复方中药益肝康抑制肝星状细胞增殖的作用机制

Inhibition of Yigankang on proliferation of rat hepatic stellate cells induced by interleukin-1β

目的:探讨活血化瘀复方中药益肝康抑制大鼠肝星状细胞(HSC)增殖的作用机制.方法:采用IL-1β激活HSC,应用Westernblot检测JNK的活化程度;应用活细胞计数试剂盒-CCK-8检测HSC增殖并观察JNK特异性阻断剂SP600125对IL-1β促HSC增殖的影响.应用凝胶电泳移动抑制法测定AP-1的活性.结果:IL-1β有明显促大鼠HSC增殖作用,IL-1β作用培养的HSC24h后,吸光值明显高于对照组(1.573±0.026vs1.339±0.073,P=0.000);经JNK特异性阻滞剂SP600125预处理后,IL-1β促HSC增殖作用受到抑制,SP600125浓度为10,20及40μmol/L时,吸光值分别为1.427±0.113,0.772±0.093,0.675±0.074,与对照组(1.560±0.110)相比其增殖反应均明显降低(P=0.03;P=0.000;P=0.000);IL-1β可激活大鼠HSCsJNK信号蛋白,并呈现出一定的时相变化.IL-1β作用HSCs后0,5,15,30,60及120min,JNK活性分别为0.982±0.299,1.501±0.720,2.133±0.882,3.360±0.452,2.181±0.789,1.385±0.368.与0(未加IL-1β)相比,15min,30min及60min均有显著差异(P=0.002,P=0.000,P=0.001).益肝康可抑制IL-1β诱导的HSCsJNK活性(1.610±0.242vs3.360±0.452,P=0.000);益肝康可抑制IL-1β诱导的HSCsAP-1活性,经益肝康预处理HSCs1h后,AP-1活性明显受到抑制(342.43±85.77vs597.70±83.96,P=0.005).结论:IL-1β可刺激HSC增殖,细胞内JNK信号转导通路参与了IL-1β促HSC增殖作用;益肝康可通过阻滞JNK/AP-1通路,抑制HSC增殖.

AIM： To study the mechanism of Yigankang in the proliferation inhibition of rat hepatic stellate ceils （HSCs）. METHODS： The activation of JNK pathway was detected by Western blot, while the proliferation of HSCs was induced by interleukin-1β（IL-1β）. The effect of JNK inhibitor SP600125 was measured by cell counting kit-8 （CCK-8） and AP-1 activity was evaluated by electrophoretic mobility shift assay. RESULTS： Interleukin-1β up-regulated the proliferation of HSCs. After stimulation of IL-1β for 24 h, HSC proliferation increased significantly as compared with that in the controls （1.573 ± 0.026 vs 1.390 ± 0.073, P = 0.000）. After treatment with different concentrations of SP600125 （10 μmol/L, 1.427 ± 0.113; 20 μmol/L, 0.772 ± 0.093; 40 μmol/ L, 0.675 ± 0.074）, HSC proliferation induced by IL-1β decreased significantly in comparison with that in the controls （1.560 ±0.110） （P = 0.03; P = 0.000; P = 0.000）. IL-1β activated JNK pathway in a time-dependent manner in rat HSCs. After stimulation of IL-1β for 0, 5, 15, 30, 60 and 120 min, the JNK activities were 0.982 ± 0.299, 1.501 ± 0.720, 2.133 ± 0.882, 3.360 ± 0.452, 2.181 ± 0.789, 1.385 ± 0.368, respectively. In comparison with those in the cells without IL-1β, JNK activities differed significantly at 15, 30 and 60 min （P = 0.002, P = 0.000, P = 0.001）. Yigankang inhibited the activities of JNK （1.610 ± 0.242 vs 3.360 ± 0.452, P = 0.000） and AP-1 （342.43 ± 85.77 vs 597.70 ± 83.96, P 〈 0.01） obviously. CONCLUSION： IL-1β can stimulate the proliferation of rat HSCs, and JNK signaling pathway was involved in the process. Yigankang can inhibit HSC proliferation induced by IL-1β through JNK/AP-1 pathway.