| Objective:Post-traumatic stress disorder (PTSD) is a serious anxiety, which is the mental disorder that is associated with individual. The PTSD symptoms are various, including traumatic experience, avoidance behavior, and emotional numbing. PTSD is considered to be high incidence and prevalence, chronic and poor effect with great trauma care and concern.The etiology and pathogenesis of PTSD is complicated. The disorder is involved the abnormal neurotransmitters levels, the enhancement of the hypothalamic pitutary adrenal (HPA) axis in the negative feedkack inhibition and the abnormal of neural plasticity and neurosteroidogenesis. These dysregulation of the neuroendocrine pathways may result in the abnormal of brain structure and function and cause a series of symptoms, such as excessive vigilance and the intrusion, and experience (flashback).PTSD is lack of specific drugs treatment in clinic. In general, antidepressants selective serotonin reuptake inhibitors (SSRIs) are the first choice for treatment of PTSD, such as paroxetine and sertraline. But effective rate is not high (about60%). The onset latency will last from2-6weeks. Also, there are side-effects including sexual dysfunction, gastrointestinal tract, and suicide. Other anxiolyic drugs were used in the treatment of PTSD. Benzodiazepine, such as midazolam, can destroy the conditioned fear memory. But the drugs also have low efficiency. Therefore, it is important to explore the new target and treatment of PTSD.The translocator protein18KDa (TSPO) is distributed on the outer membrane of mitochondria in the central and peripheral tissue. TSPO transports the cholesterol into the mitochondrial membrane to promote the biosynthesis of neurosteroids. It had been found that TSPO is associated with mood and stress response. This target has potential value in the treatment of anxiety, depression and other stress-related psychiatric disorders. Reports have shown that TSPO is closely associated with PTSD. The TSPO ligand, such as AC-5216, has obvious antidepressant, anxiolytic and anti-panic effect in animal models without sedation, muscle relaxation tolerance and withdrawal symptoms. The mechanism may be that transport cholesterol into membrane phospholipids, which increased pregnenolone formation and its downstream neurosteroids, including progesterone and allopregnenolone. Neurosteroids enhanced the function of gamma amino-butyric acid type A receptor (GABAA) through GABAA receptors on the excitatory neurons, which inhibit the hippocampal hypothalamic pituitary adrenal (HHPA) axis function and regulation of emotion and stress response. It is indicated that TSPO can be used as a potential therapeutic target for PTSD.However, AC-5216is difficult to dissolve in water. Based on this situation, we synthesis its derivative YL-IPA08. It had been reported that YL-IPA08have higher affinity to TSPO than AC-5216and have antidepressant and anxiolytic effects by regulating neurosteroids synthesis. Also, we had applied the patent in this area. Although the anti-stress effect of AC-5216and YL-IPA08is obvious, there are few studies on anti-PTSD. It is important to clarify whether TSPO ligands have great value on anti-PTSD effect.The animal models (e.g. time-dependent sensitization (TDS) and inescapable electric foot shock, single prolonged stress (SPS), exposure to a predators, and social isolation) have been well established to resemble the clinical features of PTSD. The inescapable electric foot shock model and the TDS model were selected in the present study. The inescapable electric foot shock model based on the concept that animals exposed to foot shocks can represent pathophysiological process and core symptomatology of PTSD, including the