| Objective:Spinal cord ischemia-reperfusion injury(SCII)is a common complication in clinical spine surgery and cardiothoracic surgery,which can cause Symptoms of severe motor nerve damage such as paraplegia,resulted in a significant economic burden to families and society.Therefore,finding potential neuroprotective strategies is of great importance.However,the pathogenesis of SCII is still unclear and may be related to neuroinflammatory responses,neuronal apoptosis,or pyroptosis.Pyroptosis is a new type of inflammation-related programmed cell death that mainly occurs in phagocytic immune cells.Microglia are essential immune cells in the central nervous system(CNS)that regulate neurological homeostasis and are the main cell type undergoing pyroptosis.In various CNS disease models,including ischemia-reperfusion injury,pyroptosis of microglia can lead to persistent inflammatory responses and oxidative stress reactions,releasing multiple inflammatory factors that can directly or indirectly affect the survival and function of neurons.Suppressing the pyroptosis of microglia could be a therapeutic target for inflammation-related CNS diseases.TREM2 is a membrane protein specifically expressed on the surface of microglia in the CNS,which is involved in inhibiting pyroptosis.The role of TREM2 in SCII has not yet been reported.Diosmetin(Dio),a small molecule compound,has significant anti-pyroptosis and anti-inflammatory effects.Our previous studies using Autodock software for molecular docking of the extracellular structure of TREM2 and the 3D structure of Diosmetin suggested that they could bind to each other.Therefore,this study firstly explores the role of pyroptosis in SCII,then investigates whether TREM2 can improve the neural injury caused by SCII through inhibiting pyroptosis and its possible mechanism.Furthermore,it also explores whether Diosmetin exerts neuroprotective functions in SCII through inhibiting the pyroptosis of microglia via TREM2,providing new insights and therapeutic targets for the clinical prevention and treatment of SCII.Methods:1.The role of pyroptosis in SCII was investigated both in vivo using Sprague Dawley(SD)rats and in vitro using rat HAPI cells.In vivo experiments were carried out to investigate the role of pyroptosis in SCII.SD rats were randomly divided into three groups: Sham group,SCII group,and SCII+VX-765 group.The SCII group simulated SCII by occluding the rat’s aorta for 14 minutes and then restoring blood flow,with the same surgical procedure but without aortic occlusion as the sham operation group(Sham group).The SCII+VX-765 group received an intraperitoneal injection of VX-765(30mg/kg/d)30 minutes before modeling and continued the medication for three days.The Tarlov scoring system and Hematoxylin-eosin(HE)staining were used to assess motor neuron function and morphology,and the expression changes of pyroptosis-related proteins in different experimental groups were detected by Western blotting.The in vitro experiment was divided into three groups: Control group,OGD/R group,and OGD/R+VX-765 group.Cells that did not undergo OGD/R treatment were classified as the Control group.The OGD/R group simulated SCII in vitro by subjecting rat HAPI cells to oxygen-glucose deprivation/reoxygenation(OGD/R).In the OGD/R+VX-765 group,HAPI cells were treated with the pyroptosis inhibitor VX-765(1μM)before undergoing OGD/R.Changes in the expression of pyroptosis proteins in each group at OGD(6h)/R(12h)were detected by Western blotting.To explore the effect of microglial pyroptosis on neurons,the culture medium of each group of HAPI cells was collected,centrifuged to obtain the supernatant,which was then used to intervene VSC4.1 neuronal cells.The VSC4.1 neuronal cells were divided into N group,N+ Control group,N+VX-765 group,N+OGD/R group,and N+OGD/R+VX-765 group.The N group did not have added microglial culture supernatant,but equal volume of fresh complete culture medium was added;the N+ Control group added Control group HAPI cell supernatant to VSC4.1 cells;the N+VX-765 group added Control+VX-765 group HAPI cell supernatant to VSC4.1 cells;the N+OGD/R group added OGD/R group HAPI cell supernatant to VSC4.1 cells;and the N+ OGD/R+VX-765 group added OGD/R+VX-765 group HAPI cell supernatant to VSC4.1 cells.The changes in the expression of the anti-apoptotic protein Bcl2 in neurons from different experimental groups were detected using immunofluorescence.The CCK-8 assay was used to detect the cell viability of neurons in different cell groupings.2.The role of TREM2 in the pyroptosis of microglia induced by SCII was investigated.In in vivo experiments,the effect of SCII on TREM2 expression was firstly determined.Rats were randomly divided into Sham group and SCII group,and the spinal cord lumbar