| Background:Acute cerebral infarction seriously endangers human health.In recent years,with the improvement of the regionalized treatment system for acute ischemic stroke and the popularization of intravenous thrombolysis and neurointervention techniques,the rate of vascular recanalization in acute ischemic stroke has gradually increased.However,in the real world,some patients have malignant edema or even hemorrhagic transformation after their ischemic injury is not alleviated after recanalization,which greatly limits the beneficiaries of recanalization therapy.Studies have found that this phenomenon is closely related to cerebral ischemia-reperfusion(I-R)injury.I-R injury refers to the phenomenon that tissue damage is aggravated after blood perfusion is restored based on ischemia.In recent years,studies have found that different death modes,such as apoptosis,necroptosis,autophagy,pyroptosis,ferroptosis,etc.,are involved in the I-R injury process.Some experimental results show that the necrotic cell death mode can lead to cell edema and disintegration.The released cellular content can activate immune cells such as microglia,further triggering an imbalance in the homeostasis of the internal environment and aggravating cerebral tissue damage.As a necrotic mode of cell death,necroptosis,there are molecular mechanisms that can be regulated.Therefore,investigating the mechanism of necroptosis in cerebral I-R injury may have greater translational value.Studies have found that ischemia and hypoxia stimulation can induce the activation of Fas/Fas-associated protein with death domain(FADD),thereby forming the death-inducing signaling complex(DISC).DISC can induce cell death through two pathways: on the one hand,DISC can mediate apoptosis through caspase cascade reaction;on the other hand,when caspase 8 activity is inhibited,necroptosis can be mediated through the receptor-interacting protein kinase(RIPK)-mixed lineage kinase domain-like(MLKL)pathway,suggesting that DISC not only regulates I-R The classic apoptotic pathway in the injury process may also participate in the I-R injury process by regulating necroptosis.Exploring the molecular switch of DISC-mediated necroptosis and interfering with the related targets of this signaling pathway may provide new ideas for clinical translational research on cerebral I-R injury.Activin A(Act A)belongs to the transforming growth factor-β(TGF-β)superfamily.Act A can not only be produced by immune system cells,but also autocrine or paracrine by neurons.The previous research found that the Act A signaling pathway is activated after cerebral ischemic injury,and the Act A derived from the autocrine of neurons after ischemia can act on the neurons themselves through a target-derived mode,and has anti-ischemic injury effects,Act A can also improve cerebral ischemia-induced apoptosis by inhibiting c-Jun N-terminal kinase(JNK).The inhibition of apoptosis by Act A signaling found in previous studies is not enough to explain its neuroprotective effect.Studies have found that the TGF-β/Smad signaling pathway can crosstalk with the Fas/FADD pathway,suggesting that Act A may interact with the Fas/FADD pathway in DISC.It is speculated that the effect of Act A on DICS also exists in the process of cerebral I-R injury,and may regulate the programmed necrosis in the process of I-R through the RIPK-MLKL pathway downstream of DISC.Another study found that RIPK3 can up-regulate the level of reactive oxygen species(ROS)and promote the production of superoxide in mitochondria,suggesting that the outcome of necroptosis may be related to mitochondrial function and mitochondrial oxidative stress.However,it is unclear whether the protective effect of Act A on I-R is related to the regulation of mitochondrial oxidative stress and improvement of mitochondrial function.This study is based on the previous work of the research group,combined with the research progress in recent years,using rat middle cerebral artery occlusion/reperfusion(MCAO/R)and PC12 cell oxygen-glucose