freezing and anxiety-like behavior. Previous studies showed that animals experiencing a traumatic event, such as multiple foot shocks, rapidly form a strong phobia while a relatively mild aversive experience occurs. This phenomenon may correspond to the emergence of phobias in PTSD patients. In addition, the TDS model has proven to resemble the clinical condition with accurate face, construct, and predictive validity. Thus, both PTSD models represent important phenomenological and biological correlates of PTSD.In the present study, AC-5216and YL-IPA08were applied in inescapable electric foot shock model and TDS model to explore the anti-PTSD effect and mechanism of TSPO ligands.Methods:(1) The affinity of AC-5216and YL-IPA08to TSPO and the regulation of both the compounds on neurosteroidsWe cultured the astrocytes that were treated with AC-5216and YL-IPA08(0.05,1,2μM)2h and4h and measured pregnenolone and progesterone in culture medium by enzyme linked immunosorbent assay (ELISA). (2) The anti-PTSD effects of AC-5216and YL-IPA08in behaviral testsWe explored the anti-PTSD effects of AC-5216and YL-IPA08by inescapable footshock model and TDS model. Briefly, after a5-min adaptation period, the mice were exposed to a total of15intermittent inescapable electric foot shocks (intensity:0.8mA, interval:10s, and duration:10s) delivered through the grid floor by an isolated shock generator. Control animals were placed in the same chamber for10min in the absence of electric foot shocks. Sertraline (15mg/kg p.o.), AC-5216(0.03,0.1,0.3, and1mg/kg p.o.) and YL-IPA08(0.03,0.1,0.3,1, and3mg/kg p.o.) were given once per day from3to17d.All the animals were exposed to reminders of the situation for5min on days5,10, and17without foot shocks, to measure the duration of contextual freezing behavior. This was achieved by placing the experimental animals in the same chamber where the foot shocks were delivered. In addition, we explore the spontaneous locoactivity by open field test and the PTSD behavior by the elevated plus maze test and the staircase test.In the TDS model, each rat was placed in a perspex restrainer for2h, with the tail-gate adjusted to keep the rat well contained without impairing limb circulation. Thereafter, rats were individually placed in a clear acrylic cylinder to perform a20-min forced swim. Following the15-min recuperation, rats were then exposed to ether vapors until loss of consciousness and were then removed. The animals were undisturbed for7days. Seven days after the initial stressor, the rats were exposed to a ’restress’session consisting of a20-min swim in clear acrylic cylinders. The control group rats were remained in a room adjacent to the TDS rats for the duration of TDS and were handled twice for several minutes. Sertraline (15mg/kg p.o.), AC-5216(0.03,0.1,0.3, and1mg/kg p.o.) and YL-IPA08(0.03,0.1,0.3,1, and3mg/kg p.o.) were given once per day from the second day. We explore the spontaneous locoactivity by open field test and the PTSD behavior by the contextual fear paradigm and the elevated plus maze test.(3) The anti-PTSD mechanism of AC-5216and YL-IPA08We explored the effects of TSPO receptor antagonist PK11195in the TDS model. The prefrontal cortex and hippocampus and blood samples were removed from the TDS model to explore the anti-PTSD mechanism of AC-5216and YL-IPA08. The expression changes of TSPO in prefrontal cortex and hippocampus were measured by western blotting; the activity of TSPO in prefrontal cortex, hippocampus and platelet were measured by binding assay; the progsterone and allopregnenolone in prefrontal cortex and hippocampus and blood samples was detect by ELISA; changes of monoamine levels (DOPAC (dihydroxy-phenyl acetic acid), DA (dopamine),5-HIAA (5-hydroxyindoleacetic acid), NE (norepinephrine)) in prefrontal cortex and hippocampus were analysis by high-performance liquid chromatography with electrochemical detection.