NF-κB在电针上调内毒素休克诱发急性肺损伤兔血红素氧合酶-1表达中的作用

Role of nuclear factor-kappaB in electro-acupuncture-induced up-regulation of heme oxygenase-1 expression in rabbits with endotoxic shock-induced acute lung injury

目的 探讨NF-κB在电针上调内毒素休克诱发急性肺脏损伤兔血红素氧合酶-1（HO-1）中的作用.方法 健康雄性新西兰大白兔60只,体重1.5 ～ 2.0kg,采用随机数字表法分为6组（n=10）：对照组（C组）、内毒素休克诱发急性肺损伤组（ALI组）、假电针＋ALI组（SEA组）、电针＋ALI组（EA组）、NF-κB抑制剂PDTC组、电针＋ALI＋PDTC组（EAP组）.ALI组、SEA组、EA组和EAP组静脉注射脂多糖5 mg/kg以制备内毒素休克诱发急性肺损伤模型,C组和PDTC组给予等容量生理盐水.静脉注射脂多糖或生理盐水前0.5h,PDTC组和EAP组脉输注PDTC 20 mg/kg,其余各组给予等容量生理盐水.EA组和EAP组于模型制备前1～4d及模型制备当天电针肺俞穴和足三里穴（疏密波,频率2/100 Hz,刺激电流1～2 mA,波宽0.2 ～ 0.6 ms,以兔出现轻微肌颤为宜,持续刺激30 min）,1次/d,未次电针结束后30 min时制备模型.给予生理盐水或LPS后6h放血处死动物,取肺组织,观察病理学结果,并进行病理学评分,计算肺湿/干重（W/D）比；采用荧光定量PCR法测定HO-1 mRNA的表达；采用Western blot法测定HO-1及NF-κBp65蛋白的表达.结果 与C组比较,ALI组、SEA组、EA组、EAP组肺组织病理学评分、W/D比、MDA含量升高,SOD活性降低,肺组织HO-1蛋白及其mRNA、总NF-κBp65蛋白、核内NF-κBp65蛋白表达上调（P＜0.05）,PDTC组上述指标差异无统计学意义（P＞0.05）；与ALI组比较,EA组肺组织病理学评分、W/D比、MDA含量降低,SOD活性升高,肺组织HO-1蛋白及其mRNA、总NF-κBp65蛋白、核内NF-κBp65蛋白表达上调,EAP组肺组织病理学评分、W/D比、MDA含量升高,SOD活性降低,肺组织HO-1蛋白及其mRNA、核内NF-κBp65蛋白表达下调（P＜0.05）,SEA组上述指标差异无统计学意义（P＞0.05）；与EA组比较,EAP组肺组织病理学评分、W/D比、MDA含量升高,SOD活性降低,肺组织HO-1蛋白及其mRNA、总NF-κBp65蛋白、核内NF-κBp65蛋白表达下调（P＜0.05）.结论 NF-κB活化后可介导电针上调HO-1表达,参与电针减轻兔内毒素休克诱发急性肺损伤的作用.

Objective To investigate the role of nuclear factor-kappaB （NF-κB） in electro-acupuncture （EA）-induced up-regulation of heme oxygenase-1 （HO-1） expression in rabbits with endotoxic shock-induced acute lung injury （ALI）.Methods Sixty New Zealand white rabbits,aged 2 months,weighing 1.5-2.0kg,were randomly divided into 6 groups （n=10 each）：control group （group C）,endotoxic shock-induced ALI group,sham EA ＋ ALI group （SEA group）,EA ＋ ALI group （EA group）,PDTC （NF-κB inhibitor） group,and EA ＋ ALI ＋ PDTC group （EAP group）.The animals were anesthetized with intramuscular xylazine hydrochloride 0.8 mg/kg and tracheostomized.Ear vein was cannulated for drug administration.Lipopolysaccharide （LPS） 5 mg/kg was injected intravenously in ALI,SEA,EA and EAP groups,while the equal volume of normal saline （NS） was given instead in C and PDTC groups.Endotoxin shock was confirmed by decrease in blood pressure by 25％ of the baseline value.PDTC 20 mg/kg was infused intravenously at 0.5 h before LPS or NS injection in PDTC and EAP groups,while the equal volume of NS was given instead in the other groups.15 min EA （wave length 0.2-0.6 ms,frequency 2/100 Hz,intensity ≤ 1-2 mA） stimulation of Zusanli and Feishu was performed once a day for 4 days before establishment of endotoxic shock model and on the day of establishment of endotoxic shock model in EA and EAP groups.The model was established at 30 min after the end of the last EA.The animals were sacrificed by blood-letting at 6h after LPS or NS administration.The lungs were removed for microscopic examination and for caculation of wat/dry weight ratio（W/D ratio）,and for determination of malondialdehyde （MDA） content,superoxide dismutase （SOD） activity,the expression of HO-1 protein and mRNA and NF-κBp65 protein.The pathological changes of the lung was scored.Results Compared with group C,the pathological scores,W/D ratio and MDA content were significantly increased,SOD activity was decreased,and the expression of HO-1 protein and mRNA,and intranuclear NF-κBp65 protein was up-regulated in ALI,SEA,EA and EAP groups （P ＜ 0.05）,and no significant changes in the parameters mentioned above were found in PDTC group （P ＞ 0.05）.Compared with group ALI,the pathological scores,W/D ratio and MDA content were significantly decreased,SOD activity was increased,and the expression of HO-1 protein and mRNA,total NF-κBp65 protein and intranuclear NF-κBp65 protein was up-regulated in EA group,and the pathological scores,W/D ratio and MDA content were significantly increased,SOD activity was decreased,and the expression of HO-1 protein and mRNA,and intranuclear NFκBp65 protein was down-regulated in group EAP （P ＜ 0.05）,and no significant changes in the parameters mentioned above were found in SEA group （P ＞ 0.05）.Compared with group EA,the pathological scores,W/D ratio and MDA content were significantly increased,SOD activity was decreased and the expression of HO-1 protein and mRNA,total NF-κBp65 protein,and intranuclear NF-κBp65 protein was down-regulated in EAP group （P ＜0.05）.Conclusion Activated NF-κB mediates EA-induced up-regulation of HO-1 expression and is involved in EA-induced reduction of endotoxic shock-induced ALI in rabbits.