川芎嗪调控miR⁃31⁃5p/Ednrb通路抑制人肺泡上皮细胞BEAS⁃2B凋亡和炎症反应  

Inhibition of human alveolar epithelial BEAS⁃2B apoptosis and inflammatory response by tetramethylpyrazine via miR⁃31⁃5p/Ednrb pathway

作　　者：刘明宗 刘于嵩[2] 龙国利[1] 张钊 江宇虹 高榕茂 LIU Ming-zong;LIU Yu-song;LONG Guo-li;ZHANG Zhao;JIANG Yu-hong;GAO Rong-mao(ICU,Eastern Hospital,Sichuan Provincial Medical Sciences Academy&Sichuan Provincial People’s Hospital,Chengdu 610100,China;Clinical Laboratory,Eastern Hospital,Sichuan Provincial Medical Sciences Academy&Sichuan Provincial People’s Hospital,Chengdu 610100,China;Clinical Medical College,Southwest Medical University,Luzhou 646000,China)

机构地区：[1]四川省医学科学院·四川省人民医院东院ICU,四川成都610100 [2]四川省医学科学院·四川省人民医院东院检验科,四川成都610100 [3]西南医科大学临床医学院,四川泸州646000

出　　处：《中成药》2021年第3期617-624,共8页Chinese Traditional Patent Medicine

基　　金：四川省卫计委科研项目(16PJ501)。

摘　　要：目的探讨川芎嗪(TMP)调控miR⁃31⁃5p/内皮素受体B(Ednrb)通路对人肺泡上皮细胞BEAS⁃2B凋亡和炎症反应的影响。方法采用1μg/mL脂多糖(LPS)处理BEAS⁃2B细胞构建炎症模型。将BEAS⁃2B细胞分为对照组、LPS组、LPS+川芎嗪5μmol/L组、LPS+川芎嗪20μmol/L组、LPS+川芎嗪80μmol/L组、LPS+miR⁃con组、LPS+miR⁃31⁃5p组、LPS+si⁃con组、LPS+si⁃Ednrb组、LPS+川芎嗪+pcDNA组、LPS+川芎嗪+pcDNA⁃Ednrb组。酶连免疫吸附实验(ELISA)试剂盒检测细胞培养液中白介素IL⁃1β、IL⁃6和TNF⁃α(肿瘤坏死因子)的含量;RT⁃qPCR和Western blot检测miR⁃31⁃5p(炎性相关微小RNA)和Ednrb(内皮素受体B)表达。双荧光素酶报告实验和Western blot验证miR⁃31⁃5p对Ednrb的靶向调控关系。结果与对照组比较,LPS组BEAS⁃2B细胞Ednrb蛋白、凋亡率以及IL⁃1β、IL⁃6和TNF⁃α的水平增加,miR⁃31⁃5p表达降低(P<0.01);与LPS组比较,LPS+川芎嗪5μmol/L组、LPS+川芎嗪20μmol/L组、LPS+川芎嗪80μmol/L组BEAS⁃2B细胞Ednrb蛋白表达、凋亡率以及IL⁃1β、IL⁃6和TNF⁃α的水平降低,miR⁃31⁃5p表达升高(P<0.05,P<0.01)。miR⁃31⁃5p靶向负性调控Ednrb表达。与LPS+miR⁃con组比较,LPS+miR⁃31⁃5p组BEAS⁃2B细胞凋亡率以及IL⁃1β、IL⁃6和TNF⁃α的水平降低(P<0.01);与LPS+si⁃con组比较、LPS+si⁃Ednrb组BEAS⁃2B细胞凋亡率以及培养液中IL⁃1β、IL⁃6和TNF⁃α的水平降低(P<0.01);与LPS+川芎嗪+pcDNA组比较,LPS+川芎嗪+pcDNA⁃Ednrb组BEAS⁃2B细胞凋亡率、培养液中IL⁃1β、IL⁃6和TNF⁃α的浓度升高(P<0.05)。结论川芎嗪通过上调miR⁃31⁃5p/Ednrb通路抑制人肺泡上皮细胞BEAS⁃2B凋亡和炎症反应。AIM To investigate the effects of tetramethylpyrazine(TMP)on the apoptosis and inflammatory response of human alveolar epithelial cells BEAS⁃2B through regulation of miR⁃31⁃5p/endothelin receptor B(Ednrb)pathway.METHODS BEAS⁃2B cells treated with 1μg/mL lipopolysaccharide(LPS)were developed into inflammatory models.BEAS⁃2B cells were divided into control group,LPS group,LPS+5μmol/L TMP group,LPS+20μmol/L TMP group,LPS+80μmol/L TMP group,LPS+miR⁃con group,LPS+miR⁃31⁃5p group,LPS+si⁃con group,LPS+si⁃Ednrb group,LPS+TMP+pcDNA group,and LPS+TMP+pcDNA⁃Ednrb group.The BEAS⁃2B cells were subjected to detections of their apoptosis by flow cytometry;their IL⁃1β,IL⁃6 and TNF⁃αcontent in cell culture medium by enzyme⁃linked immunosorbent assay(ELISA)kit;their expression of miR⁃31⁃5p and Ednrb by RT⁃qPCR and Western blot;and verification of the targeted regulation of miR⁃31⁃5p to Ednrb by dual luciferase reporter assay and Western blot.RESULTS Compared with the NC group,the LPS group shared significantly increased Ednrb protein expression,apoptosis rate,and IL⁃1β,IL⁃6 and TNF⁃αlevels,significantly decreased miR⁃31⁃5p expression(P<0.01).Compared with the LPS group,LPS+5μmol/L TMP group,LPS+20μmol/L TMP group,and LPS+80μmol/L TMP group demonstrated significantly decreased Ednrb protein expression,apoptosis rate,and IL⁃1β,IL⁃6 and TNF⁃αlevels,significantly increased expression of miR⁃31⁃5p(P<0.05,P<0.01)which targeted and negatively regulated Ednrb expression.Compared with LPS+miR⁃con group,LPS+miR⁃31⁃5p group had significantly reduced cell apoptosis rate and levels of IL⁃1β,IL⁃6 and TNF⁃α(P<0.01).Compared with LPS+si⁃con group,LPS+si⁃Ednrb group was observed with significantly decreased apoptosis rate and levels of IL⁃1β,IL⁃6 and TNF⁃α(P<0.01).Compared with LPS+TMP+pcDNA group,LPS+TMP+pcDNA⁃Ednrb group displayed significantly increased apoptosis rate and levels of IL⁃1β,IL⁃6 and TNF⁃α(P<0.05).CONCLUSION TMP inhibits t

关 键 词：川芎嗪 miR⁃31⁃5p 内皮素受体B 肺泡上皮细胞凋亡 炎症反应 

分 类 号：R285.5[医药卫生—中药学]