Effects Of Serum Containing Qijishenkang Decoction On Proliferation Of Human Glomerular Mesangial Cells Via P38-p53-p21 Pathwa

Purpose:Based on the theory of collateral diseases,this experiment explored the effect of Qiji Shenkang Decoction containing serum on the proliferation of human glomerular mesangial cells induced by AngiotensinⅡthrough the p38-p53-p21 pathway in vitro.Further reveal and enrich the mechanism of Qiji Shenkang Decoction in preventing and treating IgAN and the scientific connotation of"kidney collateral disease"theory in treating IgAN.Materials and Methods:1.To prepare drug serum in legislative prescription of"tonifying,clearing and eliminating kidney collaterals":40 male Sprague-Dawley（SD）rats were randomly divided into 5 groups,each group of 8,including Normal group（normal saline）,Western medicine group（telmisartan）,Chinese medicine low-dose group（low-dose Qiji Shenkang Decoction）,Chinese medicine medium-dose group（medium-dose Qiji Shenkang Decoction）,Chinese medicine high-dose group（high-dose Qiji Shenkang Decoction）.After 3 days of adaptive feeding,the rats were gavaged Qiji Shenkang Decoction prepared by decoction room of Department of Traditional Chinese Medicine,Affiliated Hospital of Liaoning University of Traditional Chinese Medicine for 7 consecutive days.One hour after the last administration,blood was collected from abdominal aorta under sterile conditions and centrifuged.The supernatant was inactivated by water bath,filtered and sterilized,and stored at-20℃for future use.2.In vitro culture and grouping of human mesangial cells:Human mesangial cells were removed from liquid nitrogen and inoculated in 75cm²culture flask,and 10%fetal bovine serum RPMI-1640 culture medium was added into the flask,and cultured in a constant temperature 37℃,5%CO₂incubator,and the fluid was changed every two days.Angiotensinⅱwas used as a stimulating factor to induce the proliferation of human mesangial cells.The cells were grouped and treated with pharmacological serum.Respectively,The blank group,Model group(10^-7mol/LAngiotensinⅡ),Western medicine group(10^-7mol/LAngiotensinⅡ+6.25mg·kg^-1telmisartan),Chinese medicine low-dose group(10^-7mol/LAngiotensinⅡ+11.8mg·g^-1low-dose Qiji Shenkang Decoction),Chinese medicine medium-dose group(10^-7mol/LAngiotensinⅡ+23.6mg·g^-1medium-dose Qiji Shenkang Decoction),Chinese medicine high-dose group(10^-7mol/LAngiotensinⅡ+47.2mg·g^-1high-dose Qiji Shenkang Decoction).3.Detection method:MTT assay was used to detect the proliferation of mesangial cells in each group for 48h.The mrna relative expression levels of CTGF,p38MAPK,p53 and P21 in human glomerular mesangial cells induced by AngⅡwere detected by real-time quantitative PCR.The protein levels of CTGF,p38MAPK and P-p38MAPK in human mesangial cells induced by angiotensinⅱwere determined by enzyme-linked immunosorbent assay（Elisa）.Results:1.MTT method showed that compared with blank group,the proliferation of human mesangial cells in model group was the most obvious,with statistical difference（P<0.01）;Compared with model group,the inhibition effect of high-dose Chinese medicine group was the most obvious（P<0.01）.2.The results of real-time quantitative PCR showed that compared with the blank group,the expression levels of CTGF and p38MAPKm RNA in the model group were significantly increased（p<0.01）,and the expression levels of p53 and p21m RNA were significantly decreased（p<0.01）;Compared with the model group,the m RNA expression levels of the above indexes in the mesangial cells induced by AngⅡwere significantly improved in the TCM group after the intervention of drug-containing serum（P<0.01 or P<0.05）.3.Enzyme linked immunosorbent assay（ELISA）showed that compared with blank group,the protein contents of CTGF,p38MAPK and P-P38MAPK in model group were increased（P<0.01）;Compared with model group,the above protein content in TCM group was significantly decreased,and the difference was statistically significant（P<0.01）.Compared with western medicine group,the protein content of P-p38MAPK in Chinese medicine high-dose group was significantly lower than that in western medicine group（P<0.05）.Conclusion:1.The legislative prescription of"tonifying,clearing and eliminating kidney collatals"has a significant inhibitory effect on the proliferation of human glomerular mesangial cells induced by AngiotensinⅡ,and the targets may be CTGF,p38MAPK,p53 and P21 proteins.2.The legislative prescription of"tonifying,clearing and eliminating kidney collaterals"to slow down mesangial hyperplasia may be to intervene in the expression of p38-p53-p21signaling pathway related proteins,then inhibit the phosphorylation of p38MAPK,reduce the expression of CTGF,regulate p53 and P21 proteins,change the cell cycle,promote the apoptosis of proliferating cells,and finally achieve the purpose of preventing and treating IgAN.3.Demonstrated the mechanism of"tonifying,clearing and eliminating kidney collaterals"for IgAN,providing theoretical and experimental support for the theoretical treatment of"kidney collaterals disease"for IgAN.