| Drug addiction is a chronic relapsing brain disease caused by a combination of genetic and environmental factors including addictive drugs acting on the body.The World Drug Report 2021,issued by the United Nations Office on Drugs and Crime,noted the continuing increase in the number of drug users,the increasing complexity of the drug market,the acceleration of underground sales and the changing patterns of drug use.The global new crown epidemic has exacerbated the expansion of the drug market,making the global drug control situation more severe.The "2020 China Poisoning Situation Report" issued by the Office of the National Drug Control Committee of China also points out that although the drug abuse situation in China continues to improve,many new problems such as strong concealment and diversified types of abuse also occur,increasing the difficulty of drug control.Drug addiction not only brings serious harm to patients themselves and their families,but also causes the epidemic of acquired immune deficiency syndrome(AIDS),tuberculosis,hepatitis and other major infectious diseases,causes illegal and criminal acts,causes huge economic losses,and seriously threatens national security and social stability.Methamphetamine(METH)belongs to the amphetamine class of psychostimulants,and according to the annual reports of drugs in the world and China,METH has become an addictive drug widely abused in China and even worldwide.Compared with traditional drugs such as opioids,METH is addictive and neurotoxic and difficult to treat.However,regrettably,there is no drug marketed for METH addiction,which makes it impossible to effectively control METH addicted patients,which undoubtedly becomes a difficult and hot spot for global health care.Cannabidiol(CBD)is a natural cannabinoid extracted from marijuana.It has been shown that CBD has rich pharmacological activities such as,antioxidant,anticonvulsant,immunomodulatory and neuroprotective;it has potential value in the treatment of a variety of neuropsychiatric diseases(epilepsy,Parkinson’s disease and Alzheimer’s disease,etc.),cancer(breast cancer,lung cancer and colon cancer,etc.),autoimmune diseases and cardiovascular system diseases.In recent years,more and more preclinical and clinical small sample scientific studies have shown that CBD has a good inhibitory effect on physical and mental dependence induced by a variety of addictive substances.In 2017,the World Health Organization has clearly stated that there is no abuse problem with CBD.In 2005 and 2018,the FDA approved Jeva’s Sativex ?(1:1 tetrahydrocannabinol with CBD oral mucosal spray)and Epidiolex ?(CBD oral solution)for the treatment of pain and spasticity caused by multiple sclerosis and two rare childhood episodic epilepsies(Lennox-Gastaut and Dravet syndromes),respectively.The marketing of this drug suggests that CBD may have high clinical safety,increasing confidence in developing antiaddiction anti-relapse drugs.Although there have been relevant reports of CBD against METH addiction,the research work is not systematic enough and the experimental results reported in different laboratories are contradictory.It has been reported in the literature that systemic or central nuclear microinjection of CBD significantly inhibits the development,expression,and reinstatement of conditioned place preference(CPP)induced by METH in rats;it significantly antagonizes the relapse behavior induced by METH in rats.The literature gives contradictory results to the above reports,and continuous simultaneous administration of CBD and low-dose METH promotes the development of CPP induced by very low-dose METH in rats.In addition,the theory of "motivational sensitization" is one of the main mechanisms of drug addiction,and regrettably to date,there is no literature on behavioral sensitization induced by CBD anti-METH in mice.Sensitization theory holds that the essence of addiction is the excessive amplification of psychological needs,especially those triggered by cues.The neurobio logical basis underpinning this theory is the activation sensitization of the dopamine nervous system,and the behavioral sensitization model is the behavioral characterization of this theory.In this model,repeated or fixed-interval administration of addictive drugs results in a gradual increase in motor behavior.Thus,the sensitization model provides a more comprehensive experimental basis for studies addressing the neural mechanisms underlying the effects of CBD against METH addiction and warrants in-depth systematic investigation.In view of the above research status,this study will focus on mice,while using two classical addiction animal models,METH-induced behavioral sensitization and CPP,to comprehensively and systematically study the effects of chronic or acute administration of CBD on the