异基因骨髓移植小鼠肠道菌群和NLRP3炎症小体改变与急性移植物抗宿主病发生发展的关系

Relationship of aGVHD with intestinal flora and NLRP3 inflammasome in mice with allogenic bone marrow transplantation

目的:探讨异基因骨髓移植小鼠肠道菌群和核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体改变与急性移植物抗宿主病(aGVHD)的关系。方法:建立C57BL/6雄性小鼠致BALB/c雌性小鼠异基因骨髓移植模型,诱导aGVHD为aGVHD组,以BALB/c雌性小鼠为对照,ELISA方法检测模型组小鼠血清白细胞介素1β(IL-1β)、IL-4、IL-10和肿瘤坏死因子α(TNF-α)的水平,免疫组化检测肠闭合蛋白(occludin)的表达,检测16S rDNA以评估肠道菌群改变,HE染色观察aGVHD靶器官(肺、肝、脾和回肠)的病理变化,Western blot分析肠组织NLRP3炎症小体的表达。结果:与正常小鼠相比,aGVHD小鼠血清IL-1β和TNF-α水平明显上升,而IL-4和IL-10水平下降(P<0.01);aGVHD小鼠肠紧密连接蛋白occludin表达下降,其肠道菌群也发生改变,表现为疣微菌门(Verrucomicrobia)等微生物物种富集,而红蝽杆菌目(Coriobacteriales)等多个微生物种类下调;aGVHD组靶器官病理损伤明显,NLRP3炎症小体表达显著增加。结论:aGVHD可能通过炎症因子影响小鼠肠道屏障功能,改变肠道菌群,进而激活NLRP3炎症小体,导致恶性循环。

AIM:To investigate the relationship between intestinal flora/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome and acute graft-versus-host disease(aGVHD)in allogeneic bone marrow transplantation mice.METHODS:aGVHD was established from donor mice C57 BL/6 to host mice BALB/c as aGVHD group,while normal BALB/c mice was used as normal group.The serum levels of interleukin-1β(IL-1β),IL-4,IL-10 and tumor necrosis factor-α(TNF-α)were measured by ELISA.The expression of tight junction protein occludin in ileal mucosa was examined by immunohistochemistry.The intestinal flora was evaluated by detection of 16 S rDNA.The pathological changes of aGVHD target organs(lung,liver,spleen,and ileum)were observed by HE staining,and the ex-pression of NLRP3 inflammasome was analyzed by Western blot.RESULTS:Compared with normal group,the serum levels of IL-1βand TNF-αwere significantly increased,while IL-4 and IL-10 were decreased in aGVHD group(P<0.01).The expression of occludin was obviously decreased in ileal mucosa.The intestinal flora was also changed,as indi-cated by the enrichment of microbial species such as Verrucomicrobia,and the decreases in some microbial species such as Coriobacteriales in aGVHD mice.In aGVHD mice,the pathological changes of target organs were observed,while the ex-pression of NLRP3 inflammasome was significantly increased in the intestine.CONCLUSION:The aGVHD changes in-testinal flora,activates NLRP3 inflammasome through decreasing intestine mucosa barrier by cytokines,leading to a vi-cious circle.