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（1 μg/mL LPS）和不同质量浓度右美托咪定组（1 μg/mL LPS+1.25、2.50、5.00和10.00 μg/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（1 μg/mL LPS+5.00 μg/mL右美托咪定）、LY294002组（1 μg/mL LPS+10 μmol/L PI3K/AKT通路抑制剂LY294002）、抑制剂组（1 μg/mL LPS+5.00 μg/mL右美托咪定+10 μmol/L LY294002）和激活剂组（1 μg/mL LPS+5.00 μg/mL右美托咪定+10 μmol/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5.00 μg/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，右美托咪定能促进LPS诱导的人结肠上皮NCM-460细胞增殖，抑制其炎症和凋亡，其作用机制可能与阻滞PI3K/PI3K信号通路信号转导有关。本研究为结肠炎的治疗提供了新的方向。
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
中图分类号：R574.62                       文献标志码：ADOI：10.3969/j.issn.1007-7146.2024.04.010

Abstract: 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/serine-threonine kinase (PI3K/AKT) signaling pathway, we cultured human colonic epithelial NCM-460 cells in vitro, they were divided into control group, LPS group (1 μg/mL LPS) and dexmedetomidine groups with different mass concentrations (1 μg/mL LPS+1.25, 2.50, 5.00 and 10.00 μg/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 (1 μg/mL LPS+5.00 μg/mL dexmedetomidine), LY294002 group (1 μg/mL LPS+10 μmol/L PI3K/AKT pathway inhibitor LY294002) and inhibitor group (1 μg/mL LPS+ 5.00 μg/mL dexmedetomidine +10 μmol/L LY294002), and activator group (1 μg/mL LPS+5.00 μg/mL dexmedetomidine +10 μmol/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, 5.00 μg/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 significantly increased (P<0.05). Dexmedetomidine and LY294002 in dexmedetomidine group and LY294002 group reversed the above-mentioned effects of LPS on human colonic epithelial NCM-460 cells (P<0.05). Compared with dexmedetomidine group, LY294002 in inhibitor group enhanced the effect of dexmedetomidine on human colonic epithelial NCM-460 cells induced by LPS (P<0.05), while SC79 in activator group weakened the effect of dexmedetomidine on human colonic epithelial NCM-460 cells induced by LPS (P<0.05). Studies have shown that dexmedetomidine can promote the proliferation of human colonic epithelial NCM-460 cells induced by LPS and inhibit its inflammation and apoptosis, and its mechanism may be related to blocking the signal transduction of PI3K/PI3K signaling pathway. This study can provide a new direction for the treatment of colitis.
Key words: ulcerative colitis; human colonic epithelial cells; dexmedetomidine; phosphatidylinositol-3-kinase/serine-threonine kinase
（Acta Laser Biology Sinica, 2024, 33(4): 377-384）