Study On The Mechanism Of Baoshen Tongluo Prescription In Intervening Mitochondrial Dysfunction In Diabetic Kidney Diseas

ObjectiveTo explore the effect mechanism of BaoShenTongLuo formula(BSTL)on renal mitochondrial dysfunction in diabetic kidney disease(DKD)based on db/db mice on the purpose of providing scientific evidence for BSTL in DKD treatment.Methods1.Male 6-week-old db/db mice were randomly divided into 3 groups including the model group,canagliflozin group and BSTL group(n=10).Non diabetic m/m mice served as normal control group(n=10).After 2 weeks of adaptive feeding,mice in canagliflozin group were given canagliflozin by gavage with 12.86mg/kg/d;mice in BSTL group were given BSTL by gavage with 16.46g/kg/d;mice in the model group and normal control group were given normal saline by gavage with 0.1mL/g/d.They were given medicine or normal saline once a day and lasted for 12 weeks.Observed the general situation of mice,measured body weight and random blood glucose every 2weeks;detected urine ACR every 4weeks.2.After 12 weeks of administration,eyeballs were taken for blood collection,kidney weight was measured and kidney tissues were fixed or frozen.Serum Cre,BUN,TC,TG,ALT and AST were detected by reagent kits.Pathological changes of renal tissues stained with HE,Masson and PAS were observed by light microscope.Observed apoptosis by TUNEL staining.Expression of Cleaved Caspase-3,Nephrin,and PGC-1α were observed through immunohistochemistry by light microscope.Western blot was used to detect the protein expression of α-SMA,Nephrin,Podocin,COX Ⅳ,PGC-1α,NRF-1,TFAM,BCL-2,BAX,Cyt C,Cleaved Caspase-3,MFN-2 and DRP-1.qRT-PCR was used to detect the relative mtDNA copy number.Activities of mitochondrial respiratory chain complex Ⅰ,Ⅱ,Ⅲand Ⅳ were measured by mitochondrial respiratory chain complex Ⅰ,Ⅱ,Ⅲ and Ⅳ activities detection kits.Results1.Curative effect evaluation(1)Body weight and ratio of kidney weight to body weight:Compared with normal control group,the level of body weight was significantly increased(P<0.01)in the model group and was decreased by BSTL treatment after 10 weeks(P<0.05).Compared with normal control group,ratio of kidney weight to body weight was significantly decreased(P<0.01)in model group while there’s no difference between BSTL group and normal control group.(2)Random blood glucose:Compared with normal control group,the level of random blood glucose was significantly increased(P<0.01)in the model group,was increased(P<0.05)by BSTL during 2 to 4 weeks and was decreased(P<0.05)by canagliflozin during 4 to 8 weeks,but there’s no difference between each group at last.(3)Urine ACR:Compared with normal control group,the level of urine ACR was significantly increased(P<0.01)in the model group,and mounted up from 4 weeks.Level of urine ACR was mounted down and was significantly decreased(P<0.01)after BSTL treatment.(4)Serum biochemical indicators:Compared with normal control group,the level of serum TC,ALT and AST were increased(P<0.05)in the model group,and decreased after BSTL treatment but failed to reach a difference with the model group.(5)Kidney pathological observation:Compared with normal control group,enlargement of glomerular volume,glomerular basement membrane thickening,mesangial matrix proliferation,narrowed renal vesicle,hypertrophied capillary loop,vacuolar degeneration of tubular epithelial cells,more collagen fiber and glycogen deposition were observed in the model group.Changes above were reduced by BSTL treatment.Compared with normal control group,the expression intensity of a-SMA was increased(P<0.05)in the model group,and was significantly decreased(P<0.01)after BSTL treatment.(6)Kidney apoptosis:Compared with normal control group,the cell nuclear fragmentation increased in the model group by TUNEL staining,and was decreased after BSTL treatment,Compared with normal control group,the expression intensity of BCL-2 was significantly decreased(P<0.01)in the model group,and was significantly increased(P<0.01)after BSTL treatment.Compared with normal control group,the expression intensity of Cyt C was increased(P<0.05)while BAX and Cleaved Caspase-3 were significantly increased(P<0.01)in the model group.Cyt C was decreased(P<0.05),BAX and Cleaved Caspase-3 were significantly decreased(P<0.01)after BSTL treatment.(7)Podocyte:Compared with normal control group,the expression intensity of Nephrin and Podocin were significantly decreased(P<0.01)in the model group,Nephrin was significantly increased(P<0.01)and Podocin was increased(P<0.05)after BSTL treatment.2.Mitochondrial funtion(1)Mitochondrial abundance:Compared with normal control group,the expression intensity of COX Ⅳ was decreased(P<0.01)in the model group,and was increased(P<0.05)after BSTL treatment.Compared with normal control group,mtDNA/nDNA was significantly decreased(P<0.01)in model group and was significantly changed(P<0.01)by BSTL treatment.(2)Respiratory chain complex activities:Compared with normal control group,the activities of mitochondrial respiratory chain complex were decreased(P<0.05)in Ⅱ,Ⅲ and significantly decreased(P<0.01)in Ⅰ,Ⅳ.Activities of mitochondrial respiratory chain complex were increased(P<0.05)in Ⅰ,Ⅱ,Ⅲ and was significantly increased in Ⅳ(P<0.01)after BSTL treatment.(3)Mitochondrial fusion:Compared with normal control group,the expression intensity of MFN-2 was significantly decreased(P<0.01)in the model group,and was increased(P<0.05)after BSTL treatment.(4)Mitochondrial fission:Compared with normal control group,the expression intensity of DRP-1 was increased(P<0.05)in the model group,and was decreased(P<0.05)after BSTL treatment.(5)Mitochondrial biogenesis:Compared with normal control group,the expression intensity of PGC-la and TFAM were decreased(P<0.05),and the expression intensity of NRF-1 was significantly decreased(P<0.01)in the model group.The expression intensity of TFAM was increased(P<0.05),and the expression intensity of PGC-1α and NRF-1 were significantly increased(P<0.01)after BSTL treatment.ConclusionBSTL could reduce body weight,attenuate urine ACR,alleviate pathological damages,apoptosis and maintain podocytes of db/db mice.BSTL could promote mitochondrial fusion and inhibit mitochondrial fission,enrich mitochondrial abundance,promote mitochondrial biogenesis,and enhance the activities of mitochondrial respiratory chain complexes.Improvement of mitochondrial dysfunction might be related to its protective effects.