The Effect Of Quercetin On Tumor Necrosis Factor-α Induced-rat Cardiac Fibroblasts Proliferation And Expression Of P38MAPK And HMGB1

Objective: Through the proliferation of cardiac fibroblasts model induced by TNF-α, to explore the effect of quercetin on the proliferation and expression of P38 MAPK and HMGB1. Furthermore, to explore the mechanism of quercetin inhibiting myocardial fibrosis and utilization of ventricular reversing remodeling. Methods: 1. The primary cardiac fibroblasts were cultured and identificated, using trypsin digestion method and differential adherence method to obtain more purified cardiac fibroblasts, using optical microscopy and immunofluorescence method for identifying cells. 2. Detection Method, the influence of quercetin on cardiac fibroblast proliferation was determined by MTT assay. The expression of P38 MAPK and HMGB1 was detected by western blot. 3. Experimental groups, the experiment according to different drugs and different drug concentrations were divided into three part. The cardiac fibroblasts during third or fourth generation of cells was used in the experiment. Part I, to research the impact of quercetin on cultured rat cardiac fibroblasts proliferation in basic state, there were four groups, blank control group and quercetin groups(25μmol/L, 50μmol/L, 75μmol/L, 100μmol/L). Part II, there were nine groups to explore with different concentration of TNF-α and reactive time the changes of cardiac fibroblast proliferation, respectively control group and different concentration of TNF-α groups(5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml, 160ng/ml, 320ng/ml, 640ng/ml), to find the best concentration. Then with the optimal concentration intervent in different time 0h, 4h, 8h, 16 h, 32 h. Part III, in order to research the impact of quercetin on tumor necrosis factor-a induced-rat cardiac fibroblast proliferation and expression of P38 MAPK and HMGB1. In this part, there were six groups, blank control group, TNF-a group, different concentration of quercetin groups and SB203580 group. Results: 1. The primary cultured cells through optical microscopy and immunoflurescence was identified as cardiac fibroblasts, the purity above 98%. 2. Quercetin has no obvious influence on normal growth state of cardiac fibroblasts proliferation in 24 hours. Compared with blank control group, the cardiac fibroblasts cells A490 values has no statistical difference(P > 0.05). 3. Quercetin intervent TNF-α-induced cardiac fibroblasts in 24 hours, compared with control group, the A490 values of different concentration groups(5ng/ml,10ng/ml,20ng/ml,40ng/ml,80ng/ml) were significantly higher, the difference was statistical significance(P < 0.05). Compared with 0h group, the A490 values of different time groups(4h, 8h) were higher, the difference was statistical significance(P < 0.05). 4. The A490 values of different concentration of quercetin groups were significantly lower than TNF-α group(P < 0.05). The A490 values decreased with increasing concentrations of quercetin. There showed statistically significant differences between different concentration groups(P < 0.05).5. Compered with TNF-a group, the expression of P38 MAPK and HMGB1 was obviously higher than various concentrations of quercetin groups and SB203580 group(P < 0.05). Different concentration of quercetin groups(25μmol/L, 50μmol/L, 100μmol/L), the expression of P38 MAPK and HMGB1 protein was gradually decreased with the concentration of quercetin increased, there were statistical difference between groups(P < 0.05). SB203580 group expressed the P38 MAPK and HMGB1 protein significantly weaker than TNF-α group. Conclusion: 1. TNF-α can be successsfully established cardiac fibroblasts proliferation model in a rang of concentration and time. 2. Quercetin has no obvious influence on the basic cardiac fibroblasts proliferation in 24 hours, but inhibite TNF-α-induced cardiac fibroblast proliferation in a concentration-dependent manner. It showed that quercetin has a certain inhibitory effect on cardiac fibroblast cells under stress. 3. TNF-a stimulate the proliferation of cardiac fibroblasts, but also increase the expression of P38 MAPK and HMGB1 protein. The expression of P38 MAPK and HMGB1 protein was decreased with the concentration of quercetin increased. SB203580 group expressed the P38 MAPK and HMGB1 protein significantly weaker than TNF-α group. It is suggested that quercetin may be through P38 MAPK pathway involved in the release of HMGB1 secretion and inhibit in the proliferation of cardiac fibroblasts.