The Effect And Mechanism Of I2R-mediated Hippocampal Neurogenesis In DEX Alleviating Chronic Pain-induced Depression

Depression,a common mood disorder,has an increasing incidence rate year by year,which not only causes serious distress to patients and families,but also increases the economic burden of society.A variety of physical ailments can trigger depression,with chronic pain being one of the most common triggers.However,at present,the mechanism of chronic pain-induced depression(CPD)is unclear,and there is a lack of effective drug treatment.Dexmedetomidine(DEX)is a sedative drug that is often used in clinical practice.Preliminary studies have found that DEX may be a potential antidepressant,but it has not been confirmed in the CPD model and the antidepressant mechanism is also unclear.In this study,DEX was first used to treat CPD model mice to observe the antidepressant effect and dose-response relationship of DEX,and to preliminarily explore the role of hippocampal neurogenesis in DEX antidepressant.Then,combined with the pharmacological characteristics of DEX,the endogenous binding sites of DEX in the mouse brain were centrally blocked,and the key receptors of DEX’s antidepressant effect and the mechanism mediated by mitochondrial dynamic proteins were discussed.Finally,the mitochondrial dynamics mechanism of DEX-promoting neurogenesis was explored in vitro.Through the above experiments,we hope to clarify the antidepressant effect of DEX and explore the new mechanism of DEX promoting hippocampal neurogenesis through key receptors to regulate mitochondrial fission to produce antidepressant effect,and to expand new clinical uses of DEX.Part Ⅰ.Dose-effect and mechanism of DEX on chronic pain-induced depression in adult miceObjective:The aim of this study is to explore the dose-response effect and the intrinsic mechanism of DEX on depression phenotype in adult mice with CPD.Methods:Adult C57BL/6 mice aged 6-8 weeks were randomly divided into sham operation group(Sham group,n=8)and chronic pain-induced depression model group(CPD group,n=72).The mice in CPD model group were divided into six subgroups:CPDcontrol group,CPD-DEX6.25 group,CPD-DEX12.5 group,CPD-DEX25 group,CPDDEX50 group,and CPD-DEX100 group.The mice in the Sham group were only skin incised,and the mice in the CPD group underwent right sciatic nerve ligation.After 14 days,the mice in the CPD group were validated by source preference test(SPT),force swimming test(FST),paw thermal withdrawal latency(PTWL)and serum corticosterone(CORT)level.A successful model was defined when mice simultaneously showed decreased sucrose preference,prolonged FST immobility time,shortened PTWL,and increased CORT.After successful modeling,the mice in the CPD group were intraperitoneal injected(ip.)with 0.1 mL/10 g of normal saline(NS),6.25 μg/kg of DEX,12.5 μg/kg of DEX,25 μg/kg of DEX,50 μg/kg of DEX,and 100 μg/kg of DEX,respectively.Mice were treated once a day for consecutively 7 days.The pyrimidine nucleotide analog BrdU was administered to label dividing cells from 5th to 7th days(50 mg/kg,once a day).Sucrose preference,FST immobility time and PTWL were detected 24 hours after the 7-day DEX intervention.Then the CORT level of mice was detected by ELISA method.The expression of BrdU+cells and DCX+cells in the dentate gyrus(DG area)were detected by immunofluorescence.Result:(1)The success rate of the CPD model was 62.5%.(2)When DEX<25 μg/kg,the sucrose preference was improved and the FST immobility time was shortened with the increase of dose;When DEX>25 μg/kg,the preference of sucrose gradually decreased,and the immobility time of FST was prolonged with the increase of dose.The antidepressant dose-response relationship of DEX showed an inverted "U" shape and 25 μg/kg could produce the best antidepressant effect.(3)The analgesic effect of DEX was inconsistent with the antidepressant effect.When DEX≥6.25 μg/kg,the PTWL was prolonged and the serum CORT was decreased,and it did not change with increasing dose of DEX.The analgesic effect showed a "cap" effect.