Biochemical Mechanism Studies Of Venlafaxine By Metabonomic Method In Rat Model Of Depression

OBJECTIVESUsing metabonomics method to study the biochemical mechanism of Venlafaxine(VEN)through determining the metabolism of serotonin (5-HT),norepinephrine(NE)and dopamine(DA)in brain tissues and plasma of rat model of depression,and to try to find out the possible mechanism of pharmacological action by analyzing the change rule in vivo of metabonome.METHODS1.The establishment and evaluation of chronic unpredicatable mild stress(CUMS)depression modelSixty male adult Sprague-Dawley rats with similar Open-field rating were equally divided in to a normal control group,a model group,high, middle,and low dose venlafaxine treated groups and a fluoxetine treated group by randomized block design,with 10 rats in each group.Normal control group rats were raised in two cages(5 each cage)and not given any stress.Isolated-living condition and chronic mild unpredictable stress are applied successively to set up depression rat model.Then,they were assessed by Open-field test and sweet water consumption test.2.Dose administration and sample collectionAfter exposed to an unpredictable sequence of mild stressors for 21 days,normal control group rats were continued to be raised commonly, and rats of other groups were still in isolated-living condition.GroupⅠandⅡbegan to be intragastrically administered 2 mL saline;GroupⅢ,Ⅳ,Ⅴ,Ⅵwere treated with low,middle and high dose VEN(8,16 and 32 mg·kg^-1),fluoxetine(FXT,2mg·kg^-1),respectively.On the morning of the 15^thday,all rats were decollated simultaneously and the whole brain was separated out quickly on ice,and then thrown into liquid nitrogen immediately.All samples were frozen at -80℃until biochemical analysis.3.Sample analysis3.1 5-HT,NE and DA were determined in whole brain by LC-MS/MS.3.2 5-HIAA,MHPG,DOPAC and HVA were determined in whole brain by LC-MS/MS.RESULTS1.Sample analysis Determination of 5-HT,NE and DA in whole brainThe calibration curves were linear in the ranges of 4.45～4450 ng·mL^-1for 5-HT,4.11～4110 ng·mL^-1for NE,4.10～4100 ng·mL^-1for DA (r≥0.999)and their limits of quantitation were 4.45,4.11 and 4.10 ng·mL^-1,respectively.The recoveries were in the range of 80.17%～100.02%,70.00%～83.44%and 80.12%～86.30%,respectively. The intra-day and inter-day RSD were all less than 4.66%.Determination of 5-HIAA,MHPG,DOPAC and HVA in whole brainThe calibration curves were all linear in the ranges of 12.5～1000 ng·mL^-1(r≥0.998)and the limit of quantitation was 12.5 ng·mL^-1.The recoveries were in the range of 90.98%～95.71%,93.93%～106.81%, 95.13%～108.36%and 86.62%～97.42%,respectively.The intra-day and inter-day RSD were all less than 8.45%.2.Effect of Venlafaxine on neurotransmitters in rat brainCompared to model group(groupⅡ),no significant difference was found in the content of neurotransmitters and ratio in the low dose venlafaxine treated group(groupⅢ).In the middle dose venlafaxine treated group(groupⅣ),the content of 5-HT and NE were significantly increased(P＜0.01);the content of 5-HIAA was significantly decreased (P＜0.05);the content of DA was increased and the content of MHPG, DOPAC and HVA were decreased,but there were no significant difference;the ratio of 5-HIAA/5-HT and MHPG/NE were significantly decreased(P＜0.01);There were no significant difference in the ratio of (DOPAC+HVA)/DA and NE/5-HT,but decreasing trends were found. In the high dose venlafaxine treated group(groupⅤ),the content of 5-HT and 5-HIAA were significantly increased(P＜0.05);the content of NE was increased and the content of DA,MHPG,DOPAC and HVA were decreased,but there were no significant difference;The ratio of MHPG/NE was significantly decreased(P＜0.01)and the decreasing trends in the ratio of 5-HIAA/5-HT and NE/5-HT were found.In the fluoxetine treated group(groupⅥ),no significant difference was found in the content of neurotransmitters and ratio except the ratio of 5-HIAA/5-HT significantly decreased(P＜0.01).CONCLUSIONS1.With previous intragastric administration of VEN for 14 days,the middle and high dose of VEN could apparently increase the contents of 5-HT and NE in the brain of depressed rats,meanwhile,could make the content of DA have a certain degree rising.The changes of neurotransmitters made by VEN are more notable than FXT.2.VEN could adjust the relationship of monoamine neurotransmitters in many ways.It could increase the contents of 5-HT and DA by reducing the metabolic rate and correct the disord of transmitters' metabolism.VEN could coordinate the balance of 5-HT,NE and DA to reach the anti-depressied function.