(4) Statical analysisUnless otherwise specified, statistical analysis was performed using SPSS13.0. All data were presented as the means±S.E.M. The statistical significance of experimental observations was determined by one-way analysis of variance (ANOVA). The body weight was determined by repeated measure analysis. The statical significances between two groups were analyzed by LSD t-test or Welch analysis followed by Dunnett’s T3test. For all tests, differences with P<0.05were considered significant.Results:(1) The affinity of AC-5216and YL-IPA08to TSPO and the regulation of both the compounds on neurosteroidsBinding assay showed that affinity IC50of AC-5216in TSPO and CBR of the outer mitochondrial membrane of glial cells were0.65±0.02nM and67.4±6.82nM, respectively; affinity IC50of YL-IPA08in TSPO and CBR of the outer mitochondrial membrane of glial cells were0.23±0.04nM and706.18±10.18nM. The results indicated that AC-5216and YL-IPA08have high affinity to TSPO, but low affinity to CBR in the mitochondrial outer membrane of glial cells. We determine the concentration of pregnenolone and progesterone that treated by AC-5216(0.5,1,2μM) and YL-IPA08(0.5,1,2μM) in astrocytes in the culture by ELISA. The result showed that after2h treatment of AC-5216and YL-IPA08, pregnenolone (F=7.892, P=0.004, AC-5216; F=101.5, P=0.000, YL-IPA08) and progesterone (F=22.41, P=0.000, AC-5216; F=23.97,P=0.000, YL-IPA08) levels were significantly increased. After4h treatment of AC-5216and YL-IPA08, pregnenolone (F=98.37, P=0.000, AC-5216; F=52.91, P=0.000, YL-IPA08) and progesterone (F=37.99,P=0.000, AC-5216; F=22.16,P=0.000, YL-IPA08) levels were significantly increased. The results suggested that pregnenolone and progesterone levels were upregulated by AC-5216and YL-IPA08.(2) The anti-PTSD effects of AC-5216and YL-IPA08in behaviral testThe footshock model in mice showed that AC-5216(0.03,0.1,0.3,1mg/kg p.o.) and YL-IPA08(0.03,0.1,0.3,1,3mg/kg p.o.) does not affect the number of crossings (F=1.181, P=0.328, AC-5216; F=1.890, P=0.084, YL-IPA08), rears (F=1.025, P=0.418, AC-5216; F=0.342, P=0.932, YL-IPA08) and fecal pellets (F=0.323, P=0.922, AC-5216; F=0.277, P=0.961, YL-IPA08). It is indicated that AC-5216and YL-IPA08do not affect the locomotor activity in mice.The body weight was not affected by AC-5216(F=0.206, P=1.000) and YL-IPA08(F=0.176, P=1.000).The footshock significantly increased the freezing time, sertraline (15mg/kg p.o.) significantly reduce the freezing time, AC-5216and YL-IPA08do not reduce the freezing time on the5th day (F=2.618, P=0.025, AC-5216; F=4.592, P=0.000, YL-IPA08); on the10th day (F=6.602, P=0.000, AC-5216; F=6.456, P=0.000), YL-IPA08), sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.) and YL-IPA08(0.3,1mg/kg p.o.) significantly reduce the freezing time; on the17th day (F=11.868, P=0.000, AC-5216; F=6.904,.P=0.000, YL-IPA08), sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.) and YL-IPA08(0.1,0.3,1mg/kg p.o.) significantly reduce the freezing time. The results suggested that the fear induced by PTSD was allivated by AC-5216and YL-IPA08in mice. The elevated plus maze test showed that total time spent in (F=0.929,P=0.779, AC-5216; F=1.868, P=0.088, YL-IPA08) and the entries into (F=0.532, P=0.490, AC-5216; F=0.570, P=0.778, YL-IPA08) the arms were not different between the groups. The time spent in (F=3.513, P=0.005, AC-5216; F=2.318,P=0.035, YL-IPA08) and the entries into (F=2.462, P0.033, AC-5216; F=2.643,P=0.018, YL-IPA08) the open arms was reduced in the model group, but both of the