二肽基肽酶-4通过CXCR4/mTOR信号通路介导小鼠肺泡巨噬细胞MH-S自噬的机制研究

DPP-4 mediates autophagy in mouse alveolar macrophages through CXCR4/mTOR signaling pathway

摘要:

目的 探究二肽基肽酶-4（DPP-4）通过趋化因子受体4（CXCR4）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路介导小鼠肺泡巨噬细胞MH-S自噬的机制。方法 体外培养小鼠肺泡巨噬细胞MH-S并分组转染。分别设置PBS组（PBS培养MH-S细胞）、LPS组（100 ng/mL LPS诱导24 h）、DPP-4组（DPP-4表达病毒转染）、si-DPP-4组（si-DPP-4病毒载体转染）、DPP-4 + BafA1组（DPP-4表达病毒转染+自噬抑制剂BafA1干预）及si-DPP-4 + BafA1组（si-DPP-4病毒载体转染+自噬抑制剂BafA1干预）。绿色荧光蛋白（GFP）检测转染效率，稳定转染后，酶联免疫吸附试验检测MH-S细胞上清液炎症因子水平，腺病毒检测MH-S细胞自噬流变化，实时荧光定量聚合酶链反应检测细胞DPP-4、CXCR4、mTOR mRNA的表达，Western blottig检测CXCR4/mTOR通路蛋白的表达。结果 LPS组IL-1β、IL-6、TNF-α、GFP、RPF、Merge数量，CXCR4 mRNA、mTOR mRNA和CXCR4蛋白、p-mTOR/mTOR蛋白相对表达量高于PBS组（P <0.05）；DPP-4组与LPS组IL-1β、IL-6及TNF-α水平比较，差异无统计学意义（P >0.05）；DPP-4组GFP、RPF、Merge数量，DPP-4 mRNA相对表达量高于LPS组（P <0.05），CXCR4 mRNA、mTOR mRNA、CXCR4蛋白、p-mTOR/mTOR蛋白相对表达量低于LPS组（P <0.05）；si-DPP-4组IL-1β、IL-6、TNF-α水平高于LPS组（P <0.05），GFP、RPF及MergeMerge数量低于LPS组（P <0.05）；si-DPP-4组和DPP-4 + BafA1组IL-1β、IL-6、TNF-α水平、CXCR4蛋白、p-mTOR/mTOR蛋白相对表达量高于DPP-4组（P <0.05），GFP、RPF及Merge数量低于DPP-4组（P <0.05）；si-DPP-4组DPP-4 mRNA相对表达量低于DPP-4组（P <0.05），CXCR4 mRNA、mTOR mRNA相对表达量高于DPP-4组（P <0.05）；si-DPP-4 + BafA1组IL-1β、IL-6水平、CXCR4 mRNA、mTOR mRNA、CXCR4蛋白、p-mTOR/mTOR蛋白相对表达量高于si-DPP-4组（P <0.05），GFP、RPF、Merge数量低于si-DPP-4组（P <0.05）。结论 DPP-4能够调节小鼠肺巨噬细胞自噬作用，调控炎症反应，其作用机制可能与CXCR4/mTOR通路有关。

Abstract:

Objective To explore whether dipeptidyl peptidase-4 (DPP-4) mediates autophagy in mouse alveolar macrophages through CXCR4/mTOR signaling pathway.Methods Mouse alveolar macrophages (MH-S cells) were cultured in vitro, and were then grouped and transfected with different viruses. Specifically, the cells were divided into PBS group (MH-S cells were cultured in PBS), LPS group (MH-S cells were stimulated via 100 ng/mL of LPS for 24 h), DPP-4 group (MH-S cells were transfected with DPP-4 overexpression viral vector), si-DPP-4 group (MH-S cells were transfected with si-DPP-4 viral vector), DPP-4 + BafA1 group (MH-S cells were transfected with DPP-4 overexpression viral vector and treated with autophagy inhibitor BafA1) and si-DPP-4 + BafA1 group (MH-S cells were transfected with si-DPP-4 viral vector and treated with autophagy inhibitor BafA1). The fluorescence intensity of GFP was used to detect the transfection efficiency. After stable transfection, ELISA was used to detect the levels of inflammatory factors in the cell culture supernatants. Adenoviruses were used to detect the changes of autophagic flux in MH-S cells. The mRNA expressions of DPP-4, CXCR4 and mTOR in MH-S cells were determined via qPCR, and the expressions of proteins associated with CXCR4/mTOR pathway were measured via Western blotting.Results Compared with the PBS group, the levels of IL-1β, IL-6 and TNF-α were higher, the number of cells expressing GFP, RFP and both was increased, and the mRNA expressions of mTOR and CXCR4 and the protein expressions of CXCR4 and p-mTOR/mTOR were up-regulated in the LPS group (P < 0.05). There was no difference in the levels of IL-1β, IL-6 and TNF-α between the DPP-4 group and the LPS group (P > 0.05). Compared with LPS group, the number of cells expressing GFP, RFP and both was increased, and the mRNA expressions of mTOR and CXCR4 and the protein expressions of CXCR4 and p-mTOR/mTOR were down-regulated (P < 0.05). In the si-DPP-4 group, the levels of IL-1β, IL-6 and TNF-α were higher, and the number of cells expressing GFP, RFP and both was lower than those in the LPS group (P < 0.05). Compared with the DPP-4 group, the levels of IL-1β, IL-6 and TNF-α were higher, the protein expressions of CXCR4 and p-mTOR/mTOR were up-regulated, and the number of cells expressing GFP, RFP and both was inhibited in the si-DPP-4 group and the DPP-4 + BafA1 group (P < 0.05). The mRNA expression of DPP-4 was lower, but the mRNA expressions of CXCR4 and mTOR were higher in the si-DPP-4 group than those in the DPP-4 group (P < 0.05). Compared with the si-DPP-4 group, the levels of IL-1β, IL-6 and TNF-α were higher, the mRNA expressions of mTOR and CXCR4 and the protein expressions of CXCR4 and p-mTOR/mTOR were up-regulated, and the number of cells expressing GFP, RFP and both was lower in the si-DPP-4 + BafA1 group (P < 0.05).Conclusions DPP-4 can regulate the autophagy and the inflammatory response in mouse alveolar macrophages, which may be achieved via the CXCR4/mTOR pathway.