Evaluation Of Novel Compounds On Depression In Mice

Depression is a common disease in clinic, patients suffered from any kinds of depression were about 11% all over the word. Depression was becoming the major cause to suicide. To develop new antidepressants in our own intellectual property rights, we investigated in three sections:①Evaluate the antidepressant activity of synthesized new 5-HT and NE reuptake inhibitors both in vivo and in vitro. Professor Yang Guangzhong from the Chinese Academy of Medical Sciences, Institute of Materia Medica analyzed the structures of 22 different types of 5-hydroxytryptamine (5-HT) reuptake inhibitors and 19 different types of norepinephrine (NE) reuptake inhibitors, used computer-aided design technology to build pharmacophore models, designed and synthesized 225 compounds belonged to 3 types of new structures. All compounds were evaluated the inhibition effect on [3H]-5-HT and [3H]-NE reuptake in rat brain synaptosomes at 10-5mol/L, and the active compounds were evaluated the inhibition effect on monoamine oxidase, and the IC50 of inhibition of [3H]-5-HT and[3H]-NE reuptake. We selected 17 compounds which showed better activity in vitro, used the tail suspension test and forced swimming test in mice to evaluate their antidepressant activity, and used locomotor activity test to identify whether they have stimulant effect on central nervous system. In vitro, among 225 new compounds,35 compounds significantly inhibited 5-HT reuptake activity,18 compounds significantly inhibited NE reuptake activity and 5 compounds significantly inhibited MAO activity; among 17 compounds evaluated in vivo,5 compounds YQF0114, YYJ0446, YQF0237, YYJ0318 and YWH05011 significantly reduced the immobility time in the mouse tail suspension or forced swimming test, and had no increases in mouse locomotor activity. It suggested that five compounds had antidepressant activity, and YQF0237 which had the most effectively antidepressant activity was potential to develop a new antidepressant.②Investigate the antidepressant effect of Cynanchum auriculatum extracts and fractions. We evaluated the inhibition effect of C. auriailatum extracts and fractions (10-5 mol/L) on [3H]-5-HT reuptake. Duloxetine was regarded as a positive control. The locomotor activity test, tail suspension test and forced swimming test in mice were used to evaluate their antidepressant effect. In vitro, only C. auriculatum glycoside A (TG-A) and C. auriculatum glycoside B (TG-B) significantly inhibit [3H]-5-HT reuptake activity. In vivo, TGC, TGC-D and TGC-E (all 80 mg/kg, i.g.) significantly decreased t(?) immobility time in tail suspension test (P<0.05). TGC(80 mg/kg), TGC-D(80 mg/kg)and TGC-E(20 mg/kg)significantly decreased the immobility time in forced swimming test(P<0.05 or P<0.01). TGC, TGC-D and TGC-E didn't increase the locomotor activity in mice. It suggested that TGC, TGC-D and TGC-E had potential antidepressant effect, neither had excited effect on central nervous system at antidepressant effective doses. Antidepressant mechanism of TGC-E (mixture of TG-A and TG-B) may be correlated with serotonin reuptake inhibiting effect, while neither TGC nor TGC-D. These results provided evidences to develop new antidepressants from C. auriculatum.③Evaluate the antidepressant activity of scopolamine and explore its effective doses in mouse tail suspension test and forced swimming test, and evaluate its effect on the central nervous system in mouse locomotor activity test, or learning and memory ability in mouse step-down passive avoidance test at its antidepressant effective doses. Compared with the vehicle control group, acute administration of scopolamine (0.1～0.4 mg/kg, i.p.) significantly decreased the immobility time (P<0.01 or P<0.001) in tail suspension test, and significantly decreased the immobility time (P<0.001) in forced swimming test. Scopolamine (0.1-0.4 mg/kg, i.p.) had no effect on the step-down latencies and error times in step-down passive avoidance test. Scopolamine (0.1 and 0.2 mg/kg, i.p.) had no influence on the locomotor activity in open field test, while significantly increased the locomotor activity at dose of 0.4 mg/kg. It suggested that scopolamine produced reliable antidepressant effect at doses of 0.1 and 0.2 mg/kg, without impairment on learning and memory, and without significantly excited or inhibited effect on central nerve system. These results provided theoretical and experimental basis for further research of new antidepressant based on scopolamine or targeted on cholinergic system. At present, gastric ulcer is a commonly chronic disease, frequently occurring, and easy to relapse. Targets for the treatment of gastric ulcer were becoming more and more widely, no limited to traditional targets such as anti-acid. Exploring the antiulcer effect of drugs (such as morphine) which were used to treat diseases of central nervous system was becoming new investigation trend.Aim To verify the antiulcer activity of antidepressants, and explore the antiulcer mechanism of duloxetine in the regulation of neurotransmitter-HPA axis-immunoregulatory network.Method We used three experimental rat models (water immersion restraint stress induced gastric ulcer, indomethacin induced gastric ulcer, and reserpine induced gastric ulcer) to evaluate the anti-ulcer activity of four antidepressant drugs (duloxetine, amitriptyline, fluoxetine, and mirtazapine) with different mechanisms. We used pathological section with hematoxylin eosin (HE) staining to observe the protective effect of duloxetine on stress gastric ulcer in rats. We used high performance liquid chromatography electrochemistry detector (HPLC-ECD) to detect the concentration of monoamines and metabolite in rat hypothalamus, cortex, stomach and platelet. We used a,β-adrenergic receptor antagonist carvedilol or 5-HT synthesis inhibitor p-CPA to antagonize the NE and 5-HT system, and observed the influence to the gastric protective effect and regulation of neurotransmitters of duloxetine. We used ELISA to detect the content of hypothalamus CRF, serum ACTH and corticosterone in rat after water immersion restraint stress. We used the blood cell counter to detect the numbers of inflammatory cells in the rat whole blood, and used cytokine antibody microarray to detect the inflammatory cytokines in gastric mucosal.Result①Duloxetine, amitriptyline, fluoxetine, and mirtazapine (5,10,20 mg/kg, i.p.) had varied degree of reduction on the gastric ulcer index and scores of ulcer bleeding in three experimental rat gastric ulcer models.