miR-216a-5p在HMGB1介导的表皮葡萄球菌感染致腹膜透析相关腹膜炎中的作用

The role of miR-216a-5p in HMGB1-mediated peritoneal dialysis-associa-ted peritonitis induced by Staphylococcus epidermidis infection

目的探讨miR-216a-5p在高迁移率蛋白B1(HMGB1)介导的表皮葡萄球菌感染致小鼠腹膜透析相关腹膜炎(PDAP)中的作用及机制。方法选取健康雄性C57BL/6J小鼠,随机分为对照组、感染组、感染+HMGB1抑制剂组,收集腹腔积液及腹膜组织分别进行白细胞计数、HE和免疫组织染色;Real-time PCR和Western Blot检测白细胞介素-1α(IL-1α)、白细胞介素-6(IL-6)、肿瘤坏死因素-α(TNF-α)、HMGB1、核转录因子-κB(NF-κB)和核转录因子抑制蛋白(I-κB)的mRNA和蛋白表达水平;生信预测发现,miR-216a-5p可能与HMGB1结合,参与其诱导的PDAP的发生,并构建感染+miR-216a-5p mimics组小鼠,通过双荧光素酶报告基因检测验证miR-216a-5p和HMGB1的关系,采用Real-time PCR、Western Blot、免疫组织染色检测HMGB1的表达变化。结果与对照组相比,感染组小鼠腹腔积液白细胞计数增多,炎性浸润明显,IL-1α、IL-6、TNF-α,以及HMGB1 mRNA和蛋白表达均升高(均P<0.05);HMGB1抑制剂(甘草素)干预后,小鼠腹腔积液白细胞计数下降,炎性浸润改善,IL-1α、IL-6、TNF-α,以及HMGB1 mRNA和蛋白表达均下降(均P<0.05)。感染组小鼠NF-κB表达高于对照组,I-κB表达低于对照组;HMGB1抑制剂干预后NF-κB表达降低,I-κB表达升高(均P<0.05)。Real-time PCR结果证实,与对照组相比,miR-216a-5p的含量在感染组中显著减少;双荧光素酶报告基因检测提示,miR-216a-5p可直接作用于HMGB1的3’UTR区;与感染组相比,感染+miR-216a-5p mimics组小鼠中HMGB1 mRNA和蛋白表达均下降(均P<0.05)。结论HMGB1在表皮葡萄球菌感染致小鼠PDAP中发挥重要作用,抑制HMGB1可改善小鼠炎症反应,miR-216a-5p可通过靶向HMGB1参与表皮葡萄球菌感染致PDAP的发生。

Objective To investigate the role and mechanism of miR-216a-5p in high mobility group protein-B1(HMGB1)-mediated peritoneal dialysis-associated peritonitis(PDAP)induced by Staphylococcus epidermidis infection in mice.Methods Healthy male C57BL/6J mice were selected and randomly divided into control group,infection group,and infection+HMGB1 inhibitor group,peritoneal effusion and peritoneal tissue was collected for detection of whit blood cell(WBC)count,HE and immunohistochemical staining;mRNA and protein expression levels of interleukin-1α(IL-1α),interleukin-6(IL-6),tumor necrosis factors-α(TNF-α),HMGB1,NF-κB,and I-κBi were detected by real-time polymerase chain reaction(RT-PCR)and Western Blot;bioinformatics prediction found that miR-216a-5p may bind with HMGB1 and participate in the occurrence of HMGB1-mediated PDAP,and construct dinfection+miR-216a-5p mimics mice group,relationship between miR-216a-5p and HMGB1 was verified by double luciferase reporter gene assay,changes in expression of HMGB1 were detected by RT-PCR,Western blot and immunohistochemical staining.Results Compared with control group,WBC count of peritoneal effusion in mice in infection group increased,inflammatory infiltration was obvious,expression of mRNA and protein of IL-1α,IL-6,TNF-αand HMGB1 all increased(all P<0.05);after intervention by HMGB1 inhibitor(glycyrrhizin),WBC count of mice peritoneal effusion decreased,inflammatory infiltration improved,expression of mRNA and protein of IL-1α,IL-6,TNF-αand HMGB1 all decreased(all P<0.05).The expression of NF-κB in mice in infection group was higher than that in control group,I-κBi was lower than that in control group;after HMGB1 inhibitor intervention,NF-κB expression decreased,I-κB expression increased(both P<0.05).RT-PCR results confirmed that the content of miR-216a-5p decreased significantly in infection group compared with control group;dual-luciferase reporter gene assay suggested that miR-216a-5p could directly act on the 3’UTR region of HMGB1;compared with infection group,the expression of mRNA and protein of HMGB1 decreased in mice in infection+miR-216a-5p mimics group(both P<0.05).Conclusion HMGB1 plays an important role in PDAP induced by Staphylococcus epidermidis infection,inhibiting HMGB1 can improve the inflammatory response in mice,miR-216a-5p can participate in the occurrence of PDAP induced by Staphylococcus epidermidis infection through targeting HMGB1.