formation,expression,regression and reconstruction of METH-induced addiction in mice,in order to provide a solid experimental basis for CBD as a potential candidate drug against METH addiction.Experimental MethodBehavioral sensitization model and CPP model are behavioral quantification of dopamine system sensitization process and addictive memory,respectively,with good predictive validity and structural validity,and easy operation,short experimental cycle,is a good model for studying drug addiction potential,drug addiction mechanism and efficacy evaluation of anti-addictive drugs,and has been widely used.In this study,mice were used to study the pharmacodynamic effects of CBD against METH addiction using two animal model systems,behavioral sensitization and CPP,by intraperitoneal administration.Before carrying out the above studies,we first determined the dose range in which CBD did not affect locomotor activity in the locomotor activity model to rule out false positive experimental results;then performed the detection of CPP or conditioned place aversion(CPA)in the selected dose range,and finally determined the dose use range to study CBD anti-METH addiction based on these two experimental results to observe the acute and chronic effects of CBD in the development period,expression period,extinction period,and drug-seeking behavior reinstatement period of METH-induced behavioral sensitization or CPP models in mice,respectively.The results of the study(1)The effect of CBD itself on mice motility and CPP or CPA.1)The effect of CBD itself on the basal activity of mice in the dose range of this experimentIn the locomotor activity test,intraperitoneal injection of different doses of CBD(5,10,20,40 and 80 mg/kg)failed to affect the basal locomotor activity of mice compared with the vehicle group(main effect of treatment:F(5,42)=1.743,P>0.05).2)CBD itself failed to induce CPP or CPA in the dose range of this experimentAccording to the training process of the conditioned place preference model,vehicle or CBD(5,20 and 80 mg/kg)and positive control drug METH(1 mg/kg)were intraperitoneally injected 30 min in advance on the training day with the chamber for 1 h,and saline(10 ml/kg)was intraperitoneally injected 30 min in advance on the training day without the chamber for 1 h in the other chamber,with alternating training of the chamber and the non-chamber for 10 days,for a total of 5 rounds.The results showed that the CPP score of mice in the positive control drug METH group was significantly higher than that before preconditioning(P<0.001),suggesting that CPP was formed.There were no significant differences in CPP scores between mice in the CBD group and the preconditioning pre-conditioning values and the vehicle group(P>0.05 compared with the preconditioning pre-conditioning values;P>0.05 compared with the vehicle group),suggesting that CBD itself does not cause CPP or CPA behavior in mice and has no addictive or malignant psychoactive potential.Eventually,20-80 mg/kg was selected as the dose range to study the pharmacodynamics of CBD against METH addiction.(2)Effects of CBD on METH-induced behavioral sensitization in mice1)Effect of CBD on METH-induced behavioral sensitization development in mice.During the development period,vehicle or different doses of CBD(20,40 and 80 mg/kg)were intraperitoneally injected 30 min before intraperitoneal injection of saline or METH(1 mg/kg)for 6 consecutive days,respectively,and then immediately placed in a locomotor activity chamber to test the movement distance within 90 min.The results showed that the activity of mice in the vehicle+METH group was significantly higher than that in the saline group(t=4.846,P<0.001);the activity of animals in this group showed a gradually increasing trend with the extension of administration time,and the movement distance on day 6 was significantly higher than that on day 1(P<0.01),suggesting that mice in the vehicle+METH group could induce typical behavioral sensitization behavior;for the CBD+METH group,different doses of CBD(20,40 and 80 mg/kg)were intraperitoneally injected 30 min earlier on day 1,respectively,with no significant difference compared with the activity of mice in the vehicle+METH treatment group(main effect of treatment:F(3,38)=0.975,P>0.05),and this result suggested that CBD(20,40 and 80 mg/kg)administration on day 1 had no significant effect on the acute hyperactivity induced by METH(1 mg/kg).However,with the extension of treatment time,there were no significant differences in the motor distance of mice in the CBD(20,40 and 80 mg/kg)+METH group on day 6 compared with the activity distance on their respective day 1(P>0.05),suggesting that concomitant administration of CBD(20,40 and 80 mg/kg)significantly reduced the development of behavioral sensitization induced by