parameters were reversed by sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.) and YL-IPA08(1mg/kg p.o.). The staircase test showed that there were no differences among the groups in steps climbing (F=0.836, P=0.547, AC-5216; F=0.827, P=0.568, YL-IPA08). The model can reduce the number of rears (F=4.504, P=0.002, AC-5216; F=2.166,P=0.047, YL-IPA08), but the reduction can be reversed by sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.) and YL-IPA08(0.3,1mg/kg p.o.). The elevated plus maze test and staircase test results suggest sertraline (15mg/kg p.o.), AC-5216(0.1,0.3,1mg/kg p.o.) and YL-IPA08(1mg/kg p.o.) has the anti-PTSD effect. The results suggested that PTSD was allivated by AC-5216and YL-IPA08in rats.The effect of AC-5216and YL-IPA08in the TDS model showed that AC-5216(0.03,0.1,0.3,1mg/kg p.o.) and YL-IPA08(0.03,0.1,0.3,1, and3mg/kg p.o.) does not affect the number of crossings (F=0.735, P=0.623, AC-5216; F=1.176, P=0.327, YL-IPA08), rears (F=0.245, P=0.959, AC-5216; F=0.514, P=0.821, YL-IPA08) and fecal pellets (F=0.388, P=0.884, AC-5216; F=0.479, P=0.847, YL-IPA08). It is indicated that AC-5216and YL-IPA08do not affect the locomotor activity in rats. AC-5216(F=0.141,P=1.000) and YL-IPA08(F=0.433,P=1.000) also do not affect the body weight in rats. Contextual fear paradigm showed that the model group significantly increased the freezing time, sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.)(F=3.913, P=0.002) and YL-IPA08(0.1,0.3mg/kg p.o.)(F=33.77, P=0.000) significantly reduce the freezing time. The results suggested that the fear induced by PTSD can be allivated by AC-5216and YL-IPA08in rats. The elevated plus maze test showed that total time spent in (F=1.281, P=0.298, AC-5216; F=1.090, P=0.379, YL-IPA08) and the entries into (F=0.216,P=0.986, AC-5216; F=1.533, P=0.171, YL-IP08) the arms were not different between the groups. The time spent in (F=2.423, P=0.036, AC-5216; F=3.491, P=0.003, YL-IPA08) and the entries into (F=2.378, P=0.039, AC-5216; F=3.916,P=0.001, YL-IPA08) the open arms was reduced in the model group, but both of the parameters were reversed by sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.) and YL-IPA08(0.1,0.3mg/kg p.o.). The results suggested that the PTSD behaviors were allivated by AC-5216and YL-IPA08in rats.(3) The anti-PTSD mechanism of AC-5216and YL-IPA08The effect of AC-5216and YL-IPA08was antagonized by PK11195in the on the time dependent sensitization model. The results showed that PK11195(1,3mg/kg i.p.) does not affect the number of crossings (F=0.352, P=0.954, AC-5216; F=0.263, P=0.982, YL-IPA08), rears (F=0.552, P=0.832AC-5216; F=0.449, P=0.904, YL-IPA08) and fecal pellets (F=0.206, P=0.993, AC-5216; F=0.155, P=0.998, YL-IPA08). It is indicated that PK11195(1,3mg/kg, i.p.) did not affect the locomotor activity. Contextual fear paradigm showed that the reduction of the freezing time by AC-5216(0.3mg/kg p.o.)(F=4.552, P=0.000) and YL-IPA08(0.3mg/kg p.o.)(F=5.738, P=0.000) were antagonized by PK11195(3mg/kg, i.p.) and the elevated plus maze test showed that total time spent in (F=0.244, P=0.987, AC-5216; F=0.310, P=0.970, YL-IPA08) and the entries into (F=0.702, P=0.705, AC-5216; F=0.898, P=0.531, YL-IPA08) the arms were not different between the groups. The time (F=2.128, P=0.035, AC-5216; F=2.097, P=0.038, YL-IPA08) spent in and the entries (F=2.252, P=0.026, AC-5216; F=2.442, P=0.016, YL-IPA08) into the open arms was increased by AC-5216(0.3mg/kg p.o.) and YL-IPA08(0.3mg/kg p.o.), which were antagonized by PK11195(3mg/kg, i.p.). The results suggested that AC-5216and YL-IPA08allivated PTSD by TSPO.Western blot showed that the expression of TSPO is down-regulated in the prefrontal cortex and hippocampus of the TDS group. The down-regulated TSPO is reversed by administration of AC-5216(0.3mg/kg p.o.)