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目的:研究洋葱中黄酮类化合物对结核分枝杆菌的影响,探讨其治疗结核的作用机制。方法:以结核分枝杆菌标准菌株(H37RV)为对照,临床结核分枝杆菌为研究对象,采用分格平板琼脂比例法观察28μg/mL、140μg/mL、280μg/mL、560μg/mL不同浓度洋葱黄酮类化合物、一线抗结核药物利福平(REP)、异烟肼(INH)、链霉素(SM)和乙胺丁醇(EMB)对结核分枝杆菌的抑菌作用。小鼠腹腔巨噬细胞与结核分枝杆菌共同孵育后,给予细胞不同浓度洋葱黄酮类化合物和INH药物干预,细胞实验分组为对照组、INH组、28μg/mL洋葱黄酮组、140μg/mL洋葱黄酮组、280μg/mL洋葱黄酮组、560μg/mL洋葱黄酮组,给药结束后,ELISA法检测细胞培养液中细胞因子水平,RT-PCR法检测巨噬细胞吞噬的结核分枝杆菌DNA水平。结果:280μg/mL洋葱黄酮组敏感率高于各一线抗结核药物(P<0.05),故后续选择280μg/mL洋葱黄酮进行研究。280μg/mL洋葱黄酮联合1.0μg/mL REP、2.0μg/mL INH、2.0μg/mL SM和5.0μg/mL EMB的敏感率均显著高于各单个抗结核药物,差异均有统计学意义(P<0.05)。与对照组比较,INH组、140μg/mL洋葱黄酮组、280μg/mL洋葱黄酮组、560μg/mL洋葱黄酮组IFN-γ、IL-1β、IL-6水平显著升高,巨噬细胞吞噬的结核分枝杆菌DNA扩增Ct值较低,DNA拷贝数较高,差异均有统计学意义(P<0.05)。与INH组比较,280μg/mL洋葱黄酮组、560μg/mL洋葱黄酮组IFN-γ、IL-1β、IL-6水平显著升高,巨噬细胞吞噬的结核分枝杆菌DNA扩增Ct值较低,DNA拷贝数较高,差异均有统计学意义(P<0.05)。与140μg/mL洋葱黄酮组比较,280μg/mL洋葱黄酮组、560μg/mL洋葱黄酮组IFN-γ、IL-1β、IL-6水平显著升高,巨噬细胞吞噬的结核分枝杆菌DNA扩增Ct值较低,DNA拷贝数较高,差异均有统计学意义(P<0.05)。结论:洋葱黄酮类化合物体外可抑制结核分枝杆菌,与各单个抗结核药物具有协同抑菌作用,促进巨噬细胞释放IFN-γ、IL-1β和IL-6细胞因子,提高巨噬细胞吞噬能力,且呈剂量依赖性。
Objective: To study the effect of flavonoids in onion on Mycobacterium tuberculosis and to explore its mechanism of action in treating tuberculosis. Methods: Mycobacterium tuberculosis was used as the control in the standard strain of Mycobacterium tuberculosis (H37RV), and the effects of 28μg / mL, 140μg / mL, 280μg / mL and 560μg / mL onions Flavonoids, first-line anti-tuberculosis drugs rifampicin (REP), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) on Mycobacterium tuberculosis. The mice peritoneal macrophages were incubated with M. tuberculosis, then the cells were treated with different concentrations of onion flavonoids and INH drugs. The cells were divided into control group, INH group, 28μg / mL onion flavone group, 140μg / mL onion flavone Group, 280μg / mL onion flavone group and 560μg / mL onion flavone group. After the drug was administered, cytokines levels in cell culture medium were detected by ELISA and DNA levels of phagocytosed Mycobacterium tuberculosis by macrophages were detected by RT-PCR. Results: The sensitivity of 280μg / mL onion flavone group was higher than that of the first-line antituberculous drugs (P <0.05). Therefore, 280μg / mL onion flavonoids were selected for further study. The sensitivities of 280μg / mL flavonoids combined with 1.0μg / mL REP, 2.0μg / mL INH, 2.0μg / mL SM and 5.0μg / mL EMB were significantly higher than those of single anti-tuberculosis drugs, the differences were statistically significant <0.05). Compared with the control group, the levels of IFN-γ, IL-1β and IL-6 in INH group, 140μg / mL onion flavone group, 280μg / mL onion flavone group and 560μg / mL onion flavone group were significantly increased, while those phagocytosed by macrophages Mycobacterium DNA amplification Ct value is low, DNA copy number is higher, the difference was statistically significant (P <0.05). Compared with INH group, the levels of IFN-γ, IL-1β and IL-6 in 280μg / mL onion flavone group and 560μg / mL onion flavone group were significantly increased, and the Ct value of M. tuberculosis phagocytosis by macrophages was lower , DNA copy number is higher, the difference was statistically significant (P <0.05). The levels of IFN-γ, IL-1β, and IL-6 in 280μg / mL onion flavone group and 560μg / mL onion flavone group were significantly higher than those in 140μg / mL onion flavone group, and M. tuberculosis phagocytosis by macrophages Ct value is low, DNA copy number is higher, the difference was statistically significant (P <0.05). CONCLUSION: Onion flavonoids can inhibit Mycobacterium tuberculosis in vitro and have synergistic antimicrobial activity with each single anti-TB drug, and promote macrophages to release IFN-γ, IL-1β and IL-6 cytokines and increase macrophage phagocytosis Ability, and in a dose-dependent manner.