Effects of Qishen Yiqi dripping pills-containing serum on KATP channel opening and PI3K/AKT signaling pathway in hypoxic/reoxygenated H9C2 cardiocytes

Objective:To investigate the effects of Qishen Yiqi dropping pills serum on KATP channel opening and PI3K/AKT signaling pathway of hypoxic/reoxygenated H9C2 cardiocytes.Methods:H9C2 cardiocytes cultured in vitro were randomly divided into five groups,A:H9C2 cell group B:H9C2 cells+H2O2 model group C:H9C2 cells+H2O2 model+Qishen Yiqi group D:H9C2 cells+H2O2 model+Qishen Yiqi+wort group E:H9C2 cells+H2O2 model+Qishenyiqi+5-HD group,the drug intervention is according to the corresponding conditions.CCK-8 method was used to detect the cell activity of each group;Western blot was used to detect the expression of AKT and P-Akt proteins in myocardial cells in each group.The current was recorded by the standard patch clamp whole cell recording method,and the current was collected and analyzed by Pclamp6.0 software.Results:CCK-8 test results showed that compared with group A,the activity of myocardial cells in group B was significantly decreased,and the difference was statistically significant(P<0.01);compared with group B,the difference in group C was statistically significant(P<0.01);compared with C,cardiomyocyte activity in D and E group were significantly decreased,and the difference was statistically significant(P<0.05);WB results showed that compared with A,p-Akt protein expression in B,C,D and E groups were significantly decreased,and the difference was statistically significant(P<0.01);compared with group B,p-Akt protein expression in C,D and E group were significantly increased,and the difference was statistically significant(P<0.01),but there was no significant difference in AKT expression among groups(P>0.05);The results of whole cell patch clamp experiment showed that the outward current of B was significantly increased compared with that of A,and the difference between groups was statistically significant(P<0.01);compared with group B,cardiomyocytes in group C further increased the outward current,and the difference between groups was statistically significant(P<0.01);compared with C,the current of D and E group were significantly decreased,with statistical significance between groups(P<0.01).Conclusion:QishenYiqi dropping pills can protect cardiomyocytes by activating p-Akt protein expression and KATP channel opening in H9C2 cardiomyocytes.

Activation of the wnt/β-catenin/CYP1B1 pathway alleviates oxidative stress and protects the blood-brain barrier under cerebral ischemia/reperfusion conditions

Accumulating evidence suggests that oxidative stress and the Wnt/β-catenin pathway participate in stroke-induced disruption of the blood-brain barrier.However,the potential links between them following ischemic stroke remain largely unknown.The present study found that cerebral ischemia leads to oxidative stress and repression of the Wnt/β-catenin pathway.Meanwhile,Wnt/β-catenin pathway activation by the pharmacological inhibito r,TWS119,relieved oxidative stress,increased the levels of cytochrome P4501B1(CYP1B1)and tight junction-associated proteins(zonula occludens-1[ZO-1],occludin and claudin-5),as well as brain microvascular density in cerebral ischemia rats.Moreove r,rat brain microvascular endothelial cells that underwent oxygen glucose deprivation/reoxygenation displayed intense oxidative stress,suppression of the Wnt/β-catenin pathway,aggravated cell apoptosis,downregulated CYP1B1and tight junction protein levels,and inhibited cell prolife ration and migration.Overexpression ofβ-catenin or knockdown ofβ-catenin and CYP1B1 genes in rat brain mic rovascular endothelial cells at least partly ameliorated or exacerbated these effects,respectively.In addition,small interfering RNA-mediatedβ-catenin silencing decreased CYP1B1 expression,whereas CYP1B1 knoc kdown did not change the levels of glycogen synthase kinase 3β,Wnt-3a,andβ-catenin proteins in rat brain microvascular endothelial cells after oxygen glucose deprivatio n/reoxygenation.Thus,the data suggest that CYP1B1 can be regulated by Wnt/β-catenin signaling,and activation of the Wnt/β-catenin/CYP1B1 pathway contributes to alleviation of oxidative stress,increased tight junction levels,and protection of the blood-brain barrier against ischemia/hypoxia-induced injury.

Cav3.2 channel regulates cerebral ischemia/reperfusion injury:a promising target for intervention

Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.

