柴胡皂苷D靶向抑制NLRP3炎症小体调控犬尿氨酸代谢的抗抑郁作用机制

Antidepressant mechanism of saikosaponin D targeting kynurenine metabolism by inhibiting NLRP3 inflammation

目的:研究柴胡皂苷D对色氨酸-犬尿氨酸代谢途径和炎症抑制的作用及与抗抑郁作用的关联。方法:通过4周慢性温和性不可预见性应激刺激建立抑郁大鼠模型,以蔗糖摄取偏好实验和强迫游泳实验评价大鼠的抑郁状态。给药8周后,采用液相色谱串联质谱法测定各组大鼠海马组织中的色氨酸及犬尿氨酸水平;酶联免疫法测定海马组织中的白介素-1β(IL-1β),肿瘤坏死因子-α(TNF-α),白介素-6(IL-6)水平;蛋白免疫印迹技术测定自噬相关蛋白(Beclin-1,LC3Ⅱ/LC3Ⅰ的值)、炎症小体相关蛋白(nucleotide-binding oligomerization domain,leucine-rich repeat and pyrin domain-containing 3,NLRP3、Caspase-1)及色氨酸限速酶吲哚胺-2,3-双加氧酶(indoleamine-2,3-dioxygenase,IDO)的含量。结果:柴胡皂苷D可显著增加抑郁大鼠的蔗糖摄取指数(P<0.05),缩短大鼠在水中的不动时间(P<0.05),改善大鼠的抑郁行为;抑制抑郁大鼠海马组织中色氨酸-犬尿氨酸途径的异常激活,显著降低促炎症细胞因子IL-1β,IL-6的浓度和IDO的含量(P<0.05),降低NLRP3、Caspase-1的蛋白含量,增加自噬标志物Beclin-1的蛋白含量与LC3Ⅱ/LC3Ⅰ的值(P<0.05)。结论:柴胡皂苷D可能通过调节自噬参与NLRP3炎症小体的形成,进一步抑制促炎症细胞因子水平,调节色氨酸-犬尿氨酸途径,发挥抗抑郁作用。

OBJECTIVE To study the effect of saikosaponin D on tryptophan-kynurenine metabolic pathway and inhibition of inflammation,as well as its correlation with antidepressant effect.METHODS In this study,the depression rat model was established by chronic mild unpredictable stress stimulation for 4 weeks.The depression state of rats was evaluated by sucrose intake preference experiment and forced swimming experiment.After 8 weeks of administration,the tryptophan and kynurenine levels in hippocampus were determined by liquid chromatography tandem mass spectrometry.The determination of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in hippocampus used enzyme-linked immunosorbent assay,and the Western blot method was used to determine the content of autophagy related protein(Beclin-1,LC3Ⅱ/LC3Ⅰratio),inflammatory corpuscle related protein(nucleotide-binding oligomerization domain,leucine-rich repeat and pyrin domain-containing 3,NLRP3,Caspase-1)and tryptophan rate limiting enzyme indoleamine 2,3 dioxygenase(IDO).RESULTS The results showed that saikosaponin D could significantly increase the sucrose intake index of depressed rats(P<0.05)and shorten the immobility time of rats in water(P<0.05).In addition,saikosaponin D could significantly reduce the proinflammatory cytokine IL-1βand IL-6 in the hippocampus(P<0.05)and the content of tryptophan metabolizing enzyme IDO(P<0.05).The content of NLRP3 and Caspase-1 in hippocampus of depression rats could be significantly reduced by saikosaponin D,and the content of autophagic markers Beclin-1and the ratio of LC3Ⅱ/LC3Ⅰwere significantly increased(P<0.05).CONCLUSION The results showed that the antidepressant mechanism of saikosaponin D may through participating in the formation of NLRP3 inflammation by regulating autophagy,further inhibit the level of proinflammatory cytokines and regulate the tryptophan-kynurenine pathway.