鞘氨醇激酶信号通路在NAFLD肝纤维化发病机制中的作用及与西北燥证的关系研究

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. The previous studies have confirmed that patients with northwest dryness syndrome(NDS) have genetic polymorphisms at PPARγ gene rs2921190 locus, which is a risk factor and clinical syndrome for NAFLD. Non-targeted metabolic fingerprinting analysis found that, as a specific differential metabolic pathway in NAFLD combined NDS group and pure NAFLD group, sphingosine kinase signaling pathway (SphK-S1P-S1PRs) plays a key role in liver fibrosis by regulating cell proliferation and apoptosis, but the regulatory mechanism is still unclear. In this study, a research on the difference of key protein expression level in the SphK-S1P-S1PRs signaling pathways among different groups of NAFLD will be made first. The effect of SphK1 low expression and over-expression on the proliferation and apoptosis of hepatic stellate cell will be further studied by building a cell model. Though animal model of hepatic fibrosis, an further investigation will be made on the effect of SphK1 inhibitor on hepatic SphK1 expression, autophagosome formation and the expression of autophagy-related protein. The ultimate purpose of this study is to reveal the role and mechanism of SphK1 in the process of formation of hepatic fibrosis in NAFLD by cell autophagy, and provide new ideas and new targets for the prevention and treatment of NAFLD.

非酒精性脂肪性肝病(NAFLD）是目前世界范围内慢性肝病最常见的原因。前期研究已证实西北燥证患者在PPARγ基因rs2921190位点具有基因多态性，是NAFLD危险因素及临床伴随证候。非靶向代谢指纹图谱分析发现，鞘氨醇激酶信号通路是NAFLD合并燥证组与单纯NAFLD组特异性差异代谢通路。该通路调控细胞增殖与凋亡，对肝纤维化疾病起关键作用，但具体调节机制不清。本课题拟首先研究NAFLD不同组别间鞘氨醇激酶信号通路中的关键蛋白表达水平差异。进一步通过构建细胞模型，研究SphK1稳定低表达及过表达对肝星状细胞增殖与凋亡的影响。通过纤维化动物模型，研究SphK1抑制剂对肝组织SphK1表达、自噬体形成及自噬相关蛋白表达的影响，最终揭示SphK1通过细胞自噬在NAFLD肝纤维化形成中的作用及机制，为NAFLD防治提供新思路和新靶点。

本课题通过非靶向代谢组学分析，发现NAFLD患者的脂质代谢产物与正常人有明显差异，其脂质代谢通路被严重扰动。同时，罹患西北燥证NAFLD患者的脂质代谢及其通路进一步被扰动，其中鞘氨醇激酶信号通路是NAFLD合并西北燥证者的特异性代谢通路。NAFLD合并西北燥证者脂肪肝与纤维化程度更重。在动物实验中，发现肝纤维化组大鼠肝组织中鞘氨醇激酶信号通路关键细胞因子SphK1、TRAF2、PTEN、ERK1和S1PR3 mRNA表达显著升高，并同时伴有纤维化标志物TGF-β你mRNA和蛋白表达显著升高。提示SphK1/S1P/S1PR信号轴在CCL4造模的纤维化大鼠中处于活化状态。SphK1抑制剂PF-543干预D7可显著降低肝纤维化及脂肪肝肝纤维化病变面积；以上结果表明，PF-543对自噬有显著抑制作用，并同时伴有纤维化面积的减少。提示靶向SphK1，可影响到肝脏的自噬水平，并进而缓解CCL4与CCL4联合高脂饮食两种造模方式所致肝纤维化模型的纤维化状态。在细胞实验中，进一步揭示了SphK1对肝纤维化的靶向调控作用，即SphK1高表达时伴有纤维化标志物的显著升高，而SphK1低表达时是纤维化的保护性因素。由于SphK1抑制剂PF-543在动物实验中虽表现出抗纤维化作用，但仅限于轻到中度纤维化，随着纤维化程度加重，即使延长疗程，抗纤维化治疗作用不在显现。因此，在本研究中，加入了课题组临床经验用方“和血柔肝方”，观察和血柔肝方是否可抑制鞘氨醇激酶信号通路的活性，并逆转肝纤维化。结果发现，该方可显著抑制SphK1/S1P/S1PR信号轴活性，并逆转肝纤维化。在细胞实验中，进一步验证了，活化的肝星状细胞中SphK1表达升高，并同时伴有纤维化相关标志物mRNA和蛋白表达水平升高，而抑制SphK1可逆转肝纤维化，和血柔肝方可抑制SphK1并逆转肝纤维化。