Inhibitory Effects And Mechanism Of Xinmaitong On Proliferation Of Cardiac Fibroblasts And The Expression Of Collagen MRNA

Objective:So many researches reported that myocardial fibrosis can occur in many cardiovascular diseases these years.Great effects can be made on the ventricular remodeling and the prognosis of cardiovascular disease by myocardial fibrosis.Xinmaitong is a compound recipe of traditional Chinese drug to prevent and cure ischemic heart diseases.Main functions of it include enhancing blood circulation,removing stasis and relieving pain.Clinical practices and experimental studies have shown that Xinmaitong can prevent injury of myocardial ischemia.DAng-â… ,a new biological agent,has the biological effect which is the reverse of angiotoninâ…¡.To study inhibitory effects and mechanism of Xinmaitong and DAng-â… on the myocardial fibrosis,we evaluated inhibitory effects of Xinmaitong and DAng-â… on prolific fibroblasts induced by NE, observed the change of TGF-β₁ in the culture medium,collagen and collagenâ… â…¢mRNA expression in the cells and discussed the function of drugs.Methods:1.Prolific effects on CFb of NE in vitro:Preparation of CFb of neonate rats was done by the enzyme digestion and adherence of unequal velocity.Cells were identified by immunocytochemistry staining.CFb from passages 2-4 were used in general.Cells were seeded into 96 well culture plates at a cell concentration of 7×10⁴/ml,and cultured in 37,5%CO℃₂,and 95%moist condition.Cells were washed after 24h, serum-deprived media added,and then washed again after the next 24h.Cells were treated with NE(including10⁴,10³,10²,10,0 nM final concentration groups),and cultured for 48h subsequently.Cell proliferation was detected by MTT.2.Inhibitory effects of Xinmaitong on CFb's proliferation and over expression of collagen mRNA of NE-induced in vitro:Early passage cells were grown to confluence in 6,24 and 96 well culture plates containing 10%NBSC.Prior to treatment,culture media was replaced with serum-deprived media for 24h. Cells were divided into 4 groups:control groupâ‘ :cells+(10%non-medicated serum+DMEM); model groupâ‘¡:cells+(10%non-medicated serum+10⁴nMNE+DMEM);â‘¢Xinmaitong group: (cells+10%Xinmaitong-medicated serum+10⁴nMNE+DMEM);â‘£DAng-â… group: (cells+5×10³ nM DAng-â… +10⁴nMNE+DMEM+10%non-medicated serum).Following 48h of treatment,culture media in the 24 well culture plates was collected and TGF-β₁ levels determined by ELISA subsequently.Cells in the 6 well culture plates were immediately used for RNA extraction.The proliferation of cell was detected of MTT in the 96 well culture plates. Results:1.Prolific effects on CFb of NE in vitro:NE can induce the proliferation of CFb.The Result of MTT showed that values of OD of each group treated with 10⁴,10³,10²,10å’Œ0nM NE were 0.33±0.02,0.30±0.01,0.28±0.03,0.27±0.02å’Œ0.27±0.01,respectively.Values of OD showed that dose-effect relationship was exist in the concentration of NE from 10 nM to 10⁴nM(r_s=0.9,P＜0.05).2.Inhibitory effects of Xinmaitong on the proliferation of CFb and the over expression of collagen mRNA induced by NE in vitro:The result of MTT showed that NE induced CFb to profile significantly(P＜0.05). Xinmaitong and DAng-â… inhibited the proliferation of CFb induced by NE,compared to the NE group(P＜0.05).The studies of molecular biology showed that Xinmaitong and DAng-â… reduced the over expression of collagen mRNA induced by NE,compared to the NE group(P＜0.05). There was no significant difference in control group,Xinmaitong group and DAng-â… group(P＞0.05).3.Effects of Xinmaitong on levels of TGF-β₁The result of ELISA showed that levels of TGF-β₁ of these drug groups were not up-regulated or down-regulated significantly(P＞0.05).Conclusion:1.NE can induce proliferation of CFb in vitro.2.Xinmaitong and DAng-â… can inhibit the proliferation of CFb and down-regulate the over expression of collagen typeâ… and collagen typeâ…¢mRNA of CFb induced by NE in vitro. Levels of TGF-β₁ of these drug groups in the culture medium were not down-regulated significantly.