Tao Yan-yan, Yan Xiu-chuan, Zhou Tao, Shen Li, Liu Zu-long, Liu Cheng-hai
Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Pudong New Area, Shanghai 201203, China.
BMC Complement Altern Med. 2014 Nov 18;14:449. doi: 10.1186/1472-6882-14-449.
What was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and HSC activation at different stage of liver fibrosis? In order to answer this question, the study was carried out to dynamically observe FZHY's effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis.
Mice were randomly divided into four groups: normal, model, FZHY, and N-acetylcystein (NAC) groups. Acute hepatic injury and liver fibrosis in mice were induced by CCl4. Three days before the first CCl4 injection, treatment with FZHY powder or NAC respectively was started. In vitro, primary hepatocytes were pretreated with FZHY medicated serum or Z-VAD-FMK and then incubated with ActD and TNF-α. Primary HSCs were treated with DNA from apoptotic hepatocytes incubated by Act D/TNF-α or FZHY medicated. Liver sections were analyzed for HE staining and immunohistochemical evaluation of apoptosis. Serum ALT and AST, Alb content and TNF-α expression in liver tissue were detected. Hyp content was assayed and collagen deposition was visualized. Expressions of α-SMA and type I collagen were analyzed by immunofluorescence and immunoblotting. Flow cytometry, immunofluorescence, and DNA ladder for hepatocyte apoptosis and immunoblotting for TNF-R1, Bcl-2 and Bax were also analyzed.
Mice showed characteristic features of massive hepatocytes apoptosis in early stage of liver injury and developed severe hepatic fibrosis in later phase. FZHY treatment significantly alleviated acute liver injury and hepatocyte apoptosis, and inhibited liver fibrosis by decreasing α-SMA expression and hepatic Hyp content. In vitro, primary hepatocytes were induced by TNF-α and Act D. The anti-apoptotic effect of FZHY was generated by reducing TNFR1 expression and balancing the expressions of Bcl-2 and Bax. Meanwhile, the nuclear DNA from apoptotic hepatocytes stimulated HSC activation in a dose dependent manner, and the DNA from apoptotic hepatocytes treated with FZHY or Z-VAD-FMK reduced HSC activation and type I collagen expression.
These findings suggested that FZHY suppressed hepatocyte apoptosis through regulating mediators in death receptor and mitochondrial pathways, and the effect of FZHY on hepatocyte apoptosis might play an important role in inhibiting liver fibrosis.
扶正化瘀方（FZHY）在肝纤维化不同阶段抑制肝细胞凋亡和肝星状细胞（HSC）激活的关系是怎样的？为回答这个问题，本研究动态观察了FZHY对肝细胞凋亡和HSC激活的影响，并进一步探究了FZHY抗肝细胞凋亡的潜在机制。
将小鼠随机分为四组：正常组、模型组、FZHY组和N-乙酰半胱氨酸（NAC）组。采用四氯化碳（CCl4）诱导小鼠急性肝损伤和肝纤维化。在首次注射CCl4前三天，分别开始用FZHY粉剂或NAC进行治疗。体外实验中，原代肝细胞先用FZHY含药血清或Z-VAD-FMK预处理，然后与放线菌素D（ActD）和肿瘤坏死因子-α（TNF-α）共同孵育。原代HSCs用经ActD/TNF-α孵育的凋亡肝细胞的DNA或FZHY含药处理。对肝组织切片进行苏木精-伊红（HE）染色及凋亡的免疫组织化学评估。检测血清谷丙转氨酶（ALT）和谷草转氨酶（AST）、白蛋白（Alb）含量以及肝组织中TNF-α的表达。测定羟脯氨酸（Hyp）含量并观察胶原沉积情况。通过免疫荧光和免疫印迹分析α-平滑肌肌动蛋白（α-SMA）和I型胶原的表达。还通过流式细胞术、免疫荧光和DNA梯状条带分析肝细胞凋亡情况，并用免疫印迹分析TNF受体1（TNF-R1）、Bcl-2和Bax。
小鼠在肝损伤早期呈现大量肝细胞凋亡的特征性表现，后期发展为严重肝纤维化。FZHY治疗显著减轻急性肝损伤和肝细胞凋亡，并通过降低α-SMA表达和肝脏Hyp含量抑制肝纤维化。体外实验中，原代肝细胞由TNF-α和ActD诱导。FZHY的抗凋亡作用是通过降低TNFR1表达以及平衡Bcl-2和Bax的表达产生的。同时，凋亡肝细胞的核DNA以剂量依赖方式刺激HSC激活，而经FZHY或Z-VAD-FMK处理的凋亡肝细胞的DNA可降低HSC激活和I型胶原表达。
这些研究结果表明，FZHY通过调节死亡受体和线粒体途径中的介质来抑制肝细胞凋亡，且FZHY对肝细胞凋亡的作用可能在抑制肝纤维化中发挥重要作用。
