Effect Of CagA On The Rat Glomerular Mesangial Cells(RGMC)

bjective:To observe the effect of Cytotoxin-associated protein(cagA)on the proliferation,apoptosisnd and the extracellular matrix secreted from Rat glomerular mesangial cells,and to investigate the relations of Helicobacter pylori infection and IgA nephropathy,to provide new ideas for IgA nephropathy pathogenesis and prevention research.Methods:1.Rat glomerular mesangial cells induced by different concentrations of cagA(Oug/ml、1ug/ml、2ug/ml、4ug/ml)cultured for 24h,48h,72h in vitro.CCK-8 assay for analyzing the effect of cagA on the proliferation of Rat glomerular mesangial cells,Enzyme-linked immunoadsordent assay(ELISA)was used to detect supernatant of cell culture including collagen type I and collagen type Ⅲ exploring the proliferation and extracellular matrix effects on RGMC cell dose,time-dependent..2.According to cagA optimum concentration and time,design was randomly divided into 3 groups,blank control group,IL-1(lOng/ml)group and cagA(4ug/ml)group,given the appropriate treatment cells cultured for 72 hours,then Rat glomerular mesangial cell’s proliferation was detected by CCK-8,the secretions of collagen type Ⅰ and collagen type Ⅲwere measured by ELISA,PCNA was detected by Immunohisto-chemistry,the expression of collagen type Ⅰ、collagen type Ⅲ、BAX and BCL-2 mRNA were detected by PCR.western-blot was used to detect the expression of collagen type I‘collagen type Ⅲ、BAX、BCL-2 protein levels.Results:1.CCK-8 Results show that RGMC proliferation was increased significantly with the concentration of cagA(0ug/ml、1ug/ml、2ug/ml、4ug/ml),and with the duration of action,RGMC proliferation was also significantly increased.The supernatant show that cagA secrete collagen type Ⅰ and collagen type Ⅲ increased with the concentration of cagA,cagA secrete collagen type Ⅰ and collagen type Ⅲ also increased with the time extension stimulating by cagA,in a time-and dose-response relationship;.2.According to 4μg/ml and 72h as the cagA optimal concentration and stimulation time,the results showed,collagen typeⅠ and collagen typeⅢ in IL-1(lOng/ml)group and cagA(4μg/ml)were significantly increased compared with control group(P<0.05);3.Immunohistochemistry showed that cells cultured 72h,IL-1(10ng/ml)group and cagA(4μg/ml)group compared with the control group,proliferating Cell Nuclear Antigen were significantly increased(P<0.05);.4 PCR and Western blot showed,IL-1(10ng/ml)group and cagA(4μg/ml)cultured cells after 72h,the mRNA and protein levels of Ⅰ,Ⅲ collagen and BCL-2 than in the control group were significantly increased(P<0.05),whereas the expression of BAX mRNA and protein was decreased compared with the control group,the difference was statistically significance(P<0.05).Conclusion:1.CagA could induce RGMC proliferation and increase the secretion of extracellular matrix,by a time and dose dependent manner.2.CagA induce RGMC proliferation through promoting the expression of proliferating cell nuclear antigen.3.cagA’s mechanisms induced mesangial cell proliferation and extracellular matrix have to be with increased inhibition of apoptosis while promoting Ⅰ,Ⅲ collagen expression.