基于T淋巴细胞表达的纤维化蛋白/微粒双向调节HSC功能研究柔肝冲剂抗肝纤维化作用机制

Activation of hepatic stellate cell(HSC) and more collagen synthesis than degradation is the core link of hepatic fibrosis(HF). The fibrosin(FBRS) expressed by T lymphocytes can promote the activation of HSC and synthesis of collagen, and microparticle(MP)derived from T lymphocytes can inhibit the activation of HSC and promote the expression of MMPs and collagen degradation. Our previous studies showed that Rougan granule play some roles in anti-hepatic fibrosis through inhibiting the synthesis of collogen and promoting its degradation. The contents of this research are as follows:① ConA induced murine model for hepatic fibrosis is used in this study,and the effects of Rougan granule on the expressions of FBRS and MP by T lymphocytes in the liver and the activation of HSC are examined, and the correlation between FBRS and MP expressions and the activation of HSC is analyzed;② the mechanism of HSC activation and collagen synthesis regulated by FBRS expressed by T cell line ;③the mechanism of HSC activation and collagen degradation regulated by MP derived from T cell line;④ the effects of Rougan granule on the expressions of FBRS and MP by T cell line and the relevant regulation mechanism of HSC activation . This research will confirm that the mechanism of Rougan granule on anti-hepatic fibrosis is through inhibiting the FBRS expression and promoting the MP expression and the dual-directional regulational roles on HSC actviation, and inhibiting the collagen synthesis and promoting degradation. This study will in further elucidate the modern biological mechanimsm of Chinese herbs on anti-hepatic fibrosis.

肝星状细胞（HSC）活化及胶原合成大于降解是肝纤维化（HF）核心环节。T淋巴细胞表达的纤维化蛋白（FBRS）促进HSC活化及其胶原合成，T淋巴细胞表达的微粒（MP）抑制HSC活化、促进基质金属蛋白酶（MMP）表达降解胶原。前期研究证实"柔肝冲剂"通过抑制胶原合成促进降解抗HF。本研究拟开展：①建立ConA肝纤维化小鼠模型，观察柔肝冲剂对肝脏内T淋巴细胞FBRS、MP表达、HSC活化的影响，并分析FBRS、MP与HSC活化的相关性；②T淋巴细胞株表达的FBRS对HSC活化及胶原合成的调节机制；③T淋巴细胞株表达的MP对HSC活化及胶原降解的调节机制；④柔肝冲剂对T淋巴细胞株分泌FBRS和MP的影响，从而调节HSC功能的机制。以期证实柔肝冲剂可通过抑制T淋巴细胞FBRS表达、促进MP表达，双向调节HSC活化，从而抑制胶原合成、促进胶原降解抗HF的作用机制，进一步明确中药抗HF的现代生物学机制。

肝星状细胞（HSC）活化及胶原合成大于降解是肝纤维化核心环节。据文献报道，T淋巴细胞表达的纤维化蛋白促进HSC活化及其胶原合成，T淋巴细胞表达的微粒抑制HSC活化、促进基质金属蛋白酶（MMP）表达降解胶原。我们前期研究证实“柔肝冲剂”通过抑制胶原合成促进降解抗肝纤维化。本研究主要进行如下工作：①建立ConA肝纤维化小鼠模型，观察柔肝冲剂对肝脏内FBRS、MMPs表达、HSC活化的影响；②FBRS对HSC活化及胶原合成的调节机制；③ConA对T淋巴细胞株表达的MP影响及其对HSC活化的调节作用；④柔肝冲剂药物血清对HSC活化的调节作用及相关机制。结果发现，1.与ConA慢型肝纤维化模型组小鼠相比，柔肝冲剂组小鼠肝纤维化的程度明显降低，肝脏内FBRS、a-SMA、TGFβ1、Col I、MMP3、TIMPs表达明显下调；2. 体外研究发现，ConA能促进Jurkat T细胞表达表达FBRS，且能上调HSC细胞系LX2的FBRS表达；3.进一步研究发现FBRS能够促进LX2活化，具有时间和剂量依赖性，该活化过程与P38、ERK和PI3K/AKT通路活化有关，应用FBRSshRNA干扰 LX2 的FBRS表达以后，LX2 的a-SMA表达明显下调；4.柔肝方含药血清对FBRS诱导的LX-2细胞表达a-SMA、TGFβ1、ColI具有抑制作用；5.柔肝方含药血清能够抑制FBRS对LX-2细胞P38信号的活化作用。此外，研究发现，ConA能促进Jurkat T 细胞表达MP，但MP的表达量很低，对HSC无明显的调节作用。 通过上述研究，我们阐明柔肝冲剂抗肝纤维化的主要机制是通过降低FBRS的表达、抑制HSC的活化、减少胶原沉积，在明确了中药抗肝纤维化的现代生物学机制的基础上，为抗纤维化提供了一个新的研究靶点。