The Effect Of Tangshenping On The Autophagy Of Kidney And TEC In KKAy Mice With Diabetic Nephropathy Was Studied Based On AMPK/mTOR Signaling Pathway

With the improvement of people’s living standard,obesity and population aging,the incidence of Diabetes mellitus(DM)has been increasing year by year.According to data from the world health organization(WHO),the number of DM patients in the world is expected to double from 2000 to 380 million by 2030.The number of DM in China also shows a rapid growth trend.In 2007,the number of DM in China was about 39.8 million.In 2010,the number of DM patients exceeded 92.4 million,and China became the first major country in the world.By 2025,the number of DM in China will reach 59.3 million[1～3].Serious micro vascular complication of Diabetic Nephropathy,diabetes incidence is 6.5%～42%,the Diabetic Nephropathy(Diabetic Nephropathy,DN)cause of end-stage renal disease is rising year by year,Diabetic Nephropathy is one of the Europe and the United States and other developed countries the Primary cause of end-stage renal disease and end-stage renal disease in China’s second largest etiology,and might replace Primary Glomerulonephritis(Primary Glomerulonephritis,PGN)and become the first cause of end-stage renal disease.Renal interstitial fibrosis is the final process of DN,and its main features are renal tubular atrophy and renal interstitial fibrosis.Renal interstitial fibrosis is the common pathway and major pathological changes of various chronic renal disease progression to end-stage renal failure[4]And now,in the study of renal interstitial fibrosis,autophagy abnormality may be one of the most important mechanisms for the occurrence of renal interstitial fibrosis[5～10].The effective repair of the intrinsic cells of diabetic nephropathy is the key to determining the disease.Autophagy is a cellular self-protection mechanism,autophagy in cellular energy shortage situation,digestion of protein and lipid to provide energy for cells,and can remove aging or abnormal protein folding,remove damaged organelles.Autophagy is the key to self-repair of all cells under stress.Hyperglycemia and glycosylation end products have caused autophagy damage in renal cells,especially in renal tubular epithelial cells,which has received extensive attention in recent years.In the physiological state,there is a basal level of autophagy in the tubular epithelial cells of the renal tubular epithelial cells.The main purpose is to remove the long-lived protein and senile damaged organelles.Studies have identified mTOR signaling pathway is one of the main inhibit autophagy pathway,in the development of the occurrence of autophagy and play an important role,and mTOR upstream AMPK pathway protein phosphorylation increase inhibits mTOR,increasing the autophagy.AMP activated protein kinase(AMP activated protein kinase,AMPK)is an important protein kinase organism,was a key enzyme of the coordination of substance and energy metabolism,it can be triggered by phosphorylation Ulkl autophagy genes that promote autophagy occurs[11].Mammaliantarget of rapamycin(mTOR)is a serine/threonine kinase that regulates cell growth,proliferation and energy metabolism,and inhibits autophagy.Intracellular mTOR is mainly composed of mTOR Complex 1(mTOR Complex 1,mTORC1)and mTOR Complex 2(mTOR Complex 2,mTORC2),and mTORC1 is sensitive to rapamycin and is considered to be a non-dependent autophagic regulator.In the case of rich nutrition,mTORCl direct phosphorylation and inhibit autophagy involved,including Atg13,ULK1 Atg protein,making them a combination of ability is abate,thus inhibiting autophagy compound ULK1 Atg13-Atg17 formation,inhibit autophagy[12].In the case of starvation,mTORC1 has no activity and is isolated from the ULK complex to induce autophagy[13]AMPK/mTOR pathway,as an energy regulator,plays an important role not only in regulating the lipid metabolism of the liver,fat and skeletal muscle,but also in regulating the level of autophagy in the kidney.In DN state,hyperglycemia and glycosylation end products can activate AMPK/mTOR signal transduction pathway,inhibit autophagy and aggravate the DN process.Tutor professor zong-jiang zhao put forward the theory of "kidney impotent",DN may increase heat,depression,phlegm dampness,blood stasis and toxin factor mutual cementation in kidney,blocking blood running,the structure of the kidney damage,"kidney" damaged,the structure of the kidney damage will eventually lead to normal kidney function is damaged or lost,namely"kidney","body" and "kidney" damage,kidney impotent without waste,finally"impotent".This research adopts the DN KKAy mice and renal tubular epithelial cells in mice as the research object,using immunofluorescence,iinmunohistochemical and in situ hybridization,Western blot,rt-pcr and Real Time PCR method,from