目的 观察月见草提取物对脂多糖(lipopolysaccharide,LPS)所致大鼠急性肺损伤(acute lung injury,ALI)的影响,并初步探讨其作用机制。方法 LPS滴鼻10 mg·kg-1建立大鼠ALI模型。40只雄性SD大鼠随机分为正常组(CON)、模型组(LPS)、月见草提取物低剂量组(YJC-L)和高剂量组(YJC-H)。造模后连续灌胃给药3 d,苏木素-伊红(hematoxylin-eosin staining, HE) 染色观察肺脏病理学变化;酶联免疫吸附(enzyme-linked immunosorbent assay, ELISA)法检测各组肺泡灌洗液中炎症因子白介素-6(interleukin-6, IL-6)、干扰素-γ(interferon-γ,IFN-γ)及肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)水平;酶法检测肺组织中谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde, MDA)含量;流式细胞术检测原代肺细胞凋亡和活性氧(reactive oxygen species, ROS)水平并用Western blot检测肺组织核转录因子E2相关因子(nuclear factor erythroid2-related factor 2, Nrf2)、NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain 3, NLRP3)、半胱氨酸天冬氨酸蛋白酶1(cysteinyl aspartate specific proteinase 1, caspase-1)、切割型半胱氨酸天冬氨酸蛋白酶-1(cleaved cysteinyl aspartate specific proteinase 1, cleaved-caspase-1)、焦孔素(gasdermin D, GSDMD)及切割型焦孔素(cleaved gasdermin D, cleaved-GSDMD)的表达。结果 与正常组相比,LPS组肺泡腔消失,肺泡壁增厚,肺泡周围炎症细胞明显增多;肺泡灌洗液中IL-6和TNF-α含量均显著升高(P<0.01),IFN-γ显著降低(P<0.01);GSH-Px、SOD含量显著下降(P<0.01),MDA含量显著升高(P<0.01);原代肺细胞凋亡率和ROS水平升高(P<0.01);Nrf2、NLRP3、caspase-1、cleaved-caspase-1、GSDMD及cleaved-GSDMD蛋白表达水平显著升高(P<0.01)。给药后,与模型组相比,月见草提取物能使肺脏病理形态有明显改善,并使IL-6和TNF-α水平显著降低(P<0.01或P<0.05),IFN-γ显著升高(P<0.01);GSH-Px、SOD水平显著升高(P<0.01或P<0.05),MDA降低;原代肺细胞凋亡率和ROS水平降低以及Nrf2蛋白升高(P<0.01或P<0.05),NLRP3、caspase-1、cleaved-caspase-1、GSDMD及cleaved-GSDMD蛋白表达水平降低(P<0.01或P<0.05)。结论 月见草提取物通过降低炎症、氧化应激与凋亡水平从而改善LPS诱导的急性肺损伤,该作用可能是介导Nrf2-NLRP3通路发挥的。
Abstract
OBJECTIVE To investigate the effect of Oenothera biennis L. extract on lipopolysaccharide-induced acute lung injury in rats and explore its mechanism. METHODS The rat model of acute lung injury(ALI) was established by intranasal inoculation of 10 mg·kg-1 lipopolysaccharide. A total of 40 male SD rats were randomly divided into control group(CON), model group(LPS), low-dose Oenothera biennis L. extract group(YJC-L) and high-dose Oenothera biennis L. extract group(YJC-H). After the model was established, test drug was continuously administered intragastrically for 3 d, hematoxylin-eosin(HE) staining was used to observe the pathological changes of lung tissue. Enzyme-linked immunosorbent assay(ELISA) was used to detect the contents of interleukin-6(IL-6), interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α) in bronchoalveolar lavage fluid of each group. Enzymatic method was used to detect the contents of glutathione peroxidase(GSH-Px), superoxide dismutase(SOD) and malondialdehyde(MDA) in rat lung tissue. Flow cytometry was used to detect the levels of apoptosis and ROS in primary lung cells. Western blot was used to detect the contents of nuclear factor E2-related factor 2(Nrf2), NOD-like receptor protein 3(NLRP3), cysteinyl aspartate specific proteinase 1(caspase-1), cleaved cysteinyl aspartate specific proteinase 1(cleaved-caspase-1), gasdermin D(GSDMD) and cleaved gasdermin D(cleaved-GSDMD). RESULTS Compared with the CON, the alveolar cavity was disappeared, the alveolar wall was thickened and the inflammatory cells around the alveolar increased significantly in the LPS group. The contents of IL-6 and TNF-α increased significantly in bronchoalveolar lavage fluid(P<0.01), while IFN-γ decreased significantly(P<0.01). The contents of GSH-Px and SOD decreased significantly(P<0.01), while the content of MDA increased significantly(P<0.01). The levels of apoptosis and ROS of primary lung cells were increased (P<0.01), and the protein expression levels of Nrf2, NLRP3, caspase-1, cleaved-caspase-1, GSDMD and cleaved-GSDMD were significantly increased(P<0.01). Compared with the LPS group, the Oenothera biennis L. extract could significantly improve the pathological morphology of lung. The levels of IL-6 and TNF-α were significantly decreased(P<0.01 or P<0.05), and IFN-γ was significantly increased(P<0.01). The levels of GSH-Px and SOD increased significantly(P<0.01 or P<0.05), and the level of MDA was decreased. The levels of apoptosis and ROS were decreased in primary lung cells, the expression level of Nrf2 protein was upregulated(P<0.01 or P<0.05), while the protein expression levels of NLRP3, caspase-1, cleaved-caspase-1, GSDMD and cleaved-GSDMD were downregulated(P<0.01 or P<0.05). CONCLUSION Oenothera biennis L. extract can improve LPS-induced ALI by reducing the levels of inflammation, oxidative stress and apoptosis. This effect might be mediated by Nrf2-NLRP3 pathway.
关键词
月见草 /
急性肺损伤 /
脂多糖 /
炎症 /
氧化应激
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Key words
Oenothera biennis L. /
acute lung injury /
lipopolysaccharide /
inflammation /
oxidative stress
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中图分类号：
R965
R285.5
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脚注
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基金
国家重点研发计划资助(2019YFC1708800);河南省重大科技专项资助(171100310500);河南省高层次人才特殊支持计划“中原千人计划”资助(ZYQR201810080)
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