| Depression,a life-threatening neurodegenerative disease,is expected to become the second leading cause of the total burden of disability by 2020.Although the scientists and clinicians have made great efforts in the treatment of depression,there are still many unsolved problems in the diagnosis method,etiology,pathological mechanism,therapeutic drugs and prognosis of it.In addition,studies have shown that cognitive disorders can worsen this disease and lead to a poor prognosis.Therefore,there is an urgent need for new antidepressants.Drugs that improve learning and memory may work better.Recent studies have proven the natural products bearing great potential for the discovery of new antidepressants.Cajaninstilbene acid(CSA),an bioactive stilbenes from Cajanus cajan,has pharmacological effects of anti-inflammatory,antioxidant and enhancing neuroplasticity.It has been reported that CSA not only alleviated depressive symptoms in mice with chronic unpredictable mild stress(CUMS)but also improved the cognitive impairment caused by Aβ1-42.Therefore,we speculate that CSA may be a candidate drug for the treatment of depression.In this study,the model of lipopolysaccharide(LPS),CUMS and ovariectomy(OVX)were used to further study the pharmacological effects of CSA in antidepressant and cognitive impairment improvement,and its possible pharmacological mechanism,providing scientific basis for the study of CSA on new antidepressant and cognitive-improvement drugs.1.Studies on the effects of CSA on the depression-like behavior induced by LPS and the pharmacological mechanismFirstly,four groups of mice were injected intraperitoneally with different doses of LPS(0,0.32,0.8,and 2 mg/kg)to establish a reliable LPS-induced depression model.The sucrose preference test(SPT),tail suspension test(TST)and open field test(OFT)were conducted to evaluate the depressive-like behavior induced by LPS.The neuroplasticity was assessed by the levels of related proteins,TrkB and PSD-95,and by the quantification of neurons using Nissl staining.The levels of the two metabolites of the kynurenine(KYN)pathway,3-hydroxykynurenine(3-HK)and kynurenic acid(KYNA),in the brain were analyzed by LC-MS/MS.The mRNA levles of TNFα,IL-1β,IL-10 and TGFβ were determined by real-time fluorescence quantification-polymerase chain reaction(RT-qPCR).Activation of microglia and astrocytes in the brain were also determined by immunohistochemistry(IHC)and western blot(WB).respectively.The results showed that,compared with the control group,the sucrose preference index of mice in three LPS-treated groups(0.32,0.8 and 2 mg/kg)was significantly reduced.And the locomotor activity of mice in these three LPS-treated groups decreased to different extent,but with no significant difference.The mice in the 0.8 mg/kg LPS-treated group exhibited a remarkable increase of immobility time in the TST.After a preliminary study of mechanism,it was found that for the mice in the 0.8 mg/kg LPS-treated group,their cortex and hippocampus exhibited a lighter Nissl staining and arranged the neurons in a loose manner.The contents of PSD-95 and TrkB proteins were significantly reduced,the content of 3-HK was significantly increased,and microglia was significantly activated.However,compared with the control group,the other two LPS-treated groups did not perform significant difference in the indicators mentioned above.In addition,we found that the content of KYNA in the mice of 2 mg/kg LPS-treated group was significantly increased.Moreover,the mRNA levels of IL-1β,TNFα,and TGFβ,as well as the protein expression levels of GFAP in the three dose groups were dose-dependent.Based on the results above,we speculated that LPS may change the metabolic level of KYN pathway by regulating the homeostasis of different glial cells,and ultimately affect the neuroplasticity of the central nervous system(CNS).In this study,it was finally determined that the dose of 0.8 mg/kg was proper to establish the LPS-induced depression model.Secondly,the antidepressant effect of CSA(7.5.15 and 30 mg/kg)on LPS-induced depressive mice was evaluated by SPT,TST and OFT tests.The quantification of neurons was observed by Nissl staining.The levels of