YAP干扰下调MARCO受体表达抑制肺泡巨噬细胞的吞噬功能

YAP Inhibits Phagocytosis of Alveolar Macrophages by Downregulation of MARCO Receptors

目的探讨Yes相关蛋白(Yes-associated protein,YAP)对肺泡巨噬细胞(alveolar macrophages,AMs)吞噬功能的影响及相关机制。方法通过鼻滴脂多糖(lipopolysaccharide,LPS)构建急性肺损伤(ALI)小鼠模型,24 h后经支气管肺泡灌洗获取原代AMs,采用Western blot检测原代AMs中YAP的表达;使用200 ng/mL的TNF-α刺激肺泡巨噬细胞株MH-S,24 h后检测YAP蛋白表达情况。MH-S细胞经YAP siRNA干扰表达或经Verteporfin抑制YAP活性48 h后再与FITC-E.coli共孵育1 h,流式细胞术检测MH-S细胞吞噬率变化。MH-S细胞经MARCO阻断抗体(MARCO Ab)作用6 h后,检测其吞噬E.coli能力的变化。YAP siRNA干扰MH-S细胞YAP表达48 h后,检测MARCO受体的表达情况。结果与对照组相比,ALI小鼠AMs总蛋白(P<0.05)和胞质蛋白(P<0.01)中YAP的表达均下降;TNF-α刺激后的MH-S细胞,其总蛋白和胞质蛋白中YAP蛋白水平均下降(均P<0.05)。与干扰对照组(Sc siRNA组)相比,YAP siRNA组MH-S细胞吞噬能力降低(P<0.01);与对照组相比,YAP活性抑制组MH-S细胞的吞噬率明显下降(P<0.01)。用MARCO Ab阻断MARCO受体后,MH-S吞噬率降低(P<0.01)。与Sc siRNA组相比,YAP siRNA组MH-S细胞MARCO受体表达降低(P<0.01)。结论ALI小鼠AMs中YAP蛋白表达下降,YAP的下调可以导致AMs表面MARCO受体表达降低和吞噬E.coli能力减弱。

Objective To explore the effect of Yes-associated protein(YAP)on the phagocytosis of alveolar macrophage(AM)and its related mechanism.Methods ALI mouse model was constructed by nasal drop of lipopolysaccharide(LPS),and primary AM was obtained by bronchoalveolar lavage(BAL)after 24 h.The expression level of YAP in primary AM was detected by Western blotting;the AM cell line MH-S was stimulated with 200 ng/mL of TNF-αand YAP protein level was detected after 24 h.MH-S cells were interfered by YAP siRNA or verteporfin for 48 h and then co-incubated with FITC-E.coli for 1 h.Changes in phagocytosis rate were detected by flow cytometry.The phagocytic ability of MH-S cells on E.coli was observed after they were blocked for 6 h by MARCO inhibitory antibodies(MARCO Ab).The expression of MARCO receptor in MH-S cells was detected after being interfered by YAP siRNA for 48 h.Results Compared with the control group,the expression levels of YAP were decreased in total protein(P<0.05)and cytoplasmic protein(P<0.01)in the ALI group.YAP levels were decreased in both total protein and cytoplasmic protein in the TNF-αstimulated group(both P<0.05).Compared with the Sc siRNA group,the MH-S phagocytosis rate was reduced in the YAP siRNA group(P<0.01);compared with the control group,the phagocytosis rate of MH-S in the YAP activity inhibition group was significantly decreased(P<0.001).After blocking MARCO receptors with MARCO Ab,the phagocytosis rate of MH-S cells was significantly reduced(P<0.01).Compared with the Sc siRNA group,the expression of MARCO receptor in MH-S cells was significantly lower in the YAP siRNA group(P<0.01).Conclusion YAP protein expression is decreased in AM of ALI mice,and downregulation of YAP could lead to reduced expression of MARCO receptors on the surface of AMs and diminished phagocytosis ability on E.coli.