香草乙酮静注对大鼠肺缺血再灌注损伤的预防作用观察

Preventive effect of intravenous injection of apocynin on rat ischemia-reperfusion injury

目的观察香草乙酮(APO)对大鼠肺缺血再灌注损伤(LIRI)的预防作用。方法将24只SD大鼠随机分为APO组、LIRI组、假手术(SO)组各8只,APO组在造模前30 min静注50 mg/kg的APO,APO组和LIRI组采用在体左肺门夹闭60 min后再灌注120 min的方法制备LIRI模型,SO组仅游离左肺门、不夹闭。各组再灌注后120min剖杀取材。切取约1/3新鲜左肺组织,计算湿干比(W/D);另取肺组织的部分石蜡包埋切片,经HE染色后在光学显微镜下观察其病理改变并进行肺病理评分;采用DHE法检测肺组织活性氧自由基(ROS),硫代巴比妥酸法和水溶性四唑盐-1法检测肺组织丙二醛(MDA)、超氧化物歧化酶(SOD);免疫荧光法检测肺组织MPO、TNF-α、IL-1β、IL-6。免疫组化法检测肺组织NAPDH氧化酶2(NOX2)、NAPDH氧化酶4(NOX4)蛋白。结果 SO组肺间质及肺泡结构清晰、完整,未见明显充血、水肿及炎性浸润;LIRI组肺间质水肿增宽,炎症细胞浸润,肺泡毛细血管扩张,肺泡内可见较多红细胞渗出,损伤明显;APO组的上述改变减轻。与SO组比较,APO组及LIRI组W/D、病理学评分、ROS平均荧光强度、MDA浓度、MPO阳性细胞数和TNF-α、IL-1β、IL-6平均荧光强度升高,SOD活性降低(P均<0. 05);与LIRI组比较,APO组W/D、病理学评分、ROS平均荧光强度、MDA浓度、MPO阳性细胞数和TNF-α、IL-1β、IL-6平均荧光强度降低,SOD活性升高(P均<0. 05)。与SO组比较,APO组和LIRI组NOX2、NOX4蛋白IOD值升高(P均<0. 05);与LIRI组比较,APO组NOX2、NOX4蛋白IOD值降低(P均<0. 05)。结论 APO能够有效预防大鼠LIRI,其机制可能是抑制肺组织NOX2、NOX4蛋白表达。

Objective To observe the preventive effect of apocynin( APO) on rat lung ischemia-reperfusion injury( LIRI). Methods Twenty-four SD rats were randomly assigned to three groups( n =8) : APO group,LIRI group,and SO group. Rats in the APO group were injected with APO at a dose of 50 mg/kg 30 minutes before surgery. In the APO group and LIRI group,rats underwent orthotopic LIRI modeling with clamping left hilum for 60 minutes and reperfusion for120 minutes;while rats in the SO group underwent surgery with separating the hilum rather than clamping it. All rats were euthanized at 120 min after reperfusion. The fresh left lung tissues( 1/3) were dissected,and the W/D ratio was calculated;part of left lung tissues was paraffin embedded and sliced for HE staining,and then was observed under an optical microscope and scored for histopathology. DHE assay was applied to detect the reactive oxygen species( ROS);thiobarbituric acid method and water soluble tetrazol-1 method were used to detect the malondialdehyde( MDA) and superoxide dismutase( SOD) in the lung tissues;immunofluorescence was used to detect the expression levels of MPO,TNF-α,IL-1β,and IL-6 in the lung tissues;immunohistochemistry was applied to detect the expression levels of NADPH oxidase 2( NOX2) and NAPDH oxidase 4( NOX4) in the lung tissues. Results SO group showed clear and intact interstitial and alveolar structure without obvious congestion,edema or inflammatory infiltration in the lung tissues;LIRI group exhibited interstitial edema,inflammatory infiltration,dilation of alveolar capillaries,exudation of erythrocytes in the alveoli and obvious damage in the lung tissues;while these changes were alleviated in the APO group. Compared with the SO group,the W/D ratio,histopathology score,mean fluorescent intensity of ROS,TNF-α,IL-1β,IL-6,MDA concentration,and MPO positive cells increased and the SOD activity decreased in the APO and LIRI groups( all P < 0. 05). Compared with the LIRI group,W/D ratio,histopathology score,mean fluorescent intensity of ROS,TNF-α,IL-1β,IL-6,MDA concentration,MPO positive cells decreased,and the SOD activity increased in the APO group( all P < 0. 05). Compared with the SO group,LIRI and APO groups showed increased IOD values of NOX2 and NOX4( both P < 0. 05). Compared with the LIRI group,APO group exhibited the decreased IOD values of NOX2 and NOX4( both P < 0. 05). Conclusion APO may prevent rats from LIRI probably by inhibiting the NOX2 and NOX4 protein expression.