HMGB1抑制剂对脓毒症小鼠T淋巴细胞和单核细胞的作用机制

The mechanism of HMGB1 inhibitor on T lymphocytes and monocytes in the sepsis mice

目的探讨高迁移率族蛋白B1(high modility groupbox 1,HMGB1)抑制剂对脓毒症T淋巴细胞和单核细胞的作用机制。方法将30只C57BL/6雄性小鼠随机分为3组,每组10只,依次为假手术组、模型组、抑制剂组。模型组、抑制剂组采用盲肠结扎穿孔手术构建脓毒症小鼠模型,假手术组小鼠仅暴露盲肠后即进行伤口缝合,不结扎。抑制剂组小鼠术后腹腔注射HMGB1特异性抑制剂甘草酸(10 mg/kg),每6 h注射1次,连续4次,假手术组和模型组腹腔注射等剂量的生理盐水。处死小鼠后,无菌分离小鼠的胸腺组织,常规提取胸腺中的T淋巴细胞和单核细胞。MTT法与流式细胞术测定T淋巴细胞的增殖活性和凋亡情况,Transwell趋化实验与ELISA法测定单核细胞的趋化活性以及分泌炎性因子TNF-α、IL-6及IL-10的情况,Western blot检测胸腺组织中HMGB1和第10号染色体同源丢失性磷酸酶-张力蛋白(phosphatase and tensin homolog deleted onchromosometen,PTEN)的表达水平。结果与假手术组相比,模型组小鼠胸腺组织T淋巴细胞的增殖活性和凋亡率、单核细胞的趋化活性和TNF-α、IL-6、IL-10蛋白的表达、HMGB1和PTEN的表达明显升高(均P<0.05);与模型组相比,抑制剂组小鼠胸腺组织T淋巴细胞凋亡率、TNF-α、IL-6蛋白的表达、HMGB1和PTEN的表达明显降低,T淋巴细胞的增殖活性、单核细胞的趋化活性、IL-10蛋白的表达明显升高(均P<0.05)。结论HMGB1抑制剂可降低脓毒症小鼠胸腺T细胞的凋亡率,增强T细胞的增殖活性和单核细胞的趋化活性,提高单核细胞抗炎因子IL-10的分泌,抑制促炎因子TNF-α、IL-6的分泌。

Objective To investigate the mechanism of the high mobility group box protein 1(HMGB1)inhibitor on T lymphocytes and monocytes in sepsis.Methods Thirty C57BL/6 male mice were randomly divided into 3 groups,10 in each group,followed by a sham operation group,a model group,and an inhibitor group.In the model group and inhibitor group,a mouse model of sepsis was replicated by cecal ligation and puncture(CLP).In the sham operation group,the wound was sutured only after exposure to the cecum,and no ligation was performed.The mice in the inhibitor group were intraperitoneally injected with glycyrrhizic acid,a specific inhibitor of HMGB1(10 mg/kg),and injected once every 6 hours for 4 times.The mice in the sham operation group and the model group were intraperitoneally injected with the same amount of normal saline.After the mice were sacrificed,the thymus tissue of the mice was aseptically isolated,and the T lymphocytes and monocytes in the thymus were routinely extracted.MTT assay and flow cytometry were used to detect the proliferation activity and apoptosis of T lymphocyte.Transwell chemotaxis assay and ELISIA method were used to detect the chemotactic activity of monocytes and the expression levels of TNF-α,IL-6 and IL-10.Western blot was used to detect the expression levels of HMGB1 and phosphatase and tensin homolog deleted onchromosometen(PTEN)in each group.Results Compared with the sham operation group,the proliferation activity and the apoptosis rate of T lymphocytes in the thymus tissue of the model group,the chemotactic activity of monocytes and the expression levels of TNF-α,IL-6,IL-10 and the expression of HMGB1 and PTEN significantly increased(all P<0.05).Compared with the model group,the apoptosis rate of T lymphocytes,the expression levels of TNF-α,IL-6,HMGB1 and PTEN significantly reduced in inhibitor group mice,the proliferation activity of T lymphocytes,the chemotactic activity of monocytes,and the expression of IL-10 protein significantly increased(all P<0.05).Conclusions HMGB1 inhibitor can reduce the apoptosis rate of thymic T cells in septic mice,enhance the proliferation activity of T cells and the chemotactic activity of monocytes,increase the secretion of monocyte antiinflammatory factor IL-10,and inhibit the pro-inflammatory factor TNF-α,IL-6 secretion.