enlargement(L1-3)tissues were taken at 6h,12 h,24h,3d,and 5d after SCII,and WB experiment was carried out to detect the expression of TREM2 protein at different time points.Secondly,to determine the role of TREM2 in pyroptosis of microglia caused by SCII,SD rats were divided into AAVNC group and AAV-TREM2 group.30 days before modeling,adeno-associated virus(AAV)empty vector and AAV overexpressing TREM2 were injected into the sheath respectively.Then SD rats were randomly divided into Sham group,SCII group,SCII+AAV-NC group and SCII+AAV-TREM2 group.The treatment measures of Sham group and SCII group were the same as before.30 days before modeling,adeno-associated virus(AAV)was injected into the sheath to overexpress TREM2 in the spinal cord of SD rats,which was the SCII+AAV-TREM2 group.Intrathecal injection of AAV empty vector was the SCII+AAV-NC group.Tarlov scoring system,HE staining,Nissl staining and other methods were used to evaluate the function and morphology of motor neurons,and WB experiment was used to detect the expression of pyroptosis-related proteins in each group.Secondly,the effect of OGD/R on TREM2 expression was tested.In in vitro experiments,HAPI cells were randomly divided into Control group and OGD/R group.Proteins were extracted after oxygen glucose deprivation for 6 hours and reoxygenation for 0h,6h,12 h,24h,and 36 h.The expression of TREM2 at different time points was detected by WB.Secondly,to explore the role of TREM2 in the pyroptosis of microglia induced by OGD/R,HAPI cells were divided into OE-NC group and OE-TREM2 group.The lentivirus carrying TREM2 and the blank lentivirus were transfected respectively,and the transfection effect was detected.Then HAPI cells were randomly divided into Control group,OGD/R group,OGD/R+OE-NC group and OGD/R+OE-TREM2 group.The treatment measures of Control group and OGD/R group were the same as before.The OGD/R+OE-NC group and OGD/R+OE-TREM2 group were transfected with the corresponding lentivirus before OGD/R.Immunofluorescence detected the expression of TREM2 and cleaved caspase-1 in each group.3.The potential mechanism of TREM2 regulating pyroptosis was investigated.FOXO1(Forkhead Box Protein O1)is an important transcription factor regulating various cellular activities,and some studies have found that FOXO1 is a downstream effector of TREM2.Therefore,the expression of FOXO1 was firstly detected at different time points 6h,12 h,24h,3d,and 5d after SCII in rats.The role of FOXO1 in pyroptosis of microglia induced by SCII was clarified.SD rats were randomly divided into 4 groups: Sham group,SCII group,SCII+siNC group and SCII+si-FOXO1 group.The treatment measures of Sham group and SCII group were the same as before.In SCII+si-FOXO1 group,small interfering RNA(si RNA)was injected intrathecally to reduce the expression of FOXO1.SCII+si-NC group was the negative control group of si RNA injection.The changes of Tarlov score,HE staining and pyroptosisrelated proteins in the lower limbs of rats in different groups were detected.In vitro experiments were used to explore the relationship between TREM2 and FOXO1.HAPI cells were randomly divided into Control group,OGD/R group,OGD/R+OE-NC group and OGD/R+OE-TREM2 group to detect the change of FOXO1 expression.The treatment methods of each group were the same as before.The change of FOXO1 expression in different groups was detected by immunofluorescence and WB.To further verify the relationship between TREM2 and FOXO1,HAPI cells were randomly divided into 3 groups: OGD/R group,OGD/R+OE-TREM2 group and OGD/R+OE-TREM2+OE-FOXO1 group.The treatment methods of OGD/R group and OGD/R+OE-TREM2 group were the same as before.In OGD/R+OE-TREM2+OE-FOXO1 group,two lentiviruses were used to transfect HAPI cells,so that they overexpressed TREM2 and FOXO1 before OGD/R.The expression of pyroptosis-related proteins in different groups was detected.To clarify whether TREM2 regulates FOXO1 through the PI3K/AKT pathway,HAPI cells were randomly divided into Control group,OGD/R group,OGD/R+OE-TREM2 group and OGD/R+OE-TREM2+LY294002 group.The treatment methods of Control group,OGD/R group and OGD/R+OE-TREM2 group were the same as before.OGD/R+OETREM2+LY294002 group was treated with PI3 K specific inhibitor LY294002(20μM)1h before the start of OGD/R on stably transfected HAPI cells overexpressing TREM2.WB was used to detect the expression of PI3 K,AKT and their phosphorylated proteins p-PI3 K,p-AKT,FOXO1 and pyroptosis-related proteins in different groups.Finally,JASPAR website was used to predict the regulatory relationship between FOXO1 and GSDMD,and CHIP-PCR experiment was used to verify the regulatory relationship between FOXO1 and GSDMD.4.Exploring the role of diosmetin in SCII and its relationship with TREM2.Firstly,the effect of diosmetin on SCII in SD