| Stroke,also known as"cerebrovascular disease",can be divided into ischemic stroke and hemorrhagic stroke according to their pathogenesis.It has the characteristics of high morbidity,high mortality,and high disability rate.Stroke has become the leading cause of death in China.It not only seriously threatens patients’health,but also brings a heavy burden to the society and country.The in-depth exploration of its injury mechanism is essential for developing new effective treatment and prevention strategies for stroke.The effective reperfusion therapy in the early stage is the most important treatment for acute ischemic stroke（AIS）.However,the AIS patients often have a doubtful prognosis although the developments of strategies of restoring blood flow stent retriever-based technology and alteplase-based technology have been achieved in recent years.A mass of reactive oxygen species（ROS）,which plays a key role in the oxidative stress injuries,is generated upon reperfusion,leading to the“effective reperfusion therapy”that seems like“uneffective”treatment strategies.Therefore,the relieving of the ischemia/reperfusion（I/R）injury is really important for improving the outcome of stroke.Adiponectin（APN）,a fat-derived hormone,has a wide range of antioxidative and anti-inflammatory effects.Moreover,it has the potential to protect against brain disorders.However,APN is a 247-amino-acid polypeptide whose molecular weight is 30 k Da.Thus,APN has restricted blood-brain barrier（BBB）permeability under physiological conditions,which seriously restricts the application of APN in the central nervous system.Therefore,in the present study,an APN peptide（APNp）（amino acid sequence:NH2-LQVYGDGDHNGLYADNVN-COOH）was designed and chemically synthesized on the basis of the functional area in APN’s globular domain.Nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3（NLRP3）is an intracellular sensor that detects changes in the microenvironment and responds to the endogenous danger signals,which leads to the formation and activation of the NLRP3 inflammasome.The NLRP3 inflammasome,consisting of NOD-like receptor,apoptosis-associated speck-like protein containing a caspase recruitment domain（ASC）and caspase-1,has been demonstrated to be involved in the inflammation responses in a variety of pathological conditions,including those caused by I/R injury.The generation of（ROS）,which are produced in mitochondria by the electron transport chain under physiological conditions and induce oxidative stress under pathological conditions,is considered one of the most important regulating factors of NLRP3 inflammasome activation.The thioredoxin（Trx）system is one of the main intracellular antioxidant systems,and it detoxifies ROS and protects cells from oxidative damage.The thioredoxin-interacting protein（TXNIP）represents the intersection of oxidative stress and inflammasome activation.As one of the critical mechanisms in I/R injury,the overload of ROS not only induces oxidative stress damage but also mediates inflammation and apoptosis.However,the role of Trx/TXNIP,ROS,and the NLRP3inflammasome in the protection of APN against cerebral I/R injury remains unclear.The present study was designed to explore the antioxidative and anti-inflammatory mechanism of APNp in an in vivo transient middle cerebral artery occlusion（t MCAO）model and an in vitro oxygen-glucose deprivation and reintroduction（OGD-R）model.The roles of AMPK/GSK-3βand Trx/TXNIP/ROS/NLRP3 were explored.This research can be divided into three parts:1.The neuroprotective effect of APNp on cerebral I/R injury.2.The role of oxidative stress and NLRP3 inflammasome in the neuroprotective effect of APNp on cerebral I/R injury.3.The effect of APNp on astrocyte after OGD-R injury.PartⅠThe neuroprotective effect of APNp on cerebral I/R injury MethodsFirst,the FITC-labeled APNp was injected intraperitoneally into mice to evaluate the penetrability of APNp to the BBB.Second,the mice were randomly divided into 4 groups:Sham group,I/R group,I/R+sham group,and I/R+APNp group.Mice were subjected to t MCAO/sham treatment and APNp/vehicle administration according to the grouping.The mortality at 14d was recorded.The neurological scores,cerebral infarct volume,brain water content were tested to evaluate the severity of the injury.The TUNELstaining and western blot were conducted to evaluate the cell apoptosis.The content of ROS,MDA,3-NT,8-OHd G,and the activity of SOD and GSH-Px were tested to evaluate the oxidative