BACKGROUND:Individuals who survive a cardiac arrest often sustain cognitive impairments due to ischemia-reperfusion injury.Mesenchymal stem cell(MSC)transplantation is used to reduce tissue damage,but exosomes are mor...BACKGROUND:Individuals who survive a cardiac arrest often sustain cognitive impairments due to ischemia-reperfusion injury.Mesenchymal stem cell(MSC)transplantation is used to reduce tissue damage,but exosomes are more stable and highly conserved than MSCs.This study was conducted to investigate the therapeutic effects of MSC-derived exosomes(MSC-Exo)on cerebral ischemia-reperfusion injury in an in vitro model of oxygen-glucose deprivation/reperfusion(OGD/R),and to explore the underlying mechanisms.METHODS:Primary hippocampal neurons obtained from 18-day Sprague-Dawley rat embryos were subjected to OGD/R treatment,with or without MSC-Exo treatment.Exosomal integration,cell viability,mitochondrial membrane potential,and generation of reactive oxygen species(ROS)were examined.Terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nickend labeling(TUNEL)staining was performed to detect neuronal apoptosis.Moreover,mitochondrial function-associated gene expression,Nrf2 translocation,and expression of downstream antioxidant proteins were determined.RESULTS:MSC-Exo attenuated OGD/R-induced neuronal apoptosis and decreased ROS generation(P<0.05).The exosomes reduced OGD/R-induced Nrf2 translocation into the nucleus(2.14±0.65 vs.5.48±1.09,P<0.01)and increased the intracellular expression of antioxidative proteins,including superoxide dismutase and glutathione peroxidase(17.18±0.97 vs.14.40±0.62,and 20.65±2.23 vs.16.44±2.05,respectively;P<0.05 for both).OGD/R significantly impaired the mitochondrial membrane potential and modulated the expression of mitochondrial functionassociated genes,such as PINK,DJ1,LRRK2,Mfn-1,Mfn-2,and OPA1.The abovementioned changes were partially reversed by exosomal treatment of the hippocampal neurons.CONCLUSIONS:MSC-Exo treatment can alleviate OGD/R-induced oxidative stress and dysregulation of mitochondrial function-associated genes in hippocampal neurons.Therefore,MSCExo might be a potential therapeutic strategy to prevent OGD/R-induced neuronal injury.展开更多
In this study,we investigated the protective effect of hyperbaric oxygen(HBO)on PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion and its possible mechanism.PC12 and H9C2 cell oxygen-glucose d...In this study,we investigated the protective effect of hyperbaric oxygen(HBO)on PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion and its possible mechanism.PC12 and H9C2 cell oxygen-glucose deprivation/reperfusion model were established.Cells were divided into a control group,model group,hyperbaric air(HBA)group and HBO group.The cell viability was detected by the CCK8 assay.Hoechst 33342 and PI staining assays and mitochondrial membrane potential(MMP)assays were used to detect cell apoptosis.The ultrastructure of cells,including autophagosomes,lysosomes,and apoptosis,were examined using a transmission electron microscope.The expression of autophagy-related proteins was detected by cellular immunofluorescence and immunocytochemistry.Our results showed that HBO can significantly improve the vitality of damaged PC12 and H9C2 cells caused by oxygen–glucose deprivation/reperfusion.HBO can significantly inhibit apoptosis of PC12 and H9C2 cells caused by oxygenglucose deprivation/reperfusion.Importantly,we found that the protective mechanism of PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion may be related to the inhibition of the autophagy pathway.In this study,the results of cellular immunofluorescence and immunocytochemistry experiments showed that the 4E-BP1,p-AKt and mTOR levels of PC12 and H9C2 cells in the model group decreased,while the levels of LC3B,Atg5 and p53 increased.However,after HBO treatment,these autophagy-related indexes were reversed.In addition,observation of the cell ultrastructure with transmission electron microscopy found that in the model group,a significant increase in the number of autophagic vesicles was observed.In the HBO group,a decrease in autophagic vesicles was observed.The study demonstrated that hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy.展开更多
