【作者】 刘燕；

【导师】 叶山东；

【作者基本信息】 安徽医科大学 ， 内科学（内分泌与代谢病）（专业学位）， 2020， 博士

【摘要】 研究背景:糖尿病是一组以糖脂代谢紊乱、高血糖为特征的内分泌代谢性疾病,是由胰岛素产生不足或胰岛素抵抗所致。长期的高血糖可以导致糖尿病并发症的发生,肾脏是主要的靶器官。大约40%的1型和2型糖尿病患者会发展为糖尿病肾病,是导致终末期肾病的主要原因。尽管目前有许多方法可预防和治疗糖尿病肾病,包括降糖、控制血压、肾素-血管紧张素-醛固酮系统阻断剂、抗氧化、抗炎等,但这些方法的单一防治效果并不令人十分满意,需要不断寻找治疗糖尿病肾病的新型治疗方法和药物。AKT信号转导通路是抑制细胞凋亡,促进细胞分化、存活的重要通路。该通路既是药物保护肾脏细胞的重要作用靶点,又是调节抑制细胞凋亡的上游通路。水飞蓟素是从乳蓟中提取的黄酮类化合物,它由水飞蓟宾,水飞蓟宁和水飞蓟亭等组成,其中水飞蓟宾是生物活性最高的成分。据报道,它具有抗氧化、抗炎、抗脂肪生成、保肝、抗菌、抗癌等多种药理作用,对心血管、中枢神经系统有保护作用。近年来,水飞蓟素在糖尿病及慢性并发症方面的治疗作用日益受到重视。尽管有报道称水飞蓟素对链脲佐菌素及四氧嘧啶诱导的糖尿病大鼠肾脏损伤有保护作用,但水飞蓟素对db/db糖尿病小鼠生化参数变化和肾脏损伤的影响,特别是水飞蓟素对db/db小鼠生物学功能的潜在分子机制尚不完全清楚。本研究通过动物实验和临床研究评估了水飞蓟素对糖尿病肾病的影响,并探讨其中潜在的作用机制。研究目的:1.通过检测db/db小鼠体重、生化参数及肾脏损伤指标的变化评估水飞蓟宾对糖尿病肾病的保护作用;2.通过检测db/db小鼠氧化应激、细胞凋亡、AKT信号通路指标探讨水飞蓟宾肾脏保护作用的潜在机制;3.进一步评价水飞蓟素对早期2型糖尿病肾脏病患者临床应用的疗效,通过检测生化指标和氧化应激指标探讨其作用机制。研究方法:1.实验一:C57BL/Ks J db/db小鼠作为研究对象,以年龄匹配野生型非糖尿病(db/m)小鼠为对照组。将血糖超过11.1mmol/l的db/db小鼠随机分为三组:db/db小鼠、db/db小鼠+水飞蓟宾(15mg/kg/day)、db/db小鼠+水飞蓟宾(30mg/kg/day)。治疗10周后,采集血液,收集尿液,并测量小鼠体重及生化参数(血糖、糖化血红蛋白、血清胰岛素、肌酐及尿素氮、尿白蛋白肌酐比值,组织病理学分析肾小球系膜扩张指数和肾小管间质损伤指数评估肾脏损伤情况。2.实验二:动物造模及分组同实验一。检测血清和肾脏组织匀浆中氧化应激指标丙二醛、超氧化物歧化酶和谷胱甘肽过氧化酶水平,免疫印迹法检测p-AKT、AKT、p-GSK3β、GSK3β、Bax、裂解Caspase-3蛋白水平。3.实验三:选择了80例早期2型糖尿病肾脏病患者,随机分为水飞蓟素组及安慰剂组。水飞蓟素组接受水飞蓟素胶囊(140mg/次,每日3次,N=40),安慰剂组(N=40)接受相同剂量的安慰剂,持续12周。治疗前后测量患者的血清生化参数、尿白蛋白肌酐比值、血清丙二醛、总抗氧化能力、超氧化物歧化酶、谷胱甘肽过氧化物酶等氧化应激指标。研究结果:1.实验一:与正常对照组db/m小鼠相比,db/db小鼠的体重、糖化血红蛋白、胰岛素、尿素氮、肌酐、尿白蛋白肌酐比值显著升高。水飞蓟宾治疗可以阻止体重增加,降低糖化血红蛋白、胰岛素水平、尿素氮、肌酐和尿白蛋白肌酐比值,且呈剂量依赖性。与正常对照组db/m小鼠相比,db/db小鼠的肾小球系膜扩张指数和肾小管损伤指数明显升高,水飞蓟素治疗可使肾小球系膜扩张指数和肾小管损伤指数降低且呈剂量依赖性。2.实验二:与正常对照组db/m小鼠相比,db/db小鼠血清及肾脏中丙二醛水平明显升高,而超氧化物歧化酶和谷胱甘肽过氧化物酶水平明显降低,给予水飞蓟宾治疗后丙二醛水平明显降低,而超氧化物歧化酶和谷胱甘肽过氧化物酶水平明显升高。与正常对照组db/m小鼠相比,db/db小鼠的p-AKT水平下降,p-GSK-3β水平增加,下游蛋白Bax和裂解Caspase-3蛋白水平增加,水飞蓟宾治疗使p-AKT水平增加,并且阻止了db/db小鼠p-GSK-3β、Bax和裂解Caspase-3蛋白水平的升高。3.实验三:两组间比较,治疗前的血压,体重指数,空腹血糖、糖化血红蛋白、总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白、尿素氮、肌酐、尿蛋白肌酐比值均无显著差异。治疗后两组血压,体重指数,空腹血糖、糖化血红蛋白、甘油三酯、尿素氮、肌酐水平差异无统计学意义,水飞蓟素组较安慰剂组治疗后总胆固醇、低密度脂蛋白水平、尿白蛋白肌酐比值及丙二醛显著降低,高密度脂蛋白水平及总抗氧化能力、超氧化物歧化酶、谷胱甘肽过氧化物酶水平显著升高。两组内比较,水飞蓟素组治疗前后血压,体重指数,空腹血糖、糖化血红蛋白、甘油三酯、尿素氮、肌酐水平差异无统计学意义,而总胆固醇、低密度脂蛋白、尿白蛋白肌酐比值及丙二醛水平治疗后显著降低,高密度脂蛋白及总抗氧化能力、超氧化物歧化酶、谷胱甘肽过氧化物酶水平显著升高。安慰剂组治疗前后的血压,体重指数,血糖、糖化血红蛋白、总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白、尿白蛋白肌酐比值、尿素氮、肌酐及丙二醛、总抗氧化能力、超氧化物歧化酶、谷胱甘肽过氧化物酶水平均无显著差异。研究结论:水飞蓟宾可以降低db/db小鼠的血糖和尿白蛋白肌酐比值,改善肾小球和小管间质损伤,具有肾脏保护作用。水飞蓟宾可预防db/db小鼠氧化应激,抗细胞凋亡。水飞蓟素对早期糖尿病肾病患者的临床应用同样显示出肾脏保护作用,同时具有改善氧化应激及调脂作用。水飞蓟素肾脏保护作用潜在的机制可能与其活化AKT信号通路、抗氧化应激、抗细胞凋亡和调脂等有关。但是需要进行更大样本量和更长干预时间的进一步研究,以确认水飞蓟素在糖尿病肾病患者临床应用中的有益作用,并探讨确切的肾脏保护机制。更多还原

【Abstract】 Background Diabetes mellitus is an endocrine and metabolic disease characterized by glucose and lipid metabolism disorders and hyperglycemia,which is caused by insulin deficiency or insulin resistance.Chronic hyperglycemia can lead to complications of diabetes,and the kidney is considered the primary target organ.Diabetic nephropathy develop in about 40% of patients with type 1 and type 2 diabetes,which is the main cause of end-stage renal disease.Although there are many methods to prevent and treat diabetic nephropathy,including standard glycemic control,blood pressure control,renin-angiotensin-aldosterone system blockers,antioxidant,anti-inflammatory,etc,the single effects of these methods are unsatisfied,demanding