deprivation/reoxygenation(OGD/R)model,to simulate the process of cerebral I-R injury in vivo and in vitro,to explore the mechanism of Act A mediating DISC component FADD to regulate necroptosis and apoptosis in the process of cerebral I-R injury and its effect on cerebral I-R injury through the FADD-RIPK pathway Mitochondrial function,the influence of mitochondrial oxidative stress,looking for possible intervention targets,and providing a new direction for translational research for the clinical treatment of acute ischemic stroke in the process of cerebral ischemia-reperfusion injury.Methods:1.Study on the protective effect of Act A on cerebral ischemia-reperfusion injury.To verify the protective effect of Act A on rat MCAO/R and PC12 cell OGD/R injury,based on successfully establishing in vivo and in vitro simulated cerebral I-R injury models with Act A.TTC staining,CCK-8 assay was used to detect cell viability in vivo,and LDH release assay was used to detect the degree of damage in vitro.1).To detect the effect of exogenous Act A on cerebral I-R injury in the MCAO/R model of rats by using the Zea-Longa scoring method,TTC staining.2).To detect the success of establishing OGD/R model of PC12 cells,the appropriate time of oxygen-glucose restoration,the effect of exogenous Act A on OGD/R injury of PC12 cells,the appropriate concentration and timing of exogenous Act A administration by using CCK-8 cell viability test and LDH release experiment.2.Act A alleviates cerebral ischemia-reperfusion injury by regulating cell death mode.To clarify whether there are different cell death modes in cerebral I-R injury and to verify the regulatory effect of Act A on different cell death modes,the simulated brain I-R injury model in vivo and in vitro with Act A targeted intervention,and immunofluorescence staining was used to detect the relationship between apoptosis and necroptosis,Western Blot detection of DISC,apoptosis,and necroptosis marker protein expression.1).To detect the effect of MCAO/R on necroptosis and apoptosis,the effect of exogenous Act A on the cell death pattern of rat by using immunofluorescence staining.2).To detect the DISC,necroptosis,and apoptosis proteins,and the effect of exogenous Act A in cerebral I-R injury by using Western Blot.3).To verify the effect of Act A-mediated DISC regulation of PC12 cell death mode on OGD/R injury by using siRNA of Act A,Western Blot.3.Act A mediates FADD to regulate cell death mode and improve cerebral ischemia-reperfusion injury.To clarify whether Act A improves cerebral I-R injury by regulating FADD to inhibit neuronal necroptosis and apoptosis,PC12 cells were used to establish an OGD/R model with Act A,silenced the main component of DISC,FADD,and overexpress necroptosis marker protein receptors.Receptor interacting protein kinase 3(RIPK3),using CCK-8 assay,LDH release assay,Western Blot to detect the degree of cell damage,DISC,apoptosis,and necroptosis marker protein expression levels.1).To detect the effect of exogenous Act A-mediated DISC on OGD/R injury of PC12 cells by using siRNA of FADD,CCK-8 cell viability test and LDH release experiment,and Western Blot.2).To detect the effect of exogenous Act A on OGD/R injury,the death pattern of X PC12 cells induced by OGD/R injury through DISC-RIPK pathway by using overexpression of RIPK 3,LDH test,and Western Blot.4.Act A regulates FADD-RIPK and affects mitochondrial function to improve cerebral ischemia-reperfusion injury.To explore the mechanism by which Act A regulates FADD to inhibit the necroptosis and apoptosis of nerve cells and improve cerebral I-R injury,from the perspective of mitochondrial function and oxidative stress,Act A was given to simulate the cerebral I-R injury model in vitro and in vivo,and fluorescence detection was used in vivo experiments.Needle detection of ROS level,mitochondrial membrane potential in situ fluorescent staining technology,ELISA method,Western Blot technology,in vitro experiments using silencing FADD and overexpression RIPK3 technology,and