OBJECTIVE TO investigate the neural protection of dehydrocostus lactone(DHL)against neuronal injury induced by oxygen and glucose deprivation/reperfusion(OGD/R)in differentiated PC12 cells.METHODS We used a cellular m...OBJECTIVE TO investigate the neural protection of dehydrocostus lactone(DHL)against neuronal injury induced by oxygen and glucose deprivation/reperfusion(OGD/R)in differentiated PC12 cells.METHODS We used a cellular model of 2 h of OGD and 24 h of reperfusion to mimic cerebral ischemia-reperfusion injury.Cell viability was used to reflect the degree of OGD/R-induced injury.Cells were treated with DHL during the reperfusion phase.Cell Counting Kit(CCK-8)and LDH assays were performed to determine the optimal dose of DHL and cell viability.Flow cytometry analysis and Monodansylcadaverine(MDC)staining were then conducted to detect apoptosis rate and autophagosome formation after OGD/R in PC12 cells.Immunofluorescence and Western blotting analyses were used to detect the expres⁃sion of proteins associated with autophagy and apoptosis.RESULTS OGD/R significantly decreased cell viability and increased apoptosis rate.The expression levels of autophagy-related proteins,namely,LC3 and Beclin-1,and apoptosisrelated proteins,namely,Bax and caspase-3 increased,but that of the anti-apoptosis Bcl-2 protein decreased.However,DHL attenuated OGD/R-induced neuronal injury through inhibition of apoptosis and autophagy properties by modulating au⁃tophagy-associated proteins(LC3 and Beclin-1)and apoptosis-modulating proteins(caspase-3 and Bcl-2/Bax).CONCLU⁃SION Our data provide an evidence for the neuroprotective effect of DHL against ischemic neuronal injury.Hence,DHL could be a promising candidate for treatment of ischemic stroke.展开更多
Shuxuetong injection composed of leech(Hirudo nipponica Whitman) and earthworm(Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotect...Shuxuetong injection composed of leech(Hirudo nipponica Whitman) and earthworm(Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotective mechanism of Shuxuetong injection remains poorly understood. Here, cerebral microvascular endothelial cells(bEnd.3) were incubated in glucose-free Dulbecco's modified Eagle's medium containing 95% N_2/5% CO_2 for 6 hours, followed by high-glucose medium containing 95% O_2 and 5% CO_2 for 18 hours to establish an oxygen-glucose deprivation/reperfusion model. This in vitro cell model was administered Shuxuetong injection at 1/32, 1/64, and 1/128 concentrations(diluted 32-, 64-, and 128-times). Cell Counting Kit-8 assay was used to evaluate cell viability. A fluorescence method was used to measure lactate dehydrogenase, and a fluorescence microplate reader used to detect intracellular reactive oxygen species. A fluorescent probe was also used to measure mitochondrial superoxide production. A cell resistance meter was used to measure transepithelial resistance and examine integrity of monolayer cells. The fluorescein isothiocyanate-dextran test was performed to examine blood-brain barrier permeability. Real-time reverse transcription polymerase chain reaction was performed to analyze mRNA expression levels of tumor necrosis factor alpha, interleukin-1β, interleukin-6, and inducible nitric oxide synthase. Western blot assay was performed to analyze expression of caspase-3, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, occludin, vascular endothelial growth factor, cleaved caspase-3, B-cell lymphoma 2, phosphorylated extracellular signal-regulated protein kinase, extracellular signal-regulated protein kinase, nuclear factor-κB p65, I kappa B alpha, phosphorylated I kappa B alpha, I kappa B kinase, phosphorylated I kappa B kinase, claudin-5, and zonula occludens-1. Our results show that Shuxuetong injection increases bEnd.3 cell viability and B-cell lymphoma 2 expression, reduces cleaved caspase-3 expression, inhibits production of reactive oxygen species and mitochondrial superoxide, suppresses expression of