| Background:Depression, one of the most common psychiatric diseases, exerts devastating impairment in quality of life and psychosocial function, for its high morbidity and recurrence rate. Misdiagnosis and missed diagnosis derived from ambiguous depression symptom criteria in depression scales made it significant to develop a new, reliable diagnose method founded on biochemical index. 5-HT is an important neurotransmitter in depression etiology. Its concentration in blood plasma turns out to be a promising candidate as a marker of depression, according to its solid ability of revealing depressive level. However, currently detection methods of 5-HT in blood like fluorospectrophotometry, ELISA, HPLC are limited in clinical application, for their multi-step pretreatment and long measuring time. In contrast with them, screen-printed electrode with electrochemical method contact possesses unapproachable merits such as low-cost, simple, rapid detection, which are inherited with electrochemical detect methods.Objective:To prepare a disposable 5-HT biosensor for biological samples analysis in research of depression etiology and therapeutic intervention. It will provide a new, rapid, reliable detect method for antidepressants discovery and clinical diagnosis.To explore the correlation between the concentration of 5-HT in central nerve system and in peripheral blood during the pathogenesis of depression. Therefore, provide scientific supports for 5-HT in peripheral as a biochemical biomarker of clinical depression diagnosis.Through observing the antidepressive effects of Mifepristone, Lentinan and Berberine, obtain experimental basis for the discovery of novel antidepressants and adjunct therapy, founded on new mechanisms.Method:1. The screen-printed technology combined with modified senor technology was employed in fabrication the modified Electrode for 5-HT determination, which is easy to prepare and detect, possessing high sensitivity and selectivity, applicable for determination of 5-HT in trace biological sample including serum, plastocytes enriched plasma, brain tissue.2. Build acute mouse depression model by depletion of neurotransmitter by reserpine, on which the concentration of 5-HT in blood, brain, depressive behavior in different doses of reserpine were observed and correlation between 5-HT in blood and in brain was tested.3. Chronic unpredictable mild stress depression model on rat was built, on which the correlation between blood 5-HT and behavior changes was valued and regulation of antidepressants with different mechanisms on 5-HT system, immune system, endocrine system, oxidation and BDNF was examined.Result:1. Nafion/CGSPE was made to detect 5-HT, The peak currents vary linearly with concentration of 5-HT in the condition of 0.1 M PBS (pH 7.4), in the range of 2×10-8 M-5×10-6 M, with a correlation coefficient of 0.9981. The Lowest Detectable Limit is 5.0 nM. This biosensor could resisting disturbance from DA at 30 times concentration of 5-HT, AA 80000 times and UA 10000 times, when used to detect the 5-HT in concentration of 1.0×10-7M. When the concentration of 5-HT is 0.10,0.30,1.00μM, the RSD of the detection is lower than 5% for accuracy,5% for within-run precision, 8% for between-run precision,8% for stability. The correlation coefficient of standard curve, when used for Hippocampus tissue (BRA), platelet rich plasma (PRP), serum (SER) of mice, PRP of rat and healthy population is, separately:0.9970,0.9979, 0.9985,0.9966,0.9912. The concentration of 5-HT in these samples is, separately, 0.8μg/g,5.0×10-7 M, 1.0×10-6 M,4.0×10-7 M,2.0×10-7 M. In our study, recovery of Nafion/CGSPE is close to HPLC-ECD, but is more approximated to 100%, and much higher than that of fluorospectrophotometry and ELISA.2. The level of 5-HT in control group slightly rise at 10:00a.m and 16:00p.m, with a deviation less than 0.2×10-7 M, and dropped into the bottom at 20:00 p.m. In reserpine group (0.25,0.50,1.00 mg/kg), the level of 5-HT rise during 0.25-1 h after injection, then dropped dramatically. In mice, the variation trend of 5-HT in PRP is consistent with that in Hippocampus, while delayed a little. The variation trend of 5-HT in SER is not always consistent with that in Hippocampus, which need more study.According to correlative graph of mice BRA, PRP and SER, we discovered that 5-HT in BRA is not constant related with that in SER (R<0.2), but is positive correlated with 5-HT in PRP. The correlation coefficient between 5-HT in BRA and PRP is approximated to 0.8, which indicates that 5-HT in PRP could be a biological index of depression by revealing the level of this neurotransmitter in brain.3. In Chronic unpredictable mild stress depression model, saccharine solution preference and spontaneous activity of rats in model group decreased alone with the process of CUMS in 1-4 weeks, while control group is unchanged (P>0.05). In 5-7 weeks, when the drugs were administrated, saccharine solution preference and spontaneous activity of each group recovered rapidly. Mifepristone has the best curative effect on saccharine solution preference, while Fluoxetine can return the spontaneous activity approximated to normal level. Rhizoma Coptidis water extract (BER) reelevated the spontaneous activity onto a higher level than control group (P< 0.001).PRP 5-HT decreased progressively in the model, while Mifepristone and BER lift it quickly, Fluoxetine a week delayed, and Lentinan (LNT) mildly and dose depended elevated it. Fluoxetine, Mifepristone, LNT and BER can elevate the hippacampus 5-HT, LNT was dose depended.The correlation coefficient in CUMS rats between 5-HT in PRP and saccharine solution preference is approximated to 0.65, between 5-HT in PRP and spontaneous activity is 0.76. Thus correlation between PRP 5-HT and spontaneous activity is more evident.During CUMS, changes of depressed rats including weight increased less than normal rats, spleen and paranephros enlarged, latentperiod of food intake prolonged, plastocytes aggregation fraction increased, TNF-a and IL-6 level in serum rise, cortisol in plasma elevated, activity of GSH-xp, SOD decreased, expression of BDNF down relagulated. Fluoxetine could treat almost all symptoms of depression down regulate cortisol to normal level, higher the activity of GSH-xp and SOD, up regulate the expression of BDNF and the deficiency is a relative long initial time for 3 week. Mifepristone relieved the symptoms significant and immediately, improved the decreased amplification of weight (P< 0.001), but has no effect on BDNF expression. LNT (10 mg/kg) deter the enlargement of paranephros (P<0.05), and higher the activity of SOD (P<0.01). LNT (20 mg/kg) decreased the spleen index. LNT (10,20 mg/kg) effect on weight gain is better than Mifepristone. LNT (10,20 mg/kg) shorten the latentperiod of food intake, against the aggregation of plastocytes, decreased the level of TNF-a and IL-6, all of these effects are dose-depended. BER (10,20,100 mg/kg) dose depended decreased amplification of weight, shorten the latentperiod of food intake, against the aggregation of plastocytes, higher the activity of GSH-xp, reduce the MAO activity, up regulate the expression of BDNF. BDNF expression in BER (100 mg/kg) group is almost the same as the control group.Conclusion:The nafion/aerosol modified 5-HT screen printing electrodes fabricated in this study are capable in 5-HT detection in biological sample, could be used to antidepressant screening and clinical diagnosis. PRP 5-HT has evident correlation with BRA 5-HT, could indicate the depressive level. According to treatment of the clinical antidepressant, PRP 5-HT shows great potential to be a biological marker of curative effect for Fluoxetine and Mifepristone. Whether it can be applied to Lentinan remains unclear. Variance of PRP 5-HT might be a result of neurotrophic effect of antidepressants. Thus, PRP 5-HT, qualified itself as a potential clinical biological marker, with its tight relationship with endocrine and immune system as well as nervous system. |
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