Study On The Protective Effect Of Tangshenping On The Kidneys Of KKAy Mice With Diabetic Nephropathy And Its Influence On The Wnt/β-catcnin Pathway

With the improvement of people’s living standards and the aging population,the incidence of diabetes(Diabetes mellitus,DM)has increased year by year.It has become one of the most important chronic diseases that threaten global human health and is rising worldwide.Trends,the International Diabetes Federation’s 2017 data show that 425 million adults worldwide have diabetes,and 212 million have not been diagnosed,and are expected to reach 629 million in 2045.Diabetic nephropathy(Diabetic Nephropathy,DN)is one of the most common and most severe microvascular diseases in many chronic complications of diabetes,and is also the main cause of end stage Renal Disease(ESRD).Clinically,proteinuria and progressive deterioration of renal function are the main features.The main pathological changes are mainly glomerular filtration rate,glomerular hypertrophy,mesangial cells and stromal hyperplasia,glomerular basement membrane.Thickening,glomerular nodule sclerosis,renal interstitial cell infiltration,partial tubular atrophy,etc.,until renal failure.Its high incidence rate and rapid progress make diabetes nephropathy a serious chronic disease that threatens human health.In the study of renal interstitial fibrosis,one of the most important mechanisms of renal interstitial fibrosis is epithelial-mesenchymal transition.Therefore,studying the transdifferentiation of renal tubular epithelial cells has a more important significance in the pathogenesis of diabetic nephropathy,and the drug for improving the transdifferentiation of renal tubular epithelial cells has become a focus and innovation for the treatment of diabetic nephropathy.Professor Zhao Zongjiang put forward the theory of "kidney spasm".When the condition of DN is delayed,the heat,stagnation,phlegm,blood stasis and poisonous evils are glued to the kidneys,blocking the blood and running,causing the structure of the kidney to be destroyed and the "kidney body" to be damaged.The structural destruction of the kidney will inevitably lead to the impaired or even loss of normal function of the kidney,that is,the "kidney" is abnormal,the "kidney body" and the "kidney" are both damaged,and the kidneys are not used up,and eventually the "kidney spasm".In this study,DN KKAy mice and mouse renal tubular epithelial cells were used as subjects.Immunohistochemistry,in situ hybridization,Western blotting and RT-PCR were used to investigate the effects of Tangshenping on DN kidneys from both in vivo and in vitro perspectives.The protective role and the expression of key molecules in the Wnt/β-catenin signaling pathway provide a new experimental basis for the "kidney spasm" theory.Objective:In the whole animal experiment,KKAy mice with spontaneous diabetic nephropathy were used to observe the effects of Tangshenping on the pharmacodynamics and oxidative stress in DNKKAy mice.HE and Mallory staining were used to observe the pathological morphology of renal tissues.In situ hybridization and immunization groups were used.The protein and mRNA expressions of Wnt4,Gsk-3β and β-catenin in renal tissues were detected.In vitro cell experiments were performed by Western blot and RT-PCR to detect the effects of Tangshenping on the expression of Wnt4,Gsk-3β and P-catenin in renal tubular epithelial cells.Methods:1.The efficacy of Tangshenping in the prevention and treatment of DN KKAy miceFemale 10-week-old SPF-class KKAy mice were induced by KK rats for 10weeks.The DN animal model was successfully determined with blood glucose>16.7mmol/L and 24-hour urine protein>0.4mg.The model mice were randomly divided into a model group,an irbesartan group,a small,medium and high dose group of sugar kidney,and 10 female C57BL/6J mice as a control group.The treatment groups were given the corresponding therapeutic drugs by intragastric administration.The normal group and the model group were given an equal volume of deionized water for intragastric administration.The intragastric dose was 0.1mL/10g body weight coefficient once a day.The general condition of the mice was recorded,and the body mass was weighed every4 weeks and24 h urine protein was quantified.At the 26th week of the experiment,the eyeballs were bled and the mice were sacrificed,and the kidney mass was measured.The kidney weight/body weight ratio was calculated after recording;the serum was measured for blood glucose,triglyceride(Triglyeride,TG),creatinine(Serum creatinine,Scr)and urea nitrogen(Blood urea,nitrogen,BUN).The