| Objective:used spontaneous diabetic kidney disease (DKD) KKAy mice as animal model, while irbesartan as the controlled drugs. To observe the effect of Tangshenping to the pharmacodynamics and oxidative stress level of DKD KKAy mice, and observe the influence of Tangshenping to Transforming growth factor beta 1 (TGF-β1), Smad6, BMP7, a-SMA and E-cadherin protein and mRNA expression of DKD KKAy mice renal tissue by the immunohistochemistry, in situ hybridization and Western blotting method.Methods:1. Tangshenping effect on DKD KKAy mice pharmacodynamics:10 female C57BL/6J mice were used as normal control group. Sixty female 10 week old KKAy mice, KK mice feed induced by 10 weeks, after blood sugar is equal to or greater than 16.7mmol/1 24h urine protein is more than or equal to 0.4 mg to call animal model of diabetic kidney disease. Mice were randomly divided into model group, irbesartan group and Tangshenping low, medium and high dose groups according to body weight and blood glucose. Then give the corresponding drug gavage treatment. The general situation of mice was observed, and body weight and the quantity of 24h urinary protein was detected. The 26th week, Tested the serum triglyceride (TG), blood urea nitrogen (BUN), serum creatinine (Scr); the mice kidney weight to body weight ratio was calculated; renal tissue paraffin sections were stained by HE, Mallory staining to observe the pathological changes of renal tissue.2. Use colorimetric assay test the serum malondialdehyde (MDA), superoxide dismutase (SOD) and nitric oxide (NO) levels.3. Immunohistochemistry:detection of the expression of TGF-β1, Smad6, BMP7, a-SMA, E-Cadherin protein in mouse renal tissue of different groups.4. In situ hybridization:detection of the expression of TGF-β1, Smad6, BMP7,-SMA, E-Cadherin mRNA in mouse renal tissue of different groups.5. Western blotting:detection of TGF-β1, Smad6, BMP7, a-SMA, E-Cadherin protein expression in mouse renal tissue of different groups.6. The experimental data using SPSS20.0 software for statistical processing.Results:1. The study of Tangshenping in DKD KKAy mice Pharmacodynamics influence:model group compared improve Tangshenping low, medium and high dose group mice general condition and body weight were reduced to a different extent, after 16 weeks of treatment of Tangshenping, high dose group mice body weight significantly reduced (P<0.01), kidney weight body weight ratio decreased significantly (P<0.01),24h urinary protein significantly reduced (P<0.01), BUN content decreased significantly (P<0.01), the content of TG and Scr decreased (P<0.05), renal pathological change significantly reduced.2 the effect of on oxidative stress in DKD KKAy mice:Compared with the model group the content of NO and SOD in low, medium and high dose group was significantly higher (P<0.01), and the content of MDA was significantly lower (P<0.01).3. the effect of Tangshenping TGF-β1, Smad6, BMP7, a-SMA and E-cadherin protein expression of DKD KKAy mice renal tissue.Results of immunohistochemistry:normal control group renal tubular epithelial cells have a small amount of TGF-β1, a-SMA protein expression, significantly less than the model group (P<0.01); and compared to model group, the treatment groups renal tubular epithelial cell TGF-β1, a-SMA protein expression decreased obviously, there was significant difference (P<0.01); and high dose group of Tangshenping reduce the most obvious. Normal control group renal tubular epithelial cells Smad6, BMP7, E-cadherin protein strong positive expression, significantly more than the model group (P<0.01); and compared to model group, the treatment groups of renal tubular epithelial cells Smad6, BMP7 protein expression significantly increased (P<0.01). The high dose group of Tangshenping of renal tubular epithelial cells E-cadherin protein expression significantly increased (P<0.01), the low and medium dose group of Tangshenping of renal tubular epithelial cells E-cadherin protein expression increased (P<0.05).Western blotting showed that normal control group a small amount of expression in renal tissue of TGF-β1 and a-SMA, compared to the model group there were significant difference (P<0.01); and compared to model group, in Tangshenping medium and high dose group the expression of TGF-β1 protein had significantly reduced(P<0.01), in Tangshenping low dose group the expression of TGF-β1 protein had reduced(P<0.05), in Tangshenping high dose group the expression of a-SMA protein had significantly reduced(P<0.01), in Tangshenping medium dose group the expression of a-SMA protein had reduced(P<0.05). Normal control group Samd6, BMP7 and E-cadherin is highly expressed in renal tissue, significantly more than the model group (P<0.01); and compared to model group, in Tangshenping high dose group the expression of Samd6 protein had significantly increased (P<0.01), in Tangshenping medium and low dose group the expression of Samd6 protein had increased (P<0.05). In the treatment groups the expression of BMP7 protein had significantly increased (P<0.01). In Tangshenping medium and high dose group the expression of E-cadherin protein had significantly increased (P<0.01), in Tangshenping low dose group the expression of E-cadherin protein had increased (P<0.05).4. The effect of Tangshenping on TGF-β1, Smad6 and BMP7, a-SMA and E-cadherin mRNA expression of DKD KKAy mice renal tissue.In situ hybridization showed:normal control group renal tubular epithelial cells with a small amount of TGF-β1, a-SMA mRNA expression, significantly lower than in the model group (P<0.01); compared with the model group, Tangshenping high and medium dose group of renal tubular epithelial cell a-SMA mRNA expression decreased obviously, with significant statistical difference (P<0.01), Tangshenping low dose group of renal tubular epithelial cella-SMA mRNA expression decreased (P<0.05). Normal control group renal tubular epithelial cells has a lot of Smad6 and BMP7, E-cadherin mRNA expression and significantly higher than that in the model group (P<0.01); and compared to model group, the treatment groups of renal tubular epithelial cells Smad6 mRNA expression were significantly increased (P<0.01) and curative effect of Tangshenping high dose group is the best. Compared to the model group, Tangshenping high dose group, the renal tubular epithelial cell BMP7, E-cadherin mRNA expression was significantly increased (P<0.01), Tangshenping medium and low dose group, the renal tubular epithelial cell BMP7, E-cadherin mRNA expression was increased (P<0.05).Conclusion:1 Tangshenping can improve the general condition of DKD KKAy mice, reduce DKD KKAy mice body weight, kidney weight to body weight ratio,24h urinary protein and reduce DKD KKAy mice kidney pathological damage, the treatment effect of Tangshenping was dose dependent.2 Tangshenping can reduce the content of serum MDA, increased the content of serum NO and SOD, reduce oxidative stress in DKD KKAy mice, and protect renal function.3 Tangshenping can reduce the expression of TGF-β1, α-SMA protein and mRNA, increase the expression of BMP7, Smad6, E-Cadherin protein and mRNA in DKD KKAy mice, thus reversing the transdifferentiation of renal tubular epithelial cells.In summary, Tangshenping can improve the general state of DKD KKAy mice, and its renal protective and reverse epithelial-mesenchymal trans differentiation of renal tubular epithelial cells effect may be achieved through a reduction of oxidative stress and may be related to TGF-β1/Samd6/BMP7 signaling pathway. And all these results will provide new experimental basis for "Consumptive kidney disease". |
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