Role Of 5-HT1A-mediated Upregulation Of Brain Indoleamine 2,3 Dioxygenase 1 In The Reduced Antidepressant And Antihyperalgesic Effects Of Fluoxetine During Maintenance Treatment

Backgroud:The symptoms of depression comorbid with chronic pain greatly limit the clinical management of chronic pain.Due to the comorbidity of chronic pain and depression,antidepressants have gradually become the first-line treatment for chronic pain in clinical practice.Tricyclic antidepressants and selective serotonin and/or norepinephrine reuptake inhibitors(SSRIs or SNRIs)have good pain relief effects.However,antidepressants can cause a decrease in efficacy during maintenance treatment,and this phenomenon is more pronounced in SSRIs(such as fluoxetine),but little is known about the molecular mechanism of this phenomenon.Objectives:In three comorbid pain and depression animal models(genetic predisposition,chronic social stress,CFA-induced arthritis),we used knockout(KO)mice and techniques such as stereotaxic injection(SI),in vivo electrophysiology,and high-performance liquid chromatography,to study the roles of 5-hydroxytryptamine 1A(5-HT1A)autoreceptors and indoleamine 2,3-dioxygenase 1(IDO1,a tryptophan-limited enzyme)in the dorsal raphe nucleus(DRN)in the development of tolerance to fluoxetine treatment in chronic pain and depression comorbidity.These results provide a new strategy to improving the therapeutic efficacy of SSRI during maintenance treatment.Methods:(1)The study involved constructing three chronic pain and depression comorbidity animal models,namely genetic(WKY rats),chronic social stress,and CFA-induced arthritis.Fluoxetine was injected intraperitoneally and changes in thermal pain threshold(measured by the paw withdrawal latency in the thermal test,TH)and depression(measured by the forced swimming test,FST)were observed in rats during maintenance treatment with fluoxetine.(2)Protein levels of 5-HT1 A autoreceptor and IDO1 were detected by Western blot in the dorsal raphe nucleus(DRN)of three kind of chronic pain and depression comorbidity rats treated with long-term fluoxetine.Additionally,the metabolites of tryptophan were analyzed by high-performance liquid chromatography(HPLC).In vivo electrophysiology techniques was used to record hippocampal CA3 pyramidal neuron activities.Manipulating the 5-HT1 A autoreceptor in the DRN by SI.In vitro experiments,RN46 A cells(an immortalized serotonergic neuronal cell line)were exposed to PBS or fluoxetine for 0.5,2,8,or 24 hours.The m RNA levels of 5-HT1 A and IDO1 in RN46 A cells were detected by real time quantitative PCR.The nuclear transcription factor level of RN46 A cells exposed to fluoxetine was also detected using Western Blot and immunofluorescence methods.Finally,the effect of fluoxetine on IDO1 m RNA expression of RN46 A cells was detected by PCR under the inhibition of nuclear transcription(ACHP,nuclear transcription inhibitor).(3)WKY rats were microinjected a selective 5-HT1 A receptor antagonist(WAY100635)into the DRN for 14 consecutive days.Changes in pain and depression behaviors were observed during the combined treatment with fluoxetine,and the m RNA and protein levels of5-HT1 A receptor and IDO1 were detected.5-HT1 A receptor KO mice combined with a CFA-induced arthritis model was used to observe fluoxetine tolerance and detect the expression of 5-HT1 A receptor and IDO1.In the CSS and CFA models,the combined use of 1-MT(IDO1inhibitor,i.p.)once daily for 28 consecutive days was used to observe its effects on the antidepressant and anti-hyperalgesic effects of fluoxetine,and detect the protein levels of 5-HT1 A receptor and IDO1 and the metabolites of tryptophan.Further validation was performed by SI of1 MT into the DRN of WKY rats.Results:(1)Compared with the control group,the WKY,CSS,and CFA-induced arthritis rat models all showed decreased thermal pain threshold and increased immobility time in the FST.In the WKY group,there were no significant changes in thermal pain threshold and forced swimming immobility time during the 14-day treatment with fluoxetine(10 mg/kg,i.p)(P>0.05).In the CSS and CFA model groups,compared with the solvent group,on the 14 th day after continuous intraperitoneal injection of fluoxetine,the immobility time in the FST was significantly reduced(P<0.01),and the thermal pain threshold was significantly increased(P<0.01)in the fluoxetine group rats,but both recovered to pre-treatment levels on the 28 th day after drug administration(P>0.05).(2)WB results showed that the expression of 5-HT1 A autoreceptor and IDO1 protein in the DRN of WKY rats was significantly higher than that of Wistar rats(P<0.01).Compared with the solvent group,fluoxetine treatment further increased the expression of 5-HT1 A autoreceptor(P<0.01)and IDO1(P<0.05)in the DRN of WKY rats.Electrical stimulation of the DRN significantly inhibited the firing rate of hippocampal CA3 pyramidal neurons(P<0.01),and intravenous injection of WAY100635(50μg/kg)reversed the inhibitory response induced by electrical stimulation(P>0.05).In cell experiments,compared with the PBS-treated group,fluoxetine treatment significantly upregulated the levels of 5-HT1A(0.5h,P<0.01)and IDO1 m RNA(2h,P<0.05)in RN46 A cells.WB and immunofluorescence results showed that fluoxetine treatment upregulated the level of NF-k B p65 in the nucleus of RN46 A cells.PCR results showed that both LPS(P<0.01)and fluoxetine(P<0.05)exposure significantly increased the expression of IDO1 m RNA.There was no significant difference in IDO1 m RNA expression between the ACHP group and the ACHP+fluoxetine group compared with the PBS group(P>0.05).(3)Fluoxetine combined with DRN-SI WAY100635 reduced immobility time(P<0.05)and increased thermal pain threshold(P<0.01)of WKY rats,and reversed the increased expression of 5-HT1 A autoreceptors and IDO1 induced by fluoxetine(P<0.01).In the CFA model,continuous fluoxetine treatment on day 14 and day 28 decreased immobility time(P<0.05)and increased thermal pain threshold(P<0.001),but did not change IDO1 protein expression in the DRN of KO mice(P>0.05).In CSS and CFA model,compared to fluoxetine alone,combined with 1-MT(i.p)reduced immobility time(P<0.05)and increased thermal pain threshold(P<0.001)on day28.Combined with DRN-SI 1-MT also reduced immobility time(P<0.05)and increased thermal pain threshold(P<0.001)of WKY rats on day14.But this combination did not change the expression of 5-HT1 A receptor in DRN(P>0.05).Conclusion:In three comorbid pain and depression animal models(genetic predisposition,chronic social stress,arthritis),we demonstrated the reduced antidepressant and antihyperalgesic effects of fluoxetine during its maintenance treatment.5-HT1 A autoreceptor in DRN may mediate the reduced antidepressant and antihyperalgesic effects of fluoxetine via NF-k B-IDO1 pathway.