| BackgroundCerebral apoplexy has become one of the main diseases endangering human life and health,including hemorrhagic apoplexy and ischemic apoplexy.Hemorrhagic apoplexy mainly includes parenchymal hemorrhage,spontaneous intracerebral hemorrhage and subarachnoid hemorrhage(SAH).Substantial cerebral hemorrhage is mainly caused by hypertension,cerebrovascular malformation,amyloidosis of blood vessels caused by blood vessel rupture hemorrhage,hematoma compression of brain tissue,and then cause secondary brain injury.At present,the treatment measures for hemorrhagic stroke mainly include active treatment of internal primary diseases,craniotomy hematoma clearance,minimally invasive hematoma puncture and so on.However,there is still no good strategy for extensive and delayed brain injury caused by SAH and primary ventricular hemorrhage into subarachnoid space,especially the poor prognosis caused by acute stage injury,which is the focus of current research.SAH is a disease with a high mortality rate,mainly caused by intracranial aneurysm rupture,accounting for about 3-5%of cerebral apoplexy,and nearly half of the patients are under the age of 55.The secondary brain injury caused by SAH is a key factor for disability and death of patients.Therefore,the research on the secondary brain injury after SAH has become a hot spot in the field of neuroscience.The injury within 72 hours after SAH is called early brain injury(EBI),which is the primary factor leading to poor prognosis of patients.The pathological process of SAH is complex,including vasospasm,cell apoptosis,intracranial hypertension,neuroinflammation,oxidative stress,cerebral edema,and destruction of the blood-brain barrier.At present,there is a lack of clinical treatment that can significantly improve the prognosis of patients.The total incidence of ischemic stroke is significantly higher than that of hemorrhagic stroke,accounting for 75%of all strokes.In the past,ischemic stroke was mainly caused by thrombosis and blood vessel obstruction.In the past,ischemic strokes were mainly caused by blood clots and blockages.Middle cerebral artery occlusion(MCAO)caused by the frontal circulating blood system accounts for most pathological types of ischemic stroke.And the pathogenesis is complex,usually accompanied by the release of various inflammatory mediators.Infiltration and accumulation of inflammatory cells.The breakdown of the blood-brain barrier;secretion of inflammatory factors and release of adhesion molecules.The main therapeutic measures for ischemic stroke include drug therapy,interventional therapy,bone flap decompression and a series of measures to restore blood flow and relieve cerebral compression.However,neurological defect caused by ischemia reperfusion injury(CIRI)after reperfusion injury caused by reperfusion injury is still a serious problem for clinicians.Strong inflammatory response and apoptosis after cerebral ischemia are the main causes of secondary brain injury,and play an important role in cerebral ischemia and CIRI.Although the time window for mechanical thrombectomy or thrombolysis has been extended,the rupture of blood vessels during thrombectomy and subsequent recovery are still the biggest problems in the neurological community.Therefore,considering the complexity of the pathophysiology of ischemic stroke,no comprehensive and effective treatment has been found.Therefore,the study on the molecular mechanism of cerebral ischemia and CIRI will provide important clinical guidance for the early intervention after cerebral ischemia.The change of brain tissue microenvironment plays an important role in the brain injury after stroke,especially the inflammatory response,which runs through the whole pathological process and seriously affects the prognosis of patients.Microglias,as resident immune cells of the central nervous system(CNS),are mainly involved in the neuroinflammatory response,and they belong to the mononuclear phagocytic system.In physiological state,microglia play a role in maintaining homeostasis by monitoring pathogens and injury-related molecular patterns and engulfing apoptotic neurons.Microglia cells can adjust their phenotypes according to changes in micro environmental signals,including classically activated M1 phenotypes and substitutively activated M2 phenotypes.M1 phenotype microglia can produce tumor necrosis factor-?