In this study, a rat model of transient focal cerebral ischemia was established by performing 100 minutes of middle cerebral artery occlusion, and an in vitro model of experimental oxygen-glucose deprivation using cul...In this study, a rat model of transient focal cerebral ischemia was established by performing 100 minutes of middle cerebral artery occlusion, and an in vitro model of experimental oxygen-glucose deprivation using cultured rat cortical neurons was established. Proprotein convertase 2 activity gradually decreased in the ischemic cortex with increasing duration of reperfusion. In cultured rat cortical neurons, the number of terminal deoxynucleotidyl transferase-mediated 2＇-deoxyuridine 5＇-triphosphate-biotin nick end labeling-positive neurons significantly increased and proprotein convertase 2 activity also decreased gradually with increasing duration of oxygen-glucose deprivation. These experimental findings indicate that proprotein convertase 2 activity decreases in ischemic rat cortex after reperfusion, as well as in cultured rat cortical neurons after oxygen-glucose deprivation. These changes in enzyme activity may play an important pathological role in brain injury.展开更多
Certain microRNAs(miRNAs)can function as neuroprotective factors after reperfusion/ischemia brain injury.miRNA-142-3p can participate in the occurrence and development of tumors and myocardial ischemic injury by negat...Certain microRNAs(miRNAs)can function as neuroprotective factors after reperfusion/ischemia brain injury.miRNA-142-3p can participate in the occurrence and development of tumors and myocardial ischemic injury by negatively regulating the activity of Rac1,but it remains unclear whether miRNA-142-3p also participates in cerebral ischemia/reperfusion injury.In this study,a model of oxygen-glucose deprivation/re-oxygenation in primary cortical neurons was established and the neurons were transfected with miR-142-3p agomirs or miR-142-3p antagomirs.miR-142-3p expression was down-regulated in neurons when exposed to oxygen-glucose deprivation/re-oxygenation.Over-expression of miR-142-3p using its agomir remarkably promoted cell death and apoptosis induced by oxygen-glucose deprivation/re-oxygenation and improved mitochondrial biogenesis and function,including the expression of peroxisome proliferator-activated receptor-γcoactivator-1α,mitochondrial transcription factor A,and nuclear respiratory factor 1.However,the opposite effects were produced if miR-142-3p was inhibited.Luciferase reporter assays verified that Rac Family Small GTPase 1(Rac1)was a target gene of miR-142-3p.Over-expressed miR-142-3p inhibited NOX2 activity and expression of Rac1 and Rac1-GTPase(its activated form).miR-142-3p antagomirs had opposite effects after oxygen-glucose deprivation/re-oxygenation.Our results indicate that miR-142-3p down-regulates the expression and activation of Rac1,regulates mitochondrial biogenesis and function,and inhibits oxygen-glucose deprivation damage,thus exerting a neuroprotective effect.The experiments were approved by the Committee of Experimental Animal Use and Care of Central South University,China(approval No.201703346)on March 7,2017.展开更多
Recent studies have shown that induced expression of endogenous antioxidative enzymes thr- ough activation of the antioxidant response element/nuclear factor erythroid 2-related factor 2 （Nrf2） pathway may be a neur...Recent studies have shown that induced expression of endogenous antioxidative enzymes thr- ough activation of the antioxidant response element/nuclear factor erythroid 2-related factor 2 （Nrf2） pathway may be a neuroprotective strategy. In this study, rat cerebral cortical neurons cultured in vitro were pretreated with 10 ktM curcumin or post-treated with 5 pM curcumin, respectively before or after being subjected to oxygen-glucose deprivation and reoxygenation for 24 hours. Both pretreatment and post-treatment resulted in a significant decrease of cell injury as indicated by propidium iodide/Hoechst 33258 staining, a prominent increase of Nrf2 protein expression as indicated by western blot analysis, and a remarkable increase of protein expression and enzyme activity in whole cell lysates of thioredoxin before ischemia, after ischemia, and after reoxygenation. In addition, post-treatment with curcumin inhibited early DNA/RNA oxidation as indicated by immunocytochemistry and increased nuclear Nrf2 protein by inducing nuclear accumulation of Nrf2. These findings suggest that curcumin activates the expression of thi- oredoxin, an antioxidant protein in the Nrf2 pathway, and protects neurons from death caused by oxygen-glucose deprivation in an in vitro model of ischemia/reperfusion. We speculate that pharmacologic stimulation of antioxidant gene expression may be a promising approach to neu- roprotection after cerebral ischemia.展开更多