(F=150.2, P=0.000, prefrontal cortex; F=11.45, P=0.006, hippocampus) and YL-IPA08(0.3mg/kg p.o.)(F=18.69, P=0.002, prefrontal cortex; F=12.66, P=0.005, hippocampus). It is indicated that the anti-PTSD effect of AC-5216and YL-IPA08is associated with up-regulation of TSPO expression.Binding assay showed that the activity of TSPO receptor is down-regulated in the prefrontal cortex, hippocampus and platelet of the TDS group. The down-regulated TSPO receptor activcty is reversed by administration of AC-5216(0.3mg/kg p.o.)(F=15.63, P=0.0042, prefrontal cortex; F=7.454, P=0.0236, hippocampus; F=6.825, P=0.0285, platelet) and YL-IPA08(0.3mg/kg p.o.)(F=11.99, P=0.0080, prefrontal cortex; F=13.68,P=0.0058, hippocampus; F=11.24, P=0.0093, platelet). It is indicated that the anti-PTSD effect of AC-5216and YL-IPA08is associated with up-regulation of TSPO receptor activity.ELISA showed that progesterone and allopregnenolone was reduced in the prefrontal cortex, hippocampus and serum in the TDS model group. However, AC-5216(0.3,1mg/kg p.o.) and YL-IPA08(0.3,1mg/kg p.o.) reversed the down-regulation of allopregnenolone in the prefrontal cortex (F=2.687,P=0.030, AC-5216; F=2.550, P=0.039, YL-IPA08), hippocampus (F=3.478, P=0.010, AC-5216; F=3.131, P=0.017, YL-IPA08) and serum (F=3.002, P=0.021, AC-5216; F=3.321,P=0.011, YL-IPA08), but not the progesterone in the prefrontal cortex (F=3.985, P=0.003, AC-5216; F=2.802,P=0.022, YL-IPA08), hippocampus (F=3.171, P=0.012, AC-5216; F=2.379, P=0.045, YL-IPA08) and serum (F=2.722, P=0.025, AC-5216; F=2.925, P=0.018, YL-IPA08). It also found that the effect of AC-5216and YL-IPA08were antagonized by PK11195(3mg/kg, i.p.) in the prefrontal cortex (F=2.439, P=0.039, AC-5216; F=2.830, P=0.018, YL-IPA08), hippocampus (F=2.296, P=0.048; F=3.587, P=0.005, YL-IPA08) and serum (F=3.035, P=0.013, AC-5216; F=3.318,P=0.007, YL-IPA08) of allopregnenolone. The result indicated that the anti-PTSD effect of AC-5216and YL-IPA08was associated with up-regulation of allopregnenolone.HPLC-ECD showed that5-HT was reduced in the TDS model group, sertraline (15mg/kg p.o.), AC-5216(1mg/kg p.o.) can reverse the down-regulation of5-HT (F=3.663, P=0.007, prefrontal cortex; F=2.473, P=0.046, hippocampus), but no effect on DOPAC (F=0.163, P=0.985, prefrontal cortex; F=0.414, P=0.864, hippocampus), DA (F=0.604, P=0.725, prefrontal cortex; F=0.591, P=0.735, hippocampus),5-HIAA (F=0.890, P=0.513, prefrontal cortex; F=0.520, P=0.789, hippocampus) and NE (F=0.181, P=0.980, prefrontal cortex; F=0.674, P=0.671, hippocampus). YL-IPA08(1mg/kg p.o.) can reverse the down-regulation of5-HT (F=2.457, P=0.045, prefrontal cortex; F=2.605, P=0.034, hippocampus), but no effect on DOPAC (F=0.277, P=0.944, prefrontal cortex; F=0.384, P=0.884, hippocampus), DA (F=0.157, P=0.986, prefrontal cortex; F=0.474, P=0.823, hippocampus),5-HIAA (F=0.199, P=0.975, prefrontal cortex; F=0.678, P=0.668, hippocampus) and NE (F=0.317, P=0.924, prefrontal cortex; F=0.534, P=0.779, hippocampus). It is indicated that anti-PTSD effects of AC-5216and YL-IPA08was associated with the up-regulation of5-HT.Conclusion:AC-5216and YL-IPA08have high affinity to TSPO, but low affinity to CBR. In addition, YL-IPA08has higher affinity to TSPO than AC-5216and dissolved in water. Neurosteriods levels were pregulated by AC-5216and YL-IPA08. AC-5216and YL-IPA08have anti-PTSD effect. The anti-PTSD effect of AC-5216and YL-IPA08was associated with up-regulation of allopregnenolone and the regulation was mediated by TSPO. The anti-PTSD effects of AC-5216and YL-IPA08was associated with the up-regulation of5-HT. |
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