和厚朴酚对哮喘小鼠肺组织炎症反应的干预作用及其机制

目的：探讨和厚朴酚（HNK）对哮喘小鼠肺组织炎症反应的影响，阐明其干预作用及相关机制。方法：选取健康雌性C57BL/6J小鼠20只，随机分为对照组、模型组、地塞米松（DXM）组和HNK组，每组5只。采用鸡卵清蛋白（OVA）联合氢氧化铝[Al（OH）₃]悬液腹腔注射致敏和OVA滴鼻激发构建哮喘小鼠模型，在激发前30 min分别对DXM组和HNK组小鼠予以腹腔注射DXM和HNK溶液，模型组小鼠予以等量生理盐水。处死小鼠后取小鼠肺组织，HE染色观察评估肺组织病理形态表现；收集支气管肺泡灌洗液（BALF），行快速迪夫染色，观察炎性细胞浸润程度；ELISA法检测各组小鼠血清中白细胞介素4（IL-4）、白细胞介素6（IL-6）和白细胞介素17（IL-17）水平，比色法检测各组小鼠肺组织匀浆中丙二醛（MDA）水平和超氧化物歧化酶（SOD）及谷胱甘肽过氧化物酶（GSH-Px）活性，Western blotting法检测各组小鼠肺组织中磷酸化c-Jun氨基末端激酶（p-JNK）、核因子κB（NF-κB）、含半胱氨酸的天冬氨酸蛋白水解酶（Caspase-3）、B淋巴细胞瘤-2（Bcl-2）和H2AX组蛋白（γH2Ax）表达水平，检测各组小鼠肺组织中γH2Ax免疫荧光强度。结果：与对照组比较，模型组小鼠气道上皮细胞脱落坏死数量明显增多，气道管壁增厚及气道旁炎症细胞浸润程度明显增加；与模型组比较，DXM组和HNK组小鼠上述表现减轻，而HNK组和DXM组小鼠肺组织病理改变表现相似。与对照组比较，模型组小鼠血清中IL-4、IL-6和IL-17水平升高（P<0.05）；与模型组比较，DXM组和HNK组小鼠血清中IL-4、IL-6和IL-17水平降低（P<0.05）；而HNK组小鼠血清中IL-4、IL-6和IL-17水平与DXM组比较差异无统计学意义（P>0.05）。与对照组比较，模型组小鼠肺组织匀浆中MDA水平升高（P<0.05），而SOD和GSH-Px活性降低（P<0.05）；与模型组比较，DXM组和HNK组小鼠肺组织匀浆中MDA水平降低（P<0.05），而SOD和GSH-Px活性升高（P<0.05）；HNK组小鼠MDA水平和SOD及GSH-Px活性与DXM组比较差异无统计学意义（P>0.05）。与对照组比较，模型组小鼠肺组织中p-JNK、NF-κB、Caspase-3和γH2Ax蛋白表达水平升高（P<0.05），而Bcl-2蛋白表达水平降低（P<0.05）；与模型组比较，DXM组和HNK组小鼠肺组织中p-JNK、NF-κB、Caspase-3和γH2Ax蛋白表达水平降低（P<0.05），而Bcl-2蛋白表达水平升高（P<0.05）；HNK组小鼠肺组织中p-JNK、NF-κB、Caspase-3、γH2Ax和Bcl-2蛋白表达水平与DXM组比较差异均无统计学意义（P>0.05）。与对照组比较，模型组小鼠肺组织中γH2Ax免疫荧光强度明显增强；与模型组比较，DXM组和HNK组小鼠肺组织中γH2Ax免疫荧光强度减弱，HNK组小鼠γH2Ax免疫荧光强度与DXM组接近。结论：HNK可在一定程度上抑制哮喘小鼠的肺组织损伤及炎症反应，其机制可能与其抑制氧化应激反应及DNA损伤有关。

Intervention effect of honokiol on inflammatory response in lung tissue of asthma mice and its mechanism

Objective: To investigate the effect of honokiol(HNK) on the inflammatory response in lung tissue of the asthma mice, and to clarify the intervention effect and its related mechanism. Methods: Twenty healthy female C57BL/6J mice were randomly divided into control group, model group, dexamethasone(DXM) group and HNK group,with 5 mice in each group.The asthma mouse model was established by intraabdominal injection of ovalbumin(OVA) combined with aluminum hydroxide[Al(OH)₃] suspension and OVA nasal stimulation.Thirty min before stimulation,the mice in DXM group and HNK group were treated with intraabdominal injection of the DXM solution and HNK solution, and the mice in model group were treated with the same amount of normal saline. After the mice were killed,the pathological changes of lung tissue were observed and evaluated by HE staining; the bronchoalveolar lavage fluid(BALF) was collected and the infiltration degree of inflammatory cells was observed by Diff-quik staining.The levels of IL-4,IL-6 and IL-17 in serum of the mice in various groups were detected by ELISA assay, and the expression levels of malondialdehyde(MDA) and the activities of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) in lung homogenate of the mice in various groups were detected by colorimetric method;the expression levels of p-JNK,nuclear factor-κB(NF-κB),Caspase-3,Bcl-2 and γH2Ax proteins in lung tissue of the mice in various groups were detected by Western blotting method.The immunofluorescence intensities of γH2Ax in lung tissue of the mice in various groups were measured. Results: Compared with control group, the number of airway epithelial exfoliation and necrosis cells of the mice in model group was increased obviously, the airway wall was thickened and the airway side inflammation cell infiltration degree was increased obviously;compared with model group,the performances mentioned above of the mice in DXM group and HNK group were alleviated;the pathological changes of lung tissue of the mice in HNK group were similar to DXM group.Compared with control group,the leves of IL-4, IL-6 and IL-17 in serum of the mice in model group were increased(P<0.05);compared with model group, the levels of IL-4,IL-6 and IL-17 of the mice in DXM group and HNK group were decreased(P<0.05);there were no significant differences in the IL-4, IL-6 and IL-17 levels in serum of the mice between HNK group and DXM group(P>0.05).Compared with control group,the level of MDA in lung homogenate of the mice in model group was increased(P<0.05),but the activities of SOD and GSH-Px were decreased(P<0.05);compared with model group,the levels of MDA of the mice in DXM group and HNK group were decreased(P<0.05),but the activities of SOD and GSH-Px were increased(P<0.05);there were no significant differences in the MDA levels and the activities of SOD and GSH-Px of the mice between HNK group and DXM group(P>0.05).Compared with control group, the expression levels of p-JNK,NF-κB,Caspase-3 and γH2Ax proteins in lung tissue of the mice in model group were increased(P<0.05),but the expression level of Bcl-2 protein was decreased(P<0.05); compared with model group, the expression levels of p-JNK,NF-κB,Caspase-3 and γH2Ax of the mice in DXM group and HNK group were decreased(P<0.05),but the expression levels of Bcl-2 protein were increased(P<0.05);there were no significant differences in the expression levels of p-JNK, NF-κB, Caspase-3, γH2Ax and Bcl-2 proteins in lung tissue of the mice between HNK group and DXM group(P>0.05).Compared with control group,the immunofluorescence intensity of γH2Ax in lung tissue of the mice in model group was significantly enhanced;compared with model group,the immunofluorescence intensities of γH2Ax in lung tissue of the mice in DXM group and HNK group were relatively weak,while the immunofluorescence intensity of γH2Ax in HNK group was similar to that in DXM group. Conclusion: HNK can inhibit the lung tissue injury and inflammation response in the asthma mice to a certain extent, and its mechanism may be related to its inhibition of oxidative stress and DNA damage.