| ObjectiveStroke is also called zhongfeng.It is a common and relatively acute cerebrovascular disease.There are many causes of stroke,mainly blockage and rupture of blood vessels in the brain,which correspond to ischemic stroke and hemorrhagic stroke respectively.Compared with hemorrhagic stroke,the incidence rate of ischemic stroke is higher.Stroke is currently one of the leading causes of death and disability among all diseases.Therefore,it is of great significance to explore the occurrence and development process of stroke related diseases and find new treatment approaches in the study of stroke related diseases.In the pathogenesis of ischemic stroke,brain neuron cells will be damaged.The main reason is the death of oxidative stress caused by cerebral ischemia,inflammation and energy metabolism.After an ischemic stroke,the oxidative stress homeostasis in the brain is quickly broken,and then a more serious inflammatory response occurs in the brain.For example,microglia and astrocytes will be activated within a few hours,and it produces inflammatory factors and chemokines,and causes the infiltration of white blood cells and macrophages,which damages the nervous system again.In many years,studies about the involvement of energy metabolism in the regulation of ischemic stroke,are not less,including AMPK/mTOR this pathway.The AMPK/mTOR signaling pathway cooperates in the cell to form a switch that controls the process of energy metabolism.The main function of mTOR signaling pathway is to regulate cell growth and proliferation.Target of rapamycin(mTOR)can form two different complexes mTORC1 and mTORC2.Among them,mTORC1 is regulated by a variety of signals,such as growth factors,amino acids and cell energy.As a key physiological energy sensor,adenylate-activated protein kinase(AMPK)can coordinate multiple metabolic pathways,balance energy supply and demand,and ultimately regulate the growth of cells and organs.It has been confirmed that AMPK can negatively regulate the activity of mTORC1 by phosphorylating Ser722 and Ser792 of Raptor in the mTORC1 complex.Previous studies have reported that the AMPK/mTOR signaling pathway plays a role in neuronal ischemia,showing that Sirt3 can protect neurons from ischemia by inducing autophagy,and the AMPK-mTOR pathway is involved in the process.Amyloid β precursor protein binding family A member 3(Mint3),early identified as Alzheimer’s disease β-amyloid precursor protein(APP)interacting factor.Mint3 interacts with several membrane proteins in the Golgi apparatus(such as APP and furin),and can regulate the transport of clathrin from the trans-Golgi apparatus.At the same time,by regulating the inhibit HIF-1,Mint3 enhances the ennergy of hypoxia-inducible factor 1(HIF-1)in macrophages and other cells.Mint3 can also enhance IFN-β expression and antiviral immune response induced by TLR3/4(Toll-like receptor 3/4)and RIG-I(retinoic acid inducible gene protein I).However,it is still unclear what role Mint3 plays in the process of ischemic stroke.Since Mint3 can regulate the expression of HIF-1,and the signal pathway is induced by hypoxia,it is similar to the pathogenesis of ischemic stroke.Therefore,we speculate that Mint3 can also play a role in the pathological regulation of ischemic stroke..We first tested whether Mint3 is involved in the pathological process of stroke by regulating immune cell function and inflammation,and found that it has no obvious effect.Therefore,we considered whether Mint3 can play an energy metabolism-related regulatory role in neuronal cells.At the same time,in the preliminary screening process,we found that Mint3 can interact with the AMPK pathway,so we focused on the regulation of Mint3 on the energy metabolism of ischemic stroke,especially the regulatory effect and molecular mechanism of Mint3 on the AMPK/mTOR pathway.This study is based on the ischemia-reperfusion model of mouse MCAO and the in vitro OGD model of mouse brain neurons.The aim is to clarify how Mint3 regulates the AMPK/mTOR pathway,thereby affecting the hypoxic neuronal cells.Energy metabolism process,which participates in nerve damage.It provides a new therapeutic target for the related damage caused by abnormal neuronal cell energy metabolism after ischemic stroke.Materials and Method1.The effect of Mint3 on the degree of nerve damage after ischemic stroke in mice1.1 Mint3+/+mice were divided into two groups,a control group and an experimental group.MCAO sham operation and MCAO operation were performed respectively.When the mice wake up,reperfusion was performed 2h after the surgery,and the brain of the surgical side of the mouse