| Background: Insomnia has become a global public health problem causing heavy social burden.At present,conventional insomnia therapy is mainly drugs,commonly used sedative hypnotic drugs have quickly and significant hypnotic effect,but there are common side-effects and potential addiction and other adverse reactions.Melatonin is considered as an endogenous sleep physiological regulator,which plays a prominent role in regulating sleep.However,melatonin has a short half-life and low absolute bioavailability greatly limit the therapeutic effect.In recent years,cannabinol is also increasingly used as a drug for the treatment of insomnia without addiction.Cannabidiol can inhibit the activity of enzymes related to melatonin metabolism at clinically relevant concentrations,delay the metabolism of melatonin in vivo,and further improve the therapeutic effect.This provided feasibility for this project to explore the combination of cannabidiol and melatonin to treat insomnia.Objective: In this study,orthogonal experimental design and pentobarbital sodium synergistic hypnosis experiment were used to investigate the synergistic sedative and hypnotic effects of cannabidiol-melatonin compound,and the prescription ratio was studied.The PCPA 5-HT depletion insomnia model and the new environment insomnia model were established,and the spontaneous activity video analysis system and wireless physiological signal telemetry system were used to evaluate the synergistic sleep regulation effect of cannabadiol-melatonin combination.Further,from the perspective of hypothalamic core clock gene and its post-translational modification regulation,SCN neuron activity,serum oxidative stress index content and melatonin metabolism,the mechanism of drug compound in sedation and hypnosis regulation of circadian rhythm disturbance and oxidation-reduction balance was explored.Methods:(1)Pentobarbital sodium combined with hypnosis experiment,righting reflex experiment,orthogonal experiment to screen the optimal ratio of formular: Fifty male ICR mice were randomly divided into 5 groups,namely blank control group and 4 groups with different doses of formular.Normal saline and formular mixed solution(20 m L/kg)were administered by gavage at 07:00,respectively.At 08:00,pentobarbital sodium solution(45 mg/kg,10 m L/kg)was injected intraperitoneally.The disappearance time and recovery time of the righting reflex were observed,and the sleep latency and sleep duration were calculated.(2)Establishment of PCPA 5-HT depletion insomnia model and hypnotic effect of drug formular: Sixty male ICR mice were randomly divided into 6 groups,namely blank control group,model group,diazepam group(3 mg/kg),compound low(5 mg/kg cannabidiol +5 mg/kg melatonin),medium(10 mg/kg cannabidiol +10 mg/kg melatonin)and high(20 mg/kg cannabidiol +20 mg/kg melatonin)dose groups,each group consisted of 10 animals.The blank control group was not treated,the other groups were intraperitoneally injected with PCPA(400 mg/kg,10 m L/kg)for 5 consecutive days to establish the insomnia model.The sleep latency and sleep duration of the mice were examined by righting reflex test.(3)The spontaneous activity experiment of mice: grouping and modeling were performed as described in method(2).Normal saline,diazepam solution and compound mixed solution were given by gavage at 08:00,respectively,and then the mice were placed in the spontaneous activity box.(4)Establishment of a new environmental insomnia model combined with EEG analysis technology to evaluate the sleep regulation effect of drug combination: Twenty-eight male Sprague-Dawley rats were randomly divided into 4 groups,namely blank control group,model group,diazepam group and cannabidiol + melatonin group,with 7 rats in each group.Baseline sleep data for 24 h were collected 7 days after recovery after implantation surgery.The rats were given diazepam and drug mixture solution by gavage at 07:30 the next day,respectively.At 08:00,10:00 and 12:00,the rats were transferred into a dirty cage where other animals had lived for at least 5 days.Sleep monitoring began at 08:00and lasted for 24 hours.(5)Materials collected and related testing work after molding : As in methods(2)and(4),the model was constructed and given intragastric administration.Blood was collected from eyeballs of mice and from posterior orbital venous plexus of rats.Then,the head was severed and the hypothalamus tissue was quickly removed from the ice for use.The expression of core clock protein in hypothalamus was detected by western blotting.Serum oxidative stress index was detected by biochemical reaction kit.ELISA kit was used to detect the melatonin concentration in rat serum.(6)Immunofluorescence experiment: Grouping,administration and modeling as shown in Method(4).After the modeling,the rats were deeply anesthetized with 10% chloral hydrate,fixed by perfusion with normal saline and 4% paraformaldehyde,and then the whole brain was dissected with decapitated head.The expression of C-FOS was observed by immunofluorescence method.Results:1.The optimal ratio and dosage of the two drugs were selected successfully.The minimum effective doses of cannabidiol and melatonin to prolong sleep time were 5mg/kg and 10 mg/kg,respectively.After the combination of the two drugs,3.75 mg/kg(1.25 mg/kg cannabidiol +2.5 mg/kg melatonin)could significantly prolong the sleep time induced by pentobarbital sodium.2.The combined administration of cannabidiol and melatonin significantly increased the plasma concentration of melatonin,suggesting that cannabidiol may prolong the plasma half-life of melatonin and improve its bioavailability by inhibiting the activities of enzymes related to melatonin metabolism in the body.3.The combination of the two drugs can improve the activity-rest rhythm of mice with insomnia,significantly regulate the levels of core clock genes in the hypothalamus SCN and the content of oxidative stress indicators in serum,and play a comprehensive therapeutic role in regulating biological rhythm disorder and improving oxidative stress injury.4.Drug compound intervention can optimize the sleep structure of insomnia rats by regulating the activity of neurons in the hypothalamic SCN region,promote deep sleep,and improve sleep quality.Conclusion: Cannabidiol and melatonin drug formular has obvious synergy sedative hypnotic effect,can regulate the oxidative stress in mice and rats insomnia index concentration in order to maintain the body’s redox equilibrium,and change the abnormal expression of hypothalamic core clock protein and its post-translational modification regulation to improve the status of circadian rhythm disturbance and insomnia.Significance: In order to solve the problems of low bioavailability and high incidence of adverse reactions in the current clinical application of cannabidiol and melatonin,a two-drug formula was formed,the synergistic effect of cannabidiol and melatonin was evaluated,the dosage of the prescription was determined,and the interaction between the two drugs was studied,so as to provide a potential treatment method for insomnia patients that can be used for a long time,is not easy to be tolerated and addictive,and has few adverse reactions. |