(tumor necrosis factor-alpha,TNF-?),interferon-?(interferon alpha,IFN-?),interleukin-1?(interleukin-1?,IL-1?),inducible nitric oxide synthase(iNOS)and other pro-inflammatory factors,which cause toxicity to neurons and tissue inflammation damage.M2 phenotype microglia can secrete anti-inflammatory factors,such as transforming growth factor-beta(TGF-beta),arginase-1(arginase-1 Arg-1),etc.Neuronal damage plays a protective role.Microglia activation state and polarization phenotypic changes after brain injury of nerve inflammation plays a crucial role,especially for the parietal cortex(parietal cortex,PC)and hippocampus(hippocampus,HP)the microglia,PC and HP in patients after stroke neurocognitive plays an important role,how to improve the early brain injury after stroke,especially the nerve inflammation caused by damage,is the focus of the present study.In neuroinflammatory reactions,M1-phenotype and M2-phenotype microglia usually coexist and can be distinguished according to different surface markers.CD 16 and CD206 are mainly expressed on the Ml and M2-phenotype microglia cell membranes,respectively.Ml-type microglia cells accounted for a higher proportion in the early stage of neuroinflammation,while M2-type microglia cells mainly appeared in the late stage of neuroinflammation.Therefore,the conversion of microglia from the M1 phenotype to the M2 phenotype in the early stage of inflammatory response,so as to restore the immune balance,is crucial to improve the neurological function of stroke patients.Among the treatment measures for stroke,stem cells and their related therapies have increasingly shown strong advantages.Stem cells can be transplanted to the host for the release of a variety of biological activity factors exert its nerve protective effect,but there are also many shortcomings stem cell therapy,local or systemic drug,stem cells really reach the number of injury is very limited,and the survival time in injury is very short,through the barrier system in the body at the same time,and tumor risk.Mesenchymal Stem cells(MSCs)can secrete a variety of bioactive substances and target to the damaged regions in the brain,including nutritional factors and EVs,which are closely related to neurogenesis,angiogenesis and neuroprotection.Extracellular vesicles are membranous vesicles that can be produced naturally by most living cells,and can be roughly divided into apoptotic bodies(800-5000 nm in diameter),microvesicles or exosomes(50-1000 nm in diameter)and exosomes(40-100 nm in diameter)according to their sizes and sources.Extracellular vesicle due to good cycle stability and compared to the stem cells with low immunogenicity,and easily through the barrier system in the body,convenient for storage and transportation,etc.A huge advantage,its scope is more extensive than stem cells,has great potential in the field of biological treatment,at the same time,it can overcome the limitations of cell therapy with relevant and potential risk.Studies have shown that extracellular vesicles derived from mesenchymal stem cells(MSCs-EVs)can well maintain the biological activity similar to that of the parent cells,such as homing to the inflammatory site to regulate the immune response and participate in tissue repair.In the treatment of stroke,MSCs-EVs has shown a strong neuroprotective effect,and the application of MSCs-EVs can promote the neurovascular remodeling and neurological function recovery after stroke.There are several important signaling pathways involved in inflammatory response and apoptosis after stroke.Nuclear factor Kappa B(NF-?B)has long been recognized as a classic inflammatory signaling pathway.Activation of NF-?B promotes the expression and secretion of cytokines,chemicals,and adhesion molecules.NF-?B,an important intracellular nuclear transcription factor,targets this pathway to reduce infiltration and apoptosis of inflammatory cells.Amp-activated protein kinase(AMPK),as an important serine/creatine protein kinase,can be rapidly activated by anti-inflammatory factors and inactivated by pro-inflammatory factors.When cellular energy supply is reduced,AMPK activation can maintain cellular metabolic balance