This study aimed to elucidate the molecular mechanisms by which berberine protects against cerebral ischemia/reperfusion(I/R)injury.The oxygen-glucose deprivation/reperfusion(OGD/R)PC12 model was established.Cell coun...This study aimed to elucidate the molecular mechanisms by which berberine protects against cerebral ischemia/reperfusion(I/R)injury.The oxygen-glucose deprivation/reperfusion(OGD/R)PC12 model was established.Cell counting kit-8(CCK-8)was used to detect the toxicity of berberine and the viability of PC12 cells.Hoechst 33258 staining and flow cytometry were used to observe the nuclear morphology,and changes of apoptosis and reactive oxygen species(ROS),respectively.Western blotting and immunofluorescence assay were employed to detect autophagy-related proteins[microtubule-associated protein 1A/1B-light chain 3(LC3),P62/SQSTM-1,Beclin-1]and endoplasmic reticulum(ER)stress-related markers[glucose-regulated protein 78(GRP78),C/EBP homologous protein(CHOP),Bcl-2-associated X(Bax)and cleaved caspase-3].The GFP-RFP-LC3 adenovirus was used to assay the change of autophagic flux.Our results showed that berberine could increase the viability of PC12 cells,decrease the concentrations of ROS after OGD/R treatment,and suppress OGD/R-induced ER stress and autophagy.Moreover,the results revealed the involvement of the mammalian target of rapamycin(mTOR)pathway in the induction of autophagy,and berberine could activate the phosphorylation of mTOR and thus mitigate autophagy.In conclusion,our study suggested that berberine may protect against OGD/R-induced apoptosis by regulating ER stress and autophagy,and it holds promises in the treatment of cerebral I/R injury.展开更多
CDGSH iron sulfur domain 2 can inhibit ferroptosis,which has been associated with cerebral ischemia/reperfusion,in individuals with head and neck cancer.Therefore,CDGSH iron sulfur domain 2 may be implicated in cerebr...CDGSH iron sulfur domain 2 can inhibit ferroptosis,which has been associated with cerebral ischemia/reperfusion,in individuals with head and neck cancer.Therefore,CDGSH iron sulfur domain 2 may be implicated in cerebral ischemia/reperfusion injury.To validate this hypothesis in the present study,we established mouse models of occlusion of the middle cerebral artery and HT22 cell models of oxygen-glucose deprivation and reoxygenation to mimic cerebral ischemia/reperfusion injury in vivo and in vitro,respectively.We found remarkably decreased CDGSH iron sulfur domain 2 expression in the mouse brain tissue and HT22 cells.When we used adeno-associated virus and plasmid to up-regulate CDGSH iron sulfur domain 2 expression in the brain tissue and HT22 cell models separately,mouse neurological dysfunction was greatly improved;the cerebral infarct volume was reduced;the survival rate of HT22 cells was increased;HT22 cell injury was alleviated;the expression of ferroptosis-related glutathione peroxidase 4,cystine-glutamate antiporter,and glutathione was increased;the levels of malondialdehyde,iron ions,and the expression of transferrin receptor 1 were decreased;and the expression of nuclear-factor E2-related factor 2/heme oxygenase 1 was increased.Inhibition of CDGSH iron sulfur domain 2 upregulation via the nuclear-factor E2-related factor 2 inhibitor ML385 in oxygen-glucose deprived and reoxygenated HT22 cells blocked the neuroprotective effects of CDGSH iron sulfur domain 2 up-regulation and the activation of the nuclear-factor E2-related factor 2/heme oxygenase 1 pathway.Our data indicate that the up-regulation of CDGSH iron sulfur domain 2 can attenuate cerebral ischemia/reperfusion injury,thus providing theoretical support from the perspectives of cytology and experimental zoology for the use of this protein as a therapeutic target in patients with cerebral ischemia/reperfusion injury.展开更多
