基于Janus激酶/信号转导和转录激活因子信号通路的乙胺丁醇片对肺结核大鼠模型的作用机制

Action Mechanism of Ethambutol Tablets on Pulmonary Tuberculosis Rat Model Based on Janus Kinase/Signal Transducer and Activator of Transcription Signaling Pathway

目的 探讨乙胺丁醇片(EMB)对肺结核(PTB)大鼠的治疗作用及其作用机制是否与Janus激酶(JAK)/信号转导和转录激活因子(STAT)信号通路有关。方法 将60只SD大鼠按照随机数字表法分为对照组、PTB组、PTB+EMB组(30 mg/kg)、PTB+EMB+Colivelin(JAK/STAT通路激活剂)组(30 mg/kg+1 mg/kg),每组15只。除对照组外,其他组大鼠均通过注射0.2 ml 5 mg/ml的结核杆菌悬液构建PTB模型。建模成功后,进行连续4周(1次/d)的给药处理,检测大鼠在给药第1、14、28天时的体重变化;计数结核分枝杆菌菌落数;HE染色检测大鼠肺组织病理变化;ELISA法检测各组大鼠血清中白细胞介素(IL)-6、肿瘤坏死因子-α、IL-1β、γ干扰素水平;流式细胞术检测各组大鼠外周血中T淋巴细胞亚群CD3^(+)、CD4^(+)、CD8^(+)、CD4^(+)/CD8^(+)水平;16S rRNA测序检测各组大鼠肠道菌群属水平的相对丰度;Western blot检测JAK/STAT通路相关蛋白表达。结果 与对照组比较,PTB组大鼠体重减轻(第14、28天时)、结核分枝杆菌菌落数增加,肺组织呈现出严重的病理学变化,血清中IL-6、肿瘤坏死因子-α、IL-1β水平、CD8^(+)、肠道菌群中拟杆菌属、消化球菌属、梭状芽孢杆菌属、放线菌属、乳酸杆菌科、疣微菌科、韦荣球菌科相对丰度及肺组织中磷酸化JAK2、磷酸化STAT3蛋白均显著升高,CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)、γ干扰素水平均显著降低(P均<0.001);与PTB组比较,PTB+EMB组大鼠体重增加(第14、28天时),结核分枝杆菌菌落数减少,肺组织病理损伤减轻,血清中IL-6、肿瘤坏死因子-α、IL-1β水平、CD8^(+)、肠道菌群中拟杆菌属、消化球菌属、梭状芽孢杆菌属、放线菌属、乳酸杆菌科、疣微菌科、韦荣球菌科相对丰度及肺组织中磷酸化JAK2、磷酸化STAT3蛋白均显著降低,CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)、γ干扰素水平均显著升高(P均<0.001);Colivelin减弱了EMB对肺结核大鼠模型感染的改善作用(P均<0.001)。结论 EMB可通过抑制JAK/STAT信号通路改善肺结核大鼠模型的感染。

Objective To explore the therapeutic effect of ethambutol tablets(EMB) on pulmonary tuberculosis(PTB) in rats and whether the action mechanism of EMB is related to Janus kinase(JAK)/signal transducer and activator of transcription(STAT) signaling pathway.Methods Sixty SD rats were assigned into a control group, a PTB group, a PTB+EMB group(30 mg/kg),and a PTB+EMB+Colivelin(JAK/STAT pathway activator) group(30 mg/kg+1 mg/kg) via the random number table method, with 15 rats in each group.The rats in other groups except the control group were injected with 0.2 ml of 5 mg/ml Mycobacterium tuberculosis suspension to establish the PTB model.After the modeling, the rats were administrated with corresponding drugs for 4 consecutive weeks(once a day).On days 1,14,and 28 of administration, the body weights of rats were measured and the Mycobacterium tuberculosis colonies were counted.Hematoxylin-eosin staining was carried out to detect the pathological changes in the lung tissue.Enzyme-linked immunosorbent assay was employed to measure the levels of interleukin(IL)-6,tumor necrosis factor-α(TNF-α),IL-1β,and interferon-γ(IFN-γ) in the serum.Flow cytometry was used to determine the levels of T lymphocyte subsets CD3^(+),CD4^(+),CD8^(+),and CD4^(+)/CD8^(+).The 16 S rRNA sequencing was performed to detect the relative abundance of the intestinal microorganisms.Western blotting was employed to determine the expression of the proteins in the JAK/STAT pathway.Results Compared with the control group, the modeling of PTB reduced the rat body weight(on days 14 and 28),increased Mycobacterium tuberculosis colonies, caused severe pathological changes in the lung tissue, and elevated the levels of IL-6,TNF-α,and IL-1β in serum and CD8^(+).Moreover, the modeling increased the relative abundance of Bacteroides,Peptococcus,Clostridium,Actinomyces,Lactobacillus,Verrucomicrobium,and Veillonella in the intestine, up-regulated the protein levels of phosphorylated JAK2 and phosphorylated STAT3 in the lung tissue, and lowered the levels of CD3^(+),CD4^(+),CD4^(+)/CD8^(+),and IFN-γ levels(all P<0.001).Compared with the PTB group, PTB+EMB increased the rat body weight(on days 14 and 28),reduced Mycobacterium tuberculosis colonies, alleviated the pathological damage in lung tissue, lowered the levels of IL-6,TNF-α,and IL-1β in serum and CD8^(+).Moreover, the treatment decreased the relative abundance of Bacteroides,Peptococcus,Clostridium,Actinomyces,Lactobacillus,Verrucomicrobium,Veillonella in the intestine, down-regulated the protein levels of phosphorylated JAK2 and phosphorylated STAT3 in the lung tissue, and elevated the levels of CD3^(+),CD4^(+),CD4^(+)/CD8^(+),and IFN-γ(all P<0.001).Colivelin weakened the alleviation effect of EMB on PTB(all P<0.001).Conclusion EMB can inhibit the JAK/STAT signaling pathway to alleviate the PTB in rat.