tumor necrosis factor alpha, interleukin-1β, interleukin-6, inducible nitric oxide synthase mRNA, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, markedly increases transepithelial resistance, decreases blood-brain barrier permeability, upregulates claudin-5, occludin, and zonula occludens-1 expression, reduces nuclear factor-κB p65 and vascular endothelial growth factor expression, and reduces I kappa B alpha, extracellular signal-regulated protein kinase 1/2, and I kappa B kinase phosphorylation levels. Overall, these findings suggest that Shuxuetong injection has protective effects on brain microvascular endothelial cells after oxygen-glucose deprivation/reperfusion. Moreover, its protective effect is associated with reduction of mitochondrial superoxide production, inhibition of the inflammatory response, and inhibition of vascular endothelial growth factor, extracellular signal-regulated protein kinase 1/2, and the nuclear factor-κB p65 signaling pathway.展开更多
Stigmasterol is a plant sterol with anti-apoptotic,anti-oxidative and anti-inflammatory effect through multiple mechanisms.In this study,we further assessed whether it exerts protective effect on human brain microvess...Stigmasterol is a plant sterol with anti-apoptotic,anti-oxidative and anti-inflammatory effect through multiple mechanisms.In this study,we further assessed whether it exerts protective effect on human brain microvessel endothelial cells(HBMECs)against ischemia-reperfusion injury and explored the underlying mechanisms.HBMECs were used to establish an in vitro oxygen and glucose deprivation/reperfusion(OGD/R)model,while a middle cerebral artery occlusion(MCAO)model of rats were constructed.The interaction between stigmasterol and EPHA2 was detected by surface plasmon resonance(SPR)and cellular thermal shift assay(CETSA).The results showed that 10μmol·L−1 stigmasterol significantly protected cell viability,alleviated the loss of tight junction proteins and attenuated the blood-brain barrier(BBB)damage induced by OGD/R in the in vitro model.Subsequent molecular docking showed that stigmasterol might interact with EPHA2 at multiple sites,including T692,a critical gatekeep residue of this receptor.Exogenous ephrin-A1(an EPHA2 ligand)exacerbated OGD/R-induced EPHA2 phosphorylation at S897,facilitated ZO-1/claudin-5 loss,and promoted BBB leakage in vitro,which were significantly attenuated after stigmasterol treatment.The rat MCAO model confirmed these protective effects in vivo.In summary,these findings suggest that stigmasterol protects HBMECs against ischemia-reperfusion injury by maintaining cell viability,reducing the loss of tight junction proteins,and attenuating the BBB damage.These protective effects are at least meditated by its interaction with EPHA2 and inhibitory effect on EPHA2 phosphorylation.展开更多
Background:Disruption of the blood–brain barrier(BBB)after a stroke can lead to brain injury and neurological impairment.Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass p...Background:Disruption of the blood–brain barrier(BBB)after a stroke can lead to brain injury and neurological impairment.Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2(LMP2)in the pathophysiology of ischemia stroke.However,the relationship between the immunoproteasome LMP2 and the BBB remains unclear.Methods:Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion(MCAO/R).Three days before MCAO,the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region.The rat brain microvascular endothelial cell(RBMVEC)line was exposed to oxygen–glucose deprivation/reperfusion(OGD/R)to mimic ischemic conditions in vitro.The RNA interference-mediated knockdown of LMP2 orβ-catenin was analysed in vivo and in vitro.Analysis of the quantity of extravasated Evans blue(EB)and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB.Immunofluorescence and Western blotting were employed to detect the expression of target proteins.Cell migration was evaluated using a scratch migration assay.The results of immunofluorescence,Western blotting and cell migration were quantified using the software ImageJ(Version 1.53).Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference(LSD)test.Results:Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins[occludin,claudin-1 and zonula occludens(ZO-1)]in the MCAO/R group compared with the sham group(P<0.001).However,inhibition of the immunoproteasome LMP2 restored the expression of these proteins,resulting in higher levels of occludin,claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group(P<0.001).In addition,inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability[e.g.,the quantity of extravasated EB:LMP2-shRNA group(58.54±7.37)μg/g vs.control-shRNA group(103.74±4.32)μg/g,P<0.001],and promoted the upregulation of Wnt-3a andβ-catenin proteins in rats following MCAO/R.In vitro experiments,OGD/R induced marked upregulation of LMP2,proapoptotic protein Bax and cleaved caspase-3,and downregulation of occludin,claudin-1,ZO-1 and Bcl-2,as well as inhibition of the Wnt/β-catenin pathway Wnt-3a andβ-catenin proteins in RBMVECs,compared with the control group under normal culture conditions(P<0.001).However,silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R.Silencing ofβ-catenin by transfection of RBMVECs withβ-catenin-si RNA aggravated the downregulation of tight junction proteins,and reduced the proliferation and migration of RBMVECs following OGD/R,compared with the control-siRNA group(P<0.001).LMP2-si RNA andβ-catenin-si RNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.Conclusions:This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia induced BBB injury,and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.展开更多
In the central nervous system,hyperpolarizationactivated cyclic nucleotide-gated(HCN)channels are essential to maintain normal neuronal function.Recent studies have shown that HCN channels may be involved in the patho...In the central nervous system,hyperpolarizationactivated cyclic nucleotide-gated(HCN)channels are essential to maintain normal neuronal function.Recent studies have shown that HCN channels may be involved in the pathological process of ischemic brain injury,but the mechanisms remain unclear.Autophagy is activated in cerebral ischemia,but its role in cell death/survival remains controversial.In this study,our results showed that the HCN channel blocker ZD7288 remarkably decreased the percentage of apoptotic neurons and corrected the excessive autophagy induced by oxygen-glucose deprivation followed by reperfusion(OGD/R)in hippocampal HT22 neurons.Furthermore,in the OGD/R group,p-mTOR,p-ULK1(Ser757),and p62 were significantly decreased,while p-ULK1(Ser317),atg5,and beclin1 were remarkably increased.ZD7288 did not change the expression of p-ULK1(Ser757),ULK1(Ser317),p62,Beclin1,and atg5,which are involved in regulating autophagosome formation.Besides,we found that OGD/R induced a significant increase in Cathepsin D expression,but not LAMP-1.Treatment with ZD7288 at 10μmol/L in the OGD/R group did not change the expression of cathepsin D and LAMP-1.However,chloroquine(CQ),which decreases autophagosome-lysosome fusion,eliminated the correction of excessive autophagy and neuroprotection by ZD7288.Besides,shRNA knockdown of HCN2 channels significantly reduced the accumulation of LC3-Ⅱand increased neuron survival in the OGD/R and transient global cerebral ischemia(TGCI)models,and CQ also eliminated the effects of HCN2-shRNA.Furthermore,we found that the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes decreased in Con-shRNAtransfected HT22 neurons exposed to OGD/R or CQ.In HCN2-shRNA-transfected HT22 neurons,the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes increased under OGD/R;however,the percentage was significantly decreased by the addition of CQ to HCN2-shRNA-transfected HT22 neurons.The present results demonstrated that blockade of HCN2 channels provides neuroprotection against OGD/R and TGCI by accelerating autophagic degradation attributable to the promotion of autophagosome and lysosome fusion.展开更多
基金supported by a grant from the National Natural Science Foundation of China(81701872)。
