黄芩素通过抑制核转录因子-κB活性减轻小鼠肠缺血／再灌注致急性肺损伤

Baicalein attenuates acute lung injury induced by intestinal ischemia/reperfusion via inhibition of nuclear factor-κB pathway in mice

目的探讨黄芩素对小鼠肠缺血／再灌注（I／R）致急性肺损伤（ALI）的作用及机制。方法按随机数字表法将24只清洁级健康雄性C57BL／6J小鼠分为假手术（Sham）组、I／R组和黄芩素＋I／R组，每组8只。采用夹闭系膜上动脉90min后恢复灌注制备肠I／R诱发小鼠肺损伤模型；黄芩素＋I／R组于制模前1h腹腔注射黄芩素100mg／kg；Sham组小鼠不钳夹血管，其余操作同I／R组。于再灌注4h后处死小鼠取下腔静脉血和肺组织标本。苏木素-伊红（HE）染色，光镜下观察小鼠肺组织病理学变化，并进行病理学评分；测定肺湿／干重（W／D）比值；采用原位末端缺刻标记试验（TUNEL）检测肺组织细胞凋亡情况；采用酶联免疫吸附试验（ELISA）检测血清肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）含量；采用实时定量反转录-聚合酶链反应（RT-PCR）检测肺组织TNF-α和IL-6的mRNA表达水平；采用蛋白质免疫印迹试验（Westem Blot）检测肺组织胞质核转录因子-κB（NF-κB）抑制因子-α（IκB-α）和胞核NF-κB的蛋白表达水平。结果光镜下观察显示，Sham组小鼠肺泡结构完整，无明显病理学改变；I／R组小鼠肺组织结构严重破坏，肺泡壁明显增厚，肺间质水肿，大量炎性细胞浸润；黄芩素＋I／R组小鼠肺泡及间质水肿程度明显减轻，有少量炎性细胞浸润。与Sham组比较，I／R组肺组织病理学评分、W／D比值明显升高，细胞凋亡指数显著增加，血清TNF-α、IL-6含量及其肺组织mRNA表达均明显增加，胞质IκB-α蛋白表达明显下降，胞核NF-κB蛋白表达明显升高；而给予黄芩素预处理可明显改善上述肠I／R诱导的肺组织损伤，表现为与I／R组比较，黄芩素＋I／R组肺组织病理学评分、W／D比值明显降低[病理学评分（分）：4．59±1．17比6．27±1．34，W／D比值：3．79±0．28比4．32±0．57]，细胞凋亡指数显著下降[（4．85±2．47）％比（8．15±2．33）％]，血清TNF—α、IL-6含量及其肺组织mRNA表达水平均明显下降[血清TNF-α（pg／L）：124．18±30．49比167．72±38．65，IL-6（ng／L）：1．65±0．69比2．43±0．57：肺组织TNF-α mRNA（2^-△△Ct：4．75±2．38比7．69±2．32，IL-6 mRNA（2^-△△Ct）：16．45±4．39比27．69±6．82]，胞质IκB-α蛋白表达明显升高（灰度值：0．47±0．11比0．27±0．09），胞核NF-κB蛋白表达明显下降（灰度值：0．57±0．13比1．07±0．34），差异均有统计学意义（均P〈0．05）。结论黄芩素可能通过抑制炎症反应和细胞凋亡，从而减轻肠I／R所致ALI。

Objective To investigate the effects of baicalein （Bai） on acute lung injury （ALI） induced by intestinal ischemia/reperfusion （I/R） and its mechanism in mice. Methods Twenty-four male C57BL/6J mice were divided into three groups by random number table： namely sham group, I/R group and Bai＋I/R group, with 8 mice in each group. Intestinal I/R induced lung injury model was reproduced by clamping superior mesenteric artery for 90 minutes, followed by reperfusion. Bai （100 mg/kg） was intraperitoneally injected 1 hour before ischemic challenge in the Bai＋I/R group. The mice in sham group underwent the similar procedure with I/R group but without vascular occlusion. All mice were sacrificed at 4 hours of reperfusion, and blood was collected from inferior vena cava and lung tissues were harvested.Lung tissues were stained with hematoxylin-eosin （HE）, and histological changes were examined under light microscope for pathological score. Lung wet/dry （W/D） ratio was calculated. Lung cell apoptosis was determined by TdT-mediated dUTP nick end labeling （TUNEL） technique. Serum levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） were determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA expressions of TNF-α and IL-6 in lung tissues were determined by real-time quantitative reverse transcription-polymerase chain reaction （RT-PCR）. The protein expression levels of cytoplasmic inhibitory factor-α of nuclear factor-κB （IκB-α） and nucleus NF-κB were determined by Western Blot. Results Under light microscope, a normal lung tissue structure was shown in the sham group and no evidence of obvious lung injury was found. In the I/R group, the alveolar structure was seriously damaged. The alveolar wall was widened and there was significant interstitial edema and leukocytes infiltration. In the Bai＋I/R group, pathological damage was significantly decreased as indicated by reduced lung tissue edema and leukocytes infiltration. Compared with the sham group, the lung pathological scores, W/D ratio and cellular apoptosis in the I/R group were significantly increased. Both serum TNF-α and IL-6 contents and lung TNF-α and IL-6 mRNA expressions were significantly increased. Furthermore, I/R significantly resulted in a decrease of IκB-α in the cytoplasm and an increase of NF-κB in the nucleus. Notably, Bai treatment significantly attenuated ALI induced by intestinal I/R injury. Compared with the I/R group, the lung pathological scores and W/D ratio in the Bai＋I/R group were significantly decreased （lung pathological score： 4.59±1.17 vs. 6.27±1.34, W/D ratio： 3.79±0.28 vs. 4.32±0.57）, cellular apoptosis was significantly decreased [（4.85 ± 2.47）% vs. （8.15 ± 2.33）%], both serum TNF-α and IL-6 contents and lung TNF-α and IL-6 mRNA expressions were significantly decreased [serum TNF-α （pg/L）： 124.18±30.49 vs. 167.72±38.65, IL-6 （ng/L）： 1.65±0.69 vs. 2.43±0.57; lung TNF-α mRNA （2^-△△Ct）： 4.75±2.38 VS. 7.69±2.32, IL-6 mRNA （2^-△△Ct）： 16.45 ±4.39 vs. 27.69±6.82], additionally, Bai pretreatment significantly increased cytoplasmic IKB-α protein expression （gray value： 0.47±0.11 vs. 0.27±0.09）, while decreased nuclear NF-κB protein expression （gray value： 0.57±0.13 vs. 1.07 ± 0.14, all P 〈 0.05）. Conclusion Bai could attenuate intestinal I/R injury induced ALl via the inhibition of inflammation and apoptosis.