柴胡-白芍对肝窦内皮细胞在刀豆蛋白A肝纤维化中的作用及机制

Liver sinusoidal endothelial cells (LSEC) are recognized as an important barrier for material exchange between hepatocytes and sinusoidal blood. As one of traditional couplet medicines, Bupleuri Radix and Paeoniae Alba couplet (Chaihu-Baishao in Chinese) has widely been applied in therapy for a variety of chronic liver diseases. Our previous investigations found that the level of plasma paeonoflorin was highest and saikosaponin(SS)d was lowest at 30 min after mice were given intragatrically with Chaihu-Baishao extract. Paeoniflorin and SSs are major ingredients of Chaihu-Baishao, respectively. Accordingly, we hypothesize that the constituent ratio of main effective ingredients of Chaihu-Baishao may play an important role in the treatment of liver fibrosis, which is closely associated with liver sinusoidal endothelial cells (LSEC), vaso-active substances and signaling transduction. The present investigations are described as follows. ① Chaihu-Baishao decoction, ethanol extract and effective fraction isolated with macroporous resins will treat liver fibrosis in mice induced by concanavalin A (ConA). ② The ratio of total glucosides of paeony to total glucosides of Bupleuri Radix, and the ratio of paeoniflorin to SSa or SSc or SSd will be analyzed in Chaihu-Baishao extracts, plasma or liver tissues in mice. ③ Primary human LSEC activated by ConA will be pretreated with Chaihu-Baishao extracts or paeoniflorin plus SSa, or paeoniflorin plus SSc or paeoniflorin plus SSd. ④ The indicators of studies include ultrastructure for LSEC; levels of transaminases, hyaluronic acid, hydroxyproline, TGF-β 1 and IL-10 in plasma; pathological changes in liver; and the expression of α-smooth muscle actin, collagen type IV, miR-10a, TGF-β1, IL-10, HIF-1α,VEGF, VEGFR, NOS, ET-1, NO, ERK1/2 and Smad2/3/4/7 in liver tissues or in LSECs. The results in this study will contribute to the effect and mechanism of Chaihu-Baisho on liver fibrosis through the regulation of LSECs.

肝窦内皮细胞(LSEC)是肝细胞与肝窦血间物质交换的屏障，柴胡-白芍药对（简称柴芍）是治疗肝纤维化相关肝病的常用药对。前研究发现用柴芍醇提物后，30分血浆芍药苷含量最高、柴胡皂苷(SS)d最低。因而推测柴芍主要活性成分构成比可能显著影响肝纤维化的防治，并与LSEC密切相关。本研究如下：①采用柴芍提取物处理刀蛋白A(ConA)肝纤维化鼠。②分析柴芍提取物、血浆或肝组织中芍药总苷/柴胡总皂苷和芍药苷/SSa/SSc/SSd比值。③柴芍提取物和芍药苷+SSa/SSc/SSd分别处理LSEC。④研究指标：肝窦内皮超微结构；转氨酶、透明质酸、TGF-β1、IL-10含量；肝组织病理；α-SMA、IV型胶原、miR-10a、TGF-β1、IL-10、HIF-1、VEGF、VEGFR、ET-1、NOS、ERK1/2及Smads表达。本项目的完成将初步阐明柴芍对小鼠ConA肝纤维化和LSEC的作用及机制。

初步结果表明，本研究将提供剂量-效应曲线（DEC）参数的标准化计算方法（有可能打破米氏方程定论和目前的半数计算法）。计算酶动力学参数的米氏曲线，被国内外广泛应用了一个多世纪。DEC广泛存在于医学各领域，本研究发现米氏曲线是DEC的一部分，故用其计算结果代表DEC的参数是不能够反映实验的真实情况。我们以SSA在HLM中的代谢为例，通过DEC模式的理论研究、比较研究和标准化计算，提供DEC参数的计算方法。第二，SSA在HLM中的体外代谢及机制研究，发现：SSA一级消除代谢动力学出现在SSA-HLM-NADPH孵育的10min内；按米氏方程计算，表观米氏常数(Km)、最大反应速度(Vmax)、半衰期(T1/2)、肝固有清除率(Clint)、肝清除率(CLh)分别为4.809μM、0.290μM/mg•min、35min、49.66mL/min•mg和14.61mL/min•kg；SSA在HLM中代谢的CYP450表型研究表明：CYP2D6和CYP2C8在SSA-HLM体外代谢中起主要作用；CYP2D6抑制剂（奎尼丁）+CYP2C8抑制剂（槲皮素）能够竞争抑制SSA在HLM中的代谢。对接结果提供了SSA/CYP2D6/CYP2C8与CYP2D6/CYP2C-相互作用的分子基础。第三，柴胡白芍抗肝纤维化作用及机制研究：①柴芍水煮液和50%乙醇提取物（柴芍）能够降低ConA小鼠肝纤维化；②降低MCP-1和IL-6释放；③抑制VEGF、VEGFR1和VEGFR2表达；④柴芍也能够降低CCL4小鼠血清ALT水平和MCP-1释放；⑤SSD经p-ERK1/2影响原代人肝窦内皮细胞释放IL-8。第四，SSD和PF与人白蛋白相(HSA)互作用研究结果，表明：SSD和+PF与HSA共结合，诱导HSA荧光淬灭和构型改变；其作用模式为静态淬灭，三元结合常数低于二元结合常数；结合力为氢键。结合位点在HSA位点I和II。