电针预处理对脓毒症急性肺损伤大鼠肺组织中 NEK7- NLRP3炎症小体激活的影响

Effect of Electroacupuncture Preconditioning on Activation of NEK7-NLRP3 Inflammasome in Lung Tissue of Rats with Sepsis Acute Lung Injury

目的观察电针预处理“足三里”穴和“尺泽”穴对脓毒症急性肺损伤(ALI)大鼠肺组织中NEK7-NLRP3炎症小体激活的影响,探讨电针预处理在脓毒症ALI中发挥的保护效应及可能机制。方法将SD大鼠随机分为对照组、电针预处理+对照组、模型组、电针预处理+模型组,每组10只。各模型组采用腹腔注射脂多糖(LPS)的方法建立脓毒症ALI大鼠模型。各电针预处理组于LPS造模前1周进行连续7 d电针预处理,疏密波,频率4 Hz/20 Hz,强度1~2 mA,持续30 min。检测各组大鼠肺功能;HE染色法观察大鼠肺组织病理学变化;测定大鼠肺组织湿/干质量比值(W/D);ELISA法检测大鼠血浆及肺组织中炎症因子IL-1β、IL-18含量;免疫荧光观察大鼠肺组织中ASC蛋白阳性表达;Western blot法检测大鼠肺组织中NEK7、NLRP3、Caspase-1及IL-1β蛋白的表达。结果与对照组相比,模型组大鼠用力肺活量(FVC)、第0.1秒用力呼气量(FEV_(0.1))、第0.3秒用力呼气量(FEV_(0.3))、FEV_(0.1)/FVC、FEV_(0.3)/FVC均显著降低(P<0.001);肺泡结构紊乱,肺组织内炎性细胞浸润明显,肺组织充血水肿;W/D比值显著升高(P<0.001);血浆及肺组织内IL-1β、IL-18含量明显升高(P<0.001);ASC蛋白阳性表达明显增多(P<0.001);肺组织中炎症小体相关蛋白NEK7、NLRP3、Caspase-1及IL-1β的表达水平均显著升高(P<0.001)。与模型组相比,电针预处理+模型组大鼠FVC、FEV_(0.1)、FEV_(0.3)、FEV_(0.1)/FVC、FEV_(0.3)/FVC均明显升高(P<0.05,P<0.001);肺组织内炎症细胞浸润及充血水肿有明显改善;W/D比值显著降低(P<0.01);血浆及肺组织内IL-1β、IL-18含量显著降低(P<0.01,P<0.001);ASC蛋白阳性表达降低(P<0.001);肺组织中炎症小体相关蛋白NEK7、NLRP3、Caspase-1、IL-1β表达水平均显著降低(P<0.05,P<0.01,P<0.001)。结论电针预处理可以减轻肺部炎性反应,改善肺功能,其机制与电针抑制脓毒症ALI大鼠肺组织中NEK7-NLRP3炎症小体激活相关。

OBJECTIVE To observe the effect of electroacupuncture(EA)pretreatment at"Zusanli"(ST 36)and"Chize"(LU 5)points on the activation of NEK7-NLRP3 inflammasome in the lung tissue of rats with sepsis acute lung injury(ALI),and to explore the protective effect and possible mechanism of EA pretreatment in septic ALI.METHODS According to the principle of randomization,40 rats were equally divided into 4 groups,namely the control group,EA preconditioning+control group,model group and EA preconditioning+model group.The septic ALI model was established by intraperitoneal injection of lipopolysaccharide(LPS).EA(4 Hz/20 Hz,1-2 mA)was applied to bilateral ST 36 and LU 5 for 30 min,once a day for 7 consecutive days before modeling.The lung function of the rats was detected;HE staining was used to observe the pathological changes of the lung,and the degree of pulmonary edema in the left lung of rats was detected by the ratio of wet/dry weight of the lung;the secretions of inflammatory factors IL-1βand IL-18 in the plasma and lung tissue were measured by ELISA kits;the positive expression of ASC protein in the lung tissue of rats was observed by immunofluorescent staining;the expression of NEK7,NLRP3,Caspase-1 and IL-1βprotein was assayed by Western blot.RESULTS Compared with the control group,the forced vital capacity(FVC),forced expiratory volume at 0.1 second(FEV_(0.1)),FEV_(0.3),FEV_(0.1)/FVC,FEV_(0.3)/FVC were significantly decreased(P<0.001);Inflammatory cell infiltration in the lung tissue was obvious and accompanied by congestion and edema,and the alveolar structure was ruptured;the ratio of W/D was significantly increased(P<0.001);the contents of IL-1βand IL-18 in plasma and lung tissue were significantly increased(P<0.001)while the ASC protein positive expression was increased significantly(P<0.001);the expression levels of inflammasome-related proteins NEK7,NLRP3,Caspase-1 and IL-1βin lung tissue were significantly increased in the model group(P<0.001).After EA intervention,the FVC,FEV_(0.1),FEV_(0.3),FEV_(0.1)/FVC,FEV_(0.3)/FVC were significantly increased(P<0.05,P<0.001),and the lung pathology has improved significantly.The other indicators mentioned above were significantly decreased in the EA preconditioning+model group(P<0.05,P<0.01,P<0.001).CONCLUSION EA pretreatment can reduce pulmonary inflammatory response and improve lung function in septic ALI rats,which is relevant with inhibiting the activation of NEK7-NLRP3 inflammasome.