葛根总黄酮对载脂蛋白E基因缺陷小鼠动脉粥样硬化病变内细胞凋亡及凋亡相关基因的影响

Effects of the total flavone of radix puerariae on apoptotic cell and apoptotic related-gene in atherosclerotic plaques of apoE gene deficency mice

目的探讨葛根总黄酮对载脂蛋白 E 基因缺陷(apoE-/-)小鼠主动脉窦动脉粥样硬化斑块抑制作用的分子机制。方法采用电镜观察、缺口末端标记(TUNEL)法检测 apoE-/-小鼠动脉粥样硬化斑块内的细胞凋亡。免疫组化方法检测平滑肌细胞、巨噬细胞及 Caspase-3蛋白的表达。结果电镜观察到凋亡细胞核形不规则,胞核内染色质浓集、边聚,附着在核膜周边,线粒体肿胀,内质网扩张,超微结构形态符合巨噬细胞早期凋亡,并可见典型凋亡小体形成。TUNEL 结果表明,凋亡细胞主要分布在粥样斑块脂质核心处,模型组动脉粥样硬化斑块内凋亡细胞数较多,而葛根总黄酮干预组凋亡细胞数明显减少,葛根高、低剂量组细胞凋亡率明显低于模型组[分别为(0.38±0.17)%,(1.95±1.02)%,(10.50±5.89)%,P<0.01],葛根高剂量组细胞凋亡率明显低于葛根低剂量组。抗 CD68抗体免疫组化染色证实在动脉粥样硬化斑块脂质坏死核心内细胞 CD68强阳性表达。葛根高、低剂量治疗组 Caspase-3蛋白免疫表达低于模型组,葛根高剂量治疗组低于葛根低剂量治疗组。结论葛根总黄酮可能通过下调 apoE-/-小鼠主动脉窦动脉粥样硬化病变内 Caspases-3蛋白的表达,显著降低了动脉粥样硬化病变内巨噬细胞凋亡,从而抑制了动脉粥样硬化斑块的进展。

Objective To investigate the antiathemsclemsis effect and related mechanisms of total flavone of radix puerariae （TFRP） on athemsclerotic plaques in apoE gene deficency （ apoE - / - ） mice. Methods apoE -/- mice were treated with saline, TFRP 15 mg · kg^-1 · d^-1 or TFRP 85 mg· kg^-1·d^-1 （n = 8 each group） respectively per gavage for 12 weeks. The apoptotic cells in atherosclemtic plaques were then detected by TUNEL analysis, transmission electron microscope （TEM）. The expression of CD-68, SMA and Caspase-3 were determined by immunochemical methods. Results Early macmphagocyte apoptosis signs were observed under TEM, TUNEL-positive and CD-68 positive cells were found in lipid cores of atherosclemtic plaques. TFRP significantly reduced the number of apoptotic cells in a dose-dependent manner [ （ 0. 38 ±0. 17 ） % , （ 1.95 ±1.02 ） % , （ 10. 50 ±5. 89 ） % , respectively, P 〈 0. 01 ] in athemsclemtic plaques. TFRP treatment also significantly reduced the immune expression of Caspase-3 protein in a dose-dependent manner. Conclusion TFRP significantly attenuated the development of advanced athemsclemtic plaques in a dose-dependent manner which might related to down-regulated expression of Caspase-3 protein and reduced macmphagocyte apoptotic cells in athemsclerotic plaques post TFRP treatment.