柔肝方调控IL-22—IL-22R1/IL-10R2—STAT3通路诱导肝祖细胞增殖和肝星状细胞凋亡抗肝纤维化的作用机制

Liver progenitor cells proliferation and hepatic stellate cells senescence played a critical role in promoting liver repair and inhibiting hepatic fibrosis. It was reported that Interleukin-22 (IL-22) could promote liver progenitor cells proliferation and induce hepatic stellate cells senescence by binding to the receptors IL-22R1 and IL-10R2, which is generally expressed in T helper (Th) 22 cells. We have shown that Rougan formula could promote Th22 cells to express IL-22 and accelerate hepatic stellate cells senescence, which the treatment principle is restoring deficiency and dissolving blood stasis. We raised a hypothesis that Rougan formula promote liver progenitor cells proliferation and hepatic stellate cells senescence for inhibiting hepatic fibrosis by regulating the IL-22—IL-22R1/IL-10R2—STAT3 signal pathway. We would carry out the following experiments : the effect of Rougan formula on expression of IL-22 in Th22 cells and IL-22R1/IL-10R2 on liver progenitor cells and hepatic stellate cells, with in the rats’ liver with hepatic fibrosis; liver progenitor cells and hepatic stellate cells with gene silencing and over expression of IL-22R1/IL-10R2 co-culture with Th22 cells in transwell respectively, which treated with Rougan formula, and observed the changes of expression of IL-22 and the gene expression of liver progenitor cells proliferation and hepatic stellate cells senescence；the molecule mechanism of Rougan formula promoting liver progenitor cells proliferation and hepatic stellate cells senescence by siRNA interference STAT3 signal pathway, and this program could give us a definite mechanism of Rougan formula.

肝祖细胞(LPC)增殖和肝星状细胞（HSC）凋亡是促进肝脏修复、抗肝纤维化(HF)的关键环节。文献报道：Th22分泌IL-22与IL-22R1/IL-10R2结合诱导LPC增殖和HSC凋亡。青年基金课题证实：养阴柔肝、活血化瘀的柔肝方促进Th-22分泌IL-22，诱导HSC凋亡。提出假说：柔肝方调控Th22分泌IL-22，通过IL-22R1/IL-10R2—STAT3通路，促进LPC增殖和HSC凋亡而抗HF。拟开展1) 建立肝纤维化小鼠模型，研究柔肝方调控肝脏Th22分泌IL-22、LPC增殖和HSC凋亡与其抗HF疗效的相关性；2）IL-22R1/IL-10R2基因沉默和过表达的LPC和HSC，分别与Th22共培养，验证柔肝方对IL-22分泌、LPC增殖和HSC凋亡相关分子表达的影响；3）siRNA干扰STAT3通路，探讨柔肝方诱导LPC增殖和HSC凋亡的分子靶点，明确柔肝方抗HF的机制。

肝纤维化发生发展的中心环节是肝星状细胞（HSCs）活化增殖并分化为肌样成纤维母细胞，合成过量的细胞外基质并在肝脏内沉积。辅助性T细胞22（Th22）分泌的白细胞介素22 （IL-22）与HSCs上表达的IL-22R1和IL-10R2结合通过STAT3信号通路诱导HSCs凋亡从而发挥抗肝纤维化的作用。本研究首先验证柔肝方对肝纤维化小鼠肝脏内Th22分泌IL-22及IL-22R1/IL-10R2表达、STAT3信号通路的影响；其次探讨柔肝方对Th22细胞分泌的IL-22诱导和HSCs凋亡相关分子表达的影响；再次研究柔肝方调控Jak-1—STAT3信号通路诱导HSCs凋亡的分子机制。研究发现：1）柔肝方可改善ConA联合DDC饮食肝纤维化小鼠模型肝功能ALT水平，减轻肝脏炎症浸润、纤维化增生和胶原增生；提高ConA联合DDC饮食肝纤维化小鼠模型肝脏Th22细胞表达的IL-22分泌水平和HSCs表达的IL-22RA1蛋白水平。2）混合淋巴反应表明，柔肝方可通过促进Th22细胞IL22分泌，进一步提升HSCs细胞IL-10R2及相关细胞因子P53、CXCL9、CXCL10、 CXCL11基因表达水平，降低HSCs中α-SMA和CollagenⅠ的mRNA和蛋白质水平，上调MMP9、IL-6、P53、P21和MIP2等衰老相关基因表达以抑制HSC增殖和活化。3）柔肝方干预后，HSCs细胞内STAT3信号分子随着时间变化磷酸化水平增高，HSCs相关趋化因子P53、CXCL10蛋白表达水平增高。Th22细胞内JAK信号分子随着时间变化磷酸化水平增高。本研究初步明确了柔肝方对调控Th22细胞分泌IL-22诱导HSCs凋亡及IL-22R1/IL-10R2表达和STAT3信号通路活化的影响，为进一步深入探讨柔肝方促进肝脏修复和逆转肝纤维化的作用机制提供前期基础和科学依据。