Effect of tubastatin A on NLRP3 inflammasome activation in macrophages under hypoxia/ reoxygenation conditions

BACKGROUND:There are currently no effective drugs to mitigate the ischemia/reperfusion injury caused by fluid resuscitation after hemorrhagic shock(HS).The aim of this study was to explore the potential of the histone deacetylase 6(HDAC6)-specific inhibitor tubastatin A(TubA)to suppress nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome activation in macrophages under hypoxia/reoxygenation(H/R)conditions.METHODS:The viability of RAW264.7 cells subjected to H/R after treatment with different concentrations of TubA was assessed using a cell-counting kit-8(CCK8)assay.Briefly,2.5μmol/L TubA was used with RAW264.7 cells under H/R condition.RAW264.7 cells were divided into three groups,namely the control,H/R,and TubA groups.The levels of reactive oxygen species(ROS)in the cells were detected using fluorescence microscopy.The protein expression of HDAC6,heat shock protein 90(Hsp90),inducible nitric oxide synthase(iNOS),NLRP3,gasdermin-D(GSDMD),Caspase-1,GSDMD-N,and Caspase-1 p20 was detected by western blotting.The levels of interleukin-1β(IL-1β)and IL-18 in the supernatants were detected using enzyme-linked immunosorbent assay(ELISA).RESULTS:HDAC6,Hsp90,and iNOS expression levels were significantly higher(P<0.01)in the H/R group than in the control group,but lower in the TubA group than in the H/R group(P<0.05).When comparing the H/R group to the control group,ROS levels were significantly higher(P<0.01),but significantly reduced in the TubA group(P<0.05).The H/R group had higher NLRP3,GSDMD,Caspase-1,GSDMD-N,and Caspase-1 p20 expression levels than the control group(P<0.05),however,the TubA group had significantly lower expression levels than the H/R group(P<0.05).IL-1βand IL-18 levels in the supernatants were significantly higher in the H/R group compared to the control group(P<0.01),but significantly lower in the TubA group compared to the H/R group(P<0.01).CONCLUSION:TubA inhibited the expression of HDAC6,Hsp90,and iNOS in macrophages subjected to H/R.This inhibition led to a decrease in the content of ROS in cells,which subsequently inhibited the activation of the NLRP3 inflammasome and the secretion of IL-1βand IL-18.

IcarisideⅡ alleviates oxygen-glucose deprivation and reoxygenation-induced PC12 celloxidative injury by activating Nrf2 / SIRT3signaling pathway

OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxygen-glucose deprivation/reoxygenation(OGD/R) 2 h/24 h in PC12 cells.N-acetyl-lcysteine(NAC),a classical anti-oxidant,was used as positive control.Pharmacodynamic experimental study groups as follows:control,control+ICS Ⅱ50 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ 12.5 μmol·L^(-1),OGD/R + ICS Ⅱ 25 μmol·L^(-1),OGD/R + ICS Ⅱ50 μmol·L^(-1),and OGD/R+NAC 100 μmol·L^(-1) groups.Cell viability and lactate dehydrogenase(LDH) leakage rate were measured by MTT assay and LDH ELISA kit,respectively.Moreover,reactive oxygen species(ROS) ELISA kit was used for detection of intracellular ROS generation,Mito-SOX fluorescence staining was used for detecting production of ROS in mitochondria and mitochondrial membrane potential(MMP)was detected by rhodamine 123 dye.In addition,PC12 cells apoptosis was detected by one-step TUNEL assay.Furthermore,the expressions of nuclear factor erythroid 2-related factors(Nrf2),Keap1,HO^(-1),NQO^(-1),silent information regulator 3(SIRT3),IDH2,Bax,Bcl-2 and caspase 3 were detected by Western blotting analysis.RESULTS The results of MTT and LDH assay showed that OGD/R reduced the cell viability and improved LDH release compared with the control or ICSⅡ 50 μmol·L^(-1) alone(P<0.01).Meanwhile,OGD/R not only increased intracellular and mitochondrial ROS generation,but also elevated the fluorescence intensity of TUNEL staining,at the same time,the MMP was declined when challenged by OGD/R.Furthermore,the Western blotting results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and cleaved-caspase 3 level,while the decrease in the expression of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).However,ICS Ⅱ significantly increased the viability of PC12 cells and reduced LDH leakage(P<0.01).Notably,ICS Ⅱ also suppressed ROS generation both in the intracellular and mitochondria,as well as restored MMP.It was also worthy to note that ICS Ⅱ decreased the expressions of cytoplasmNrf2,Keap1,Bax and the level of cleaved-caspase3,whereas,it increased the expressions of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).CONCLUSION ICSⅡ reduced OGD/Rinduced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.