②With stress time lasting (from 15 min to 6 h), injury and bleeding of rat gastric mucosa were gradually increasing. In microscopic, mucosa and gland loss, inflammatory cells infiltration and crater-like ulcers were observed in stress rat gastric mucosa. Duloxetine can effectively reverse these mucosal damage induced by water immersion restraint stress 6 h, and maintain the structural integrity of gastric mucosa.③Water immersion restraint stress 6 h significantly decreased the content of NE, and significantly increased the contents of 5-HT and 5-HIAA in rat hypothalamus, while duloxetine increased the contents of NE and 5-HT, decreased the 5-HIAA content in stress rat hypothalamus.④Water immersion restraint stress 6 h did not influence the contents of NE and 5-HT, but increased the 5-HIAA level in rat cortex. Duloxetine increased the 5-HT content and decreased the 5-HIAA content, although still had no influence on NE content.⑤Stress 6 h significantly decreased the contents of 5-HT and 5-HIAA in rat stomach, and duloxetine can reverse these changes induced by stress. Stress 6 h had no influence on 5-HT content in rat platelet, and duloxetine had no influence on 5-HT content in stress rat platelet.⑥Carvedilol had weak gastric protective effect and it can not antagonize the protective effect of duloxetine. Carvedilol had no influence the contents of NE and 5-HT, but decreased the 5-HIAA content in stress rat hypothalamus and cortex, while duloxetine or combination of duloxetine and carvedilol significantly increased the contents of NE and 5-HT, decreased the 5-HIAA content in stress rat hypothalamus and cortex.⑦p-CPA had no gastric protective effect, and it can not antagonize the protective effect of duloxetine. p-CPA or combination of p-CPA and duloxetine had no influence on content of NE in stress rat hypothalamus, but decreased the NE level in stress rat cortex, and significantly decreased the contents of 5-HT and 5-HIAA in stress rat hypothalamus and cortex. While duloxetine increased the contents of NE and 5-HT, decreased the content of 5-HIAA in stress rat hypothalamus and cortex.⑧Stress 6 h significantly increased the CRF level in rat hypothalamus, duloxetine can partly reverse this increase induced by stress. Compared with nonstress rat, the serum ACTH level significantly increased in rat after stress 15 min,30 min,1 h,3 h or 6 h, and the increase rates were from 315% to 365%; the serum corticosterone level significantly decreased in rat after stress 15 min,30 min,1 h,3 h or 6 h, and the decrease rates were from 46% to 58%. Duloxetine can partly reverse this increase of serum ACTH content in rat after 6 h stress, and decreased the content of serum corticosterone.⑨Compared with vehicle control, the white blood cells, lymphocytes and intermediate cells in whole blood significantly increased after stress 6 h in rats, while the plateletcrit, mean platelet volume, platelet distribution width, hematocrit, red blood cell volume distribution width had no significant changes. Duloxetine can significantly reverse the increase of inflammatory cells caused by stress 6 h.⑩Cytokine antibody array results showed that stress 6 h significantly increased the expression of Thymus Chemokine-1, CINC-2a, CINC-3 and Fas Ligand in gastric mucosa, and duloxetine can effectively reverse the increase caused by stress. Expression of ICAM-1, L-Selectin and Prolactin R significantly reduced in stress rat, and duloxetine can effectively reverse the decrease caused by stress.Conclusion Antidepressants duloxetine, amitriptyline, fluoxetine, and mirtazapine had significantly protective effect on water immersion restraint stress induced gastric ulcer, indomethacin induced gastric ulcer, or reserpine induced gastric ulcer in rat. Duloxetine effectively reversed these mucosal damage induced by water immersion restraint stress 6 h, and maintained the structural integrity of gastric mucosa, this identified its potential gastric protective effect. Antidepressant drug duloxetine performed protective effect on stress induced gastric ulcer may through the regulation of neurotransmitter-HPA axis- immunoregulatory network. Firstly, duloxetine enhanced NE and 5-HT system through increase the contents of NE and 5-HT in rat hypothalamus and cortex, attenuation of 5-HT metabolism, reversing the decrease of contents of 5-HT and 5-HIAA in stomach, were partly involved in gastric protective mechanisms of duloxetine. Secondly, duloxetine attenuated the over-activation of HPA during stress response through the reduction of stress induced upregulation of hypothalamus CRF content and serum ACTH content. Thirdly, duloxetine lightened the local gastric inflammation by inhibiting gastric mucosa cells apoptosis through the reduction of Fas Ligand and inhibiting the recruitment of inflammatory cells through the reduction of chemotactic cytokines in the gastric mucosa, and reduced the inflammatory reaction through partly reversing the increase of inflammatory cells induced by stress. All these results provided evidences for the antiulcer activity of antidepressants and suggested some clues for the development of new antiulcer drugs.