(4)DEX promotes neurogenesis in the DG region of the hippocampus.Compared with the Sham group,the percentages of BrdU+and DCX+cells in the hippocampus of the CPD-control group were significantly decreased.When DEX<25 μg/kg,the percentage of BrdU+ and DCX+ cells gradually increased with the increase of dose;when DEX>25 μg/kg,the percentage of BrdU+and DCX+cells gradually decreased with the increase of dose.DCX at a dose of 25 μg/kg showed the best promoting effect on hippocampal DG neurogenesis.Conclusion:The dose-effect of DEX in alleviating chronic pain-induced depression showed an inverted "U" shape,and 25 μg/kg was the optimal dose.Adult mice hippocampal DG neurogenesis played a role in DEX alleviating chronic pain-induced depression.Part Ⅱ.Mechanism of DEX improving chronic pain-induced depression by regulating mitochondrial dynamin via I2RObjective:By centrally blocking the endogenous binding sites of DEX,which include α2 adrenergic receptors(α2-AR),imidazoline 1 receptors(I1R)and imidazoline 2 receptors(I2R),to explore the key receptors of DEX’s antidepressant effect and the mechanism mediated by mitochondrial dynamic proteins.Methods:C57BL/6 mice aged 6 to 8 weeks were randomly divided into Sham group(n=6)and CPD group(n=64).Only the skin in the Sham group was incised,and the right sciatic nerve was ligated in the CPD group.14 days after the operation,the mice that were verified to be successfully modeled were included in the CPD group and further divided into six subgroups(n=6 in each group):CPD-control group,CPD-DEX group,CPD-DEX+artificial cerebrospinal fluid(aCSF)group,CPD-DEX+Yohimbine(Yoh,a2-AR antagonist)group,CPD-DEX+Efaroxan(Efa,I1R antagonist)group,and CPD-DEX+Idazoxan(Ida,I2R antagonist)group.The mice in the CPD group underwent lateral ventricle puncture.The Sham group was administered NS 0.1mL/10g ip.According to grouping,CPD mice were given 0.1mL/10g NS ip.,25 μg/kg of DEX ip.,25 μg/kg of DEX ip.+3 μL aCSF injected by Lateral ventricular(icv.),25 μg/kg of DEX ip.+ 5 μg Yoh icv.,25 μg/kg of DEX ip.+10 μg Efa icv.,25 μg/kg of DEX ip.+4 μg Ida icv,respectively.These drugs were injected once a day for consecutive 7 days.From day 5th to 7th of intervention,the CPD group was injected with 50 mg/kg BrdU(ip.)once a day for consecutive 3 days.Sucrose preference,FST immobility time and PTWL were detected 24 h after the 7-day intervention.Then the CORT level of mice was detected by ELISA method.Before the brain tissue isolation,methylene blue was injected(icv.)to identify the location of the lateral ventricle intubation.The BrdU+and DCX+cells in the hippocampal DG region were detected by immunofluorescence.Western blot was used to detect mitochondrial kinetic protein in the hippocampus which include optic atrophy protein 1(OPA1),mitochondrial fusion protein 1(Mfnl),mitochondrial fusion protein 2(Mfn2)and mitochondrial dynamics-related protein 1(Drpl).Result:(1)Model validation:Compared with the Sham group,the sucrose preference of CPD mice decreased,the FST immobility time was prolonged,the PTWL was shortened,and the CORT was increased,indicating the success of the modeling.(2)Depression phenotype:After DEX intervention,compared with the CPD-control group,the mice in the CPD-DEX group and the CPD-DEX+aCSF group had increased sucrose preference and shortened FST immobility time;there was no statistical difference between the CPD-DEX group and the CPD-DEX+aCSF group.Compared with CPD-DEX+aCSF group,the sucrose preference in the CPD-DEX+Ida group was decreased and the immobility time of FST was prolonged.There was no statistical difference between the other groups injected with antagonists and the CPD-DEX+aCSF group.(3)Pain phenotype:There were no statistical difference in PTWL and CORT among CPD-DEX,CPD-DEX+aCSF,CPDDEX+Yoh,CPD-DEX+Efa,CPD-DEX+Ida groups.(4)Regeneration of neurons in the hippocampal DG area:compared with the Sham group,the percentages of BrdU+and DCX+cells in the hippocampal DG area of the CPD-control group were decreased.Compared with the CPD-control group,the percentages of BrdU+ cells and DCX+ cells in the hippocampus of the CPD-DEX group and the CPD-DEX+aCSF group increased,but there was no statistical difference between the CPD-DEX group and the CPD-DEX+aCSF group.Compared with the CPD-DEX+aCSF group,the percentages of BrdU+cells and DCX+cells in the hippocampal DG area of the CPD-DEX+Ida group decreased;there was no significant difference between the other antagonist groups compared with the CPDDEX+aCSF group.