文摘BACKGROUND:Individuals who survive a cardiac arrest often sustain cognitive impairments due to ischemia-reperfusion injury.Mesenchymal stem cell(MSC)transplantation is used to reduce tissue damage,but exosomes are more stable and highly conserved than MSCs.This study was conducted to investigate the therapeutic effects of MSC-derived exosomes(MSC-Exo)on cerebral ischemia-reperfusion injury in an in vitro model of oxygen-glucose deprivation/reperfusion(OGD/R),and to explore the underlying mechanisms.METHODS:Primary hippocampal neurons obtained from 18-day Sprague-Dawley rat embryos were subjected to OGD/R treatment,with or without MSC-Exo treatment.Exosomal integration,cell viability,mitochondrial membrane potential,and generation of reactive oxygen species(ROS)were examined.Terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nickend labeling(TUNEL)staining was performed to detect neuronal apoptosis.Moreover,mitochondrial function-associated gene expression,Nrf2 translocation,and expression of downstream antioxidant proteins were determined.RESULTS:MSC-Exo attenuated OGD/R-induced neuronal apoptosis and decreased ROS generation(P<0.05).The exosomes reduced OGD/R-induced Nrf2 translocation into the nucleus(2.14±0.65 vs.5.48±1.09,P<0.01)and increased the intracellular expression of antioxidative proteins,including superoxide dismutase and glutathione peroxidase(17.18±0.97 vs.14.40±0.62,and 20.65±2.23 vs.16.44±2.05,respectively;P<0.05 for both).OGD/R significantly impaired the mitochondrial membrane potential and modulated the expression of mitochondrial functionassociated genes,such as PINK,DJ1,LRRK2,Mfn-1,Mfn-2,and OPA1.The abovementioned changes were partially reversed by exosomal treatment of the hippocampal neurons.CONCLUSIONS:MSC-Exo treatment can alleviate OGD/R-induced oxidative stress and dysregulation of mitochondrial function-associated genes in hippocampal neurons.Therefore,MSCExo might be a potential therapeutic strategy to prevent OGD/R-induced neuronal injury.
基金supported by the National Natural Science Foundation of China(81960246,81701089,81560044 and 30860113)the Guangxi Natural Science Foundation(2020GXNSFAA238003 and 2017GXNSFBA198010)+1 种基金the Guangxi Medical and Health Appropriate Technology Research and Development Project(S2020076,S201422-01 and S2019087)the Shanxi Health Research Project(2019165).
文摘In this study,we investigated the protective effect of hyperbaric oxygen(HBO)on PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion and its possible mechanism.PC12 and H9C2 cell oxygen-glucose deprivation/reperfusion model were established.Cells were divided into a control group,model group,hyperbaric air(HBA)group and HBO group.The cell viability was detected by the CCK8 assay.Hoechst 33342 and PI staining assays and mitochondrial membrane potential(MMP)assays were used to detect cell apoptosis.The ultrastructure of cells,including autophagosomes,lysosomes,and apoptosis,were examined using a transmission electron microscope.The expression of autophagy-related proteins was detected by cellular immunofluorescence and immunocytochemistry.Our results showed that HBO can significantly improve the vitality of damaged PC12 and H9C2 cells caused by oxygen–glucose deprivation/reperfusion.HBO can significantly inhibit apoptosis of PC12 and H9C2 cells caused by oxygenglucose deprivation/reperfusion.Importantly,we found that the protective mechanism of PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion may be related to the inhibition of the autophagy pathway.In this study,the results of cellular immunofluorescence and immunocytochemistry experiments showed that the 4E-BP1,p-AKt and mTOR levels of PC12 and H9C2 cells in the model group decreased,while the levels of LC3B,Atg5 and p53 increased.However,after HBO treatment,these autophagy-related indexes were reversed.In addition,observation of the cell ultrastructure with transmission electron microscopy found that in the model group,a significant increase in the number of autophagic vesicles was observed.In the HBO group,a decrease in autophagic vesicles was observed.The study demonstrated that hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy.
