烟酰胺预防全反式维甲酸诱导小鼠腭裂的初步实验研究

Preliminary experimental study on the prevention of all⁃trans retinoic acid induced cleft palate in mice by nico⁃tinamide

目的探讨烟酰胺(nicotinamide,NAM)对全反式维甲酸(all-trans retinoic acid,RA)诱导小鼠腭裂的预防作用,为腭裂的预防提供研究依据。方法采用70 mg/kg的RA于胚胎发育(embryonic,E)10.5 d(E10.5)灌胃诱导的小鼠腭裂模型为对照组,采用20 mg/kg的NAM于E8.5~E13.5尾静脉注射干预上述腭裂模型为实验组(1),采用40 mg/kg的NAM于E8.5~E13.5尾静脉注射干预上述腭裂模型为实验组(2),于E16.5剖腹观察胎鼠腭裂情况并进行统计分析。对鼠胚腭突间质(mouse embryonic palatal mesenchyme,MEPM)细胞进行分组干预,共分4组:对照组(CONTROL)、RA 1μmol/L组(RA 1)、NAM 200μmol/L组(NAM 200)、NAM 200μmol/L+RA 1μmol/L组(NAM 200+RA 1)。各组药物干预24 h后采用Annexin V-FITC/PI双染色法进行细胞凋亡检测并比较凋亡率。结果动物实验中对照组小鼠腭裂率为98%;实验组(1)腭裂率为87%,与对照组差异无统计学意义(P>0.05);实验组(2)腭裂率为63%,与对照组差异具有统计学意义(P<0.01)。细胞实验中CONTROL组细胞凋亡率为16.53%±2.89%,RA 1组细胞凋亡率为22.9%±1.85%,凋亡率上升(P<0.01);NAM 200组细胞凋亡率9.23%±1.39%,凋亡率下降(P<0.01);与RA 1组相比较,NAM 200+RA 1组细胞凋亡率为14.9%±7.67%,凋亡率下降(P<0.01)。结论40 mg/kg是NAM预防RA诱导小鼠腭裂的有效浓度;其预防腭裂作用的机制可能是NAM抑制了RA诱导MEPM细胞凋亡所致。

Objective To explore the preventive effect of nicotinamide(NAM)on cleft palate induced by all-trans retinoic acid(RA),to provide research evidence for the prevention of cleft palate.Methods The mouse cleft palate model was induced by intragastric administration of 70 mg/kg all-trans retinoic acid at embryonic day 10.5(E10.5)in the control group.The mouse cleft palate model was treated by caudal vein injection of 20 mg/kg NAM at E8.5 to E13.5 in the experimental group(1).The cleft palate model was treated by caudal vein injection of 40 mg/kg NAM at E8.5-E13.5 in the experimental group(2).The cleft palate of fetal rats was observed by laparotomy on E16.5 and statistically analyzed.Annexin V-FITC/PI double staining was used to detect the apoptosis of mouse embryonic palatal mesenchyme(MEPM)cells treated with RA 1μmol/L(RA 1 group),NAM 200μmol/L(NAM 200 group),and both NAM 200μmol/L and RA 1μmol/L(NAM 200+RA 1 group)for 24 hours by flow cytometry and the apoptosis rate in groups were compared.Culture without RA or NAM was used as a control.Results The cleft palate rate in the control group was 98%.The cleft palate rate in experimental group(1)was 87%.There was no significant difference between groups(P>0.05).The cleft palate rate in the experimental group(2)was 63%,compared with the control group,there was a significant dif-ference(P<0.01).The cell apoptosis rate was 16.53%±2.89%in the CONTROL group.The cell apoptosis rate was 22.9%±1.85%in the RA 1 group,which was a significant increase compared with the CONTROL group(P<0.01).The apoptotic rate of the NAM 200 group was 9.23%±1.39%,which was a significant decrease compared with NA 1 group(P<0.01).The apoptosis rate of the NAM 200+RA 1 group was 14.9%±7.67%,which was a significant decrease compared with the RA 1 group(P<0.01).Conclusion NAM can prevent cleft palate.40 mg/kg nicotinamide during pregnancy is an effective concentration for the prevention of RA-induced cleft palate.The mechanism by which NAM prevents cleft palate may be that NAM inhibits RA-induced apoptosis of MEPM cells.