桃红四物汤调控HAS-2/NOTCH3信号通路抗肝纤维化的机制研究

Anti-fibrosis treatment can inhibit the development of chronic liver disease to cirrhosis, Traditional Chinese medicine is the focus of anti-liver fibrosis research. The classic prescription Tao Hong Si Wu Decoction(THSWD) can inhibit the hepatic stellate cell activation to against liver fibrosis, but the specific mechanism is not clear. Our previous studies found that hyaluronan acid synthase 2 (HAS-2) is closely related to the development of liver fibrosis. When the HAS-2 were inhibited in the LX2, NOTCH3 expression decreased, LX2 cell activation also was inhibited; The HAS-2 and NOTCH3 expression decreased when THSWD inhibit LX2 activation. Therefore, this project is based on the TCM theory of "promoting blood circulation and removing blood stasis", combined with the classic signal pathway NOTCH3 and the preliminary study of THSWD on the expression of HAS-2 and NOTCH3, to propose the anti-fibrosis hypothesis that the THSWD regulates the HAS-2/NOTCH3 signaling pathway. In order to prove this hypothesis, we continue to expand the clinical sample volume, replication of carbon tetrachloride liver fibrosis model, combined with in vitro cell culture, from the in vivo-cell-molecule-gene multilevel to elucidate the mechanism that THSWD inhibit HAS-2 regulation of NOTCH3 to anti-liver fibrosis. It will provide a theoretical basis for THSWD treatment of liver fibrosis in the clinical.

抗肝纤维化治疗可抑制慢性肝病向肝硬化发展，中医药是目前抗肝纤维化研究的焦点。经典方药桃红四物汤可抑制肝星状细胞激活以抗肝纤维化，而具体机制尚不明确。前期研究发现透明质酸合成酶2(HAS-2)与肝纤维化密切相关。在LX2细胞中抑制HAS-2表达，NOTCH3表达降低，LX2细胞活化被抑制；且桃红四物汤抑制LX2活化时HAS-2、NOTCH3表达降低。因此，本项目基于桃红四物汤“扶正补虚，活血化瘀”的中医理论，结合经典信号通路NOTCH3及前期研究桃红四物汤对HAS-2、NOTCH3表达的初步探索，提出“桃红四物汤调控HAS-2/NOTCH3信号通路抗肝纤维化”的科学假说。为此，扩大临床样本量，复制四氯化碳肝纤维化模型，结合体外细胞培养，从体内-细胞-分子-基因多层次阐明桃红四物汤抑制HAS-2调控NOTCH3以抗肝纤维化的机制，为桃红四物汤临床防治肝纤维化提供理论依据。

桃红四物是活血化瘀的经典方剂，肝纤维化的基本病机是血淤，负责人在项目研究中发现桃红四物汤调控HAS-2/NOTCH3信号抑制HSC活化抗肝纤维化机制。通过临床肝活检石蜡切片、冻存组织及血清样本，检测HAS-2、NOTCH3蛋白和mRNA表达及血清含量；运用CCl4肝纤维化模型检测HAS-2在桃红四物汤在抗肝纤维化中的作用；体外培养LX2和分离小鼠原代HSC，通过基因敲除等等技术，从临床-小鼠-细胞-分子多平层面揭示：1）在肝纤维化进展中HAS-2与NOTCH3表达上调，两者共定位于纤维间隔，且在肝纤维化进展中相互影响；2）桃红四物汤可显著抑制CCl4诱导的肝纤维化形成。采用腺病毒过表达HAS-2显著加重CCl4诱导的肝纤维化形成。腺病毒敲减HAS-2表达，可减轻CCl4诱导的肝纤维化，而敲减HAS-2后，桃红四物汤对CCl4诱导的肝纤维化无明显治疗作用。3）HAS-2、NOTCH3在肝星状细胞激活中表达升高，HAS-2因子处理LX2，加重肝纤维化，抑制HAS-2表达后TGFβ不能诱导HSC活化，抑制NOTCH3表达后HAS-2不能诱导HSC活化；项目解析桃红四物汤通过调控HAS-2/NOTCH3信号抗肝纤维化机制，为临床应用提供理论基础。