| Purpose:1.To observe the intervention effect of Fushen granules on TGF-β1-induced fibrosis model in vitro;2.To explore the possible mechanism of Fushen granules in the intervention of peritoneal fibrosis.Method:1.Production of drug-containing serum for Fushen granules: 50 SD male rats were cultured in this experiment,25 rats were given Fushen granules and 25 rats were given normal saline as blank group.After 7 days of intragastric administration,blood was taken from the abdominal aorta and the drug-containing serum was centrifuged.Human peritoneal mesothelial cells(HMr SV5)were cultured in vitro and divided into five groups:(1)Blank group: M199medium+10% blank serum;(2)Model group: M199 medium +10% blank serum+TGF-β1(10ng/ml);(3)Drug-containing serum group: M199 medium+TGF-β1+10% drug-containing serum of Fushen granule;(4)BMP7 group: M199 medium+10% blank serum +TGF-β1+BMP7(100ng/ml);(5)Gremlin group: M199 medium+TGF-β1+10% drug-containing serum of Fushen granule+Gremlin(400ng/ml).The optimum concentration of Fushen granules in serum was determined by CCK-8 cell activity detection.2.The morphological changes of peritoneal mesothelial cells after TGF-β1 intervention were observed intuitively under the microscope,and the expressions of E-cadherin and ZO-1in peritoneal mesothelial cells were detected by immunofluorescence to determine whether the modeling was successful.The protein expressions of E-cadherin,FN,Vimentin and α-SMA in peritoneal mesenchymal cells were detected by Western Blot.Immunofluorescence was used to detect the expression of EMT-related indexes FN and α-SMA in peritoneal mesenchymal cells.The m RNA expressions of E-cadherin,FN,α-SMA and Vimentin in peritoneal mesothelial cells were detected by real-time fluorescence quantitative PCR(q RT-PCR).The protein expressions of TGF-β1,BMP7,Gremlin and P-smad1/5/9 in BMP7/Gremlin pathway were detected by WB.The m RNA expressions of BMP7/Gremlin pathway related indicators TGF-β1,BMP7 and Gremlin in peritoneal mesenchymal cells were detected by PCR.Result:1.Microscopically,it was shown that after TGF-β1 was added,the cell morphology changed into a spindle shape,showing a fibrous change;Immunofluorescence results showed that compared with the blank group,the expression of E-cadherin and ZO-1 protein decreased after TGF-β1 stimulation,indicating the formation of EMT and successful modeling.2.Western Blot results: Compared with the blank group,the protein expression levels of FN,α-SMA and Vimentin in peritoneal mesothelial cells in model group were significantly increased(P<0.001);Compared with the model group,protein expression levels of FN,α-SMA and Vimentin in drug-containing serum group,BMP7 group and Gremlin group were decreased to varying degrees(P<0.05,P<0.01,P<0.001,P>0.05).Compared with the blank group,the expression level of E-cadherin protein in model group was significantly decreased(P<0.001);Compared with the model group,e-cadherin protein expression levels in drug-containing serum group,BMP7 group and Gremlin group were increased in different degrees(P<0.05,P<0.01,P<0.001).Immunofluorescence results: The expression of FN protein in the extracellular matrix of peritoneal mesothelial cells was low in the blank group,while the expression of FN protein in the model group was significantly increased after TGF-β1 stimulation.The intervention of Fushen granules could significantly reduce the expression of FN protein in the extracellular matrix of peritoneal mesenchymal cells.The expression of α-SMA protein in the peritoneal mesothelial cells was low in the blank group,while the expression of α-SMA protein in the model group was significantly increased after TGF-β1 stimulation,while the expression of α-SMA protein in the peritoneal mesothelial cells was significantly decreased by Fushen granule intervention.PCR results: Compared with the blank group,the m RNA expression levels of FN,α-SMA and Vimentin in peritoneal mesothelial cells in model group were significantly increased(P<0.001);Compared with the model group,m RNA expression levels of FN,α-SMA and Vimentin in drug-containing serum group,BMP7 group and Gremlin group were decreased to varying degrees(P<0.05,P<0.01,P<0.001,P>0.05).Compared with the blank group,the m RNA expression level of E-cadherin in peritoneal mesothelial cells in model group was significantly decreased(P<0.001);Compared with the model group,the m RNA expression level of E-cadherin in drug-containing serum group,BMP7 group and Gremlin group increased in different degrees(P<0.01,P<0.001).3.Western Blot results: Compared with the blank group,the protein expression levels of TGF-β1 and Gremlin in peritoneal mesothelial cells in model group were significantly increased(P<0.001);Compared with the model group,TGF-β1 and Gremlin protein expression levels in drug-containing serum group,BMP7 group and Gremlin group were decreased to varying degrees(P<0.05,P<0.01,P<0.001,P>0.05).Compared with the blank group,the protein expression levels of BMP7 and P-smad1/5/9 in peritoneal mesothelial cells in model group were significantly decreased(P<0.001).Compared with the model group,protein expression levels of BMP7 and P-smad1/5/9 in drug-containing serum group,BMP7 group and Gremlin group were increased to varying degrees(P<0.05,P<0.01,P<0.001,P>0.05).PCR results: Compared with blank group,m RNA expression levels of TGF-β1 and Gremlin in peritoneal mesothelial cells in model group were significantly increased(P<0.001);Compared with model group,m RNA expression levels of TGF-β1 and Gremlin in drugcontaining serum group,BMP7 group and Gremlin group were decreased to varying degrees(P<0.05,P<0.01,P<0.001,P>0.05).Compared with blank group,the m RNA expression level of BMP7 in model group was significantly decreased(P<0.001);Compared with model group,the m RNA expression levels of BMP7 in drug-containing serum group,BMP7 group and Gremlin group were increased in different degrees(P<0.05,P<0.01,P<0.001).Conclusion:Fushen granules can reduce extracellular matrix formation of peritoneal cells and inhibit TGF-β1-induced fibrosis progression of peritoneal mesothelial cells,the mechanism of which may be related to its regulation of BMP7/Gremlin signaling pathway,and then regulate EMT process. |
PDF Full Text Request