| Major depressive disorder is currently the most common mental illness.It is becoming more and more common in modern society,which in turn brings a heavy burden to society and families.Although monoamine reuptake inhibitor antidepressants have been used for more than 50 years,they have disadvantages such as slow onset,poor patient compliance,and easy to relapse after stopping the drug,which is very dangerous for patients with suicidal tendencies.In recent years,rapid antidepressant has been widely concerned.With ketamine as the representative,a single administration of ketamine produces an antidepressant effect after 2 hr,and this effect can last about 1 week.However,the extensive clinical application was limited by its addictive and neurotoxic.Chinese medicine has a significant advantage in the treatment of depression.As a classic formulation,Yueju Pills have been proved to have significant antidepressant characteristics,the results of previous studies have shown that they are dependent on pituitary adenylate cyclase activating peptide(PACAP)Activation of signal pathways.However,the unique intracellular signal transduction of Yueju Pills,the specific signal mechanism of its antidepressant effects,and the antidepressant efficacy of effective compounds are all worth of further investigating.Aims1.To clarify the effect of Yueju Pill depends on the activation of PACAP in the hippocampus to produce rapid antidepressant;2.To clarify that Yueju Pills produce rapid antidepressant effects on the regulation of CaMKII signal by PACAP in the hippocampus;3.To clarify that the active ingredients in Yueju Pills(Geniposide and Shanzhiside methyl ester)produce rapid antidepressant effects and the neural mechanism of rapid antidepressant effects.Methods1.Using chronic unpredictable mild stress(CMS)model combined with behavior tests and Western blot detection to detective whether Yueju Pills depend on the activation of PACAP in the hippocampus to produce rapid antidepressant effects,and further detective the downstream PACAP related to its antidepressant effects Signal CaMKⅡ;2.Using RNAi virus and microinjection of PACAP antagonist in brain area to specifically knock out or block the activity of PACAP in the hippocampus,and to further prove the relationship between the rapid antidepressant effect of Yueju Pill and PACAP and its down-stream CaMKⅡ;3.Using opto-genetic,behavioral and Western blot techniques to detective whether the rich components in Yueju pills(Geniposide and Shanzhiside methyl ester)have coordinated and rapid antidepressant effects and whether the antidepressant mechanism of them is tsimilar to Yueju pills;4.Using Elisa technology to detect the expression of PACAP in the serum of patients with MDD,to further investigate the correlation between the changes of PACAP and the occurrence and development of depression.Results1.In chronic mild stress(CMS)mice and single administration of Yueju pills,the relationship between hippocampal PACAP levels and downstream signals and depression and antidepression was evaluated.In the tail suspension test(TST),forced swimming test(FST)and sucrose preference test(SPT),mice showed depression-like responses.A single administration of Yueju pills can reverse these behavioral defects(30 minutes,2 hours,4 hours and 2 days measurement).In the hippocampus of CMS mice,the expression of PACAP was significantly reduced,and it was reversed by a single dose of Yueju Pill 2 hours or 2 days after the occurrence of a single dose of Yueju Pill.CMS reduced the expression of PACAP,PKA,pCREB/CREB,PSD95 and pCaMKⅡ/CaMKⅡ,and up-regulated the expression of peEF2/eEF2,which was reversed 2 hours or 2 days after single administration of Yueju Pill;2.Microinjection of PACAP antagonist into the hippocampus weakened the rapid antidepressant activity of Yueju Pills.In addition,the deliberate knockout of PACAP in the hippocampus also blocked the rapid antidepressant effect of Yueju Pills.Yueju pills increased the expression of PACAP,PKA,pCREB/CREB,and decreased eEF2 and CaMKⅡ signals at 30 minutes.Without affecting PKA levels,PACAP gene knockout down-regulated the expression levels of PACAP,pCREB/CREB and CaMKⅡ signals,but up-regulated eEF2 signals.Yueju pills for PACAP gene knockout mice can significantly increase PACAP and PKA,at the same time increase the expression of pCREB/CREB,and reduce eEF2 signal,but compared with Yueju pills,the effect on blank virus control mice was reduced.In contrast,Yuejuwan’s regulation of CaMKⅡ signal was reversed by knockout,but unlike the downregulation of blank virus control mice,it was upregulated in knockout mice on calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)signaling,which was initially inhibited;3.We tested the effects of geniposide(44 mg/kg;G)and shanzhiside methyl ester(4.7 mg/kg,S)alone or in combination on the antidepressant response and its relationship with hippocampal PACAP signal.The dosage of Yueju pills is proportional.In the combined group(G+S),the immobility time of TST was significantly reduced within 30 minutes,and this effect lasted for 3 days,and there was no effect on every behavior alone.In NSF,after a single combination therapy for 2 days,the food ingestion latency was significantly reduced,and food consumption increased 24 hours after administration.Antidepressant effects were found in FST,but no longer worked after 5 days of administration.AAV-mediated eNPHR3.0 or control vector was used to evaluate the optogenetic inhibition of bilateral hippocampal PACAPergic neurons in PACAP knock-in mice.15 minutes after the administration,the neurons expressing PACAP were photo-inhibited.TST was performed during optogenetic suppression,and the antidepressant effect of the combination group(G+S)was weakened due to suppression.After 1 day of photoinhibition,the antidepressant effect of the combination group(G+S)in other behavioral tests was not affected by the inhibition.At 30 minutes,the combination group(G+S)quickly activated the expression of PACAP in the hippocampus,activated pCREB/CREB,and inhibited CaMKⅡsignaling.Its effect was comparable to that of Yueju pills.However,the combination group(G+S)did not change the expression of PKA,PSD95 or PEEF2/EF2.The ERK1/2 signal is also downstream of PACAP and upstream of CREB,and is activated by the combination group(G+S)or Yueju Wan.Regarding the upstream regulator of eEF2,the combination group(G+S)and Yueju pills had a better inhibitory effect on eEF2k,while 4EBP1 was only activated by Yueju pills.In the expression of PDE4B,PDE4D and PDE4E,no significant effect was found between the combination group(G+S)and Yueju pills;4.To test the relationship between PACAP levels and major depression,we surveyed a group of subjects composed of MDD patients and age-matched healthy controls.None of the patients took any medication.In the healthy control group,the PACAP level of women was significantly higher than that of men.Compared with women’s serum PACAP levels,the patients were significantly lower than those of healthy people.But there was no significant difference between male patients and healthy people.Among women,chronic MDD patients with the disease for more’ than 2 years showed significantly lower PACAP levels than age-matched healthy controls.In addition,serum PACAP levels in female patients with chronic MDD were significantly negatively correlated with the Hamilton Depression Scale(24 items)score.Conclusion1.The rapid antidepressant effect of Yueju Pill is achieved by activating PACAP to affect downstream PKA/CREB and CaMKⅡ/eEF2;2.PACAP antagonist or knockout in the hippocampus can weaken the fast antidepressant effect of Yueju Pill,and block the effect of Yueju Pill on PACAP and its downstream CaMKⅡ and neurosynaptic-related proteins;3.The synergistic effect of geniposide and mountain gardenia methyl ester can produce fast and lasting antidepressant effect.Inhibiting PACAP neurons in the hippocampus can block the synergistic antidepressant effect and regulate fewer signal pathways than Yueju pills;4.The serum PACAP level of patients with chronic depression is reduced,and there are obvious gender differences. |
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