by reducing energy consumption and increasing energy utilization,thereby promoting neural function repair after stroke.Meanwhile,AMPK activation has been shown to be a negative regulator of NF-?B and an effective counterregulator of key transcription factors regulating proinflammatory cytokine expression and macrophage in flammatory signaling pathways.In addition,Janus kinase/signal transduction and transcriptional activation son(Janus kinase/signal transducers and transcriptional activators,JAK/STAT)family of signaling pathway activation is most cytokines play an important way of its biological function,widely expressed in the brain,the nerve inflammation,and apoptosis plays a vital role.In the JAK/STAT family,the JAK2/STAT3 pathway is the most conserved and closely related to the pathophysiology of inflammation and oxidative stress in CNS.Activation of JAK2 protein leads to phosphorylation of STAT3.The JAK2/STAT3 signaling pathway plays an important role in neurological function recovery after traumatic brain injury and cerebral ischemia.This topic with MSCs-EVs as a research center,AMPK as research target,using the vascular puncture method and line bolt blockage MCA method to establish experimental SD rats respectively SAH and MCAO model,with PC and HP area as the main research area,follow the general pathology,histopathological and molecular pathological research route,a detailed study of MSCs-EVs for SAH and nerve protection after MCAO,revealing the MSCs-EVs by reducing nerve inflammation,promote the microglia to M2 phenotypes polarization,alleviate nerve cell apoptosis play a role of neural protection mechanisms.Through the study of this topic,it can provide a brand-new intervention means and targets for the early neurological function protection after the occurrence of SAH and MCAO,which has a distinct guiding value for the development of clinical medicine.Part ? Neuroprotective effects and mechanisms of MSCs-derived extracellular vesicles on SAH in rats by regulating AMPK pathway1.1 Research purposes:(1)The basic pathological changes and neurological damage in the EBI stage after SAH are clarified.At this stage,MSCs-EVs are used to intervene in experimental SAH rat models to detect the occurrence of SAH by MSCs-EVs.Take PC and HP regions as representatives to detect the ameliorating effect of MSC-EVs on brain tissue pathological damage after SAH.(2)Analyze the molecular mechanism and related pathways of MSCs-EVs to improve brain injury after SAH.(3)It is clear that MSCs-EVs can improve neurological damage by affecting EBI,and clarify changes in related signaling pathways.1.2 Research method(1)Construction of a stable experimental sah rat model198 four-week-old healthy adult male SD rats of SPF grade were selected,and their weights were in the range of 280-350 g.The SAH animal model was established by modified vascular puncture method.The SAH model established by modified vascular puncture method can simulate the aneurysm rupture and bleeding caused by SAH.Extensive neurological damage.(2)Extraction and identification of MSCs-EVsThe mesenchymal stem cells(MSCs)in the femur and tibia bone marrow cavity of rats were extracted and cultured.After the third passage,the EVs-free medium was replaced and the culture was continued for 48 h.High-purity MSCs-EVs were obtained by gradient centrifugation of the culture medium.MSCs-EVs were identified by transmission electron microscopy,qNano and Western blot.(3)Animal grouping method,MSCs-EVs injection and animal executionUsing random number method,in this study,rats were randomly divided into 5 groups:(1)Sham group,(2)SAH 24 h group,(3)SAH 24 h+MSCs-EVs group,and(4)SAH 48 Group h,(5)SAH 48 h+MSCs-EVs group.Each rat added to the MSCs-EVs group was injected with a 100 ?g MSCs-EVs PBS suspension through its tail vein within 10 minutes after successful SAH modeling.The other groups were injected with the same volume of sterile PBS as a control.At the corresponding time points,the experimental animals were sacrificed by a lethal dose of chloral hydrate intraperitoneally.