血管活性肠肽对肺泡巨噬细胞活化和p38MAPK信号通路影响的实验研究

Vasoactive intestinal peptide inhibits alveolar macrophage activation by p38MAPK signaling pathway

目的研究血管活性肠肽(vasoactive intestinal peptide,VIP)对肺泡巨噬细胞活化的影响。方法原代小鼠肺泡巨噬细胞分为Normal组(正常对照)、LPS组(脂多糖刺激巨噬细胞活化)、LPS+VIP组(脂多糖刺激巨噬细胞活化后,10^(-6)mol/L VIP干预)。3 C57BL/6小鼠随机分为Normal组(正常对照),ALI组(急性肺损伤),ALI+VIP组(急性肺损伤后,10^(-8)mol/L VIP气管滴入)。免疫荧光法检测肺泡巨噬细胞标记物CD86和白细胞介素-6(interleukin-6,IL-6)的表达;ELISA法检测细胞培养上清和血清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、IL-6的含量;RT-PCR法检测巨噬细胞和肺组织内TNF-α、IL-6、p38丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)mRNA的表达水平;Western blot法检测各组巨噬细胞和肺组织内TNF-α、IL-6、p38MAPK、p-p38MAPK蛋白的表达。结果与Normal组比较,经LPS刺激后,ALI组小鼠肺组织肺泡巨噬细胞和LPS组原代肺泡巨噬细胞活化增强,小鼠血清和细胞培养上清中TNF-α、IL-6含量升高,肺组织和巨噬细胞中TNF-α、IL-6、pp38MAPK、p38MAPK蛋白和TNF-α、IL-6、p38MAPK mRNA的相对表达增高。与ALI和LPS组比较,VIP干预后,ALI+VIP和LPS+VIP组肺泡巨噬细胞活化被抑制,血清和细胞培养上清中TNF-α、IL-6含量降低,肺组织和细胞中TNF-α、IL-6、p38MAPK、p-p38MAPK蛋白和TNF-α、IL-6、p38MAPKmRNA的相对表达降低。结论VIP能抑制肺泡巨噬细胞活化,减少炎症因子IL-6、TNF-α的激活和释放,其机制可能与p38MAPK信号通路相关。

To observe the effect of vasoactive intestinal peptide(VIP)on the alveolar macrophages,the primary mouse alveolar macrophages were recruited and divided into normal group(normal control),LPS group(lipopolysaccharide stimulation),LPS+VIP group(lipopolysaccharide stimulation+10^(-6)mol/L VIP intervention).C57BL/6 mice were randomly divided into normal control group,ALI group(acute lung injury),and ALI+VIP group(acute lung injury+10^(-8)mol/L VIP tracheal drip).Immunofluorescence method was used to detect the expression of alveolar macrophage marker CD86 and IL-6;ELISA was applied to measure the levels of TNF-αand IL-6 in supernatant and serum;RT-PCR method was used to detect the mRNA expression of TNF-α,IL-6,p38 mitogen activation protein kinase(MAPK)in each group of cells and lung tissue;Western blot method was used to detect the expression of TNF-α,IL-6,p38MAPK,pp38MAPK proteins in macrophages and lung tissues of each group.Compared with the normal group,the activation of alveolar macrophages in the ALI group and the LPS group were enhanced,the levels of TNF-αand IL6 in the mouse serum and the cell culture supernatant were increased,and the relative expression of TNF-α,IL-6,p-p38MAPK,p38MAPK proteins and TNF-α,IL-6,p38MAPK mRNA were increased in the lung tissue and macrophages.Compared with the ALI and LPS groups,the activation of alveolar macrophages in the ALI+VIP and LPS+VIP groups were inhibited,the levels of TNF-αand IL-6 in the serum and the cell culture supernatant were reduced,the relative expression of TNF-α,IL-6,p38MAPK,p-p38MAPK proteins and TNF-α,IL-6,p38MAPK mRNA in the lung tissues and cells were reduced.In conclusion,VIP can suppress the activation of alveolar macrophages and reduce the activation and release of inflammatory factors IL-6 and TNF-α,and the mechanism may relate to the activation of p38MAPK signaling pathway.