【作者】 王红伟； 张陆勇； 王欣之；

【机构】 中国药科大学多靶标天然药物全国重点实验室新药筛选和药效评价中心；

【摘要】 目的:雌激素是敏感的妊娠妇女、口服避孕药或绝经后激素治疗人群中诱发肝内胆汁淤积(IHC)的高危因素,其诱导的IHC有较高的发病率和潜在的危害性。IL-17与IHC炎症密切相关。胆汁酸(BA)代谢产物影响RORγt活性。靶向IL-17和RORγt可改善小鼠胆汁淤积。然而,IL-17在IHC中的来源、作用、信号通路和潜在药物靶点仍不清楚。本文首先研究i NKT17细胞在炔雌醇(EE)诱导IHC中的作用机制,论证i NKT17细胞功能调控作为IHC治疗靶点的可行性。雷公藤红素(CEL)具有抗炎保肝作用,因而进一步探究CEL对i NKT细胞亚型的调控在其改善IHC中的作用机制。方法:给以EE后检测肝脏i NKT细胞分泌的Th1/Th2/Th17型细胞因子及其表面CXCR3和CXCR6的表达;将EE和RORγt抑制剂地高辛联用,采用i NKT细胞缺陷的Jα18-/-小鼠给以EE、EE和重组IL-17联用,EE和IL-17-/-i NKT细胞过继转输,EE和野生型i NKT细胞过继转输,考察和比较IHC和肝脏炎性细胞浸润;采用CXCL10-/-小鼠给以EE,考察IHC和肝脏招募i NKT细胞比例。CEL体内给以EE和ANIT诱导的IHC,体外给以牛磺胆酸(TCA)诱导的IHC模型以评估其药效,检测i NKT细胞亚型的变化。体外构建两种(RAW264.7或AML12)共培体系,探究与i NKT细胞亚型的互作细胞。ANIT和CEL治疗的小鼠肝脏i NKT细胞经流式分选后,转录组测序、分析,并通过QPCR、WB验证。采用抑制剂进行干预,对IHC的改善,以及对i NKT细胞亚型的调控。结果:EE诱导肝脏i NKT17细胞特异性活化、扩增,形成新型i NKT17/Treg失衡。Jα18-/-小鼠和地高辛减轻IHC,下调IL-17信号通路。给Jα18-/-小鼠EE和重组IL-17,则再次诱发IHC,增加肝脏中性粒细胞和单核细胞的浸润。与回输野生型小鼠i NKT细胞相比,Jα18-/-小鼠回输IL-17-/-i NKT细胞后给以EE,减轻IHC、减少肝脏中性粒细胞和单核细胞的招募。i NKT17细胞的招募和活化归因于其表面CXCR3的高水平表达。缺乏CXCL10可改善EE诱导的IHC,减少肝脏CXCR3+i NKT细胞。CEL在体内外均对IHC具有显著的改善作用,恢复BA代谢稳态。CEL在体内外减少损伤性的i NKT17细胞,诱导保护性的i NKT1细胞。测序结果显示,CEL诱导i NKT细胞中差异基因富集在吞噬体、抗原递呈和溶酶体等。吞噬体和溶酶体参与自噬过程。半胱氨酸组织蛋白酶S(CTSS)对于抗原递呈和自噬的降解、调节至关重要。CEL在体内外下调i NKT细胞中CTSS表达,上调线粒体自噬通路。CTSS抑制剂可通过抑制i NKT细胞中CTSS,上调线粒体自噬通路,在体内外减少损伤性的i NKT17细胞,诱导保护性的i NKT1细胞,改善IHC及BA代谢稳态。结论:i NKT17细胞通过CXCR3介导的招募和激活在药物性胆汁淤积中起着关键作用,CTSS通过调控i NKT细胞亚型为IHC和CEL药理作用的潜在靶点。本文为药物性胆汁淤积的临床防治和药物开发提供支撑。更多还原