(4)Effect of MSCs-EVs on the gross pathology of SAH animalsObserve the animal's survival at any time,eliminate dead rats in time,at the corresponding time node,before killing each group of animals,use the modified neurological score scale to detect the changes in the neurobehavior of each group of animals,the amount of subarachnoid hemorrhage in the skull base and brain water Content,compare the difference between SAH+MSCs-EVs group and SAH group.(5)MSCs-EVs improve neuronal damage after SAHAfter the experimental animals were sacrificed,paraffin sections of whole brain tissues were made,and the activation of microglia in the PC and HP CA1 areas of each group and morphological analysis were observed by immunohistochemistry;the main observation target was ionized calcium binding adaptor molecule-1(Iba-1).Analysis of the relative number of positive microglia and the area,diameter and circumference of the microglia.(6)Detection of MSCs-EVs at the gene and protein levels to regulate indicators of inflammatory factors after SAH,and identify related signaling pathwaysAfter the animals were sacrificed,the brain tissues from the PC and HP regions were collected,total mRNA was extracted,and brain tissue protein homogenates were made.Reverse transcription PCR(RT-PCR)and Western blot techniques were used to detect M1 and M2 phenotype microglia surface markers.And the expression of inflammatory factors such as IL-1? in various animal models.Western blot was used to detect the expression of key proteins in the possible signaling pathways that MSCs-EVs regulate inflammation after SAH.(7)Statistical analysisSPSS software(version 22,IBM,New York,USA)is used for data analysis.The data are expressed as mean±standard deviation,and the data are all analyzed using one-way ANOVA and post-Bonferroni analysis.P<0.05 is considered statistically significant.1.3 Results(1)Identification of MSCs-EVsObservation under a transmission electron microscope showed that MSCs-EVs had a cup-shaped uniform spherical vesicle-like structure.The expression of TSG101 and CD9,which are characteristic markers of MSCs-EVs,can be detected by Western blot,while the expression of endoplasmic reticulum stress protein(Celnexin)is very small.The particle size analysis results showed that the diameter of most MSCs-EVs was between 60-150 nm,and the results showed that the extracted MSCs-EVs met the experimental requirements.(2)The effect of MSCs-EVs on neurological deficit and brain water content after SAH in ratsThe improved Longa scoring system was used to score the neurobehavior of each group of rats by two experimenters who were unaware of the experiment and recorded independently,compared with the Sham group,the neurological function scores of the SAH group were significantly reduced at 24 h and 48 h after surgery(P<0.05)Compared with the SAH group,the neurological function score of the MSCs-EVs group increased at 48 h,and the difference was statistically significant(P<0.05).At the same time,further detection of brain water content found that the SAH group had significant brain water content in each brain area after surgery Higher than that in the Sham group(P<0.01,P<0.05,P<0.01),especially in the left cerebral hemisphere.The cerebral water content of the left cerebral hemisphere in the SAH group was 1.08 and 1.15 of that of the Sham group at 24 h and 48 h after surgery.Times.However,in SAH rats with intervention of MSCs-EVs,the brain water content of each brain area was significantly reduced after operation(P<0.01,P<0.05,P<0.01).(3)The effect of MSCs-EVs on the amount of subarachnoid hemorrhage after SAH in ratsCompared with the Sham group,the amount of subarachnoid hemorrhage in the SAH group increased significantly at 24 h(P<0.001)and 48 h(P<0.01)after the operation;at 48 h after the operation,compared with the SAH group,MSCs-The amount of subarachnoid hemorrhage in the EVs group was significantly reduced(P<0.05).(4)MSCs-EVs reduce the activation of microglia in the parietal cortex and hippocampusCompared with the Sham group,the number of activated microglia in the PC area of the SAH group increased significantly at 24 h and 48 h after SAH(P<0.001).Compared with the Sham group,the activation of microglia in the HP CA1 area of the SAH group the number increased significantly at 24 and 48 h after SAH(P<0.01;P<0.001).Compared with the SAH group,the activation of microglia in the PC area and HP CA1 area in the MSCs-EVs group was significantly lower than that in the SAH group(48 h:P<0.001).