紫草素调控TLRs信号通路对结核分枝杆菌感染小鼠肺部炎症、免疫功能及炎性因子的作用

Role of Shikonin modulation of TLRs signalling pathway on lung inflammation,immune function and inflammatory factors in Mycobacterium tuberculosis-infected mice

目的分析紫草素对结核分枝杆菌感染小鼠肺部炎症、免疫功能及炎性因子的作用及作用机制。方法随机选取SPF级C57BL/6小鼠87只,随机抽取15只作为对照组,将剩余72只小鼠建立结核分枝杆菌感染小鼠模型,建模成功45只,成功率62.50%,随机分为模型组、紫草素组、紫草素+Toll样受体4(toll like receptor 4,TLR4)、髓样分化因子88(myeloid differentiation factor 88,MyD88)、核转录因子κB(nuclear transcription factorκB,NF-κB)p65信号通路激动剂组(紫草素+LPS组)各15只,紫草素组腹腔注射40 mg/kg紫草素;紫草素+LPS组腹腔注射40 mg/kg紫草素及0.25 mg/kg LPS;对照组及模型组腹腔注射等量生理盐水。末次给药后,观察各组小鼠肺重指数及肺部组织结核菌荷菌量、肺组织病理学形态改变、免疫功能指标、炎性因子及通路相关因子表达水平。结果与对照组对比,模型组小鼠肺重指数、肺病变指数、肺部组织结核菌荷菌量均明显增多(P均<0.05);与模型组对比,紫草素组小鼠肺重指数、肺病变指数、肺部组织结核菌荷菌量均明显减少(P均<0.05);与紫草素组对比,紫草素+LPS组小鼠肺重指数、肺病变指数、肺部组织结核菌荷菌量均明显增多(P均<0.05)。对照组小鼠肺组织结构正常,细胞排列紧密,肺泡结构完整,未见炎性细胞浸润情况;模型组小鼠肺组织结构受损严重,肺泡数量减少,可见大量炎性细胞浸润,可见结核结节形成;紫草素组肺组织结构基本完整,偶见炎性细胞浸润;紫草素+LPS组肺组织结构、细胞排列、炎性细胞浸润及结核结节情况与模型组相似,但较优于模型组。与对照组对比,模型组小鼠CD3^(+)T淋巴细胞、CD4^(+)T淋巴细胞、CD4^(+)/CD8^(+)、白细胞介素10(interleukin-10,IL-10)水平均明显降低,CD8^(+)T淋巴细胞、白细胞介素6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及TLR4、MyD88、NF-κB p65表达水平均明显升高(P均<0.05);与模型组对比,紫草素组小鼠CD3^(+)T淋巴细胞、CD4^(+)T淋巴细胞、CD4^(+)/CD8^(+)、IL-10表达水平均明显升高,CD8^(+)T淋巴细胞、IL-6、TNF-α及TLR4、MyD88、NF-κB p65表达水平均明显降低(P均<0.05);与紫草素组对比,紫草素+LPS组小鼠CD3^(+)T淋巴细胞、CD4^(+)T淋巴细胞、CD4^(+)/CD8^(+)、IL-10表达水平均明显降低,CD8^(+)T淋巴细胞、IL-6、TNF-α及TLR4、MyD88、NF-κB p65表达水平均明显升高(P均<0.05)。结论紫草素可能通过抑制TLR4/MyD88/NF-κB p65信号通路改善结核杆分枝菌感染小鼠肺部组织结核分枝杆菌荷菌量,抑制肺部炎症反应,提高免疫功能,从而发挥一定的肺保护作用。

Objective To analyse the effects and mechanism of action of Shikonin on pulmonary inflammation,immune function and inflammatory factors in Mycobacterium tuberculosis-infected mice.Methods Eighty-seven SPF C57BL/6 mice were randomly selected,and 15 mice were randomly selected as the control group.The remaining 72 mice were established with Mycobacterium tuberculosis infection,and 45 mice were successfully established,with a success rate of 62.50%.They were randomly divided into model group,shikonin group,shikonin+Toll-like receptor 4(Toll-like receptor 4,TLR4),Myeloid differentiation factor 88(MyD 88),nuclear transcription factorκB(nuclear transcription factorκB,NF-κB,NF-κB)P65 signal pathway agonist group(shikonin+LPS group)with 15 rats in each group,and shikonin group was intraperitoneally injected with 40 mg/kg shikonin.In shikonin+LPS group,40 mg/kg shikonin and 0.25 mg/kg LPS;were injected intraperitoneally;The control group and model group were injected with the same amount of normal saline intraperitoneally.After the last administration,the lung weight index,the amount of tuberculosis bacteria in lung tissue,the pathological changes of lung tissue,the indexes of immune function,the expression levels of inflammatory factors and pathway-related factors were observed.Results Compared with the control group,the lung weight index,lung lesion index and the amount of tuberculosis bacteria in lung tissue in the model group were significantly increased(P<0.05).Compared with the model group,the lung weight index,lung lesion index and pulmonary tuberculosis load in shikonin group were significantly reduced(P<0.05).Compared with the shikonin group,the lung weight index,lung lesion index and pulmonary tuberculosis load in the shikonin+LPS group were significantly increased(P<0.05).In the control group,the lung tissue structure is normal,the cells are closely arranged,the alveolar structure is complete,and no inflammatory cell infiltration is found.In the model group,the lung tissue structure was seriously damaged,the number of alveoli decreased,a large number of inflammatory cells infiltrated,and tuberculosis nodules were formed.In shikonin group,the lung tissue structure was basically complete,and inflammatory cell infiltration was occasionally seen.The lung tissue structure,cell arrangement,inflammatory cell infiltration and tuberculosis nodules in shikonin+LPS group are similar to those in model group,but better than those in model group.Compared with the control group,the levels of CD3^(+)T lymphocytes,CD4^(+)T lymphocytes,CD4^(+)/CD8^(+)and interleukin-10(IL-10)in the model group were significantly decreased,while CD8^(+)T lymphocytes and interleukin-6(IL-6),Tumor necrosis factor-α(TNF-α),TLR4,MyD88 and NF-κB p65 all increased significantly(P<0.05).Compared with the model group,the expression levels of CD3^(+)T lymphocytes,CD4^(+)T lymphocytes,CD4^(+)/CD8^(+)and IL-10 in shikonin group were significantly increased,while the expression levels of CD8^(+)T lymphocytes,IL-6,TNF-α,TLR4,MyD88 and NF-κB p65 were significantly decreased(P<0.05).Compared with shikonin group,the expression levels of CD3^(+)T lymphocytes,CD4^(+)T lymphocytes,CD4^(+)/CD8^(+)and IL-10 in shikonin+LPS group were significantly decreased,while the expression levels of CD8^(+)T lymphocytes,IL-6,TNF-α,TLR4,MyD88 and NF-κB p65 were significantly increased(P<0.05).Conclusion Comfreyin may play a certain lung-protective role by inhibiting the TLR4/MyD88/NF-κB p65 signalling pathway to improve Mycobacterium tuberculosis charge in the lung tissues of Mycobacterium tuberculosis-infected mice,suppressing inflammatory responses in the lungs,and improving immune function.