背景:T细胞浸润和分化在狼疮性肾炎(LN)的发展中起重要作用。我们之前的研究表明,蛋白质,虫草(WCP)的主要活性成分,中药,具有可以增强肾脏纤维化并提供肾脏保护的特性。尽管如此,LN中WCP与T细胞浸润和分化之间的联系仍然知之甚少。
目的:本研究的目的是评估WCP对LN小鼠的免疫调节作用,并通过体内和体外研究阐明其潜在机制。
方法:为了研究WCP对MRL/lpr狼疮易感小鼠的影响和机制,WCP(1.5g/kg/d),百令胶囊(BC,0.75g/kg/d),和等量的盐水在8周的时间内给予小鼠。治疗效果,经由过程ELISA验证WCP对MRL/lpr小鼠的T细胞浸润和分化,苏木精-伊红(H&E),高碘酸希夫(PAS)染色,免疫荧光,Luminex分析和流式细胞术。使用小鼠组织研究了WCP减轻LN的机制,T细胞和小鼠足细胞克隆-5(MPC-5)细胞的转录组学,Westernblot(WB),和实时定量聚合酶链反应(RT-qPCR)。
结果:我们发现WCP通过减少尿蛋白改善MRL/lpr小鼠的LN,肌酐,和血清自身抗体,增加补体3(C3)水平,改善肾脏免疫病理和下调血清细胞因子,包括IFN-γ,IL-12和RANTES。值得注意的是,WCP减少了肾脏中CD4和CD8T细胞的浸润。同样,细胞transwell共培养研究表明,WCP处理的MPC-5细胞在诱导T细胞迁移方面较弱。与这一发现一致,我们的观察表明,WCP可以抑制肾脏和MPC-5细胞中T细胞相关趋化因子的表达,以及降低TLR4,MYD88,磷酸化-p38,磷酸化-ERK的水平,和磷酸化-JNK。另一方面,发现WCP在体内和体外极大地抑制Th1细胞分化。基于差异表达基因(DEGs)富集分析,细胞因子受体诱导的Th1细胞分化途径和PI3K-AKT途径是最富集的途径。RT-qPCR和WB结果显示WCP显著降低IL-12、p-STAT4、IFN-γ水平,p-STAT1,p-PI3K,和T细胞中的p-AKT。
结论:WCP对LN疾病表现出积极的免疫调节作用,通过TLR4/MYD88/MAPK信号通路减少T细胞浸润,通过IL-12-STAT4和IFN-γ-STAT1通路抑制Th1细胞分化,除了PI3K-AKT途径。
BACKGROUND: T cell infiltration and differentiation play a central part in the development of lupus nephritis (LN). Our prior research has indicated that protein, the primary active component of cordyceps (WCP), a traditional Chinese medicine, possesses properties that can enhance renal fibrosis and provide kidney protection. Nonetheless, the connection between WCP and T cell infiltration and differentiation in LN remains poorly understood.
OBJECTIVE: The objective of this research was to assess the immunomodulatory impacts of WCP in LN mice and elucidate the underlying mechanism through in vivo and in vitro investigations.
METHODS: To investigate the impact and mechanism of WCP in MRL/lpr lupus-prone mice, WCP (1.5 g/kg/d), Bailing capsules (BC, 0.75 g/kg/d), and saline in equivalent quantities were administered to the mice over a period of 8 weeks. The therapeutic effects, T cell infiltration and differentiation of WCP on MRL/lpr mice were verified through ELISA, Hematoxylin-eosin (H&E), Periodic Acid Schiff (PAS) staining, immunofluorescence, Luminex analysis and flow cytometry. The mechanism by which WCP alleviates LN was investigated using tissues of mice, T cells and Mouse Podocyte Clone-5 (MPC-5) cells by transcriptomics, Western blot (WB), and Real-time quantitative polymerase chain reaction (RT-qPCR).
RESULTS: We found that WCP improved LN in MRL/lpr mice by reducing urinary protein, creatinine, and serum auto antibodies, increasing complement 3 (C3) level, improving renal immunopathology and downregulating serum cytokines, including IFN-γ, IL-12, and RANTES. Notably, the infiltration of CD4+ and CD8+ T cells in the kidney was reduced by WCP. Similarly, the cell transwell co-culturation study showed that the WCP treated MPC-5 cells were weaker in inducing T cell migration. Consistent with this finding, our observations revealed that WCP could inhibit T cell-related chemokine expression in kidney and MPC-5 cells, as well as reduce the levels of TLR4, MYD88, phosphorylated-p38, phosphorylated-ERK, and phosphorylated-JNK. On the other hand, WCP was found to greatly inhibit the Th1 cells differentiation in vivo and in vitro. Cytokine-receptor induced Th1 cell differentiation pathway and PI3K-AKT pathway were the most enriched pathways based on differentially expressed genes (DEGs) enrichment analysis among different cell groups. Results from RT-qPCR and WB showed that WCP notably reduced the levels of IL-12, p-STAT4, IFN-γ, p-STAT1, p-PI3K, and p-AKT in T cells.
CONCLUSIONS: WCP demonstrated positive immunomodulatory effects on LN disease, by decreasing the T cells infiltration through TLR4/MYD88/MAPK signaling pathway and inhibiting Th1 cells differentiation via IL-12-STAT4 and IFN-γ-STAT1 pathways, in addition to the PI3K-AKT pathway.