基金National Natural Science Foundation of China(8166070081260679)Ningxia Col ege First-Class Discipline Construction Project(Chinese Medicine)Funded Project(NXYLXK2017A06)
文摘OBJECTIVE TO investigate the neural protection of dehydrocostus lactone(DHL)against neuronal injury induced by oxygen and glucose deprivation/reperfusion(OGD/R)in differentiated PC12 cells.METHODS We used a cellular model of 2 h of OGD and 24 h of reperfusion to mimic cerebral ischemia-reperfusion injury.Cell viability was used to reflect the degree of OGD/R-induced injury.Cells were treated with DHL during the reperfusion phase.Cell Counting Kit(CCK-8)and LDH assays were performed to determine the optimal dose of DHL and cell viability.Flow cytometry analysis and Monodansylcadaverine(MDC)staining were then conducted to detect apoptosis rate and autophagosome formation after OGD/R in PC12 cells.Immunofluorescence and Western blotting analyses were used to detect the expres⁃sion of proteins associated with autophagy and apoptosis.RESULTS OGD/R significantly decreased cell viability and increased apoptosis rate.The expression levels of autophagy-related proteins,namely,LC3 and Beclin-1,and apoptosisrelated proteins,namely,Bax and caspase-3 increased,but that of the anti-apoptosis Bcl-2 protein decreased.However,DHL attenuated OGD/R-induced neuronal injury through inhibition of apoptosis and autophagy properties by modulating au⁃tophagy-associated proteins(LC3 and Beclin-1)and apoptosis-modulating proteins(caspase-3 and Bcl-2/Bax).CONCLU⁃SION Our data provide an evidence for the neuroprotective effect of DHL against ischemic neuronal injury.Hence,DHL could be a promising candidate for treatment of ischemic stroke.
基金supported in part by the National Natural Science Foundation of China,No.81573644(to LMH),81573733(to SWX)the Tianjin 131 Innovative Team Project,China(to HW)+5 种基金the National Major Science and Technology Project of China,No.2012ZX09101201-004(to SWX)the Science and Technology Plan Project of Tianjin of China,No.16PTSYJC00120(to LMH)the Applied Foundation and Frontier Technology Research Program of Tianjin of China(General Project),No.14JCYBJC28900(to SXW)the National International Science and Technology Cooperation Project of China,No.2015DFA30430(to HW)the Key Program of the Natural Science Foundation of Tianjin of China,No.16ICZDJC36300(to HW)the Scientific Research and Technology Development Plan Project of Guangxi Zhuang Autonomous Region of China,No.14125008-2-5(to SXW)
文摘Shuxuetong injection composed of leech(Hirudo nipponica Whitman) and earthworm(Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotective mechanism of Shuxuetong injection remains poorly understood. Here, cerebral microvascular endothelial cells(bEnd.3) were incubated in glucose-free Dulbecco's modified Eagle's medium containing 95% N_2/5% CO_2 for 6 hours, followed by high-glucose medium containing 95% O_2 and 5% CO_2 for 18 hours to establish an oxygen-glucose deprivation/reperfusion model. This in vitro cell model was administered Shuxuetong injection at 1/32, 1/64, and 1/128 concentrations(diluted 32-, 64-, and 128-times). Cell Counting Kit-8 assay was used to evaluate cell viability. A fluorescence method was used to measure lactate dehydrogenase, and a fluorescence microplate reader used to detect intracellular reactive oxygen species. A fluorescent probe was also used to measure mitochondrial superoxide production. A cell resistance meter was used to measure transepithelial resistance and examine integrity of monolayer cells. The fluorescein isothiocyanate-dextran test was performed to examine blood-brain barrier permeability. Real-time reverse transcription polymerase chain reaction was performed to analyze mRNA expression levels of tumor necrosis factor alpha, interleukin-1β, interleukin-6, and inducible nitric oxide synthase. Western blot assay was performed to analyze expression of caspase-3, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, occludin, vascular endothelial growth factor, cleaved caspase-3, B-cell lymphoma 2, phosphorylated extracellular signal-regulated protein kinase, extracellular signal-regulated protein kinase, nuclear factor-κB p65, I kappa B alpha, phosphorylated I kappa B alpha, I kappa B kinase, phosphorylated I kappa B kinase, claudin-5, and zonula occludens-1. Our results show that Shuxuetong injection increases bEnd.3 cell viability and B-cell lymphoma 2 expression, reduces cleaved caspase-3 expression, inhibits production of reactive oxygen species and mitochondrial superoxide, suppresses expression of tumor necrosis factor alpha, interleukin-1β, interleukin-6, inducible nitric oxide synthase mRNA, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, markedly increases transepithelial resistance, decreases blood-brain barrier permeability, upregulates claudin-5, occludin, and zonula occludens-1 expression, reduces nuclear factor-κB p65 and vascular endothelial growth factor expression, and reduces I kappa B alpha, extracellular signal-regulated protein kinase 1/2, and I kappa B kinase phosphorylation levels. Overall, these findings suggest that Shuxuetong injection has protective effects on brain microvascular endothelial cells after oxygen-glucose deprivation/reperfusion. Moreover, its protective effect is associated with reduction of mitochondrial superoxide production, inhibition of the inflammatory response, and inhibition of vascular endothelial growth factor, extracellular signal-regulated protein kinase 1/2, and the nuclear factor-κB p65 signaling pathway.
