目的:观察复方五凤草液治疗结核性溃疡的临床疗效，并基于干扰素-γ(INF-γ)/信号转导和转录激活因子1(STAT1)、白细胞介素-4(IL-4)/信号转导和转录激活因子6(STAT6)信号通路和卡介苗(BCG)诱导巨噬细胞极化模型探讨其作用机制。方法:(1)临床试验:将135例结核性溃疡患者分为观察组、中医对照组和西医对照组。在规范抗结核治疗基础上，观察组予复方五凤草液，中医对照组予康复新液，西医对照组予异烟肼液局部外用治疗，观察周期28 d。分别观察三组患者安全性指标、创面愈合总有效率、创面组织病理学形态及创面组织、创面组织中Ifnr、Il4、Stat1、Stat6 mRNA的表达，填写中医评分量表(Traditional Chinese Medicine Syndrome Score Scale，TCMSSS)病例报告表(Case Report Form，CRF)。(2)细胞实验:建立 BCG 诱导 THP-1 巨噬细胞模型，分别予不同药物处理后分为BCG组、BCG+康复新液组、BCG+异烟肼液组、BCG+复方五凤草液组。镜下观察各组BCG菌落情况，收集细胞上清液并离心，Western blot、RT-qPCR技术检测IFN-γ、IL-4、STAT1、STAT6的蛋白和mRNA表达，流式细胞术检测药物对CD11b、CD86、CD163、CD206百分率的影响。结果:(1)临床试验:治疗期间，3组患者安全性指标无明显差异;观察组总有效率(95。56％)高于中医对照组(82。22％)、西医对照组(75。56％);治疗28 d后，与中、西医对照组比较，观察组创面组织病理学形态改善更为明显，观察组患者Ifnr、Stat1 mRNA的表达低于中、西医对照组，Il4、Stat6 mRNA的表达高于中、西医对照组;与中、西医对照组比较，观察组中医证候积分显著降低。(2)细胞实验:THP-1巨噬细胞在感染BCG后吞噬泡明显增多，自噬小体增多;各组药物干预后胞内结核分枝杆菌的存活率降低。与对照组相比，BCG组M1型巨噬细胞所占比例较多，另3组M2型巨噬细胞所占比例较多。其中，BCG+复方五凤草液组中M2巨噬细胞占比显著高于其他3组。BCG+复方五凤草液组IFN-γ、STAT1表达下调，IL-4、STAT6表达上调，CD86百分率明显降低，CD206的百分率显著增加。结论:复方五凤草液治疗结核性溃疡临床疗效显著，其作用机制可能与抑制病原菌，干预IFN-γ/STAT1、IL-4/STAT6信号通路以介导M1/M2巨噬细胞极化平衡有关。
Mechanism Research of Compound Wufengcao(五凤草)Liquid Regulating Macrophage Polarization in Tuberculous Ulcer Based on IFN-γ/STAT1 and IL-4/STAT6 Signaling Pathway
Objective:To observe the clinical efficacy of compound Wufengcao(五凤草)liquid in the treatment of tuberculous ulcer,and to explore its mechanism based on interferon-γ(IFN-γ)/signal transducer and activator of transcription 1(STAT1),interleukin-4(IL-4)/signal trans-ducer and activator of transcription 6(STAT6)signaling pathway and Bacillus Calmette-Guerin(BCG)-induced macrophage polarization model.Methods:(1)Clinical trial:135 patients with tuberculous ulcer were divided into observation group,traditional Chinese medicine(TCM)control group and Western medicine control group.On the basis of standardized anti-tuberculosis treatment,the patients in the obser-vation group were treated with compound Wufengcao liquid,the patients in the TCM control group were treated with Kangfuxin solution,and the patients in the Western medicine control group were treated with isoniazid solution for external use.The observation period was 28 days.The safety indexes,total effective rate of wound healing,histopathological morphology of wound,expression levels of IFN-γ,IL-4,STAT1 and STAT6 in wound tissue and blood were measured,and Case Report Form(CRF)and Traditional Chinese Medicine Syndrome Score Scale(TCMSSS)were filled in.(2)Cell experiment:The THP-1 macrophage model induced by BCG was established and divided into BCG group,BCG+Kangfuxin solution group,BCG+isoniazid solution group,and BCG+compound Wufengcao liquid group after treated with different drugs.The BCG colony condition in each group was observed under microscope,and the cell culture supernatants were collected and centri-fuged.Western blotting and RT-qPCR were used to detect the expression levels of IFN-γ,IL-4,STAT1 and STAT6.Flow cytometry was used to detect the effect of the drug on the expressions of CD11b,CD86,CD163 and CD206.Results:(1)Clinical trial:During the treatment,there was no significant difference in safety indexes among the three groups.The total effective rate in observation group(95.56％)was higher than that of the TCM control group(82.22％)and Western medicine control group(75.56％).After 28 days of treatment,compared with the TCM and Western medicine control groups,the histopathological morphology of wounds in the observation group was improved more obviously.The expression levels of IFN-γ and STAT1 in the observation group were lower than those in the TCM and Western medicine control groups,while the expression levels of IL-4 and STAT6 in the observation group were higher than those in the TCM and Western medicine control groups.Compared with the TCM and Western medicine control groups,the TCM syndrome score in observation group was significantly reduced.(2)Cell experiment:The phagocytic vesicles and autophagosomes in THP-1 macrophages were significantly increased after BCG infection.The sur-vival rate of intracellular mycobacterium tuberculosis(MTB)was decreased after drug intervention in each group.The results of Western blot-ting,RT-qPCR and flow cytometry showed that,compared with the control group,the proportion of M1 macrophages in BCG group was higher,while the proportion of M2 macrophages in other three groups was higher.Among them,the proportion of M2 macrophages in BCG+compound Wufengcao liquid group was significantly higher than that in other three groups.In BCG+compound Wufengcao liquid group,the expressions of IFN-γ and ST AT1 were decreased,while the expressions of IL-4 and STAT6 were increased,the CD86 expression was significantly down-regulated and CD206 expression was up-regulated.Conclusion:Compound Wufengcao liquid has a significant clinical effect in the treatment of tuberculous ulcer,and its mechanism may be related to the inhibition of pathogenic bacteria and the intervention of IFN-γ/STAT1 and IL-4/STAT6 signaling pathways to mediate the polarization balance of M1/M2 macrophages.
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