血必净通过上调血红素氧合酶-1减轻脓毒症诱导肺损伤

Xuebijing Attenuates Septic Lung Injury Via Heme Oxygenase-1 Up-regulation

目的探讨血必净(XBJ)是否能通过上调血红素氧合酶-1(heme oxygenase-1,HO-1)的表达,来减轻脓毒症小鼠的肺损伤(ALI)。方法本实验在哈尔滨医科大学附属第一医院肝脾外科中心实验室进行。采用改良盲肠结扎穿孔法(CLP)制作脓毒症小鼠模型。48只雄性C57BL/6小鼠,随机分为假手术组(Sham组,n=12)、模型组(CLP组,n=12)、血必净组(XBJ+CLP,n=12)和HO-1抑制剂组(XBJ+Zn PP+CLP组,n=12)。血必净进行干预时,造模前2h经尾静脉注射血必净(2ml/kg),HO-1特异抑制剂锌卟啉Zn PP(40μmol/kg)在给予血必净1h后经腹腔注射,其余各组给予等体积的生理盐水。术后12h后处死,光镜下观察肺组织的形态学改变,检测肺组织的湿干重比值(W/D),测定小鼠血清中的炎性介质(TNF-ɑ、IL-6),采用Western blot法以及免疫组化法检测肺组织HO-1的表达情况,Western blot法检测HO-1上游转录因子——核因子E2相关因子2(Nrf-2)的表达情况。用单因素方差分析(ANOVA)进行4组间比较,再进行Turkey检验,进行两组间多重比较差异有统计学意义(P<0.05)。结果与参照组(Sham组)相比,CLP组血清中TNF-ɑ、IL-6水平均明显增加(32.45±9.62 vs573.51±105.26,47.38±11.84 vs 853.72±198.56pg/ml,P<0.05),光镜下观察肺损伤及炎性细胞浸润程度明显加重(肺损伤评分:3.24±1.38 vs 12.32±3.45,P<0.05),肺组织W/D比值增加(4.52±0.34 vs 5.98±0.85,P<0.05),蛋白印迹法及免疫组化法检测结果显示肺组织中HO-1、Nrf-2的蛋白表达含量均轻度的增加(P<0.05)。给予血必净(XBJ)干预后,与CLP组相比,血清中TNF-ɑ、IL-6释放均减少(573.51±105.26 vs 327.45±68.52,853.72±198.56 vs 504.72±113.28pg/ml,P<0.05),肺组织病理损伤及炎性细胞浸润程度减轻(肺损伤评分:12.32±3.45 vs 6.34±1.68,P<0.05),肺组织W/D比值减少(5.98±0.85 vs 4.75±0.56,P<0.05),肺组织中HO-1、Nrf-2蛋白表达含量均明显增加(P<0.05)。XBJ对于肺组织的上述保护作用被HO-1抑制剂Zn PP所逆转,HO-1抑制剂组的肺损伤程度与CLP组差异无统计学意义(肺损伤评分:12.32±3.45vs 11.45±2.85,P>0.05;肺W/D值:5.98±0.85 vs 5.73±0.64,P>0.05)。结论血必净对于脓毒症小鼠所诱发的急性肺损伤具有保护作用,其可能机制为上调HO-1通路减轻炎性反应。

Objective To investigate the effects of Xuebijing（XBJ） on septic lung injury in mice and its modulation of heme oxygenase-1（HO-1） in lung. Methods The experiment was carried out in the Key Hepatosplenic Surgery Laboratory of First Affiliated Hospital of Harbin Medical University. A model of sepsis in mice was made by using the modified method of Cecal ligation and puncture（CLP）. 48 male C57BL/6 mice were randomly divided into four groups：Sham group（n=12）, CLP group（n=12）, XBJ＋CLP（n=12） and XBJ＋ZnPP＋CLP group（n=12）. XBJ（2ml/kg） was injected via tail vein 2 hours before the CLP procedures, and ZnPP IX,a inhibitor of HO-1, was intraperitoneally injected 1 hour after the XBJ injection. The mice in other groups were intreperitoneally injected with the same volume of normal saline. The mice were sacrificed 12 hours after the CLP procedures.The changes of morphology of lung tissue was observed with optical microscope, the lung injury score and wet/dry ratio（W/D） were measured. The TNF-α, IL-6 in serum were assayed by ELISA. The Western blot and immunohistochemistry were used to detect the expression of HO-1 in the lung tissues. The protein expression of nuclear factor E2 related factor 2（Nrf-2）, a HO-1 upstream transcription factor, was determined by Western blot.Comparison of continuous variables among four groups was performed using One-way analysis of variance（ANOVA） followed by Tukey＇s post hoc test for multiple comparisons. P〈0.05 was considerd as statistically significant. Results Compared with the sham group, the TNF-α and IL-6 in serum were both increased（32.459.62 vs 573.51105.26, 47.3811.84 vs 853.72198.56pg/ml, P〈0.05） by CLP, the pathological injury and pulmonary edema were both aggravated（lung injury score：3.241.38 vs 12.323.45, wet/dry weight ratio：4.520.34 vs 5.980.85, P〈0.05） under light microscope. The HO-1 and Nrf-2 proteins were both slightly up-regulated in lung tissue in CLP group（P〈0.05）. After the intervention of XBJ, the TNF-α and IL-6 in serum（573.51105.26 vs 327.4568.52,853.72198.56 vs 504.72113.28pg/ml, P〈0.05）, pulmonary pathology injury（lung injury score：12.323.45 vs 6.341.68, P〈0.05） and lung pulmonary edema（W/D weight ratio：5.980.85 vs 4.750.56, P〈0.05） were all reduced, the HO-1 and Nrf-2 proteins were both significantly increased（P〈0.05）. However, the protective role of XBJ on the lung injury induced by sepsis was partly reversed by ZnPP. No significant difference was detected between the XBJ＋CLP＋ZnPP and CLP group（LIS：12.323.45 vs 11.452.85, P〉0.05;W/D weight ratio：5.980.85 vs 5.730.64,P〉0.05）. Conclusion XBJ has a protective effect on septic lung injury, reducing lung inflammation and alleviating lung injury, which may be related to heme oxygenase-1 up-regulation.