基金supported by the Key Research Project of the Science&Technology Department of Sichuan Province,China(Nos.2021YFS0131 and 2020YFS0414).
文摘Stigmasterol is a plant sterol with anti-apoptotic,anti-oxidative and anti-inflammatory effect through multiple mechanisms.In this study,we further assessed whether it exerts protective effect on human brain microvessel endothelial cells(HBMECs)against ischemia-reperfusion injury and explored the underlying mechanisms.HBMECs were used to establish an in vitro oxygen and glucose deprivation/reperfusion(OGD/R)model,while a middle cerebral artery occlusion(MCAO)model of rats were constructed.The interaction between stigmasterol and EPHA2 was detected by surface plasmon resonance(SPR)and cellular thermal shift assay(CETSA).The results showed that 10μmol·L−1 stigmasterol significantly protected cell viability,alleviated the loss of tight junction proteins and attenuated the blood-brain barrier(BBB)damage induced by OGD/R in the in vitro model.Subsequent molecular docking showed that stigmasterol might interact with EPHA2 at multiple sites,including T692,a critical gatekeep residue of this receptor.Exogenous ephrin-A1(an EPHA2 ligand)exacerbated OGD/R-induced EPHA2 phosphorylation at S897,facilitated ZO-1/claudin-5 loss,and promoted BBB leakage in vitro,which were significantly attenuated after stigmasterol treatment.The rat MCAO model confirmed these protective effects in vivo.In summary,these findings suggest that stigmasterol protects HBMECs against ischemia-reperfusion injury by maintaining cell viability,reducing the loss of tight junction proteins,and attenuating the BBB damage.These protective effects are at least meditated by its interaction with EPHA2 and inhibitory effect on EPHA2 phosphorylation.
