| Objective:In vivo observe the effect of Yougui pill on renal interstitial fibrosis induced by adenine and in vitro observe the effect of EMT induced by TGF-?1 to discuss the effect and mechanism of renal interstitial fibrosis between Yougui pill intervention.Methods:1.100 rats were randomly divided into 15 days and 30 days two large groups, each group 50 rats.Then,each large group divided into control,model and three different doses of Yougui pill(2.25 g·kg-1 of low,4.50 g·kg-1 of middle and 9.00 g·kg-1 of high) these five sub-groups,each group 10 rats.Every day at 8:00 am besides control group was given normal saline (NS), the other groups were fed with 150 mg·kg-1 of Adenine.Every day at 17:00 pm except control group and model group were given NS, the other groups were orally given the appropriate dosage of Yougui pill.The dose were 10mL·kg-1, the two large groups were administered continuously 15days and 30days.From time to time during the administration,rats should measured weight.Until the end of the administration,abdominal aortic blood, and separate the serum.Then,detect serum creatinine (Cr), blood urea nitrogen (BUN); malondialdehyde (MDA), superoxide dismutase (SOD); tumor necrosis factor (TNF-a), interleukin-1? (IL-1?) and interleukin-6 (IL-6).Weighing the kidneys,left kidney preserved in 4% paraformaldehyde for histopathological detection (HE staining) and collagen staining (Masson staining).Alpha smooth muscle actin (?-SMA) and E-cadherin (E-cad) detecting of immunohistochemistry (IHC).Right kidney stored at-80? prepared for test. WB is mainly detect fibronectin (FN), Collagen I (Col-I), smooth muscle actin (a-SMA), E-cadherin (E-cad),?-catenin protein (?-catenin) and transforming growth factor-?1 (TGF-?1) expression of the protein in renal tissue.Quantitative Real-time PCR is mainly detect Col-I,?-SMA, E-cadherin, TGF-?1,?p-catenin, and Wnt4 of mRNA in renal tissue.2.MTT assay different concentrations of TGF-?1 (0,1.25,2.5,5.0,10,20,40,80ng·mL-1) on the HK-2 cells cultivate for 24h to observe the proliferation of HK-2 cells.MTT assay different concentrations of Yougui pill serum on HK-2 cells cultivate for 24h to observe the inhibition of HK-2 cells.MTT assay different volume fractions serum of Yougui pill administered to lOng·mL-1 TGF-?1 effect on HK-2 cells cultivate for 24h to observe the inhibition rate and observe the cell morphology of HK-2 cells after inhibition. WB is mainly detect FN, Col-I, ?-SMA, E-cadherin, ?-catenin and TGF-?1 expression of the protein on HK2 cells. Real-time PCR is mainly detect Col-I,?-SMA, E-cadherin, TGF-?1,?-catenin, and Wnt4 of mRNA on HK2 cells.Immunofluorescence (IF) detect ?-SMA,?-catenin and E-cadherin expression of the protein on HK2 cells.Results:1. After rats intragastrically administrator Adenine 15days and 30days, compared with the control group:Adenine rats weight growth slowed down, or even reduce,kidney weight and kidney index increased significantly (P<0.01, P<0.001),Cr and BUN also significantly increased (P<0.01),Cr and BUN of serum increased more significantly in 30 days compared with 15 days.MDA significantly increased (P<0.05, P<0.01),SOD significantly decreased (P <0.01, P<0.001); TNF-?, IL-1? and IL-6 were also significantly increased like SOD(P<0.05, P<0.01, P<0.001).After oral administration of different concentrations of Yougui pill, compared with the model group:Body weight of rats in each group have increased, but no significantly different (P>0.05);Kidney weight and kidney index decreased significantly, in which middle and high dose group more significantly(P<0.05, P<0.01);Cr and BUN of serum significantly reduced, which 15days group with the high dose group has significantly difference(P<0.05),30days group of Yougui pill three dose groups were all have significant differences(P<0.001);MDA significantly decreased in the middle and high dose groups (P <0.05),SOD increased significantly middle and high dose groups (P<0.05);TNF-?, IL-1? and IL-6 significantly reduced especially with middle and high dose groups(P<0.05, P <0.01).After administration of Adenine 15days and 30days,observe the kidneys by eyes compared with the control group:Adenine rat kidneys shape and color changed significantly,the kidneys and the renal capsule easy to peel,volume increased significantly, commonly known as "white kidney".30days group changed is more significant.Pathology results shows that Adenine Rats glomerular damaged, renal tubular epithelial severe necrosis, a large number of inflammatory cell infiltration, crystal deposition within the lumen;Masson staining shows that Adenine rats' kidneys appear sever renal fibrosis,glomerular severe necrosis,and 30days group is sever than 15days group.IHC results shows that E-cadherin expression was significantly decreased in Adenine rats' kidneys and fracture increases while the expression of a very small amount of brown particulate matter; a-SMA expression was significantly increased in Adenine rats' kidneys and fracture increases while the expression of a large number of brown particulate matter.The expression of FN,Col-I,?