补肾活血方对血管性痴呆大鼠海马神经细胞自噬的影响＊

目的 探究补肾活血方对血管性痴呆（Vascular Dementia, VD）大鼠模型自噬的影响。方法 52周龄SD雄性大鼠50只,随机分为假手术组（Sham组）、模型组（VD组）、模型+补肾活血方组（BSHX组）、模型+雷帕霉素组（Rap组）和模型+3-甲基腺嘌呤组（3-MA组）,每组10只大鼠。除Sham组外其余各组采用两血管阻断法（2-VO）建立VD模型。BSHX组中药灌胃治疗28天;自噬干预组于造模前30 min侧脑室给药。运用Morris水迷宫测试各组大鼠的学习记忆能力;通过尼氏染色和透射电子显微镜（Transmission electron microscope,TEM）观察大鼠海马区病理形态及自噬变化;采用蛋白免疫印迹（Western blot）法和反转录实时荧光定量PCR（RT-qPCR）检测大鼠海马Beclin-1、P62以及微管相关蛋白1轻链3（LC3）蛋白及mRNA表达情况。结果 与VD组比较,BSHX组大鼠学习记忆能力显著提升（P<0.05）,镜下观察BSHX组和3-MA组的细胞形态及数量均有改善,自噬小体鲜见,Beclin-1以及LC3蛋白和mRNA表达水平显著降低（P<0.05）,P62蛋白和mRNA表达水平明显升高（P<0.05）结论 补肾活血方可以降低VD大鼠海马区Beclin-1和LC3蛋白及mRNA的表达,提高P62的表达,通过抑制自噬的发生,减轻对神经细胞的损伤,改善学习记忆能力。

Effects of Bushenhuoxue Formula on Autophagy of Hippocampal Nerve Cells in Rats of Vascular Dementia

Objective To explore the effect of Bushenhuoxue formula on autophagy in rat model of vascular dementia (VD).Methods Fifty 52 weeks SD male rats randomly were divided into sham operation group (Sham group), model group (VD group), Bushenhuoxue group (BSHX group), rapamycin group (Rap group) and 3-methyladenine group (3-group MA), with 10 rats in each group. The two-vessel occlusion method (2- VO) was used to establish the VD model except Sham groups. BSHX group was given traditional Chinese medicine intragastric administration for 28 days; Autophagy intervention group was administered 30 min before modeling in lateral ventricle. Morris water maze was used to test the learning and memory ability of rats in each group; The pathological morphology and autophagy in the hippocampus of rats were observed by Nissl staining and Transmission electron microscopy (TEM). The protein and mRNA expressions of Beclin-1, P62 and microtubule-associated protein 1 light chain 3(LC3) in the hippocampus of rats were detected by Western blot and RT-qPCR.Results Compared with VD group, the learning and memory ability of rats in BSHX group was significantly improved (P<0.05). Microscopically, the cell morphology and quantity of BSHX group and 3-MA group were improved, and autophagosomes were rare. Beclin-1 and LC3 protein and mRNA expression levels were significantly down-regulated (P<0.05). The expression of P62 protein and mRNA increased significantly (P<0.05).Conclusion Bushenhuoxue formula can reduce the expression of Beclin-1 and LC3 protein and mRNA in hippocampus of VD rats, increase the expression of P62, reduce the nerve cells'' damage by inhibiting autophagy, and improve the ability of learning and memory.