Protective Effects Of Silymarin On Diabetic Nephropathy And Its Mechanisms

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.