for the new treatments and drugs for diabetic nephropathy.The AKT signaling pathway is an important pathway that inhibits apoptosis and promotes cell differentiation and survival.Consequently,this pathway is not only an important target for drugs to protect kidney cells,but also an upstream pathway to regulate and inhibit apoptosis.Silymarin,a flavonoid compound extracted from milk thistle,is mainly composed of silibinin,silydianin and silicristin.Among them,silibinin is the main bioactive component.It is reported that it has a variety of pharmacological effects,such as anti-oxidation,anti-inflammatory,anti-adipogenic effects,liver protection,antibacterial,anti-cancer,etc.In addition,it has protective effects on cardiovascular and central nervous system.In recent years,silymarin plays an increasingly important role in the treatment of diabetes and chronic complications.Although silymarin has been reported to exert a protective effect against renal injury in streptozotocin and alloxan-induced diabetic rats,the effects of silymarin on the biochemical parameters changes and kidney injury,especially the potential molecular mechanism involved in the biological function of silymarin on db/db mice are not fully understood.In this thesis,the effects of silymarin on diabetic nephropathy were evaluated through animal and clinical experiments and its potential mechanisms were explored.Objectives 1.To evaluate the reno-protective effects of silibinin by detecting changes of body weight,biochemical parameters and kidney injury indexes in db/db mice;2.To explore the potential mechanisms of silibinin’s reno-protective effects by detecting oxidative stress,apoptosis,and AKT signaling pathway indicators in db/db mice;3.To further evaluate the clinical efficacy of silymarin in patients with early type 2 diabetic nephropathy,and explore its potential mechanisms by detecting biochemical and oxidative stress indicators.Methods 1.PART ONE C57BL/Ks J db/db mice were used as subjects,and age-matched wildtypenon-diabetic(db/m)mice were used as control groups.db/db mice with blood glucose over 11.1mmol/L were randomly allocated into three groups: db/db mice,db/db mice + silibinin(15mg/kg/day),and db/db mice + silibinin(30mg/kg/day).After 10 weeks of treatment,blood samples and spot urines were collected,and the body weights(BW)and biochemical parameters of the mice(fasting blood glucose(FBG),glycated hemoglobin A1c(Hb A1c),fasting serum insulin(FINS),serum creatinine(SCr),blood urea nitrogen(BUN),and urine albumin to creatinine ratio(UACR))were measured.The glomerular mesangial expansion index and the tubulointerstitial injury index were used to evaluate renal damage.2.PART TWO Animal modeling and grouping were similar to part one.The levels of oxidative stress indicators malonic dialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in serum and kidney tissue were detected,and the levels of p-AKT,AKT,p-GSK3β,GSK3β,Bax,and cleaved caspase-3 protein were determined by western blot.3.PART THREE Eighty patients with early type 2 diabetic nephropathy were randomly divided into silymarin group and placebo group.The silymarin group(N=40)were dosed with silymarin(140 mg capsule,three times daily),while the placebo group(N=40)received the