Background: Cerebral ischemia-reperfusion injury(CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury re...Background: Cerebral ischemia-reperfusion injury(CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive.Methods: The 150 male C57 mice underwent middle cerebral artery occlusion(MCAO) for 1 h and reperfusion for 24 h,Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1(Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine(NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA(mtDNA) copy number, intracellular and mitochondrial reactive oxygen species(ROS),autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3 II/I, TNF-α,IL-1β, etc., were detected under normal or Drp1 interference conditions.Results: The mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI(P<0.05). However, the high cytoplasmic LC3 II/I ratio and increased aggregation of p62 could be reversed by 44%and 88% by Drp1 short hairpin RNA(shRNA)(P<0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC(P<0.05). RIP1/RIP3inhibitor Necrostatin-1(Nec-1) restored 75% to a low LC3 II/I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation(P<0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes(P>0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4–5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1(P<0.05). Furthermore, TNF-α and IL-1βincreased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen–glucose deprivation/reoxygenation(OGD/R) conditions(P<0.05).Conclusions: CIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation,triggering a vicious cycle.展开更多
Objective:To observe the protective effect and mechanism of hydroxyl safflower yellow A(HsYA)from myocardial ischemia-reperfusion injury on human umbilical vein endothelial cells(HUVECs).Methods:HUVECs were treated wi...Objective:To observe the protective effect and mechanism of hydroxyl safflower yellow A(HsYA)from myocardial ischemia-reperfusion injury on human umbilical vein endothelial cells(HUVECs).Methods:HUVECs were treated with oxygen-glucose deprivation reperfusion(OGD/R)to simulate the ischemia reperfusion model,and cell counting kit-8 was used to detect the protective effect of different concentrations(1.25-160μmol/L)of HSYA on HUVECs after OGD/R.HSYA 80μmol/L was used for follow-up experiments.The contents of inflammatory cytokines interleukin(IL)-18,IL-1β,monocyte chemotactic protein 1(MCP-1),tumor necrosis factorα(TNF-α)and IL-6 before and after administration were measured by enzyme-linked immunosorbent assay.The protein expressions of toll-like receptor,NOD-like receptor containing pyrin domain 3(NLRP3),gasdermin D(GSDMD)and GSDMD-N-terminal domain(GSDMD-N)before and after administration were detected by Western blot.NLRP3 inflammasome inhibitor cytokine release inhibitory drug 3 sodium salt(CRID3 sodium salt,also known as MCC950)and agonist were added,and the changes of NLRP3,cysteine-aspartic acid protease 1(Caspase-1),GSDMD and GSDMD-N protein expressions were detected by Western blot.Results:HSYA inhibited OGD/R-induced inflammation and significantly decreased the contents of inflammatory cytokines IL-18,IL-1β,MCP-1,TNF-αandIL-6(P<0.01or P<0.05).At the same time,by inhibiting NLRP3/Caspase-1/GSDMD pathway,HSYA can reduce the occurrence of pyroptosis after OGD/R and reduce the expression of NLRP3,Caspase-1,GSDMD and GSDMD-N proteins(P<0.01).Conclusions:The protective effect of HSYA on HUVECs after OGD/R is related to down-regulating the expression of NLRP3 inflammasome and inhibiting pyroptosis.展开更多
基金supported by the National Natural Science Foundation of China,No.81070999the foundation of Xi’an Jiaotong University,No.95,2009+2 种基金Foundation of the Second Affiliated Hospital of Xi’an Jiaotong University,No.RC(GG)201109the US National Institutes of Health,No.NS046560the American Heart Association,No.0450142Z
文摘In this study, a rat model of transient focal cerebral ischemia was established by performing 100 minutes of middle cerebral artery occlusion, and an in vitro model of experimental oxygen-glucose deprivation using cultured rat cortical neurons was established. Proprotein convertase 2 activity gradually decreased in the ischemic cortex with increasing duration of reperfusion. In cultured rat cortical neurons, the number of terminal deoxynucleotidyl transferase-mediated 2＇-deoxyuridine 5＇-triphosphate-biotin nick end labeling-positive neurons significantly increased and proprotein convertase 2 activity also decreased gradually with increasing duration of oxygen-glucose deprivation. These experimental findings indicate that proprotein convertase 2 activity decreases in ischemic rat cortex after reperfusion, as well as in cultured rat cortical neurons after oxygen-glucose deprivation. These changes in enzyme activity may play an important pathological role in brain injury.
