Based On The TGF-β1/Smad Pathway To Explore The Study Of Xiaokeshen Decoction In The Treatment Of Mice With Early And Mid-term Diabetic Nephropathy

BackgroundDiabetic kidney disease(DKD)is the most common microvascular complication of diabetes.In the past 30 years,the continuous surge of diabetes incidence rate in China has provided a huge base for DKD,making DKD the main cause of CKD.The pathogenesis of DKD is complex,TGF-β1 was up-regulated under various pathological stimulation,and ECM deposition was induced by TGF-β1/Smad signal transduction,which accelerated the process of renal fibrosis.In recent years,more and more studies have shown that the intervention of TGF-β1/Smad signaling pathway plays an important role in the treatment of DKD.At present,western medicine mainly delays the progress of DKD by treating primary disease.Traditional Chinese medicine can not only improve clinical symptoms,but also effectively reduce proteinuria and delay the progress of DKD.It is urgent to carry out the research of early intervention of traditional Chinese medicine on DKD.The tutor suggested that we should seize the opportunity in early or middle period to treat DKD and attack the " Xiaokeshen heat" directly,which is of great significance in delaying the course of DKD and improving the prognosis.On the basis of previous research,this study hypothesize that the renal protective effect of Xiaokeshen formula on DKD db/db mice model in the early or middle stage is achieved by inhibiting the TGF-β1/Smad signal pathway,which promote renal fibrosis in the early stage of DKD.Through animal experiments,this study use Xiaokeshen formula to treat DKD db/db mice model in early or middle period of DKD,and observe weight,blood glucose,urinary protein,liver and kidney function,blood lipid,renal tissue structure and TGF-β1/Smad pathway related proteins to provide theoretical basis for the clinical use of Xiaokeshen formula.ObjectiveThrough animal experiment,we observed the effects of Xiaokeshen formula on blood glucose,urinary protein,renal pathology,TGF-β1/Smad-related proteins and fibrotic marker proteins in db/db mice with early or middle stage DKD,and explore the mechanism of Xiaokeshen formula,which provided theoretical basis for the clinical treatment of DKD.Method1.50 male SPF grade C57BL/KSJ db/db mice of 8 weeks old and 10 male SPF grade db/m mice of the same genetic background were selected.After 4-weeks’ adaptive feeding,50 db/db mice were randomly divided into five groups:model group(db/db group),western medicine group(db/db+A group),Xiaokeshen formula of low(db/db+L group),middle(db/db+M group),high dose group(db/db+H group),with 10 mice in each group and 10 db/m mice as normal control group(db/m group).2.Each group was administrated by gavage 12 weeks continuously.The western medicine group was given losartan potassium tablets 45.5 mg/kg/d,the low dose group was given Xiaokeshen formula 1.06 g/kg/d,the middle dose group was given Xiaokeshen formula 2.12 g/kg/d,the high dose group was given Xiaokeshen formula 4.24 g/kg/d,the normal control group and the model group were given normal saline.3.Observe the general situation of mice in each group,and measure its random blood glucose and 24h microalbuminuria regularly.At the end of gavage,the blood and kidney samples of mice were collected to detect the related indexes.Detect the blood biochemical indexes,observe renal pathological changes under light and electron microscope,detected the expression of TGF-β1/Smad-related proteins(TGF-β1,p-Smad2,p-Smad3,Smad7)and fibrotic marker proteins(E-cadherin,α-SMA)by Western blot.4.SPSS 22.0 was used for data statistical analysis,and p<0.05 was used to represent the difference.Results1.General condition and weightXiaokeshen formula was good for the abnormal states of db/db mice in spirit,body type,hair,reaction,diet,excretion and urine.Compared with db/m mice,db/db mice are always obesity(p<0.01).The weight of db/db mice in each group fluctuated in a small range after treatment.2.Random blood glucoseIn db/m group,blood glucose fluctuated steadily at low level,which was always at high level of db/db mice.Compared with db/db group,db/db+M group decreased