目的 探讨磷脂酰肌醇3-激酶δ(PI3 Kδ)-Ras同源基因家族成员A(RhoA)通路在慢性阻塞性肺疾病(慢阻肺)小鼠肺泡巨噬细胞(AM)吞噬功能低下中的作用.方法 40只SPF级8周龄雄性BALA/c小鼠,20只使用烟草烟雾暴露法建立慢阻肺模型,20只常规饲养.根据随机数字表分为健康对照组、慢阻肺组、健康IC87114(PI3 Kδ抑制剂)组及慢阻肺IC87114组.其中IC87114组AM培养基中加入终浓度1 nmol/L IC87114培养24 h.不连续密度梯度离心法分离AM,流式细胞术测AM吞噬异硫氰酸荧光素标记的大肠杆菌(FITC-E.coli的能力,以平均荧光强度(MFI)和吞噬FITC-E.coli阳性细胞百分比(吞噬％)表示;实时荧光定量PCR及蛋白印迹法(Western blot)测AMPI3Kδ、RhoA mRNA和蛋白表达;小G蛋白活性试剂测量AM RhoA活性;激光共聚焦显微镜观察细胞骨架.结果 AM吞噬能力:慢阻肺组MFI和吞噬％[(4 512 ±517)、(32.2±4.6)％]均低于健康对照组[(9857±1 042)、(68.0±4.0)％,均P＜0.01];慢阻肺IC87114组[(6 894±472)、(50.6±2.1)％]均高于慢阻肺组(均P ＜0.01).AM PI3Kδ mRNA和蛋白表达:慢阻肺组(3.14 ±0.54、0.84 ±0.08)均高于健康对照组(1.00±0.00、0.57±0.07,均P＜0.01);慢阻肺IC87114组(1.52±0.28、0.66 ±0.13)均低于慢阻肺组(均P＜0.01).AM RhoA mRNA、蛋白表达及活性:慢阻肺组(0.70 ±0.07、0.41 ±0.10、0.70 ±0.06)均低于健康对照组(1.00±0.00、0.56 ±0.09、1.19 ±0.09,均P＜0.01);慢阻肺IC87114组(0.91 ±0.08、0.48±0.06、0.86 ±0.06)均高于慢阻肺组(均P＜0.05).AM细胞骨架变化:健康对照组和健康IC87114组伪足伸出良好,吞噬FTTC-E.coli能力较强;慢阻肺组伪足伸出不良甚至缺失,吞噬FITC-E.coli能力受损;慢阻肺IC87114组伪足伸出情况及吞噬能力较慢阻肺组改善.相关性分析:基础状态和IC87114干预后,AM的PI3Kδ mRNA、蛋白表达与RhoAmRNA、蛋白表达及活性均呈负相关,PI3Kδ mRNA、蛋白表达与MFI均呈负相关,RhoA mRNA、蛋白表达及活性与MFI均呈正相关.结论 慢阻肺小鼠AM吞噬功能低下,细胞骨架异常重排;PI3 Kδ特异性调控RhoA,且两者呈负相关;慢阻肺小鼠AM PI3Kδ过度活化致RhoA活性降低从而使AM细胞骨架异常重排与其吞噬功能低下有关;PI3 Kδ抑制剂IC87114抑制慢阻肺小鼠AM PI3Kδ活化,使RhoA活性改善,部分恢复慢阻肺AM吞噬功能.%Objective To investigate effects of Phosphoinositide3-Kinases (PI3Kδ)-Ras homolog gene family member A (RhoA) pathway on phagocytosis deficiency of alveolar macrophages (AMs) in a mouse model of chronic obstructive pulmonary disease (COPD).Methods Twenty mice were exposed to cigarette smoking to establish the COPD model,with 20 mice as the control group.AMs were isolated fromlung tissue by discontinuous density gradient centrifugation and then divided into a healthy control group,aCOPD group,a healthy IC87114 group and a COPD IC87114 group.The culture of IC87114 group wasmixed with a final concentration of 1 nmol/L IC87114 for 24 hours.Mean fluorescence intensity (MFI) andthe positive percent of AMs engulfing flurescein isothiocyanate-labeled Escherichina coli (FITC-E.coli)(AM％) were detected by flow cytometry.Real-Time PCR (RT-PCR)and Western blot were applied todetect mRNA and protein.G-LISA RhoA Kit was used to detect the activity of RhoA,and laser scanningconfocal microscopy was used to observe the cytoskeleton structure of AMs.Results Phagocytosis of AM:MFI and AM ％ in the COPD group [(4 512 ± 517),(32.2 ± 4.6)％] were decreased than those in thehealthy control group [(9 857 ± 1 042),(68.0 ±4.0)％,all P ＜0.01].Compared with the COPD group,MFI and AM％ in the COPD IC87114 group [(6 894 ± 472),(50.6 ± 2.1) ％] were increased (all P ＜0.01).The expressions of mRNA and protein of PI3Kδ in the COPD group (3.14 ±0.54,0.84 ±0.08)were increased than those in the healthy control group (1.00 ± 0.00,0.57 ± 0.07) (all P ＜ 0.01).Compared with the COPD group,the expressions of mRNA and protein of PI3Kδ in the COPD IC87114 group (1.52 ± 0.28,0.66 ± 0.13) were decreased (all P ＜ 0.01).The RhoA mRNA,protein and activity in the COPD group (0.70 ±0.07,0.41 ±0.10,0.70 ±0.06) were decreased compared to those in the healthy control group (1.00 ± 0.00,0.56 ± 0.09,1.19 ± 0.09) (all P ＜ 0.01).Compared with the COPD group,the expression of mRNA,protein and activity of RhoA in the COPD IC87114 group(0.91 ± 0.08,0.48 ± 0.06,0.86 ± 0.06) were increased (P ＜ 0.01,P ＜ 0.05).Cytoskeleton of AM:The pseudopods of the healthy control group and the healthy IC87114 group extended well,and the ability of phagocytosing FITCE.coli was intact,but there were some defects in the COPD group.Compared with the COPD group,the COPD IC87114 group was better,both in phagocytosing and extending of pseudopods.Negative correlations existed between the mRNA,protein of PI3Kδ with mRNA,protein and activity of RhoA.Negative correlations also existed between the mRNA,protein of PI3Kδ with MFI,but positive correlations between RhoA and MFI were observed in all groups.Conclusion The phagocytosis of AMs in COPD mice was defective,with abnormal rearrangement of the cytoskeleton.PI3Kδ negatively regulated RhoA,while PI3Kδ over activation resulted in decreasing activity of RhoA and then induced abnormal cytoskeleton rearrangement in AMs,which led to phagocytosis deficiency.IC87114 inhibited PI3Kδ activation,improved the activity of RhoA and partly recovered phagocytosis of AMs.
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