stress.Results1.The APNp was observed in the brain after intraperitoneal administration.2.APNp alleviated the neurological deficit and brain edema and decreased the infarct size after cerebral I/R in mice.3.APNp ameliorated cellular apoptosis and oxidative stress after cerebral I/R in mice.APNp administration significantly alleviated the increase of apoptosis index and the increase of protein levels of Bax/Bcl-2 and cleaved caspase-3 after I/R in the penumbra.APNp also significantly alleviated the increase of molecular markers of oxidative stress injury and the decrease of the activities of SOD and GSH-Px.ConclusionAPNp has BBB penetrability and protects against cerebral I/R injury.PartⅡThe role of NLRP3 inflammasome and oxidative stress in the neuroprotective effect of APNp against cerebral I/R injuryMethodsFirst,the mice were randomly divided into 4 groups:Sham group,I/R group,I/R+sham group,and I/R+APNp group.Mice were subjected to t MCAO/sham treatment and APNp/vehicle administration according to the grouping.The protein levels of Trx,TXNIP,NLRP3,ASC and caspase-1 p20 were tested by western blot to evaluate the activity of NLRP3 inflammasome in the penumbra.The phosphorylation of AMPK and Gsk-βin the penumbra were also tested by western blot.Second,the mice were randomly divided into 5groups:Sham group,I/R group,I/R+APNp group,I/R+APNp+PX-12,and I/R+APNp+CC group.PX-12 or CC was administered through tail vein injection immediately after occlusion in the t MCAO+APNp+PX-12 group and the t MCAO+APNp+CC group.The activity of NLRP3 inflammasome and the phosphorylation of AMPK and Gsk-βin each group were tested by western blot.Then the oxidative stress,cellular apoptosis,and neurological function in each group were tested by immunostaining,DHE staining,transmission electron microscopy observations,western blot,etc..Results1.The protein levels of TXNIP,NLRP3,ASC,and caspase-1 p20 increased significantly,while the protein level of Trx decreased significantly in the penumbra after I/R.APNp administration reversed this trend to promote Trx downregulation and NLRP3inflammasome activation.The protein levels of p-AMPK（Thr172）/AMPK and p-GSK-3β（Ser9）/GSK-3βin the penumbra were also significantly upregulated by APNp.2.The suppression effects of APNp against the upregulation of TXNIP,NLRP3,ASC,and caspase-1 p20 were reversed by the addition of PX-12,a selective Trx inhibitor.Moreover,CC,a selective AMPK inhibitor,significantly decreased the phosphorylation of GSK-3β,downregulated the protein level of Trx,and upregulated the protein level of TXNIP.3.APNp’s protective effect of alleviating cerebral infarction and brain edema,promoting the survival of neurons,alleviating oxidative stress injury and mitochondrial dysfunction against I/R injury can be reversed by AMPK inhibition and Trx inhibition.ConclusionThe NLRP3 inflammasome is activated and oxidative stress is increased in the penumbra after cerebral I/R.APNp promotes the phosphorylation of AMPK and GSK-3β,regulates the protein levels of Trx and TXNIP,and inhibits oxidative stress and the activation of NLRP3 inflammasome in the penumbra after cerebral I/R.Part Ⅲ The effect of APNp on astrocyte after OGD-R injuryMethodsThe cellular localization of Adipo R1 in the penumbra was investigated in the present study.Most of Adipo R1 was expressed in astrocytes after I/R.Thus,the effects of APNp on AMPK phosphorylation and NLRP3 inflammasome activation were investigated in primary astrocytes using an OGD-R injury model.The astrocytes were randomly divided into 5groups:Control group,OGD-R group,OGD-R+APNp group,OGD-R+APNp+PX-12group,and OGD-R+APNp+CC group.The treatment of vehicle,APNp,TX-12,and/or CC was given according to the grouping.The protein levels of p-AMPK/AMPK、p-Gsk-3β/Gsk-3β,Trx,TXNIP,NLRP3,ASC,caspase-1 p20,IL-1βand IL-18 in each group were tested.And the nuclear translocation of Nrf2 was also tested.Results1.The phosphorylations of AMPK（Thr172）and GSK-3β（Ser9）were notably promoted by APNp and were downregulated by CC.2.The nuclear translocation of Nrf2 was notably promoted by APNp and was downregulated by CC.3.APNp administration ameliorated the increase in the protein levels of TXNIP,NLRP3,ASC,caspase-1 p20,IL-1β,and IL-6,and notably alleviated the decrease in Trx after OGD-R.PX-12 and CC inhibited this effect of APNp.ConclusionAPNp promotes the phosphorylation of AMPK（Thr172）and GSK-3β（Ser9）in astrocyte and inhibits the activation of NLRP3 inflammasome after OGD-R. |
