右美托咪定下调PI3K/AKT信号通路改善脂多糖诱导的结肠炎结肠上皮细胞损伤  

Dexmedetomidine Regulates the PI3K/AKT Signaling Pathway to Improve Lipopolysaccharide-induced Colitis Colon Epithelial Cell Injury

作　　者：董江龙[1] 宋万军[1] 单新[1] 仝烨峰[2] DONG Jianglong;SONG Wanjun;SHAN Xin;TONG Yefeng(The Third Hospital of Hebei Medical University,Department of Anesthesia I,Shijiazhuang 050051,China;The Third Hospital of Hebei Medical University,Department of Anesthesia II,Shijiazhuang 050051,China)

机构地区：[1]河北医科大学第三医院麻醉一科,石家庄050051 [2]河北医科大学第三医院麻醉二科,石家庄050051

出　　处：《激光生物学报》2024年第4期377-384,共8页Acta Laser Biology Sinica

基　　金：河北省医学科学研究课题计划项目(20210381)。

摘　　要：为研究右美托咪定对脂多糖(LPS)诱导的人结肠上皮NCM-460细胞的炎症、增殖和凋亡的影响及磷脂酰肌醇-3-激酶/丝氨酸-苏氨酸激酶(PI3K/AKT)信号通路的调控作用,本研究体外培养人结肠上皮NCM-460细胞,将其分为对照组、LPS组(1μg/mL LPS)和不同质量浓度右美托咪定组(1μg/mL LPS+1.25、2.50、5.00和10.00μg/mL右美托咪定),干预24 h,筛选出合适的右美托咪定作用质量浓度用于后续试验。随后,将人结肠上皮NCM-460细胞分为对照组、LPS组、右美托咪定组(1μg/mL LPS+5.00μg/mL右美托咪定)、LY294002组(1μg/mL LPS+10μmol/L PI3K/AKT通路抑制剂LY294002)、抑制剂组(1μg/mL LPS+5.00μg/mL右美托咪定+10μmol/L LY294002)和激活剂组(1μg/mL LPS+5.00μg/mL右美托咪定+10μmol/L PI3K/AKT通路激动剂SC79),干预24 h。用细胞计数试剂盒-8(CCK-8)检测细胞活力;通过酶联免疫吸附试验(ELISA)检测肿瘤坏死因子-α(TNF-α)和白细胞介素-8(IL-8)的表达水平;用5-乙炔基-2'脱氧尿嘧啶核苷(EdU)测定细胞增殖率;用Hoechst 33258染色法测定细胞凋亡率;用蛋白免疫印迹(WB)法测定细胞周期蛋白D1(Cyclin D1)、剪切的半胱氨酸蛋白酶-3(cleaved Caspase 3)和PI3K/AKT信号通路关键蛋白的表达水平。根据细胞活力和炎症因子TNF-α的表达水平选择5.00μg/mL右美托咪定用于后续试验。结果显示,与对照组相比,LPS组细胞增殖率和Cyclin D1的表达水平显著降低(P<0.05),细胞凋亡率、TNF-α、IL-8、cleaved Caspase 3的表达水平、p-PI3K/PI3K及p-AKT/AKT的比值显著升高(P<0.05);右美托咪定组和LY294002组中的右美托咪定和LY294002扭转了LPS对人结肠上皮NCM-460细胞的上述作用(P<0.05);与右美托咪定组相比,抑制剂组中的LY294002增强了右美托咪定对LPS诱导的人结肠上皮NCM-460细胞的作用(P<0.05),激活剂组中的SC79则削弱了右美托咪定对LPS诱导的人结肠上皮NCM-460细胞的作用(P<0.05)。研究表明,右美托咪定能促�In order to study the effects of dexmedetomidine on inflammation,proliferation and apoptosis of human colonic epithelial NCM-460 cells induced by lipopolysaccharide(LPS)and the regulation of phosphatidylinositol-3-kinase/serinethreonine kinase(PI3K/AKT)signaling pathway,we cultured human colonic epithelial NCM-460 cells in vitro,they were diimmunovided into control group,LPS group(1μg/mL LPS)and dexmedetomidine groups with different mass concentrations(1μg/mL LPS+1.25,2.50,5.00 and 10.00μg/mL dexmedetomidine).After 24 hours’intervention,the appropriate mass concentration of dexmedetomidine was screened out for subsequent experiments.Then,The human colonic epithelial NCM-460 cells were divided into control group,LPS group,dexmedetomidine group(1μg/mL LPS+5.00μg/mL dexmedetomidine),LY294002 group(1μg/mL LPS+10μmol/L PI3K/AKT pathway inhibitor LY294002)and inhibitor group(1μg/mL LPS+5.00μg/mL dexmedetomidine+10μmol/L LY294002),and activator group(1μg/mL LPS+5.00μg/mL dexmedetomidine+10μmol/L PI3K/AKT pathway agonist SC79)were intervened for 24 hours.Cell viability was detected by live cell counting kit-8(CCK-8).The expression levels of tumor necrosis factor-α(TNF-α)and interleukin-8(IL-8)were detected by enzyme-linked immunosorbent assay(ELISA).The cell proliferation rate was determined by 5-ethynyl-2'deoxyuridine(EdU).The apoptosis rate was determined by Hoechst 33258 staining.The expression levels of cyclin D1,cleaved cysteine protease 3(cleaved Caspase 3)and key proteins of PI3K/AKT signaling pathway in cells were determined by Western blot(WB).According to the cell viability and the expression level of inflammatory factor TNF-α,5.00μg/mL dexmedetomidine was selected for the follow-up experiment.The results showed that compared with the control group,the cell proliferation rate and the expression level of Cyclin D1 in LPS group significantly decreased(P<0.05),while the cell apoptosis rate,the expression levels of TNF-α,IL-8,and cleaved Caspase 3,the ratio of p-PI3K/PI3K and p-AKT/AKT significa

关 键 词：溃疡性结肠炎 人结肠上皮细胞 右美托咪定 磷脂酰肌醇-3-激酶/丝氨酸-苏氨酸激酶 

分 类 号：R574.62[医药卫生—消化系统]