Hyodeoxycholic acid protects the neurovascular unit against oxygen-glucose deprivation and reoxygenation-induced injury in vitro

Calculus bovis is commonly used for the treatment of stroke in traditional Chinese medicine. Hyodeoxycholic acid(HDCA) is a bioactive compound extracted from calculus bovis. When combined with cholic acid, baicalin and jas-minoidin, HDCA prevents hypoxia-reoxygenation-induced brain injury by suppressing endoplasmic reticulum stress-mediated apoptotic signaling. However, the effects of HDCA in ischemic stroke injury have not yet been studied. Neurovascular unit(NVU) dysfunction occurs in ischemic stroke. Therefore, in this study, we investigated the effects of HDCA on the NVU under ischemic conditions in vitro. We co-cultured primary brain microvascular endothelial cells, neurons and astrocytes using a transwell chamber co-culture system. The NVU was pre-treated with 10.16 or 2.54 μg/mL HDCA for 24 hours before exposure to oxygen-glucose deprivation for 1 hour. The cell counting kit-8 assay was used to detect cell activity. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were used to assess apoptosis. Enzyme-linked immunosorbent assay was used to measure the expression levels of inflammatory cytokines, including interleukin-1β, interleukin-6 and tumor necrosis factor-α, and neurotrophic factors, including brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. Oxidative stress-related factors, such as superoxide dismutase, nitric oxide, malondialdehyde and γ-glutamyltransferase, were measured using kits. Pretreatment with HDCA significantly decreased blood-brain barrier permeability and neuronal apoptosis, significantly increased transendothelial electrical resistance and γ-glutamyltransferase activity, attenuated oxidative stress damage and the release of inflammatory cytokines, and increased brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression. Our findings suggest that HDCA maintains NVU morphological integrity and function by modulating inflammation, oxidation stress, apoptosis, and the expression of neurotrophic factors. Therefore, HDCA may have therapeutic potential in the clinical management of ischemic stroke. This study was approved by the Ethics Committee of Experimental Animals of Beijing University of Chinese Medicine(approval No. BUCM-3-2016040201-2003) in April 2016.

Propofol postconditioning ameliorates hypoxia/reoxygenation induced H9c2 cell apoptosis and autophagy via upregulating forkhead transcription factors under hyperglycemia

Background:Administration of propofol,an intravenous anesthetic with antioxidant property,immediately at the onset of post-ischemic reperfusion(propofol postconditioning,P-PostC) has been shown to confer cardioprotection against ischemia–reperfusion(I/R) injury,while the underlying mechanism remains incompletely understood.The forkhead box O(FoxO) transcription factors are reported to play critical roles in activating cardiomyocyte survival signaling throughout the process of cellular injuries induced by oxidative stress and are also involved in hypoxic postconditioning mediated neuroprotection,however,the role of FoxO in postconditioning mediated protection in the heart and in particular in high glucose condition is unknown.Methods:Rat heart-derived H9c2 cells were exposed to high glucose(HG) for 48 h,then subjected to hypoxia/reoxygenation(H/R,composed of 8 h of hypoxia followed by 12 h of reoxygenation) in the absence or presence of postconditioning with various concentrations of propofol(P-PostC) at the onset of reoxygenation.After having identified the optical concentration of propofol,H9c2 cells were subjected to H/R and P-PostC in the absence or presence of FoxO1 or FoxO3a gene silencing to explore their roles in P-PostC mediated protection against apoptotic and autophagic cell deaths under hyperglycemia.Results:The results showed that HG with or without H/R decreased cell viability,increased lactate dehydrogenase(LDH) leakage and the production of reactive oxygen species(ROS) in H9c2 cells,all of which were significantly reversed by propofol(P-PostC),especially at the concentration of 25 μmol/L(P25)(P<0.05,NC vs.HG;HG vs.HG+HR;HG+HR+P12.5 or HG+HR+P25 or HG+HR+P50 vs.HG+HR).Moreover,we found that propofol(P25) decreased H9c2 cells apoptosis and autophagy that were concomitant with increased FoxO1 and FoxO3a expression(P<0.05,HG+HR+P25 vs.HG+HR).The protective effects of propofol(P25) against H/R injury were reversed by silencing FoxO1 or FoxO3a(P<0.05,HG+HR+P25 vs.HG+HR+P25+siRNA-1 or HG+HR+P25+siRNA-5).Conclusions:It is concluded that propofol postconditioning attenuated H9c2 cardiac cells apoptosis and autophagy induced by H/R injury through upregulating FoxO1 and FoxO3a under hyperglycemia.

