丹皮酚抑制血管平滑肌细胞增殖和对增殖细胞抗原及P27kip1表达的影响

Paeonol inhibits vascular smooth muscle cell proliferation by regulating proliferating cell nuclear antigen and P27kip1 expression

目的 探讨丹皮酚对兔血管成形术后再狭窄的作用及其机制. 方法 选用清洁级雄性新西兰大白兔,采用腹主动脉球囊损伤和高脂饲料喂养方法建立腹主动脉粥样硬化模型,对模型兔腹主动脉狭窄段行2次球囊扩张后分为4组,分别为对照组、低剂量丹皮酚组、高剂量丹皮酚组、雷帕霉素组,分别给予生理盐水2 ml· kg-1·d^-1、低剂量丹皮酚75 mg·kg-1·d^-1、高剂量丹皮酚150mg·kg-1·d^-1、雷帕霉素1.25 mg· kg-1·d^-1灌胃,每天灌胃1次,连续6周给药.6周后取出腹主动脉,使用苏木精-伊红（HE）染色和Masson染色观察狭窄动脉的大体形态学及内膜增生厚度.免疫组化检测增殖细胞抗原（PCNA）表达水平,免疫印迹法（Western-blot）检测P27kip1蛋白表达水平.结果 使用丹皮酚、雷帕霉素后内膜增生程度降低.对照组、低剂量丹皮酚组、高剂量丹皮酚组和雷帕霉素组新生内膜面积分别为0.16±0.03、0.09±0.01、0.05±0.01、0.04±0.01;新生内膜面积/中膜面积分别为90.62±3.43、42.30±2.94、33.72±3.19、28.78±6.67;内膜面积增生率分别为（33.3±3.1）％、（23.2±2.2）％、（17.5±1.5）％、（16.2±1.7）％,差异有统计学意义（F值分别为47.98、257.25、74.60,均P＜0.05）.对照组、低剂量丹皮酚组、高剂量丹皮酚组和雷帕霉素组PCNA的表达水平分别为0.20±0.04、0.06±0.02、0.05±0.02、0.08±0.02,差异均有统计学意义（F=37.22,P＜0.01）;P27kip1表达水平分别为0.47±0.09、0.78±0.20、1.29±0.12、1.83±0.13,差异有统计学意义（F=107.69,P＜0.01）. 结论 丹皮酚可能通过抑制PCNA表达,在细胞周期中上调P27kip1蛋白的表达,起到抗血管平滑肌细胞增殖的作用.

Objective To study the preventive effect of paeonol on restenosis after angioplasty in rabbit balloon injury model and its mechanism.Methods Male New Zealand white rabbits with clean grade were selected.Balloon injury rabbit abdominal aorta combined with high cholesterol diet were used to establish rabbit atherosclerosis model.We used balloon to dilate the narrow abdominal aorta.The animals were randomly divided into four groups （control group,low concentration paeonol group,high concentration paeonol group and rapamycin group）.Animals in different group were orally given normal saline 2 ml · kg^-1 · d^-1,low concentration of paeonol 75 mg · kg^-1 ·d^-1,high concentration of paeonol 150 mg·kg^-1 · d^-1,rapamycin administrated with 1.25 mg · kg^-1 · d^-1 for six weeks respectively.After 6 weeks,the animals were killed by air embolism,and immediately dissected to remove the abdominal aorta.We used hematoxylin and eosin（HE） staining and Masson staining to detect the thickness of the arterial intimal hyperplasia and the gross morphological changes of the narrow artery.Immunohistochemical staining was used to detect changes of proliferating cell nuclear antigen （PCNA） expression level.Western blotting was used to discover P27kip1 protein expression in the narrow artery.Results Paeonol and rapamycin reduced the degree of intimal hyperplasia.The neointima areas in the control group,low concentration paeonol group,high concentration paeonol group and rapamycin group were （0.16±0.03）,（0.09±0.01）,（0.05±0.01） and （0.04±0.01） respectively; the ratio of neointimal area to media area was （90.62 ±3.43）,（42.30 ±2.94） （33.72±3.19） and （28.78±6.67） respectively; the intimal area proliferation rate was （33.3 ± 3.1） %,（23.2 ± 2.2） %,（17.5 ± 1.5） % and （16.2 ± 1.7） % respectively.There were significant differences in the neointima area,ratio of neointimal area to media area,and the intimal area proliferation rate among the four groups （F=47.98,257.25,74.60,all P〈0.05）.The expression level of PCNA and P27kip1 protein were （0.20±0.04）,（0.06±0.02）,（0.05±0.02） and （0.08± 0.02）,and （0.47±0.09）,（0.78±0.20）,（1.29±0.12） and （1.83±0.13） in the control group,low concentration paeonol group,high concentration paeonol group and rapamycin group respectively,and the differences were statistically significant between the four groups （F=37.22 and 107.69,both P〈0.01）.Conclusions Paeonol plays a role in anti-vascular smooth muscle cell proliferation.This effect may be through inhibiting the expression of PCNA,and increasing the expression of P27kip1 in the cell cycle.