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.

Anisodine hydrobromide alleviates oxidative stress caused by hypoxia/reoxygenation in human cerebral microvascular endothelial cells predominantly via inhibition of muscarinic acetylcholine receptor 4

Background:Anisodine hydrobromide(AT3),an anti-cholinergic agent,could be delivered to the brain across the blood-brain barrier and has been used clinically for the treatment of cerebral ischemia/reperfusion injury.Endothelial dysfunction can be caused by hypoxia/reoxygenation(H/R)via oxidative stress and metabolic alterations.The present study investigated whether AT3 regulates the production of nitric oxide(NO)and reactive oxygen species(ROS),and the HIF-1αpathway via regulation of muscarinic acetylcholine receptors(mAChRs)in brain microvascular endothelial cells after H/R exposure.Methods:Under H/R conditions,hCMEC/D3 cerebral microvascular endothelial cells were treated with AT3.Specific inhibitors of M2-and M4-mAChRs were used to explore the mechanism by which AT3 influences oxidative stress in endothelial cells.Then,mAChRs expression was detected by western blotting and NO production was detected by Greiss reaction.The intracellular ROS level was measured using DCFH-DA probes.The expression of hypoxia-inducible transcription factor 1α(HIF-1α)was also detected.Results:While H/R induced the expression of M2-and M4-mAChRs,AT3 suppressed the H/R-upregulated M2-and M4-mAChRs.H/R also induced the production of NO,ROS,and apoptosis.AT3 and M4-mAChR inhibitors inhibited the H/R-induced production of NO and ROS and apoptosis.HIF-1αwas induced by H/R,but was suppressed by AT3.Conclusion:Thus,the in vitro evidence shows that AT3 protects against H/R injury in cerebral microvascular endothelial cells via inhibition of HIF-1α,NO and ROS,predominantly through the downregulation of M4-mAChR.The findings offer novel understandings regarding AT3-mediated attenuation of endothelial cell apoptosis and cerebral ischemia/reperfusion injury.

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.

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.

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.

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.

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.

TIR/BB-loop mimetic AS-1 protects vascular endothelial cells from injury induced by hypoxia/reoxygenation

Morphological and functional abnormalities of vascular endothelial cells(VECs) are risk factors of ischemiareperfusion in skin flaps.Signaling pathway mediated by interleukin-1 receptor(IL-1 R) is essential to hypoxia/reoxygenation(H/R) injury of VECs.While the TIR/BB-loop mimetic(AS-1) disrupts the interaction between IL-1 R and myeloid differentiation primary-response protein 88(MyD88),its role in the VECs dysfunction under H/R is unclear.In this study,we first showed that there was an infiltration of inflammatory cells and the apoptosis of VECs by using a skin flap section from patients who received flap transplantation.We then showed that the H/R treatment induced apoptosis and loss of cell migration of endothelial cell line H926 were attenuated by AS-1.Furthermore,our data suggested that AS-1 inhibits the interaction between IL-1 R and MyD88,and subsequent phosphorylation of IκB and p38 pathway,as well as the nuclear localization of NF-κB subunit p65/p50.Thus,this study indicated that the protective role of AS-1 in H/R induced cellular injury may be due to the AS-1 mediated down-regulation of IL-1 R signaling pathway.

Interventional effect of phycocyanin on mitochondrial membrane potential and activity of PC12 cells after hypoxia/reoxygenation

