The Research Of Amelioratory Effects Of Testosterone Propionate On Age-related Kidney Fibrosis By Inhibiting TGF-?/Smad Signaling Pathway

Objective: Renal interstitial fibrosis is considered to cause a variety of different causes of chronic kidney disease progress to end-stage irreversible common pathological pathway.Thus,how to prevent the renal fibrosis is very important.Renal fibrosis,characterized by the accumulation of extracellular matrix(ECM)proteins.Matrix metalloproteinases(MMPs)may be described as multifunctional enzymes capable of cleaving the basal membrane and extracellular matrix components.MMPs activity is regulated via a number of mechanisms,including inhibition by tissue inhibitors of metalloproteinases(TIMPs).The downregulation of MMPs or upregulation of TIMPs leads to an imbalance in the MMP/TIMP ratio,which results in ECM accumulation.With advancing age,the males experience a decrease in the circulating level of testosterone.Tumor growth factor ?(TGF-?)signaling pathway is known to play a pivotal role in the renal fibrosis.TGF-?1 has been long considered as a key mediator in renal fibrosis by activating its downstream Smad signaling pathway.To date,it is not known whether exogenous testosterone intervention to aged male rats ameliorates the age-related kidney fibrosis.To explore the effects of long-term testosterone propionate(TP)treatment on aging rat kidney morphological structure and the kidney function,and the mechanism of TGF-?/Smad signal pathway involved in kidney fibrosis.Methods: Male Wistar rats were randomly divided into three groups.6 months of age group(6Mon,n=10),24 months of age group(24Mon,n=10)and 24 months of age following treatment with TP group(24Mon-TP,n=10).24Mon-TP rats received subcutaneous injection of TP at the age of 21 months.The dosage was 2mg/kg.Once a day.The supplement of TP continued for 12 weeks(84-day).6Mon and 24 Mon rats received sesame oil at the same time.5 rats in each group were used for HE stains and Masson's trichrome.The venous blood was collected for enzyme-linked immuno sorbent assay(ELISA)determination.Another 5 rats in each group were used for real-time fluorescence quantitative PCR and Western blot analysis.Results:1 HE staining results: The 6Mon group showed regular morphology with no evidence of histopathological changes.The 24 Mon group showed prominent degenerative changes with tubular degeneration,tubular cell swelling and cellular vacuolization as well as glomerular degeneration in the renal cortical tissues.TP supplement was able to restore age-related histopathological changes.2 Masson's trichrome results: The average percentage positive stain in 24 Mon rats was significantly increased compared to 6Mon rats(P<0.01).The average percentage positive stain of 24Mon-TP rats was decreased after TP treatment than that of 24 Mon rats(P<0.01)and was not restored to the level of 6Mon rats3 ELISA results: The serum level of CysC,TIMP-1 and TIMP-2 in 24 Mon rats was significantly increased and the serum level of MMP-2 and MMP-9 was significantly decreased compared to 6Mon rats(P<0.01).The level of CysC,TIMP-1 and TIMP-2 of 24Mon-TP rats were lower and the level of MMP-2 and MMP-9 were increased after TP treatment than that of 24 Mon rats(P<0.01).The serum level of CysC,MMP-2,MMP-9,TIMP-1 and TIMP-2 of 24Mon-TP rats were not restored to the level of 6Mon rats(P<0.01).4 The effect of TP on expression of Collagen IV in the renal tissue of aged rats: Real-time quantitative PCR detection results: The expression of Collagen IV mRNA in 24 Mon rats was significantly increased compared to 6Mon rats(P<0.01).The expression of Collagen IV mRNA of 24Mon-TP rats was lower than that of 24 Mon rats(P<0.01)and was not restored to the level of 6Mon rats(P<0.05).Western blot test results: The expression of Collagen IV protein in 24 Mon rats was significantly increased compared to 6Mon rats(P<0.01).The expression of Collagen IV protein of 24Mon-TP rats was lower than that of 24 Mon rats(P<0.01)and was not restored to the level of 6Mon rats(P<0.01).5 The effect of TP on expression of MMP-2,MMP-9,TIMP-1 and TIMP-2 in the renal tissue: Real-time quantitative PCR detection results: The expression of MMP-2 mRNA and MMP-9 mRNA in 24 Mon rats was significantly decreased and the expression of TIMP-1 mRNA and TIMP-2 mRNA in 24 Mon rats was significantly increased compared to 6Mon rats(P<0.01).The expression of MMP-2 mRNA and MMP-9 mRNA of 24Mon-TP rats were increased(P<0.05)and the expression of TIMP-1 mRNA and TIMP-2 mRNA of 24Mon-TP rats lower(P<0.01)than that of 24 Mon rats.The expression of MMP-2 mRNA(P<0.01),MMP-9 mRNA(P<0.01),TIMP-1 mRNA(P<0.05)and TIMP-2 mRNA(P<0.01)of 24Mon-TP rats was not restored to the level of 6Mon rats.Western blot test results: The expression of MMP-2 and MMP-9 in 24 Mon rats was significantly decreased and the expression of TIMP-1 and TIMP-2 in 24 Mon rats was significantly increased compared to 6Mon rats(P<0.01).The expression of MMP-2 and MMP-9 of 24Mon-TP rats were increased and the expression of TIMP-1 and TIMP-2 of 24Mon-TP rats lower than that of 24 Mon rats(P<0.01).The expression of MMP-2,MMP-9,TIMP-1 and TIMP-2 of 24Mon-TP rats was not restored to the level of 6Mon rats(P<0.01)6 The effect of TP on expression of TGF-?/Smad signaling pathway in the renal tissue: Real-time quantitative PCR detection results: The expression of TGF-?1 mRNA,Smad 2 mRNA,Smad 3 mRNA and Smad 4 mRNA in 24 Mon rats was significantly increased compared to 6Mon rats(P<0.01).The expression of TGF-?1 mRNA,Smad 2 mRNA,Smad 3 mRNA and Smad 4 mRNA of 24Mon-TP rats lower than that of 24 Mon rats(P<0.01)and was not restored to the level of 6Mon rats(P<0.01).Western blot test results: The expression of TGF-?1,Smad 2/3,p-Smad 2/3 and Smad 4 in 24 Mon rats was significantly increased compared to 6Mon rats(P<0.01).The expression of TGF-?1,Smad 2/3,p-Smad 2/3 and Smad 4 of 24Mon-TP rats lower than that of 24 Mon rats(P<0.01)and was not restored to the level of 6Mon rats(P<0.01)Conclusions: Long-term TP treatment inhibited the TGF-?/Smad signaling pathway and ameliorated the renal fibrosis,increased MMP-2 and MMP-9,decreased TIMP-1 and TIMP-2.The results provided a theoretical basis for TP adjuvant treatment of age-related kidney fibrosis.