The Study Of The Sedative-hypnotic Effects Of NHBA And Its Analogue B2and Their Mechanisms

Insomnia is an extremely common symptom in modern society. It results in serious daytime consequences, such as impaired quality of life, reduced energy and memory problems, and causes impairment of both physical and mental health. At present, there is no satisfying drug for treating insomnia in market. Thus, it is necessary to search for new therapeutics for insomnia.The search for novel pharmacotherapy for psychiatric illness from medicinal plants has progressed significantly in the past decade. A considerable number of herbal constituents whose behavioral effects and pharmacological actions have been well characterized may be good leading compounds for further modification that may ultimately lead to clinical use. The rhizomes of Gastrodia elata have been extensively used in traditional Chinese medicine for the treatment of headaches, dizziness, vertigo, insomnia and convulsive illnesses, such as epilepsy and tetanus. We collaborated with Professor Shi Jiangong in the department of Phytochemistry and isolated a trace ingredient, N6-(4-hydroxybenzyl) adenine riboside, from Gastrodia elata. We got a new chemical entity-B2via modifying the chemical structure of NHB A. This study aimed to investigate the sedative and hypnotic effects and possible mechanisms of NHB A and its analogue B2.The sedative and hypnotic effects of NHBA and B2were investigated in the locomotor activity test and sodium pentobarbital-hypnosis test. In locomotor activity test, compared with control, NHBA (1mg/kg, i.p.) decreased locomotor activity by79%(p<0.01) in mice. B2(1mg/kg, i.p.) decreased locomotor activity by93%vs. control. In pentobarbital-hypnosis test, NHBA (1and5mg/kg, i.p.) decreased latency of sleep from4.6±0.3min to3.8±0.1min (p<0.05) and3.2±0.1min (p<0.01), prolonged pentobarbital-induced sleeping time from42.2±1.9min to68.3±6.4min (p<0.05) and84.3±8.5min (p<0.01) respectively. B2(1and5mg/kg, i.p.) reduced latency of sleep from4.6±0.3min to3.3±0.2min (p<0.05) and3.1±0.1min (p<0.01), elongated pentobarbital-induced sleeping time from42.2±1.9min to99.1±9.2min (p<0.05) and 106.3±9.2min (p<0.01) respectively. Both NHBA (1and5mg/kg, i.p.) and B2(1and5mg/kg, i.p.) increased the rate of sleep onset induced by a sub-hypnotic dose of sodium pentobarbital in mice. The present results showed that NHBA and B2have sedative and hypnotic effects in mice.Moreover, the side effects of NHBA and B2on muscle strength, motor coordination and body temperature in mice were observed in the traction test and rota-rod test. In the traction test, NHBA (5mg/kg, i.p.) and B2(1mg/kg, i.p.) had no effect on muscle strength. In the rota-rod test, compared with control group, NHBA (5mg/kg, i.p.) decreased the latency falling from the rota-rod by19.1%. B2(1mg/kg, i.p.) decreased the latency by67.1%(p<0.001). NHBA(5mg/kg, i.p.)and B2(1mg/kg, i.p.) decreased body temperature by1.9±0.1℃(p<0.001) and3.2±0.3℃(p<0.001) in mice respectively. These results indicate that NHBA and B2have no effect on muscle strength, but they impaire motor coordination and reduce body temperature in mice.Sleep-wake cycle is a physiological process which refers to many systems and centers. It has a complicated neural regulation mechanism. We have comfirmed the sedative and hypnotic effects of NHBA and B2, then we decide to investigate possible mechanisms of NHBA and B2on sleep.1. NHBA, as an adenosine analog, has been demonstrated the ability to prevent serum deprivation-induced apoptosis in PC12cells and to bind adenosine A2A receptor. Previous studies reported that NHBA displayed affinity to adenosine A1receptor with Ki=1.78μM and adenosine A2A receptor with K;=1.39μM. B2also displayed affinity to adenosine A1receptor with Ki=0.42μM and adenosine A2A receptor with Ki=0.37μM. Adenosine has been proposed to be an endogenous sleep factor. Several lines of evidence indicate that both adenosine A1receptor and A2A receptor are involved in sleep induction. Therefore, we have investigated whether sedative and hypnotic effects of NHBA and B2were mediated through adenosinergic system. The contents of adenosine and inosine in brain and adenosine deaminase activity of thalamus were determined by high performance