褪黑素在电针减轻肢体缺血再灌注诱发兔肺损伤中的作用

Role of melatonin in electroacupuncture-induced reduction of lung injury induced by limb ischemia-reperfusion in rabbits

目的:评价褪黑素在电针减轻肢体缺血再灌注诱发兔肺损伤中的作用。方法:清洁级健康雄性新西兰大白兔50只,体重2.0~2.5 kg,3月龄,采用随机数字表法分为5组(n=10):假手术组(Sham组)、肢体缺血再灌注组(IR组)、电针刺激穴位组(EA组)、电针刺激非穴位组(SEA组)和电针刺激穴位+褪黑素受体拮抗剂Luzindole组(EA+L组)。采用夹闭股动脉3 h后再灌注4 h的方法制备兔肢体缺血再灌注损伤模型。EA组和EA+L组选取双侧足三里和肺俞穴(进针深度4~6 mm),SEA组选取双侧足三里和肺俞穴旁开0.5 cm处(进针深度3 mm),于模型制备前1~7 d及模型制备过程中给予电针刺激,刺激参数设置:频率2/15 Hz,刺激电流l^2 mA,疏密波,以兔出现轻微肌颤为宜,每次持续30 min,1次/d。EA+L组于模型制备前30 min时腹腔注射Luzindole 30 mg/kg。分别于缺血前即刻(T 0)、缺血3 h(T 1)和再灌注4 h(T 2)时,收集右侧颈内静脉血样,采用ELISA法检测血清褪黑素浓度。再灌注4 h时,收集支气管肺泡灌洗液(BALF),采用ELISA法检测BALF中TNF-α、IL-1β和IL-6浓度,采用黄嘌呤氧化酶法测定BALF中SOD活性,采用硫代巴比妥酸法测定BALF中MDA浓度。随后处死兔,取肺组织,测定湿重/干重(W/D)比值,光镜下观察病理学结果并进行肺损伤评分,透射电镜下观察超微结构,计算线粒体数量和线粒体相对横截面积。结果:与Sham组比较,其余4组肺损伤评分和肺组织W/D比值升高,线粒体数量减少,线粒体相对横截面积增大,BALF中TNF-α、IL-1β、IL-6和MDA水平升高,SOD活性降低,IR组T 1和T 2时血清褪黑素浓度降低,EA组和EA+L组T 0时血清褪黑素浓度升高(P<0.05)。与IR组比较,EA组肺损伤评分和肺组织W/D比值降低,线粒体数量增加,线粒体相对横截面积减小,BALF中TNF-α、IL-1β、IL-6和MDA水平降低,SOD活性升高,EA组和EA+L组各时点血清褪黑素浓度升高(P<0.05),SEA组上述各指标差异无统计学意义(P>0.05)。与EA组比较,SEA组和EA+L组肺损伤评分和肺组织W/D比值升高,线粒体数量减少,线粒体相对横截面积增大,BALF中TNF-α、IL-1β、IL-6和MDA水平升高,SOD活性降低,SEA组各时点血清褪黑素浓度降低(P<0.05)。结论:电针可通过提高血清褪黑素水平减轻肢体缺血再灌注诱发兔肺损伤。

Objective To evaluate the role of melatonin in electroacupuncture(EA)-induced reduction of lung injury induced by limb ischemia-reperfusion(I/R)in rabbits.Methods Fifty clean-grade healthy male New Zealand white rabbits,weighing 2.0-2.5 kg,aged 3 months,were divided into 5 groups(n=10 each)using a random number table method:sham operation group(group Sham),limb I/R group(group IR),EA group,sham EA group(group SEA)and EA plus melatonin receptor antagonist luzindele group(group EA+L).The model of limb I/R injury was established by clamping the femoral artery for 3 h followed by 4-h reperfusion in anesthetized animals.In group EA and group EA+L,bilateral Zusanli and Feishu acupoints(4-6 mm depth)were stimulated with constant voltage(2/15 Hz,l-2 mA,disperse-dense waves)for 30 min once a day during 1-7 days before establishing the model and during establishment of the model.EA was performed at the points(3 mm depth)0.5 cm lateral to the acupoints of Zusanli and Feishu instead in group SEA.Luzinole 30 mg/kg was intraperitoneally injected at 30 min before establishing the model in group EA+L.Blood samples from the right internal jugular vein were collected before ischemia(T0),at 3 h of ischemia(T1)and 4 h of reperfusion(T2)for determination of the serum melatonin concentrations by enzyme-linked immunosorbent assay.Bronchoalveolar lavage fluid(BALF)was collected at 4 h of reperfusion for measurement of tumor necrosis factor-alpha(TNF-α),interleukin-1beta(IL-1β)and IL-6 concentrations(by enzyme-linked immunosorbent assay),superoxide dismutase(SOD)activity(by xanthine oxidase method),and malondialdehyde(MDA)concentration(by thiobarbituric acid method).Then the rabbits were sacrificed,and the lung tissues were taken for determination of wet to dry weight ratio(W/D ratio)and for microscopic examination of the pathological changes(with a light microscope)which were scored and ultrastructure(with a transmission electron microscope).The number of mitochondria and relative cross-sectional area of mitochondria were calculated.Results Compared with group Sham,lung injury scores and W/D ratio were significantly increased,the number of mitochondria was decreased,the relative cross-sectional area of mitochondria was increased,levels of TNF-α,IL-1β,IL-6 and MDA in BALF were increased,and activities of SOD in BALF were decreased in the other four groups,and the serum melatonin concentration was decreased at T1 and T2 in group I/R and increased at T0 in EA and EA+L groups(P<0.05).Compared with group IR,the lung injury score and W/D ratio were significantly decreased,the number of mitochondria was increased,the relative cross-sectional area of mitochondria was decreased,levels of TNF-α,IL-1β,IL-6 and MDA in BALF were decreased,and activities of SOD in BALF were increased in group EA,the serum melatonin concentration was increased at each time point in EA and EA+L groups(P<0.05),and no significant change was found in the parameters mentioned above in group SEA(P>0.05).Compared with group EA,lung injury scores and W/D ratio were significantly increased,the number of mitochondria was decreased,the relative cross-sectional area of mitochondria was increased,levels of TNF-α,IL-1β,IL-6 and MDA in BALF were increased,and activities of SOD in BALF were decreased in SEA and EA+L groups,and the serum melatonin concentration was decreased at each time point in group SEA(P<0.05).Conclusion EA can reduce lung injury induced by limb I/R by increasing serum melatonin level in rabbits.