mouse kidney was embedded in paraffin and sectioned,and HE and Mallory staining were performed to analyze the effect of Tangshenping on the pathological morphology of the mouse kidney.2.Colorimetric method:The serum levels of malondialdehyde(MDA),nitric oxide(NO)and superoxide dismutase(SOD)were measured.3.Immunohistochemistry:The expression levels of Wnt4,Gsk-3β and β-catenin proteins in mouse kidney tissues were detected.4.In situ hybridization:The expression levels of Wnt4,Gsk-3β and β-catenin in mouse kidney tissues were detected.Western blot:The expression levels of Wnt4,Gsk-3β and β-catenin in mouse renal tubular epithelial cells were detected.6.RT-PCR:The expression levels of Wnt4,Gsk-3β and β-catenin in mouse renal tubular epithelial cells cultured in vitro were detected.7.All experimental data were statistically analyzed using SPSS 20.0,Results:1.Effect of Tangshenping on pharmacodynamics of DN KKAy miceCompared with the model group,the mice in the small,medium and large doses of Tangshen Ping were generally improved in state and their body weights were alleviated.After 16 weeks of administration,the body weight of the mice in the middle and high dose groups was significantly reduced(P<0.01),the ratio of kidney mass to body weight decreased significantly(P<0.01),24h urine protein quantitation decreased significantly(P<0.01),serum BUN content decreased significantly(P<0.01),TG and Scr content decreased(P<0.05).),renal pathological changes were significantly reduced.2.Effect of Tangshenping on oxidative stress in DN KKAy miceCompared with the model group,the MDA content in the serum of mice with small,medium and large doses of Tangshen was significantly decreased(P<0.01),and the contents of NO and SOD were significantly increased(P<0.01).3.Effect of Tangshen Ping on the expression of Wnt4,Gsk-3β and β-catenin in renal tissues of DN KKAy miceImmunohistochemistry:(1)wnt4 protein:There was a small amount of Wnt4 protein expression in the cytoplasm of renal tubular epithelial cells in the normal group,which was significantly lower than that in the model group(P<0.01).Compared with the model group,wnt4 and protein expression in the irbesartan group and the glycoside group.The decrease was significant except for the low-dose group(P<0.01).The difference of the large dose of Tangshenping was the most significant in the treatment group(P<0.01).(2)Gsk-3β protein:There was a small amount of Gsk-3β protein expression in the cytoplasm of renal tubular epithelial cells in the normal control group,which was significantly lower than that in the model group(P<0.01).Compared with the model group,Gsk-3β protein in renal tubular epithelial cells in each treatment group The expression was significantly reduced,except for the low-dose group(P<0.01).(3)β-catenin protein:the normal group of renal tubular epithelial cell membrane and cytoplasmic β-catenin has a very small expression,significantly lower than the model group(P<0.01);compared with the model group,except for the small dose of Tangshen Ping,each The expression of β-catenin protein in renal tubular epithelial cells was decreased in the treatment group,and it was statistically significant(P<0.01).4.Effect of Tangshiping on the expression of Wnt4,Gsk-3β and β-catenin mRNA in kidney of DN KKAy miceIn situ hybridization:Wnt4 mRNA:Compared with the normal control group,Wnt4 mRNA expression was more in the model group(P<0.01).Compared with the model group,the expression of Wnt4 mRNA in the renal tubular epithelial cells of irbesartan group,Tangshenping medium dose group and high dose group was less,and the reduction of glycosaminoglycan group was the most significant(P<0.01).Gsk-3β mRNA:The expression of Gsk-3β mRNA in renal tubular epithelial cells in the normal control group was less than that in the model group(P<0.01).Compared with the normal group,a large number of Gsk-were found in the renal tubular epithelial cells in the model group.The expression of 3β mRNA was significantly different(P<0.01).Compared with the model group,the expression of Gsk-3β mRNA was decreased in each treatment group,and the decrease in the high dose of Tangshenping group was the most significant(P<0.01)..