was taken 24h later for processing.The tissue was wiped evenly,and then RNA and protein were extracted.The protein and mRNA expression of Mint3 were detected by Western blot and RT-PCR.1.2 Mint3-/-mice and Mint3+/+mice were used for MCAO surgery.Reperfusion was performed after 2h.Whole brain tissue was taken after 24h,then frozen,sectioned,and then stained with TTC.Observe the effects of Mint3 on the cerebral infarction of mice after MCAO The effect of area.1.3 Mint3-/-mice and Mint3+/+mice were used for MCAO surgery.Reperfusion was performed 2h later,and their behavioral scores were recorded 24h later to compare the effects of Mint3 on the behavioral scores of mice after MCAO.1.4 MintS-/-mice and Mint3+/+mice were used for MCAO surgery,reperfusion was performed 2h later,and the whole brain was taken 24h later,paraffin section was performed,and immunofluorescence staining was performed.Use NEUN to label neuronal cells to make them appear red.The macrophages were labeled with CD68 to make them appear green.Through immunofluorescence staining experiments,observe the effect of Mint3 on the changes in the number of neurons and macrophages in the brain tissue of mice after MCAO.2.The effect of Mint3 on the mortality of mouse neuronal cells treated with OGD2.1 Mint3-/-mice and Mint3+/+mice were divided into two groups,the control group underwent MCAO sham operation and the experimental group underwent MCAO operation.2h later,reperfusion was performed.24h later,the cerebral tissue on the side of the rat cerebral stem was taken for processing and RNA was extracted.They were also able to make up the mRNA levels of IL-6 and TNF-alpha.They were also able to make up the mRNA levels of IL-6 and TNF-alpha.They were also able to make up the mRNA levels of TNF-alpha.2.2 Mint3-/-mice and Mint3+/+mice were used to extract primary neuronal cells,cultured until the primary neuronal cells matured,treated with OGD for 2h,and reoxygenated for 24h,then collected the cell supernatant to detect LDH release.The LDH release value was read by a microplate reader to detect the effect of Mint3 on the LDH release of primary neuronal cells after OGD treatment.2.3 Mint3-/-mice and Mint3+/+mice were used to extract primary neuronal cells,cultured until the primary neuronal cells matured,treated with OGD for 2h,and reoxygenated for 24h,then collected the cell supernatant to detect the release of CCK8.The CCK8 release value was read by a microplate reader to detect the effect of Mint3 on the CCK8 release of primary neuronal cells after OGD treatment.2.4 Use Mint3+/+mice to extract primary neuronal cells,culture them until the primary neuronal cells mature,and use transfection reagent INTERFERIN(?)to transfect Ctrl-siRNA and Mint3-siRNA into mouse neuronal cells,respectively.The culture was continued for 48 hours,OGD treatment was performed for 2 hours,and after reoxygenation for 24 hours,the cell supernatant was collected to detect the release of CCK8.The CCK8 release value was read by a microplate reader to detect the effect of Mint3 on the CCK8 release of primary neuronal cells after OGD treatment.3.The effect of ischemic stroke on the expression of core molecules in AMPK/mTOR pathway3.1 Mint3+/+mice were divided into two groups,the control group underwent MCAO sham operation,and the experimental group underwent MCAO operation.Reperfusion was performed after 2h.After 24h,the cerebral infarct side of the mouse was taken for processing,and protein was extracted.Western blot was used to detect the expression of molecular in the pathway we need,like p-mTOR,p-p70,p-4EBP1,p-AMPK,AMPKα,and Mint3 at the protein level Variety.Observe the effect of stroke on the expression of core molecules in AMPK/mTOR pathway in brain tissue.3.2 Mint3+/+ mice were used to extract primary neuronal cells and cultured until the primary neuronal cells matured.The experimental group was subjected to OGD treatment for 2 hours and reoxygenation for 24 hours.The control group was left untreated.The protein was extracted,and Western blot was used to detect the expression changes of p-mTOR,p-p70,p-4EBP1,p-AMPK,AMPKα,and Mint3 at the protein level.Observe the effect of OGD on the expression of core molecules in AMPK/mTOR pathway in primary neuronal cells.3.3 Transfect Ctrl-siRNA and Mint3-siRNA into PC 12 cells with the transfection reagent,and continue to culture for 48h,OGD treatment for 2h,reoxygenation for 24h,protein extraction,Western blot detection of p-mTOR,p-p70,P-4EBP1,p-AMPK,AMPKα,Mint3 expression changes at the protein level.Observe the effect of OGD on the expression of core molecules in AMPK/mTOR pathway in