Renal Protective Effect Of Tangshenping On Diabetic Nephropathy KKAy Mice And Its Effect On Notch/snail1 Pathway

Purpose:To investigate the effect of TangShenPing on Epithelial-Myofibroblast Tr ansdifferentiation via Inhibition of Notch/Snail1 Pathway in Diabetic Nephropathy KKAy Mice.Use new method to establish DN animal model with renal tubular E MT and renal interstitial fibrosis,and to investigate the effect of TangShenPing on renal tubular EMT and renal interstitial fibrosis tubular induced by diabetic nephr opathy by in vitro and in vivo experiments。Study the mechanism of the effect of Tangshenping on inhibiting renal tubular epithelial cell transdifferentiation and ren al interstitial fibrosis in diabetic nephropathy by inhibiting the activation of Notch/snail1 signal pathway.To explore the mechanism of Tangshenping inhibiting renal tubular epithelial cells EMT and renal interstitial fibrosis in diabetic nephropathy KKAy mouse from multi-level.To provide experimental proofs for the further stud y of the efficacy and mechanism of Tangshenping.Method:1.Animal experiment1.1 Sixty female KKAy mice（8^-10 weeks of age）weighing 25-28 g were used in thecurrent experiments.Ten female C57BL/6J mice(8^-10 weeks of age)weighing 23-25 g were used as age-matched controls.All mice were raised in the animal houses of the Beijing University of Chinese Medicine;1.2 After all the mice were fed adaptively for 10 week,blood samples were collected from the tail vein to measure the blood glucose,The criteria for identification as a suitable DM animal model was random blood glucose（RBG）>16.7 mmol/L;1.3 the DM KKAy mice were allowed access to KK feed and water ad libitum for ten weeks,then 24 h urine was collected by the metabolic cage.The criteria for identification as a suitable DN animal model was 24 h urinary protein>0.4 mg.To serve as a control,the C57BL/6J mice were fed a normal diet and allowed ad libitum access to water until the end of the experiment;1.4 The DN model KKAy mice were randomLy divided into the model group,irbesartan group and Tangshenping low,medium and high dose groups,with an equivalent distribution of average body weights and blood glucose levels.Each treatment group was gavage administrated for 16 weeks,while the model group received an equivalent volume of saline for 16 weeks.The mice were housed individually in plastic cages in a clean animal cabinet with ad libitum access to food and water throughout the experimental periods.;1.5 Body weight measurements and 24h urine collection were conducted every four weeks.Blood samples for the determination of blood glucose levels were taken from the tip of the tail in the 26th week using the Roche blood glucose meter and test strips（Roche,Germany）.In the 26th week,all the mice were anaesthetised with an intraperitoneal injection of 4%hydrazine acid（0.1 mL/10 g）.Blood was retro-orbitally collected,stored for 2 h at 4℃,and centrifuged（2,500 rpm）for 20 min at 4℃;the upper serum was collected and stored at-20℃ until analysis.The mouse kidney was sterilely removed,followed by sector slitting,and the kidney tissues were separately fixed in 10%neutral formalin for HE,Mallory and PAS stain,and special 10%neutral formalin for ISH.The remaining tissues were quickly frozen in liquid nitrogen and then transferred to-80℃,in preservation for the western blotting assays.2.In vitro experiment2.1 We use high glucose stimulated mouse renal tubular epithelial cells（mRTECs）to build DN cell model;2.2 mRTECs were maintained in 1640 medium,in a humidified atmosphereatmoshoere containing 5%C02 at 37℃.After growing to approximately 80%confluence,mRTECs were cultured in serum-free DMEM for 24 h at 37℃ to arrest and synchronise cell growth.In vitro,the cells were divided into the following seven groups:normal control group,high glucose group,irbesartan serum group and Tangshenping low,medium and high dose serum groups and DAPT group.The mRTECs of the seven groups were treated for 48 h and then were used for the purposed experiments;2.3 MTT Assay.Cells were cultured in a 96-well plate at 3000 cells/well.After incubation with serum-free DMEM,mRTECs were divided into normal control group,high glucose group,irbesartan serum group,Tangshenping low,medium