淫羊藿苷对补体旁路激活诱导的小鼠急性肺损伤的保护作用
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摘要
目的探讨淫羊藿苷(ICA)对补体旁路激活造成的小鼠急性肺损伤的保护作用及作用机制。方法 32只昆明小鼠随机分为正常对照组、模型组、PDTC组、淫羊藿苷组,预防给药7 d后,用特异性补体旁路激活蛋白眼镜蛇毒因子(CVF),尾静脉注射复制小鼠急性肺损伤模型。测定小鼠支气管肺泡灌洗液(BALF)中蛋白含量和炎性细胞数目;肺组织匀浆后测定髓过氧化物酶(MPO)活性;ELISA法检测BALF和血清中IL-6、TNF-α、P-selectin、ICAM-1的含量;HE染色法观察肺组织病理形态学变化;免疫组化法测定肺组织中NF-κB p65的磷酸化水平,并采用双萤光素酶报告基因检测系统,在细胞水平上测定补体旁路激活对微血管内皮细胞中NF-κB的核内转录活性的影响。结果 ICA可以明显降低小鼠肺组织匀浆液中MPO含量和BALF中炎性细胞数目,并同时降低BALF中IL-6、TNF-α、P-selectin含量和血清中TNF-α、P-selectin、ICAM-1水平,减轻小鼠肺部炎性细胞浸润,明显抑制小鼠肺组织中NF-κB p65的磷酸化,并有效抑制NF-κB核内转录活性的上调。结论 ICA能有效减轻补体旁路激活诱导的小鼠肺部急性炎症反应,其机制可能与抑制肺组织炎性细胞浸润、NF-κB p65的磷酸化及核内转录活性,从而降低炎症反应水平有关。
Aim To explore the protective effect and mechanism of icariin( ICA) on acute lung injury( ALI) in mice induced by activation of the complement alternative pathway. Methods 32 healthy KM mice were randomly divided into four groups: the normal group,the model group,the PDTC group and the Icariin group,which received 7-day intragastric administration respectively. Then cobra venom factor( CVF)was used to activate specifically complement alternative pathway to induce acute lung injury in mice by intravenous injection. Myeloperoxidase( MPO) activity of lung homogenate,the cell count and the protein content of bronchoalveolar lavage fluid( BALF) were measured. The concentration of IL-6,TNF-α,P-selectin and ICAM-1 in BALF and serum were determined by ELISA. The pathological change of lung tissue was observed by HE staining. The phosphorylation of NF-κB p65 in lung tissues was checked by immunohistochemistry. The effect of the transcriptional activity of NF-κB signal pathway in microvascular endothelial cells was measured by employing dual-luciferase reporter assay system. Results ICA reduced MPO activity of lung homogenate,the cell count and the content of IL-6,TNF-α,P-selectin in BALF obviously.The level of TNF-α,P-selectin and ICAM-1 in serum was decreased,the pulmonary inflammatory cell infiltration was reduced,the phosphorylation of NF-κB p65 in lung was inhibited significantly and the transcriptional activity of NF-κB was also down-regulated. Conclusion ICA can alleviate acute inflammatory response of ALI mice induced by activation of the complement alternative pathway. The mechanism may be highly related to the inhibition of inflammatory cell infiltration in lung tissue,the down-regulation of phosphorylation of NF-κB p65 and nuclear transcriptional activity.
Aim To explore the protective effect and mechanism of icariin( ICA) on acute lung injury( ALI) in mice induced by activation of the complement alternative pathway. Methods 32 healthy KM mice were randomly divided into four groups: the normal group,the model group,the PDTC group and the Icariin group,which received 7-day intragastric administration respectively. Then cobra venom factor( CVF)was used to activate specifically complement alternative pathway to induce acute lung injury in mice by intravenous injection. Myeloperoxidase( MPO) activity of lung homogenate,the cell count and the protein content of bronchoalveolar lavage fluid( BALF) were measured. The concentration of IL-6,TNF-α,P-selectin and ICAM-1 in BALF and serum were determined by ELISA. The pathological change of lung tissue was observed by HE staining. The phosphorylation of NF-κB p65 in lung tissues was checked by immunohistochemistry. The effect of the transcriptional activity of NF-κB signal pathway in microvascular endothelial cells was measured by employing dual-luciferase reporter assay system. Results ICA reduced MPO activity of lung homogenate,the cell count and the content of IL-6,TNF-α,P-selectin in BALF obviously.The level of TNF-α,P-selectin and ICAM-1 in serum was decreased,the pulmonary inflammatory cell infiltration was reduced,the phosphorylation of NF-κB p65 in lung was inhibited significantly and the transcriptional activity of NF-κB was also down-regulated. Conclusion ICA can alleviate acute inflammatory response of ALI mice induced by activation of the complement alternative pathway. The mechanism may be highly related to the inhibition of inflammatory cell infiltration in lung tissue,the down-regulation of phosphorylation of NF-κB p65 and nuclear transcriptional activity.