(5)MSCs-EVs promote the polarization of microglia in the M2 direction and inhibit neuroinflammatory responseCompared with the Sham group,in the SAH group,the surface marker of M1 microglia in the PC area was iNOS(24 h:P<0.01,48 h:P<0.001);CD16(24 h:P<0.05,48 h:P<0.01);CDllb(24 h:P<0.01,48 h:P<0.01)and inflammation index IL-1?(24 h:P<0.01,48 h:P<0.001)expression levels at 24 h and after SAH The expression levels of the surface markers of M1 microglia in HP area,iNOS;CD 16;CDllb,and inflammation index IL-1? were also significantly increased after 48 h after SAH(P<0.001).Compared with the SAH group,the expression levels of the above-mentioned microglia surface markers in the PC area in the MSCs-EVs group were significantly reduced,iNOS(24 h:P<0.05;48 h:P<0.001);CD16(24 h:P<0.05;48 h:P<0.01);CD11b(24 h:P<0.01;48 h:P<0.001);IL-1?(24 h:P<0.001;48 hP<0.001),compared with SAH group The expression levels of the above-mentioned microglia surface markers in the HP area in the MSCs-EVs group were significantly reduced,iNOS(24 h:P<0.001;48 h:P<0.001);CD16(24 h:P<0.001;48 h:P<0.001);CD11b(24 h:P<0.01;48 h:P<0.001);IL-1?(24 h:P<0.001,48 h:P<0.05).Compared with the SAH group,the expression levels of M2 microglia in the PC area in the MSCs-EVs group were significantly increased after 24 h and 48 h.TGF-?(24 h:P<0.001;48 h:P<0.001),Arg-1(24 h:P<0.001;48 h:P<0.001),and CD206(24 h:P<0.01;48 h:P<0.001).Compared with SAH group,MSCs The expression level of M2 microglia surface markers in the HP area in the EVs group was significantly increased after 24 h and 48 h,TGF-?(24 h:P<0.05;48 h:P<0.01);Arg-1(24 h:P<0.001;48 h:P<0.001);CD206(24 h:P<0.05;48h:P<0.01).It shows that MSCs-EVs can inhibit the neuroinflammatory response induced by SAH in PC and HP area and promote the transformation of microglia to M2 phenotype.The immunohistochemical results showed that compared with the Sham group,the number of CD16+microglia in the PC area of the SAH group was significantly increased(24 h:P<0.001;48h:P<0.01),and MSCs-EVs could reduce SAH for 24 h and 48 h The number of CD16+microglia(P<0.05).Compared with the SAH group,the number of Arg+microglia in the PC area of the MSCs-EVs group was significantly increased(24h:P<0.05;48h:P<0.01).Compared with the Sham group,the number of CD16+microglia in the HP CA1 area of the SAH group was significantly increased(24 h:P<0.001;48h:P<0.01).MSCs-EVs can reduce the number of CD 16+microglia(24h:P<0.05;48h:P<0.01).Compared with the SAH group,the number of Arg+microglia in the HP CA1 area of the MSCs-EVs group was significantly increased(24 h:P<0.01).(6)The effect of MSCs-EVs on the morphology of microglia in the parietal cortex and hippocampal CA1 area after SAHCompared with the Sham group,the area,diameter and circumference of the microglia in the PC area of the SAH group increased significantly at 24 h and 48 h after SAH(24 h:P<0.001,P<0.01,P<0.001;48 h:P<0.001,P<0.001,P<0.001).Compared with the Sham group,the area,diameter(P<0.001)and circumference(P<0.001)of microglia in the hippocampal CA1 area of the SAH group increased significantly at 24 h after SAH.Compared with the Sham group,the SAH group hippocampal CA1 the area(P<0.05),diameter(P<0.001),and circumference(P<0.001)of microglia in the region increased significantly at 48 h after SAH.Compared with the SAH group,the intervention of MSCs-EVs can increase the area(24 h:P<0.01;48 h:P<0.05),diameter(48 h:P<0.001)and circumference(24 h:P<0.001;48 h:P<0.001)significantly reduced after SAH.Compared with SAH group,MSCs-EVs intervention can increase the area of microglia in hippocampal CA1 area(48 h:P<0.05),Diameter(24 h:P<0.001;48 h:P<0.001)and circumference(24 h:P<0.001;48 h:P<0.001)are significantly reduced.The above experimental results further show that the intervention of MSCs-EVs can reduce the activation of microglia after SAH.