Study On Mechanism Of Dopamine/Norepinephrine Reuptake Inhibitor Amfebutamone In Treating Neuropathic Pain

PartⅠ:Changes of dopamine D2/α2 adrenergic receptors expression in the spinal dorsal horn of central sensitization rats with neuropathic painStudy background and objectiveNeuropathic pain(NP)is an intractable pain based on neuroplastic changes caused by somatosensory nervous system injury or lesions,with a high incidence and accompanied by anxiety,depression and sleep disorders,which seriously threatens the people’s health.Due to its numerous etiologies,various clinical manifestations,and complex pathogenesis,there are great challenges in the diagnosis and treatment of neuropathic pain in clinical practice.Therefore,elucidating its pathogenesis and finding effective therapeutic targets are currently the focus of attention.In recent years,a wealth of data has been accumulated on the molecular and cellular mechanisms of neuropathic pain,especially at the level of primary sensory neurons and the spinal cord.It is now generally accepted that peripheral sensitization is a major cause of the development of neuropathic pain and is the basis of central sensitization.Central sensitization,which occurs at the level of the spinal cord as well as above,is an important factor in the development and persistence of neuropathic pain.The spinal dorsal horn is the primary integrative center for pain and the primary center for central sensitization of neuropathic pain.Nociceptive information is converted into electrochemical signals by primary sensory neurons and transmitted here,followed by being further processed and uploaded to higher centers above the spinal cord to eventually form pain sensation.The central sensitization mechanism of spinal dorsal horn is complex,and the imbalance of descending inhibitory system and descending facilitatory system plays an important role in the occurrence and development of neuropathic pain.Studies have shown that the weakening of the function of the descending inhibitory system is an important cause of imbalance,and the main factor is the disinhibitory effect caused by serotonin and norepinephrine deficiency,especially norepinephrine.Alternatively,in addition to norepinephrine and serotonin,dopamine also plays an important role in the process of central sensitization.In our previous clinical research,we used radiofrequency therapy and drugs to treat neuropathic pain with dopamine dysfunction as the main cause,and achieved good therapeutic effects and fewer adverse reactions.In this study,we used the SNL animal model to observe the changes in the number of dopamine D2/α2 adrenergic receptors in the descending inhibitory system of neuropathic pain rats from the perspective of behavior,pathology,protein expression and gene expression,so as to investigate the role of the two receptors in the development and maintenance of neuropathic pain and provide a theoretical basis for the clinical treatment of neuropathic pain.Study methodThirty-six SD rats were randomly divided into three groups:control group,sham operation group and model group.Among them,the rat spinal nerve selective ligation model(SNL)was prepared in the model group.All rats were tested by von Frey method for mechanical withdrawal threshold(MWT)and hot plate test for paw withdrawal latency(PWL)for behavior test,and the occurrence and development of neuropathic pain were observed.HE stained sections of the sciatic nerve were used to observe the changes of neuronal cells after nerve injury.After successful modeling,the protein expression levels of dopamine D2 receptor(Drd2)and α2 adrenergic receptor(Adrα2)in the spinal dorsal horn of rats were detected by immunoblotting(Western blot),and the gene expression levels of dopamine D2 receptor and α2 adrenergic receptor in the spinal dorsal horn of rats were detected by real-time PCR.The changes of the two receptors during the development and maintenance of neuropathic pain were observed.Results1.After the establishment of SNL model in rats,the MWT and PWL were significantly decreased,reflecting the typical behavioral manifestations of neuropathic pain,while the neurons in the sciatic nerve were loosely arranged and neuronal cell injury occurred.However,none of the above findings appeared in the corresponding control group and sham operation group.2.The dopamine D2 receptor and α2 adrenergic receptor protein were significantly down-regulated in SNL model rats compared with the control and sham operation groups.3.The dopamine D2 receptor and α2 adrenergic receptor mRNA levels were significantly down-regulated in SNL model rats compared with the control and sham operation groups.Part Ⅱ:Effects of amfebutamone on dopamine and norepinephrine concentrations in the spinal dorsal horn of rats with neuropathic painStudy background and objectiveNeuropathic pain is pain resulting from