rats was investigated in vivo.SD rats were randomly divided into three groups: the Sham group,the SCII group,and the SCII+Dio group.The treatment methods for the Sham group and SCII group were as described previously.For the SCII+Dio group,diosmetin was injected intraperitoneally for three consecutive days prior to SCII induction,at 80 mg/kg/day.The behavioral Tarlov scores and morphological changes of neurons observed through H&E staining were assessed in different groups.In vitro experiments were conducted to verify the impact of diosmetin on microglial pyroptosis.HAPI cells were randomly divided into the Control group,OGD/R group,and OGD/R+Dio group.The treatment methods for the Control group and OGD/R group were as previously described.For the OGD/R+Dio group,complete culture medium containing diosmetin(10μM)was added one hour before OGD/R.Changes in pyroptosis-related proteins were assessed in the different groups.Further research was conducted to explore the relationship between diosmetin and TREM2.Firstly,Autodock software was utilized to dock the 3D structures of the extracellular domain of TREM2 and diosmetin.Secondly,in vitro observations were made of the expression levels of pyroptosis-related proteins in cell groups treated with diosmetin when TREM2 was knocked down.To verify the predictive results of molecular docking,HAPI cells were divided into the Control group,OGD/R group,OGD/R+Dio group,and OGD/R+Dio+si-TREM2 group.The treatment methods for the Control group,OGD/R group,and OGD/R+Dio group were as described previously.For the OGD/R+Dio+si-TREM2 group,after si-TREM2 transfection,HAPI cells were switched to complete culture medium containing diosmetin(10μM)for one hour prior to OGD/R.The expression levels of pyroptosis-related proteins were observed in different groups.Results:1.In vitro experiments revealed that OGD/R induced microglial pyroptosis,and the use of the pyroptosis inhibitor VX-765 could suppress the increase in pyroptosis-related proteins caused by OGD/R.After OGD/R in HAPI cells,immunofluorescence detection found that compared with the Control group,IL-1β expression was significantly increased in the OGD/R group(p<0.01).After applying VX-765,the expression of IL-1β in the OGD/R+VX-765 group was decreased compared to the OGD/R group(p<0.01).WB results revealed an increase in the expression of NLRP3,IL-1β,and cleaved caspase-1 in the OGD/R group,while the OGD/R+VX-765 group reversed this trend(p<0.01).Further research found that the supernatant of microglia after OGD/R could cause a decrease in the anti-apoptotic protein Bcl2 in neurons,which VX-765 could reverse.The expression of Bcl2 in neurons in different groups was detected by immunofluorescence after treating neuronal cells with the culture supernatant from different experimental groups of microglia.Compared with the Control,Bcl2 in the N+OGD/R group was significantly decreased(p<0.01),while the expression of Bcl2 in the N+OGD/R+VX-765 group was increased compared to the N+OGD/R group(p<0.01).Using CCK-8 to detect cell viability,it was found that compared to the N+Control group,cell viability in the N+OGD/R group was significantly reduced(p<0.01),whereas cell viability in the N+OGD/R+VX-765 group was increased compared to the N+OGD/R group(p<0.01).In vivo experiments found that VX-765 could alleviate neuronal injury caused by SCII in rats.The results showed that compared with the Sham group,the SCII group had lower Tarlov scores,reduced numbers of intact neurons,and increased numbers of TUNEL-positive cells(p<0.01).Comparatively,the SCII+VX-765 group showed increased Tarlov scores,increased numbers of intact neurons,and reduced numbers of TUNEL-positive cells(p<0.01)compared to the SCII group.2.SCII and OGD/R induced a time-dependent increase in TREM2 expression,and overexpression of TREM2 could inhibit behavioral changes in rats and the increase in pyroptosis-related proteins caused by SCII and OGD/R.In vitro experiments with HAPI cells found that after OGD/R treatment,TREM2 expression levels were increased compared to the Control group,peaking 12 h after reoxygenation(p<0.01).Lentiviral transfection to upregulate TREM2 expression could alleviate pyroptosis in microglia caused by OGD/R.Immunofluorescence results showed that compared with the Control group,the expression of TREM2 and cleaved caspase-1 in the OGD/R group and OGD/R+OE-NC group was increased(p<0.05).In the OE-TREM2 group,which underwent lentiviral transfection,TREM2 expression was significantly higher than in the LV-NC group(p<0.01),and cleaved caspase-1expression in the LV-TREM2 group was significantly lower than in the LV-NC group(p<0.01).In vivo experiments with SD rats after SCII showed an increase in TREM2 expression,which began to rise at 12 h and peaked at 