罗哌卡因减轻LPS诱导的人结肠上皮细胞系NCM-460凋亡  

Ropivacaine alleviates LPS-induced apoptosis of ulcerative colitis cell line NCM-460

作　　者：周灵琴 王伟娟 任玲玲 朱军来 陈光兰 ZHOU Lingqin;WANG Weijuan;REN Lingling;ZHU Junlai;CHEN Guanglan(Department of Anesthesiology,the Second People′s Hospital of Lishui,Lishui 323000,China;Department of Gastroenterology,the Second People′s Hospital of Lishui,Lishui 323000,China)

机构地区：[1]丽水市第二人民医院麻醉科,浙江丽水323000 [2]丽水市第二人民医院消化科,浙江丽水323000

出　　处：《基础医学与临床》2024年第10期1368-1375,共8页Basic and Clinical Medicine

基　　金：国家卫生健康委员会“十四五”规划全国重点课题(YYWS4176)。

摘　　要：目的探究罗哌卡因对脂多糖(LPS)诱导的人结肠上皮细胞系NCM-460凋亡和对核苷酸寡聚化结构域(NOD)样受体蛋白3(NLRP3)炎性小体活性的影响。方法体外培养人结肠上皮细胞系NCM-460,将细胞分组:对照组(control,不干预)、模型组(model,10μg/mL的LPS处理)、低/中/高浓度组(0.5、1、1.5 mmol/L罗哌卡因干预模型组)。然后将细胞分为对照组、模型组、罗哌卡因组(1.5 mmol/L罗哌卡因干预模型组)、罗哌卡因+抑制剂组(1μmol/L NF-κB通路抑制剂BAY 11-7082干预罗哌卡因组)、抑制剂组(1μmol/L NF-κB通路抑制剂BAY 11-7082干预罗哌卡因组)和罗哌卡因+激活剂组(1μmol/L NF-κB通路激活剂Prostratin干预罗哌卡因组),干预24 h。采用酶联免疫吸附试验(ELISA)检测IL-6、IL-8及肿瘤坏死因子-α(TNF-α)水平;EdU掺入法检测增殖率、Hoechst 33258染色法检测凋亡率;Western blot检测周期蛋白D1(cyclinD1)、半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、NLRP3和NF-κB通路相关蛋白水平。结果与对照组相比,模型组细胞活力降低,高浓度罗哌卡因组活力升高(P<0.05)。与对照组相比,模型组细胞炎性因子IL-6、IL-8、TNF-α浓度、凋亡率和caspase-3、NLRP3、磷酸化(p)-NF-κB蛋白水平上升(P<0.05),增殖率和cycilnD1蛋白水平下降(P<0.05);与模型组相比,罗哌卡因组和抑制剂组IL-6、IL-8、TNF-α浓度、凋亡率和caspase-3、NLRP3、p-NF-κB蛋白水平下降(P<0.05),增殖能力和cycilnD1蛋白水平上升(P<0.05);与罗哌卡因组相比,罗哌卡因+抑制剂组上述指标趋势变化更加显著(P<0.05),罗哌卡因+激动剂组则显著扭转了这些指标趋势(P<0.05)。结论罗哌卡因抑制LPS诱导的NCM-460细胞凋亡,促进增殖,NF-κB通路可能参与起作用。Objective To investigate the impact of ropivacaine on apoptosis of lippolysaccharide(LPS)-induced ulcerative colitis cell line NCM-460 and on activity of nucleotide oligomerization domain(NOD)-like receptor protein-3(NLRP3)inflammatome.Methods Human colon epithelial cell line NCM-460 was cultured in vitro and divided into control group(no intervention),model group(10μg/mL LPS treatment),low/medium/high concentration ropivacaine group(10μg/mL LPS and 0.5,1,1.5 mmol/L ropivacaine co-treatment,respectivoly).Cell viability was determined by cell counting kit 8(CCK-8)and the appropriate concentration was selected.The cells were then divided into control group,model group,ropivacaine group(10μg/mL LPS and 1.5 mmol/L ropivacaine treatment)and ropivacaine+inhibitor group(10μg/mL LPS,1.5 mmol/L ropivacaine and 1μmol/L NF-κB pathway inhibitor BAY 11-7082 treatment),inhibitor group(10μg/mL LPS+1μmol/L NF-κB pathway inhibitor BAY 11-7082 treatment)and ropivacaine+activator group(10μg/mL LPS,1.5 mmol/L ropivacaine and 1μmol/L NF-κB pathway activator Prostratin),all groups were treated for 24 h.The level of IL-6,IL-8 and TNF-αwere detected by enzyme-linked immunosorbent assay(ELISA).The proliferation rate was detected by EdU incorporation.Hoechst 33258 staining microscopy was used to detect the apoptosis rate.Level of cyclinD1,caspase-3,NLRP3 and NF-κB pathway-related proteins were detected by Western blot.Results Compared with the control group,the cell viability of the model group was significantly decreased and the cell viability of high-concentration experimental group was increased after adding ropivacaine(P<0.05).So,1.5 mmol/L ropivacaine was selected for the follow-up experiment.Compared with the control group,the concentration of inflammatory cytokines IL-6,IL-8,TNF-α,apoptosis rate and the protein expression of caspase-3,NLRP3 and phosphorylated p-NF-κB in model group were all significantly increased(P<0.05),while the proliferation rate and cycilnD1 protein expression were decreased(P<0.05).Compared wi

关 键 词：人结肠上皮细胞系 脂多糖 罗哌卡因 增殖 凋亡 

分 类 号：R318.14[医药卫生—生物医学工程]