冬凌草甲素抗结核病理损伤的作用机制研究  

Study on the mechanism of Oridonin against pathological damage of tuberculosis

作　　者：李银虹 刘芳琳 鹿振辉[3] 姜昕[1] Li Yinhong;Liu Fanglin;Lu Zhenhui;Jiang Xin(Department of Immunology and Pathology,School of Basic Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;School of Acupuncture and Tuina,Shannxi University of Traditional Chinese Medicine,Xianyang 712046,China;Department of Pulmonary Disease,Longhua Hospital Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学基础医学院免疫学与病原生物学教研室,上海201203 [2]陕西中医药大学针灸推拿学院,咸阳712046 [3]上海中医药大学附属龙华医院肺病科,上海200032

出　　处：《中国防痨杂志》2022年第8期849-854,共6页Chinese Journal of Antituberculosis

基　　金：国家自然科学基金(81873069);“十三五”国家科技重大专项(2018ZX10725-509)。

摘　　要：目的:通过冬凌草甲素对结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染巨噬细胞NOD样受体家族蛋白3(NOD-like receptor protein 3,NLRP3)炎症小体及内质网应激的影响,探讨冬凌草甲素抗结核病理损伤的作用机制。方法:利用MTB标准株H37Ra建立小鼠巨噬细胞Raw264.7感染模型,设置空白对照组、MTB感染模型组、冬凌草甲素作用不同浓度(0.5、1.0、2.0、4.0μmol/L)及作用不同时间点(6、12、24 h)干预组。应用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法检测冬凌草甲素的可用药物浓度。应用蛋白免疫印迹法检测NLRP3、硫氧还蛋白互作蛋白(thioredoxin-interacting protein,TXNIP)表达情况;检测内质网应激相关蛋白免疫球蛋白结合蛋白(Bip)、CCAAT/增强子结合蛋白同源蛋白(CHOP)、真核生物翻译起始因子2α(peIF2α)、磷酸化肌醇需要酶1α(pIRE1α)、肌醇需要酶1α(IRE1α)的表达情况;检测内质网应激下游核因子κB/磷酸化应激活化蛋白激酶/p38(NF-κB/pJNK/pp38)通路的变化,并采用Image J软件做蛋白定量分析。结果:冬凌草甲素在4.0μmol/L浓度以下细胞生存率在90%左右,对细胞毒性较小。在不同时间点(12、24 h),与MTB感染模型组相比,冬凌草甲素可明显降低NLRP3蛋白表达(4.35±0.13 vs.5.95±0.15;1.90±0.05 vs.3.93±0.09),明显降低TXNIP蛋白表达(1.14±0.05 vs.1.73±0.04;0.78±0.05 vs.1.33±0.02),差异均有统计学意义(F值分别为508.308和166.278,P值均为0.000)。在不同时间点(6、12 h),与MTB感染模型组相比,冬凌草甲素可明显降低Bip蛋白表达(1.85±0.07 vs.2.27±0.07;0.97±0.03 vs.2.28±0.17),明显降低peIF2α蛋白表达(1.75±0.42 vs.1.75±0.03;1.31±0.04 vs.2.45±0.17),明显降低IRE1α蛋白表达(10.48±0.40 vs.14.19±0.45;6.15±0.15 vs.15.76±1.27),明显降低pp65蛋白表达(0.69±0.01 vs.1.07±0.03;0.28±0.01 vs.0.39±0.02),差异均有统计学意义(F值分别为149.510、10.489、10.294、288.194,P值均<0.01)。结论:MTB感染�Objective:To explore the mechanism of Oridonin against pathological damage of tuberculosis through its effect on the NOD-like receptor protein 3(NLRP3)inflammasomes and endoplasmic reticulum stress in Mycobacterium tuberculosis(MTB)-infected macrophages.Methods:H37Ra strain was used to establish macrophage infection model,meanwhile,blank control group,MTB infection model group and Oridonin intervention groups with different concentrations(0.5,1.0,2.0,4.0μmol/L)and different time points(6,12,24 h)were also set up.Methyl thiazolyl tetrazolium(MTT)method was used to detect the safe dosage range of Oridonin.Western blot was used to detect the expression level of NLRP3 and thioredoxin-interacting protein(TXNIP)protein,immunoglobulin binding protein(Bip),CCAAT/enhancer binding protein homologous protein(CHOP),phosphorylated eukaryotic translation initiation factor 2α(peIF2α)and phosphorylated inositol Requires protein expression of enzyme 1α(pIRE1α)which related to endoplasmic reticulum stress.Western blot was also used to detect the changes of NF-κB/pJNK/pp38 pathway located on downstream of endoplasmic reticulum stress.Protein quantitative analysis was performed with Image J software.Results:The survival rate of infected macrophage treated with oridonin below 4.0μmol/L was about 90%,indicating minor toxic to cells.Western blot showed that compared with the model group at different time points(12,24 h),Oridonin could significantly reduce expression levels of NLRP3 protein(4.35±0.13 vs.5.95±0.15;1.90±0.05 vs.3.93±0.09)and TXNIP protein(1.14±0.05 vs.1.73±0.04;0.78±0.05 vs.1.33±0.02),the differences were statistically significant(F=508.308 and 166.278;all P=0.000).Compared with the model group at different time points(6 h,12 h),Oridonin could significantly reduce expression levels of Bip protein(1.85±0.07 vs.2.27±0.07;0.97±0.03 vs.2.28±0.17),peIF2αprotein(1.75±0.42 vs.1.75±0.03;1.31±0.04 vs.2.45±0.17),IRE1αprotein(10.48±0.40 vs.14.19±0.45;6.15±0.15 vs.15.76±1.27),pp65 protein(0.69±0.01 vs.1.

关 键 词：分枝杆菌 结核 巨噬细胞 冬凌草属 炎症介导素类 内质网 

分 类 号：R96[医药卫生—药理学] R378.911[医药卫生—药学]