基于斑马鱼模型不同提取方法人源胎盘冻干粉抗抑郁活性及作用机制研究

Comparison of anti-depressant activity and mechanism investigation among human placenta freeze-dried powder extracted by different methods by zebrafish model

目的明确人源胎盘冻干粉是否具有抗利血平诱发斑马鱼抑郁症的活性,比较不同提取方法胎盘冻干粉抗抑郁活性并探索其作用机制。方法将6月龄野生型斑马鱼AB随机分为8组:对照组、模型组、丁螺环酮(阳性药,3 mg·L^(-1))组和胎盘冻干粉酶解、水提、50%醇提、醇提、二氯甲烷提(均为5mg·L^(-1))组,每组12条。除对照组外,各组以利血平处理20 min造模,而后更换相应含药养鱼用水,至14 d,每天换水。14 d时用Zebrabox斑马鱼行为分析仪对各组斑马鱼进行新型水槽测试,分析其行为变化。收集鱼脑,实时荧光定量PCR(qRT-PCR)法检测抑郁、神经调控相关基因(bdnf、pomc、hcrt、mao、nf-κb、pparγ)的mRNA表达;试剂盒法检测氧化应激相关超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性。结果与对照组比较,模型组斑马鱼进入新型水槽上部次数显著减少(P<0.001),上部游动距离及时间比例均显著减少(P<0.001);与模型组比较,丁螺环酮、胎盘冻干粉酶解物组斑马鱼进入上部次数显著增加(P<0.05),上部游动距离及时间比例均显著增加(P<0.05)。与对照组比较,模型组中的bdnf、pomc、hcrt和mao基因表达显著上调(P<0.01、0.001),nf-κb和pparγ基因表达显著下调(P<0.05、0.01);与模型组比较,经不同提取方法胎盘冻干粉处理后bdnf、pomc和mao基因表达显著下调(P<0.05、0.01、0.001),酶解、水提、醇提组hcrt mRNA水平显著降低(P<0.05、0.001),酶解、水提组nf-κb mRNA水平和酶解组pparγmRNA水平显著上调(P<0.01)。与对照组比较,模型组CAT活性显著升高(P<0.001),SOD活性升高,但没有统计学意义;与模型组比较,经不同提取方法胎盘冻干粉处理后CAT活性显著降低(P<0.001),SOD活性有不同程度的降低。结论人源胎盘冻干粉具有抗利血平诱发斑马鱼抑郁症的活性,且胎盘冻干粉酶解抗抑郁症活性最好,作用机制可能与改善氧化应激状态有关。

Objective To determine whether human placenta freeze-dried powder has the activity of anti-depressant in zebrafish depression model induced by reserpine, as well as compare the anti-depressant activity of dried human placenta extracted by different methods and explore the underlying mechanism. Methods The 6-month-old wild-type zebrafish AB were divided into eight groups including blank control group, model group, buspirone(positive control, 3 mg·L^(-1))group, dried human placenta enzymatic hydrolysate, water extract, alcohol extract, 50% alcohol water extract and dichloroethane extract(5 mg·L^(-1))group, with 12 fish in each group. Among them, the model group was treated with reserpine for 20 min, and the other groups were subsequently treated with samples for 14 days. The medium was changed every day. After 14 days treatment, the novel tank assay was conducted in adult zebrafish to analyze the behavior changes of each group. The zebrafish brains were collected after behavior assays, which were used to test the expression of depression and neuromodulation related genes(bdnf、 pomc、 hcrt、 mao、 nf-κb、 pparγ) by real-time fluorescence quantitative PCR(qRT-PCR) method. In addition, the activities of anti-oxidative enzymes superoxide dismutase(SOD)and catalase(CAT) were detected by kits. Results Compared with control group, the number of zebrafish entering the upper part of the new tank was significantly reduced(P < 0.001), and the proportion of swimming distance and time in the upper part was significantly decreased(P < 0.001) in model group. Compared with model group, the number of zebrafish entering the upper part in buspiroone and placenta lyophilized powder enzymolysis groups was significantly increased(P < 0.05), and the proportion of upper swimming distance and time was significantly increased(P < 0.05). Compared with control group, bdnf, pomc, hcrt and mao gene expressions were significantly up-regulated(P < 0.01, 0.001), while nf-κb and pparγ gene expressions were significantly decreased(P < 0.05, 0.01) in model group. Compared with model group, the expressions of bdnf, pomc and mao genes were significantly down-regulated(P < 0.05, 0.01, 0.001) after human placenta freeze-dried powder treatment with different extraction methods, and hcrt mRNA levels were significantly decreased in hydrolysate, water extract and alcohol extract groups(P < 0.05, 0.001). The mRNA levels of nf-κb and pparγ of hydrolysate and water extract were significantly up-regulated(P < 0.01). Compared with the control group, CAT activity in model group was significantly increased with statistical significance(P < 0.001), while SOD activity was increased with no statistical significance. Compared with model group, CAT activity decreased significantly(P < 0.001) and SOD activity decreased to different degrees after human placenta freeze-dried powder treatment with different extraction methods.Conclusion human placenta freeze-dried powder had the activity against depression induced by reserpine in zebrafish, among which the enzymatic hydrolysate of dried human placenta has the best anti-depressant activity. Mechanism investigation suggested that antidepressant activity may be related to the anti-oxidative action.