same dose of placebo for 12 weeks.Before and after treatment,the levels of serum biochemical parameters,UACR,serum MDA,TAC,SOD and GSH-Px were measured.Results 1.PART ONE Compared with db/m mice,the BW of db/db mice increased significantly,accompanying by the increase of the levels of Hb A1 c,FINS,BUN,SCr and UACR.Silibinin could prevent BW increase and reduce the levels of Hb A1 c,FINS,BUN,SCr and UACR in a dose-dependent manner.The levels of the glomerular mesangial expansion index and the tubulointerstitial injury index in db/db mice were significantly higher than those in db/m mice.The levels of the glomerular mesangial expansion index and the tubulointerstitial injury index decreased in silibinin-treated db/db mice in a dose-dependent manner.2.PART TWO Compared with db/m mice,the levels of MDA in serum and kidneys of db/db mice were significantly higher,while the levels of SOD and GSH-Px were significantly decreased.After treatment with silibinin,the levels of MDA decreased significantly,while the levels of SOD and GSH-Px levels increased significantly.Additionally,p-AKT levels in db/db mice decreased,the levels of p-GSK-3β,downstream Bax and caspase-3 protein increased.After silibinin treatment,p-AKT levels increased and the increased levels of p-GSK-3β,Bax and caspase-3 protein were prevented in db/db mice.3.PART THREE There were no significant difference in the levels of blood pressure(BP),body mass index(BMI),FBG,Hb A1 c,total cholesterol(TCH),triglyceride(TG),high-density lipoprotein(HDL),low-density lipoprotein(LDL),BUN,SCR,UACR,MDA,total antioxidant capacity(TAC),SOD and GSH-Px between the silymarin group and placebo group before treatment.After treatment,there were no significant difference in BP,BMI,FBG,Hb A1 c,TG,BUN and SCR between the two groups.Compared with the placebo group,the levels of TC,LDL,UACR and MDA decreased significantly,while the levels of HDL,TAC,SOD,GSH-Px increased significantly in the silymarin group after treatment.Compared with prior treatment,there were no significant change in BP,BMI,FBG,Hb A1 C,BUN,SCR and TG.However,the levels of TC,LDL,UACR and MDA declined significantly,accompanying by significant increase in the levels of HDL,TAC,SOD and GSH-Px after treatment in the silymarin group.There were no significant difference in the levels of BP,BMI,FBG,Hb A1 C,TC,TG,HDL,LDL,BUN,SCR,UACR,MDA,TAC,SOD and GSH-Px before and after treatment in the placebo group.Conclusion Silibinin can reduce blood glucose and UACR in db/db mice and improve glomerular and tubulointerstitial injury,suggesting that silibinin has a reno-protective effect.Silibinin can prevent oxidative stress and apoptosis in db/db mice.The clinical application of silymarin in patients with early diabetic nephropathy also shows a reno-protective effect,accompanying by the improvement of oxidative stress and lipid regulation.The underlying mechanisms could be related to anti-oxidative stress,activation of AKT signaling pathway,anti-apoptosis and lipid regulation.However,further studies with larger sample sizes and longer intervention time are needed to confirm the beneficial role of silymarin in the clinical application in patients with diabetic nephropathy and to investigate the protective mechanisms in detail.更多还原