Activated Drp1 regulates p62-mediated autophagic flux and aggravates inflammation in cerebral ischemia-reperfusion via the ROS-RIP1/RIP3-exosome axis

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.

Scutellarin protects oxygen/glucose-deprived astrocytes and reduces focal cerebral ischemic injury

Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxida- tive effects and has been used to treat cardiovascular and cerebrovascular diseases in China. However, the mechanisms by which scutellarin mediates neuroprotection in cerebral ischemia remain unclear. The interaction between scutellarin and nicotinamide adenine dinucleotide phosphate oxidase 2 （NOX2） was assessed by molecular docking study, which showed that scutellarin selectively binds to NOX2 with high affinity. Cultures of primary astrocytes isolated from the cerebral cortex of neonatal Sprague-Dawley rats were pretreated with 2, 10 or 50 μM scutellarin for 30 minutes. The astrocytes were then subjected to oxygen/glucose deprivation by incubation for 2 hours in glucose-free Dulbecco＇s modified Eagle＇s medium in a 95% N2/5% CO2 incubator, followed by simulated reperfusion for 22 hours. Cell viability was assessed by cell counting kit-8 assay. Expression levels of NOX2, connexin 43 and caspase-3 were assessed by western blot assay. Reactive oxygen species were measured spectrophotometrically. Pretreatment with 10 or 50 μM scutellarin substantially increased viability, reduced the expression of NOX2 and caspase-3, increased the expression of connexin 43, and diminished the levels of reactive oxygen, species in astrocytes subjected to ischemia-＇reperfusion. We also assessed the effects of scutellarin in vivo in the rat transient middle cerebral artery occlusion model of cerebral ischemia-reperfusion injury. Rats were given intraperitoneal injection of 100 mg/kg scutellarin 2 hours before surgery. The Bederson scale was used to assess neurological deficit, and 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct size. Western blot assay was used to assess expression of NOX2 and connexin 43 in brain tissue. Enzyme-linked immunosorbent assay was used to detect 8-hydroxydeoxyguanosine （8-OHdG）, 4-hydroxy-2-nonenal （4-HNE） and 3-nitrotyrosin （3-NT） in brain tissue. Immunofluorescence double staining was used to determine the co-expression of caspase-3 and NeuN. Pretreatment with scutellarin im- proved the neurological function of rats with focal cerebral ischemia, reduced infarct size, diminished the expression of NOX2, reduced levels of 8-OHdG, 4-HNE and 3-NT, and reduced the number of cells co-expressing caspase-3 and NeuN in the injured brain tissue. Furthermore, we examined the effect of the NOX2 inhibitor apocynin. Apocynin substantially increased connexin 43 expression in vivo and in vitro. Collectively, our findings suggest that scutellarin protects against ischemic injury in vitro and in vivo by downregulating NOX2, upregulating connexin 43, decreasing oxidative damage, and reducing apoptotic cell death.

LncRNA SNHG12 ameliorates brain microvascular endothelial cell injury by targeting miR-199a

Long non-coding RNAs regulate brain microvascular endothelial cell death, the inflammatory response and angiogenesis during and after ischemia/reperfusion and oxygen-glucose deprivation/reoxygenation（OGD/R） insults. The long non-coding RNA, SNHG12, is upregulated after ischemia/reperfusion and OGD/R in microvascular endothelial cells of the mouse brain. However, its role in ischemic stroke has not been studied. We hypothesized that SNHG12 positively regulates ischemic stroke, and therefore we investigated its mechanism of action. We established an OGD/R mouse cell model to mimic ischemic stroke by exposing brain microvascular endothelial cells to OGD for 0, 2, 4, 8, 16 or 24 hours and reoxygenation for 4 hours. Quantitative real-time polymerase chain reaction showed that SNHG12 levels in brain microvascular endothelial cells increased with respect to OGD exposure time. Brain microvascular endothelial cells were transfected with pc DNA-control, pc DNA-SNHG12, si-control, or si-SNHG12. After exposure to OGD for 16 hours, these cells were then analyzed by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide, trypan blue exclusion, western blot, and capillary-like tube formation assays. Overexpression of SNHG12 inhibited brain microvascular endothelial cell death and the inflammatory response but promoted angiogenesis after OGD/R, while SNHG12 knockdown had the opposite effects. miR-199a was identified as a target of SNHG12, and SNHG12 overexpression reversed the effect of miR-199a on brain microvascular endothelial cell death, the inflammatory response, and angiogenesis. These findings suggest that SNHG12 suppresses endothelial cell injury induced by OGD/R by targeting miR-199a.

