Study On The Mechanism Of Dapagliflozin Alleviating Depressive-like Behavior Of Diabetes Mellitus Rats Through Inhibiting Lateral Habenula

Objectives:The incidence of diabetes mellitus(DM)is high and the harm is great.The incidence of depression in DM patients is 2-3 times of the general population,which seriously affects the life of DM patients and even is one of the important causes of death.Due to its unknown pathogenesis,effective clinical treatment is limited.Dapagliflozin is a novel oral hypoglycemic agent,belonging to the class of sodium-glucose cotransporter 2(SGLT2)inhibitors.It is noteworthy that dapagliflozin can improve depressive symptoms of DM patients,but the mechanism is unknown,and the central nucleus involved is little known.Studies have shown that the level of5-hydroxytryptamine(5-HT)in the brain of DM animal models is significantly reduced,and the reduction of 5-HT neurotransmitter plays an important role in the onset of depression.The dorsal raphe nucleus(DRN)is a key site for the synthesis and release of 5-HT in the brain,but some antidepressants do not target the DRN,suggesting that its upstream structure may play a more important role in the pathogenesis of depression.The lateral habenula(LHb)is a key brain nucleus controlling DRN,and its important role in the pathogenesis of depression has been confirmed in animal experiments and clinical treatment.There is SGLT2 distributed in the LHb.Therefore,we speculated that dapagliflozin may change the level of 5-HT in DRN by influencing the activity of LHb neurons,thereby improving the depressive-like behavior of DM rats.The purpose of this study was to investigate the role of LHb in depressive-like behavior of DM rats and the mechanism of dapagliflozin in improving depressive-like behavior of DM rats through LHb,and provid new targets for the prevention and treatment of depression in DM patients.Methods:This study mainly used brain stereotaxic technology,cannula implantation and microinjection technology,chemogenetic technology,high performance liquid chromatography,molecular biology technologies,bioinformatics analysis,patch clamp electrophysiological recording and so on.(1)Establishment of DM depressed model and behavioral detectionThe DM model was established by 6-week high fat diet(HFD)combined with low-dose streptozotocin(STZ,35 mg/kg).Diabetes mellitus depressed(DMD)model was screened by forced swim test(FST).FST,open field test(OFT)and sucrose preference test(SPT)were used to detect the behavior of rats and verify the DMD model.(2)Dapagliflozin was given by intragastric administrationDMD rats were intragastric administrated dapagliflozin(1 mg/kg/d)for 6 weeks,and blood glucose,depressive-like behavior,the expression of c-Fos,SGLT2 and AMPK-GABABR signaling pathway related protein in LHb rats were observed.(3)Microinjection of dapagliflozin in LHbCannula was implanted in LHb of DMD rat by brain stereotaxic technique,and then dapagliflozin was injected into LHb(500 ng/ml,0.2 μl/side).After 30 min later,blood glucose,depressive-like behavior,c-Fos expression in LHb,5-HT activity in DRN,SGLT2 and AMPK-GABABR signaling pathway related protein expression in LHb of DMD rats were observed.(4)Manipulation of LHb neuronal activity by chemogeneticsThe inhibitory virus r AAV2/9-Ca MKIIa-h M4D(Gi)-m Cherry-WPREs was injected into LHb of DMD rats,and h M4 D was statically expressed in LHb after 3weeks.Glutaminergic neurons of LHb was inhibited by intraperitoneal injection of CNO(2 g/kg),and the changes of blood glucose and depressive-like behavior of DMD rats were observed after 45 min.(5)The levels of 5-HT and its metabolites in DRN were detected by high performance liquid chromatography5-HT and its metabolite 5-hydroxy-indoleacid(5-HIAA)in DRN of DMD rats were detected after microinjection of dapagliflozin in LHb,and 5-HIAA/5-HT ratio was calculated to evaluate 5-HT activity in DRN.(6)Bioinformatics analysis screened relevant signal pathwaysThe core risk genes of DM complicated with depression were screened,and KEGG pathway enrichment analysis was performed to identify the signaling pathways that may affect depressive-like behavior of DM rats.(7)Molecular biological techniques were used to detect the expression of related proteinsThe expression of c-Fos in LHb was detected by immunohistochemical staining.The expression of c-Fos,SGLT2,p-AMPK,AMPK,GABABR2 and p-GABABR2(S783)in the LHb was detected by Western blot.