the angles of animal experiment and in vitro cell experiment to explore the protection effect of TangshenPing on DN kidney and AMPK/mTOR signaling pathways critical molecular expression,the influence of provides the new experiment basis for the theory of "kidney impotent".Objective:To observe the TangshenPing against oxidative stress and renal protection DN KKAy mice and the effect of using immunohistochemical and in situ hybridization technique in sugar renal LC3 in kidney tissues,P62,P-AMPK,AMPK,P-mTOR,the influence of the mTOR protein and mRNA expression;In vitro cell experiments were performed using immunofluorescence technology,Western blot and Real Time PCR to observe the effects of glucose renping on LC3,P62,p-AMPK,AMPK,P-mTOR,mTOR protein and mRNA expression in renal tubular epithelial cells.Methods:1.The pharmacodynamics effect of sugar and kidney in the control of DN KKAy mice.There were 60 KKAy mice aged 8～10 weeks and 10 C57BL/6J mice.KKAy mice were fed high-fat and high-protein feed and C57BL/6J mice were fed normal feed.KKAy mice were fed with KK mice and C57BL/6J mice were fed normal feed.Blood glucose was greater than 16.7mmol/L 24h,urine protein was more than 0.4mg,and it was successful in inducing diabetic nephropathy animal model.The successful KKAy mice were randomly divided into model group,erbesartan group,and the small,medium and large dose group,with gastric administration,according to blood glucose and weight.The general condition of the experimental mice was recorded,the body mass was weighed,the urine volume was collected for 24 hours,and the urine protein was measured.All the mice in each group collected Blood in 26 weeks,and measured Blood glucose,Triglyeride(TG),creatinine(Scr),and Blood urea nitrogen(BUN).The kidney weight was calculated and the weight ratio was calculated.The mouse kidney paraffin was embedded and sectioned,and HE,Mallory and PAS staining were used to analyze the pathological changes of the mouse kidney.2.Colorimetry:detection of serum Malondialdehyde(MDA),Nitric oxide(NO)and Superoxide dismutase(SOD)content in mice.3.Immunofluorescence:the expression level of LC3 and P62 protein in mouse renal tubular epithelial cells was detected.4.Immunohistochemistry:the expression levels of LC3,P62,P-AMPK,AMPK,P-mTORand mTOR protein were detected in mouse kidney tissues.5.In situ hybridization:to detect the expression level of LC3 and P62 mRNA in mouse kidney tissues.6.Western blot:the expression levels of LC3,P62,P-AMPK,AMPK,P-mTOR and mTOR protein were detected in mouse renal tubular epithelial cells cultured in vitro.8.Real Time PCR:the expression level of LC3,P62,P-AMPK,AMPK,P-mTORand mTOR mRNA in mouse renal tubular epithelial cells cultured in vitro.9.All experimental data were statistically analyzed with SPSS 22.0.Results:1.Effects of glycolidine on pharmacodynamics of DN KKAy mice.Compared with model group,sugar renal small,medium,large dose group mice general condition improved,body quality is different extent reduce degree is reduced,16 weeks after treatment,TangshenPing body quality significantly reduce large dose group mice(P<0.01),renal mass and body quality ratio significantly decreased(P<0.01),24 h urine protein quantitative decreased significantly(P<0.01),serum BUN content significantly decreased(P<0.01),TG,Scr were decreased(P<0.05),kidney pathological changes significantly reduce.2.The effect of sugar kidney on oxidative stress in DN KKAy mice.The MDA level in the model group was significantly higher than that in the control group(P<0.01),and the level of NO and SOD in the model group was significantly lower than that in the normal control group(P<0.01).Compared with the model group,the content of NO and SOD in the serum of the small,medium and large dose group was significantly increased(P<0.01),while the MDA content decreased significantly(P<0.01).3.The effect of sugar kidney ping on the expression of LC3,P62,P-AMPK,AMPK,P-mTOR and mTOR protein in renal tissues of DN KKAy mice.Immunohistochemistry:(1)LC3 protein:the expression quantity of LC3 protein in the cytoplasm of the normal group of renal tubular epithelial cells was higher,which was significantly higher than that in the model group(P<0.01).Compared with the model group,the expression of LC3 protein in renal tubular epithelial cells(P<0.01)was significantly increased in the treatment group,except in the small dose group of the sugar kidney.The increase in the dose and the large dose group was the most obvious in the treatment group.(2)P62 protein:a small number of P62 protein expressions in the renal tubular epithelial cells of the normal control group were significantly lower than that in the model group(P<0.01).Compared with the model group,the expression of P62 protein in renal tubule epithelial cells was significantly decreased in the treatment group(P<0.05).