PC 12 cells.3.4 Use the transfection reagent to transfect Ctrl-siRNA,Mint3-siRNA and AMPKa-siRNA into PC12 cells respectively,and continue to culture for 48h.Collect the cell supernatant and detect the release of CCK8.The release value of CCK8 was read by a microplate reader to detect the effect of Mint3 and AMPKa on the proliferation rate of PC 12 cells.4.The effect of Mint3 on the expression of core molecules in AMPK/mTOR pathway after ischemic stroke4.1 Mint3-/-mice and Mint3+/+mice were used for MCAO surgery,and reperfusion was performed 2h later.After 24h,the cerebral infarct side of the mouse was taken for processing,and the protein was extracted.Western blot was used to detect p-mTOR and p-p70,P-4EBP1,p-AMPK,AMPKa expression changes at the protein level.Observe the effect of Mint3 on the expression of core molecules in the AMPK/mTOR pathway in the brain tissue of stroke mice.4.2 Mint3-/-mice and Mint3+/+mice were used to extract primary neuronal cells,treated with OGD for 2h,and reoxygenated for 24h,then protein was extracted.Western blot was used to detect the expression changes of p-mTOR,p-p70,p-4EBP1,p-AMPK,AMPKa at the protein level.Observe the effect of Mint3 on the expression of core molecules in AMPK/mTOR pathway in primary neuronal cells after OGD treatment.4.3 Transfect Ctrl-siRNA and Mint3-siRNA into PC 12 cells with the transfection reagent,and continue to culture for 48 hours.After OGD treatment for 2 hours and reoxygenation for 24 hours,the protein was extracted.Western blot was used to detect the expression changes of p-mTOR,p-p70,p-4EBP1,p-AMPK,AMPKa at the protein level.Observe the effect of Mint3 on the expression of core molecules in AMPK/mTOR pathway in PC 12 cells after OGD treatment.5.How Mint3 regulates AMPK/mTOR pathway5.1 PC 12 cells were used for non-OGD treatment and OGD treatment.After 2 hours of OGD treatment,reoxygenation was performed to extract the cell contents and perform co-immunoprecipitation experiments.Detect the binding of Mint3 to AMPKa and AMPKβ in AMPK pathway.5.2 Mouse primary neuronal cells were cultured to maturity and then subjected to non-OGD treatment and OGD treatment.After 2 hours of OGD treatment,reoxygenation was carried out,and immunofluorescence confocal experiments were carried out to detect the localization of Mint3,AMPKa and nucleus.Results1.Mint3 slows down the nerve damage of ischemic stroke in mice1.1 MCAO treatment reduce the expression of Mint3Mint3+/+mice were divided into two groups,one group was the control group,the other group was the experimental group,the control group was treated with sham surgery,the experimental group was treated with MCAO surgery,after the surgery,the mice woke up,2 hours after the stroke reperfusion,24 hours after the operation of the mouse brain tissue for further treatment,RNA and protein extraction of the mouse brain tissue.The associated changes were detected by Western blotting.Changes in expression of Mint3 at protein level.RNA was extracted and reverse transcribed into cDNA,and the expression changes of Mint3 in mRNA and level were detected by RT-PCR.1.2 Mint3 reduces cerebral infarct area in mice after MCAO treatmentMCAO surgery was performed with Mint3-/-mice and Mint3+/+mice.Reperfusion was performed after 2h.Whole brain tissue was taken after 24h.Then,it was frozen and sliced.The thickness of each brain slice was 1mm.Add 2%TTC solution and place it in a 37℃ water bath for staining.Observe the cerebral infarct area after MCAO.The white area represents the area of cerebral infarction after surgery,and the red area represents the normal area without stroke.TTC staining results showed that Mint3 decreased the cerebral infarction area of mice treated with MCAO.1.3 Mint3 reduces the behavioral score of cerebral infarction in mice after MCAO treatmentMint3-/-ice and Mint3+/+mice were used for MCAO surgery,reperfusion was performed 2h later,and their behavioral scores were recorded 24h later.From 1-5 represents the increasing severity of cerebral infarction.Statistics showed that Mint3 reduced the behavioral score of cerebral infarction in mice after MCAO treatment.1.4Mint3 reduces neuronal cell death and macrophage infiltration in the mouse brain after MCAO treatmentMint3-/-mice and Mint3+/+mice were used for MCAO surgery,reperfusion was performed 2h later,and the whole brain was taken 24h later,paraffin section was performed,and immunofluorescence staining was performed.Use NEUN to label neuronal cells to make them appear red.The macrophages were labeled with CD68 to make them appear green.Through the immunofluorescence staining experiment,the slices were