引文
[1]龚其海,杨丹莉,石京山,等.淫羊藿苷的神经药理作用及分子机制研究进展[J].中国新药与临床杂志,2011,30(7):481-6.[1]Gong Q H,Yang D L,Shi J S,et al.Advances in neuroharmacological effects and molecular mechanisms of icariin[J].Chin J New Drugs Clin Rem,2011,30(7):481-6.
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[8]孙黔云,李敏,叶巧玲,李红玲.补体旁路激活导致内皮细胞的活化和损伤[J].中国药理学通报,2012,28(7):925-9.[8]Sun Q Y,Li M,Ye Q L,Li H L.Endothelial cell activation and injury induced by complement alternative pathway[J].Chin Pharmacol Bull,2012,28(7):925-9.
[9]李敏,孙玲,李红玲,孙黔云.阿托伐他汀减轻氧化型低密度脂蛋白导致的人微血管内皮细胞活化和损伤[J].中国药理学通报,2014,30(5):679-83.[9]Li M,Sun L,Li H L,Sun Q Y.Atorvastatin attenuates activation and injury of human microvascular endothelial cells induced by oxidized low density lipoprotein[J].Chin Pharmacol Bull,2014,30(5):679-83.
[10]吴金峰,董竞成,徐长青,等.淫羊藿苷拮抗脂多糖炎症模型的体内和体外研究[J].中国中西医结合杂志,2009,29(4):330-4.[10]Wu J F,Dong J C,Xu C Q,et al.Effects of icariin on inflammation model stimulated by lipopolysaccharide in vitro and in vivo[J].Chin J Integr Tradit West Med,2009,29(4):330-4.
[11]徐长青.淫羊藿苷对卵蛋白和内毒素诱导炎症反应的影响及机制研究[D].上海:复旦大学,2010.[11]Xu C Q.The effects and mechanism of icariin on OVA and endotoxin-induced inflammation[D].Shanghai:Fudan University,2010.
[2]李梨,周岐新,石京山.淫羊藿苷药理作用研究进展[J].中国药房,2005,16(12):952-4.[2]Li L,Zhou Q X,Shi J S.Progress of pharmacological research on icariin[J].China Pharm,2005,16(12):952-4.
[3]Pan L H,Zhang Y H,Chen N,Yang L.Icariin regulates cellular functions and gene expression of osteoarthritis patient-derived human fibroblast-like synoviocytes[J].Int J Mol Sci,2017,18(12):2656.
[4]孙玉姣,李祎群,李莉.淫羊藿苷对慢性阻塞性肺疾病模型的抗炎和抗氧化作用[J].湖北中医药大学学报,2015,17(4):4-7.[4]Sun Y J,Li Y Q,Li L.Anti-inflammatory and antioxidant effects of icariin on chronic obstructive pulmonary disease model[J].J Hubei Univ Chin Med,2015,17(4):4-7.
[5]Xu C Q,Le J J,Duan X H,et al.Molecular mechanism of icariin on rat asthmatic model[J].Chin Med J,2011,124(18):2899-906.
[6]Bosmann M,Ward P A.Role of C3,C5 and anaphylatoxin receptors in acute lung injury and in sepsis[J].Adv Exp Med Biol,2012,946:147-59.
[7]郭静,李敏,杨付梅,孙黔云.补体旁路激活致小鼠急性肺损伤的炎症病理机制研究[J].中国药理学通报,2016,32(11):1521-6.[7]Guo J,Li M,Yang F M,Sun Q Y.Inflammatory mechanism of acute lung injury in mice induced by activation of complement alternative pathway[J].Chin Pharmacol Bull,2016,32(11):1521-6.
[8]孙黔云,李敏,叶巧玲,李红玲.补体旁路激活导致内皮细胞的活化和损伤[J].中国药理学通报,2012,28(7):925-9.[8]Sun Q Y,Li M,Ye Q L,Li H L.Endothelial cell activation and injury induced by complement alternative pathway[J].Chin Pharmacol Bull,2012,28(7):925-9.
[9]李敏,孙玲,李红玲,孙黔云.阿托伐他汀减轻氧化型低密度脂蛋白导致的人微血管内皮细胞活化和损伤[J].中国药理学通报,2014,30(5):679-83.[9]Li M,Sun L,Li H L,Sun Q Y.Atorvastatin attenuates activation and injury of human microvascular endothelial cells induced by oxidized low density lipoprotein[J].Chin Pharmacol Bull,2014,30(5):679-83.
[10]吴金峰,董竞成,徐长青,等.淫羊藿苷拮抗脂多糖炎症模型的体内和体外研究[J].中国中西医结合杂志,2009,29(4):330-4.[10]Wu J F,Dong J C,Xu C Q,et al.Effects of icariin on inflammation model stimulated by lipopolysaccharide in vitro and in vivo[J].Chin J Integr Tradit West Med,2009,29(4):330-4.
[11]徐长青.淫羊藿苷对卵蛋白和内毒素诱导炎症反应的影响及机制研究[D].上海:复旦大学,2010.[11]Xu C Q.The effects and mechanism of icariin on OVA and endotoxin-induced inflammation[D].Shanghai:Fudan University,2010.