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    巨噬细胞XBP1调控肝脏衰老的机制研究

    Mechanism of macrophage XBP1 in regulating liver aging

    • 摘要: 目的 探究巨噬细胞X盒结合蛋白 1(XBP1)对肝脏衰老的影响及作用机制。方法 选取2月龄(年轻组)和20月龄(老年组)SPF级C57BL/6J雄性小鼠各10只,另选取20月龄的Xbp1髓系特异性敲除小鼠(Xbp1ΔMφ组)和同窝对照小鼠(Xbp1FL/FL组)各10只,留取各组小鼠肝脏组织标本。采用β-半乳糖苷酶染色检测肝脏组织衰老相关-β-半乳糖苷酶(SA-β-gal)的表达;免疫组化检测肝脏组织p16表达;实时荧光定量PCR(RT-qPCR)检测衰老相关分泌表型(SASP,Il1b、Il6、Il8、Cxcl10、Ifnb1)及Xbp1、Nlrp3、Yap1的mRNA表达水平。通过Western blot检测XBP1剪接体(XBP1s)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)、Yes相关蛋白(YAP)及磷酸化YAP(p-YAP)的蛋白表达水平。通过转录组测序分析20月龄Xbp1FL/FLXbp1ΔMφ小鼠肝脏基因表达差异。提取各组小鼠肝脏巨噬细胞后,通过RT-qPCR和Western blot分别检测Nlrp3 mRNA及XBP1s、NLRP3 蛋白水平。结果 相较于年轻组,老年组小鼠肝脏SA-β-gal、p16表达水平升高,SASP相关基因mRNA水平升高,且肝脏组织和肝脏巨噬细胞中XBP1s的mRNA与蛋白水平均显著升高。与Xbp1FL/FL组相比,Xbp1ΔMφ组上述指标表达均明显降低,同时肝脏组织及肝巨噬细胞中NLRP3的mRNA和蛋白水平明显降低,且肝脏Hippo信号通路激活,YAP的mRNA和蛋白水平升高,p-YAP蛋白水平降低。结论 巨噬细胞XBP1可促进肝脏衰老,Xbp1ΔMφ可通过降低肝脏炎症水平、激活Hippo信号通路中的YAP蛋白减缓肝脏衰老。

       

      Abstract: Objective To investigate the effects of macrophage X-box binding protein 1 (XBP1) on liver aging and its underlying mechanisms. Methods Ten SPF-grade male C57BL/6J mice aged 2 months (young group) and ten aged 20 months (aged group) were selected. Another 20-month-old cohort included myeloid-specific Xbp1 knockout mice (Xbp1ΔMφ group) and their littermate control mice (Xbp1FL/FL group), with 10 mice in each group. Liver tissue samples were collected from all groups. Senescence-associated β-galactosidase (SA-β-gal) expression in liver tissues was detected by β-galactosidase staining, and p16 expression was detected by immunohistochemistry. The mRNA expression levels of senescence-associated secretory phenotype (SASP) factors (Il1b, Il6, Il8, Cxcl10, and Ifnb1), Xbp1, Nlrp3, and Yap1 were determined by real-time quantitative PCR (RT-qPCR). Western blot was performed to measure protein expression of XBP1 spliced form (XBP1s), NOD-like receptor family pyrin domain-containing 3 (NLRP3), Yes-associated protein (YAP), and phosphorylated YAP (p-YAP). Transcriptome sequencing (RNA-seq) was conducted to analyze gene expression differences between liver tissues from 20-month-old Xbp1FL/FL and Xbp1ΔMφ mice. Macrophages were isolated from liver tissues of each group, and Nlrp3 mRNA and XBP1s/NLRP3 protein levels were analyzed by RT-qPCR and Western blot, respectively. Results Compared with the young group, the aged group exhibited increased hepatic expression of SA-β-gal and p16, elevated mRNA levels of SASP-related genes, and significantly increased levels of XBP1s mRNA and protein in both liver tissues and hepatic macrophages. Compared with the Xbp1FL/FL group, the Xbp1ΔMφ group showed markedly reduced expression of the above indicators, as well as decreased NLRP mRNA and protein levels in both liver tissues and macrophages. Additionally, the Hippo signaling pathway was activated in the liver of Xbp1ΔMφ mice, as evidenced by increased YAP mRNA and protein levels and decreased p-YAP protein levels. Conclusions Macrophage XBP1 promotes liver aging. Xbp1ΔMφ attenuates hepatic aging by reducing liver inflammation and activating YAP in the Hippo signaling pathway.

       

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