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.

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.

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.

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.

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.

Antiapoptotic Mechanism of Insulin in Reoxygenation-induced Injury in Cultured Cardiomyocytes of Neonatal Rats

To examine the protective effect of insulin on reoxygenation-induced injury and explore the underlying mechanisms, the model of anoxia/reoxygenation （A/R） injury was established by inducing anoxia for 2 h and reoxygenation for 4 h in cultured cardiomyocytes of neonatal rats. The rats were randomized to four groups receiving vehicle, insulin, LY294002, insulin plus LY294002 at the onset of reoxygenation after 2 h of anoxia. At the end of reoxygenation of 4 h, activity of lactate dehydrogenase （LDH） and content of malondialdehyde （MDA） were spectrophotometrically determined, apoptosis of cardiomyocytes were detected by using TUNEL and DNA Ladder, and Western blotting was employed to examine the expression of phosphorylated Akt in all groups. Our results showed that compared with vehicle-treated group, activities of LDH, contents of MDA, apoptosis index （AI） were significantly decreased, and expression of phosphorylated Akt was increased significantly in insulin-treated group. However, changes in LDH, MDA, AI and phosphorylated Akt resulting from insulin were attenuated or abolished by LY294002 （PI3K inhibitor）. These data strongly suggest that early administration of insulin at reoxygenation protects cardiomyocytes from reoxygenation-induced apoptosis through PI3K/Akt signaling pathway.

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.

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.

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.

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.

The antiapoptotic effect of insulin against anoxia/reoxygenation injury in cultured cardiomyocyte of neonatal rat

Objective： To study protective effect of insulin against cardiomyocyte apoptosis in anoxia/reoxygenation （A/R） injury of neonatal rat. Methods： The model of A/R injury was finished through receiving anoxia for 2h and reoxygenation for 4h in cultured cardiomyocytes of neonatal rat. The cardiomyocytes were divided randomly into 3 groups： control group （CON）, anoxia/reoxygenation group （A/R） and insulin-treated group （INS）. At the end of reoxygenation of 4 hours, activities of lactate dehydrogenase （LDH）, contents of malondiaidehyde （MDA）, were assessed through spectrophotometric procedures, myocyte apoptosis were detected through TUNEL and DNA Ladder. Results： MDA, LDH, and Apoptosis Index were significantly decreased in INS group compared with A/R group （P〈0.01）. Conclusion： Insulin has a protective effect against A/R injury in cultured cardiomyocyte of neonatal rat; the protective mechanism may contribute to antiapoptosis of insulin.

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

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

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.

Effect of emulsified isoflurane on apoptosis of anoxia-reoxygenation neonatal rat cardiomyocytes

Objective:To explore the effect of emulsified isoflurane(EI)on apoptosis of anoxia-reoxygenation neonatal rat cardiomyocytea and relevant protein expression.Methods:Cardiac muscle anoxiareoxygenation damage model was established with culture in vitro neonatal rat cardiomyocytes.The cardiomyocytes were divided into control group,model group,fat emulsion group and EI group.The cardiomyocytes apoptosis rates and lactic dehydrogenase(LDH),superoxide dismutase(SOD)and malondialdehyde(MDA)index standardization were detected after relevant treatment The expression of apoptosis-related proteins Bel-2,Bax and Caspase-3 were detected with Western blot approach.Results:After hypoxia/reoxygenation(H/R)model was treated by EI,the cells apoptosis rate decreased and was dramatically below the fat emulsion group(P<0.05),Cardiomyocytes biochemical index detection presented that,compared with the control group that the LDH activity and MDA content dramatically increased(P<0.05),while the SOD activity notably decreased(P<0.05);compared with the H/R group,the SOD activity of the fat emulsion group and EI group increased(P<0.05);while the LDH activity and MDA content decreased(P<0.05).And the change of the EI group was more remarkable than the fat emulsion group(P<0.05).The Western blot analysis presented that,compared with the control group,the Bcl-2 protein expression of the other groups significantly decreased(P<0.05),the expressions of Bax protein and Caspase-3protein increased significantly(P<0.05);compared with H/R group,cardiomyocytes Bc1-2protein expression of EI group increased significantly(P<0.05),the expressions of Bax protein and Caspase-3 protein decreased significantly(P<0.05),and the change of EI group was more remarkable than the fat emulsion group(P<0.05).Conclusions:EI can inhabit the apoptosis of anoxia-reoxygenation damage model cardiomyocytes,and may he related to the up-regulation of expression of Bcl-2 and down-regulation of expression of Caspase-3 protein.