基金supported by the National Natural Science Foundation of China,No.81771422(to ZY)
文摘Certain microRNAs(miRNAs)can function as neuroprotective factors after reperfusion/ischemia brain injury.miRNA-142-3p can participate in the occurrence and development of tumors and myocardial ischemic injury by negatively regulating the activity of Rac1,but it remains unclear whether miRNA-142-3p also participates in cerebral ischemia/reperfusion injury.In this study,a model of oxygen-glucose deprivation/re-oxygenation in primary cortical neurons was established and the neurons were transfected with miR-142-3p agomirs or miR-142-3p antagomirs.miR-142-3p expression was down-regulated in neurons when exposed to oxygen-glucose deprivation/re-oxygenation.Over-expression of miR-142-3p using its agomir remarkably promoted cell death and apoptosis induced by oxygen-glucose deprivation/re-oxygenation and improved mitochondrial biogenesis and function,including the expression of peroxisome proliferator-activated receptor-γcoactivator-1α,mitochondrial transcription factor A,and nuclear respiratory factor 1.However,the opposite effects were produced if miR-142-3p was inhibited.Luciferase reporter assays verified that Rac Family Small GTPase 1(Rac1)was a target gene of miR-142-3p.Over-expressed miR-142-3p inhibited NOX2 activity and expression of Rac1 and Rac1-GTPase(its activated form).miR-142-3p antagomirs had opposite effects after oxygen-glucose deprivation/re-oxygenation.Our results indicate that miR-142-3p down-regulates the expression and activation of Rac1,regulates mitochondrial biogenesis and function,and inhibits oxygen-glucose deprivation damage,thus exerting a neuroprotective effect.The experiments were approved by the Committee of Experimental Animal Use and Care of Central South University,China(approval No.201703346)on March 7,2017.
基金supported by grants from the National Natural Science Foundation of China,No.81171090Natural Science Foundation of Chongqing Education Committee of China,No.KJ110313+1 种基金Foundation of Key State Laboratory of Neurobiology of Fudan University in China,No.10-08Foundation of Key Laboratory of Ministry of Education of the Third Medical Military University in China
文摘Recent studies have shown that induced expression of endogenous antioxidative enzymes thr- ough activation of the antioxidant response element/nuclear factor erythroid 2-related factor 2 （Nrf2） pathway may be a neuroprotective strategy. In this study, rat cerebral cortical neurons cultured in vitro were pretreated with 10 ktM curcumin or post-treated with 5 pM curcumin, respectively before or after being subjected to oxygen-glucose deprivation and reoxygenation for 24 hours. Both pretreatment and post-treatment resulted in a significant decrease of cell injury as indicated by propidium iodide/Hoechst 33258 staining, a prominent increase of Nrf2 protein expression as indicated by western blot analysis, and a remarkable increase of protein expression and enzyme activity in whole cell lysates of thioredoxin before ischemia, after ischemia, and after reoxygenation. In addition, post-treatment with curcumin inhibited early DNA/RNA oxidation as indicated by immunocytochemistry and increased nuclear Nrf2 protein by inducing nuclear accumulation of Nrf2. These findings suggest that curcumin activates the expression of thi- oredoxin, an antioxidant protein in the Nrf2 pathway, and protects neurons from death caused by oxygen-glucose deprivation in an in vitro model of ischemia/reperfusion. We speculate that pharmacologic stimulation of antioxidant gene expression may be a promising approach to neu- roprotection after cerebral ischemia.
