OBJECTIVE: The primary toxic elements and specific mechanisms PM causing liver damage are still not thoroughly clear. Our study aimed to investigate the influences of TSG on the immune response in idiosyncratic hepatotoxicity of PM.
METHODS: The male C57BL/6 mice were treated with different doses of TSG and the alterations in liver histology, serum liver enzyme levels, proportions of T cells and cytokines secretion were evaluated by hematoxylin and eosin (HE), RNA sequencing, quantitative real time polymerase chain reaction (qRT-PCR), Flow cytometry (FCM), and enzyme-linked immunosorbent assay (ELISA), respectively. Then, primary spleen cells from drug-naive mice were isolated and cultured with TSG in vitro. T cell subsets proliferation and cytokines secretion after treated with TSG were assessed by CCK8, FCM and ELISA. In addition, mice were pre-treated with anti-CD25 for depleting regulatory T cells (Tregs), and then administered with TSG. Liver functions and immunological alterations were analyzed to evaluate liver injury.
RESULTS: Data showed that TSG induced liver damage, and immune cells infiltration in the liver tissues. FCM results showed that TSG could activate CD4+T and CD8+T in the liver. Results further confirmed that TSG notably up-regulated the levels of inflammatory cytokines including TNF-α, IFN-γ, IL-18, perforin and granzyme B in the liver tissues. Furthermore, based on transcriptomics profiles, some immune system-related pathways including leukocyte activation involved in inflammatory response, leukocyte cell-cell adhesion, regulation of interleukin-1 beta production, mononuclear cell migration, antigen processing and presentation were altered in TSG treated mice. CD8+T/CD4+T cells were also stimulated by TSG in vitro. Interestingly, increased proportion of Tregs was observed after TSG treatment in vitro and in vivo. Foxp3 and TGF-β1 mRNA expressions were up-regulated in the liver tissues. Depletion of Tregs moderately enhanced TSG induced the secretion of inflammatory cytokines in serum.
CONCLUSIONS: Our findings showed that TSG could trigger CD4+T and CD8+T cells proliferation, promote cytokines secretion, which revealed that adaptive immune response associated with the mild liver injury cause by TSG administration. Regulatory T cells (Tregs) mainly sustain immunological tolerance, and in this study, the progression of TSG induced liver injury was limited by Tregs. The results of our investigations allow us to preliminarily understand the mechanisms of PM related idiosyncratic hepatotoxicity.
目的:PM引起肝损伤的主要毒性因素和具体机制尚不完全清楚。我们的研究目的是探讨TSG对PM特异性肝毒性免疫反应的影响。
方法:用不同剂量的TSG治疗雄性C57BL/6小鼠,观察肝脏组织学的改变,血清肝酶水平,通过苏木精和伊红(HE)评估T细胞和细胞因子分泌的比例,RNA测序,定量实时聚合酶链反应(qRT-PCR),流式细胞术(FCM),和酶联免疫吸附测定(ELISA),分别。然后,分离未用药小鼠的原代脾细胞,用TSG体外培养。通过CCK8,FCM和ELISA评估TSG处理后的T细胞亚群增殖和细胞因子分泌。此外,小鼠用抗CD25预处理以消耗调节性T细胞(Tregs),然后用TSG管理。分析肝功能和免疫学改变以评估肝损伤。
结果:数据显示TSG引起肝损伤,和免疫细胞在肝脏组织中的浸润。FCM结果显示TSG能激活肝脏中的CD4+T和CD8+T。结果进一步证实,TSG显著上调炎症细胞因子,包括TNF-α,IFN-γ,肝组织中的IL-18,穿孔素和颗粒酶B。此外,根据转录组学概况,一些免疫系统相关途径,包括参与炎症反应的白细胞激活,白细胞-细胞粘附,调节白细胞介素-1β的产生,单核细胞迁移,TSG处理小鼠的抗原加工和呈递发生了改变。TSG还在体外刺激CD8+T/CD4+T细胞。有趣的是,在体外和体内TSG处理后观察到Tregs的比例增加。Foxp3和TGF-β1mRNA在肝组织中表达上调。Tregs的消耗中度增强TSG诱导血清中炎症细胞因子的分泌。
结论:我们的研究结果表明,TSG可以触发CD4+T和CD8+T细胞增殖,促进细胞因子分泌,这表明,适应性免疫反应与TSG引起的轻度肝损伤有关。调节性T细胞(Tregs)主要维持免疫耐受,在这项研究中,TSG诱导的肝损伤进展受Tregs限制。我们的研究结果使我们能够初步了解PM相关的特异性肝毒性的机制。