基于NLRP3-IL-1β-NF-κB信号轴研究野菊花总黄酮对脂多糖诱导HK-2细胞炎性反应及自噬的影响  

Effects of total flavonoids of chrysanthemum indicum on inflammatory response and autophagy of HK-2 cells induced by lipopolysaccharide based on NLRP3-IL-1β-NF-κB signal axis

作　　者：杨彬[1] 黄俊卿[1] 孟庆良[2] 袁威起 梁新 YANG Bin;HUANG Jun-qing;MENG Qing-liang;YUAN Wei-qi;LIANG Xin(Department of Pain,Henan Hospital of Traditional Chinese Medicine,Zhengzhou 450002,China;Department of Rheumatology,Henan Hospital of Traditional Chinese Medicine,Zhengzhou 450002,China;Bone Injury Major of Traditional Chinese Medicine,Henan University of Traditional Chinese Medicine)

机构地区：[1]河南省中医院疼痛科,450002 [2]河南省中医院风湿免疫科,450002 [3]河南中医药大学

出　　处：《天津医药》2020年第2期81-87,I0001,共8页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81874456);河南省中医药科学研究专项课题(2019ZY2054)。

摘　　要：目的探究野菊花总黄酮(TFC)对脂多糖(LPS)诱导HK-2细胞炎性反应及自噬的影响,并初步探究其可能的作用机制。方法体外培养人肾小管上皮HK-2细胞,将细胞分为阴性对照(Control)组、LPS处理(LPS)组、TFC预处理(TFC)组、NLRP3激活预处理(4-MET)组、TFC联合4-MET预处理(TFC+4-MET)组。CCK-8法检测细胞增殖情况;Hoechst 33342染色观察各组细胞形态变化;免疫印迹法检测各组细胞核苷酸结合域样受体蛋白3(NLRP3)、凋亡相关斑点样蛋白(Asc)、半胱氨酸天冬氨酸蛋白酶1(caspase-1)、p-IκB、IκB、自噬相关蛋白(Beclin1)、微管相关蛋白轻链3Ⅱ(LC3Ⅱ)、LC3Ⅰ、蛋白表达情况;ELISA法检测细胞中白细胞介素(IL)-1β、IL-18水平;透射电镜观察细胞自噬情况。结果与Control组相比,LPS组细胞增殖率降低,细胞核破碎情况加重,细胞凋亡率升高,NLRP3、Asc、caspase-1蛋白表达升高、IκB蛋白磷酸化水平升高,IL-1β、IL-18水平升高,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达降低(P<0.05)。与LPS组相比,TFC组细胞增殖率升高,细胞核破碎减轻,细胞凋亡率降低,NLRP3、Asc、caspase-1蛋白表达降低、IκB蛋白磷酸化水平降低,IL-1β、IL-18水平降低,细胞自噬程度增加,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达升高;4-MET组细胞增殖率降低,细胞凋亡率升高,细胞核破碎情况加重,NLRP3、Asc、caspase-1蛋白表达升高、IκB蛋白磷酸化水平升高,IL-1β、IL-18水平升高,细胞自噬程度降低,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达降低(P<0.05)。TFC+4-MET组细胞增殖率、细胞自噬程度、LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达高于4-MET组,细胞核破碎情况减轻,细胞凋亡率、NLRP3、Asc、caspase-1蛋白表达、IκB蛋白磷酸化、IL-1β、IL-18水平低于4-MET组(P<0.05)。结论野菊花总黄酮可能通过抑制NLRP3-IL-1β-NF-κB信号通路活化,进而抑制脂多糖诱导HK-2细胞炎性反应,诱导细胞自噬。Objective To investigate the effects of total flavonoids of chrysanthemum indicum(TFC)on inflammatory response and autophagy of HK-2 cells induced by lipopolysaccharide(LPS),and to explore its possible mechanism.Methods HK-2 cells of human renal tubular epithelium were cultured in vitro and divided into control group,LPS treatment group,TFC pretreatment group,NLRP3 activator(4-MET)group and TFC combined with 4-MET pretreatment(TFC+4-MET)group.CCK-8 method was used to detect cell proliferation,Hoechst 33342 staining was used to observe the morphological changes of cells in each group.The expressions of apoptosis-associated speek-like protein containing CARD(Asc),caspase-1,p-IκB,IκB,autophagy-related protein(Beclin1),microtubule-related protein light chain 3Ⅱ(LC3Ⅱ)/LC3Ⅰwere detected by Western blot assay.The levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)were detected by ELISA method,and autophagy was observed by transmission electron microscopy.Results Compared with the control group,the proliferation rate decreased in LPS group,the fragmentation of cell nucleus increased,the apoptosis rate increased,the expressions of NLRP3,Asc,caspase-1 protein,the phosphorylation level of IκB protein increased,the levels of IL-1βand IL-18 increased,and the expressions of LC3Ⅱ/Ⅰand Beclin-1 protein decreased(P<0.05).Compared with LPS group,cell proliferation rate increased in TFC group,cell nuclear fragmentation decreased,apoptosis rate decreased,NLRP3,Asc,caspase-1 protein expression and IκB protein phosphorylation level decreased,IL-1β and IL-18 levels decreased,autophagy increased,LC3Ⅱ/Ⅰlevel and Beclin-1 protein expression increased.The cell proliferation rate decreased in 4-MET group,apoptosis rate and nuclear fragmentation increased.The expression levels of NLRP3,Asc,caspase-1,IκB protein phosphorylation,IL-1β and IL-18 increased,autophagy,LC3Ⅱ/Ⅰand Beclin-1 protein decreased(P<0.05).The cell proliferation rate,autophagy degree,LC3Ⅱ/Ⅰlevel and Beclin-1 protein expression were higher

关 键 词：痛风 脂多糖类 自噬 白细胞介素1β NF-κB 野菊花总黄酮 核苷酸结合域样受体蛋白3 HK-2细胞 

分 类 号：R589.7[医药卫生—内分泌]