羟基类固醇硫酸基转移酶SULT2B1在中毒性肝损伤修复中的作用和机制

The occurrence of drug/toxin-induced liver injury is getting increasing recently. The dynamic interaction between liver injury and compensatory tissue repair plays a pivotal role in determining the ultimate outcome of hepatotoxicity initiated by drugs or toxins. Our previous work found the expression of hydrosteroid sulfotransferase SULT2B1 was dramatically upregulated in the liver of hepatotoxicity model induced by CCL4、ANIT、DDC and TAA. However, the role of SULT2B1 in the tissue repair during the drug/toxin-induced liver injury remains to be elucidated. Based on our previous study, we propose that SULT2B1 may regulate the progression of tissue repair in drug/toxin-induced liver injury by participating in the following three facets: the proliferation of hepatocytes, activation of fat storing stellate cells (HSCs) and the cell proliferation and differentiation of hepatic oval cells. To clarify the proposal, we design the following experiments. Firstly, Lenti-SULT2B1-siRNA and Lenti-SULT2B1 will be delivered into C57BL/6J mice with liver injury induced by DDA or TAA via tail vein injection. The cell proliferation of hepatocytes, the activation of HSCs and the number of HOCs will be detected. Secondly, the primary mouse hepatocytes and HSCs will be co-cultured. The effect of upregulated SULT2B1 induced by DDC/TAA in the hepatocytes on the activation of HSCs will be studied. The underlying mechanisms will be further explored. Thirdly, the effect of upregulated SULT2B1 induced by DDC/TAA in the hepatocytes or the primary hepatic oval cells on the cell proliferation and differentiation of hepatic oval cells will be investigated. The underlying mechanisms will be further explored. The present project will clarify the role of SULT2B1 in the tissue repair of the drug/toxin-induced liver injury, and then provide a novel target for treating drug/toxin-induced liver injury.

药物/毒物诱导的中毒性肝损伤进程中肝组织损伤与修复并存。本课题前期发现，在多种中毒性肝损伤模型中，肝脏羟基类固醇硫酸基转移酶2B1(SULT2B1) 表达显著增高。本项目据前期工作提出：SULT2B1通过影响肝细胞增殖、星状细胞（HSCs）活化和卵圆细胞（HOCs）增殖分化在中毒性肝损伤修复中发挥重要作用。本项目拟首先，在DDC和TAA两种毒物诱导的肝损伤模型中尾静脉注射SULT2B1干扰/过表达慢病毒，研究SULT2B1对肝细胞增殖、HSCs活化、HOCs数目等的影响及机制；其次，利用小鼠肝细胞和HSCs共培养模型，研究DDC/TAA诱导肝细胞中SULT2B1增高对HSCs活化状况的影响及机制；最后，研究DDC/TAA诱导肝细胞/HOCs中SULT2B1增高对小鼠HOCs增殖分化的影响及机制。本课题将阐明SULT2B1在中毒性肝损伤修复中的作用及机制，为防治中毒性肝损伤提供新的作用靶点。

肝损伤修复过程中伴随着肝卵圆细胞的大量增殖，而调控肝卵圆细胞增殖的分子机制尚未完全阐明。羟基类固醇硫酸基转移酶2B1b（SULT2B1b）能高度选择性的硫酸化3β-羟基类固醇，如胆固醇、氧化固醇、DHEA、孕酮等。本研究发现，在DDC或者其他毒物诱导的肝损伤模型中，肝卵圆细胞大量增殖，同时SULT2B1b在肝组织中的表达显著升高，且主要表达在肝卵圆细胞中，而非肝细胞中。与WT小鼠相比，SULT2B1-/-小鼠的肝损伤程度明显减轻、肝卵圆细胞标志物的表达明显减低、SULT2B1-/-小鼠来源的肝卵圆细胞体外增殖能力明显降低。鼠尾静脉注射过表达SUT2B1b腺病毒可促进肝卵圆细胞的增殖；转染过表达SUT2B1b腺病毒可促进肝卵圆细胞系WB-F344细胞的增殖，而转染SULT2B1b-siRNA干扰SUT2B1b则抑制WB-F344细胞的增殖。以上结果证实在肝损伤过程中，过表达的SULT2B1b可促进肝卵圆细胞增殖和肝损伤进展。继而我们又进一步研究了SULT2B1b促进肝卵圆细胞增殖和肝损伤进展的机制。我们发现SULT2B1-/-小鼠肝脏LXRs降低幅度较WT小鼠小，提示SULT2B1b可抑制LXRs的激活；通过LC-MS检测DDC诱导的肝组织中22-羟化固醇、25-羟化固醇和24,25-环氧胆固醇等氧化固醇的含量，结果显示氧化固醇在SULT2B1-/-小鼠肝组织中的含量较WT小鼠显著升高，进而导致SULT2B1-/-小鼠肝脏LXRs活性增高。我们进一步证实氧化固醇不仅可以促进LXRs的激活，而且可以抑制WB-F344细胞中STAT3的磷酸化。在肝损伤时，我们在体内激活LXRs可抑制肝卵圆细胞的增殖，而转染LXRs-siRNA干扰LXRs则促进WB-F344细胞的增殖，同时提高STAT3的磷酸化水平， LXR-siRNA介导的低表达LXRs可促进WB-F344细胞增殖，而SULT2B1b可减弱LXRs对WB-F344细胞的抑增殖作用。这些结果提示SULT2B1b可通过降低LXRs活性进而激活IL-6/gp130/STAT3信号通路，从而促进肝卵圆细胞的增殖。综上所述，在DDC诱导的肝损伤过程中，上调表达的SUT2B1b通过硫酸化氧化固醇以降低LXRs的活性，继而激活IL-6/gp130/STAT3信号传导通路，促进肝卵圆细胞的增殖，卵圆细胞进而分化为大量的胆管细胞增殖且无