(8)Patch-clamp electrophysiological recording was used to record neuronal dischargeThe difference of discharge frequency of LHb neurons in Control group and DMD group was compared,and the effect of dapagliflozin(20 μM)perfusion on the discharge frequency of LHb neurons in DMD rats was observed.Results:(1)DMD model was successfully establishedCompared with the normal rats,DMD rats showed elevated blood glucose and depressive-like behavior: the central duration of OFT was significantly reduced,the immobility time of FST was significantly increased,and the sucrose preference index of SPT was significantly decreased.(2)The effects of intragastric administration of dapagliflozin on blood glucose and depressive-like behavior of DMD ratsCompared with the control group,dapagliflozin administration significantly improved the blood glucose and depressive-like behavior of DMD rats: the central duration of OFT was increased,the immobility time of FST was decreased,and the sucrose preference index of SPT was increased.(3)The role of LHb in depressive-like behavior in DMD ratsImmunohistochemical staining and Western blot showed that compared with the control group,the expression of c-Fos in LHb of DMD rats was significantly increased,and dapagliflozin administration could significantly reduce the expression of c-Fos.Compared with the control group,specific inhibition of glutaminergic neurons in LHb significantly improved blood glucose and depressive-like behavior of DMD rats:the central duration of OFT was increased,the immobility time of FST was decreased,and the sucrose preference index of SPT was increased.(4)Dapagliflozin alleviating depressive-like behavior of diabetes mellitus rats through inhibiting LHbCompared with the control group,local microinjection of dapagliflozin in LHb significantly improved the depressive-like behavior of DMD rats: the central duration of OFT was increased,the immobility time of FST was decreased,and the sucrose preference index of SPT was increased.Compared with the control group,local microinjection of dapagliflozin in LHb can significantly reduce c-Fos expression in LHb of DMD rats,and significantly increase 5-HIAA/5-HT in DRN,which means the 5-HT activity was increased,but there is no improvement in blood glucose.The electrophysiological results showed that compared with normal rats,the firing frequency of LHb neurons of DMD rats was significantly increased,and dapagliflozin perfusion could inhibit the firing frequency of LHb neurons of DMD rats.(5)The core risk genes and signaling pathways affecting depressive-like behavior of DM rats were explored by bioinformatics analysisAccording to bioinformatics analysis,a total of 576 risk genes of DM complicated with depression were obtained.PPI network of DM complicated with depression was constructed.GABABR was found to be one of the core risk genes of DM complicated with depression through two screening.A total of 194 KEGG pathways were identified in the enrichment analysis of risk genes of DM complicated with depression,and AMPK signaling pathway was the main pathway involved in the regulation of DM complicated with depression.These suggested that AMPK-GABABR may be involved in the regulation of LHb neuronal activity.Verify the results of bioinformatics analysis: compared with the normal rats,the expression of SGLT2 in LHb of DMD rats was up-regulated,the expression of p-AMPK and p-GABABR2(S783)was down-regulated,and the AMPK-GABABR signaling pathway was inactivated.The differences were statistically significant.Dapagliflozin was administrated by gavage or local microinjected in LHb could down-regulate SGLT2 and up-regulate the expression of p-AMPK and p-GABABR2(S783)in LHb of DMD rats,and the AMPK-GABABR signaling pathway was activated.The difference was statistically significant.Conclusion:The depressive-like behavior of DM rats is related to the increase of LHb neuron activity,and inhibition of LHb neuron activity can improve the depressive-like behavior of DM rats.LHb is the target of dapagliflozin and mediates the antidepressant effect of dapagliflozin in DM rats.Dapagliflozin inhibits LHb neuronal activity by activating AMPK-GABABR signaling pathway of LHb,and then increases the 5-HT activity of DRN to realize its antidepressant effect.