4,5,7-三羟基异黄酮对肝纤维化大鼠肝窦内皮细胞窗孔、增殖及合成一氧化氮的影响

目的 探讨酪氨酸蛋白激酶抑制剂4、5、7-三羟基异黄酮(genistein)对肝窦内皮细胞(sinusoidalendothelial cell, SEC)窗孔、增殖及合成一氧化氮(nitric oxide, NO)的影响。方法 用胶原酶原位灌注,Percoll不连续密度梯度离心法分离正常及CCl_4实验性肝纤维化大鼠的SEC并进行体外培养。采用扫描电镜技术,MTT法及硝酸还原酶法,分别观察genistein对SEC细胞窗孔、增殖及合成NO的影响。结果 Genistein对肝纤维化各级 SEC的窗孔数目及大小均无明显的影响。经不同浓度的genistein作用24h后,肝纤维化各级大鼠SEC的生长均受抑制,以100 μmol/L浓度的genistein对肝纤维化Ⅰ级大鼠SEC的作用最为明显[细胞增殖率为-(15.38±6.26)% vs(4.91±2.16)%,t=13.7. P＜0.05]。100 μmol/L浓度的genistein作用24h,可明显促进肝纤维化Ⅰ级大鼠SEC NO的合成[NO合成为(25.4±3.8)μmol/L vs(16.6±3.3)μmol/L,t=6.79,P＜0.05];但对肝纤维化Ⅱ级、Ⅲ级大鼠SEC的NO合成的影响却不明显。结论 体外实验中genistein可以抑制肝纤维化Ⅰ级SEC增殖,促进SEC细胞NO的合成;对肝纤维化SEC细胞的功能有一定的调节作用。

Effects of genistein on the fenestrae, proliferation and nitric oxide synthesis of liver sinusoidal endothelial cells from carbon tetrachioride-induced experimental hepatic fibrosis rats

OBJECTIVE: To investigate the effects of genistein, a tyrosine protein kinase inhibitor, on the fenestrae, proliferation and nitric oxide (NO) synthesis of the liver sinusoidal endothelial cells from CCl(4)-induced hepatic fibrosis rats in vitro. METHODS: By in situ collagenase perfusion and two-step percoll gradient centrifugation, SECs were isolated and cultured from normal and CCl(4)-treated Wistar rats. The fenestrae of SECs were observed by the scanning electron microscopy, and the SECs cell proliferation was determined by the MTT assay. The concentrations of NO in the cultured medium of SECs were detected indirectly by measurement of nitrates and nitrites (the stable products of NO) using the nitrate reduction method. RESULTS: Scanning electron microscopic studies revealed that the number of fenestrae in SECs from all stage of hepatic fibrotic rats was decreased markedly as compared with the SECs from normal controls; however, no obvious changes in the fenestrae of SECs were observed after treated with genistein (100 mumol/L) for 24 hours. After treated with 100 mumol/L genistein for 24 hours, the cell proliferation rates of SECs from all stages of hepatic fibrosis were decreased significantly was compared with the control group (P<0.05). The synthesis of NO by SECs from all stages of hepatic fibrosis was markedly lower than those of normal controls. Treatment with 100 mumol/L genistein for 24 hours could increase the synthesis of NO by SECs from the early stage (stage I) of fibrosis; however, this effect of genistein was not observed in SECs from stage II or III of fibrosis at this concentration. CONCLUSIONS: The results suggest that genistein may play an important role in regulating the function of SECs.