基于NLRP3炎症通路研究通窍方对腺样体上皮细胞焦亡的影响  

Effect of Tongqiao Decoction on Pyroptosis of Adenoid Epithelial Cells Based on NLRP3 Inflammatory Pathway

作　　者：王彤 李永春 刘航 文武林 崔瑞琴[1,3] WANG Tong;LI Yongchun;LIU Hang;WEN Wulin;CUI Ruiqin(School of Traditional Chinese Medicine,Ningxia Medical University,Yinchuan 750004,China;the First People’s Hospital of Yinchuan,Second Clinical Medical College of Ningxia Medical University,Yinchuan 750004,China;Ningxia Regional Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Regional High Incidence Disease,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学中医学院,银川750004 [2]银川市第一人民医院,宁夏医科大学第二临床医学院,银川750004 [3]宁夏区域高发病中西医结合防治研究重点实验室,银川750004

出　　处：《宁夏医科大学学报》2024年第2期153-160,共8页Journal of Ningxia Medical University

基　　金：宁夏自然科学基金项目(2022AAC03214);宁夏区域高发病中西医结合防治研究重点实验室开放课题(20023301)。

摘　　要：目的探索通窍方含药血清抑制腺样体上皮细胞焦亡的作用机制。方法选择2022年3月至2023年3月在银川市第一人民医院耳鼻喉科行腺样体切除术的腺样体肥大(adenoidal hypertrophy,AH)患儿,收集术后切除的腺样体组织,采用组织块贴壁法分离上皮细胞,并进行培养及鉴定。用透射电镜观察腺样体上皮细胞的超微结构;用CCK-8法筛选通窍方含药血清的最佳干预浓度。将培养至适宜浓度的人原代腺样体上皮细胞分为4组,分别是空白对照组、孟鲁司特钠组、通窍方组、MCC950组(MCC950,10μmol·L^(-1))。各组给予相应的含药血清处理24 h后,采用RT-qPCR法和Western blot法分别检测NOD样受体蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白酶(Caspase-1)、白细胞介素(IL)1β、IL-18基因及蛋白表达水平;Tunnel染色法检测细胞凋亡情况;ELISA法检测IL-18、IL-1β、IL-6和肿瘤坏死因子(TNF-α)含量。结果AH患儿的原代腺样体上皮细胞呈不规则型,轮廓不清,折光性较差;免疫荧光细胞鉴定泛角蛋白阳性率高。透射电镜下腺样体上皮细胞可见到明显焦亡征象。通窍方低剂量组细胞存活率最高(P<0.05)。与空白对照组相比,通窍方组、孟鲁司特钠组、MCC950组的NLRP3、Caspase-1、IL-1β、IL-18基因及蛋白表达量均降低(P均<0.05);细胞凋亡率下降(P均<0.05);细胞上清液中IL-18、IL-1β、IL-6和TNF-α含量均下降(P均<0.01)。结论通窍方含药血清可通过抑制腺样体上皮细胞焦亡而下调炎症因子表达,其机制或与调节NLRP3介导的细胞焦亡信号通路有关。Objective To explore the mechanism of Tongqiao Decoction containing serum in inhibiting pyroptosis of adenoid epithelial cells in children with adenoid hypertrophy.Methods The tissue was derived from children with adenoid hypertrophy who underwent adenoidectomy in the Department of Otorhinolaryngology,the First People’s Hospital of Yinchuan from March 2022 to March 2023.The discarded adenoid tissue after adenoidectomy was collected,and the epithelial cells were isolated by tissue block adherence method,cultured and identified.The ultrastructure of adenoid epithelial cells was observed by transmission electron microscope.CCK-8 method was used to screen the optimal serum intervention concentration of Tongqiao Decoction.Human primary adenoid epithelial cells cultured to appropriate concentration were divided into four groups:blank control group,montelukast sodium group,Tongqiao Decoction group and MCC950 group(MCC950,10μmol·L^(-1)).After 24 hours of treatment with corresponding drug-containing serum,the expression levels of NOD-like receptor protein 3(NLRP3),Caspase-1,IL-1β,IL-18 gene and protein were detected by RT-qPCR and Western blot,respectively.Tunnel staining was used to detect cell apoptosis.ELISA method was applied to detect IL-18,IL-1β,IL-6 and TNF-αcontents.Results The primary adenoid epithelial cells of children with AH were irregular,unclear in outline,and had poor refractive activity.High positive rate for immunofluorescence cell identification.Transmission electron microscopy showed obvious signs of pyroptosis in adenoid epithelial cells.The cell survival rate was the highest in the low-dose group(P<0.05).Compared with the blank control group,the Tongqiao Decoction group,montelukast sodium group,and MCC950 group had significant reductions in the gene and protein expression of NLRP3,Caspase-1,IL-1β,and IL-18(P all<0.05).The apoptosis rate was decreased(P all<0.05).The levels of IL-18,IL-1β,IL-6 and TNF-αin the cell supernatant were significantly decreased(P all<0.01).Conclusion Tongqiao Decoc

关 键 词：细胞焦亡 含药血清 通窍方 腺样体肥大 NOD样受体蛋白3 

分 类 号：R322.25[医药卫生—人体解剖和组织胚胎学]