目的 研究丹酚酸B对香烟烟雾提取物(CSE)诱导的支气管上皮细胞凋亡的影响及机制.方法 将人支气管上皮细胞16HBE分成对照组、模型组(香烟烟雾提取物诱导)、实验低剂量组(烟雾诱导+11 μmol·L-1丹酚酸B处理)、实验中剂量组(烟雾诱导+22 μmol·L-1丹酚酸B处理)、实验高剂量组(烟雾诱导+44 μmol·L-1丹酚酸B处理)、实验高剂量+Compound C组(烟雾诱导+44 μmol·L-1丹酚酸B+AMPK信号通路抑制剂Compound C处理).用细胞计数试剂盒-8(CCK-8)实验检测细胞增殖情况,以蛋白质印迹法检测磷酸化腺苷—磷酸活化蛋白激酶(p-AMPK)、CCAAT/增强子结合蛋白同源蛋白(CHOP)、活化转录因子4(ATF4)蛋白表达,用PI单染法检测细胞周期,用Annexin V-FITC/PI双染法检测细胞凋亡情况,用分光光度法检测胱天蛋白酶-3(caspase-3)活性.结果 对照组、模型组、实验低剂量组、实验中剂量组、实验高剂量组、实验高剂量+Compound C组的细胞增殖活性(OD值)分别为0.86±0.07、0.38±0.03、0.45±0.03、0.54±0.04、0.68±0.03 和0.42±0.04,支气管上皮细胞中 p-AMPK/AMPK蛋白表达量比值分别为0.41±0.03、0.13±0.03、0.20±0.02、0.28±0.04、0.36±0.04 和 0.22±0.02,G0/G1 期细胞比例 分 别 为(54.40±5.84)％、(82.93±4.50)％、(75.45±4.73)％、(67.41±2.70)％、(59.15±3.73)％和(69.80±6.59)％,细胞凋亡率分别为(3.21±0.49)％、(24.90±3.35)％、(20.56±1.73)％、(13.55±1.68)％、(9.20±1.07)％和(18.04±1.79)％;实验低剂量组、实验中剂量组、实验高剂量组与模型组相比,在统计学上差异均有统计学意义(均P＜0.05);实验高剂量+Compound C组与实验高剂量组相比,在统计学上差异均有统计学意义(均P＜0.05).结论 丹酚酸B通过激活AMPK信号通路影响内质网应激,减少香烟烟雾提取物诱导的支气管上皮细胞凋亡.
Objective To explore the effect and mechanism of salvianolic acid B on bronchial epithelial cell apoptosis induced by cigarette smoke extract.Methods Human bronchial epithelial cells 16HBE were divided into control group,model group(induced by cigarette smoke extract),experimental low-dose group(induced by cigarette smoke extract+11 μmol·L-1 salvianolic acid B treatment),and experimental medium-dose group(cigarette smoke extract Induction+22 μmol·L-1 salvianolic acid B treatment),experimental high-dose group(cigarette smoke extract induction+44 μmol·L-1 salvianolic acid B treatment),experimental high-dose+Compound C group(cigarette smoke extract induction+44 μmol·L-1 Salvianolic acid B+AMPK signaling pathway inhibitor Compound C treatment).Cell counting kit-8(CCK-8)assay was used to detect proliferation;Western blot was used to detect the phosphorylated adenosine monophosphate activates protein kinase(p-AMPK),CCAAT/enhancer-binding protein C/EBP(CHOP),activating transcription factor 4(ATF4)protein expression;PI single staining was used to detect cell cycle;Annexin V-FITC/PI double staining was used to detect apoptosis,and spectrophotometry was used to detect caspase-3 activity.Results The cell proliferation activity(OD value)in the bronchial epithelial cells of the control group,model group,experimental low-dose group,experimental medium-dose group,experimental high-dose group,and experimental high-dose+Compound C group were 0.86±0.07,0.38±0.03,0.45±0.03,0.54±0.04,0.68±0.03 and 0.42±0.04;the expression levels of p-AMPK/AMPK protein were 0.41±0.03,0.13±0.03,0.20±0.02,0.28±0.04,0.36±0.04 and 0.22±0.02;G0/G1 phase were(54.40±5.84)％,(82.93±4.50)％,(75.45±4.73)％,(67.41±2.70)％,(59.15±3.73)％and(69.80±6.59)％;apoptosis rate were(3.21±0.49)％,(24.90±3.35)％,(20.56±1.73)％,(13.55±1.68)％,(9.20±1.07)％and(18.04±1.79)％.Compared experimental low-dose group,experimental medium-dose group,experimental high-dose group with model group,the difference of above indicators were all statistically significant(all P＜0.05);compared the experimental high-dose+Compound C group with the experimental high-dose group,the difference of above indicators were all statistically significant(all P＜0.05).Conclusion Salvianolic acid B affects endoplasmic reticulum stress by activating AMPK signaling to reduce bronchial epithelial cell apoptosis induced by cigarette smoke extract.
胡秋芳;吕秋菊
乐山职业技术学院基础医学教研室,四川乐山 614000乐山市人民医院内分泌科,四川乐山 614000
中医学
丹酚酸B;腺苷一磷酸活化蛋白激酶信号;内质网应激;支气管上皮细胞;细胞周期阻滞
salvianolic acid B;adenosine monophosphate activated protein kinase signal;endoplasmic reticulum stress;bronchial epithelial cell;cell cycle arrest
《中国临床药理学杂志》 2024 (006)
825-829 / 5
10.13699/j.cnki.1001-6821.2024.06.009
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