Activation Of TLR4/STAT3 Signaling In VTA Contributes To The Acquisition And Maintenance Of Morphine-induced Conditioned Place Preference

Background and Objective:Morphine is an opiate alkaloid that is extracted from opium and an opioid that is one of the most effective drugs for clinical reduction of acute or chronic pain.But morphine abuse will result in a strong reward effect through the mesolimbic dopamine system(MLDS)leading to morphine addiction.After the use of morphine,the individuals produce euphoria,and when they stop using it there are a variety of functional disorders and withdrawal syndrome.In order to pursue euphoria and avoid pain,the individual will get morphine at all costs.It is also easy to relapse after morphine withdrawal.Morphine addiction not only affects physical and mental health of individuals,but also causes serious harm to social security.The study of morphine addiction mechanism not only helps health management effectively prevent and treat morphine addiction,reduce the medical and social problems,but also contributes to the further management of clinical narcotic drugs.Morphine addiction is closely related to multiple brain regions of the central nervous system(CNS).MLDS plays an important role in morphine addiction,including the ventral tegmental area(VTA)and the nucleus accumbens(NAc).Addiction-related behavior is closely related to proinflammatory immune response.Recent studies have shown that long-term morphine treatment activates the immune signaling pathway in the dopaminergic neurons of the VTA region,suggesting that the proinflammatory response in the VTA region is critical in the morphine reward effect,but the mechanism remains unclear.Toll-like receptor family is the important immune receptors of the central nervous system and is involved in resisting the invasion of pathogens on the body and the aggregation of adaptive immune response.TLR4 is the Toll-like receptor of the mammal first discovered in 1997.Studies have shown that the TLR4 signaling pathway in the VTA region is involved in the regulation of cocaine-induced addiction and drug-seeking behavior,but its mechanism in morphine addiction remains unclear.STAT3 is one of the most important transcription factors in the family of signal transducers and activators(STAT),which is closely related to proinflammatory and immune signaling pathways.Studies have shown that LPS/TLR4 can induce proinflammatory response,activate STAT3 signaling pathway and regulate the occurrence of immune inflammatory disease.It has been reported that activation of STAT3(p-STAT3)in VTA play a critical role for rewarding effects of running and food intake.This study clarifies that the molecular mechanism of the VTA region in morphine addiction is the basis for prevention and treatment of addiction.This study was to investigate the mechanism of TLR4-mediated central immune proinflammatory signaling pathway in the process of morphine addiction,and to investigate whether TLR4 in the VTA region is involved in the acquisition and maintenance of morphine reward by activating its downstream STAT3 signaling pathway.Subjects and Methods:The present study chose male Sprague-Dawley adult rats and STAT3flox/flox mice as the research object,and used a randomized double-blind method.Conditioned place preference test,the brain region trace administration,immunofluorescence histochemical technique and western-blot were used to study the role of activating TLR4/STAT3 signaling pathway in VTA in morphine-induced CPP.Results:1.Injecting TLR4 antagonist LPS-RS or STAT3 activity inhibitor S3I-201 into the VTA inhibited the acquisition of morphine-induced CPP;2.Following the acquisition of morphine CPP,a single LPS-RS or S3I-201 injection into the VTA failed to block the expression of morphine CPP.But administration of LPS-RS or S3I-201 into the VTA for consecutive 5 days following the CPP induction blocked the maintenance of morphine CPP.3.The phosphorylated STAT3 in VTA was considerably increased following chronic morphine treatment.4.After injecting AAV-Cre-GFP into the VTA,the expression of AAV-Cre-GFP is colocalized with TH-positive cell in the VTA area of STAT3flox/flox mice,and the expression of both STAT3 protein and mRNA in VTA area were significantly reduced.And conditioning scores was greatly attenuated.Local knockout of STAT3 in the VTA area impaired the acquisition of morphine CPP.5.Following the establishment of CPP the TLR4 expression is colocalized with p-STAT3-positive cell in VTA.And the inhibition of TLR4 significantly reduced the level of phosphorylated STAT3.Conclusion:Activation of TLR4/STAT3 signaling pathway in VTA plays an important role in the acquisition and maintenance of morphine-induced conditioned place preference.And this signaling pathway might be a potential target for treatment of morphine addiction.