基于HSC的ER介导NOX/ROS氧化应激探讨化浊解毒补肾法抗绝经后女性肝纤维化机制

Postmenopausal women with estrogen receptor (ER) and reduced, antioxidant ability cause oxidative stress has led to an increased incidence of hepatic fibrosis (HF). The activation of hepatic stellate cells (HSC) is a central part of the HF. Studies show that ER has suppressed HSC activation, NOX/ROS mediated oxidative stress is the key to the HSC activation, Effectively improve the level of ER, resistance to oxidative stress is the key to the HF treatment of postmenopausal women. Li diangui, a master of traditional Chinese medicine, believes that "kidney deficiency is the basis and turbid toxin is the standard" is the basic pathogenesis of HF in postmenopausal women. Using the method of Huazhuo jiedu bushen effect is remarkable. But the mechanism is unclear. Is it possible for the Huazhuo jiedu bushen to be used to enhance the effects of the "ER" effect on NOX/ROS? In this study, plasmid transfection was used to observe the effect of Huazhuo jiedu bushen prescription on ER transcriptional activation. The gene and protein expressions of -sma, p22phox, gp91phox, p67phox, p22pho, ER, ER, NOX2 and NOX4 were detected by Real time PCR and Western Bolt; .ELISA tests the content of MDA, SOD, gsh-px, GSH,To observe the effect of the Huazhuojiedubushen prescription on NOX/ROS-mediated oxidative stress in HSC. Moreover, ER blocker was used to further reveal the mechanism of the intervention of Huazhuo-jiedu bushenfang in HSC through ER mediated NOX/ROS. To provide evidence for the prevention and treatment of HF in postmenopausal women.

绝经后女性因雌激素及受体（ER）降低，抗氧化能力下降导致肝星状细胞（HSC）活化引起肝纤维化（HF）发病率增加。NOX/ROS是氧化应激关键通路。国医大师李佃贵将“肾虚为本，浊毒为标”作为绝经后女性HF的基本病机，化浊解毒补肾法可抑制氧化应激抗HF，但该方是否通过提高ER影响NOX/ROS发挥抗HF的作用？本课题采用质粒转染技术观察该方对ER转录激活影响；免疫染色观察α-SMA，Real time PCR、Western Bolt分别检测α-SMA、p22phox、gp91phox、p67phox、p22 pho、ERα、ERβ、NOX2及NOX4基因及蛋白表达，ELISA检测MDA、SOD、GSH-Px、GSH含量，并采用ER阻断剂方式进一步揭示化浊解毒补肾方通过ER介导NOX/ROS干预HSC抗HF的机制,为其防治绝经后女性HF用药提供依据。

绝经后女性雌激素及受体数量减少，导致机体抗氧化能力下降，引起氧化应激反应为肝纤维化发生的关键。而氧化应激主要由NOX/ROS介导，且参与调控多条重要的肝纤维化相关信号通路，以NOX为特异性靶点的药物对于阻断肝纤维有重要意义，但是对于雌激素受体变化与NOX/ROS介导氧化应激之间的关系尚不清楚。国医大师李佃贵将“肾虚为本，浊毒为标”作为绝经后女性肝纤维化的基本病机，化浊解毒补肾法可抑制氧化应激抗肝纤维化。基于上述理论和科学假设，本课题采用不同浓度化浊解毒补肾方含药血清分别处理HSCs，通过MTT法检和流式细胞仪分别检测HSCs细胞增殖和凋亡，筛选出了作用于HSCs细胞的最佳给药浓度为10%，H2O2诱导HSCs建立氧化应激模型，与模型组对比，经过10%化浊解毒补肾方含药血清干预后，细胞中ROS、MDA水平和α-SMA、Collagen Ⅰ蛋白表达以及α-SMA平均荧光强度降低，而SOD、 Cytochrome C、COX-Ⅰ均升高，表明含药血清可提高抗氧化能力，抑制氧化应激从而抗肝纤维化；运用雌激素受体阻断剂干预HSCs模拟绝经后女性雌激素减少的内环境，采用RT-PCR及Western Blot检测含药血清对ER及 NOX/ROS通路的影响，发现细胞内SOD水平和Cytochrome C、COX-Ⅰ、ER及NOX等表达均显著降低，相反，ROS、MDA、α-SMA及Collagen Ⅰ表达均升高。表明化浊解毒补肾含药血清通过上调ER阻断NOX/ROS信号通路抑制H2O2诱导的HSCs细胞线粒体氧化应激，从而抑制HSCs增殖和活化，减少细胞ECM沉积，从而起到抗纤维化作用。本课题揭示了化浊解毒补肾方通过ER介导NOX/ROS发挥抗氧化应激效应，抑制肝星状细胞增殖及活化，从而发挥抗肝纤维化的机制，为绝经后女性肝纤维化患者应用化浊解毒补肾方提供理论依据。