组蛋白修饰对COPD大鼠肺泡Ⅱ型上皮细胞趋化因子表达的影响

Effects of Histone Modification on Chemokines Expression in Alveolar Epithelial TypeⅡ Cells in a Rat Model of COPD

目的探讨COPD大鼠肺泡Ⅱ型上皮细胞(AECⅡ)炎症因子表达是否与组蛋白修饰有关。方法熏烟法建立大鼠COPD模型。取大鼠肺组织进行病理观察,提取AECⅡ进行碱性磷酸酶染色鉴定和电镜鉴定;实时定量PCR检测AECⅡ三种趋化因子:单核细胞趋化蛋白1(MCP-1)、IL-8及巨噬细胞炎性蛋白2α(MIP-2α)的mRNA表达;Westernblot检测组蛋白去乙酰化酶2(HDAC2)蛋白表达;染色质免疫共沉淀(ChIP)检测趋化因子启动子区组蛋白H3、H4乙酰化和H4K9甲基化修饰情况。结果 COPD组IL-8、MCP-1、MIP-2αmRNA表达较正常对照组分别增加4.48倍、3.14倍、2.83倍;COPD组AECⅡ细胞HDAC2蛋白表达较正常对照组降低(0.25±0.15比0.66±0.15,P<0.05),且HDAC2的下降程度与IL-8、MCP-1、MIP-2αmRNA表达增高程度呈负相关(r值分别为-0.960、-0.914、-0.928,P均<0.05)。COPD组趋化因子基因启动子区H3、H4乙酰化水平较正常对照组均升高,H4K9甲基化水平则降低(P均<0.05)。结论 COPD模型大鼠AECⅡ的IL-8、MCP-1、MIP-2α三种趋化因子基因表达增加,HDAC2介导的组蛋白修饰在COPD炎症反应中发挥重要作用。

Objective To investigate the effects of histone modification on the expression of chemokines in alveolar epithelial typeⅡ cells （AECⅡ） in a rat model of chronic obstructive pulmonary disease （COPD）.Methods 20 SD rats were randomly assigned to a normal control group and a COPD group.The rat model of COPD was established by cigarette smoking.Lung histological changes were observed by HE staining.AECⅡ cells were isolated and identified by alkaline phosphatase staining and electron microscopic.The mRNA expressions of monocyte chemoattractant protein （MCP）-1,IL-8,and macrophage inflammatory protein （MIP）-2α were detected by real-time quantitative PCR.The expression of histone deacetylase （HDAC）2 was measured by western blot.Chromatin immunoprecipitation （ChIP） was used to detect H3 and H4 acetylation,and H4K9 methylation in the promoter region of chemokine gene.Results Compared with the control group,the mRNA expressions of MCP-1,IL-8,and MIP-2α in the COPD group increased 4.48,3.14,and 2.83 times,respectively.The expression of HDAC2 protein in the COPD group was significantly lower than in the control group （0.25±0.15 vs.0.66±0.15,P0.05）.The expression of HDAC2 had a negative correlation with the gene expressions of IL-8,MCP-1,and MIP-2α （r=-0.960,-0.914,-0.928,respectively,all P0.05）.The levels of H3 and H4 acetylation were higher,and H4K9 methylation level was lower in the promoter region of chemokine gene in the COPD group compared with the control group （all P0.05）.Conclusions MCP-1,IL-8,and MIP-2α participate and promote the lung inflammatory response in COPD.HDAC2-mediated histone modification may play an important role in COPD inflammation.