BACKGROUND： Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation. OBJECTIVE： To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation. DESIGN： Randomized controlled study SETTING ： Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University MATERIALS： The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells, were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide （MTT） and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA. METHODS： ① Culture of PC12 cells： PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37℃. Number of cells was regulated to 4 × 10^5 L 1, and cells were inoculated at 96-well culture plate. The final volume was 100μL. ② Model establishing and grouping： Cultured PC12 cells were randomly divided into three groups： phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L , but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group （the final concentration of 3 g/L） and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non-hypoxia group were cultured with oxygen and were measured at 1 hour after hypoxia/reoxygenation. ③ Detecting items： At 1, 2 and 3 hours after reoxygenation, absorbance （A value） of PC12 cells was measured with MTT technique so as to observe activity and quantity of cells. Fluorescence intensity of PC12 cells marked by rhodamine 123 was measured with confocal microscope in order to observe changes of mitochondrial membrane potential. MAEN OUTCOME MEASURES： Comparisons between quantity and activity of PC12 cells and mitochondria membrane potential at 1, 2 and 3 hours after reoxygenation. RESULTS： ① Effect of phycocyanin on quantity and activity of PC12 cells： A value was 0.924±0.027 in adding phycocyanin group and 0.924±0.033 in non-adding phycocyanin group. A value was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after reoxygenation （0.817±0.053, 0.838±0.037, 0.875±0.029; 0.842±0.029, 0.872±0.025, 0.906±0.023, P 〈 0.05）. A value was higher in phycocyanin group than that in model control group at 1, 2 and 3 after culture （P 〈 0.05）. With culture time being longer, A value was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. ~ Effect of phycocyanin on mitochondrial membrane potential of PC12 cells： Fluorescence intensity was 2.967±0.253 in adding phycocyanin group and 2.962±0.294 in non-adding phycocyanin group. Fluorescence intensity was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after hypoxia/reoxygenation （1.899±0.397, 2.119±0.414, 2.287±0.402; 2.191±0.377, 2.264±0.359, 2.436±0.471, P 〈 0.05）; but it was higher in phycocyanin group than that in model control group at 1, 2 and 3 after reoxygenation （P 〈 0.05）. With culture time being longer, fluorescence intensity was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. CONCLUSION： Phycocyanin and reoxygenation can protect PC12 cells after hypoxia injury through increasing mitochondrial membrane potential and cellular activity, and the effect is improved gradually with prolonging time of reoxygenation.

Triptolide protects astrocytes from hypoxia/reoxygenation injury

Astrocytes in an in vitro murine astrocyte model of oxygen and glucose deprivation/hypoxia and reoxygenation were treated with different concentrations of triptolide （250, 500, 1 000 ng/mL） in a broader attempt to elucidate the protection and mechanism underlying triptolide treatment on astrocytes exposed to hypoxia/reoxygenation injury. The results showed that the matrix metalloproteinase-9, interleukin-1β, tumor necrosis factor α and interleukin-6 expressions were significantly decreased after triptolide treatment in the astrocytes exposed to hypoxia/ reoxygenation injury, while interleukin-10 expression was upregulated. In addition, the vitality of the injured astrocytes was enhanced, the triptolide＇s effect was apparent at 500 ng/mL. These experimental findings indicate that triptolide treatment could protect astrocytes against hypoxia/ reoxygenation injury through the inhibition of inflammatory response and the reduction of matrix metalloproteinase-9 expression.

Effects of Crocin on Nox2 Expression and ROS Level of Hypoxia/Reoxygenation-induced Injury of Cardiomyocytes

[Objectives]To explore the protection mechanism of crocin against ischemia-reperfusion injury of myocardial cells.[Methods]Newborn male SD rats were selected,left ventricular cardiomyocytes(CMs)were isolated,and a hypoxia/reoxygenation model of CMs was established to simulate the process of ischemia/reperfusion injury.The cells were randomly divided into four groups:normal cell group(control group),crocin group),hypoxia/reoxygenation group(H/R group),hypoxia/reoxygenation+crocin group(H/R+crocin group).H/R+crocin group selected the concentration of crocin 1,10,and 100μmol/L,and determined the optimal concentration of crocin by detecting the cell proliferation ability.After the cells were pretreated using the optimal concentration of crocin,the levels of superoxide anion,cell proliferation,apoptosis and Nox2 levels in each group of cells were detected.[Results]Compared with the control group,the proliferation ability of CMs after hypoxia-reoxygenation injury was reduced(P<0.05),while cell apoptosis and intracellular superoxide anion levels were significantly increased(P<0.01);the CMs pretreated with crocin can reduce the level of Nox2(P<0.01),increase the cell proliferation ability of CMs,reduce cell apoptosis,and accordingly reduce the level of superoxide anion in the cell(P<0.05).[Conclusions]Crocin protects CMs from hypoxia/reoxygenation injury through down-regulating the level of Nox2 and reducing oxidative stress injury.

