从巨噬细胞角度探讨复方五凤草液干预结核性溃疡的机制  

Therapeutic Effect of Compound Wufengcao Liquid on Tuberculous Ulcer from Perspective of Macrophages

作　　者：钱佳燕 黄子慧[1] 孙佳玥 许费昀[1] 王裕玲 余洋[1] QIAN Jiayan;HUANG Zihui;SUN Jiayue;Xu Feiyun;Wang Yuling;Yu yang(Nanjing Integrated Traditional Chinese and Western Medicine Hospital Affiliated to Nanjing University of Chinese Medicine,Nanjing 210016,China)

机构地区：[1]南京中医药大学附属南京市中西医结合医院,江苏210014

出　　处：《中国实验方剂学杂志》2023年第4期86-96,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：江苏省中医药重点科技项目(ZD202105);南京市卫计委重点科技项目(ZKX18042);南京市卫计委科技发展项目(YKK21192);许芝银全国名中医专家工作室项目([No.宁卫财务2019]26号);南京市卫健委青年人才项目(QRX17030)。

摘　　要：目的:探讨复方五凤草液治疗结核性溃疡的临床疗效及对巨噬细胞极化的影响。方法:(1)临床实验。将南京市中西医结合医院145例结核性溃疡患者按随机数字表法分为观察组、对照Ⅰ组和对照Ⅱ组。3组均予以基础抗结核化学治疗的同时,观察组予以复方五凤草液,对照Ⅰ组予以康复新液,对照Ⅱ组予以异烟肼液局部外用治疗,疗程4周。分别观察3组患者创面愈合总有效率、中医证候积分、创面组织病理学形态及创面组织中诱导型一氧化氮合酶(iNOS)、精氨酸酶-1(Arg-1)的表达水平。(2)细胞实验。RAW264.7细胞在完全培养基DMEM(10%的胎牛血清,1%青-链酶素溶液)于37℃、5%CO_(2)的细胞培养箱中培养。经佛波酯(PMA)诱导后向巨噬细胞分化,分别用脂多糖(LPS)诱导成M1巨噬细胞、白细胞介素-4(IL-4)诱导成M2巨噬细胞,分别予康复新溶液、异烟肼液、复方五凤草液处理以上细胞模型36 h,收集细胞上清液并离心,蛋白免疫印迹法(Western blot)检测肿瘤坏死因子-α(TNF-α)、转化生长因子-β(TGF-β)、iNOS和Arg-1的蛋白量的表达,流式细胞术(FCM)检测药物对CD86、CD206表达的影响。结果:(1)临床实验。与对照Ⅰ组[87.5%(42/48),χ^(2)=3.962,P<0.05]和对照Ⅱ组[83.3%(40/48),χ^(2)=6.162,P<0.05]比较,复方五凤草液组的总有效率[98.0%(48/49)]明显较高;治疗28 d后,与对照Ⅰ组、对照Ⅱ组比较,复方五凤草液组的中医证候积分明显降低(P<0.05),复方五凤草液组创面组织病理学形态改善更为明显;免疫组化结果显示,治疗28 d后,与对照Ⅰ组、对照Ⅱ组比较,复方五凤草液组局部病灶组织样本中的iNOS表达量明显降低(P<0.05),Arg-1表达量显著升高(P<0.05,P<0.01)。(2)细胞实验。Western blot实验结果显示,与M0组比较,LPS组中iNOS、TNF-α表达升高(P<0.01)。与LPS组比较,LPS+异烟肼组、LPS+康复新液组、LPS+复方五凤草液组中iNOS、TNF-α表达下降(P<0.05);与LObjective:To explore the clinical efficacy of compound Wufengcao liquid(CWL)on tuberculous ulcer and the influence on macrophage polarization.Method:(1)Clinical experiment:A total of 145 patients with tuberculous ulcer who were treated in Nanjing Integrated Traditional Chinese and Western Medicine Hospital were randomized into observation group,control groupⅠ,and control groupⅡaccording to the random number table method.In addition to the basic anti-tuberculosis chemotherapy,CWL,Kangfuxin liquid,and isoniazid solution(local external application)were respectively used in the observation group,control groupⅠ,and control groupⅡ.The treatment lasted 4 weeks for each group.The total effective rate in wound healing,traditional Chinese medicine(TCM)syndrome score,and histopathological morphology of wound were observed and the expression of inducible nitric oxide synthase(iNOS)and arginase-1(Arg-1)in wound tissue was measured.(2)Cell experiment:RAW264.7 cells were cultured in DMEM(10%fetal bovine serum,1%double-antibody solution)in a cell incubator(37°C,5%CO_(2)).Phorbol 12-myristate 13-acetate(PMA)was used to induce the differentiation of RAW264.7 cells into macrophages.Lipopolysaccharide(LPS)was employed to stimulate polarization of macrophages into M1 type and interleukin-4(IL-4)to induce the polarization into M2 type.Kangfuxin solution,isoniazid solution,and CWL were respectively applied to the above cell model for 36 h.The cell supernatant was collected and centrifuged.Western blot was used to detect the protein expression of tumor necrosis factor-α(TNF-α),transforming growth factor-β(TGF-β),iNOS,and Arg-1,and flow cytometry(FCM)to detect the expression of CD86 and CD206.Result:(1)Clinical experiment:The total effective rate in the CWL group[98.0%(48/49)]was higher than that in the control groupⅠ[87.5%(42/48),χ^(2)=3.962,P<0.05]and control groupⅡ[83.3%(40/48),χ^(2)=6.162,P<0.05].After 28days of treatment,compared with control groupⅠand control groupⅡ,CWL decreased the TCM syndrome score(P<0.05

关 键 词：复方五凤草液 结核性溃疡 巨噬细胞极化 慢性创面 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学] R2-031R285.5R287