(7)MSCs-EVs increase the phosphorylation level of AMPK after SAH and its influence on NF-?B signalingAfter SAH,the activation of key signal molecules in AMPK and NF-?B pathways was activated.The experimental results showed that compared with the Sham group,the expression of p-AMPK in the PC area increased in the SAH group at 24 h(P<0.01);The expression of p-AMPK in the HP area increased(24 h:P<0.01;48 h:P<0.05);the expression of p-IKBa in the PC and HP area increased after 24 h and 48 h in the SAH group(PC:P<0.001;HP:P<0.01);compared with the Sham group,the expression of p-NF-?B in the HP area increased after 24 h and 48 h in the SAH group(24 h:P<0.01;48 h:P<0.01).After the application of MSCs-EVs,the phosphorylation level of AMPK in SAH rats was significantly increased.Compared with SAH group,MSCs-EVs further up-regulated the PC area(24 h:P<0.001;48 h:P<0.05)and HP the expression of p-AMPK in the region(24 h:P<0.05;48 h:P<0.001).The activation of key signal molecules in the downstream NF-?B pathway was significantly inhibited.Compared with the SAH group,MSCs-EVs could significantly reduce the expression of p-IKBa in the PC and HP areas(PC 24 h:P<0.01;48 h:P<0.001;HP 24 h:P<0.001;48 h:P<0.001).MSCs-EVs can also significantly reduce the expression of p-NF-?B in PC and HP area(PC 24 h:P<0.01;48 h:P<0.01;HP 24 h:P<0.05;48 h:P<0.001).The results of immunohistochemistry showed that the intervention of MSCs-EVs can increase the number of p-AMPK+cells in the PC area and HP CA1 area of rats after SAH(PC 48 h:P<0.001;HP 24 h:P<0.001;48 h:P<0.05).Compared with the Sham group,the number of p-NF-?B+cells in the PC area and HP CA1 area after 24 h and 48 h in the SAH group(P<0.001).The intervention of MSCs-EVs can reduce the PC area and the number of p-NF-?B+cells in HP CA1 area(PC 24 h:P<0.01;48 h:P<0.001;HP 48 h:P<0.001).The above experimental results show that after SAH,MSCs-EVs can inhibit neuroinflammatory response by activating AMPK and inhibiting NF-?B signaling pathway.1.4 Conclusion(1)MSCs-EVs can reduce brain water content and neuroinflammation,improve the degree of EBI caused by SAH,increase neurological deficit scores,and improve neural function.(2)The molecular mechanism by which MSCs-EVs exert neuroprotective effects is by up-regulating AMPK and down-regulating the NF-?B signaling pathway,thereby reversing SAH-induced neuroinflammation,promoting microglial polarization to the M2 phenotype,and exerting neuroprotective effects.Part? Neuroprotective effects and mechanisms of MSCs-derived extracellular vesicles on MCAO in rats by regulating AMPK pathway2.1 Research purpose(1)To clarify the basic pathological changes and nerve function damage in rats after MCAO,and use MSCs-EVs to intervene in the experimental MCAO rat model,with PC and HP CA1 areas as representatives,to detect the effects of MSCs-EVs on the brain tissue pathology after MCAO Improvement of injury.(2)Analyze the molecular mechanism and related pathways of MSCs-EVs to improve brain injury after MCAO.(3)Clarify the mechanism by which MSCs-EVs improve rat nerve function damage and clarify the changes in the expression of key proteins in related signaling pathways.With the help of AMPK pathway specific blockers(compound C,CC),further study the mechanism of AMPK signaling pathway in neuroprotection after MCAO.2.2 Experimental methods(1)Rats were randomly divided into 8 groups:(1)Sham 24 h group,(2)MCAO 24 h group,(3)MCAO 24 h+MSCs-EVs group,(4)MCAO 24 h+MSCs-EVs+CC group,(5)Sham 48 h group,(6)MCAO 48 h group,(7)MCAO 48 h+MSCs-EVs group and(8)MCAO 48 h+MSCs-EVs+CC group.Each rat in group(4)and group(8)30 min before MCAO surgery,use a Hamilton microsyringe to slowly inject CC into the right lateral ventricle by stereotactic technique 30 minutes before MCAO surgery.Each rat in the MSC s-EVs group Within 10 min after MCAO,a PBS suspension containing 100 ?g MSCs-EVs was injected via the tail vein.Complete the preparation of experimental animals in each model group.(2)At 24 h and 48 h after MCAO,the rats in each group were evaluated for neurological deficits using the modified Garcia scoring system and the scores were recorded.The animals were then sacrificed,and brain water content was measured in the left and right hemispheres of some animals.(3)The cerebral infarction volume of rats in each group was observed with TTC staining.(4)HE staining was used to observe the morphological changes of the brain tissue of each group of rats under the microscope,and to explore the histopathological level.(5)The Nissl body loss in the brain tissue of each group of rats was observed under a microscope using Nissl staining,and the histopathological level was discussed.