Protective Effects And Mechanism Of Salidroside On Diabetic Nephropathy

Background and Aim:Diabetic nephropathy(DN)is a very serious and the most harmful chronic complication caused by diabetes mellitus,The main pathological change is that hyperglycemia leads to micro vascular disease and then leads to glomerulosclerosis.In our country,with the continuous improvement of the economic level,the way of life is becoming more and more westernized,and the incidence and mortality of diabetes are increasing year by year,Treatment methods and techniques have also developed rapidly,and fewer patients die of acute diabetic complications.However,Cardiovascular disease and kidney disease caused by diabetes have become the main cause of death and disability in recent years,and diabetic nephropathy is the leading cause of death in patients with type 1 diabetes.Rhodiola is an important health food and Chinese medicine,which can invigorate the circulation of the blood,stop the bleeding,clear lung cough,be as the antipyretic and so on.Salidroside is one of the main components of Rhodiola with the function of reducing blood glucose,anti-inflammation,anti-oxidation and a certain therapeutic effect on diabetes.Studies have shown that Rhodiola can alleviate the damage of DN on renal tissue,but its mechanism is still unclear.In this paper,we mainly studied the protective effect of salidroside on diabetic renal tissue injury,and took the endoplasmic reticulum stress and Akt/GSK-3β signaling pathway as a target for mechanism studyMethods:30 male SD rats with the weight of 200-250g were injected intraperitoneally with streptozotocin(STZ)according to the dose of 65mg/kg,The fasting blood glucose(FBG)and urine protein were measured after 72 hours,When FBG is greater than or equal to 16.7mmol/L,the urine protein is greater than or equal to 30mg/kg/d,The model was successfully prepared.After successful modeling,the rats were randomly divided into two groups:diabetic nephropathy model group(DN group)and salidroside treatment group(DN+Sal group),each group has 15 rats.At the same time,10 normal rats were taken as normal control group.Rats in DN+Sal group were treated with salidroside(Po,50mg/kg/d),while DN group and control group were given equal volume of normal saline each day.8 weeks treatment later,all rats were executed under deep anesthesia,blood,urine and renal tissue were collected.The concentration changes of fasting blood glucose(FBG),lipid metabolism(triglyceride:TC,total cholesterol,TG)blood urea nitrogen(BUN),serum creatinine(Scr)were detected by automatic biochemical analyzer.HE staining,Masson staining and transmission electron microscope were used to observe the change of basic structure,interstitial fibrosis and ultrastructural in renal tissue.TUNEL fluorescence staining was used to observe the number and extent of renal cell apoptosis.Enzyme linked immunosorbent assay method(ELISA)and Colorimetric analysis were used to detect the change of interleukin 1β(IL-1β),tumor necrosis factor α(TNF-α),superoxide dismutase(SOD)and malondialdehyde(MDA)in serum and renal tissue.Western Blot method was used to detect the expression of Bcl-2,Bax,Akt,p-Akt,GSK-3β,p-GSK-3β,GRP-78,CHOP and other proteins in renal tissue.Results:1.Basic situation performance:Compared with the normal control group,Diabetic nephropathy(DN)group rats daily showed the symptoms of polydipsia,polyphagia,polyuria,weight loss("three increasing and one decreasing"),yellow fur,dry and no light,poor mental state,less activity,eyeball white,no focus of vision.But the salidroside treatment group(DN+Sal group)rats significantly gained weight and had good spirit.2.Blood biochemistry and urine test results:Compared with normal control group,Diabetic nephropathy(DN)group rats;blood glucose significantly increased,;their blood lipids largely accumulated;their blood urea nitrogen(BUN)and serum creatinine(Scr)levels increased significantly and their urine volume also increased significantly.The difference was considered to be statistically significant(P<0.05).But compared with the diabetic nephropathy(DN)group,Salidroside treatment group(DN+Sal group)rats’ blood glucose and blood lipid levels decreased;their urine volume was significantly reduced(P<0.05)and their glomerular injury also improved significantly.3.Morphological