placed under a confocal laser microscope for observation.It can be concluded that Mint3 reduces the number of neuronal cell deaths and the number of macrophages infiltrated in the mouse brain after MCAO treatment.2.Mint3 reduces the mortality of OGD-treated mouse neuronal cells2.1 Mint3 does not affect the inflammatory response of mouse brain tissue after MCAO treatmentMint3-/-mice and Mint3+/+mice were divided into two groups,the control group underwent MCAO sham operation,and the experimental group underwent MCAO operation.After 2h,reperfusion was performed.After 24h,the cerebral infarct side of the mouse was taken for processing and RNA was extracted.RNA was reverse transcribed into cDNA,and the expression of IL-6 and TNF-α at the mRNA level was detected by RT-PCR,and it was found that there was no significant change in IL-6 and TNF-α.2.2 Mint3 reduces LDH release from OGD-treated mouse neuronal cellsMint3-/-mice and Mint3+/+mice were used to extract primary neuronal cells,cultured until the primary neuronal cells matured,treated with OGD for 2h,reoxygenated for 24h,collected the cell supernatant and passed the LDH detection reagent The box detects LDH release.Read the LDH release value through a microplate reader.The results showed that Mint3 reduced the LDH release of primary n2.3 Mint3 enhances cell viability of OGD-treated mouse neuronal cellsMint3-/-mice and Mint3+/+mice were used to extract primary neuronal cells,cultured until the primary neuronal cells matured,treated with OGD for 2h,reoxygenated for 24h,collected the cell supernatant,and used CCK8 detection reagent The box detects the release of CCK8.Read the release value of CCK8 with a microplate reader.Use Mint3+/+mice to extract primary neuronal cells,culture them until the primary neuronal cells mature,and use transfection reagent to transfect Ctrl-siRNA and Mint3-siRNA into mouse neuronal cells,respectively.The culture was continued for 48 hours,OGD treatment was performed for 2 hours,and after reoxygenation for 24 hours,the cell supernatant was collected to detect the release of CCK8.Read the release value of CCK8 with a microplate reader.In summary:Mint3 enhances the release of CCK8 from mouse neuronal cells after OGD treatment,and enhances the cell viability of neuronal cells.3.The effect of ischemic stroke on the expression of core molecules in AMPK/mTOR pathway3.1 MCAO inhibits the expression of mTOR pathway in brain tissue and enhances the expression of AMPK pathwayMint3+/+mice were divided into two groups,the control group underwent MCAO sham operation,and the experimental group underwent MCAO operation.Reperfusion was performed after 2h.After 24h,the cerebral infarct side of the mouse was taken for processing,and protein was extracted.Western blot was used to detect the expression of p-mTOR,p-p70,p-4EBP1 p-AMPK,AMPKα,and Mint3 at the protein level Variety.Western blot results showed that after MCAO treatment,the expression of p-mTOR,p-p70,p-4EBP1,AMPKα,and Mint3 decreased,and the expression of p-AMPK increased.It means that the mTOR pathway is inhibited,and the AMPK pathway is activated.3.2 OGD inhibits the expression of mTOR pathway in primary neuronal cells and enhances the expression of AMPK pathwayPrimary neurons were extracted from Mint3+/+mice and cultured until the primary neurons matured.The experimental group was treated with OGD model for 2 hours and reoxygenated for 24 hours.The control group was not treated.The proteins were extracted and Western blot was used to detect the levels of proteins involved,including p-mTOR,p-P70,p-4EBP1,p-AMPK,AMPK alpha,and Mint3.Western blot analysis revealed that the levels of P-mTOR,P-P70,P-4EBP1,AMPK,and Mint3 were down,while the levels of P-AMPK were up.3.3 OGD inhibited the expression of mTOR pathway and enhanced the expression of AMPK pathway in PC 12 cellsTransfect Ctrl-siRNA and Mint3-siRNA into PC12 cells with the transfection reagent,and continue to culture for 48h,OGD treatment for 2h,reoxygenation for 24h,protein extraction,Western blot detection of p-mTOR,p-p70,P-4EBP1,p-AMPK,AMPKa,Mint3 expression changes at the protein level.Western blot results showed that after MCAO treatment,the expression of p-mTOR,p-p70,p-4EBP1,AMPKα,and Mint3 decreased,and the expression of p-AMPK increased.In summary,in the brain tissue,primary neuronal cells,and PC 12 cells,the expression of p-mTOR,p-p70,p-4EBP1,AMPKα,and Mint3 decreased,and the expression of p-AMPK was affected by the symptoms of stroke The increase indicates that the expression of mTOR pathway is suppressed,while the expression of AMPK pathway is enhanced.4.The effect of Mint3 