Sevoflurane pretreatment inhibits the myocardial apoptosis caused by hypoxia reoxygenation through AMPK pathway:An experimental study

Objective:To study whether sevoflurane pretreatment inhibits the myocardial apoptosis caused by hypoxia reoxygenation through AMPK pathway.Methods:H9c2 myocardial cell lines were cultured and divided into control group(C group),hypoxia reoxygenation group(H/R group),sevoflurane pretreatment+hypoxia reoxygenation group(SP group) and sevoflurane combined with Compound C pretreatment+hypoxia reoxygenation group(ComC group),and the cell proliferation activity and apoptosis rate,myocardial enzyme levels in culture medium as well as the expression of apoptosis genes and p-AMPK in cells were determined.Results:p-AMPK expression in cells of H/R group was significantly lower than that of C group,SP group was significantly higher than that of H/R group;cell proliferation activity value and Bcl-2 expression in cells of H/R group were significantly lower than those of C group,SP group were significantly higher than those of H/R group,Com C group were significantly lower than those of SP group;apoptosis rate,LDH,CK and AST levels as well as the Bax and Caspase-3 expression in cells of H/R group were significantly higher than those of C group,SP group were significantly lower than those of H/R group,ComC group were significantly higher than those of SP group.Conclusions:Sevoflurane pretreatment can activate AMPK signaling pathway to inhibit the myocardial apoptosis caused by hypoxia reoxygenation.

Hypoxia inducible factor-1α mediates protective effects of ischemic preconditioning on ECV-304 endothelial cells

AIM: To investigate whether hypoxia inducible factor-1α (HIF-1α) is linked to the protective effects of ischemic preconditioning (IP) on sinusoidal endothelial cells against ischemia/reperfusion injury. METHODS: Sinusoidal endothelial cell lines ECV-304 were cultured and divided into four groups: control group, cells were cultured in complete DMEM medium; cold anoxia/warm reoxygenation (A/R) group, cells were preserved in a 4℃ UW solution in a mixture of 95% N2 and 5% CO2 for 24 h; anoxia-preconditioning (APC) group, cells were treated with 4 cycles of short anoxia and reoxygenation before prolonged anoxia- preconditioning treatment; and anoxia-preconditioning and hypoxia inducible factor-1α (HIF-1α) inhibitor (I-HIF-1) group, cells were pretreated with 5 μm of HIF-1α inhibitor NS398 in DMEM medium before subjected to the same treatment as group APC. After the anoxia treatment, each group was reoxygenated in a mixture of 95% air and 5% CO2 incubator for 6 h. Cytoprotections were evaluated by cell viabilities from Trypan blue, lactate dehydrogenase (LDH) release rates, and intracellular cell adhesion molecule-1 (ICAM-1) expressions. Expressions of HIF-1α mRNA and HIF-1α protein from each group were determined by the RT-PCR method and Western blotting, respectively. RESULTS: Ischemia preconditioning increased cell viability, and reduced LDH release and ICAM-1 expressions. Ischemia preconditioning also upregulated the HIF-1α mRNA level and HIF-1α protein expression. However, all of these changes were reversed by HIF-1α inhibitor NS398.CONCLUSION: Ischemia preconditioning effectively inhibited cold hypoxia/warm reoxygenation injury to endothelial cells, and the authors showed for the first time HIF-1α is causally linked to the protective effects of ischemic preconditioning on endothelial cells.

Vav1 promotes inflammation and neuronal apoptosis in cerebral ischemia/reperfusion injury by upregulating microglial and NLRP3 inflammasome activation