Nonhematopoietic erythropoietin derivative protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis

Objective：Carbamylated EPO（CEPO） is a derivative of erythropoietin（EPO） by subjecting it to carbamylation. It does not stimulate erythropoiesis, but effectively protects tissue from injury. The present study was to investigate the effect of CEPO treatment using in vitro models of hypoxia/reoxygenation（H/R）. Methods：Cardiomyocytes were exposed to hypoxia（95% N2 and 5% CO2） for 1 hour followed by 4 hours of reoxygenation（95% O2 and 5% CO2）. CEPO was administered after hypoxia, just before reoxygenation. The apoptotic cardiomyocytes were determined by flow cytometry. The level of protein was assessed by western blot analysis. Results： CEPO treatment significantly decreased the apoptotic cardiomyocytes by 54.20% compared with H/R group. Western blot analysis showed that CEPO administration increased the level of Bcl-2（an antiapoptotic protein） by 62.22% compared with H/R group. Conclusion： Acute administration of CEPO protected cardiomyocytes from H/R-induced apoptosis. CEPO protected cardiomyocytes with a concomitant upregulation of Bcl-2 after H/R injury.

Role of NF-κB in protection of EPO pretreatment on neonatal rat cardiac myocytes with hypoxia/reoxygenation injury

Objective：To observe the protective effects of erythropoietin （EPO） pretreatment on cardiac myocyte with hypoxia/reoxygenation （H/R） injury and the role of NF-κBin this effects. Methods：After the H/R model of cardiac myocytes of neonatal rats was established, the cultured cardiac myocytes were divided into 4 groups, including EPO pretreatment group （ EPO 10 U/ml 24 h before H/R）, EPO pretreatment ＋ PDTC group（EPO 10 U/ml and PDTC 5 μg/ml 24 h before H/R）, PDTC group （PDTC 5 μg /ml 24 h before H/R） and eomrolgroup. Before and after the H/R, assay of LDH concentration in the culture medium, the survival rate of the myocytes tested by MTT chromatometry and the apoptosis by flow cytometry were undertaken. Activation of NF-κB was determined by EMSA before and after H/R. Results：EPO pretreatment markedly reduced the LDH concentration in the medium, elevated the survival rate of myocytes and inhibited the apoptosis after H/R. Addition of PDTC during the pretreatment abol- ished the protective effects of EPO pretreatment. NF-κB was markedly activated during EPO pretreatment and PDTCinhibited the activation. However, after H/R, the activity of NF-κB in myocytes with EPO pretreatment was significantly inhibited compared to the other myocytes. Conclusion：NF-κB is significantly activated during EPO pretreatment, but is inhibited after H/R, which is correlated with the protective effects of EPO pretreatment on cardiac myocytes with H/R. This phenomenon can be explained as the negative feedback mechanism of the activation of NF-κB.

Protection of ghrelin postconditioning on hypoxia/ reoxygenation in gastric epithelial cells

AIM: To investigate the protective effect and mechanisms of ghrelin postconditioning against hypoxia/reoxygenation (H/R)-induced injury in human gastric epithelial cells. METHODS: The model of H/R injury was established in gastric epithelial cell line (GES-1) human gastric epithelial cells. Cells were divided into seven groups: normal control group (N); H/R postconditioning group; DMSO postconditioning group (DM); ghrelin postconditioning group (GH); D-Lys3-GHRP-6 + ghrelin postconditioning group (D + GH); capsazepine + ghrelin postconditioning group (C + GH); and LY294002 + ghrelin postconditioning group (L + GH). 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect GES-1 cell viability. Hoechst 33258 fluorochrome staining and flow cytometry were conducted to determine apoptosis of GES-1cells. Spectrophotometry was performed to determine release of lactate dehydrogenate (LDH). Protein expression of Bcl-2, Bax, Akt, and glycogen synthase kinase (GSK)-3β was determined by western blotting. Expression of vanilloid receptor subtype 1 (VR1), Akt and GSK-3β was observed by immunocytochemistry. RESULTS: Compared with the H/R group, cell viability of the GH group was significantly increased in a dosedependent manner (55.9% ± 10.0% vs 69.6% ± 9.6%, 71.9% ± 17.4%, and 76.3% ± 13.3%). Compared with the H/R group, the percentage of apoptotic cells in the GH group significantly decreased (12.38% ± 1.51% vs 6.88% ± 0.87%). Compared with the GH group, the percentage of apoptotic cells in the D + GH group, C + GH group and L + GH groups significantly increased (11.70% ± 0.88%, 11.93% ± 0.96%, 10.20% ± 1.05% vs 6.88% ± 0.87%). There were no significant differences in the percentage of apoptotic cells between the H/R and DM groups (12.38% ± 1.51% vs 13.00% ± 1.13%). There was a significant decrease in LDH release following ghrelin postconditioning compared with the H/R group (561.58 ± 64.01 U/L vs 1062.45 ± 105.29 U/L). There was a significant increase in LDH release in the D + GH, C + GH and L + GH groups compared with the GH group (816.89 ± 94.87 U/L, 870.95 ± 64.06 U/L, 838.62 ± 118.45 U/L vs 561.58 ± 64.01 U/L). There were no significant differences in LDH release between the H/R and DM groups (1062.45 ± 105.29 U/L vs 1017.65 ± 68.90 U/L). Compared with the H/R group, expression of Bcl-2 and Akt increased in the GH group, whereas expression of Bax and GSK3β decreased. Compared with the GH group, expression of Bcl-2 decreased and Bax increased in the D + GH, C + GH and L + GH groups, and Akt decreased and GSK-3β increased in the L + GH group. The H/R group also upregulated expression of VR1 and GSK-3β and downregulated Akt. The number of VR1-positive and Akt-positive cells in the GH group significantly increased, whereas the number of GSK-3β-positive cells significantly decreased. These effects of ghrelin were reversed by capsazepine and LY294002.CONCLUSION: Ghrelin postconditioning protected against H/R-induced injury in human gastric epithelial cells, which indicated that this protection might be associated with GHS-R, VR1 and the PI3K/Akt signaling pathway.

