摘要
目的 观察沉默信息调节因子5(Silent information regulator 5,SIRT5)介导的丙酮酸激酶M2受体(Pyruvate kinase type 2,PKM2)通过调节巨噬细胞极化对香烟烟雾诱导的炎症水平的影响,探讨其可能存在的作用机制。方法 以小鼠巨噬细胞为研究对象,香烟烟雾提取物(Cigarette smoke extract, CSE)作为刺激因素,诱导体外炎症模型,分为空白对照组(正常培养RAW264.7细胞,不进行任何干预)、CSE干预组(培养细胞,并进行CSE干预)、SIRT5-NC组(CSE干预细胞,只用于感染过程中的对照)、SIRT5-siRNA-2组(CSE干预细胞,转染SIRT5-siRNA)和SIRT5-siRNA-2+OE-PKM2组(CSE干预细胞,转染SIRT5-siRNA后,加入PKM2激动剂)。实时荧光定量逆转录聚合酶链式反应(RT-qPCR)检测SIRT5及PKM2 mRNA的表达。蛋白质印迹(Western blot)法检测各组细胞模型SIRT5和PKM2的蛋白表达。流式细胞术检测各组细胞M1、M2型巨噬细胞表型分布。酶联免疫吸附测定(ELISA)法检测细胞上清中白细胞介素-6(Interleukin-6,IL-6)、IL-10、IL-12和肿瘤坏死因子-α(TNF-α)表达水平。结果 在细胞模型中,与空白对照组和siRNA-NC组相比,SIRT5-siRNA-2组的PKM2表达水平明显降低,差异有统计学意义。CSE干预后M1型促炎巨噬细胞表达增高,M2型抑炎巨噬细胞减少。M1型巨噬细胞随着SIRT5沉默而减少,过表达PKM2后又增多,差异有统计学意义(P<0.01)。与空白对照组相比,M2型巨噬细胞则在炎症模型中均降低,差异有统计学意义(P<0.05)。ELISA结果显示,与空白对照组相比,CSE干预组促炎因子IL-6、IL-12、TNF-α表达增高,抑炎因子IL-10表达降低,差异有统计学意义(P<0.01)。与CSE干预组相比,SIRT5-siRNA-2组下调了促炎因子表达同时也上调了抑炎因子水平,差异具有统计学意义(P<0.01)。SIRT5-siRNA-2+OE-PKM2组较SIRT5-siRNA-2组相比,又上调了IL-6、IL-12、TNF-α,同时IL-10的表达也增高,差异有统计学意义(P<0.01)。结论 SIRT5介导的PKM2通过降低IL-6、IL-12、TNF-α表达,促进IL-10表达,从而减轻香烟烟雾提取物诱导的巨噬细胞极化产生的炎症反应。
Objective The effect of pyruvate kinase type 2(PKM2)mediated by silent information regulator 5(SIRT5)on cigarette smoke-induced inflammation was observed,and its possible mechanism was explored.Methods Mouse macrophages were used as the research object,and cigarette smoke extract(CSE)was used as the stimulus factor to induce ina blank control group(normally cultured RAW264.7 cells without any intervention),CSE intervention group(cultured cells,and CSE intervention),SIRT5-NC group(CSE intervention cells,only used for control during infection),SIRT5-siRNA-2 group(CSE intervention cells,transfected with SIRT5-siRNA),SIRT5-siRNA-2+OE-PKM2 group(CSE intervention cells,transfected with SIRT5-siRNA,Add PKM2 agonist).Real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the expression of SIRT5 and PKM2 mRNA.Western blot was used to detect the protein expression of SIRT5 and PKM2 in each group of cell models.Flow cytometry was used to detect the phenotypic distribution of M1 and M2 in macrophage phenotypes in each group of cells.ELISA was used to detect the expressions levels of IL-6,IL-10,IL-12,and TNF-αin the cell supernatant.Results In the cell model,compared with the blank control group and the siRNA-NC group,the expression level of PKM2 in the SIRT5-siRNA-2 group was significantly decreased,and the difference was statistically significant.After CSE intervention,the expression of M1 pro-inflammatory macrophages increased,while that of M2 anti-inflammatory macrophages decreased.M1-type macrophages decreased with SIRT5 silencing and increased after PKM2 overexpression,and the difference was statistically significant(P<0.01).Compared with the blank control group,M2-type macrophages were all decreased in the inflammatory model,and the difference was statistically significant(P<0.05).The results of ELISA showed that compared with the blank control group,the expression of pro-inflammatory factors IL-6,IL-12,and TNF-αin the CSE intervention group increased,while the expression of IL-10 decreased in the CSE intervention group,and the difference was statistically significant(P<0.01).Compared with the CSE intervention group,the SIRT5-siRNA-2 group down-regulated the expression of pro-inflammatory factors and up-regulated the levels of anti-inflammatory factors,and the difference was statistically significant(P<0.01).Compared with the SIRT5-siRNA-2 group,the SIRT5-siRNA-2+OE-PKM2 group further up-regulated the expression of IL-6,IL-12,and TNF-α,and the expression of IL-10 also increased,and the difference was statistically significant(P<0.01).Conclusion SIRT5-mediated PKM2 promotes IL-10 expression by decreasing IL-6,IL-12,and TNF-αexpression,thereby mitigating the inflammatory response caused by macrophage polarization induced by cigarette smoke extract.
作者
崔萌萌
徐丹
李风森
CUI Mengmeng;XU Dan;LI Fengsen(The Fourth Clinical Medical College of Xinjiang Medical University,Xinjiang Medical University,Urumqi 830000,China;The Affiliated Hospital of Traditional Chinese Medicine,Xinjiang Medical University,Urumqi 830000,China)
出处
《新疆医科大学学报》
2026年第1期9-15,共7页
Journal of Xinjiang Medical University
基金
新疆维吾尔自治区重点实验室开放课题项目(2021D04023)。
关键词
慢性阻塞性肺疾病
SIRT5
PKM2
巨噬细胞
炎症
chronic obstructive pulmonary disease(COPD)
silent information regulator 5(SIRT5)
pyruvate kinase type 2(PKM2)
macrophages
inflammation
