清燥救肺汤对肺炎支原体感染小鼠NLRP3炎性小体相关因子表达的影响
|
摘要
目的探讨清燥救肺汤对肺炎支原体(MP)感染后NLRP3炎性小体相关因子的动态变化,试图进一步明确清燥救肺汤防治肺炎支原体肺炎(MPP)的效应靶点。方法将72只Balb/c小鼠随机分成正常组、模型组、清燥救肺汤组、阿奇霉素组,每组18只。除正常组小鼠外,其余3组采用滴鼻法对实验Balb/c小鼠进行MP感染。造模后,清燥救肺汤组每只给予15 g/kg的对应方剂进行灌胃,1次/d,连续14 d;阿奇霉素组,每只给予阿奇霉素90 mg/kg灌胃,1次/d,连续3 d,停药4 d,2个循环,停药期间蒸馏水灌胃;正常组、模型组每只给予0.3 mL蒸馏水灌胃。在感染后的第3、7、10天进行取材,采用透射电镜观察肺组织超微结构改变,运用免疫组织化学SP法和Western blot法检测肺组织NLRP3、caspase-1蛋白的表达,并采用ELISA法检测血清中白细胞介素-1β(IL-1β)水平。结果 MP感染后,小鼠肺组织出现炎症改变,肺泡壁增厚、肺泡上皮细胞破坏、细胞浸润。MP感染后,小鼠肺组织中的NLRP3、caspase-1的表达明显升高,血清中IL-1β明显上升;与模型组比较,各用药组均可以下调NLRP3、caspase-1及IL-1β的表达(均P<0.05)。结论清燥救肺汤可以有效地下调caspase-1、NLRP3及IL-1β的表达,可能为其治疗MPP的效应靶点。
Objective To investigate the dynamic changes of NLRP3-related inflammatory molecules in mice infected with Mycoplasma pneumoniae(MP) treated with Qingzao Jiufei(Dryness-rectifying Lung-saving, QZJF) Decoction, and to explore target of QZJF Decoction on the prevention and treatment of Mycoplasma pneumoniae pneumonia(MPP). Methods Seventy-two Balb/c mice were randomly divided into normal group(0.3 mL distilled water, gastric perfusion), model group(0.3 mL distilled water, gastric perfusion), QZJF Decoction(15 g/kg, once daily for 14 consecutive days) group, and azithromycin(90 mg/kg, once daily for 3 consecutive days, at 4-day interval of distilled water intragastric administration) group(n =18). All mice were infected with MP with nasal inoculation method except the ones in normal group. After establishing the model, 15 g/kg of QZJF decoction were given intragastrically once a day for 14 consecutive days. Samples were taken on the 3 rd, 7 th and 10 th day after infection. Ultrastructural changes of lung tissue were observed by transmission electron microscopy. The expressions of NLRP3 and caspase-1 protein in lung tissue were detected by SP immunohistochemistry and Western blot, and the levels of IL-1β in the serum were measured by ELISA. Results After MP infection, the lung tissue of the mice showed inflammatory changes, such as the alveolar wall thickening, alveolar epithelial cell destruction, and cell infiltration. After MP infection, the expression of NLRP3 and caspase-1 in lung tissue of mice increased significantly, and IL-1β in serum increased significantly, too. Compared with the model group, the expressions of NLRP3, caspase-1 and IL-1β were down-regulated in each drug group. Conclusion QZJF decoction can effectively down-regulate the expression of caspase-1, NLRP3 and IL-β, which may be the therapeutic target for the treatment of MPP.
Objective To investigate the dynamic changes of NLRP3-related inflammatory molecules in mice infected with Mycoplasma pneumoniae(MP) treated with Qingzao Jiufei(Dryness-rectifying Lung-saving, QZJF) Decoction, and to explore target of QZJF Decoction on the prevention and treatment of Mycoplasma pneumoniae pneumonia(MPP). Methods Seventy-two Balb/c mice were randomly divided into normal group(0.3 mL distilled water, gastric perfusion), model group(0.3 mL distilled water, gastric perfusion), QZJF Decoction(15 g/kg, once daily for 14 consecutive days) group, and azithromycin(90 mg/kg, once daily for 3 consecutive days, at 4-day interval of distilled water intragastric administration) group(n =18). All mice were infected with MP with nasal inoculation method except the ones in normal group. After establishing the model, 15 g/kg of QZJF decoction were given intragastrically once a day for 14 consecutive days. Samples were taken on the 3 rd, 7 th and 10 th day after infection. Ultrastructural changes of lung tissue were observed by transmission electron microscopy. The expressions of NLRP3 and caspase-1 protein in lung tissue were detected by SP immunohistochemistry and Western blot, and the levels of IL-1β in the serum were measured by ELISA. Results After MP infection, the lung tissue of the mice showed inflammatory changes, such as the alveolar wall thickening, alveolar epithelial cell destruction, and cell infiltration. After MP infection, the expression of NLRP3 and caspase-1 in lung tissue of mice increased significantly, and IL-1β in serum increased significantly, too. Compared with the model group, the expressions of NLRP3, caspase-1 and IL-1β were down-regulated in each drug group. Conclusion QZJF decoction can effectively down-regulate the expression of caspase-1, NLRP3 and IL-β, which may be the therapeutic target for the treatment of MPP.
引文
[1] Odeh AN,Simecka JW.Regulatory CD4+CD25+ T cells dampen inflammatory disease in murine myco plasma pneumonia and promote IL-17 and IFN-γ responses[J].PLoS ONE,2016,11(5):e0155648.
[2] Davis BK,Wen H,Ting PY.The inflammasome NLRs in immunity,inflammation,and associated diseases[J].Annual Review of Immunology,2011,29(1):707-712.