METH(1 mg/kg)in mice.In the process of sensitization,the statistics of pairwise comparison of separate groups showed that the movement distance was significantly reduced in the CBD(40 mg/kg)+METH vehicle+METH group compared with the CBD(40 mg/kg)+METH vehicle+METH group on days 3,4,5 and 6,respectively(P<0.05;P<0.01);similarly,the movement distance was significantly reduced in the CBD(80 mg/kg)+METH vehicle+METH group compared with the CBD(80 mg/kg)+METH vehicle+METH group on days 3,4,5 and 6,respectively(P<0.01;P<0.001).Next,mice in each group were returned to the housing room for a 7-day conversion period without any drug treatment.Finally,all mice were kindled by intraperitoneal injection of a small dose of METH(0.5 mg/kg),and the results showed that the motor distance of mice in the vehicle+METH group was significantly increased compared with the saline group,suggesting that the sensitizing behavior of mice in the METH model group was primed(t=4.429,P<0.001);compared with the vehicle+METH group,the motor distance of mice was dose-dependently decreased after concomitant treatment with CBD(20,40 and 80 mg/kg)for 6 consecutive days during the formation period(main effect of treatment:F(3,38)=5.852,P<0.01),of which CBD(40 and 80 mg/kg)had a significant inhibitory effect on kindling after withdrawal(P<0.01;P<0.05).The above results suggest that concomitant administration of CBD(20,40 and 80 mg/kg)for 6 consecutive days during the development period significantly counteracted the development of METH-induced behavioral sensitization in mice;it also effectively blocked the priming behavior induced by low-dose METH(0.5 mg/kg)under the premise of inhibiting the development of behavioral sensitization,showing that CBD may have a preventive effect on METH-induced behavioral sensitization.2)Effect of CBD on METH-induced behavioral sensitization transformation in mice.No CBD treatment was administered during the development of behavioral sensitization evoked by daily intraperitoneal injections of METH in mice for 6 consecutive days.However,during the 7-day transformation period,vehicle or different doses of CBD(20,40 and 80 mg/kg)treatment were intraperitoneally injected daily,and finally the effects of the above treatment on METH(0.5 mg/kg)-induced behavioral sensitized priming in mice were observed during the challenge period.It was found that administration of CBD for 7 consecutive days during the transformation period had no significant intervention effect on the priming behavior provoked by low-dose METH compared with the vehicle+METH group(main effect of treatment:F(3,36)=0.256,P>0.05).3)Effect of CBD on METH-induced behavioral challenge in mice.After METH induced typical behavioral sensitization in mice,balanced grouping was performed according to motor distance,and CBD treatment was intraperitoneally injected only 30 min before priming test,and the results revealed that CBD administration(20,40 and 80 mg/kg)significantly reduced the increase in motor distance induced by low-dose METH(0.5 mg/kg)in a dosedependent manner(main effect of treatment:F(3,35)=25.511,P<0.001),with CBD(40 and 80 mg/kg)having a significant inhibitory effect compared with the vehicle+METH group(P<0.01;P<0.001).(3)Effect of CBD on METH-evoked CPP in mice.1)Effect of concomitant administration of CBD during the development period on METHinduced CPP development in mice.In the study of the formation phase of METH-induced CPP in mice,two administration treatment modalities were used for CBD to observe its effect on METH-induced CPP development in mice.One processing modality was intraperitoneal injection of CBD(20,40 and 80 mg/kg)30 min before METH administration,followed by training in the companion chamber immediately after METH administration to investigate the effect of CBD on the process of METH-evoked reward and environment association memory development;the other processing modality was intraperitoneal injection of CBD(20,40 and 80 mg/kg)immediately after METH administration training to investigate the effect of CBD on METH-evoked reward memory consolidation.The specific results were as follows:First,in the CBD treatment before METH training,the CPP score was significantly increased in the vehicle+METH pretest,and METH could induce CPP behavior in mice,suggesting that the model was successfully established(P<0.01).However,there was also a significant difference in each dose of CBD(20,40 and 80 mg/kg)compared with the respective pretest,and there was no significant difference compared with the vehicle+METH group(P>0.05).Second,in the CBD treatment after METH training,vehicle+METH pretest could establish significant preference behavior compared with METH pretest,suggesting that METH could induce CPP behavior in mice,and the model was successfully established(P<0.001).However,there