作者：李倩，李婧娥，李媛，高笑宇，孙德俊

单位：

1.内蒙古科技大学包头医学院2.内蒙古自治区人民医院国家卫生健康委员会慢阻肺诊治重点实验室内蒙古自治区呼吸疾病重点实验室

Units：

1.Baotou Medical College, Inner Mongolia University of Science & Technology, Baotou 014000, China 2.People's Hospital of Inner Mongolia Autonomous Region/Key Laboratory of COPD Diagnosis and Treatment of the National Health Commission/Key Laboratory of Respiratory Diseases of Inner Mongolia, Hohhot 010017, China

关键词：

炎症; 上皮细胞; 尼古丁; 转录组学;

Keywords：

Inflammation; Epithelial cells; Nicotine; Transcriptomics

CLC：

DOI：

10.12114/j.issn.1008-5971.2023.00.117

Funds：

国家自然科学基金资助项目（81960013）； 中央引导地方科技发展资金项目（ZYZ20200486）； 中国医学科学院中央级公益性科研院所基本科研业务费项目（2019PT350001）； 内蒙古自治区自然科学基金项目（2021MS08165）； 内蒙古自治区科技计划项目（内财科【2022】662号）；

摘要：

目的 基于转录组学分析尼古丁诱导气道上皮细胞炎症反应的机制。方法 本实验时间为2021年6月至2022年10月。取对数生长期的BEAS-2B细胞，将其随机分为A组（空白对照）、B组（1 nmol/L尼古丁处理24 h）、C组（10 nmol/L尼古丁处理24 h）、D组（100 nmol/L尼古丁处理24 h）、E组（1μmol/L尼古丁处理24 h）、F组（10μmol/L尼古丁处理24 h）、G组（100μmol/L尼古丁处理24 h）、H组（1 mmol/L尼古丁处理24 h）、I组（5 mmol/L尼古丁处理24 h）、J组（10 mmol/L尼古丁处理24 h）、K组（15 mmol/L尼古丁处理24 h），采用细胞计数试剂盒8(CCK-8)检测各组细胞增殖活性。取对数生长期的BEAS-2B细胞，将其随机分为空白对照组与5 mmol/L尼古丁组（5 mmol/L尼古丁处理24 h），采用实时荧光定量PCR检测各组IL-1β、IL-8 mRNA表达水平。取对数生长期的BEAS-2B细胞，将其随机分为空白对照组与5 mmol/L尼古丁组（5 mmol/L尼古丁处理24 h），采用ELISA检测各组IL-1β、IL-8蛋白表达水平。取对数生长期的BEAS-2B细胞，将其随机分为空白对照组与5 mmol/L尼古丁组（5 mmol/L尼古丁处理24 h），收集各组细胞后进行转录组学测序。采用实时荧光定量PCR验证差异表达基因。采用clusterProfiler工具包对差异表达基因进行GO功能富集分析和KEGG通路富集分析。结果 I组、J组、K组细胞增殖活性低于A组、B组、C组、D组（P<0.05）;J组、K组细胞增殖活性低于E组、F组、G组、H组、I组（P<0.05）;K组细胞增殖活性低于J组（P<0.05）。5 mmol/L尼古丁组IL-1β、IL-8 mRNA表达水平高于空白对照组（P<0.05）。5 mmol/L尼古丁组IL-1β、IL-8蛋白表达水平高于空白对照组（P<0.05）。转录组学测序结果显示，共检出24 751个基因，其中7 539个差异表达基因（3 771个基因表达上调，占50.02%;3 768个基因表达下调，占49.98%）。选出P值最小的19个差异表达基因，实时荧光定量PCR检测结果显示，EGR3、IL-24、NOV、CXCL8、CXCL2、JUN、FASN、HSPA5、AXL、IGFBP4、FADS1、ANXA3、TUBA1A、ACSS2、EZR、STMN3、CPA4、HMGCS1、UCP2表达水平与转录组学检测结果一致。GO功能富集分析结果显示，在生物过程（BP）方面，上述19个差异表达基因主要与DNA复制、辅因子生物合成过程、粒细胞激活等相关；在细胞组分（CC）方面，上述19个差异表达基因主要与黏着斑、细胞-基质黏附结、细胞基质结等相关；在分子功能（MF）方面，上述9个差异表达基因主要与钙黏着蛋白结合、细胞黏附分子结合、单链RNA结合等相关。KEGG通路富集分析结果显示，共富集到322条KEGG通路，前5条分别为肌动蛋白细胞骨架调节、癌症中miRNA、细胞周期、碳代谢、溶酶体。结论 尼古丁诱导气道上皮细胞炎症反应的机制可能与EGR3、IL-24、NOV、CXCL8、CXCL2、JUN、FASN、HSPA5、AXL、IGFBP4、FADS1、ANXA3、TUBA1A、ACSS2、EZR、STMN3、CPA4、HMGCS1、UCP2的异常表达有关，涉及粒细胞激活、中性粒细胞脱颗粒与相关免疫、细胞-基质黏附结、肌动蛋白结合等细胞功能及肌动蛋白细胞骨架调节、癌症中miRNA、细胞周期等通路。