Effects of Cornus Officinal Is Total Glycoside on Cardiomyocyte Apoptosis and Calcium Overload after Hypoxia/Reoxygenation Injury in Rats

Objective: To investigate the mechanism of Cornus officinalis Total Glycosides (COTG) on myocardial protection, by studying effects of COTG on cardiomyocyte apoptosis induced by hypoxia/reoxygenation and calcium concentration in rats. Methods: The myocardial cells of born 1-3d SD rats were isolated by enzyme digestion, cultured for 3 days. Cells were divided into five groups: Control group, H/R group, Cornus officinalis Total Glycosides low-dose group (LDG), Cornus officinalis Total Glycosides middle-dose group (MDG) and Cornus officinalis Total Glycosides high-dose group (HDG). Three drug groups were pretreated with different doses of Cornus officinalis Total Glycosides before hypoxia/reoxygenation treatment. The apoptotic rate was determined by flow cytometry assay, the intracellular free calcium concentration was examined by flow cytometry, and the ultrastructure of myocardial cells was observed under transmission electron microscope. Results: The results revealed that Cornus officinalis Total Glycosides pretreatment decreased apoptosis rate, but the effect of lower dosage is not significant. Furthermore, Cornus officinalis Total Glycosides can attenuate mitochondrial calcium overload, improve mitochondrial morphology and inhibit cardiomyocyte apoptosis caused by H/R. Conclusion: Cornus officinalis Total Glycosides pretreatment can inhibit cardiomyocyte apoptosis and calcium overload during H/R injury. However, the underlying mechanisms require us to further study.

Effects of L-Tetrahydropalmatine on NOSⅢ Gene Expression in Hypoxia and Cultured Porcine Cerebral Arterial Endothelial Cells during Reoxygenation

To investigate the expression of NOSⅢ mRNA and protein in cultured porcine cerebral arterial endothelial cells (CAEC) during hypoxia and reoxygenation and the effects of L-Tetrahydropalmatine (L-THP) on the gene expression of NOSⅢ in CAEC during hypoxia and reoxygenation. The cultured CAEC were divided into 5 groups: control, hypoxia, hypoxia+reoxygenation, hypoxia+L-THP and reoxygenation+L-THP groups. NOSⅢ mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemistry was used to detect the level of NOSⅢ protein. The expression of NOSⅢ mRNA and protein were increased when CAEC were exposed to hypoxia for 1 h, and significantly decreased during reoxygenation 2, 6 and 12 h after 1-h of hypoxia. L-THP from 10 -8 mol/L to 10 -3 mol/L could inhibit the up-regulation of NOSⅢ gene expression during hypoxia and down-regulation of NOSⅢ gene expression during reoxygenation.