【Abstract】 Objective: Estrogen is a high-risk factor for inducing intrahepatic cholestasis(IHC) in sensitive pregnant women, oral contraceptives or postmenopausal hormone treated people. IHC has a high incidence rate and potential harm. Interleukin-17(IL-17) is closely related to inflammation in IHC. Bile acid(BA) metabolites directly bind to the key transcription factor retinoid-related orphan receptor-γt(RORγt). Targeting IL-17 and RORγt can ameliorate IHC in mice. However, the source, effect, signaling pathway, and potential drug targets of IL-17 in IHC remain unclear. In the present study, we investigated the mechanism of iNKT17 cells in ethinylestradiol(EE)-induced IHC, demonstrating the feasibility of regulating iNKT17 cell function as a therapeutic target for IHC. Meanwhile, celastrol(CEL) shows antiinflammatory and hepatoprotective effects, thus further exploring the role of CEL in regulating iNKT cell subtypes and its immune mechanism in improving IHC. Methods: After EE administration, the secretion of Th1/Th2/Th17 cytokines by liver iNKT cells and the expression of CXCR3 and CXCR6 on their surface were detected by flow cytometry. Several administration groups were established to regulation iNKT17 cell function:(1) EE combined with the RORγt inhibitor digoxin,(2) i NKT cell-deficient Jα18-/-mice were administered with EE,(3) a coadministration of EE with recombinant IL-17 to Jα18-/-mice,(4) a combination of EE and adoptive transfer of IL-17-/-or wild-type i NKT cells,(5) CXCL10-/-mice were administered with EE. After administration, IHC and hepatic inflammatory cell infiltration were investigated and compared. Moreover, CEL was administered to IHC models in vivo induced by EE and alpha-naphtylisothiocyanate(ANIT), and in vitro induced by taurocholic acid(TCA) to evaluate its efficacy and its effects on liver iNKT cell subtypes. Two co-culture systems(RAW264.7 or AML12) were constructed in vitro to explore the interaction between iNKT cells and macrophages or hepatocytes. After flow sorting, the transcriptome of liver iNKT cells from mice treated with ANIT and CEL was sequenced and analyzed, and further verified by QPCR and WB. Pharmacological inhibitors were used for intervention to evaluate its improvement for IHC and its regulation for iNKT cell subtypes. Results: EE promoted the specific activation and expansion of hepatic i NKT17 cells, which contributed to a novel hepatic iNKT17/Treg imbalance. Both iNKT cell-deficient Jα18-/-mice and digoxin alleviated EE-induced cholestatic hepatotoxicity and downregulated the IL-17 signaling pathway. The coadministration of EE with recombinant IL-17 to Jα18-/-mice induced cholestatic hepatotoxicity and increased the infiltration of hepatic neutrophils and monocytes. Compared with the adoptive transfer of wild-type iNKT cells, the adoptive transfer of IL-17-/-iNKT cells to Jα18-/-mice resulted in reduced cholestatic hepatotoxicity and decreased recruitment of neutrophils and monocytes in the liver. The recruitment and activation of iNKT17 cells could be attributed to the high expression of CXCR3 on their surface. Knockout of CXCL10 improved EE-induced cholestatic liver injury, and reduced liver CXCR3+i NKT cells. CEL exhibited significant improvement on IHC both in vitro and in vivo and restored BA metabolic homeostasis. CEL reduced damaging iNKT17 cells and induced protective iNKT1 cells. Sequencing results indicated that the differentially expressed genes in iNKT cells induced by CEL are enriched in phagosomes, antigen presentation, and lysosomes. Phagosomes and lysosomes are involved in the autophagy process. Cathepsin S(CTSS) plays a crucial role in antigen presentation and autophagy regulation. CEL downregulates CTSS expression in i NKT cells and upregulates the mitochondrial autophagy pathway both in vitro and in vivo. CTSS inhibitors can reduce damaging iNKT17 cells and induce protective iNKT1 cells by inhibiting CTSS and upregulating the mitochondrial autophagy pathway in iNKT cells, and can improve IHC and BA metabolic homeostasis. Conclusion: iNKT17 cells play a key role in EE-induced cholestatic liver injury through CXCR3-mediated recruitment and activation. CTSS serves as a potential target for the pharmacological effects of IHC and CEL by regulating iNKT cell subtypes. Our study provides support for the clinical prevention and treatment of drug-induced cholestasis, as well as drug development.更多还原