补体旁路激活致小鼠急性肺损伤的炎症病理机制研究
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摘要
目的研究补体旁路激活所导致的小鼠肺部急性炎症的发生发展及相关指标的变化,为药物筛选及干预研究提供理想的小鼠肺部急性炎症病理模型。方法 SPF级昆明小鼠尾静脉注射眼镜蛇毒因子(CVF)激活血清补体旁路途径,根据注射后取样时间不同,分为15 min、30 min、1 h、2 h、6 h组,同时平行设置PBS对照组。取肺组织测定肺系数、肺含水量,并行病理切片检查,肺组织匀浆测定髓过氧化物酶(MPO)活性;制备支气管肺泡灌洗液(BALF)和血清,测定BALF中的细胞数和蛋白含量,采用ELISA法分别测定BALF和血清中的IL-6、TNF-α、P-selectin和ICAM-1含量。结果小鼠尾静脉注射CVF后可致肺部炎性细胞明显浸润,MPO活性明显上调,BALF中细胞总数和蛋白浓度明显增加。BALF和血清中的IL-6、TNF-α、P-selectin水平及血清中ICAM-1的含量均明显升高,其中,BALF中P-selectin含量在30 min时间点出现1个小高峰,IL-6和TNF-α在1 h时间点出现1个高峰,在2 h时间点均无进一步上升,但在6 h时间点各指标均又明显升高;血清中IL-6和TNF-α含量在1 h时间点达到峰值,随后浓度降低,P-selectin和ICAM-1水平随着时间的延长持续上升。而肺系数、肺含水量及BALF中ICAM-1的含量与PBS组相比无明显变化。结论补体旁路激活可导致小鼠肺部急性炎症的发生,以30 min至1 h的炎症反应最为明显,该实验可以为药物筛选及干预研究提供理想的动物肺部急性炎症病理模型。
Aim To study the development of acute lung inflammation in mice induced by activation of the complement alternative pathway and the changes of the related indicators,and to provide an ideal pathologicalmodel of acute lung inflammation in mice for drug screening and intervention. Methods Cobra venom factor( CVF) was used to activate complement alternative pathway of SPF Kunming mice by intravenousinjection. According to different sampling time,the mice were divided into 15 min,30 min,1 h,2 h,6 h group,and the parallel PBS control groups were set at the same time. Lung coefficient,lung water content,myeloperoxidase( MPO) activity,BALF cell number and protein content were tested. The pathological changes of lung tissue were observed by HE staining.The concentration of IL-6, TNF-α, P-selectin and ICAM-1 in bronchoalveolar lavage fluid( BALF) and serum were determined by ELISA. Results CVF caused pulmonary inflammatory cell infiltration in mice obviously. Compared with PBS groups,MPO activity of lung tissue,BALF cell and the protein concentration were significantly increased. The contents of IL-6,TNF-α, P-selectin in BALF and serum were increased,and the content of ICAM-1 in serum was also increased. The content of P-selectin in BALF reached the first peak at 30 min point,the content of IL-6 andTNF-α in BALF reached the first peak at 1 h point,but the indicators had no further changes at 2 h point,and all the indicators rose again at 6 h point. The levels of IL-6 and TNF-α in serum reached peak at 1 h point,then the content showed lower levels at the subsequent time points. The levels of P-selectin and ICAM-1 in serum increased along the time. Lung coefficient,lung water content and ICAM-1 of the BALF showed no significant alteration. Conclusion The activation of the complement alternative pathway can lead to acute lung inflammation in mice and the inflammatory response is the most obvious at 30 min to 1 h. The study could provide an ideal pathological model of acute lung inflammation in mice for drug screening and intervention.