Effect of hypoxia/reoxygenation on the viability expression of ROS and MAPKs in myocardial cells

Objective:To investigate to the expression effect of hypoxia and hypoxia/reoxygenation on ROS,MAPKs and cell apoptosis in H9c2 cardiomyocytes.Methods:H9c2 cells were treated with cobalt chloride(CoCl2)to establish the chemical hypoxia and hypoxia/reoxygenation-induced cardiomyocyte injury model.CoCl2 was used to process cells at different concentrations from 150-2400μmol/L and different time from4-24 h;H9c2 cells viability was detected by MTT,and the intracellular ROS level was measured by 2’,7’-dichlorflμoresceindiacetate(DCFH-DA)and dihydroethidiμm(DHE)staining and photoflurography.The active expression level of mitogen-activated protein kinases(MAPKs)(including JNK,ERK and p38)and caspase-3.Results:At the concentration from 300 to 1200μmol/L,CoCl2 does/time-dependently inhibited the cell viability in H9c2 cells(P<0.01).Compared with control group,the ROS levels in hypoxia group were significantly increased(P<0.05).In hypoxia group,the active expression levels of p-JNK,p-p38 and caspase-3 was higher than those in control group(P<0.05).However,the expression of p-ERK wasn’t significant differernce.Furthermore,all the expression levels of ROS,p-JNK,p-ERK,p-p38 and caspase-3 in H/R group were significantly raised compared with hypoxia group(P<0.01).Conclusions:Reoxygenation further aggravate chemical hypoxia induced cardiomyocyte oxidative stress injury by activating ROS/MAPKs signals,suggesting the role of myocardial ischemia/reperfusion injury in the pathogenesis of ischemic heart disease.

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.

Relation or Influence of RVOTO in the Inflammatory Response to Reoxygenation in Patients with Tetralogy of Fallot

Background:This study evaluated differential inflammatory response to cardiopulmonary bypass reoxygenation in tetralogy of Fallot repair.Methods:We performed a retrospective study at a cardiovascular center from 2012 to 2018,including 500 patients aged 1 week–18 years who received complete repair of tetralogy of Fallot.Patients were grouped according to tertiles of preoperative RVOT gradient on echocardiography into mild,moderate,and severe stenosis.We measured the highest perfusate oxygenation(PpO_(2))during aortic occlusion as independent variable.Primary outcome was systemic inflammatory response syndrome(SIRS)within 7 days postoperatively or the time of death or discharge.Results:Overall,rate of SIRS was 24.2% without significant differences among three groups(P>0.05).Older age,male,and smaller indexed left ventricular end-diastolic volume is independent risk factor of SIRS.There were significant interactions between RVOT stenosis and PpO2 on SIRS(P interaction=0.011):higher PpO_(2) was associated with a greater SIRS risk among combined moderate and severe stenotic children(OR 1.46395%CI[1.080,1.981]per-SD increase,P=0.014)but not among mild stenotic children(OR 0.900[0.608,1.333]per-SD increase;P=0.600),independent of covariates.Conclusion:The association of PpO_(2) with SIRS was modified by RVOT obstruction severity in tetralogy of Fallot repair.

Calreticulin Translocation Aggravates Endoplasmic Reticulum Stress-associated Apoptosis during Cardiomyocyte Hypoxia/Reoxygenation

Background： Calreticulin （CRT） is major Ca^2＋-binding chaperone mainly resident in the endoplasmic reticulum （ER） lumen. Recently, it has been shown that non-ER CRT regulates a wide array of cellular responses. We previously found that CRT was up-regulated during hypoxia/ reoxygenation （H/R） and this study was aimed to investigate whether CRT nuclear translocation aggravates ER stress （ERS）-associated apoptosis during H/R injury in neonatal rat cardiomyocytes. Methods： Apoptosis rate and lactate dehydrogenase （LDH） leakage in culture medium were measured as indices of cell injury. lmmunofluorescence staining showed the morphological changes of ER and intracellular translocation of CRT. Western blotting or reverse transcription polymerase chain reaction was used to detect the expression of target molecules. Results： Compared with control, H/R increased apoptosis rate and LDH activity. The ER became condensed and bubbled, and CRT translocated to the nucleus. Western blotting showed up-regulation of CRT, Nrf2, activating transcription factor 4 （ATF4）, CHOP and caspase-12 expression after H/R. Exogenous CRT overexpression induced by plasmid transfection before H/R increased cell apoptosis, LDH leakage, ER disorder, CRT nuclear translocation and the expression of ERS-associated molecules. However, administration of the ERS inhibitor, taurine, or CRT siRNA alleviated cell injury, ER disorder, and inhibited ERS-associated apoptosis. Conclusions： Our results indicated that during H/R stress, CRT translocation increases cell apoptosis and LDH leakage, aggravates ER disorder, up-regulates expression of nuclear transcription factors, Nrf2 and ATF4, and activates ERS-associated apoptosis.