24h(p<0.01,p<0.05).Compared to the Sham group,the SCII group and SCII+AAV-NC group had significantly lower Tarlov scores and fewer intact neurons(p<0.01).Comparatively,the SCII+AAV-TREM2 group had increased Tarlov scores and more intact neurons compared to the AAV-NC group(p<0.01).WB results of the spinal cord samples suggested that compared to the Sham group,the expression of GSDMD,IL-1β,and cleaved caspase-1 was significantly increased in the SCII group(p<0.01).Compared to the SCII group,the expression of GSDMD,IL-1β,and cleaved caspase-1 was reduced in the SCII+AAVTREM2 group which overexpressed TREM2 through AAV(p<0.01).3.Overexpression of TREM2 promotes the phosphorylation of the PI3K/AKT/FOXO1 pathway,thereby inhibiting the expression of pyroptosis-related proteins.First,in vivo experiments found that the expression of FOXO1 increased 24 hours after SCII(p<0.01),peaking at 24 hours(p<0.05).Further investigation of the role of FOXO1 in SCII showed a decrease in Tarlov scores and the number of intact neurons in both the SCII and SCII+si-NC groups(p<0.01).However,compared with the SCII+si-NC group,the SCII+si-FOXO1 group showed an increase in Tarlov scores and the number of intact neurons(p<0.01).WB detection of changes in pyroptosis-related proteins in different groups found that the expressions of NLRP3,GSDMD,and IL-1β were significantly reduced in the SCII+si-FOXO1 group compared to the SCII+si-NC group(p<0.01).In vitro experiments investigating the relationship between TREM2 and FOXO1 found that the expression of FOXO1 in the OE-TREM2 group was lower than in the OE-NC group(p<0.01)as per immunofluorescence and WB tests.Further restoration experiments confirmed the regulatory relationship between TREM2 and FOXO1,showing lower expressions of NLRP3,GSDMD,IL-1β,and cleaved caspase-1 in the OGD/R+OE-TREM2+OE-FOXO1 group co-expressing TREM2 and FOXO1 compared to the OGD/R+OE-TREM2 group(p<0.01,p<0.01,p<0.05,p<0.05).To validate whether TREM2 regulates FOXO1 via the PI3K/AKT pathway,WB detection results found that the levels of PI3 K and AKT phosphorylation were higher in the OGD/R+LV-TREM2 group than in the OGD/R group(p<0.01,p<0.05),the expression of FOXO1 was reduced(p<0.01),and the pyroptosis-related proteins were also reduced(p<0.01).The use of LY294002 in pretreating overexpressed TREM2 HAPI cells reversed this phenomenon.The Jaspar website predicted that FOXO1 could regulate the transcription of GSDMD(Jaspar score=13.605804,relative score=0.949397120016357).Further CHIP-PCR experiments showed that the %INPUT in the IP group was significantly higher than in the Ig G group,proving that FOXO1 regulates the transcription of GSDMD.4.Diosmetin can inhibit SCII-induced changes in rat motor function and increase in pyroptosis-related proteins,possibly related to the binding to the membrane protein TREM2.To explore the role of diosmetin in SCII,the results showed that compared with the SCII group,the Tarlov scores and the number of intact neurons in the HE staining of rats in the SCII+Dio group that were pre-treated with diosmetin were both increased(p<0.01),suggesting that diosmetin can improve the neuronal damage caused by SCII.In vitro experiment results showed that the expressions of FOXO1,NLRP3,IL-1β,and cleaved caspase-1 in the OGD/R+Dio group were significantly lower compared with the OGD/R group(p<0.01).Autodock software results of molecular docking of the extracellular domain structure of TREM2 and the 3D structure of diosmetin showed a binding energy of-5.46kcal/mol,suggesting a potential target binding relationship.Further investigation into the relationship between diosmetin and TREM2 found that the expressions of FOXO1,GSDMD,IL-1β,and cleaved caspase-1 in the OGD/R+Dio+si-TREM2 group were all increased compared to the OGD/R+Dio group(p<0.05).These results suggest that diosmetin exerts anti-pyroptotic and neuroprotective effects via TREM2.Conclusions:Following spinal cord ischemia-reperfusion injury(SCII)in rats,significant pyroptosis occurs in microglia.Inhibiting cell pyroptosis can improve the damage to motor neurons in rat hind limbs caused by SCII.Overexpression of TREM2 can alleviate cell pyroptosis and neuronal damage caused by SCII and oxygen-glucose deprivation/reoxygenation(OGD/R).Further research revealed that TREM2 regulates the phosphorylation of FOXO1 via the PI3K/AKT pathway,thereby controlling GSDMD,and hence inhibiting microglial pyroptosis.The above studies unveil the mechanism of TREM2 in inhibiting cell pyroptosis induced by SCII and protecting neurons.In addition,we found that diosmetin may inhibit cell pyroptosis caused by SCII and improve neuronal function through TREM2,offering promise for translation from basic research to clinical application. |
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