Microglia,which are the resident macrophages of the central nervous system,are an important part of the inflammatory response that occurs after cerebral ischemia.Vav guanine nucleotide exchange factor 1(Vav1) is a guanine nucleotide exchange factor that is related to microglial activation.However,how Vav1 participates in the inflammato ry response after cerebral ischemia/reperfusion inj ury remains unclea r.In this study,we subjected rats to occlusion and repe rfusion of the middle cerebral artery and subjected the BV-2 mic roglia cell line to oxygen-glucose deprivatio n/reoxygenation to mimic cerebral ischemia/repe rfusion in vivo and in vitro,respectively.We found that Vav1 levels were increased in the brain tissue of rats subjected to occlusion and reperfusion of the middle cerebral arte ry and in BV-2 cells subjected to oxygen-glucose deprivation/reoxygenation.Silencing Vav1 reduced the cerebral infarct volume and brain water content,inhibited neuronal loss and apoptosis in the ischemic penumbra,and im p roved neurological function in rats subjected to occlusion and repe rfusion of the middle cerebral artery.Further analysis showed that Vav1 was almost exclusively localized to microglia and that Vav1 downregulation inhibited microglial activation and the NOD-like receptor pyrin 3(NLRP3) inflammasome in the ischemic penumbra,as well as the expression of inflammato ry facto rs.In addition,Vov1 knoc kdown decreased the inflammatory response exhibited by BV-2 cells after oxygen-glucose deprivation/reoxyge nation.Taken together,these findings show that silencing Vav1 attenuates inflammation and neuronal apoptosis in rats subjected to cerebral ischemia/repe rfusion through inhibiting the activation of mic roglia and NLRP3 inflammasome.

Protective effects of erythropoietin pretreatment on myocardium with hypoxia/reoxygenation injury in rats

Objective: To establish the rat model with myocardial hypoxia/reoxygenation (H/R) injury, and investigate the protective effect of EPO pretreatment on the myocardium. Methods: Sixty male adult Wistar rats were randomly divided into 3 groups: control group, H/R group, and EPO group, 20 in each group. The rats in EPO group accepted injection of 5 000 U/kg recombinant human erythropoietin (RHuEPO) through vein, and the other rats accepted the injection of the same volume of saline. Twenty-four hours after the injection, rats in the EPO and H/R groups were put into the hypoxia environment for 12 h and then returned to the normoxic environment for 2 h, and then the samples of blood and myocardium were collected. Serum myocardial enzyme activity, apoptosis, ultrastructure, myocardial MDA contents, EPO receptor (EPOR) expression in cardiac myocytes and cardiac functions were tested. Results: EPOR expression was positive in cardiac myocytes of adult rat according to the result of immunonistochemitry assaying. Compared to those in H/R group, rats in EPO group presented lighter injury of myocardial ultrastructure, the reduction of serum myocardial enzyme activity, inhibition of apoptosis, the better recovery of cardiac functions, and the less production of oxygen-derived free radicals. Conclusion: Adult rat cardiac myocytes could express EPOR, and EPO pretreatment produced protective effects on myocardium with H/R injury.

Long non-coding RNA MEG3 regulates autophagy after cerebral ischemia/reperfusion injury

Severe cerebral ischemia/reperfusion injury has been shown to induce high-level autophagy and neuronal death.Therefore,it is extremely important to search for a target that inhibits autophagy activation.Long non-coding RNA MEG3 participates in autophagy.However,it remains unclear whether it can be targeted to regulate cerebral ischemia/reperfusion injury.Our results revealed that in oxygen and glucose deprivation/reoxygenation-treated HT22 cells,MEG3 expression was obviously upregulated,and autophagy was increased,while knockdown of MEG3 expression greatly reduced autophagy.Furthermore,MEG3 bound mi R-181 c-5 p and inhibited its expression,while mi R-181 c-5 p bound to autophagy-related gene ATG7 and inhibited its expression.Further experiments revealed that mir-181 c-5 p overexpression reversed the effect of MEG3 on autophagy and ATG7 expression in HT22 cells subjected to oxygen and glucose deprivation/reoxygenation.In vivo experiments revealed that MEG3 knockdown suppressed autophagy,infarct volume and behavioral deficits in cerebral ischemia/reperfusion mice.These findings suggest that MEG3 knockdown inhibited autophagy and alleviated cerebral ischemia/reperfusion injury through the mi R-181 c-5 p/ATG7 signaling pathway.Therefore,MEG3 can be considered as an intervention target for the treatment of cerebral ischemia/reperfusion injury.This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University,China(approval No.XF20190538)on January 4,2019.