(5)Mitochondrial dynamic protein:compared with the Sham group,the expression of Drpl in the CPD-control group was down-regulated;compared with the CPD-control group,the expression of Drp1 in the CPD-DEX group and the CPDDEX+aCSF group was up-regulated.However,there was no statistical difference between the CPD-DEX+aCSF group and the CPD-DEX+aCSF group.Compared with the CPDDEX+aCSF group,the expression of Drp1 in the CPD-DEX+Ida group was downregulated,and there was no statistical difference between the other antagonist groups and the CPD-DEX+aCSF group.There was no statistical difference in the data of OPA1,Mfn1 and Mfn2 among the groups.Conclusion:I2R is a key receptor for the antidepressant effect of DEX at a dose of 25 μg/kg;I2R promotes hippocampal neurogenesis through the upregulation of Drpl to alleviate chronic pain-induced depression.Part Ⅲ.Mechanism of DEX promoting eurogenesis in the hippocampus mediated by Drpl mitochondrial translocationObjective:The aim of this study is to investigate the mechanism of mitochondrial dynamics-mediated DEX pro-neurogenesis in hippocampal neural stem cells(NSCs)transfected with mitochondrial red fluorescent protein autophagic adenovirus(HBADMito-DsRed).Methods:The hippocampal NSCs of C57BL/6 mice were used in the experiment.The NSCs grown in the state of suspension neurospheres were processed into single suspension NSCs,which were seeded on PDL/Laminin-coated 24-well plates/slides at a density of 2×15 cells/well or seeded on PDL/Laminin-coated 6-well plates at a density of 8×105 cells/well,then incubated overnight in NSCs complete medium.The plates were incubated overnight with NSCs complete medium.After adhered,NSCs were transfected at a multiplicity of infection(MOI)of 200.NSCs were divided into three groups:Control group,DEX group and DEX+KN93(CaMK Ⅱ specific antagonist)group,and each group was repeated three times.NSCs complete medium,10 μM of DEX with NSCs complete medium,and 10 μM of DEX with 0.37 μM of KN93 added in NSCs complete medium were used to incubate cells respectively.After 7 days,the cell slides were taken for immunofluorescence detection,and the proportion of betaⅢ-tubulin+cells and the length of intracellular mitochondria co-expressed by betaⅢ-tubulin and mito-DsRed were observed.After the adherent cells were lysed,the expressions of CaMKⅡ and Drp1 were determined by ELISA.Adherent cells were assayed for the expression of Drpser616 and Drpser637 by Western blot.Result:(1)Compared with the Control group,percentage of betaⅢ-tubulin+cell in the DEX group were significantly increased;compared with the DEX group,the percentage in the DEX+KN93 group decreased.However,there was no statistical difference between the DEX+KN93 group and the Control group.(2)Compared with the Control group,the mitochondria of the betaⅢ-tubulin cells in the DEX group were in a split state,and the length of mitochondrion was significantly shorter than that in the Control group;while the mitochondria of the beta Ⅲ-tubulin+cells in the KN93 group were in a fusion state,and the length was longer than that in the DEX group.However,there was no significant difference in mitochondrial length between DEX+KN93 group and Control group.(3)Compared with the Control group,the expressions of Drpl and CaMKⅡ in the DEX group were up-regulated;the expressions of Drp1 and CaMKⅡ in the DEX+KN93 group were lower than those in the DEX group,and there was no statistical difference between the DEX+KN93 group and the Control group.(4)Compared with the Control group,the expression of p-Drplser616 in the DEX group was increased;compared with the DEX group,the expression of p-Drp1ser616 in the DEX+KN93 group was decreased,but the there was no statistical difference between DEX+KN93 group and the Control group.There was no significant difference in the expression of p-Drp1ser637 among the three groups.Conclusion:DEX promotes the mitochondrial translocation of Drp1 by phosphorylating Drp1ser616 through the Ca2+/CaMKⅡ signaling pathway,thereby helping to mediate mitochondrial fission and upregulating the ability of neurogenesis.This study clarified that DEX has an antidepressant effect,and the optimal dose is 25μg/kg and neurogenesis in the hippocampal DG area is one of the antidepressant mechanisms of DEX.It is also confirmed that DEX affects the expression of mitochondrial dynamic protein Drpl through the key receptor I2R,then further promote neurogenesis to alleviate depression.Finally,the results of this study propose a mechanism by which DEX regulates mitochondrial fission and promotes neurogenesis through the phosphorylation of Drp1ser616 mediated by the Ca2+/CaMKⅡ signaling pathway.This study not only provide a new mechanism theory for the effect of DEX on promoting neurogenesis,but also expand the research field of new clinical uses of DEX.