微炎症环境中肠上皮细胞与巨噬细胞共培养后MAPK、NF-κB通路改变及意义

Activation changes of MAPK and NF-κB pathway after co-culture of intestinal epithelial cells and macro⁃phages in micro-inflammatory environment and their significance

目的观察微炎症环境中肠上皮细胞与巨噬细胞共培养后MAPK、NF-κB通路改变,探究固有免疫系统维持肠道免疫平衡的细胞间交流机制。方法将人结肠腺癌上皮细胞株Caco2、HCA7分别与人白血病单核巨噬细胞株THP1构建Transwell小室共培养体系,加入肿瘤坏死因子α(TNF-α)5 ng/mL刺激不同时间后,采用Western blotting法检测三种细胞单独及共培养后的MAPK通路[磷酸化细胞外信号调节激酶(p-ERK)、磷酸化丝裂原活化蛋白激酶(p-p38)]及NF-κB通路[磷酸化NF-κB抑制蛋白α(p-IκBα)、磷酸化核蛋白(p-p65)]相关蛋白表达。将对数生长期的THP1细胞分为Caco2培养基组和DMEM培养基组,离心后分别重悬于Caco2条件培养基和DMEM完全培养基,加入TNF-α5 ng/mL刺激不同时间后,采用Western blotting法检测细胞p-ERK、p-p38、p-IκBα、p-p65蛋白表达,采用RT-PCR法检测细胞TNF-α相关细胞因子[白细胞介素(IL)-8、IL-6、IL-1β]和CC亚族趋化性细胞因子2(CCL2)、CCL3 mRNA表达。结果在单独培养的细胞中,Caco2细胞在TNF-α刺激30 min后p-ERK、p-p38、p-p65表达均升高,THP1细胞在TNF-α刺激90 min后p-p38、p-IκBα、p-p65表达均升高(P均<0.05);与单独培养细胞TNF-α刺激相同时间比较,共培养后的Caco2细胞在TNF-α刺激90 min后p-ERK、p-p38、p-IκBα表达均降低(P均<0.05),共培养后的THP1细胞在TNF-α刺激90 min后p-ERK、p-p38、p-IκBα、p-p65表达均降低(P均<0.05)。在单独培养的细胞中,HCA7细胞在TNF-α刺激120 min后p-ERK、p-p38、p-IκBα、p-p65表达均升高,THP1细胞在TNF-α刺激30、120 min后p-ERK、p-p38、p-p65表达均升高(P均<0.05);与单独培养细胞TNF-α刺激相同时间比较,共培养后的HCA7细胞在TNF-α刺激120 min后p-ERK、p-IκBα、p-p65表达均降低,共培养后的THP1细胞在TNF-α刺激30、120 min后p-ERK、p-p38、p-p65表达均降低(P均<0.05)。与DMEM培养基组TNF-α刺激相同时间比较,Caco2培养基组TNF-α刺激120 min后p-ERK、p-p38、p-IκBα、p-p65表达均降低,TNF-α刺激60 min后IL-8、IL-1β、CCL2 mRNA相对表达量均降低(P均<0.05);两组TNF-α刺激相同时间后的IL-6、CCL3 mRNA相对表达量比较均无明显差异(P均>0.05)。结论微炎症环境中肠上皮细胞Caco2、HCA7和巨噬细胞THP1共培养后各细胞MAPK和NF-κB通路激活及促炎因子分泌均被抑制,这种细胞间的信号交流是通过分泌可溶性介质介导的。

Objective To investigate the changes of MAPK and NF-κB pathways after co-culture of intestinal epithe-lial cells and macrophages in micro-inflammatory environment,and to explore the inter-cell communication mechanism of innate immune system to maintain intestinal immune balance.Methods Human colon adenocarcinoma epithelial cell lines Caco2 and HCA7 were cultured with human leukemia mononuclear macrophage cell line THP1 in Transwell cham-ber,respectively.After stimulation of tumor necrosis factor-α(TNF-α,5 ng/mL)for different time,the MAPK pathwayrelated proteins including phosphorylated extracellular signal-regulated kinase(p-ERK)and phosphorylated mitogen-acti-vated protein kinase(p-p38),and NF-κB pathway-related proteins including phosphorylated NF-κB inhibitor-α(p-IκBα),and phosphorylated nucleoprotein(p-p65)were detected by Western blotting.THP1 cells in the logarithmic growth phase were divided into the CaCo2 medium group and DMEM medium group.After centrifugation,they were sus-pended in CaCo2 conditioned medium and DMEM complete medium,respectively.After adding TNF-α5 ng/mL for differ-ent time,the protein expression levels of p-ERK,p-p38,p-IκBαand p-p65 were detected by Western blotting.The mRNA expression levels of TNF-α-related cytokines[interleukin(IL)-8,IL-6,and IL-1β]and chemotactic cyto-kines CCL2 and CCL3 of CC subfamily were detected by RT-PCR.Results In the mono-cultured cells,the expression levels of p-ERK,p-p38 and p-p65 in CaCo2 cells increased after TNF-αstimulation for 30 min,and the expression levels of p-p38,p-IκBα,and p-p65 in THP1 cells increased after TNF-αstimulation for 90 min(all P<0.05).Compared with the mono-cultured cells with the same time of TNF-αstimulation,the expression levels of p-ERK,p-p38 and p-IκBαin CaCo2 cells after co-culture decreased after TNF-αstimulation for 90 min(all P<0.05);the expression levels of p-ERK,p-p38,p-IκBαand p-p65 in the THP1 cells after co-culture decreased after 90 min of TNF-αstimulation(all P<0.05).In the mono-cultured cells,the expression levels of p-ERK,p-p38,p-IκBαand p-p65 in HCA7 cells increased after 120 min of TNF-αstimulation,and the expression levels of p-ERK,p-p38 and p-p65 in THP1 cells increased after 30 and 120 min TNF-αstimulation(all P<0.05).Compared with the mono-cultured cells with the same time of TNF-αstimulation,the ex-pression levels of p-ERK,p-IκBαand p-p65 in HCA7 cells after co-culture decreased after TNF-αstimulation for 120 min,and the expression levels of p-ERK,p-p38 and p-p65 in the co-cultured ThP1 cells decreased after TNF-αstimu-lation for 30 and 120 min(all P<0.05).Compared with DMEM medium group with the same time of TNF-αstimulation,the expression levels of p-ERK,p-p38,p-IκBαand p-p65 decreased after 120 min of TNF-αstimulation in CaCO2 condi-tioned medium group.Meanwhile,the mRNA relative expression levels of IL-8,IL-1βand CCl2 decreased after TNF-αstimulation for 60 min(all P<0.05).However,the relative expression levels of IL-6 and CCL3 mRNA did not significant-ly change between these two groups(all P>0.05).Conclusions The activation of MAPK and NF-κB pathways and the secretion of pro-inflammatory factors were inhibited after co-culture of intestinal epithelial cells CaCO2,HCA7 and macro-phages THP1 in a micro-inflammatory environment.The signal communication between these cells was mediated by the se-cretion of soluble mediators.