基金supported by the National Natural Science Foundation of China(81771250)the Natural Science Foundation of Fujian Province,China(2020J011059,2020R1011004)+1 种基金the Joint Funds for the Innovation of Science and Technology of Fujian Province,China(2017Y9065)the High-level hospital foster grants from Fujian Provincial Hospital,Fujian Province,China(2020HSJJ07)。
文摘Background:Disruption of the blood–brain barrier(BBB)after a stroke can lead to brain injury and neurological impairment.Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2(LMP2)in the pathophysiology of ischemia stroke.However,the relationship between the immunoproteasome LMP2 and the BBB remains unclear.Methods:Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion(MCAO/R).Three days before MCAO,the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region.The rat brain microvascular endothelial cell(RBMVEC)line was exposed to oxygen–glucose deprivation/reperfusion(OGD/R)to mimic ischemic conditions in vitro.The RNA interference-mediated knockdown of LMP2 orβ-catenin was analysed in vivo and in vitro.Analysis of the quantity of extravasated Evans blue(EB)and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB.Immunofluorescence and Western blotting were employed to detect the expression of target proteins.Cell migration was evaluated using a scratch migration assay.The results of immunofluorescence,Western blotting and cell migration were quantified using the software ImageJ(Version 1.53).Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference(LSD)test.Results:Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins[occludin,claudin-1 and zonula occludens(ZO-1)]in the MCAO/R group compared with the sham group(P<0.001).However,inhibition of the immunoproteasome LMP2 restored the expression of these proteins,resulting in higher levels of occludin,claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group(P<0.001).In addition,inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability[e.g.,the quantity of extravasated EB:LMP2-shRNA group(58.54±7.37)μg/g vs.control-shRNA group(103.74±4.32)μg/g,P<0.001],and promoted the upregulation of Wnt-3a andβ-catenin proteins in rats following MCAO/R.In vitro experiments,OGD/R induced marked upregulation of LMP2,proapoptotic protein Bax and cleaved caspase-3,and downregulation of occludin,claudin-1,ZO-1 and Bcl-2,as well as inhibition of the Wnt/β-catenin pathway Wnt-3a andβ-catenin proteins in RBMVECs,compared with the control group under normal culture conditions(P<0.001).However,silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R.Silencing ofβ-catenin by transfection of RBMVECs withβ-catenin-si RNA aggravated the downregulation of tight junction proteins,and reduced the proliferation and migration of RBMVECs following OGD/R,compared with the control-siRNA group(P<0.001).LMP2-si RNA andβ-catenin-si RNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.Conclusions:This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia induced BBB injury,and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.
基金supported by grants from the National Natural Science Foundation of China (85100929)the Natural Science Foundation of Hubei Province,China (2018CFB302 and 2019CFB446)the Youth Fund of Health and Family Planning Commission of Wuhan Municipality,Hubei Province,China (WX18Q13 and WX18Q22)。
文摘In the central nervous system,hyperpolarizationactivated cyclic nucleotide-gated(HCN)channels are essential to maintain normal neuronal function.Recent studies have shown that HCN channels may be involved in the pathological process of ischemic brain injury,but the mechanisms remain unclear.Autophagy is activated in cerebral ischemia,but its role in cell death/survival remains controversial.In this study,our results showed that the HCN channel blocker ZD7288 remarkably decreased the percentage of apoptotic neurons and corrected the excessive autophagy induced by oxygen-glucose deprivation followed by reperfusion(OGD/R)in hippocampal HT22 neurons.Furthermore,in the OGD/R group,p-mTOR,p-ULK1(Ser757),and p62 were significantly decreased,while p-ULK1(Ser317),atg5,and beclin1 were remarkably increased.ZD7288 did not change the expression of p-ULK1(Ser757),ULK1(Ser317),p62,Beclin1,and atg5,which are involved in regulating autophagosome formation.Besides,we found that OGD/R induced a significant increase in Cathepsin D expression,but not LAMP-1.Treatment with ZD7288 at 10μmol/L in the OGD/R group did not change the expression of cathepsin D and LAMP-1.However,chloroquine(CQ),which decreases autophagosome-lysosome fusion,eliminated the correction of excessive autophagy and neuroprotection by ZD7288.Besides,shRNA knockdown of HCN2 channels significantly reduced the accumulation of LC3-Ⅱand increased neuron survival in the OGD/R and transient global cerebral ischemia(TGCI)models,and CQ also eliminated the effects of HCN2-shRNA.Furthermore,we found that the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes decreased in Con-shRNAtransfected HT22 neurons exposed to OGD/R or CQ.In HCN2-shRNA-transfected HT22 neurons,the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes increased under OGD/R;however,the percentage was significantly decreased by the addition of CQ to HCN2-shRNA-transfected HT22 neurons.The present results demonstrated that blockade of HCN2 channels provides neuroprotection against OGD/R and TGCI by accelerating autophagic degradation attributable to the promotion of autophagosome and lysosome fusion.