Neuroprotective effects of neural stem cells pretreated with neuregulin1β on PC12 cells exposed to oxygen-glucose deprivation/reoxygenation

Studies on ischemia/reperfusion(I/R)injury suggest that exogenous neural stem cells(NSCs)are ideal candidates for stem cell therapy reperfusion injury.However,NSCs are difficult to obtain owing to ethical limitations.In addition,the survival,differentiation,and proliferation rates of transplanted exogenous NSCs are low,which limit their clinical application.Our previous study showed that neuregulin1β(NRG1β)alleviated cerebral I/R injury in rats.In this study,we aimed to induce human umbilical cord mesenchymal stem cells into NSCs and investigate the improvement effect and mechanism of NSCs pretreated with 10 nM NRG1βon PC12 cells injured by oxygen-glucose deprivation/reoxygenation(OGD/R).Our results found that 5 and 10 nM NRG1βpromoted the generation and proliferation of NSCs.Co-culture of NSCs and PC12 cells under condition of OGD/R showed that pretreatment of NSCs with NRG1βimproved the level of reactive oxygen species,malondialdehyde,glutathione,superoxide dismutase,nicotinamide adenine dinucleotide phosphate,and nuclear factor erythroid 2-related factor 2(Nrf2)and mitochondrial damage in injured PC12 cells;these indexes are related to ferroptosis.Research has reported that p53 and solute carrier family 7 member 11(SLC7A11)play vital roles in ferroptosis caused by cerebral I/R injury.Our data show that the expression of p53 was increased and the level of glutathione peroxidase 4(GPX4)was decreased after RNA interference-mediated knockdown of SLC7A11 in PC12 cells,but this change was alleviated after co-culturing NSCs with damaged PC12 cells.These findings suggest that NSCs pretreated with NRG1βexhibited neuroprotective effects on PC12 cells subjected to OGD/R through influencing the level of ferroptosis regulated by p53/SLC7A11/GPX4 pathway.

Aldehyde dehydrogenase 2 preserves mitochondrial morphology and attenuates hypoxia/reoxygenationinduced cardiomyocyte injury

BACKGROUND:Disturbance of mitochondrial fi ssion and fusion(termed mitochondrial dynamics)is one of the leading causes of ischemia/reperfusion(I/R)-induced myocardial injury.Previous studies showed that mitochondrial aldehyde dehydrogenase 2(ALDH2)conferred cardioprotective effect against myocardial I/R injury and suppressed I/R-induced excessive mitophagy in cardiomyocytes.However,whether ALDH2 participates in the regulation of mitochondrial dynamics during myocardial I/R injury remains unknown.METHODS:In the present study,we investigated the effect of ALDH2 on mitochondrial dynamics and the underlying mechanisms using the H9c2 cells exposed to hypoxia/reoxygenation(H/R)as an in vitro model of myocardial I/R injury.RESULTS:Cardiomyocyte apoptosis was significantly increased after oxygen-glucose deprivation and reoxygenation(OGD/R),and ALDH2 activation largely decreased the cardiomyocyte apoptosis.Additionally,we found that both ALDH2 activation and overexpression significantly inhibited the increased mitochondrial fission after OGD/R.Furthermore,we found that ALDH2 dominantly suppressed dynamin-related protein 1(Drp1)phosphorylation(Ser616)and adenosine monophosphate-activated protein kinase(AMPK)phosphorylation(Thr172)but not interfered with the expression levels of mitochondrial shaping proteins.CONCLUSIONS:We demonstrate the protective effect of ALDH2 against cardiomyocyte H/R injury with a novel mechanism on mitochondrial fission/fusion.

Cystic fibrosis transmembrane conductance regulator prevents ischemia/reperfusion induced intestinal apoptosis via inhibiting PI3K/AKT/NF-κB pathway

BACKGROUND Intestinal ischemia/reperfusion(I/R)injury is a fatal syndrome that occurs under many clinical scenarios.The apoptosis of intestinal cells caused by ischemia can cause cell damage and provoke systemic dysfunction during reperfusion.However,the mechanism of I/R-induced apoptosis remains unclear.Cystic fibrosis transmembrane conductance regulator(CFTR)is a cAMP-activated chloride channel.Few researchers have paid attention to its role in intestinal I/R injury,or the relationship between CFTR and intestinal apoptosis induced by hypoxia/reoxygenation(H/R).AIM To investigate the effects of CFTR on I/R-induced intestinal apoptosis and its underlying molecular mechanisms.METHODS An intestinal I/R injury model was established in mice with superior mesenteric artery occlusion, and Caco2 cells were subjected to H/R for the simulation of I/R in vivo.RESULTSThe results suggested that CFTR overexpression significantly increased the Caco2 cell viability anddecreased cell apoptosis induced by the H/R. Interestingly, we found that the translocation of p65,an NF-κB member, from the cytoplasm to the nucleus after H/R treatment can be reversed by theoverexpression of CFTR, the NF-κB P65 would return from the nucleus to the cytoplasm asdetermined by immunostaining. We also discovered that CFTR inhibited cell apoptosis in theH/R-treated cells, and this effect was significantly curbed by the NF-κB activator BA, AKTinhibitor GSK690693 and the PI3K inhibitor LY294002. Moreover, we demonstrated that CFTRoverexpression could reverse the decreased PI3K/AKT expression induced by the I/R treatment invivo or H/R treatment in vitro.CONCLUSIONThe results of the present study indicate that the overexpression of CFTR protects Caco2 cells fromH/R-induced apoptosis;furthermore, it also inhibits H/R-induced apoptosis through thePI3K/AKT/NF-κB signaling pathway in H/R-treated Caco2 cells and intestinal tissues.

