黄连解毒汤含药血清调控中性粒细胞功能的实验研究  

Effect of Huanglian Jiedu Decoction Containing Serum on the Function of Neutrophils

作　　者：孙慧娟 朱鏐娈[2,3] 李蕊 曾辉[2,3] 马雅銮 SUN Huijuan;ZHU Liu-luan;LI Rui;ZENG Hui;MA Ya-luan(Institute of Basic Theory,China Academy of Chinese Medical Sciences,Beijing,100700;Institute of Infectious Diseases,Beijing Ditan Hospital,Capital Medical University,Beijing,100015;Beijing Key Laboratory of Emerging Infectious Diseases,Beijing,100015)

机构地区：[1]中国中医科学院中医基础理论研究所,北京100700 [2]首都医科大学附属北京地坛医院传染病研究所,北京100015 [3]新发突发传染病研究北京市重点实验室,北京100015

出　　处：《中国中西医结合杂志》2021年第2期212-217,共6页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：中国中医科学院自主选题研究项目(No.YZ-1702;No.YZ-1814)。

摘　　要：目的观察黄连解毒汤含药血清对体外调控中性粒细胞功能的影响。方法20只SD大鼠每天给予黄连解毒汤5 g/kg灌胃,每12小时1次,共5次,制备黄连解毒汤含药血清。采用流式细胞仪分选C57 BL/6小鼠骨髓和外周血中性粒细胞,给予不同剂量的黄连解毒汤含药血清和脂多糖(LPS)刺激3 h,采用实时荧光定量PCR检测中性粒细胞炎性细胞因子肿瘤坏死因子-α(TNF-α)、IL-1β、IL-6及IL-12b表达,确定10%黄连解毒汤含药血清为最佳剂量。将10%黄连解毒汤含药血清用于干预活化的中性粒细胞功能:(1)中性粒细胞给予佛波醇12-十四酸酯13-乙酸酯(PMA)刺激5 min,流式细胞仪检测骨髓中性粒细胞产生活性氧(ROS)情况;(2)中性粒细胞给予LPS刺激3 h,采用PicoGreen dsDNA定量检测试剂盒检测培养上清游离DNA(cf-DNA)的含量;(3)免疫荧光法检测中性粒细胞形成的中性粒细胞胞外诱捕网(NETs)情况;(4)中性粒细胞给予趋化因子CXCL2、CXCL10、CCL3诱导3 h,流式细胞仪检测趋化的中性粒细胞数量;(5)中性粒细胞给予LPS刺激20 h,流式细胞仪检测外周血中性粒细胞凋亡情况。结果PMA刺激中性粒细胞使ROS显著增多(P<0.05);LPS刺激3 h,中性粒细胞产生的cf-DNA含量增多,NETs形成增加,CXCL2,CXCL10、CCL3使中性粒细胞趋化增多(P<0.05,P<0.01);LPS刺激20 h后,外周血中性粒细胞凋亡减少(P<0.05);10%黄连解毒汤含药血清可减少ROS产生,降低cf-DNA含量和NETs形成,减少中性粒细胞趋化,促进中性粒细胞凋亡(P<0.05,P<0.01)。结论黄连解毒汤可调节中性粒细胞功能。Objective To observe the effect of Huanglian Jiedu Decoction(HLJDD)containing serum on neutrophil function in vitro.Methods Totally 20 SD rats were administered with HLJDD(5 g/kg)by gastrogavage once every 12 h for a total of 5 times to prepare HLJDD containing serum.The neutrophils of bone marrow and peripheral blood of C57BL/6 mice were sorted by flow cytometry.The neutrophils were stimulated with different doses of HLJDD containing serum and lipopolysaccharide(LPS)for 3 h.Real-time PCR was used to detect the expressions of TNF-α,IL-1β,IL-6,and IL-12 of neutrophils,and the optimal dose of HLJDD containing serum(10%)was obtained.10%HLJDD containing serum was used to intervene the function of activated neutropils.(1)After stimulated with phorbol 12-myristate 13-acetate(PMA)for 5 min,reactive oxygen species(ROS)of bone marrow granulocytes were detected by flow cytometry.(2)After stimulated with LPS for 3 h,cf-DNA content in the culture supernatant was detected by PicoGreen dsDNA method.(3)The formation of neutrophil extracellular traps(NETs)of granulocytes was detected by immunofluorescence.(4)After induced with chemokines CXCL2,CXCL10,and CCL3 for 3 h,the numbers of chemotactic neutrophils were detected by flow cytometry.(5)After stimulated with LPS for 20 h,the apoptosis of peripheral blood neutrophils was detected by flow cytometry.Results PMA stimulated neutrophils to significantly increase ROS production(P<0.05).After stimulation by LPS for 3 h,the production of cf-DNA in neutrophils and the formation of NETs were increased.CXCL2,CXCL10 and CCL3 increased neutrophils chemotaxis(P<0.05,P<0.01).After stimulation by LPS for 20 h,the apoptosis of peripheral blood neutrophils was decreased(P<0.05).And 10%HLJDD containing serum reduced ROS production,decreased cf-DNA content and NETs formation,reduced the chemotaxis,and promoted apoptosis of neutrophils(P<0.05,P<0.01).Conclusion HLJDD regulated the function of neutrophils.

关 键 词：黄连解毒汤 炎症因子 中性粒细胞 

分 类 号：R285.5[医药卫生—中药学]