neuroplasticity related proteins(PSD-95,AP2,BDNF and TrkB),inflammation related proteins(p-IκB/IκB,p-p65/p65 and GFAP)and autophagy related proteins(p-mTOR/mTOR,p-ULK1/ULK1,LC3Ⅱ/LC3Ⅰ,p62 and Beclinl)were determined by WB test.The contents of Trp and its metabolites(5-HT,KYN,3-HK and KYNA)were determined by LC-MS/MS method.The mRNA levels of TNα,IL-1β,TGFβ,IL-10,IDO and TLR4 were determined by RT-qPCR.The results showed that CSA had no significant effect on the sucrose preference index of LPS-induced depressive mice at three doses.While the immobility time of LPS-induced depressive mice could be significantly shorten by CSA(7.5 and 15 mg/kg).The proteins and related metabolites in the cortex of mice in the control,model,and CSA(15 mg/kg)groups were then detected.It was found that,compared with LPS model group,the neurons of CSA-treated mice became tighter alignment.Protein expressions of PSD-95,AP2,BDNF and TrkB increased significantly,while the activation of astrocytes was significantly suppressed.Futhermore,TLR4/NF-κB signalling pathways,mRNA levels of TNFa,IL-1β,TGFβ,IL-10 and IDO were down-regulated remarkably.The content of 5-HT increased,while the content of KYN and 3-HK reduced.The protein expressions of Beclinl and LC3Ⅱ/LC3Ⅰ in the autophagy pathway were up-regulated,the phosphorylation of mTOR and ULK1 was down-regulated,and the protein expression of p62 was significantly down-regulated.Based on the results above,we believe that CSA plays an antidepressant role in the LPS model,possibly by enhancing neuroplasticity,balancing Trp homeostasis,reducing neuroinflammation and enhancing autophagy.2.Studies on the effects of CSA on the depression induced by CUMS and the pharmacological mechanismBalb/c mice were given CUMS stimulation with daily oral administration of CSA(7.5,15,and 30 mg/kg).After 6 weeks,the depression behaviors of the mice were evaluated by SPT,TST,forced swimming test(FST)and novelty suppressed feeding test(NSF).The quantification of neurons was observed by Nissl staining.The levels of neuroplasticity related proteins(PSD-95,AP2,BDNF and TrkB),inflammation related proteins(p-IκB/IκB,p-p65/p65 and GFAP)and autophagy related proteins(p-mTOR/mTOR,p-ULK1/ULK1,LC3Ⅱ/LC3Ⅰ,p62 and Beclinl)were determined by WB test.Activation of microglia in the cortex were determined by immunohistochemistry.The contents of Trp and its metabolites(5-HT,KYN,3-HK and KYNA)were determined by LC-MS/MS method.The mRNA levels of TNFα,IL-1β,TGFβ,IL-10,IDO and TLR4 were determined by RT-qPCR.The metabolomic profile of mice serum was analyzed by UPLC-QTOF-MS method.The results showed that the sucrose preference index of CUMS model mice was significantly increased by CSA(15 and 30 mg/kg),the immobility time in TST was significantly reduced by CSA(7.5 and 15 mg/kg),the the immobility time in FST and the feeding latency in NSF were significantly shorten by CSA(15 mg/kg),During some certain periods in the TST and FST,the CSA group exhibited a shorter immobility time and a more stable change trend than the amitriptyline(10 mg/kg)group did.The proteins and related metabolites in the cortex of mice in the control,CUMS model,and CSA(15 mg/kg)groups were then detected.It was found that,compared with CUMS model group,the neurons of CSA-treated mice became less damaged.Protein expressions of PSD-95,AP2,BDNF and TrkB increased significantly,while the activation of microglia and astrocytes were significantly suppressed.Futhermore,TLR4/NF-κB signalling pathway,mRNA levels of TNFα,TGFβ,IL-10 and IDO were down-regulated remarkably.The content of 5-HT,Trp and KYNA/3-HK increased,while the content of KYN reduced.The protein expressions of Beclinl and LC3Ⅱ/LC3Ⅰ in the autophagy pathway were up-regulated,the phosphorylation of mTOR and ULK1 was down-regulated,and the protein expression of p62 was significantly down-regulated.According to the results of metabonomics,CSA can significantly reverse the changes in serum metabolite profile induced by CUMS.Sixteen biomarkers were identified,including norepinephrine,dopamine,dihydrogen progesterone,alanine,xanthine,epoxyeicosatrienoic acids,taurine,hypotaurine,galactose,glucocyamine,etc.Moreover,neurotransmitter metabolism,arachidonic