光甘草定抑制p38MAPK／ERK信号通路减轻脂多糖致大鼠肺损伤的实验观察

Glabridin reduces lipopolysaccharide-induced lung injury in rats by inhibiting p38 mitogen activated protein kinase/extracellular regulated protein kinases signaling pathway

目的探讨中药光甘草定（Glabridin，GLA）对脂多糖（LPS）致急性呼吸窘迫综合征（ARDS）大鼠的保护作用及可能机制。方法32只Wistar大鼠按随机数字表法分为对照组、模型组（LPS组）、光甘草定（GLA）组、乌司他丁（UTI）组，每组8只。采用腹腔注射LPS（10mg／kg）的方法制备大鼠ARDS模型，对照组注射同等剂量的生理盐水，GLA组将光甘草定灌胃（30mg／kg），UTI组腹腔注射乌司他丁（20000U／kg），12h后各组大鼠留肺组织及血浆标本。计算肺组织湿／干质量（Wet／Dry，W／D）比值；光镜下观察肺组织病理改变；用酶联免疫吸附法（ELISA）法检测各组血浆中肿瘤坏死因子-α（TNF-α）、白细胞介素。18（IL-18）含量，检测各组肺匀浆中丙二醛（MDA）、超氧化物歧化酶（SOD）和一氧化氮（NO）含量；免疫组织化学法检测p38丝裂原活化蛋白激酶（p38MAPK）、细胞外信号调节蛋白激酶（ERK）蛋白表达情况；Western印迹法检测肺组织中磷酸化p38MAPK（p-p38MAPK）及磷酸化ERK（pERK）蛋白表达的变化。结果光镜下观察对照组肺组织结构完整，肺泡腔清晰；LPS组肺泡壁增厚，渗出明显，肺组织出现损伤性变化，GLA组及u_rI组病理改变较LPS组明显减轻。与对照组比较，LPS组肺组织W／D比值，血浆TNF-α、IL-18含量，肺匀浆MDA、NO含量均明显升高（P〈0．05），而血浆SOD含量明显降低；与LPS组比较，GLA组肺组织W／D比值，血浆TNF-α、IL-18含量，肺匀浆MDA、NO含量均明显降低，而血浆SOD含量明显增高[（TNF-α（μg/L）：51．7±10．3±105．7±30．5，IL-18（μg／L）：37．9±13．9比49．2±14．5，MDA（nmo/mg蛋白（prot）：2．87±0．62比3．81±0．42，NO（μmoL／L）：18．96±0．79比28．58±2．51，SOD（U／mgprot）：115．5±15．2比75．9±14．0）]；免疫组化结果显示：与对照组比较，LPS组p-p38MAPK、pERK蛋白在胞核表达均明显增加，而GLA组与UTI组较LPS组表达明显减少；Western印迹检测结果显示：与对照组比较，LPS组肺组织p-p38MAPK蛋白表达明显增高，而GLA及UTI干预组蛋白表达明显受抑制。结论传统中药光甘草定通过抑制p38MAPK／ERK信号通路、抗氧化作用而减轻炎症，发挥对LPS致大鼠ARDS的保护作用。

Objective To investigate whether glabridin has a beneficial effect on lipopolysaccharide （LPS） induced acute respiratory distress syndrome （ARDS） in rats, and to explore the possible underlying mechanisms. Methods Thirty-two Wistar rats were randomly assigned into control group, model group （LPS group ）, glabridin group （ GLA group ）, and ulinastatin group （ UTI group ）, with 8 rats in each group. ARDS rat model was reproduced by intraperitoneal injection of LPS （ 10 mg/kg）. The rats in the control group received an equal volume of normal saline at the same times. The rats in GLA group were gavaged by glabridin （30 mg/kg）. The rats in UTI group were injected ulinastatin （20 000 U/kg）. Animals were sacrificed 12 hours after LPS challenge. Plasma and lung tissue samples were collected. Histopathological evaluation, lung wet/dry （W/D） ratio, tumor necrosis factor-α（ TNF-α）, interleukin-18 （ IL-18）, malondialdehyde （ MDA）, nitric oxide （ NO ） and superoxide dismutase （ SOD ） were analyzed. Immunohistochemical method was used to detect the protein expression of p38MAPK and ERK. Western blot method was used to detect the changes of p38 mitogen activated protein kinase （p-p38MAPK） and phosphorylated extracellular regulated protein kinases （pERK） protein expression in lung tissues. Result In the control groups, lung tissue showed a normal structure and clear pulmonary alveoli under a light microscope. In the model group, ARDS characters such as extensive thickening of the alveolar wall, significant infiltration of inflammatory cells, demolished structure of pulmonary alveoli, and hemorrhage were found. In the GLA and UTI treatment group, these pathological changes in lung were markedly alleviated compare with LPS-induced ARDS group. Compared with control groups, lung W/D ratio, TNF-ct and IL-18 in plasma, and lung MDA, NO levels in lung homogenates of the LPS group were increased significantly, while the lung SOD levels of the LPS group were decreased. Compared with the LPS group, lung W/D ratio, TNF-ct and IL-18 in plasma , and lung MDA, NO levels in lung homogenates of the GLA group and UTI group were decreased significantly, while the lung SOD levels of the GLA and ulinastatin groups were increased [TNF-α（μg/L） ： 51.7 ± 10.3 vs 105.7± 30.5, IL-18（μg/L） ： 37.9 ± 13.9 vs 49.2± 14.5, MDA （nmol/mgprot）： 2. 87 ±0.62 vs 3.81 ±0.42, NO（μmol/L）： 18. 96 ±0.79 vs 28.58 ±2.51, SOD （U/mgprot） ： 115.5± 15.2 vs 75.9 ± 14. 0, all P 〈 0. 05 ]. Immunohistochemistry showed that the positive expressions of p38MAPK and ERK in cytoplasm and nucleus of the glabridin and ulinastatin treatment group were significantly lower than those of the model group. Western blot showed that compared with the control group, the p-p38MAPK and pERK protein expression in LPS group were significantly increased. And the glabridin and ulinastatin inhibited the protein expressions compared with model group. Conclusion Traditional Chinese medicine glabridin significantly ameliorated the lung injury induced by LPS in rats via reducing inflammation which caused by the inhibition of p38MAPK and ERK signaling pathway and antioxidant effect.