天津医药

天津医药 ›› 2020, Vol. 48 ›› Issue (2): 81-87.doi: 10.11958/20192697

• 细胞与分子生物学 •    下一篇

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

杨彬 1，黄俊卿 1△，孟庆良 2，袁威起 3，梁新 3   

  1. 1河南省中医院疼痛科（邮编450002），2风湿免疫科；3河南中医药大学
  • 收稿日期:2019-08-30 修回日期:2019-11-15 出版日期:2020-02-15 发布日期:2020-02-15
  • 通讯作者: 黄俊卿 E-mail:13939003396@139.com
  • 基金资助:
    基于中性粒细胞胞外诱捕网（NETs）和中性粒细胞源的外泌体研究泄浊化瘀法对急性痛风性关节炎的作用及机制;脊柱定点旋转复位法配合弧度牵引治疗神经根型颈椎病的疗效观察

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

YANG Bin1, HUANG Jun-qing1△, MENG Qing-liang2, YUAN Wei-qi3, LIANG Xin3   

  1. 1 Department of Pain, 2 Department of Rheumatology, Henan Hospital of Traditional Chinese Medicine, Zhengzhou 450002, China; 3 Bone Injury Major of Traditional Chinese Medicine, Henan University of Traditional Chinese Medicine
  • Received:2019-08-30 Revised:2019-11-15 Published:2020-02-15 Online:2020-02-15

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摘要： 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

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

Abstract: Abstract: 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)/ LC3Ⅰ were 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-1 β and IL-18 increased, and the expressions of LC3Ⅱ/Ⅰ and 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, LC3 Ⅱ/Ⅰ level 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/Ⅰ and Beclin-1 protein decreased (P＜0.05). The cell proliferation rate, autophagy degree, LC3 Ⅱ/Ⅰ level and Beclin-1 protein expression were higher in TFC+4-MET group than those in 4-MET group. The nuclear fragmentation was reduced, the apoptosis rate, NLRP3, Asc, caspase-1 protein expression, I κ B protein phosphorylation, IL-1 β and IL-18 levels were lower in TFC+4-MET group than those in 4-MET group (P＜0.05). Conclusion Total flavonoids of chrysanthemum indicum may inhibit the activation of NLRP3-IL-1β - NF - κB signaling pathway, thereby inhibiting the inflammatory response of HK-2 cells induced by lipopolysaccharide and inducing autophagy.

Key words: gout, lipopolysaccharides, autophagy, interleukin-1β, nuclear factor κB, total flavonoids of chrysanthemum indicum, nucleotide binding domain-like receptor protein 3, HK-2 cells