月华胶囊对耐多药结核分支杆菌感染自噬的影响

目的:以月华胶囊含药血清对耐多药结核分支杆菌感染小鼠单核巨噬细胞(RAW264.7)进行干预,探讨其对耐多药结核分支杆菌感染巨噬细胞自噬的作用及其机制。方法:低温冷冻干燥法制备月华胶囊,大鼠按3.02 g·kg^-1灌胃给药7 d,制备月华胶囊含药血清。体外培养RAW264.7,耐多药结核分子杆菌。以细胞增殖毒性检测试剂盒(CCK-8)检测含药血清对RAW264.7的增殖能力并选定其有效浓度。细胞分为模型组(10%胎牛血清);月华胶囊含药血清组(10%月华胶囊);自噬抑制剂3-甲基腺嘌呤(3-MA)+月华胶囊含药血清组(5 mg·L^-1的3-MA+10%月华胶囊含药血清);雷帕霉素(Rap)组(200 mg·L^-1的Rap+10%胎牛血清);正常组(10%胎牛血清)。除正常组外,各组细胞培养24 h后,按细胞-细菌1∶10感染4 h。透射电镜观察细胞自噬体的出现和形成;蛋白免疫印迹法(Western blot)检测细胞中I型微管相关蛋白轻链3(LC-3Ⅰ),Ⅱ型微管相关蛋白轻链3/I型微管相关蛋白1轻链3(LC-3Ⅱ/LC-3Ⅰ),Beclin-1蛋白的表达;间接免疫荧光染色法观察细胞中LC3B荧光颗粒、斑点及亮度;实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞中LC3,Beclin-1 mRNA的表达。结果:与正常组比较,模型组细胞未见自噬体,细胞中LC-3Ⅱ,LC-3Ⅱ/LC-3Ⅰ,Beclin-1蛋白及LC3,Beclin-1 mRNA的相对表达量均无显著变化,细胞无荧光颗粒、斑点,无荧光亮度。与模型组比较,月华胶囊含药血清组及Rap组细胞,均可观察到自噬体的形成,细胞LC-3Ⅱ,LC-3Ⅱ/LC-3Ⅰ,Beclin-1蛋白及LC3,Beclin-1 mRNA的相对表达量值均明显升高(P<0.05),月华胶囊含药血清组细胞荧光颗粒和荧光斑点明显增多,Rap组细胞荧光颗粒和荧光斑点增多非常明显,荧光闪亮,可判定细胞荧光呈现为阳性;自噬抑制剂3-MA+月华胶囊含药血清组细胞未见自噬体,细胞中LC-3Ⅱ,LC-3Ⅱ/LC-3Ⅰ,Beclin-1蛋白及LC3,Beclin-1 mRNA的相对表达量值均无明显升高。结论:月华胶囊含药血清能使耐多药结核分支杆菌感染细胞产生自噬而发挥抗结核的免疫效应,其机制是通过调控自噬相关蛋白LC3,Beclin-1蛋白及其mRNA的表达水平,促使LC3-I趋向LC3-Ⅱ的转化加快而实现的。

Effect of Yuehua Capsule on Autophagy of Macrophage Infected with Multi-drug Resistant Mycobacterium Tuberculosis

Objective: This study aims to explore the effect and mechanism of Yuehua capsule serum for autophagy of macrophages infected with multi-drug resistant mycobacterium tuberculosis.Method: The rats were undertaken intragastric gavage with Yuehua capsule by 3.02 g·kg^-1 once a day which was produced through low temperature condensation drying method.After 7 days,blood of abdominal aorta of rats was collected to prepare Yuehua capsule serum.RAW264.7 andmultidrug resistant tuberculosis were cultured in vitro.According to cell counting kit-8(CCK-8),10% drug-containing serum was considered as the effective concentration.The cultured cells were divided into four groups: model groups(10% fetal bovine serum).Yuehua capsule serum(10%Yuehua capsule serum).Autophagy inhibitor group + 3-MA + Yuehua capsule medicated serum(3-MA + 10%Yuehua capsule serum).Rapamycin(Rap) positive control group(200 mg·L^-1 Rap + 10% Yuehua capsule serum).Except for the normal group,the cells of each group were cultured for 24 h and infected for 4 h according to cell-bacteria 1∶10.Testing index: observation of autophagosomes under transmission electron microscope,the test of expression of microtubule-associated protein light chain-3Ⅱ(LC-3Ⅱ),microtubule-associated protein LC3-Ⅱ/microtubule-associated protein light chain 3-Ⅰ(LC3-Ⅰ) and Beclin-1 with Western blot, indirect immunofluorescence staining for LC3 B,and mRNA of Beclin-1 as well as LC3 with real-time fluorescent quantitative polymerase chain reaction(Real-time PCR).Result: Compared with normal group,model group did not see autophagy body cells,cells in the LC-3 Ⅱ,LC-3 Ⅱ/LC-3 Ⅰ,Beclin-1 protein and LC3,Beclin-1 mRNA gene expression level had no significant change,the cells without fluorescent particles,spots,no fluorescence intensity.Compared with model group,Yuehua capsules serum group and Rap positive control group can be observed the formation of phage,mRNA andprotein expression levelof LC-3 Ⅱ,LC-3 Ⅱ/LC-3 Ⅰ,Beclin-1 and LC3,Beclin-1 were significantly increased(P<0.05).Autophagy inhibitor group + 3-MA + Yuehua capsule medicated serum did not see autophagy,the mRNA and protein expression level of LC-3 Ⅱ,LC-3Ⅱ/LC-3 Ⅰ,Beclin-1 and LC3,Beclin-1 were no significantly increased.Conclusion: Yuehua capsule medicated serum could induce autophagy of macrophages of RAW264.7.The mechanism was probably accomplished through regulating the expression level of autophagy key protein LC3, autophagosome mature protein Beclin-1 and relevant gene,meanwhile the conversion of LC3-I to LC3-Ⅱ was accelerated.