-SMA,TGF-?1 and P-catenin protein significantly increased in Adenine rats' kidneys(P<0.01,P<0.001),and significant decrease in expression of E-cadherin protein (P<0.001);The expression of Col-1,?-SMA, TGF-?1, P-catenin and Wnt4 mRNA significantly increased in Adenine rats' kidneys (P<0.01, P<0.001),and significant decrease in expression of E-cadherin mRNA (P <0.001).After oral administration of different concentrations of Yougui pill,rats compared with Adenine group:Yougui pill rats kidneys'shape and color have improved significantly, the volume of kidneys significantly reduced;Pathology results shows that Yougui pill groups rats'kidneys glomerular morphology improved,tubular epithelial necrosis reduced, inflammatory cell infiltration and crystallization also reduced;Yougui pill different doses groups can varying degrees to improved pathological conditions, especially with high doses.Masson staining shows that Yougui pill groups can significantly improve rats renal fibrosis and reduce renal necrosis.IHC results shows that the expression of E-cadherin was significantly increased,cracks decreased and appear brown particulate matter in kidneys;While a-SMA expression was significantly reduced,fracture decreased and brown particulate matters'expression decreased in kidneys.The expression of FN, Col-I,?-SMA, TGF-?1 and P-catenin protein significantly decreased(P<0.05,P<0.01,P<0.001),the expression of E-cadherin protein increased significantly(P<0.01) in kidneys.The expression of Col-I,?-SMA,TGF-?1,?-catenin and Wnt4 mRNA significantly decreased (P<0.05,P<0.01,P<0.001),the expression of E-cadherin mRNA increased significantly (P<0.05) and the changes show dose-dependent.2. MTT results shows that the concentration at 10 ng·mL-1 of TGF-?1 can significantly promote HK-2 cell proliferation (P<0.05).Different volume fractions of Yougui pill's serum can inhibit the proliferation of HK-2 cells.At 10% serum has a significant suppression on HK2 cell and the changes show dose-dependent.In the inverted microscope TGF-?1 at 10 ng·mL-1 effect on HK-2 cells cultivate for 24h,compared with control group:The manifestations of TGF-?1 at 10 ng·mL-1 were significantly shrunken, a large number of cells added;The expression of FN,Col-I,a-SMA,TGF-?1 and ?-catenin protein significantly increased(P<0.01,P<0.001),at the same time,the expression of E-cadherin protein significantly decreased(P<0.001) in HK2 cells;The expression of Col-I,a-SMA, TGF-?1,?-catenin and Wnt4 mRNA significantly increased(P<0.01,P<0.001),and the expression of E-cadherin mRNA decreased significantly(P<0.01);Detected by IF,discover that the expression of ?-catenin and ?-SMA significantly increased and the expression of E-cadherin decreased significantly. After administration of different concentrations of Yougui pill serum, HK-2 cells shrink significantly improved, and the proliferation is suppressed also;The expression of FN,Col-I,?-SMA,TGF-?1 and ?-catenin protein significantly decrease(P<0.001),the expression of E-cadherin protein significantly increased(P<0.001);The expression of Col-I,a-SMA,TGF-?1,?-catenin and Wnt4 mRNA significantly decreased(P<0.001),and the expression of E-cadherin mRNA increased significantly(P<0.001);Detected by IF,the expression of ?-catenin and a-SMA protein significantly decreased and the expression of E-cadherin increase significantly,what's more,the changes show dose-dependent.Conclusion:1. In vivo experiments confirmed that Yougui pill improving renal function in rats induced by adenine intervention between renal interstitial fibrosis through anti-inflammatory, anti-oxidation with time-dependent manner, which may related to the regulation of Wnt/?-catenin signal pathway.2.In vitro experiments confirmed that Yougui pill improving renal interstitial fibrosis through inhibiting tubular epithelial-mesenchymal transdifferentiation between induced by TGF-?1, reducing ECM aggregation,which may related to the regulation of Wnt/?-catenin signal pathway. |
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