Aim To study the development of acute lung inflammation in mice induced by activation of the complement alternative pathway and the changes of the related indicators,and to provide an ideal pathologicalmodel of acute lung inflammation in mice for drug screening and intervention. Methods Cobra venom factor( CVF) was used to activate complement alternative pathway of SPF Kunming mice by intravenousinjection. According to different sampling time,the mice were divided into 15 min,30 min,1 h,2 h,6 h group,and the parallel PBS control groups were set at the same time. Lung coefficient,lung water content,myeloperoxidase( MPO) activity,BALF cell number and protein content were tested. The pathological changes of lung tissue were observed by HE staining.The concentration of IL-6, TNF-α, P-selectin and ICAM-1 in bronchoalveolar lavage fluid( BALF) and serum were determined by ELISA. Results CVF caused pulmonary inflammatory cell infiltration in mice obviously. Compared with PBS groups,MPO activity of lung tissue,BALF cell and the protein concentration were significantly increased. The contents of IL-6,TNF-α, P-selectin in BALF and serum were increased,and the content of ICAM-1 in serum was also increased. The content of P-selectin in BALF reached the first peak at 30 min point,the content of IL-6 andTNF-α in BALF reached the first peak at 1 h point,but the indicators had no further changes at 2 h point,and all the indicators rose again at 6 h point. The levels of IL-6 and TNF-α in serum reached peak at 1 h point,then the content showed lower levels at the subsequent time points. The levels of P-selectin and ICAM-1 in serum increased along the time. Lung coefficient,lung water content and ICAM-1 of the BALF showed no significant alteration. Conclusion The activation of the complement alternative pathway can lead to acute lung inflammation in mice and the inflammatory response is the most obvious at 30 min to 1 h. The study could provide an ideal pathological model of acute lung inflammation in mice for drug screening and intervention.
引文
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[9]李朝胜,孙黔云.三种化学小分子对补体旁路激活致内皮细胞黏附分子表达的干预及机制研究[J].中国药理学通报,2015,31(10):1421-6.[9]Li C S,Sun Q Y.Effect of three chemical molecules on adhesion molecules expression in HEMCs induced by activated complement alternative pathway[J].Chin Pharmacol Bull,2015,31(10):1421-6.
[10]Zhang L,Zhao S,Yuan L,et al.Knockdown of placental growth factor(PLGF)mitigates hyperoxia-induced acute lung injury in neonatal rats:suppressive effects on NF-κB signaling pathway[J].Int Immunopharmacol,2016,38:167-74.
[11]Huang R,Li M.Protective effect of AstragalosideⅣagainst sepsis-induced acute lung injury in rats[J].Saudi Pharm J,2016,24(3):341-7.
[2]Thurman J M,Holers V M.The central role of the alternative complement pathway in human disease[J].J Immunol,2006,176(3):1305-10.
[3]孙黔云.眼镜蛇毒因子在生命科学中的应用[J].生命科学,2016,28(1):22-6.[3]Sun Q Y.Application of cobra venom factor in life sciences[J].Chin Bull Life Sci,2016,28(1):22-6.
[4]路青瑜,李敏,孙黔云.补体旁路激活致内皮细胞纤溶凝血相关分子表达变化及干预研究[J].中国药理学通报,2015,31(8):1142-6.[4]Lu Q Y,Li M,Sun Q Y.Expression of coagulation-and fibrinolysis-related molecules of endothelial cells induced by activated complement alternative pathway and intervention[J].Chin Pharmacol Bull,2015,31(8):1142-6.
[5]孙黔云,李敏,叶巧玲,李红玲.补体旁路激活导致内皮细胞活化和损伤[J].中国药理学通报,2012,28(7):925-9.[5]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.
[6]孙黔云,叶巧玲,闫银萍.小鼠血清补体替代途径溶血活性测定的新方法[J].中国药理学通报,2011,27(11):1619-22.[6]Sun Q Y,Ye Q L,Yan Y P.A new method for measurement of the alternative pathway activity of mouse serum complement[J].Chin Pharmacol Bull,2011,27(11):1619-22.
[7]Kral J B,Schrottmaier W C,Salzmann M,Assinger A.Platelet interaction with innate immune cells[J].Transfus Med Hemother,2016,43(2):78-88.
[8]Rafiee L,Hajhashemi V,Javanmard S H.Maprotiline inhibits LPSinduced expression of adhesion molecules(ICAM-1 and VCAM-1)in human endothelial cells[J].Res Pharm Sci,2016,11(2):138-44.
[9]李朝胜,孙黔云.三种化学小分子对补体旁路激活致内皮细胞黏附分子表达的干预及机制研究[J].中国药理学通报,2015,31(10):1421-6.[9]Li C S,Sun Q Y.Effect of three chemical molecules on adhesion molecules expression in HEMCs induced by activated complement alternative pathway[J].Chin Pharmacol Bull,2015,31(10):1421-6.
[10]Zhang L,Zhao S,Yuan L,et al.Knockdown of placental growth factor(PLGF)mitigates hyperoxia-induced acute lung injury in neonatal rats:suppressive effects on NF-κB signaling pathway[J].Int Immunopharmacol,2016,38:167-74.
[11]Huang R,Li M.Protective effect of AstragalosideⅣagainst sepsis-induced acute lung injury in rats[J].Saudi Pharm J,2016,24(3):341-7.