柴胡疏肝散中色氨酸-2,3-双加氧酶抑制剂的筛选及抗抑郁药效研究

Screening of tryptophan-2,3-dioxygenase inhibitors in Chaihu Shugan Powder and its antidepressant efficacy

目的筛选柴胡疏肝散中抑制色氨酸-2,3-双加氧酶(tryptophan-2,3-dioxygenase,TDO)表达的活性组分,探寻其抗抑郁药效作用。方法双荧光素酶报告基因法检测化合物对TDO表达的影响;分子对接虚拟评价化合物与TDO的结合能力,TDO过表达人肝癌HepG2细胞株检测其抑制活性;制备柴胡疏肝散各单味药的含药血清,验证抑制TDO的显效药味;将吲哚胺-2,3-双加氧酶(indoleamine-2,3-dioxygenase,IDO)过表达慢病毒感染人源结肠癌Caco-2细胞,检测活性组分的IDO抑制活性。采用三联一复合应激法构建抑郁样小鼠模型,通过行为学观察、LC-MS/MS检测血浆中犬尿氨酸含量、Western blotting和qRT-PCR检测TDO、IDO表达等技术评价活性组分的抗抑郁作用。结果双荧光素酶报告基因法筛选出芍药苷、橙皮苷、辛弗林、刺芒柄花苷均可抑制TDO表达(P<0.05、0.01);分子对接发现上述4种化合物对TDO有亲和性;过表达细胞株证实芍药苷可抑制TDO和IDO的表达(P<0.01、0.001),刺芒柄花苷选择性抑制TDO表达(P<0.001);抑郁样小鼠在给予芍药苷、刺芒柄花苷或两药联用后,糖水偏嗜显著升高(P<0.001),悬尾不动时间明显减少(P<0.001),游泳不动时间明显减少(P<0.05、0.001),自主活动显著增多,TDO表达明显降低(P<0.05、0.01),血中犬尿氨酸含量显著减少(P<0.05)。结论芍药苷和刺芒柄花苷均可通过抑制TDO表达发挥抗抑郁作用。

Objective To screen the active components that inhibit the expression of tryptophan-2,3-dioxygenase(TDO)in Chaihu Shugan Powder(柴胡疏肝散),and explore its anti-depressant effects.Methods Dual-luciferase reporter gene assay was used to detect the effect of compounds on TDO expression.The binding ability of the compound to TDO was evaluated by molecular docking,and the inhibitory activity of TDO-overexpressed human hepatoma HepG2 cell line was detected.The serum containing Chaihu Shugan Powder was prepared to verify its effective inhibition on TDO.Human colon cancer Caco-2 cells were infected by indoleamine-2,3-dioxygenase(IDO)overexpression of lentivirus to detect the IDO inhibitory activity of the active components.A depression-like mouse model was constructed by triple-combination stress method.Behavioral observation,LC-MS/MS detection of plasma canine urine content,Western blotting and qRT-PCR were used to detect the expression of TDO and IDO to evaluate the antidepressant effect of the active components.Results Paeoniflorin,hesperidin,synephrine and formononetin could inhibit the expression of TDO by dualluciferase reporter gene method(P<0.05,0.01).Molecular docking showed that the four compounds had affinity to TDO.The overexpressed cell lines showed that paeoniflorin could inhibit the expression of TDO and IDO(P<0.01,0.001),and formononetin selectively inhibited the expression of TDO(P<0.001).After the treatment of paeoniflorin,formononetin or the combination of the two drugs,the sugar water bias of the mice was significantly increased(P<0.001),the tail suspension immobility time was significantly decreased(P<0.001),and the swimming immobility time was significantly decreased(P<0.05,0.001),the autonomic activity was significantly increased,the expression of TDO was significantly decreased(P<0.05,0.01),and the content of kynurenine in blood was significantly decreased(P<0.05).Conclusion Both paeoniflorin and formononetin can play an antidepressant role by inhibiting the expression of TDO.