(3)AMPK,P-AMPK protein and P-AMPK/AMPK:there was no significant difference in the expression of AMPK protein(P>0.05).The expression of P-AMPK protein in normal renal tubular epithelial cells was higher than that in the model group(P<0.01).Compared with the model group,the expression of P-AMPK protein in the renal tubular epithelial cells was increased(P<0.01),and the dose and large dosage groups in the treatment group were significantly increased.P-AMPK/AMPK:compared with the control group,P-AMPK/AMPK decreased in the model group(P<0.01).Compared with the model group,P-AMPK/AMPK(P<0.01)in the renal tubular epithelial cells of the treatment group was increased(P<0.01),and the dose and high-dose group were significantly increased in the treatment group.(4)mTOR,p-mTOR protein and p-mTOR/mTOR:there was no significant difference in expression of mTOR protein groups(P>0.05).There was a small amount of p-mTOR protein expression in the 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,the expression of P62 protein in renal tubule epithelial cells was significantly decreased in the treatment group(P<0.05).P-mTOR/mTOR:compared with normal control group,P-mTOR/mTOR increased(P<0.01).Compared with the model group,the ratio of each treatment group decreased(P<0.05).The large dosage group was the most obvious.4.The effect of sugar and kidney on the expression of LC3 mRNA in kidney tissues of DN KKAy mice.In situ hybridization:compared with the normal control group,LC3 mRNA expression in the model group decreased(P<0.01).In comparison with the model group,the expression of LC3 mRNA in renal tubular epithelial cells in the irbexan group,the sugar kidney level and the high-dose group was increased(P<0.01).Compared with erbesartan group,LC3 mRNA expression was reduced(P<0.01).The expression of LC3 mRNA in the small dose group was reduced(P<0.01)compared with the large dose group.5.Immunofluorescence assay was used to detect the expression of LC3 and P62 in renal tubular epithelial cells in mice with high glucose.Immunofluorescence:the expression of LC3 protein in the normal group of renal tubular epithelial cells was significantly higher than that in the high-sugar group(P<0.01).Compared with the high-sugar group,the expression of LC3 protein was increased in both the dose and the large dose(P<0.05).Compared with the normal group,the expression of P62 protein in the high-sugar group was increased(P<0.01).Compared with the high sugar group,the expression of P62 protein in the group,rapamycin group and irbesartan group decreased(P<0.01).6.The effect of glycolidine on the expression of LC3,P62,P-AMPK,AMPK,p-mTOR,and mTOR protein in the renal tubular epithelial cells.Western blot.(1)LC3 protein:the high expression of LC3 in the normal renal tubular epithelial cells was significantly higher than that in the high-sugar group(P<0.01).The expression of LC3 protein in renal tubular epithelial cells was significantly increased(P<0.05),compared with the high-sugar group,the large dose group,the rapamycin group,and the irbexan group.(2)P62 protein:compared with the normal group,P62 protein expression in the renal tubular epithelial cells of the high-sugar group was increased(P<0.01).Compared with the high-sugar group,the other treatment group and rapamycin group showed less expression(P<0.01),with the highest dose and the large dose group.(3)AMPK,P-AMPK and P-AMPKk/AMPK:the expression of AMPK protein showed no significant difference(P>0.05).Compared with the normal group,P-AMPK decreased in the high-sugar group(P<0.01).Compared with the high-sugar group,the expression of P-AMPK protein was increased in the group and the high-dose group,the rapamycin group and the urbersartan group(P<0.05).Compared with the normal group,the ratio of P-AMPK/AMPK protein expression in the high-sugar group decreased(P<0.01).The ratio of P-AMPK/AMPK protein expression in the dose group and the high-dose group was increased(P<0.05)compared with the high-sugar group.(2)mTOR,p-mTOR and p-mTOR/mTOR:the expression of mTOR protein was not significantly different(P>0.05)..Compared with the normal group,the expression of p-mTOR protein in the high-sugar group increased(P<0.01).Compared with the high-sugar group,the expression of the other treatment group and rapamycin group decreased(P<0.01)in addition to the small dose group,and the reduction was most obvious in the treatment group.Compared with the normal group,the expression of p-mTOR/mTOR in the high-sugar group was increased(P<0.01).Compared with the high-sugar group,the expression of p-mTOR/mTOR protein was decreased in the group of the sugar kidney,the high dose group,the high dose group,the rapamycin group,and the irbesartan group(P<0.01).6.Glycolidine was used to induce renal tubular epithelial cells LC3,P62 and AMP in vitro.