--catenin mRNA:There was a trace amount of β-catenin mRNA in the cytoplasm of renal tubular epithelial cells in the normal group,which was significantly lower than that in the model group(P<0.01).Compared with the model group,the renal group in each treatment group except the small dose of Tangshenping The expression of β-catenin mRNA in tubular epithelial cells was significantly increased(P<0.01).The expression ofβ-catenin mRNA in renal tubular epithelial cells was significantly increased in each group of Glucagon(P<0.01).5.Effect of Tangshenping on the expression of Wnt4,Gsk-3β and β-catenin in renal tubular epithelial cells induced by high glucose in vitroWestern blotting:(1)Wnt4 protein:The expression of Wnt4 protein in renal tubular epithelial cells in normal group was less,which was significantly lower than that in high glucose group(P<0.01).Compared with high glucose group,middle and high dose group of glucosinolate,renal tubule in irbesartan group The expression of Wnt4 protein in epithelial cells was significantly attenuated,and there was a significant difference in the high-dose group of Tangshenping(P<0.01).(2)Gsk-3β protein:Gsk-3β protein in the normal group of renal tubular epithelial cells is only slightly expressed.Compared with the normal group,the expression of Gsk-3β protein in the high glucose group was significantly increased(P<0.01).Compared with the model group,except for the small dose of Tangshenping group,the expression of Gsk-3β protein in the other treatment groups was less(P<0.01).The most significant doses were in the medium dose and high dose groups.(3)β-catenin protein:β-catenin protein is weakly expressed in the normal group of renal tubular epithelial cells.Compared with the normal group,the expression ofβ-catenin protein in the high glucose group was significantly increased(P<0.01).Compared with the model group,the expression of β-catenin protein in the irbesartan group was decreased in the middle dose group and the high dose group(P<0.05).There was a significant difference in the large dose group of Tangshenping(P<0.01).6.Effects of Tangshenping on the expression of Wnt4,Gsk-3β and β-catenin mRNA in renal tubular epithelial cells induced by high glucose in vitroRT-time PCR:(1)Wnt4 mRNA:There was a slight expression of Wnt4 mRNA in renal tubular epithelial cells in normal group.Compared with normal group,Wnt4 mRNA in high glucose group was significantly higher than that in normal group(P<0.01).Compared with high glucose group,medium and large dose of glucosinolate The expression of Wnt4 mRNA in renal tubular epithelial cells was significantly lower in the irbesartan group(P<0.05),and was most obvious in the medium dose and high dose groups.(2)Gsk-3β mRNA:Gsk-3β mRNA was expressed in a small amount in the normal group.Compared with the normal group,the expression of Gsk-3β mRNA in the renal tubular epithelial cells of the high glucose group was significantly increased(P<0.01).Compared with the high-sugar group,except for the low-dose group of Tangshenping,the other treatment groups showed less expression(P<0.01),and the decrease was the most significant in the high-dose group of Glucosamine(P<0.01).(3)β-catenin mRNA:β-catenin mRNA is weakly expressed in the renal tubular epithelial cells of the normal group.Compared with the normal group,β-catenin mRNA was significantly increased in the high glucose group(P<0.01).Compared with the high-sugar group,the expression of each drug intervention group was decreased,and the comparison between the two groups was the most significant in the high-dose group of Glycopya(P<0.01).Conclusion:1.Tangshenping can improve the general state of DN KKAy mice,reduce the symptoms of diabetes,reduce body weight,kidney weight to body weight ratio,significantly reduce 24h urine protein quantitation,blood glucose and serum BUN,Scr,TG indicators,effective Protecting renal function,its efficacy is dose dependent;2.Tangshenping can improve the oxidative stress state of DN KKAy mice by decreasing the serum MDA content of DN KKAy mice,increasing the content of NO and SOD;3.Tangshenping can reverse the Wnt/β-catenin signal transduction pathway in DN KKAy mice,down-regulate the expression of Wnt4,Gsk-3β,β-catenin protein and mRNA,thereby reversing the renal tubular epithelial cell turnover in DN KKAy mice.Differentiate and delay the progression of renal interstitial fibrosis;4.Tangshenping can inhibit the expression of Wnt4,Gsk-3β,β-catenin protein and mRNA by interfering with Wnt/β-catenin signal transduction pathway in renal tubular epithelial cells,inhibiting high glucose-induced renal tubular epithelial cellsTransdifferentiation.Tangshenping can improve the general state of DN KKAy mice,reduce the level of oxidative stress,reverse the transdifferentiation of renal tubular epithelial cells in DN KKAy mice,and delay renal interstitial fibrosis.Its mechanism may be related to the regulation of the Wnt/β-catenin signaling pathway.This provides a basis for the DN"kidney spasm"doctrine.