acid metabolism,taurine and hypotaurine metabolism,galactose metabolism,energy metabolism and fatty acid metabolism,etc.were involved.These altered metabolites were significantly associated with postpartum depression,dopamine hydroxylase deficiency,gaba-aminotransferase deficiency,xanthinuria and other diseases.Based on the results above,we believe that CSA may play an antidepressant role in CUMS model by enhancing neuroplasticity,balancing Trp metabolic homeostasis,reducing neuroinflammation,enhancing autophagy,and reversing the metabolic pathways such as neurotransmitter metabolism and energy metabolism.3.Studies on the effects of CSA on the cognitive imparement induced by OVX and the pharmacological mechanismFirstly,in order to establish an OVX-induced model of cognitive impairment,the changes in the cognitive function of mice at different time points after OVX were investigated.The female ICR mice underwent OVX surgery under anesthesia.At 2,4 and 8 weeks after surgery.The novel object recognition(NOR)and Morris water maze(MWM)were used to evaluate the cognitive ability of the mice.The levels of neuroplasticity related proteins(BDNF and TrkB)and autophagy related proteins(ULK1 and LC3Ⅱ/LC3Ⅰ)in hippocampus were determined by WB test.The results showed that the recognition index and performance of spatial reference memory were not significantly influenced at 2 and 4 weeks after OVX,while the recognition index was significantly reduced and spatial working memory was impaired at 8 weeks after OVX.It was found that the BDNF/TrkB signaling pathway was significantly down-regulated at 4 and 8 weeks after OVX,while the levels of autophagy-related proteins were reduced at 8 weeks after OVX.Therefore,we believe that OVX modeling for 8 weeks can induce the cognitive impairment of mice.Secondly,the mice were given CSA(7.5,15 and 30 mg/kg)orally every day after OVX surgery.The effects of CSA on cognitive impairment in OVX model mice were studied by NOR and MWM tests at 8 weeks after OVX.The levels of neuroplasticity related proteins(PSD-95,AP2,BDNF and TrkB),estrogen receptor α(ERα),inflammation related proteins(p-IκB/IκB,p-p65/p65 and GFAP)and autophagy related proteins(p-mTOR/mTOR,p-ULK1/ULK1,LC3Ⅱ/LC3Ⅰ,p62 and Beclinl)in hippocampus of mice were determined by WB method.The contents of Trp and its metabolites(5-HT,KYN,3-HK and KYNA)were determined by LC-MS/MS method.The mRNA levels of TNFα,IL-1,TGFβ,IL-10,IDO and TLR4 were determined by RT-qPCR.The results showed that the recognition index of OVX model mice was significantly improved and the latency to find the platform in MWM working memory test was significantly shortened by CSA administration.The proteins and related metabolites in the hippocampus of mice in the control,OVX model,and CSA(15 mg/kg)groups were then detected.It was found that protein expressions of PSD-95,AP2,BDNF and TrkB increased significantly,while the activation astrocytes were significantly suppressed.The protein expression of ERa was upregulated by CSA.Futhermore,TLR4/NF-κB signalling pathway,mRNA levels of TNFα,IL-10 and IDO were down-regulated remarkably.The content of 5-HT increased,while the content of KYN and its metabolites(3-HK and KYNA),the ratio of 3-HK to KYN reduced.The protein expressions of Beclinl and LC3Ⅱ/LC3Ⅰ in the autophagy pathway were up-regulated,the phosphorylation of mTOR and ULK1 was down-regulated,and the protein expression of p62 was significantly down-regulated.In conclusion,we believe that CSA plays a role in improving cognitive impairment in the OVX model,possibly by enhancing neuroplasticity,balancing Trp homeostasis,reducing neuroinflammation and enhancing autophagy.In conclusion,CSA exerts antidepressant and cognitive-improvement effects on depressive mice induced by LPS and CUMS and cognitive-impaired mice induced by OVX respectively.Its mechanism are related to enhancing neuroplasticity,homeostasis of Trp metabolism,reducing neuroinflammation,enhancing the level of autophagy and increasing the expression of ERα.Metabolic pathways such as neurotransmitter metabolism,energy metabolism and hormone synthesis are involved.Our study provides a basis for further new drug research of CSA. |
