Neuroprotective Mechanism Of Activin A/Smads Pathway Mediating Mfn2 Regulation Of Microglia Polarization Against Hypoxic Injury

Background.Stroke is one of the most common causes of death worldwide.Due to its rapid onset,multiple complications,and narrow time window,effective treatment options for ischemic stroke are still limited up to now.Therefore,therapeutic options targeting the regulation of the brain microenvironment during stroke and the repair of neural and tissue damage after stroke are particularly important.Microglia are immune cells resident in the CNS that can alter their phenotype in response to changes in the CNS microenvironment,exerting pro-or anti-inflammatory effects and affecting the prognosis of ischemic stroke.It has been shown that excessive activation of microglia contributes to neurodegenerative processes that result from the production of various neurotoxic factors such as pro-inflammatory cytokines and nitric oxide.Activin A,a member of the TGF-? superfamily,plays a key role in the regulation of the immune response.Recent studies have shown that the Act A/Smads signaling pathway plays an important role in the repair of neurological injury,but whether it can influence the direction of microglia polarization and its mechanism of action are not yet clear.Furthermore,as active and highly dynamic organelles,mitochondria are able to change their shape and distribution by fission or fusion in response to physiological or metabolic conditions to achieve intracellular and intra-organismal energy requirements and maintain organismal homeostasis,a process called mitochondrial dynamics.It is essential to determine the morphology and volume of mitochondria for their immediate adaptation to cellular energy demands,and the proper functioning of multiple systems in the body depends on their kinetic balance.It has been shown that mitochondrial dynamics are involved in the expression of microglial cytokines during hypoxic injury,and that increased expression of division proteins induces pro-inflammatory polarization of microglia.It is hypothesized that imbalance of mitochondrial fusion and division can have serious effects on microglia,and that controlling fusion and division to maintain dynamic balance may be important for the phenotypic polarization of microglia.However,the functional role played by mitochondrial dynamics in microglia polarization remains unknown.In this study,we established an in vitro oxygen-glucose deprivation(OGD)model for microglia to simulate the in vivo ischemic process,and explored the mechanism by which the Act A/Smads signaling pathway regulates microglia polarization behavior in cerebral ischemic injury by regulating mitochondrial fusion proteins and its effects on neuronal cells,providing new therapeutic ideas for acute ischemic stroke.Methods.1.Detect the success of establishing OGD model of BV2 cells by using CCK-8to measure cell viability and flow cytometry to measure apoptosis;Detect M1 and M2 markers of microglia by ELISA and Western Blot,and the effect on pathway changes.2.Establish microglia-neuron co-culture system by Transwell,CCK-8,flow cytometry,MDA,ROS,Western Blot were used to detect the establishment of microglia-neuron co-culture system and the effect on apoptosis,iron death GPX4,x CT,NRF2 protein of the system.3.Exogenous Act A and transfected small interfering RNA were administered to knock down Smad2,and the effect of intervention and the change of pathway activity were detected by Western Blot.Western Blot,CCK-8,flow cytometry,ELISA,Western Blot,MDA,ROS were used to detect the effect of Act A/ Smad2 pathway activity on BV2 and co-culture system.4.Western Blot to detect the effect of OGD injury on the expression of mitochondrial fusion proteins Mfn1,Mfn2,and Opa1,and the effect of differential activity of Act A/ Smads pathway on the expression of Mfn1,Mfn2,and Opa1 produced by OGD injury.5.Transfect BV2 with small interfering RNA to knock down Mfn2,Western Blot,CCK-8,flow cytometry,ELISA,Western Blot,MDA,ROS to detect the effect of si-RNA transfection of Mfn2 on apoptosis and iron death of BV2 and co-culture system.6.Explore the linkage between Smad2 and Mfn2 using biotin tool gepia2,immunofluorescence staining,dual luciferase reporter gene assay,q PCR to further determine whether Mfn2 is the mechanism by which the Act A/Smads pathway regulates the direction of microglia polarization in OGD injury.Results.1.With the extension of OGD time,BV2 viability gradually decreased,apoptosis increased,M1 markers of BV2 showed a time-dependent gradual increase,while M2 markers showed a trend of first increase and then decrease,and were consistent with the expression of pathway markers,indicating that the cellular Act A/ Smad2 pathway in the early stage of hypoxia presents a short time course protective mechanism against hypoxic injury,but the degree of damage caused by hypoxia still increased with time.This was reflected in the gradual decrease of PC12 cell viability and increase of apoptosis in the co-culture system,the increase of MDA and ROS levels with OGD time,and the decrease of iron death inhibitory protein expression.2.The apoptotic damage of BV2 cells by OGD was reduced and cell viability was increased after administration of exogenous Act A.Knockdown of smad2 in BV2 cells exacerbated the hypoxic damage,as reflected by increased expression of M1 markers and decreased expression of M2.The results of co-culture system western blot,MDA,ROS,cck-8,and flow-through further validated the above results,indicating that activation of the pathway can have a protective effect in hypoxia,while inhibition of the pathway exacerbates this injury.3.Among OGD injuries,Mfn1,Mfn2,and Opa1 expression positively correlated with the expression of key markers of the Act A/ Smads pathway,and exogenous Act A administration increased the expression of fusion proteins Mfn1,Mfn2,and Opa1 in BV2 cells,while knockdown of Smad2 decreased the expression of fusion proteins,suggesting that Act A/ Smads pathway activity could affect fusion protein expression levels.4.Knockdown of Mfn2 increased the proportion of M1 type BV2 and exacerbated the damage of BV2 and co-culture system PC12,as reflected by increased apoptosis,reduced viability and increased iron death of PC12,suggesting that Mfn2 can regulate the polarization phenotype of microglia and affect itself as well as the co-culture system.5.Gepia2,immunofluorescence staining,dual luciferase reporter gene assay,and q PCR predicted and validated Smad2 as a transcription factor for Mfn2 and its transcriptional regulation,indicating that the Act A/ Smads pathway regulates the polarization behavior of microglia by regulating the expression of Mfn2,which in turn has a protective effect on the nervous system.Conclusions.1.The microglia OGD model and microglia-neuron co-culture model were successfully established,and the alteration of Act A/Smads pathway activity during hypoxia to regulate BV2 phenotype polarization could affect microglia and neuron status.2.Act A/Smads pathway activity protects neuronal cells from hypoxic injury by regulating the polarization phenotype of microglia,as evidenced by reduced neuronal ROS and MDA levels and increased GPX4,x CT and NRF2 protein expression levels,possibly by reducing microglia-mediated inflammatory neurological injury.3.Act A/Smads may induce BV2 polarization toward M2 by regulating changes in Mfn2 expression,and the protective effect of Act A/Smads in hypoxic injury may affect BV2 polarization by regulating Mfn2 activity,which in turn has a protective effect on the neural microenvironment.