal muscle (P <.01) and the pioglitazone group showed similar effects;moreover,the protein expression levels of CK,Ca2+ATPase,α-Actin,AMPK,PGC-1α and CPT-1 in skeletal muscle significantly decreased (P <.01),however,pioglitazone had no significant effect on CK and α-Actin (P >.05).Conclusion:The possible molecular mechanism of YYHQ granule improving skeletal muscle insulin resistance in a type 2 diabetic rat model may be related to the stimulation of energy metabolism in skeletal muscle via the AMPK/SIRT/PGC-1α signalling pathway.