fluorescent probe detection of ROS level,mitochondrial membrane potential in situ fluorescent staining technology,ELISA The degree of cell injury,the expression of MDA,SOD,ATP and p-JNK were detected by Western Blot.1).To detect the oxidative stress reaction,p-JNK in brain of MCAO/R rats by using fluorescent probe,mitochondrial membrane potential in situ fluorescence staining technique,MDA,SOD,ATP,and Western Blot.2).To detect the effect of OGD/R and exogenous Act A on the level of mitochondrial oxidative stress in PC12 cells induced by OGD/R by using siRNA FADD,fluorescence probe,mitochondrial membrane potential in situ fluorescence staining,MDA,SOD,ATP.3).To detect the effect of OGD/R and exogenous Act A on mitochondrial function,oxidative stress,p-JNK expression in PC12 cells induced by OGD/R by using overexpression of RIPK3,fluorescent probe,mitochondrial membrane potential in situ fluorescence staining technique,MDA,SOD,ATP,and Western Blot.Results:1.Act A alleviates cerebral ischemia-reperfusion injury.1).Compared with the Sham group,the distribution area of the middle cerebral artery in the MCAO/R+Vehicle group was significantly positive for TTC staining,indicating that the model was established successfully;Intervention with Act A improve neurological function and reduce infarct volume,and the neuroprotective effect of 3 d and 7 d is stronger than that of 1 d(P<0.05),suggesting that Act A alleviates cerebral I-R injury.2).Compared with the Control group,OGD/R can reduce the viability of PC12 cells and increase the release of LDH(P<0.05),suggesting that the OGD/R model of PC12 cells was successfully established,and Act A increases the viability of PC12 cells and reduces the release of LDH(P<0.05).2.Act A inhibits necroptosis and apoptosis of nerve cells and alleviates cerebral ischemia-reperfusion injury.1).Immunofluorescence staining found that with the prolongation of reperfusion time,the number of p-RIPK1/3 positive cells in the brain tissue of rats in MCAO/R+Vehicle group increased compared with that in Sham group,suggesting that the rats in MCAO/R+Vehicle group were reperfused.Necroptosis exists in the early stage of perfusion;the number of p-RIPK1/3 positive cells in the MCAO/R+Act A group was less than that in the MCAO/R+Vehicle group,suggesting that Act A inhibits the necroptosis of rat nerve cells induced by MCAO/R necrosis and neuroprotection.2).Western Blot showed that compared with the Sham group and the Control group,the expressions of the major component proteins Fas,Fas L,and FADD of DISC in the MCAO/R+Vehicle group and the OGD/R group were up-regulated,and the expression of apoptosis marker proteins cleaved caspase 3 and cleaved caspase 8 was up-regulated(P<0.05),the expression of necroptosis marker proteins p-RIPK1/3 and pMLKL was up-regulated(P<0.05),and Act A inhibits the expression of DISC and necrotosis marker proteins(P<0.05),suggesting that Act A inhibits the necroptosis and apoptosis of nerve cells regulated by DISC and alleviates cerebral I-R injury.3).Western Blot found that,compared with the OGD/R+Vector group,the OGD/R+si-Act A group had DISC main proteins,apoptosis marker proteins,and necroptosis marker proteins were both up-regulated(P<0.05),suggesting that the regulatory effect of Act A on the main component proteins of DISC was inhibited after Act A was silenced.3.Act A regulates FADD,inhibits necroptosis and apoptosis of nerve cells,and alleviates cerebral ischemia-reperfusion injury.1).CCK-8 cell viability detection and LDH release assay showed that compared with OGD/R+Vector group,silencing FADD increased PC12 cell viability and decreased LDH release level(P<0.05).After silencing FADD with Act A,the cell viability was higher and the LDH release level was lower(P<0.05),suggesting that Act A may be involved in the OGD/R injury of PC12 cells through DISC;Western Blot found that compared with the OGD/R+Vector group,silencing FADD inhibit OGD /R XII induced the expression of the necroptosis marker proteins p-RIPK1/3 and p-MLKL