| Renal interstitial fibrosis is characterized by appearance of massive myofibroblats, which derived from tubular epithelial cells by an epithelial-to-mesenchymal transition, epithelial-to-mesenchymal transition has recently become one of the most intense research topics, and might be involved in the progression of chronic kidney disease (CKD). This experiment puts a perspective on the EMT, discusses the treatment mechanism of Astragalus injection combined Sulfotanshinone Sodium Injection on RIF, looks forward to providing basis for traditional Chinese medicine on RIF.Objective:To observe the effects of Astragalus injection combined Sulfotanshinone Sodium Injection on biochemical changes and morphological on rat renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). At the same time, observe the effects of the treatment on epithelial-to-mesenchymal transition related factors such as E-cadherin, a-SMA and TGF-β1 in rats. And to further explore effects of Astragalus injection combined Sulfotanshinone Sodium Injection in treatment of RIF, reveal the mechanism of Astragalus injection combined Sulfotanshinone Sodium Injection in adjusting epithelial-to-mesenchymal transition, provide a experimental basis for Astragalus injection combined Sulfotanshinone Sodium Injection in the treatment of RIF.Methods:1. in vivo:(1) Sixty male SD rats were randomly divided into sham operation group, UUO group and the treatment group. Treatment group were given Astragalus injection combined Sulfotanshinone Sodium Injection by intraperitoneal injection. These rats were sacrificed respectively on day 14 and 28 after operation. Urea, creatinine and uric acid were detected to assess renal function.(2) After the rats were sacrificed, HE and Masson staining were used to observe histological changes of the rat kidneys.(3) Immunohistochemistry was used to detect the expression of E-cadherin, a-SMA and TGF-β1 protein in kidney tissue of rat.(4) E-cadherin, a-SMA and TGF-β1 mRNA were detected in kidney tissue of rat by Real-time PCR.2. in vitro:(1) Using MTT assays to observe the influence of astragaloside and Tanshione IIA on human renal tubular epithelial cells proliferation.(2) Human renal tubular epithelial cells were divided into different groups:Control group: Normal cultured human kidney tubule epithelial cells. Model group:Cells were stimulated by recombinant TGF-β1 (8ng/ml). Treatment group:Cells were treated with recombinant TGF-β1 (8ng/ml) Plus Astragalus injection and Sulfotanshinone Sodium Injection. Cells were detected by inverted phase contrast microscope for 24h,48h,72h.(3) Detect expression of TGF-β1, E-cadherin, a-SMA,TGFP-R1 and Smad3 mRNA in human kidney tubule epithelial cells by Real-time PCR.Result:1. in vivo:(1) In comparison with that in sham operation group, the urea, creatinine and uric acid of model group were higher; The urea, creatinine and uric acid of the 28th day treatment group rats was significantly lower than model group. While there was no significant differences of the 14th day treatment group rats.(2) Renal pathology ①The macroscopic observation:Kidneys of the sham group were normal size form; Kidneys of model group got bigger, expansion of the renal pelvis, renal cortical thinning; The treatment group with different degree of ease. ②Through HE staining, we can observe the glomerular structure is complete and clear in sham group; at the same time, model group could be find renal tubular epithelial cells degeneration, necrosis, renal tubular expansion or contraction, interstitial inflammatory cell infiltration and hyperplasia of fibrous tissue change; compared with model group, the pathological injury is not seriously in treatment group. ③Masson staining:We can see obvious hyperplasia of collagen fiber in model group; Compared with model group, renal interstitial pathological changes in the treatment group was obviously improved.(3) Immunohistochemistry showed that expression of TGF-β1 and a-SMA proteins in model group kidney was significantly higher than sham operation group, but E-cadherin protein were reduced. After the treatment of Astragalus injection combined Sulfotanshinone Sodium injection, the expression of TGF-β1 and a-SMA protein in kidney were inhibited, E-cadherin was upregulated.(4) The result of Real-time PCR showed that the expression of TGF-β1, TGFβ-R1, Smad3 and a-SMA mRNA in model group was higher than sham operation group, and E-cadherin mRNA gradually reduced. Compared with model group, the expression of TGF-β1, TGFβ-R1, Smad3 and a-SMA mRNA in the treatment group gradually reduced, and E-cadherin mRNA increased.2. in vitro:(1) Compared to normal control group, different concentrations of astragaloside and Sulfotanshinone Sodium can increase the cell viability.(2) The expression of TGF-β1 and TGFβ-R1 mRNA in model group was higher than control group, and E-cadherin mRNA gradually reduced. Compared with model group, the expression of TGF-β1 and TGFβ-R1 mRNA in the treatment group gradually reduced in the time 48h and 72h hours, and E-cadherin mRNA increased in the time 72h hours. Compared with control group, the expression of Smad3 mRNA gradually reduced in the time 24h hours. There is no statistical differences between groups in α-SMA mRNA.Conclusion:(1) Astragalus injection combined Sulfotanshinone Sodium injection can ameliorate the tubulointerstitial fibrosis in UUO model and protect renal function.(2) The possible mechanism of treating RIF by Astragalus injection combined Sulfotanshinone Sodium injection:Inhibiting the expression of mRNA and Protein of smads singling mediated by TGF prevent RTF in UUO model.
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