liquid chromatography-electrochemistry detector (HPLC-ECD). The effects of selective adenosine receptor antagonist on hypnotic activity of NHBA and B2 were also studied. Our results showed that compared with control group, NHBA (5mg/kg, i.p.) increased the contents of adenosine by23.0%,14.7%and10.4%in striatum, brain stem and thalamus respectively. It had no effects on activity of adenosine deam-inase in thalamus. Pretreatment with adenosine A1receptor antagonist DPCPX (0.2and0.5mg/kg, i.p.) or A2A receptor antagonist SCH58261(0.2and1mg/kg, i.p.), the increase in sleeping time induced by NHBA (1mg/kg, i.p.) was significantly reversed. These results suggest that both adenosine A1and A2A receptor may be involved in the central inhibitory activity of NHBA. Compared with control group, B2(1mg/kg, i.p.) increased the contents of adenosine by21.3%(p<0.05),14.8%and12.4%in thalamus, brain stem and striatum respectively. It had no effects on the activity of adenosine deaminase in thalamus. Pretreatment with DPCPX (0.2and0.5mg/kg, i.p.) or SCH58261(0.2and1mg/kg, i.p.), the increase in sleeping time induced by B2(0.5mg/kg, i.p.) was significantly reversed. These results suggest that the sedative and hypnotic effects of B2may be attributed to the increased contents of adenosine in thalamus. Meanwhile, both adenosine A1and A2A receptor may be involved in the central inhibitory activity of B2.2. VLPO is a sleep center in the anterior hypothalamus. The VLPO sends inhibitory GABAergic afferents to induce sleep. Several lines of evidence indicate that adenosine activates A1/A2A receptor-expressing neurons and excites the VLPO subsequently. Our results indicated that both adenosine A1and A2A receptor may be involved in the central inhibitory effects of NHBA and B2. C-Fos expression in the GABAergic neurons of the VLPO was detected using immunohitochemistry stain. GABA is one of the important neurotransmitters mediating inhibitory postsynaptic potentials. It is well known that activation of GABAA receptors favors sleep. Up to now, insomnia has been principally treated by compounds acting at the GABAA receptor. The effects of different site-targeted GABAA receptor antagonists on hypnotic activity of NHBA and B2were studied. The contents of GABA and glutamate in brain were determined by HPLC-UV detector. Cl-exflux were investigated in cultured cerebellar granule cells using MQAE, a Cl--sensitive florescent dye. The results showed that NHBA (5mg/kg, i.p.) increased c-Fos expression in GABAergic neurons of the VLPO. There is no antagonism effect of picrotoxin (2mg/kg, i.p.) on hypnotic activity of NHBA (2and5mg/kg, i.p.). These results suggest that activiation of GABAergic neurons of the VLPO may be involved in the central inhibitory effect of NHBA. B2(1mg/kg, i.p.) increased c-Fos expression in GABAergic neurons of the VLPO. Picrotoxin (1and2mg/kg, i.p.), a GABA receptor antagonist, showed a significant antagonism effect on the hypnotic activity of B2(15mg/kg, ig). Bicuculline (2and4mg/kg, i.p.) and flumazenil (3.5and7mg/kg, i.p.) have no effects on the hypnotic activity of B2(15mg/kg, ig). Compared with control group, B2(1mg/kg, i.p.) had no effect on the contents of GABA in rat brain tissue homogenate. In addition, B2at contentration of3×10-6and3×10-5mol/L significantly increased Cl-exflux by91.1%(p<0.05) and113.7%(p<0.01) respectively in the primary cultured cerebellar granule cells. These results suggest that activiation of GABAergic neurons of the VLPO and chloride ion channel may be involved in the central inhibitory activity of B2.3. A mechanism that may help stabilize sleep-waking transitions arises from mutually inhibitory interactions between VLPO neurons and the noradrenalinergic arousal systems. The contents of monoamine neurotransmitters in rat brain were determined by HPLC-ECD. The results showed that NHBA (5mg/kg, i.p.) significantly decreased the contents of NA by12.7%(p<0.01) and9.1%(p<0.05) respectively in hypothalamus and brain stem. B2(1mg/kg, i.p.) significantly decreased the contents of NA by23.6%(p<0.001) and14.8%(p<0.01) respectively in hypothalamus and brain stem. These results suggest that the sedative and hypnotic effects of NHBA and B2may be attributed to the decreased contents of NA in hypothalamus and brain stem.