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.

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.

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.

Iptakalim ameliorates relaxation to acetylcholine in thoracic aortic rings impaired by microvesicles derived from hypoxia/reoxygenation-treated HUVECs

Objective: To investigate the effect of Iptakalim(Ipt) preventing injury of endothelial microvesicles(EMVs) derived from hypoxia/reoxygenation(H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and explore the underlying mechanism. Methods: H/R injury model was established to release H/R-EMVs from HUVECs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized by using Transmission Electron Microscope(TEM). Thoracic aortic rings of rats were incubated with 10^(-7)-10^(-3 )mol/L Ipt and co-cultured with 10 μg/ml H/R-EMVs for 4 hours, and their endothelium- dependent relaxation in response to acetylcholine(ACh) was recorded in vitro. The nitric oxide(NO) production of ACh-treated rat thoracic aortic rings was measured by using Griess reagent. The expression of endothelial NO synthase(e NOS), phosphorylated e NOS(p-e NOS, Ser-1177), serine/threonine kinas(Akt) and phosphorylated Akt(p-Akt, Ser-473) in the thoracic aortic rings of rats was detected by Western blotting. Results: H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The isolated H/R-EMVs subjected to TEM revealed small, rounded vesicles(100–1 000 nm) surrounded by a membrane. H/R-EMVs impaired relaxation induced by ACh of rat thoracic aortic rings significantly. Compared with H/R-EMVs treatment individually, relaxation and NO production of rat thoracic aortic rings were increased by Ipt treatment in a concentration-dependent manner(P<0.05, P<0.01). The expression of total e NOS(t-e NOS) and total Akt(t-Akt) was not affected by Ipt or H/R-EMVs. However, the expression of p-e NOS and p-Akt increased after treated with Ipt(P<0.01). Conclusion: Based on H/R-EMVs treatment, ACh induced endothelium-dependent relaxation of rat thoracic aortic rings was ameliorated by Ipt in a concentration-dependent manner. The mechanisms involved the increase in NO production, p-e NOS and p-Akt expression.

Microvesicles derived from hypoxia/reoxYgenation-treated human umbilical vein endothellal cells impair relaxation of rat thoracic aortic rings

Objective To investigate the effects of microvesicles(MVs) derived from hypoxia/reoxygenation(H/R)-treated human umbilical vein endothelial cells(HUVECs) on endothelium-dependent relaxation of rat thoracic aortic rings.Methods H/R injury model was established to induce HUVECs to release H/R-EMVs.H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium.H/R-EMVs were characterized using 1 urn latex beads and anti-PE-CD144 by flow cytometry.Thoracic aortic rings of rats were incubated with 2.5,5,10,20 μg/ml H/R-EMVs derived from H/R-treated HUVECs for 4 hours,and their endothelium-dependent relaxation in response to acetylcholine(ACh) or endothelium-independent relaxation in response to sodium nitroprusside(SNP) was recorded in vitro.The nitric oxide(NO) production of ACh-treated thoracic aortic rings of rats was measured using Griess reagent.The expression of endothelial NO synthase(eNOS) and phosphorylated eNOS(p-eNOS,Ser-1177) in the thoracic aortic rings of rats was detected by Western blotting.Furthermore,the levels of SOD and MDA in H/R-EMVs-treated thoracic aortic rings of rats were measured using SOD and MDA kit.Results H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation.The membrane vesicles(< 1 urn) induced by H/R were CD144 positive.ACh-induced relaxation and NO production of rat thoracic aortic rings were impaired by H/R-EMVs treatment in a concentration-dependent manner(P<0.05,P<0.01).The expression of total eNOS(t-eNOS)was not affected by H/R-EMVs.However,the expression of p-eNOS decreased after treated with H/R-EMVs.The activity of SOD decreased and the level of MDA increased in H/R-EMVs treated rat thoracic aortic rings(P<0.01).Conclusion ACh induced endothelium-dependent relaxation of thoracic aortic rings of rats was impaired by H/R-EMVs in a concentration-dependent manner.The mechanisms included a decrease in NO production,p-eNOS expression and an increase in oxidative stress.