(6)The tunel detection kit was used to detect apoptosis in the infarcted area of each group of rats,and to explore the histopathological level.(7)Analyze the molecular mechanism and related pathways of MSCs-EVs to improve brain injury after MCAO.(8)Data analysis:SPSS software(version 22,IBM,New York,USA)was used.Average Standard deviation means that the data are analyzed using one-way ANOVA combined with post-Bonferroni.P<0.05 considered the difference to be statistically significant.2.3 Experimental results(1)The effect of MSCs-EVs on the reduction of neurological deficits and brain water content in rats after MCAOThe neurological deficit score of rats in the MCAO group was lower than that of the Sham group at 24 h and 48 h(P<0.001).The intervention of MSCs-EVs can increase the neurological deficit score induced by MCAO(24 h:P<0.05,48 h:P<0.01).The brain water content of rats was significantly increased 24 h after MCAO(P<0.001),while MSCs-EVs could significantly reduce brain water content(P<0.01).The above results indicate that MSCs-EVs exert a neuroprotective effect after CIRI.(2)MSCs-EVs reduce cerebral infarction volume after MCAOThe results of TTC staining showed that the normal brain tissue was red,and the brain tissue of the cerebral infarction area was white.Compared with the Sham group,the cerebral infarct volume was significantly increased at 24 h and 48 h after MCAO(P<0.001),while MSCs-EVs significantly reduced the cerebral infarct volume(P<0.001).Experimental results show that MSCs-EVs can reduce the volume of cerebral infarction caused by CIRI in MCAO rats and exert neuroprotective effects.(3)MSCs-EVs reduce the neuropathological damage induced by MCAOMSCs-EVs can reduce the histopathological response and apoptosis rate after MCAO.Compared with the clear contours and uniform cytoplasmic coloration of normal cortical neurons in the Sham group,in the MCAO group,the cortical cells in the ischemic area were damaged to varying degrees.The specific manifestations are neuronal atrophy,fuzzy outline,deep staining of cytoplasm,unclear boundary between nucleus and cytoplasm,and a significant decrease in the number of Nissl bodies.The number of TUNEL-positive cells increased significantly at 24 and 48 h after MCAO(P<0.001),and the intervention of MSCs-EVs can improve the pathological damage of brain tissue caused by MCAO and the relative number of TUNEL-positive cells(P<0.001).(4)MSCs-EVs increase the phosphorylation levels of AMPK,JAK2,and STAT3 after MCAO and their influence on NF-?B signalingAfter MCAO,the activation of key signal molecules of AMPK pathway is activated,and MSCs-EVs are used.Later,the phosphorylation level of AMPK in the MCAO group was further increased(P<0.001),and the activation of key signal molecules in the downstream JAK2/STAT3 pathway was significantly inhibited(P<0.001),it shows that the intervention of MSCs-EVs can exert anti-apoptosis and neuroprotective effects after MCAO.(5)The effect of AMPK signal pathway blocker(compound C,CC)on the neuroprotective effect of MCAO-induced brain injuryAfter injection of the AMPK pathway specific blocker CC into the lateral ventricle,the neuroprotective effect of MSCs-EVs in MCAO was reversed to a certain extent.The results showed that after using CC to block the AMPK signaling pathway,compared with the MSCs-EVs group,the cerebral infarction volume could be increased to a certain extent(P<0.01),and the expression of p-AMPK in the infarcted cortex was down-regulated(P<0.001).The above results confirm that MSCs-EVs can reduce the release of inflammatory factors after CIRI by activating AMPK signaling pathway.2.4 Conclusion(1)MSCs-EVs can reduce the nerve damage induced by MCAO by reducing the neurological deficit,brain water content,cerebral infarction volume,histopathological damage,and the relative number of apoptotic cells in rats after MCAO,thereby reducing the degree of nerve damage induced by MCAO,thereby improving nerve function.(2)The molecular mechanism of MSCs-EVs exerting the neuroprotective effect after MCAO is to up-regulate AMPK,down-regulate JAK2/STA3 and NF-?B signal pathways,thereby reversing the neuronal apoptosis induced by MCAO and exerting neuroprotective effects. |
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