转染DPP-4 siRNA或(和)加入SP600125的小鼠肺泡巨噬细胞极化及JNK/AP-1信号通路激活情况观察

Observation on alveolar macrophage polarization and activation of JNK/AP-1 signaling pathway in mice transfected with DPP-4 siRNA or(and)added with SP600125

目的观察转染二肽基肽酶-4(DPP-4)siRNA或(和)加入c-Jun N-末端激酶(JNK)抑制剂(SP600125)的小鼠肺泡巨噬细胞极化情况、JNK/AP-1信号通路激活情况。方法体外培养小鼠肺泡巨噬细胞(MH-S),并随机分组:Control组转染空载siRNA、siDPP-4组转染DPP-4 siRNA、LPS组转染空载siRNA+LPS、siDPP-4+LPS组转染DPP-4 siRNA+LPS、LPS+SP600125组转染空载siRNA+LPS联合SP600125、siDPP-4+LPS+SP600125组转染DPP-4 siRNA+LPS联合SP600125。采用Western boltting法检测巨噬细胞中M1型和M2型极化标志物CD86、CD206蛋白及JNK、激活蛋白-1(AP-1)转录蛋白(c-Jun、c-Fos)磷酸化,RT-qPCR法检测巨噬细胞中M1型标志物(CD86、TNF-α、iNOS、IL-1β)和M2型标志物[CD206、精氨酸激酶-1(ARG-1)、IL-4、IL-10]mRNA,一氧化氮(NO)测定试剂盒、免疫荧光检测巨噬细胞上清液中M1型促炎因子NO和细胞内活性氧(ROS)生成情况。结果与Control组比较,LPS组M1型促炎型因子NO、ROS生成含量及M1型极化标志物(CD86蛋白及CD86、TNF-α、iNOS、IL-1βmRNA)表达升高,M2型极化标志物(CD206蛋白及CD206、ARG-1、IL-4、IL-10 mRNA)表达降低,P均<0.05。与LPS组比较,siDPP-4+LPS组M1型促炎型因子NO、ROS生成含量及M1型极化标志物(CD86蛋白及CD86、TNF-α、iNOS、IL-1βmRNA)表达升高,M2型极化标志物(CD206蛋白及CD206、ARG-1、IL-4、IL-10 mRNA)表达降低,P均<0.05。与LPS组比较,siDPP-4+LPS组p-JNK/JNK、p-c-Jun/c-Jun、p-c-Fos/c-Fos蛋白磷酸化相对表达量升高,P均<0.05。与siDPP-4+LPS组比较,siDPP-4+LPS+SP600125组p-JNK/JNK、p-c-Jun/c-Jun、p-c-Fos/c-Fos蛋白磷酸化相对表达量降低,P均<0.05。结论转染DPP-4 siRNA可促进LPS诱导的小鼠肺泡巨噬细胞M1型极化,抑制肺泡巨噬细胞M2型极化,并增加JNK、c-Jun、c-Fos蛋白磷酸化表达;加入JNK抑制剂后,可降低由转染DPP-4 siRNA引起的JNK、c-Jun、c-Fos蛋白磷酸化表达升高。转染DPP-4 siRNA促进LPS诱导的小鼠肺泡巨噬细胞M1型极化的作用机制可能与激活JNK/AP-1信号通路有关。

Objective To observe the polarization of alveolar macrophages and the activation of JNK/AP-1 signaling pathway in mice transfected with dipeptidyl peptidase-4(DPP-4)siRNA or(and)added with c-Jun N-terminal kinase(JNK)inhibitor(SP600125).Methods Mouse alveolar macrophages(MH-S)were cultured in vitro and randomly grouped into:control group(transfected with empty siRNA),siDPP-4 group(transfected with DPP-4 siRNA),lipopolysaccharide(LPS)group(transfected with empty siRNA+LPS),siDPP-4+LPS group(transfected with DPP-4 siRNA+LPS),LPS+SP600125 group(transfected with empty siRNA+LPS combined with SP600125),and siDPP-4+LPS+SP600125 group(transfected with DPP-4 siRNA+LPS combined with SP600125),respectively.The expression levels of M1 and M2 polarization markers CD86 and CD206 proteins and the phosphorylation of JNK and activator protein-1(AP-1)transcription proteins(c-Jun and c-Fos)in macrophages were detected by Western blotting,the mRNA levels of M1 markers[CD86,tumor necrosis factor(TNF)-α,iNOS,IL-1β]and M2 markers[CD206,arginine kinase-1(ARG-1),IL-4,IL-10]in macrophages were detected by RT-qPCR,and M1 proinflammatory factor NO and intracellular reactive oxygen species(ROS)production in macrophage supernatant were detected by nitric oxide(NO)assay kit and immunofluores-cence,respectively.Results Compared with the control group,the production of M1 proinflammatory factors NO and ROS,and the expression levels of M1 polarization markers(CD86 protein and CD86,TNF-α,iNOS,IL-1βmRNA)in-creased,and the expression levels of M2 polarization markers(CD206 protein and CD206,ARG-1,IL-4,IL-10 mRNA)decreased in the LPS group(all P<0.05).Compared with the LPS group,the production of M1 proinflammatory factors NO and ROS,and the expression levels of M1 polarization markers(CD86 protein and CD86,TNF-α,iNOS,IL-1βmRNA)increased,and the expression levels of M2 polarization markers(CD206 protein and CD206,ARG-1,IL-4,IL-10 mRNA)decreased in the siDPP-4+LPS group(all P<0.05).Compared with the LPS group,the relative expression lev-els of p-JNK/JNK,p-c-Jun/c-Jun and p-c-Fos/c-Fos protein phosphorylation increased in the siDPP-4+LPS group(all P<0.05).Compared with the siDPP-4+LPS group,the relative expression levels of p-JNK/JNK,p-c-Jun/c-Jun,p-c-Fos/c-Fos protein phosphorylation decreased in the siDPP-4+LPS+SP600125 group(all P<0.05).Conclusions Transfection of DPP-4 siRNA can promote the M1 polarization of mouse alveolar macrophages induced by LPS,inhibit the M2 polariza-tion of alveolar macrophages,and increase the phosphorylation expression of JNK,c-Jun and c-Fos proteins.Addition of JNK inhibitor may reduce the increase in phosphorylation expression of JNK,c-Jun and c-Fos proteins caused by transfec-tion of DPP-4 siRNA.The mechanism of DPP-4 siRNA transfection promoting M1 polarization in mouse alveolar macro-phages induced by LPS may be related to the activation of JNK/AP-1 signaling pathway.