摘要
目的研究熊果苷通过调控巨噬细胞Toll样受体4(TLR4)/核因子κB(NF-κB)通路抑制结核分枝杆菌活性的机制。方法将小鼠单核巨噬细胞白血病细胞RAW264.7分为对照组和熊果苷组,对照组正常培养,不干预;熊果苷组给予100μg/mL熊果苷干预24 h后,再用1000 ng/mL脂多糖培养24 h。通过定量聚合酶链式反应(PCR)法和蛋白质印迹法检测两组细胞TLR4和NF-κB表达的影响,并通过酶联免疫吸附试验(ELISA)检测熊果苷对脂多糖(LPS)诱导的RAW264.7细胞分泌肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β和IL-6的影响。40只昆明雄性小鼠经尾静脉注射H37Rv结核分枝杆菌毒株建立结核菌感染小鼠模型,随机将其均分为4组:不进行任何治疗的结核组,低剂量组、中剂量组和高剂量组分别接受腹腔注射10、25和50 mg/kg熊果苷治疗1次/周,共4周。结核感染模型建立4周后,处死小鼠,收集心脏外周血和肺组织,抗酸染色检测肺组织结核分枝杆菌数目;ELISA法检测小鼠外周血TNF-α、IL-1β和IL-6含量;流式细胞仪检测小鼠肺泡巨噬细胞凋亡率和外周血CD68阳性细胞TLR4/NF-κB表达。结果与对照组相比,熊果苷组RAW264.7细胞TLR4和NF-κB表达显著增高,培养基中TNF-α、IL-1β和IL-6含量均显著升高,差异均有统计学意义(P<0.05)。与结核组小鼠相比,经不同剂量熊果苷治疗(低剂量组、中剂量组和高剂量组)的结核杆菌感染的小鼠体重、外周血巨噬细胞TLR4/NF-κB表达水平以及外周血TNF-α、IL-1β和IL-6含量均显著增高,肺组织结核分枝杆菌数目和肺泡巨噬细胞凋亡率均显著降低,差异均有统计学意义(P<0.05),且以上指标均存在剂量依赖。结论熊果苷可以通过促进巨噬细胞TLR4/NF-κB通路的激活而促进巨噬细胞的存活和炎症因子的分泌,进而发挥抗结核的功能。
Objective To study the mechanism of arbutin inhibiting the activity of Mycobacterium tuberculosis by regulating the Toll-like receptor 4(TLR4)/nuclear factor kappa-B(NF-κB)pathway of macrophages.Methods Mouse mononuclear macrophage leukemia cells RAW264.7 were divided into control group and arbutin group.The control group was cultured normally without intervention;the arbutin group was treated with 100μg/mL arbutin for 24 h,and then cultured with 1000 ng/mL lipopolysaccharide for 24 h.The effects of TLR4 and NF-κB expression in the two groups of cells were detected by quantitative polymerase chain reaction(PCR)and Western blotting,and enzyme-linked immunosorbent(ELISA)was used to detect the effect of arbutin on the secretion of tumor necrosis factor alpha(TNF-α),interleukin(IL)-1βand IL-6 in RAW264.7 cells induced by lipopolysaccharide(LPS).Forty Kunming male mice were injected with the H37Rv Mycobacterium tuberculosis strain through the tail vein to establish a tuberculosis infection mouse model.They were randomly divided into 4 groups:tuberculosis group without any treatment,low-dose group,medium-dose group and the high-dose group received intraperitoneal injection of 10,25 and 50 mg/kg arbutin once.Four weeks after the tuberculosis infection model was established,the mice were sacrificed,peripheral blood of the heart and lung tissues were collected,and acid-fast staining was used to detect the number of Mycobacterium tuberculosis in lung tissues;ELISA method was used to detect the levels of TNF-α,IL-1βand IL-6 in peripheral blood of mice;Flow cytometry to detect the apoptotic rate of alveolar macrophages in mice and the expression of TLR4/NF-κB in CD68-positive cells in peripheral blood.Results Compared with the control group,the expression of TLR4 and NF-κB in RAW264.7 cells in the arbutin group was significantly increased,and the contents of TNF-α,IL-1βand IL-6 in the medium were significantly increased,and the differences were statistically significant(P<0.05).Compared with mice in the tuberculosis group,the body weight,the expression level of TLR4/NF-κB in peripheral blood macrophages,and the expression levels of TLR4/NF-κB in peripheral blood macrophages of mice infected with Mycobacterium tuberculosis treated with different doses of arbutin(low-dose,medium-dose and high-dose groups),the levels of TNF-α,IL-1βand IL-6 in peripheral blood were significantly increased,and the number of Mycobacterium tuberculosis in lung tissue and the apoptosis rate of alveolar macrophages were significantly decreased,and the differences were statistically significant(P<0.05).And the above indicators are all dose-dependent.Conclusion Arbutin promotes the survival of macrophages and the secretion of inflammatory factors by promoting the activation of the TLR4/NF-κB pathway of macrophages,thereby exerting anti-tuberculosis function.
作者
李卫鸿
韩芸
师成玲
龙海青
邓晓珍
李明霖
张松达
LI Wei-hong;HAN Yun;SHI Cheng-ling(Department of Dermatology,STD and AIDS,Fourth People's Hospital of Qinghai Province,Xining Qinghai 810000,China.)
出处
《临床和实验医学杂志》
2021年第18期1940-1944,共5页
Journal of Clinical and Experimental Medicine
基金
青海省科技计划项目(编号:2019-ZJ-7084)。