Abstract：

　Objective To analyze the mechanism of nicotine-induced airway epithelial cell inflammation based on transcriptomics. Methods This experiment was conducted from June 2021 to October 2022. BEAS-2B cells at logarithmic growth stage were randomly divided into group A (blank control) , group B (treated with 1 nmol/L nicotine for 24 hours) , group C (treated with 10 nmol/L nicotine for 24 hours) , group D (treated with 100 nmol/L nicotine for 24 hours) , group E (treated with 1 μmol/L nicotine for 24 hours) , group F (treated with 10 μmol/L nicotine for 24 hours) , group G (treated with 100 μmol/L nicotine for 24 hours) , group H (treated with 1 mmol/L nicotine for 24 hours) , group I (treated with 5 mmol/L nicotine for 24 hours) , group J (treated with 10 mmol/L nicotine for 24 hours) and group K (treated with 15 mmol/L nicotine for 24 hours) , and cell proliferation activity of each group was detected by cell counting kit 8 (CCK-8) . BEAS-2B cells at logarithmic growth stage were randomly divided into blank control group and 5 mmol/L nicotine group (treated with 5 mmol/L nicotine for 24 h) , and realtime fluorescence quantitative PCR was used to detect mRNA expression levels of IL-1β and IL-8 in each group. BEAS-2B cells at logarithmic growth stage were randomly divided into blank control group and 5 mmol/L nicotine group (treated with 5 mmol/L nicotine for 24 h) , and the expression levels of IL-1β and IL-8 protein in each group were detected by ELISA. BEAS2B cells at logarithmic growth stage were randomly divided into blank control group and 5 mmol/L nicotine group (treated with 5 mmol/L nicotine for 24 h) , and cells from each group were collected for transcriptomic sequencing. The differentially expressed genes were verified by real-time quantitative PCR. GO functional enrichment analysis and KEGG pathway enrichment analysis of differentially expressed genes were performed using clusterProfiler toolkit. Results The cell proliferation activity of groups I, J and K was lower than that of groups A, B, C and D (P < 0.05) . The cell proliferation activity of groups J and K was lower than that of groups E, F, G, H and I (P < 0.05) . The cell proliferation activity of group K was lower than that of group J (P < 0.05) . The mRNA expression levels of IL-1β and IL-8 in 5 mmol/L nicotine group were higher than those in blank control group (P < 0.05) . The expression levels of IL-1β and IL-8 protein in 5 mmol/L nicotine group were higher than those in blank control group (P < 0.05) . Transcriptomic sequencing results showed that a total of 24 751 genes were detected, including 7 539 differentially expressed genes (3 771 genes were upregulated, accounting for 50.02%; 3 768 genes were downregulated, accounting for 49.98%) . The 19 differentially expressed genes with the smallest P value were selected, and the real-time quantitative PCR detection results showed that, the expression levels of EGR3, IL-24, NOV, CXCL8, CXCL2, JUN, FASN, HSPA5, AXL, IGFBP4, FADS1, ANXA3, TUBA1A, ACSS2, EZR, STMN3, CPA4, HMGCS1 and UCP2 were consistent with the results of transcriptomic detection. GO functional enrichment analysis showed that in terms of biological processes (BP) , the 19 differentially expressed genes were mainly related to DNA replication, cofactor biosynthesis, granulocyte activation, etc; in terms of cell components (CC) , the 19 differentially expressed genes were mainly related to adhesion plaques, cell-matrix adhesion junctions, cell matrix junctions, etc; in terms of molecular function (MF) , the 19 differentially expressed genes were mainly related to calcium adhesion protein binding, cell adhesion molecule binding, single stranded RNA binding, etc. The results of KEGG pathway enrichment analysis showed that 322 KEGG pathways were enriched, and the first 5 pathways were actin cytoskeleton regulation, miRNA in cancer, cell cycle, carbon metabolism, and lysosome. Conclusion The mechanism of nicotine-induced inflammatory response in airway epithelial cells may be related to the abnormal expression of EGR3, IL-24, NOV, CXCL8, CXCL2, JUN, FASN, HSPA5, AXL, IGFBP4, FADS1, ANXA3, TUBA1A, ACSS2, EZR, STMN3, CPA4, HMGCS1 and UCP2, involving cellular functions such as granulocyte activation, neutrophilic granulocyte degranulation and associated immunity, cell-matrix adhesion junctions and actin binding, and pathways such as actin cytoskeleton regulation, miRNA in cancer, and cell cycle.

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