observation results:in control group,the glomerulus volume was uniform;the cytoplasm of the cells was homogeneous and the nuclear volume were uniform,arranged regularly,in a circular or oval shape,located in the central region of the cell.No fibrosis was found in the mesenchyme and the structure of Mitochondria,endoplasmic reticulum and other organelles are normal.But in diabetic nephropathy(DN)group,glomerular volume increased significantly;the staining of cytoplasm was shallow and uneven;interstitial fibrosis was obvious;glomerular basement membrane thickened and renal mesangial cells also thickened;Mitochondrial expanded,swelled and vacuolization appeared;ridge ruptured and disappeared.Endoplasmic reticulum expanded and degranulated.After treatment with salidroside,the glomerular volume was decreased,the area of fibrosis was significantly reduced,and the damage of mitochondria and endoplasmic reticulum was significantly reduced4.Enzyme linked immunosorbent assay show that:In normal control group,The levels of IL-1β and TNF-α in serum were 12.65 ± 0.48 ng/L and 152.50± 9.87 ng/L respectively,which were belong to the normal range.In diabetic nephropathy(DN)group,The levels of IL-1 and TNF-α in serum were 17.78± 0.35 ng/L and 280.80 ±9.34 ng/L,which were higher than those in the normal group(P<0.05).In salidroside treatment(DN+Sal)group,The levels of IL-1 and TNF-α in serum were 14.92±0.46ng/L and 209.30 ±13.14ng/L respectively,which were lower than those in diabetic nephropathy(DN)group(P<0.05).These results proved that salidroside can alleviate the renal inflammatory response in diabetic rats.5.Colorimetry and spectrophotometry results:Compared with the normal group,the SOD activity in serum of diabetic nephropathy group(DN)rats significantly reduced,while the content of MDA significantly increased(P<0.05),which suggest that diabetic nephropathy causes severe oxidative stress.After salidroside treatment,the activity of SOD increased obviously and the content of MDA decreased significantly(P<0.05),which showed that salidroside can protect diabetic nephropathy by resisting oxidative stress.6.Apoptosis detection results:In normal control group,the number of apoptotic cells in renal tissue was less and limited,and the fluorescence intensity was weak,which belonged to physiological death.In DN group,the apoptosis was large and the fluorescence intensity was strong.At the same time,WB results showed that the Bcl-2/Bax ratio was significantly lower than that in the normal group(P<0.05).In DN+Sal group,the number of apoptotic cells was decreased and the ratio of Bcl-2/Bax was higher than that of DN(P<0.05).7.Western Blot results:Compared with the control group,the expression of Phosphorylation of Akt(p-Akt)in renal tissue in DN group rats was significantly down regulated,,and accompanied with the down regulation expression of AKT downstream target molecule GSK-3b(p-GSK-3b)(P<0.05).After DN rats were treated with salidroside,the expression of p-Akt protein in rat renal tissue is increased significantly,and accompanied with upregulation of the expression of p-GSK-3β protein(P<0.05)in DN rats,which suggest that salidroside can activate Akt/GSK-3β signaling pathway.In addition,the expression of GRP 78 and CHOP protein in renal tissue was increased significantly in diabetic nephropathy group(P<0.05).After salidroside treatment,the expression was significantly decreased,which further suggest salidroside can reduce apoptosis induced by endoplasmic reticulum stress in renal tissue caused by diabetic(P<0.05).Conclusion1.The pathogenesis of diabetic nephropathy is various,including oxidative stress,inflammatory response and endoplasmic reticulum stress.,the inactivation of Akt/GSK-3β signaling pathway plays an important role in this process.2.Salidroside has obvious hypoglycemic,hypolipidemic,anti-inflammatory,antioxidant and anti-fibrosis effects on diabetic nephropathy rats.3.Salidroside can reduce endoplasmic reticulum stress,down regulate the expression of GRP-78,CHOP and so on,and reduce the apoptosis of diabetic nephropathy.4.Salidroside can activate Akt/GSK-3β signaling pathway and improve the inflammation and oxidative stress in diabetic nephropathy,and then prevent renal fibrosis and renal cell apoptosis,and delay or prevent the development of diabetic nephropathy.