基金This work was supported by grants from the National Natural Science Foundation of China(No.81360199)Science and Technology Department of Guizhou Province(Guizhou Specific Grant[2019]4008)+2 种基金Science and Technology Plan Project of Guizhou Province(Basic Science and Technology Cooperation[2020]1Z060)Science and Technology Fund Project of Guizhou Health and Health Commission(No.gzwjkj2019-1-039)Science and Technology Fund Project of Southwest Guizhou Autonomous Prefecture(2019-1-10).
文摘This study aimed to elucidate the molecular mechanisms by which berberine protects against cerebral ischemia/reperfusion(I/R)injury.The oxygen-glucose deprivation/reperfusion(OGD/R)PC12 model was established.Cell counting kit-8(CCK-8)was used to detect the toxicity of berberine and the viability of PC12 cells.Hoechst 33258 staining and flow cytometry were used to observe the nuclear morphology,and changes of apoptosis and reactive oxygen species(ROS),respectively.Western blotting and immunofluorescence assay were employed to detect autophagy-related proteins[microtubule-associated protein 1A/1B-light chain 3(LC3),P62/SQSTM-1,Beclin-1]and endoplasmic reticulum(ER)stress-related markers[glucose-regulated protein 78(GRP78),C/EBP homologous protein(CHOP),Bcl-2-associated X(Bax)and cleaved caspase-3].The GFP-RFP-LC3 adenovirus was used to assay the change of autophagic flux.Our results showed that berberine could increase the viability of PC12 cells,decrease the concentrations of ROS after OGD/R treatment,and suppress OGD/R-induced ER stress and autophagy.Moreover,the results revealed the involvement of the mammalian target of rapamycin(mTOR)pathway in the induction of autophagy,and berberine could activate the phosphorylation of mTOR and thus mitigate autophagy.In conclusion,our study suggested that berberine may protect against OGD/R-induced apoptosis by regulating ER stress and autophagy,and it holds promises in the treatment of cerebral I/R injury.
基金supported by the National Natural Science Foundation of China,No.81402930Natural Science Foundation of Universities in Anhui Province,No.KJ2021A0688+2 种基金National College Students Innovation and Entrepreneurship Program,No.202110367071Key projects of science and technology projects of Bengbu Medical College,No.2020byzd017512 Talents Training Program of Bengbu Medical College,No.BY51201104(all to SYD).
文摘CDGSH iron sulfur domain 2 can inhibit ferroptosis,which has been associated with cerebral ischemia/reperfusion,in individuals with head and neck cancer.Therefore,CDGSH iron sulfur domain 2 may be implicated in cerebral ischemia/reperfusion injury.To validate this hypothesis in the present study,we established mouse models of occlusion of the middle cerebral artery and HT22 cell models of oxygen-glucose deprivation and reoxygenation to mimic cerebral ischemia/reperfusion injury in vivo and in vitro,respectively.We found remarkably decreased CDGSH iron sulfur domain 2 expression in the mouse brain tissue and HT22 cells.When we used adeno-associated virus and plasmid to up-regulate CDGSH iron sulfur domain 2 expression in the brain tissue and HT22 cell models separately,mouse neurological dysfunction was greatly improved;the cerebral infarct volume was reduced;the survival rate of HT22 cells was increased;HT22 cell injury was alleviated;the expression of ferroptosis-related glutathione peroxidase 4,cystine-glutamate antiporter,and glutathione was increased;the levels of malondialdehyde,iron ions,and the expression of transferrin receptor 1 were decreased;and the expression of nuclear-factor E2-related factor 2/heme oxygenase 1 was increased.Inhibition of CDGSH iron sulfur domain 2 upregulation via the nuclear-factor E2-related factor 2 inhibitor ML385 in oxygen-glucose deprived and reoxygenated HT22 cells blocked the neuroprotective effects of CDGSH iron sulfur domain 2 up-regulation and the activation of the nuclear-factor E2-related factor 2/heme oxygenase 1 pathway.Our data indicate that the up-regulation of CDGSH iron sulfur domain 2 can attenuate cerebral ischemia/reperfusion injury,thus providing theoretical support from the perspectives of cytology and experimental zoology for the use of this protein as a therapeutic target in patients with cerebral ischemia/reperfusion injury.
