The Mechanisms Underlying Analgesic And Anti-depression Of Gentiopicroside

Background:Epidemiological studies demonstrate that pain has consistently been strongly correlated with depression. People who suffer from one disease have more frequence to suffer from the other one. These observations have led to some authors to label the comorbidity as the pain-depression syndrome or pain-depression dyad. The comorbid nature of depression and pain may reduce the drug effects, leading to treatment failure in clinic. There are only two drugs approved for the treatment of both pain and depression,and mechanism-based preclinical studies are rare.N-Methyl-d-aspartate(NMDA) receptors are the most extensively characterized ionotropic glutamate receptors. Evidence is mounting that agents targeting the NMDA receptor may be the key to developing a new generation of improved therapeutics for depression and pain. But the psychotomimetic side effects and the route of delivery precludes the clinical use of present NMDA antagonists. So it is necessary to search for other safe chemicals targeting to NMDA receptor.The monoamine theory of depression has dominated the neuropharmacology of depression for several decades. According to this theory, major depressive disorders are caused by a deficit in monoaminergic neurotransmission that can be treated by administering drugs that correct this deficit. However, many patients did not experience a response to these drugs, and up to 60% of individuals have recurrent episodes, which suggest our understanding of the pathophysiology of depression remains incomplet.Recently, the hypothesis that depression is a primary‘inflammatory’disorder is popular. The evidences include(a) patients with depression show elevated inflammatory biomarkers in clinic;(b) inflammatory cytokines can induce depression-like behaviors both in animals and people;(c) anti-inflammatory intervention is beneficial for depression.Objectives:This study is designed to evaluate the potential effects of Gent on reserpine-induced pain-depression dyad, LPS-induced depression and neuroinflammation.Moreover, the pharmacokinetic characters and blood brain barrier permeability are also investigated. Our findings in the study can help further elucidate the mechanisms underlying the effects of Gent and assist the clinical treatment of pain/depression dyad and neuroinflammation using traditional herbs.Experiments:Part One Behavioral tests associated with pain and depression were used to investigate the effects of Gent on reserpine-induced pain-depression dyad; the levels of biogenic amines in BLA were determined by HPLC; MDA and CAT activity were measured by commercially available kits; apoptosis-related proteins and Glu N2B-containing NMDA receptors in BLA were estimated by western blot.Part Two Behavioral tests including FST and TST were performed to investigate the effects of Gent on LPS-induced depression; Enzyme activity of IDO was assayed by determination of kynurenine/tryptophan using LC-MS/MS method; TNF-α and IL-1β in plasma and brain were estimated by ELISA; Glu N2 B expression was determinated by western blot.Part threeThe effects of Gent on neuroinflammation were evaluated by in vitro and in vivo experiments. MTT, ELISA, Western blot and Hoechst33258-PI double staining were used.Part four Concentrations of Gent in mouse plasma, brain homogenate and cerebrospinal fluid were determined by LC-MS/MS; DAS 2.0 software was used to calculate the pharmacokinetic parameters; blood-brain permeability was evaluated by Gent concentrations in brain homogenate and cerebrospinal fluid.Results:Part One1. Behavioral detection indicated that Gent administration dose dependently ameliorated the behavioral deficits associated with reserpine-induced pain/depression dyad.2. Gent significantly reversed the changes in the levels of biogenic amines,caspase-3, and Glu N2B-containing NMDA receptors in amygdala. However, Gent did not affect the expression of Glu N2A-containing NMDA receptors.3. The inhibitory effects of Gent on oxidative stress were occluded by simultaneous treatment of Glu N2 B receptors antagonist Ro25-6981.Part two1. 24 h after LPS administration, sickness behaviors of the LPS-induced mice disappeared while depression behaviors occurred. Gent administration could ameliorate the behavioral deficits associated with LPS-induced depression.2. Anti-inflammatory action and down-regulating the expression of Glu N2 B contributed to its effects on LPS-induced depression.Part three1. Gent could inhibit NO, TNF-α and IL-1β productions in LPS-induced astrocytes;attenuate LPS-activated astrocyte-induced neuronal death;2. Gent could down-regulate i NOS/COX-2 expressions by inhibit MAPK signaling pathway and NF-κB signaling pathway in LPS-induced astrocyte;3. Gent could ameliorate LPS-induced neuroinflammation in mice.Part four1. A rapid and sensitive liquid chromatography/tandem mass spectrometry(LC-MS/MS) method was developed to determine Gent in mouse plasma, brain homogenate and cerebrospinal fluid.2. The main pharmacokinetic parameters are summaried as follows: C0=57.4 μg/m L;t1/2=69.3 min; Cl=0.043 L/min/kg; AUC 0-∞=1995.36 mg/L*min;3. Gent could enter the CNS directly through the blood-brain-barrier.4. Conclusions:1. The results of the present study raised the possibility that Gent inhibits the pain/depression dyad by inhibiting Glu N2B-containing NMDA receptors as well as its antioxidant properties.2. Gent could ameliorate LPS-induced depression by anti-flammation and down-regulating Glu N2B-containing NMDA receptors.3. Gent could effectively suppress LPS-induced neuroinflammatory responses, at least partly, via NF-κB and MAPK signaling pathways, resulting in inhibition of i NOS,COX-2 expressions and NO, TNF-α productions in astrocytes; Gent possesses bene?cial effects in regulating immune responses in CNS.4. Gent was distributed and eliminated fast after one dose of i.v administration; it could enter the CNS directly through the blood-brain-barrier.