and high dose serum groups and DAPT group,after treated for 12,24,48,60h,cells were cultured with MTT solution at a concentrationof 0.5mg/mL for 4 h.The MTT solution was removed and DMSO was added to dissolve the purple crystals of formazan.The optical density（OD）was detected by a spectrophotometer at 492nm.Results:1.Animal experiment1.1 The effect of Tangshenping on DN KKAy mice:Compared with model group,general condition and body weight of mouse in Tangshenping low,medium and high dose group were all improved,after 16 weeks of treatment with Tangshenping,body weight of mouse in high dose group were significantly reduced（P<0.01）,kidney weight body weight ratio also decreased significantly（P<0.01）,while 24h urinary protein were significantly reduced too（P<0.01）.the levels of TG were dramatically reduced in the Tangshenping-treatment mice relative to the DN mice（P<0.01）,the levels of Cr and BUN decreased dramatically in Tangshenping high dose group（P<0.05）,Renal pathological change significantly reduced.TEM observation revealed that the normal group displayed GBMs with defined structures and normal foot processes,while the DN model KKAy mice showed irregular thickening of the GBM（P<0.01）,effacement of the foot processes,and accumulation of the mesangial and renal interstitial matrix.The severity of all of the morphological changes listed above decreased to a certain extent after treatment with Tangshenping.Compared with the model group,MDA contents of Tangshenping group significantly decreased（P<0.01）,NO、SOD contents significantly increased（P<0.01）.1.2 Effects of Tangshenping on the protein and mRNA expression of Notch/snail1 pathway,a-SMA and E-cadherin in renal tissue of DN KKAy mice:Compared with the model group,The protein and mRNA expression of jagged1,Notch1,hes1,snail1 and a-SMA in the treatment group were significantly decrease,（P<0.01）.The protein and mRNA expression of E-cadherin and were significantly higher in the Tangshenping high dose group（P<0.01）,Tangshenping high dose group had a much more better effect.2.Cell experiment2.1 Effect of Tangshenping on proliferation of high glucose induced renal tubular epithelial cells:Compared with the high glucose group,the absorbance of each treatment group was lower than that in the high glucose group at 24h（P<0.05）,Compared with the high glucose group,the proliferation of the Tangshenping high dose group was significantly lower than that of the high glucose group at 48h（P<0.01）.Compared with the high glucose group,the absorbance of each treatment group was significantly decreased at 60h（P<0.05）.2.2 Effects of Tangshenping on the expression of Notch/snaill signal transduction pathway and α-SMA,E-Cadherin in renal tubular epithelial cells induced by high glucose:Compared with the normal group,the protein and mRNA levels of Jaggedl,Notch1 Hes1,Snail1,α-SMA were significantly increased in the high glucose group（P<0.01）.The protein and mRNA expression of E-Cadherin decreased significantly（P<0.01）.Compared with the high glucose group,the protein and mRNA levels of Jagged1,Notch1,Hes1,Snail1,α-SMA were significantly decreased in Tangshenping large dose group（P<0.01）.while the expression of E-Cadherin protein and mRNA increased significantly（P<0.01）.Compared with the low dose group,the large dose group improved more significantly（P<0.05）.Conclusion:1.Tangshenping can improve the symptoms of diabetes in DN KKAy mice,reduce diabetic kidney damage,reduce proteinuria and protect renal function.2.Tangshenping can prevent diabetic nephropathy renal tubular EMT and renal interstitial fibrosis.The effect was dose dependent.3.Tangxianping can inhibit the occurrence of renal tubular epithelial cell transdifferentiation and further develop into renal interstitial fibrosis by inhibit the expression of Notch/snail1 signal transduction pathway in renal tissue of DN KKAy mice.4.Tangshenping can inhibit proliferation and apoptosis of mouse renal tubular epithelial cells induced by high glucose,and inhibit high glucose induced EMT of renal tubular epithelial cells.5.Tangshenping can inhibit high glucose-induced renal tubular epithelial cell EMT via down-regulate the expression of Notch/snail1 signal pathway in mouse renal tubular epithelial cells.