基金supported by the National Natural Science Foundation of China (81700429)the China Postdoctoral Science Foundation (2021MD703924)+1 种基金the Chongqing Postdoctoral Innovative Talents Support Program (CQBX2021018)the Kuanren Talents Program of the second affiliated hospital of Chongqing Medical University。
文摘Background: Cerebral ischemia-reperfusion injury(CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive.Methods: The 150 male C57 mice underwent middle cerebral artery occlusion(MCAO) for 1 h and reperfusion for 24 h,Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1(Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine(NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA(mtDNA) copy number, intracellular and mitochondrial reactive oxygen species(ROS),autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3 II/I, TNF-α,IL-1β, etc., were detected under normal or Drp1 interference conditions.Results: The mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI(P<0.05). However, the high cytoplasmic LC3 II/I ratio and increased aggregation of p62 could be reversed by 44%and 88% by Drp1 short hairpin RNA(shRNA)(P<0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC(P<0.05). RIP1/RIP3inhibitor Necrostatin-1(Nec-1) restored 75% to a low LC3 II/I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation(P<0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes(P>0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4–5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1(P<0.05). Furthermore, TNF-α and IL-1βincreased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen–glucose deprivation/reoxygenation(OGD/R) conditions(P<0.05).Conclusions: CIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation,triggering a vicious cycle.
基金Supported by the Youth Planning Project of Beijing Scienceand Technology Development Fund for Chinese Medicine(No.QN-2020-14)innovation Fund of China Academy of ChineseMedical Sciences(No.CI2021A00912)Scientific Fund ofNational Clinical Research Center for Chinese Medicine Cardiology(No.CMC2022005)。
文摘Objective:To observe the protective effect and mechanism of hydroxyl safflower yellow A(HsYA)from myocardial ischemia-reperfusion injury on human umbilical vein endothelial cells(HUVECs).Methods:HUVECs were treated with oxygen-glucose deprivation reperfusion(OGD/R)to simulate the ischemia reperfusion model,and cell counting kit-8 was used to detect the protective effect of different concentrations(1.25-160μmol/L)of HSYA on HUVECs after OGD/R.HSYA 80μmol/L was used for follow-up experiments.The contents of inflammatory cytokines interleukin(IL)-18,IL-1β,monocyte chemotactic protein 1(MCP-1),tumor necrosis factorα(TNF-α)and IL-6 before and after administration were measured by enzyme-linked immunosorbent assay.The protein expressions of toll-like receptor,NOD-like receptor containing pyrin domain 3(NLRP3),gasdermin D(GSDMD)and GSDMD-N-terminal domain(GSDMD-N)before and after administration were detected by Western blot.NLRP3 inflammasome inhibitor cytokine release inhibitory drug 3 sodium salt(CRID3 sodium salt,also known as MCC950)and agonist were added,and the changes of NLRP3,cysteine-aspartic acid protease 1(Caspase-1),GSDMD and GSDMD-N protein expressions were detected by Western blot.Results:HSYA inhibited OGD/R-induced inflammation and significantly decreased the contents of inflammatory cytokines IL-18,IL-1β,MCP-1,TNF-αandIL-6(P<0.01or P<0.05).At the same time,by inhibiting NLRP3/Caspase-1/GSDMD pathway,HSYA can reduce the occurrence of pyroptosis after OGD/R and reduce the expression of NLRP3,Caspase-1,GSDMD and GSDMD-N proteins(P<0.01).Conclusions:The protective effect of HSYA on HUVECs after OGD/R is related to down-regulating the expression of NLRP3 inflammasome and inhibiting pyroptosis.