[3] Lamkanfi M,Dixit VM.Mechanisms and functions of inflammasomes[J].Cell,2014,157(5):1013-1022.
[4] Liu X,Zhang Z,Ruan J,et al.Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores[J].Nature,2016,535(7610):153-159.
[5] Narita M.Pathogenesis of extrapulmonary manifesta tions of Mycoplasma pneumoniae,infection with special reference to pneumonia[J].Journal of Infection & Chemotherapy Official Journal of the Japan Society of Chemotherapy,2010,16(3):162-169.
[6] 罗征秀,罗健,徐秀娟,等.儿童肺炎支原体肺炎急性期气道黏膜损害与预后的关系[J].第三军医大学学报,2011,33(2):190-192.Luo ZX,Luo J,Xu XJ,et al.Relationship between airway mucosal lesions and prognosis in children with myco plasm pneumoniae pneumonia in children[J].Journal of Third Military Medical University,2011,33 (2):190-192.
[7] Wood PR,Hill VL,Burks ML,et al.Mycoplasma pneu moniae in children with acute and refractory asthma[J].Annals of Allergy Asthma & Immunology,2013,110(5):328-334.
[8] Cruz CM,Rinna A,Forman HJ,et al.ATP activates a re active oxygen species-dependent oxidative stress re sponse and secretion of proinflammatory cytokines in macrophages[J].Journal of Biological Chemistry,2007,282(5):2871-2879.
[9] Kebaier C,Chamberland RR,Allen IC,et al.Staphylo coccus aureus α-hemolysin mediates virulence in a mu rine model of severe pneumonia through activation of the NLRP3 inflammasome[J].Journal of Infectious Disea ses,2012,205(5):807-817.
[10] 徐玥瑾,万迎新.清燥救肺汤在肺系疾病中的应用[J].世界中医药,2014,9(11):1509-1511.Xu YJ,Wan YX.Application of Qingzaojiufei decoction in treating pulmonary diseases[J].World Traditional Chi nese Medicine,2014,9(11):1509-1511.
[11] 吴振起,杨璐,敏娜,等.清燥救肺汤及其拆方对肺炎支原体感染小鼠Bax、Bcl-2、caspase-3蛋白的影响[J].中草药,2018,49(2):389-395.Wu ZQ,Yang L,Min N,et al.Effects of Qingzao Jiufei Decoction and its disassembled prescriptions on Bax,Bcl-2 and caspase-3 proteins in mice infected with My coplasma pneumoniae[J].Chinese Traditional and Herbal Drugs,2018,49 (2):389-395.
[12] Segovia JA,Chang TH,Winter VT,et al.NLRP3 is a critical regulator of inflammation and innate immune cell response during mycoplasma pneumoniae infection[J].Infection & Immunity,2018,86(1):548-562.
[2] Davis BK,Wen H,Ting PY.The inflammasome NLRs in immunity,inflammation,and associated diseases[J].Annual Review of Immunology,2011,29(1):707-712.
[3] Lamkanfi M,Dixit VM.Mechanisms and functions of inflammasomes[J].Cell,2014,157(5):1013-1022.
[4] Liu X,Zhang Z,Ruan J,et al.Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores[J].Nature,2016,535(7610):153-159.
[5] Narita M.Pathogenesis of extrapulmonary manifesta tions of Mycoplasma pneumoniae,infection with special reference to pneumonia[J].Journal of Infection & Chemotherapy Official Journal of the Japan Society of Chemotherapy,2010,16(3):162-169.
[6] 罗征秀,罗健,徐秀娟,等.儿童肺炎支原体肺炎急性期气道黏膜损害与预后的关系[J].第三军医大学学报,2011,33(2):190-192.Luo ZX,Luo J,Xu XJ,et al.Relationship between airway mucosal lesions and prognosis in children with myco plasm pneumoniae pneumonia in children[J].Journal of Third Military Medical University,2011,33 (2):190-192.
[7] Wood PR,Hill VL,Burks ML,et al.Mycoplasma pneu moniae in children with acute and refractory asthma[J].Annals of Allergy Asthma & Immunology,2013,110(5):328-334.
[8] Cruz CM,Rinna A,Forman HJ,et al.ATP activates a re active oxygen species-dependent oxidative stress re sponse and secretion of proinflammatory cytokines in macrophages[J].Journal of Biological Chemistry,2007,282(5):2871-2879.
[9] Kebaier C,Chamberland RR,Allen IC,et al.Staphylo coccus aureus α-hemolysin mediates virulence in a mu rine model of severe pneumonia through activation of the NLRP3 inflammasome[J].Journal of Infectious Disea ses,2012,205(5):807-817.
[10] 徐玥瑾,万迎新.清燥救肺汤在肺系疾病中的应用[J].世界中医药,2014,9(11):1509-1511.Xu YJ,Wan YX.Application of Qingzaojiufei decoction in treating pulmonary diseases[J].World Traditional Chi nese Medicine,2014,9(11):1509-1511.
[11] 吴振起,杨璐,敏娜,等.清燥救肺汤及其拆方对肺炎支原体感染小鼠Bax、Bcl-2、caspase-3蛋白的影响[J].中草药,2018,49(2):389-395.Wu ZQ,Yang L,Min N,et al.Effects of Qingzao Jiufei Decoction and its disassembled prescriptions on Bax,Bcl-2 and caspase-3 proteins in mice infected with My coplasma pneumoniae[J].Chinese Traditional and Herbal Drugs,2018,49 (2):389-395.
[12] Segovia JA,Chang TH,Winter VT,et al.NLRP3 is a critical regulator of inflammation and innate immune cell response during mycoplasma pneumoniae infection[J].Infection & Immunity,2018,86(1):548-562.