Wogonin Improves Renal Inflammation And Fibrosis In Diabetic Nephropathy

Background and purpose:Diabetic nephropathy(DN)which has become the main cause of end stage renal disease(ESRD)is the complication of diabetes.A variety of mechanisms are involved in the pathogenesis of DN,among which the fibrosis and inflammation lead to persistent renal damage.Hyperglycemia which increases the secretion of fibrosis factors,proliferates mesangial matrix,and thickens the glomerular basement membrane,can eventually lead to glomerular sclerosis.Inflammation plays an important role in the progress of DN,and the overexpression of inflammatory factors can be detected in renal tissues,serum and urine of DN patients.The activation of these inflammatory factors aggravates the hemodynamic abnormality caused by high glucose environment in DN patients,and exacerbates renal metabolic disorders.Several studies have shown that flavonoid can effectively restrain the progression of DN,wogonin shows the characteristics of anti-inflammatory and anti-fibrosis,and it is proved to be curative in other animal models of kidney disease.Therefore,we chose wogonin to explore whether it can have the effect of anti-fibrosis and anti-inflammatory to delay the disease progression of diabetic nephropathy.Methods:Animal experiment: Diabetic model were induced by intraperitoneal injection with STZ at 50mg/kg·d for 5 days in C57/BL6 J mice,the diabetic model was established by the standard of blood glucose > 16.7mmol/L.The diabetic mice were gavaged with wogonin,and were fed once every other day for 12 weeks.Mice were divided intoNormal group(NC).Normal+40mg/kg wogonin group(NC+40mg/kg);Diabetic group(DN);Diabetic+wogonin group(10mg/kg,20mg/kg,40mg/kg),there were 8 mice in each group.Blood glucose of mice was detected,renal weight and body weight were weighed,and the urine was collected for 24 hours for detecting the urinary albumin,blood was collected to measure serum creatinine value.Pathological changes of the mouse kidney were observed by general microscope and electron microscope.Expression of p-Smad3,Smad3,NF-?Bp65,NF-?Bp-p65 in renal tissue was detected by Western blot.The expression of inflammatory cytokines TNF-?,IL-1? and MCP-1 in the mRNA of mice were detected by real-time fluorescent quantitative PCR.In vitro study: Mesangial cells of mice SV40 were selected for vitro experiment.and the optimal concentration of wogonin was determined by MTT experiment.Different concentrations of wogonin was added to high glucose medium to stimulate the mesangial cells for 24 hours to observe the effect of wogonin.The experimental groups :the mannitol control group(M),the low glucose control group(LG),the low glucose group with wogonin(LG+6.25 ?g/ml),the high glucose group(HG),the high glucose group with wogonin(HG+0.78125 ?g/ml,HG+1.5625 ?g/ml,HG+6.25 ?g/ml).Western blot was used to detect the expression changes of FN,Col-IV,?-SMA,TGF?1,p-Smad3,Smad3,NF-?Bp65,NF-?Bp-p65 in mesangial cells.The mRNA of inflammatory cytokines TNF-?,IL-1? and MCP-1 in mesangial cells were detected by real-time fluorescent quantitative PCR.Results:Animal experiment: compared with NC group,blood glucose,kidney weight/body weight ratio,24 hours urinary albumin,serum creatinine value were significantly increased in DN group,after the treatment of wogonin,blood glucose is no difference between DN group and treatment groups while the relative kidney weight were decreasedsignificantly,the levels of 24 hours urinary albumin and serum creatinine value among10mg/kg,20 mg/kg and 40 mg/kg groups decreased significantly;The glomerular volume increased,mesangial matrix proliferated,basement membrane thickened in DN group,while pathological lesions were significantly alleviated in 10mg/kg,20mg/kg,and 40mg/kg groups.MASSON staining showed that the secretion of collagen in DN group increased compared with NC group,while the it was inhibited in the three dose groups.Immunohistochemical staining and Western blot showed that,compared with the NC group,the levels of FN,Col-IV,?-SMA and TGF?1 in the kidney tissues of the DN group were significantly increased,while in the 10mg/kg,20mg/kg and 40mg/kg groups the levels were significantly decreased.The protein expression of p-Smad3 and NF-?Bp-p65 in renal tissues of mice showed that DN group were significantly increased compared with NC group,and the indicators were significantly decreased in the three dose groups after treatment with wogonin.The mRNA levels of TNF-?,IL-1? and MCP-1 in kidney tissues in DN group were higher than those in the NC group,while the inflammatory factors were significantly decreased in the diabetic mice fed with wogonin.There was no significant difference between the NC group and the NC+40mg/kg control group among all indicators.In vitro study: The results of MTT experiment showed that 6.25 ?g/ml,3.125 ?g/ml and1.5625 ?g/ml were the appropriate concentrations of wogonin.Western blot results showed that the expressions of FN,Col-IV,?-SMA,TGF?1,p-Smad3,and NF-?Bp-p65 in mesangial cells in HG group were significantly higher than those in LG group,the expression of HG+0.78125 ?g/ml group,HG+1.5625?g/ml group,and HG+6.25?g/ml group were significantly lower than those in HG group(P<0.05).The mRNA expression of TNF-?,IL-1? and MCP-1 in mesangial cells showed that inflammatory factors in HG group was significantly increased compared with LG group,while it was inhibited in low,medium and high concentration groups with wogonin.There were no statisticallydifference among the control groups.Conclusion:Wogonin may reduce the expression of inflammatory factors in the kidney of mice and inhibit the renal fibrosis through the NF-?B signaling pathway,TGF?1 /Smad3 pathway to play a protective role in the kidney of diabetic mice.