significantly from the 4th week(p<0.05),which decreased continuously at the 8th and 12th week(p<0.01).And db/db+L group decreased significantly from the 8th week(p<0.01).Compared with db/db+A group,db/db+M group decreased significantly at the 8th week(p<0.01),db/db+L and db/db+M group decreased significantly at the 12th week(p<0.01).However,until the end of the experiment,the blood glucose of DKD mice did not decrease to normal level.With the extension of the intervention time,the blood glucose in each group showed different trends in db/db mice.Blood glucose of all of db/db mice tended to be the same before treatment.At 0-4 weeks,there was a different degree of rise in each group.At 4-8 weeks,db/db and db/db+A group continued to increase,while all of Xiaokeshen formula groups showed a decreasing trend in varying degrees.At 8-12 weeks,all of Xiaokeshen formula groups were lower than db/db and db/db+A group,and db/db+M group was the most obvious.The random blood glucose increased continuously in db/db group and db/db+A group,which increased first and then decreased in db/db+L and db/db+M group.3.24h microalbuminuriaCompared with db/m group,24h-mALB of db/db mice in each group increased before and after treatment(p<0.05,p<0.01).Compared with db/db group,db/db+A,db/db+L and db/db+M group decreased significantly(p<0.01).db/db+L group and db/db+M group decreased more significantly than db/db+A group(p<0.05,p<0.01).4.Biochemical indicatorsThere was no significant difference in CRE,BUN and TG in each group(p>0.05).Compared with db/m group,ALT and ALB were significantly increased in db/db group(p<0.01),but there was no change in AST levels(p>0.05),and the level of TC in db/db+A and db/db+L groups increased significantly(p<0.01).Compared with db/db group and db/db+A group,the levels of AST decreased significantly in db/db+M group(p<0.01),the level of ALB in db/db+L and db/db+M groups increased significantly(p<0.01).There was no significant difference in ALT and TC in each treatment group compared with db/db group(p>0.05).5.Kidney histopathological observationLight microscopy observation:Compared with db/m group,db/db group showed disordered glomerular structure,hypertrophied capillary loops,thickened basement membrane,proliferated mesangial matrix,narrowed renal vesicles,vacuolized and degenerated epithelial cells of renal tubules,and more glycogen deposition and collagen fiber hyperplasia.Electron microscope observation:Compared with db/m group,db/db group showed thickened basement membrane with fuzzy structure,widely fused foot process,disappearance of podocyte space and fenestra,proliferated mesangial cells and the mesangial matrix.The above changes in db/db+A、db/db+L and db/db+M groups are improved in varying degrees.6.Expression of TGF-β1/Smad pathway related proteinsCompared with db/m group,TGF-β1,p-Smad2 and p-Smad3 in db/db group increased,the increasing of TGF-β1、p-Smad2 were significant(p<0.01).Compared with db/db group,TGF-β1,p-Smad2 and p-Smad3 in different doses of Xiaokeshen formula groups decreased in different degrees,among which the expression of p-Smad2 in db/db+M and db/db+H groups decreased significantly(p<0.05),which was more significant than that in db/db+A group(p<0.05).There was no significant difference in Smad7(p>0.05).7.Expression of E-cadherin and α-SMACompared with db/m group,E-cadherin decreased and α-SMA increased in db/db group(p<0.01).Compared with db/db group,E-cadherin in all Xiaokeshen formula groups increased,especially in db/db+H group(p<0.01),which was more significant than db/db+A group(p<0.05).At the same time,α-SMA in all Xiaokeshen formula groups decreased,especially in db/db+L group and db/db+H group(p<0.05,p<0.01).ConclusionThrough animal experiments,we found that Xiaokeshen formula can effectively reduce the random blood glucose and 24h microalbuminuria of DKD db/db mice in the early or middle stage under the premise of ensuring safety,improve renal pathological damage and repair renal ultrastructure.Its renal protective effect may be related to the inhibition of TGF-β1/Smad pathway mediated renal fibrosis in early stage.The effect of middle dose Xiaokeshen formula on DKD is more prominent than others.