下调细胞膜调控蛋白Paralemmin-3表达对肺泡巨噬细胞极化的调控作用

Regulation effect of downregulated expression of cell membrane regulatory protein Paralemmin-3 on alveolar macrophage polarization

目的探讨下调细胞膜调控蛋白Paralemmin-3(PALM3)表达对肺泡巨噬细胞极化的影响。方法(1)取大鼠肺泡巨噬细胞NR8383细胞,采用免疫荧光法检测其PALM3的表达情况。(2)采用100 ng/mL脂多糖、20 ng/mL白细胞介素(IL)-4分别刺激NR8383细胞极化为M1型、M2型肺泡巨噬细胞,并取未干预的正常NR8383细胞作为M0型肺泡巨噬细胞。检测NR8383细胞中的PALM3 mRNA、蛋白表达水平。(3)将NR8383细胞分为Control-siRNA组、PALM3-siRNA组和正常对照组。将Control-siRNA、PALM3-siRNA分别转染至Control-siRNA组、PALM3-siRNA组的NR8383细胞,不对正常对照组进行干预。评估转染效果后,使用100 ng/mL脂多糖、20 ng/mL IL-4刺激各组NR8383细胞。检测各组上清液中肿瘤坏死因子α(TNF-α)及IL-10含量,以及细胞中CD80、诱导型一氧化氮合成酶(iNOS)、CD206、精氨酸酶1(ARG1)的mRNA表达水平。结果(1)PALM3分子在NR8383细胞中有表达并定位于细胞膜。(2)M1型肺泡巨噬细胞、M0型肺泡巨噬细胞、M2型肺泡巨噬细胞中PALM3的mRNA和蛋白表达水平依次降低(P<0.05)。(3)经转染siRNA及脂多糖刺激后,与正常对照组及Control-siRNA组相比,PALM3-siRNA组NR8383细胞M1型表面标志物CD86和iNOS的mRNA表达水平下调,上清液TNF-α含量降低(P<0.05)。经转染siRNA及IL-4刺激后,与正常对照组及Control-siRNA组相比,PALM3-siRNA组NR8383细胞M2型表面标志物CD206和ARG1 mRNA表达水平上调,上清液IL-10含量升高(P<0.05)。结论PALM3表达于肺泡巨噬细胞的细胞膜,且在M1型肺泡巨噬细胞中呈高表达,下调PALM3的表达可抑制肺泡巨噬细胞的M1型极化并促进其M2型极化,有助于抑制促炎因子及促进抗炎因子的释放。

Objective To investigate the effect of downregulated expression of cell membrane regulatory protein Paralemmin-3(PALM3)on alveolar macrophage polarization.Methods(1)NR8383 cells of alveolar macrophages in rats were obtained,and their PALM3 expression was detected by using the immunofluorescence method.(2)NR8383 cells were stimulated to polarize into M1 and M2 alveolar macrophages by employing 100 ng/mL lipopolysaccharide,and 20 ng/mL interleukin(IL)4,respectively,and non intervened normal NR8383 cells were obtained as MO alveolar macrophages.The mRNA and protein expressions of PALM3 in NR8383 cells were detected.(3)NR8383 cells were assigned to Control-siRNA group,PALM3-siRNA group,or normal control group.Control-siRNA and PALM3-siRNA were transfected into NR8383 cells in the Control-siRNA group and PALM3-siRNA group,respectively,and no intervention was performed in the normal control group.After evaluating the transfection effct,NR8383 cells in various groups were stimulated with 100 ng/mL lipopolysaccharide and 20 ng/mL IL4.The contents of tumor necrosis factor a(TNF-a)and IL-10 in the supernatant of various groups were detected,and the mRNA expressions of CD80,indueible nitric oxide synthase(iNOS),CD206,and arginase 1(ARG1)in cells were detected.Results(1)PALM3 molecule was expressed in NR8383 cells and located in the cell membrane.(2)The mRNA and protein expressions of PALM3 in M1 alveolar macrophages,M0 alveolar macrophages,and M2 alveolar macrophages were decreased successively(P<0.05).(3)After transfection with siRNA and lipopolysaccharide stimulation,compared with the normal control group and the Control-siRNA group,the mRNA expressions of M1 surface markers CD86 and iNOS in NR8383 cells of the PALM3-siRNA group were down-regulated,and the content of TNF-c in the supernatant was decreased(P<0.05).After transfection with siRNA and IL4 stimulation,the mRNA expressions of M2 surface markers CD206 and ARG1 in NR8383 cells were up-regulated in the PALM3-siRNA group as compared with the normal control group and the Control-siRNA group,and the content of IL-10 in the supernatant was elevated(P<0.05).Conclusion PALM3 is expressed on the membrane of alveolar macrophages,and is highly expressed in MI alveolar macrophages.Down-regulation of PALM3 expression can inhibit M1 polarization of alveolar macrophages and promote their M2 polarization,which helps to inhibit the release of pro-inflammatory factors and promote the release of anti-inflammatory factors.