Effects of electroacupuncture pretreatment on M1 polarization of alveolar macrophages in rats with acute lung injury

电针预处理对急性肺损伤大鼠肺泡巨噬细胞M1极化的影响

Objective:To observe the effects of electroacupuncture(EA)pretreatment on M1 polarization of alveolar macrophages(AMs)in rats with acute lung injury(ALI)induced by lipopolysaccharide(LPS),and to explore the potential protective mechanism of EA.Methods:Forty Sprague-Dawley rats were randomly divided into a normal group,a model group,and three groups of EA pretreatment[including a Chize(LU5)group,a Zusanli(ST36)group and a Chize(LU5)plus Zusanli(ST36)group],with eight rats in each group.The model rats of ALI were established by instilling LPS[2 mg/(kg·bw)]into the trachea of rats for 3 h.The rats in each EA pretreatment group were pretreated with EA for 30 min per day at the corresponding bilateral acupoints 6 d before instilling LPS.Three hours after modeling,the pulmonary function of the rats was tested,and the lung tissue was taken to calculate the ratio of lung wet weight to dry weight(W/D).The pathological lung changes and the injury score were observed by hematoxylin-eosin staining.The contents of tumor necrosis factor(TNF)-α,interleukin(IL)-1β,and myeloperoxidase(MPO)in rat's bronchoalveolar lavage fluid(BALF)were detected by enzyme-linked immunosorbent assay.The mRNA and protein expression levels of M1 macrophage markers clusters of differentiation 86(CD86),inducible nitric oxide synthase(iNOS),and its signaling pathway factor Toll-like receptor(TLR)4,and nuclear factor-κB(NF-κB)p65 in the alveoli were detected by fluorescence quantitative polymerase chain reaction and Western blot,respectively.Results:After being induced by LPS,the pulmonary function of the model rats showed that the forced expiratory volume in 0.1 s(FEV0.1),forced expiratory volume in 0.3 s(FEV0.3),and their respective ratios of FEV to forced vital capacity(FVC)(including FEV0.1/FVC and FEV0.3/FVC)were significantly decreased(P<0.01),while the W/D of lung tissue was increased(P<0.01).The score of lung injury was significantly higher(P<0.01).The contents of TNF-α,IL-1β,and MPO in the BALF and the mRNA and protein expression levels of CD86,iNOS,TLR4,and NF-κB p65 in the lung tissue were significantly increased(P<0.01).After EA pretreatment,the FEV0.1,FEV0.3,FEV0.1/FVC,and FEV0.3/FVC were significantly increased,the lung injury score decreased significantly,and the contents of TNF-α,IL-1β,and MPO in the BALF and the expression levels of CD86,iNOS,TLR4,and NF-κB p65 mRNAs and proteins in the alveoli decreased significantly(P<0.05 or P<0.01).Compared with the other two single acupoint groups,the contents of TNF-α,IL-1β,and MPO in the BALF and the expression levels of CD86,iNOS,TLR4,and NF-κB p65 mRNAs in the alveoli in the Chize(LU5)plus Zusanli(ST36)group were significantly lower(P<0.01).Conclusion:EA pretreatment at Chize(LU5)and Zusanli(ST36)can inhibit inflammation and reduce pulmonary injury in ALI rats induced by LPS.The effect of the combination of Chize(LU5)and Zusanli(ST36)is better than that of using these two acupoints separately,and its mechanism may be related to the inhibition of AMs'M1 polarization by down-regulation TLR4/NF-κB signaling pathway.

目的:观察电针(EA)预处理对脂多糖(LPS)所致急性肺损伤(ALI)大鼠肺泡巨噬细胞(AMs)M1极化的影响,探讨EA保护ALI的可能机制.方法:将40只Sprague-Dawley大鼠随机分为正常组、模型组和3个电针预处理组(包括尺泽组、足三里组和尺泽加足三里组),每组8只.以LPS[2 mg/(kg·bw)]滴注大鼠气管复制ALI大鼠模型.各电针预处理组均于模型制备前6 d于相应双侧穴位行电针预处理(1次/d,30 min/次).模型制备3 h后大鼠进行肺功能检测,取肺组织计算肺的湿重/干重比值(W/D),苏木素-伊红染色观察肺组织病理改变,并进行肺损伤评分;酶联免疫吸附法检测大鼠支气管肺泡灌洗液(BLAF)中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β及髓过氧化物酶(MPO)的含量,荧光定量聚合酶链反应法和免疫印迹法分别检测肺泡M1型巨噬细胞标志物分化抗原簇86(CD86)、诱导型一氧化氮合酶(iNOS)及其信号通路Toll样受体(TLR)4、核因子-κB(NF-κB)p65 mRNA和蛋白表达.结果:LPS诱导3 h后大鼠肺功能显示第0.1秒用力呼气量(FEV0.1)、第0.3秒用力呼气量(FEV0.3)以及各自占用力肺活量(FVC)之比(FEV0.1/FVC;FEV0.3/FVC)均显著下降(P<0.01);肺组织W/D增高(P<0.01);肺损伤评分显著升高;BALF中TNF-α、IL-1β、MPO含量及AMs中CD86、iNOS、TLR4、NF-κB p65的mRNA及蛋白表达明显升高(P<0.01).经电针预处理后,FEV0.1、FEV0.3、FEV0.1/FVC及FEV0.3/FVC均显著上升(P<0.01);肺损伤评分降低(P<0.01);BALF中TNF-α、IL-1β、MPO含量及肺组织中CD86、iNOS、TLR4、NF-κB p65的mRNA及蛋白表达明显下降(P<0.05或P<0.01).尺泽加足三里组BALF中TNF-α、IL-1β、MPO含量及AMs中CD86、iNOS、TLR4、NF-κB p65的mRNA表达比其他两单穴组下降明显(P<0.01).结论:电针预处理尺泽和足三里对LPS所致大鼠ALI起到抑制炎症和减轻肺损伤的作用,且两穴合用优于单一穴位,其效应机制可能与电针下调TLR4/NF-κB信号通路从而抑制AMs向M1型极化有关.