in PC12 cells(P<0.05),and the intervention of Act A had a stronger inhibitory effect on necroptosis(P<0.05),suggesting that Act A may be involved in the OGD/R injury of PC12 cells through the necroptosis pathway downstream of DISC.2).The LDH release test found that compared with the OGD/R+Vector group,the overexpression of RIPK3 increased the LDH release level of PC12 cells induced by OGD/R(P<0.05),and Act A intervention on the basis of overexpression of RIPK3 made LDH The release level decreased(P<0.05),suggesting that RIPK3 overexpression can aggravate the OGD/R injury of PC12 cells,and Act A intervention inhibit the OGD/R injury of PC12 cells aggravated by RIPK3 overexpression;Western Blot detection found that RIPK3 overexpression can up-regulate OGD /R induced the expression of p-RIPK1/3 and p-MLKL in PC12 cells(P<0.05),Act A intervention could inhibit the expression of up-regulated necroptosis marker proteins induced by RIPK3overexpression(P<0.05),suggesting that the overexpression of RIPK3 can aggravate the necroptosis in the process of OGD/R,and the intervention of Act A inhibit the necroptosis in the process of OGD/R in PC12 cells exacerbated by the overexpression of RIPK3.4.Act A inhibits FADD-RIPK to maintain mitochondrial function and alleviate cerebral ischemia-reperfusion injury.1).Compared with the Sham group,the oxidative stress response of brain tissue nerve cells in the MCAO/R+Vehicle group was enhanced,the mitochondrial membrane potential(MMP)was decreased,the generation of ATP was reduced,and the expression of p-JNK was up-regulated(P<0.05),Act A intervention can alleviate the mitochondrial oxidative stress response induced by MCAO/R,promote the recovery of MMP,increase the generation of ATP,and down-regulate the expression of p-JNK(P<0.05),suggesting that in the simulated brain I-R injury model in vivo,Act A can reduce mitochondrial oxidative stress and maintain mitochondrial function.2).Compared with the Control group,OGD/R injury induces the accumulation of ROS,the loss of MMP,the decrease of ATP production,the increase of MDA level,and the decrease of SOD level(P<0.05).Compared with the OGD/R+Vector group,silencing of FADD can alleviate ROS accumulation,restore MMP,increase ATP production,reduce MDA level,and increase SOD level(P<0.05).Compared with OGD/R+si FADD group,Act A can strengthen the effect of si FADD on ROS,MMP,ATP,MDA,the role of SOD suggests that OGD/R injury can induce mitochondrial oxidative stress and mitochondrial dysfunction in PC12 cells,and silencing FADD and/or Act A intervention can alleviate OGD/R-induced mitochondrial oxidative stress and maintain normal mitochondrial function.3).Compared with the OGD/R+Vector group,RIPK3 overexpression can aggravate OGD/R-induced PC12 cell ROS accumulation,MMP loss,ATP production decrease,MDA level increase,SOD level decrease(P<0.05),Act A intervention can alleviate ROS accumulation caused by overexpression of RIPK3,restore MMP,promote ATP production,reduce MDA,and increase SOD level(P<0.05),suggesting that overexpression of RIPK3 can aggravate OGD/R injury-induced mitochondrial oxidative stress in PC12 cells and damage mitochondria Function;Act A intervention can alleviate mitochondrial oxidative stress aggravated by RIPK3 overexpression and maintain normal mitochondrial function.Conclusions:1.Experiments in vivo and in vitro confirmed that exogenous Act A has a protective effect on cerebral ischemia-reperfusion injury.2.Necroptosis and apoptosis exist in the process of cerebral ischemia-reperfusion injury,and the protective effect of exogenous Act A on cerebral ischemia-reperfusion injury is mainly by inhibiting necroptosis and apoptosis.3.Exogenous Act A attenuates cerebral ischemia-reperfusion injury by inhibiting necroptosis and apoptosis via regulating FADD.4.Exogenous Act A inhibits necroptosis by attenuating oxidative stress and regulating mitochondrial function via FADD-RIPK to improve cerebral ischemiareperfusion injury. |
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