knockout on the expression of core molecules in AMPK/mTOR pathway after ischemic stroke4.1 Mint3 knockout inhibits the expression of mTOR pathway in brain tissue and enhances the expression of AMPK pathwayMint3-/-mice and Mint3+/+mice were prepared in the same way.All mice were used for MCAO surgery.Mint3-/-mice were awake after surgery and reperfusion was performed 2 hours later.Twenty-four hours later,the brains on the surgical side of the mice were removed for processing and protein extraction.Western blotting was used to detect the expression of related molecules.Including P-mTOR and P-P70,P-4EBP1,P-AMPK,and AMPK alpha,which were also known as alpha beta proteins.Western blot results indicated that the Mint3 knockdown induced p-mTOR,p-P70,P-4EBP1,and AMPK expression levels to decrease and p-AMPK expression to increase after MCAO treatment.4.2 Mint3 knockout inhibited the expression of mTOR pathway in primary neuronal cells and enhanced the expression of AMPK pathwayMint3-/-mice and Mint3+/+mice were used to extract primary neuronal cells,treated with OGD for 2h,and reoxygenated for 24h,then protein was extracted.Western blot was used to detect the expression changes of p-mTOR,p-p70,p-4EBP1,p-AMPK,AMPKa at the protein level.Western blot results showed that after OGD treatment,the knockout of Mint3 resulted in a decrease in the expression of p-mTOR,p-p70,p-4EBP1,AMPKα,and an increase in the expression of p-AMPK.4.3 Mint3 silencing inhibited the expression of mTOR pathway in PC12 cells and enhanced the expression of AMPK pathwayTransfect Ctrl-siRNA and Mint3-siRNA into PC 12 cells with the transfection reagent,and continue to culture for 48 hours.After OGD treatment for 2 hours and reoxygenation for 24 hours,the protein was extracted.Western blot was used to detect the expression changes of p-mTOR,p-p70,p-4EBP1,p-AMPK,AMPKa at the protein level.Western blot results showed that after OGD treatment,the knockout of Mint3 resulted in a decrease in the expression of p-mTOR,p-p70,p-4EBP1,AMPKα,and an increase in the expression of p-AMPK.In summary:in mouse brain tissues treated with MCAO,primary neuronal cells treated with OGD,and PC 12 cells,the knockout of Mint3 enhanced the activation of AMPK pathway and inhibited the activation of mTOR pathway.5.Mint3 regulates AMPK/mTOR pathway by combining with AMPKα5.1 Mint3 binds AMPKaPC12 cells were treated with non-OGD and OGD.After 2h OGD treatment,the cells were reoxygenated and the immunoprecipitation test was carried out.Put the extract into the IP buffer solution and conduct cracking.The supernatant was collected by centrifugation and incubated with protein G plus-Arose immunoprecipitation reagent and Mint3 specific antibody.After incubation for 6 h,the magnetic beads were washed with IP buffer for five times.After that,the immunoprecipitate was eluted with sample buffer of 1%SDS to prepare protein samples,and the binding status was detected by Western blot.Western blot tests revealed that Mint3 had been able to bind to AMPKa,but had failed to bind to AMPKβ.5.2 Mint3 and AMPKa co-localize in the nucleus and cytoplasmUse mouse primary neuronal cells to culture and mature,and then perform immunofluorescence confocal experiments.The primary neuronal cells transiently transfected with the plasmid encoding Mint3-V5 were cultured for 24 hours,and subjected to non-OGD treatment and OGD treatment,and reoxygenation was performed after OGD treatment 2.Add Mint3 and AMPKα specific primary antibody,stain with Rabbit Alexa Fluor 633 or Mouse Alexa Fluor 488 coupled secondary antibody,and stain the cell nucleus with DAPI.Check the cells with a confocal laser microscope.The results of confocal microscopy showed that Mint3 and AMPKa co-localized in the nucleus and cytoplasm.Conclusion1.Mint3 by combining with AMPKa,competitively inhibits the phosphorylation and activation of AMPK,thereby enhancing the activation of the mTOR pathway and helping to regulate the energy steady state of neuronal cells after ischemic stroke.2.Mint3 regulates the AMPK/mTOR pathway,reduces the death of neuronal cells after ischemic stroke,and slows down the damage caused by cerebral ischemia.Innovation and significance1.This study revealed the role of Mint3 in neuronal cell death after ischemic stroke,suggesting that Mint3 can participate in the regulation of nerve damage after ischemic stroke by regulating the expression of AMPK/mTOR pathway.2.This study revealed the role of Mint3 in the process of ischemic stroke,suggesting new possible targets for the treatment of stroke and related diseases. |
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