LAMC2对巨噬细胞极化及NF-κB和ERK1/2信号通路调控的作用  

Effects of LAMC2 on macrophage polarization and regulation of NF-κB and ERK1/2 signaling pathways

作　　者：季振中[1] 胡正国[1] Ji Zhenzhong;Hu Zhengguo(Department of Geriatric Medicine,Zhongnan Hospital of Wuhan University,Wuhan 430071,China)

机构地区：[1]武汉大学中南医院老年医学科,武汉430071

出　　处：《中国医师杂志》2024年第11期1669-1676,共8页Journal of Chinese Physician

摘　　要：目的探索在糖尿病肾病(DKD)中层粘连蛋白γ2(LAMC2)在调控巨噬细胞极化中的作用及其潜在机制。方法 GEO数据库明确LAMC2作为主要研究对象。体外培养小鼠单核巨噬细胞RAW264.7, 分为以下6组:Control组(空白对照组)、HG组(高糖组, 采用30 mmol/L葡萄糖处理)、si-NC组(采用30 mmol/L葡萄糖处理并转染阴性对照)、si-LAMC2组-1#(采用30 mmol/L葡萄糖处理并转染1号shRNA-LAMC2构建体)、si-LAMC2组-2#(采用30 mmol/L葡萄糖处理并转染2号shRNA-LAMC2构建体)、si-LAMC2组-3#(采用30 mmol/L葡萄糖处理并转染3号shRNA-LAMC2构建体)。实时荧光定量聚合酶链反应(qRT-PCR)和蛋白免疫印迹法(Western Blot)实验检测巨噬细胞LAMC2表达, 使用小干扰RNA(siRNA)技术抑制LAMC2表达, EdU染色评价细胞增殖情况, Transwell实验评价细胞侵袭能力, qRT-PCR检测iNOS、IL-12、TNF-α、Arg-1、CD163和IL-10表达, 细胞免疫荧光染色检测CD86和CD206的表达, Western Blot检测NF-κB和ERK信号通路关键蛋白表达。结果 GEO数据库基因分析发现, LAMC2在DKD及M1型巨噬细胞中的表达显著上调(均P<0.01)。相比于空白对照组, HG组的巨噬细胞中LAMC2的mRNA和蛋白表达均更高(均P<0.01)。EdU染色和Transwell统计结果显示, HG组和si-NC组EdU阳性率少于Control组, 侵袭细胞数量明显多于Control组(均P<0.05)。si-LAMC2组EdU阳性率多于si-NC组, 侵袭细胞数量显著少于si-NC组(均P<0.05)。与Control组比较, HG组和si-NC组M1巨噬细胞标志物iNOS、IL-12和TNF-α的mRNA表达水平均更高(均P<0.01)。与si-NC组比较, si-LAMC2组上述三种标志物的mRNA表达水平均更低(均P<0.01)。与Control组比较, HG组和si-NC组M2巨噬细胞标志物Arg-1、CD163和IL-10的mRNA表达水平显著降低(均P<0.01)。与si-NC组比较, si-LAMC2组上述三种标志物的mRNA表达水平均更高(均P<0.01)。相比于Control组, HG组和si-NC组CD86荧光强度显著更高, CD206荧光强度更低。与si-NC组比较, Objective To explore the role and potential mechanism of middle layer adhesionγ2(LAMC2)in the regulation of macrophage polarization in diabetic nephropathy(DKD).Methods Using GEO database,LAMC2 was identified as the main research object.Mouse mononuclear macrophages RAW264.7 were cultured in vitro and divided into the following 6 groups:Control group(blank control group),HG group(high glucose group),Treated with 30 mmol/L glucose,si-NC group(treated with 30 mmol/L glucose and transfected with negative control),si-LAMC2 group-1#(treated with 30 mmol/L glucose and transfected with shRNA-LAMC2 construct 1),si-LAMC2 group-2#(treated with 30 mmol/L glucose and transfected with SHRnA-LAMC2 construct 1),and SI-LAMC2 group-2#(treated with 30 mmol/L glucose)Group 3#of si-LAMC2 was treated with mmol/L glucose and transfected with shRNA-LAMC2 construct 2,and Group 3#of Si-LAMC2 was treated with 30 mmol/L glucose and transfected with shRNA-LAMC2 construct 3.Real-time quantitative fluorescent polymerase chain reaction(qRT-PCR)and Western Blot assay were used to detect LAMC2 expression in macrophages,small interfering RNA(siRNA)technique was used to inhibit LAMC2 expression,cell proliferation was evaluated by EdU staining,and cell invasion ability was evaluated by Transwell assay.The expressions of inducible nitric oxide synthase(iNOS),interleukin(IL)-12,Tumor necrosis factor-α(TNF-α),Arg-1,CD163 and IL-10 were detected by qRT-PCR,the expressions of CD86 and CD206 were detected by cellular immunofluorescence staining,and the expressions of key proteins in nuclear factor-κB(NF-κB)and extracellular signal regulated kinase(ERK)signaling pathways were detected by western blot assay.Results Gene analysis in CEO database showed that the expression of LAMC2 in DKD and M1 macrophages was significantly up-regulated(all P<0.01).Compared with the blank Control group,the mRNA and protein expression of LAMC2 in macrophages of HG group were higher(all P<0.O1).EdU staining and transwell statistics showed that,the EdU positive rate in HG

关 键 词：糖尿病肾病 巨噬细胞极化 层粘连蛋白γ2 NF-ΚB信号通路 ERK信号通路 

分 类 号：R587.2[医药卫生—内分泌] R692.9[医药卫生—内科学]