Rapamycin Protects Cardiomyocytes against Anoxia/Reoxygenation Injury by Inducing Autophagy through the PI3k/Akt Pathway

The purpose of this study was to investigate the potential cardioprotection roles of Rapamycin in anoxia/reoxygenation（A/R） injury of cardiomyocytes through inducing autophagy, and the involvement of PI3k/Akt pathway. We employed simulated A/R of neonatal rat ventricular myocytes（NRVM） as an in vitro model of ischemial/reperfusion（I/R） injury to the heart. NRVM were pretreated with four different concentrations of Rapamycin（20, 50, 100, 150 μmol/L）, and pretreated with 10 mmol/L 3-methyladenine（3MA） for inhibiting autophagy during A/R. Then, Western blot analysis was used to examine variation in the expression of LC3-Ⅱ, LC3-Ⅰ, Bim, caspase-3, p-PI3KⅠ, PI3KⅠ, p-Akt and Akt. In our model, Rapamycin had a preferential action on autophagy, increasing the expression of LC3-Ⅱ/ LC3-Ⅰ, whereas decreasing the expression of Bim and caspase-3. Moreover, our results also demonstrated that Rapamycin inhibited the activation of p-PI3KⅠ and enhanced the activation of p-Akt. It is concluded that Rapamycin has a cardioprotection effect by inducing autophagy in a concentration-dependent manner against apopotosis through PI3K/Akt signaling pathway during A/R in NRVM.

Protective role of retinoid X receptor in H9c2 cardiomyocytes from hypoxia/reoxygenation injury in rats

BACKGROUND: Retinoid X receptor(RXR) plays a central role in the regulation of intracellular receptor signaling pathways. The activation of RXR has protective effect on H2O2-induced apoptosis of H9c2 ventricular cells in rats. But the protective effect and mechanism of activating RXR in cardiomyocytes against hypoxia/reoxygenation(H/R)-induced oxidative iniury are still unclear.METHODS: The model of H/R injury was established through hypoxia for 2 hours and reoxygenation for 4 hours in H9c2 cardiomyocytes of rats. 9-cis-retinoic acid(9-cis RA) was obtained as an RXR agonist, and HX531 as an RXR antagonist. Cultured cardiomyocytes were randomly divided into four groups: sham group, H/R group, H/R+9-cis RA-pretreated group(100 nmol/L 9-cis RA), and H/R+9-cis RA+HX531-pretreated group(2.5 μmol/L HX531). The cell viability was measured by MTT, apoptosis rate of cardiomyocytes by flow cytometry analysis, and mitochondrial membrane potential(ΔΨm) by JC-1 fluorescent probe, and protein expressions of Bcl-2, Bax and cleaved caspase-9 with Western blotting. All measurement data were expressed as mean±standard deviation, and analyzed using one-way ANOVA and the Dunnett test. Differences were considered signif icant when P was <0.05.RESULTS: Pretreatment with RXR agonist enhanced cell viability, reduced apoptosis ratio, and stabled ΔΨm. Dot blotting experiments showed that under H/R stress conditions, Bcl-2 protein level decreased, while Bax and cleaved caspase-9 were increased. 9-cis RA administration before H/R stress prevented these effects, but the protective effects of activating RXR on cardiomyocytes against H/R induced oxidative injury were abolished when pretreated with RXR pan-antagonist HX531.CONCLUSION: The activation of RXR has protective effects against H/R injury in H9c2 cardiomyocytes of rats through attenuating signaling pathway of mitochondria apoptosis.