damage or disease in the somatosensory system.Neuropathic pain is not a single disease,but a syndrome caused by many different diseases and damages,manifested as a series of symptoms and signs,covering more than 100 clinical diseases,seriously affecting the quality of life of patients.The pathogenesis of neuropathic pain is complex.Among the many mechanisms,the imbalance between the descending inhibitory system and the descending facilitatory system plays an important role.It has been shown that the cause of the imbalance between the descending inhibitory system and the descending facilitatory system is the weakening of the inhibitory system function.Inhibitory neurotransmitters involved in these pathways include norepinephrine,serotonin,dopamine,and endogenous opioid peptides.The multiple effects of these neurotransmitters on pain,mood,and sleep may also partially explain the high co-prevalence of pain with depression,anxiety,sleep disorders,etc.In clinical practice,treatment is usually performed with antidepressants,mainly including tricyclic antidepressants and norepinephrine/serotonin reuptake inhibitors.Amfebutamone,a second-generation non-tricyclic antidepressant,is a norepinephrine and dopamine reuptake inhibitor with no significant affinity for muscarinic receptors,histaminergic receptors,or adrenergic receptors,and its target of action and adverse reactions are very different from those of other antidepressants.It is a new therapeutic agent.In this study,we used the SNL animal model to evaluate the analgesic effect of amfebutamone and explored the mechanism of action of amfebutamone in the treatment of neuropathic pain from the perspectives of behavior,pathology,and norepinephrine and dopamine concentrations in the spinal dorsal horn,providing a theoretical basis for its clinical application.Study methodThirty SD rats were randomly divided into 5 groups:control group,sham operation group,model group,treatment group and drug control group.The rat spinal nerve selective ligation model(SNL)was prepared in the model group and treatment group.On the basis of the model group,the rats in the treatment group were intragastrically administered with amfebutamone 38 mg/kg every day,while the drug control group did not receive any modeling treatment and received amfebutamone 38 mg/kg intragastrically every day.All rats were tested by von Frey method for MWT and hot plate test for PWL for behavior test,and the occurrence,development and treatment effects of neuropathic pain were observed.HE stained sections of the sciatic nerve were used to observe the changes of neuronal cells after nerve injury,with and without treatment.After successful modeling,the protein expression levels of dopamine D2 receptor(Drd2)and α2 adrenergic receptor(Adra2a)in the spinal dorsal horn of rats were detected by immunoblotting(Western blot),and the gene expression levels of dopamine D2 receptor andα2 adrenergic receptor in the spinal dorsal horn of rats were detected by real-time PCR.At the same time,dopamine and norepinephrine levels in rat spinal dorsal horn tissues were measured by enzyme-linked immunosorbent assay(ELISA).The possible mechanism of action of amfebutamone in the treatment of neuropathic pain was observed.Results1.After the establishment of SNL model in rats,the MWT and PWL in the model group and treatment group were significantly decreased,reflecting the typical behavioral manifestations of neuropathic pain.2.The arrangement structure of neurons in the sciatic nerve tissue of rats in the normal group,the sham operation group and drug control group was intact,without obvious pathological changes.The neurons in the sciatic nerve of rats in the model group were loosely arranged,and neuronal cell injury occurred.Neuronal cell injury also occurred in the histopathology of the sciatic nerve of rats in the treatment group,but the changes were alleviated compared with those in the model group.3.Compared with the normal group,the sham operation group and drug control group,the levels of dopamine and norepinephrine in the model group and treatment group were significantly downregulated,but the levels of transmitters in the treatment group were higher than those in the model group.Conclusion1.Neuropathic pain induced by SNL model resulted in the down-regulation of dopamine D2 receptor and α2 adrenergic receptor in the spinal dorsal horn,revealing that the two receptors might be involved in the occurrence and development of neuropathic pain。2.Amfebutamone has a significant analgesic effect on neuropathic pain resulting from nerve injury.Its mechanism of action may be related to the increase of dopamine and norepinephrine concentrations in the spinal dorsal horn.In summary,amfebutamone,as a second-generation non-tricyclic antidepressant,has a good potential application value for the clinical treatment of neuropathic pain.