基于抗抑郁活性的百合地黄汤中生地黄与百合的配比优选及其机制

Optimal Ratio of Rehmanniae Radix and Lilii Bulbus in Baihe Dihuangtang Based on Antidepressant Activity and Its Mechanism

目的:基于抗抑郁活性优选百合地黄汤中生地黄与百合的最佳配比并初探其可能的机制。方法:100只雄性小鼠随机分为正常组、模型组、生地黄单煎液组、百合单煎液组、5种不同配比的百合地黄汤组(生地黄-百合5∶6、5∶5、5∶4、5∶3、5∶2)、盐酸氟西汀组。除正常组外,其余9组,连续21 d,采用慢性不可预知性温和应激法(CUMS)制备抑郁小鼠模型。生地黄单煎液组、百合单煎液组和5种不同配比的百合地黄汤组均以2 g·kg^(-1)分别灌胃相应的药物,盐酸氟西汀组灌胃盐酸氟西汀0.01 g·kg^(-1),正常组和模型组分别灌胃等体积生理盐水,连续21 d。通过行为学实验评价不同配比的百合地黄汤的抗抑郁作用;蛋白免疫印迹法(Western blot)检测海马组织脑源性神经营养因子(BDNF)和酪氨酸激酶受体B(TrkB)的表达水平;羟胺法检测海马组织总超氧化物歧化酶(SOD)的水平;分光光度法检测海马组织还原型谷胱甘肽(GSH)的水平;比色法检测海马组织谷胱甘肽过氧化物酶(GPX)和谷胱甘肽S转移酶(GST)的水平;硫代巴比妥酸法检测海马组织丙二醛(MDA)的水平,初探其抗抑郁机制。结果:CUMS引起了糖水偏爱率显著下降及强迫游泳和悬尾不动时间均显著延长(P<0.01);而不同配比的百合地黄汤及生地黄、百合的单煎液均在不同程度上明显逆转了上述指标的异常(P<0.05,P<0.01),尤以配比为5∶6时对上述指标的影响最为明显;此外,CUMS引起了小鼠海马BDNF和TrkB的表达水平及SOD、谷胱甘肽抗氧化相关指标水平均显著降低(P<0.01),脂质过氧化产物MDA水平显著升高(P<0.01),而较优比的百合地黄汤(生地黄-百合5∶6、5∶5、5∶4)及生地黄、百合的单煎液均在不同程度上明显逆转了以上指标的异常(P<0.05,P<0.01)。结论:5种不同配比的百合地黄汤及生地黄、百合的单煎液均能产生抗抑郁作用,尤以生地黄-百合配比为5∶6时抗抑郁作用最强,且随着百合在方中占比减少,抗抑郁作用逐渐减弱,其机制可能涉及增强海马中枢神经营养水平和抗氧化防御能力。

Objective:Based on the antidepressant activity,the optimal ratio of Rehmanniae Radix and Lilii Bulbus in Baihe Dihuangtang was optimized and its possible mechanism was preliminarily explored.Method:A total of 100 male mice were randomly divided into normal group,model group,Rehmanniae Radix single decoction group,Lilii Bulbus single decoction group,and 5 different ratios of Baihe Dihuangtang group(Rehmanniae Radix-Lilii Bulbus 5∶6,5∶5,5∶4,5∶3,5∶2),fluoxetine hydrochloride group.Except for the normal group,the remaining 9 groups were treated with chronic unpredictable mild stress method(CUMS)for21 consecutive days to establish the depression mouse model.The Rehmanniae Radix single decoction group,Lilii Bulbus single decoction group and 5 different ratios of Baihe Dihuangtang group were administered with corresponding drugs at a dose of 2 g·kg^(-1)respectively,and fluoxetine hydrochloride group was administered fluoxetine hydrochloride at a dose of 0.01 g·kg^(-1),the normal group and the model group were respectively given an equal volume of normal saline by gavage for 21 consecutive days.The antidepressant effect of Baihe Dihuangtang with different ratios was evaluated by behavioral experiments.Western blot was used to detect the expression levels of brain-derived neurotrophic factor(BDNF) and tyrosine kinase receptor B(TrkB) in hippocampus.The level of total superoxide dismutase(SOD)in hippocampus was detected by hydroxylamine method.The level of reduced glutathione(GSH)in hippocampus was detected by spectrophotometric method.The levels of glutathione peroxidase(GPX)and glutathione S-transferase(GST)in hippocampus was detected by colorimetric method.The level of malondialdehyde(MDA)in hippocampus was detected by thiobarbituric acid method,and preliminary exploration of its antidepressant mechanism.Result:CUMS caused a significant decrease in the preference rate of sugar water and a significant prolongation of forced swimming and tail suspension immobility time(P<0.01),while the different ratios of Baihe Dihuangtang and the single decoction of Rehmanniae Radix and Lilii Bulbus were significantly reversed to varying degrees.Abnormalities of the above indicators were observed(P<0.05,P<0.01),especially when the ratio of 5∶6 had the most obvious effect on the above indicators,Moreover,CUMS caused the hippocampal BDNF and the expression of TrkB,SOD and glutathione antioxidant-related indexes were significantly decreased(P<0.01),and the levels of lipid peroxidation product MDA was significantly increased(P<0.01).However,the superior ratio of Baihe Dihuangtang(Rehmanniae Radix-Lilii Bulbus 5∶6,5∶5,5∶4)and the single decoction of Rehmanniae Radix and Lilii Bulbus significantly reversed the abnormality of the above indicators to varying degrees(P<0.05,P<0.01).Conclusion:In summary,5 different ratios of Baihe Dihuangtang and the single decoction of Rehmanniae Radix and Lilii Bulbus can produce antidepressant effect,especially when the ratio of 5∶6 had the most obvious effect on the above indicators,and the antidepressant effect gradually weakens with the proportion of Lilii Bulbus in the recipe decreases,and its mechanism may involve enhancing the nutritional level and antioxidant defense ability of hippocampal central nervous system.