were also significant differences in the pretest comparison between each dose treatment of CBD(20,40 and 80 mg/kg),and there were no significant differences in the comparison between each dose treatment of CBD and vehicle+METH group(P>0.05).Therefore,in the development period,CBD had no significant effect on the formation of METH-induced CPP,suggesting that CBD had no effect on both METH-induced CPP development and consolidation.2)The effect of CBD on the expression of CPP induced by METH in mice.No CBD treatment was administered during the development period of METH-induced CPP in mice.However,during the CPP expression test period,vehicle or CBD(20,40 and 80 mg/kg)was intraperitoneally injected 30 min in advance,and the results revealed that the preference score of the vehicle+METH group was significantly higher than the value before formation training,suggesting that METH could induce the formation of CPP behavior in mice(P<0.001),but there was no significant difference between the vehicle+METH group treated with CBD administration(P>0.05).The results suggest that CBD administration during the expression period has no significant effect on the expression of CPP induced by METH in mice.3)Effect of multiple doses of CBD during the extinction period on the reinstatement of CPP induced by METH in mice.CBD treatment was not administered during both the development and expression periods of METH-induced CPP in mice.After the expression test,two different extinction training modalities were employed for the CBD administration intervention.One is the extinction mode of continuous exposure to environmental cues,that is,after 24 hours of expression test,mice were exposed to the drug box for 10 minutes and then removed,and immediately treated with vehicle CBD(20,40 and 80 mg/kg)intraperitoneally,which was performed for 7 consecutive days to observe whether CBD could exert an intervention effect during the reconsolidation stage of reward memory and thus affect the reinstatement of CPP behavior;the other is the mode of extinction of drug box training,that is,after 24 hours of expression test,4 rounds of 8-day matching training mode of vehicle or CBD in the drug box were used to observe whether CBD played an intervention role during METH reward memory extinction learning and thus affected the reinstatement of CPP behavior.The specific results were as follows:① In the low-dose METH(0.5 mg/kg)induced mice reinstatement test,the CPP score of mice in the vehicle+METH group was significantly higher than that in the pretest,suggesting successful reinstatement(t=4.488,P<0.01).However,the preference scores of all dose treatment groups of CBD(20,40 and 80 mg/kg)were significantly lower than those of the vehicle+METH group(t=2.315,P<0.05;t=2.286,P<0.05;t=2.128,P<0.05),suggesting that CBD administration treatment at this stage could significantly reduce the reinstatement of CPP.② In the way of concomitant extinction training administration treatment,low-dose METH(0.5 mg/kg)induced reinstatement in mice,and the CPP score of mice in the vehicle+METH group was significantly higher than that in the pretest,suggesting successful CPP reinstatement(t=3.738,P<0.01).The preference score of each dose treatment group of CBD(20,40 and 80 mg/kg)was significantly different from the inhibitory effect of 80 mg/kg CBD compared with the vehicle+METH group(t=2.750,P<0.05).In summary,both administration modalities employed by CBD during the extinction period could significantly inhibit the reinstatement of CPP evoked by METH in mice.4)Effect of CBD administration during priming period on METH-induced CPP reinstatement in mice.No CBD treatment was given during the development period,expression period and extinction period of METH-induced CPP in mice.CBD treatment was injected intraperitoneally 30 min before METH(0.5 mg/kg)priming,and the results revealed that the CPP score of mice in the vehicle+METH group was significantly higher than that of the pretest,suggesting successful CPP reinstatement.There was no significant difference in the preference score between the vehicle+METH group treated with each dose of CBD(20,40 and 80 mg/kg)(t=1.909,P>0.05;t=1.497,P>0.05;t=0.536,P>0.05),indicating that CBD administration during the priming period had no significant effect on the reinstatement of CPP in METH-induced mice.ConclusionIn summary,the formation and kindling of METH-induced behavioral sensitization were significantly reduced by either concomitant administration of CBD,while the reinstatement of CPP was significantly inhibited by administration after exposure or concomitant administration during the extinction period of CPP.These results suggest that CBD has anti-relapse effect against METH addiction and is worthy of in-depth study. |
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