柔肝方通过调控Kupffer细胞Dectin-1/TLR4表达抑制HSC活化抗肝纤维化的机制研究

In the course of the occurrence and development of hepatic fibrosis (HF), Dectin-1 of the innate pattern recognition receptor, which was expressed in Kupffer cells, inhibited the activation of hepatic HSC by down-regulating the TLR4 expression via the M-CSF—CD115—CD14 pathway. Rougan recipe, which the prescription is yang yin and activating blood, is an effective treatment of HF by inhibited of the activation of hepatic HSC. Previous studies confirmed the up-expression of Dectin-1 in HF model rat liver, and Rougan recipe can inhibit the TLR4 expression in Kupffer cells of HF model rat. Therefore, the research hypothesis is proposed that the expression of Dectin-1 is up-regulated and TLR4 is down-regulate of Kupffer cells following Rougan recipe treatment，which can inhibit the activation of hepatic HSC on anti- hepatic fibrosis. In this study, to observe the effect on the expression of Dectin-1 and TLR4 in Kupffer cells and its correlation with the activation of HSC on hepatic fibrosis rats induced by CCl4 in rats. Then, through the co-culture system of primary rat Kupffer cells and HSC, to investigate the anti- hepatic fibrosis target point of Rougan recipe by inhibited the activation of hepatic HSC via Dectin-1—M-CSF—CD115—CD14—TLR4 pathway. To further clarify the partial immunological mechanism of Rougan recipe in hepatic fibrosis.

在肝纤维化（HF）发生发展的过程中，Kupffer细胞表达的先天模式识别受体Dectin-1可通过M-CSF—CD115—CD14通路下调TLR4表达，抑制肝内HSC活化。基于养阴活血治法组方的柔肝方是抑制HSC活化治疗HF的有效验方，前期研究证实HF模型大鼠肝内Dectin-1高表达，柔肝方可抑制HF模型大鼠Kupffer细胞TLR4表达。据此提出假说：柔肝方可通过上调Kupffer细胞Dectin-1、下调其TLR4的表达，抑制HSC活化发挥抗HF的作用。本研究拟：1.观察柔肝方对CCl4染毒大鼠肝内Kupffer细胞Dectin-1、TLR4表达的影响及其与HSC活化的相关性。2.应用原代大鼠Kupffer细胞和HSC共培养体系，探讨柔肝方调控Kupffer细胞Dectin-1—M-CSF—CD115—CD14—TLR4通路抑制HSC活化的靶点，进一步明确柔肝方治疗HF的免疫学机制。

在肝纤维化（HF）发生发展的过程中，Kupffer细胞表达的先天模式识别受体Dectin-1可通过M-CSF—CD115—CD14通路下调TLR4表达，抑制肝内肝星状细胞（HSC）活化。基于养阴活血治法组方的柔肝方是临床治疗肝纤维化（HF）的有效验方，本课题组前期研究发现HF模型大鼠肝内Dectin-1高表达，柔肝方能显著抑制HF模型大鼠肝内TLR4表达，抑制HSC活化，抑制胶原合成，促进胶原纤维降解，因此提出柔肝方可通过上调Kupffer细胞Dectin-1、下调其TLR4的表达，抑制HSC活化发挥抗肝纤维化作用这一假说。为验证这一假说，本实验首先建立CCl4诱导肝纤维化小鼠模型，观察柔肝方对肝组织Dectin-1、TLR4及通路中各相关因子表达的影响及其与HSC活化的相关性，结果表明柔肝方能显著改善CCl4诱导肝纤维化小鼠的肝功能；明显减轻肝脏假小叶形成和纤维化程度；明显降低肝脏羟脯氨酸含量及I型胶原蛋白水平；明显上调肝组织Dectin-1表达，明显下调TLR4表达水平，且显著下调通路中各因子包括M-CSF、CD115、CD14的表达；明显改善HSC活化；且Dectin-1与TLR4、α-SMA、TGF-β1存在显著线性相关。后应用原代大鼠Kupffer细胞和HSC共培养体系，探讨柔肝方调控Kupffer细胞Dectin-1—M-CSF—CD115—CD14—TLR4通路抑制HSC活化的靶点。结果显示Dectin-1的上调可抑制HSC的活化；Dectin-1的抗纤维化机制是通过M-CSF—CD115—CD14—TLR4通路及其下游蛋白发挥作用；柔肝方可上调Kuppfer细胞Dectin-1，通过M-CSF—CD115—CD14通路，负调控TLR4的表达，减少肝脏内HSC活化。在体及体外研究结果与国内外文献报道相吻合。本课题丰富了柔肝方抗纤维化的免疫学机制。