藏药名方七十味珍珠丸减重金属后干预D-半乳糖/亚硝酸钠诱导痴呆小鼠的作用及机制研究

Effect and Mechanism of Famous Tibetan Prescription Qishiwei Zhenzhu (七十味珍珠) Pills in Intervening in Dementia Mice Induced by D-galactose/Sodium Nitrite after Heavy Metal Removal

目的:藏药名方七十味珍珠丸中含多种重金属问题是藏药现代化研究的热点和难点,本研究旨在探明藏药名方七十味珍珠丸减重金属后对痴呆小鼠模型的促智作用及机制。方法:以D-半乳糖/亚硝酸钠诱导复制C57/BL6小鼠的阿尔茨海默病模型,动物分为正常对照组、模型对照组、七十味珍珠丸60 mg/kg组、无重金属七十味珍珠丸60 mg/kg组,灌胃给药8 w后,进行行为学评价,用HE染色、ELISA试剂盒、Western blot法检测脑组织关键因子和蛋白的表达;并利用氧糖剥夺/再复氧诱发的BV2细胞损伤模型,评价无重金属七十味珍珠丸处理对细胞活力的影响。结果:与正常对照组相比,OGD/R处理后,模型对照组BV2细胞数目减少,形态改变,出现触角和突触,发生细胞激活,且出现较多细胞死亡,BV2细胞活力显著降低(P<0.01),模型对照组小鼠自发交替反应率显著降低,小鼠进入新臂的次数显著降低,小鼠僵直总次数和僵直时间所占百分比显著降低,小鼠脑组织Glu活力、AchE含量显著增加(P<0.01),脑组织BDNF、BCL-2蛋白表达显著下调,Caspase-3蛋白表达显著上调(P<0.01);与模型对照组相比,无重金属七十味珍珠丸60 mg/kg组显著提高小鼠的认知功能、逆转脑组织形态损伤;降低脑组织Glu、AchE含量,上调脑组织BDNF、BCL-2表达,下调Caspase-3表达,显著增加细胞活力,明显减轻BV2细胞损伤(P<0.05或P<0.01)。结论:无重金属七十味珍珠丸组对D-半乳糖/亚硝酸钠诱导的痴呆小鼠有一定的保护作用,对氧糖剥夺/再复氧诱导的BV2细胞损伤也有保护作用,且在活性方面无重金属七十味珍珠丸样品类似原方。其机制可能与改善大脑海马齿状回功能神经元结构紊乱、逆转模型动物脑内Glu和AchE紊乱,调控海马区BDNF、BCL-2和Caspase-3蛋白表达,减轻胶质细胞过度活化有关。

Objective:The problem of heavy metal contamination in the famous Tibetan medicine prescription Qishiwei Zhenzhu(七十味珍珠)Pills is a hot and challenging topic in modernization research on Tibetan medicine.This study aimed to investigate the cognitive-enhancing effects and mechanism of the Qishiwei Zhenzhu Pills after removing heavy metals in a mouse model of dementia.Methods:The C57/BL6 mouse model of Alzheimer's disease was induced by D-galactose/sodium nitrite,and the animals were divided into a normal control group,a model control group,a Qishiwei Zhenzhu Pills 60 mg/kg group,and a heavy metal-free Qishiwei Zhenzhu Pills 60 mg/kg group.After 8 weeks of oral administration,behavioral assessments were conducted,and HE staining,ELISA assay,and Western blot analysis were performed to evaluate the expression of key factors and proteins in brain tissues.The impact of heavy metal-free Qishiwei Zhenzhu Pills treatment on cell viability was evaluated using an oxygen-glucose deprivation/reoxygenation(OGD/R)-induced BV2 cell injury model.Results:Compared with the normal control group,the model control group showed a decrease in the number of BV2 cells,morphological changes,the presence of dendrites and synapses,cell activation,and increased cell death after OGD/R treatment.The model control group also exhibited significantly reduced BV2 cell viability and decreased spontaneous alternation response rate,number of entries into the new arm,total freezing time,and percentage of freezing time in mice.Moreover,the brain tissue showed increased Glu activity and AchE content(P<0.01),significantly downregulated expression of BDNF and BCL-2,and upregulated expression of CASPASE-3(P<0.01).Compared with the model control group,the heavy metal-free Qishiwei Zhenzhu Pills 60 mg/kg group showed significant improvement in cognitive function,reversal of brain tissue morphological damage,increased Glu activity,decreased AchE content,upregulated expression of BDNF and BCL-2,downregulated expression of CASPASE-3,increased cell viability,and reduced BV2 cell injury(P<0.05 or P<0.01).Conclusion:The heavy metal-free Qishiwei Zhenzhu Pills have a certain protective effect on D-galactose/sodium nitrite-induced dementia mice and also protect against BV2 cell injury induced by OGD/R.Furthermore,they exhibit similar activity to the original prescription.The mechanism may be associated with the improvement of neuronal structural disorder in the hippocampal dentate gyrus,reversal of Glu and AchE imbalance in the animal model's brain,regulation of BDNF,BCL-2,and CASPASE-3 protein expression in the hippocampal region,and alleviation of excessive glial cell activation.