Effect of hydrogen sulfide on myocardial hypoxia/reoxygenation injury in vivo

AIM: To investigate the changes of endogenous cystathionine-γ-lyase/hydrogen sulfide pathway on hypoxia/reoxygenation injury in vivo and to explore the relationship between this pathway and hypoxia/reoxygenation injury. METHODS: Primary myocardial cell culture in vivo came from Wistar baby rats born less than 48 h. The cells were under the conditions of hypoxia (2% O2, 5% CO2) for 3 h and reoxygenation (21% O2, 5% CO2) for 2 h to induce hypoxia/reoxygenation injury. MTT was used to detect the cell survival in every group. The activities of lactate dehydrogenase (LDH) in culture medium, malondialdehyde (MDA) and superoxide dismutase (SOD) in myocardial cells were measured with colorimetry method. RT-PCR method was used to test CSE mRNA expression in myocardial cells. RESULTS: Compared to IR group, the cells in NaHS+IR and IR+NaHS groups had a higher survival rate, lower LDH concentration in culture medium, higher SOD activity and lower MDA in myocardial cells. At the same time, the results of RT-PCR displayed that CSE mRNA were down-regulated in myocardial cells after hypoxia/reoxygenation injury, and if CSE inhibitor PPG was added into the culture medium before hypoxia, no protective effects were detected. CONCLUSION: NaHS might protect the myocardial cells from hypoxia/reoxygenation injury through decreasing oxygen free radical production and stabilizing the cell membrane.     

Effect of Ginsenoside Rb1 on apoptosis induced by hypoxia/hypoglycemia and reoxygenation in cultured rat hippocampal neurons

AIM: The aim of this work is to investigate the protective effects of Ginsenoside Rb₁ (Rb₁) on apoptosis induced by hypoxia/hypoglycemia and reoxygenation in cultured rat hippocampal neurons. METHODS: Apoptosis were measured by flow cytometry; Morphological changes and neuronal necrosis were examined under microscope; The leakage of lactic dehydrogenase (LDH) and the product of nitric oxide (NO) were measured. RESULTS: hypoxia/hypoglycemia cultures for 5 h and reoxygenation induced neuronal apoptosis and necrosis,and significantly increased the leakage of LDH and the product of NO. The effects were enhanced with the extending time of reoxygenation. Rb₁ could significantly decrease the percentage of neuronal apoptosis and necrosis, and reduce the leakage of LDH and the product of NO. CONCLUSION: Rb₁ had an effect of anti-neuronal apoptosis. This effect might be related to the inhibition of the activity of NO synthase and NO production.     

Effects of phytoestrogen α-zearalanol on hypoxia/reoxygenation injury in HUVECs

AIM:To investigate the effects of phytoestrogen α-zearalanol (ZAL) on hypoxia/reoxygenation (H/R) injury and mechanism involved in human umbilical vein endothelial cells (HUVECs). METHODS:HUVECs were exposed to hypoxia for 3 hours and then reoxygenation 1 hour. ZAL or 17β-estradiol (E₂) at concentrations of 10-9-10-6 mol/L were pretreated before hypoxia. The survival rate of HUVECs was detected by MTT. Either the activities of LDH and SOD or the level of MDA in supernatant was detected by spectrophotometry. RESULTS:The survival rate of HUVECs and the activity of SOD were significantly decreased (P<0.01), while the activity of LDH and the level of MDA were significantly increased (P<0.01) after H/R. These changes were reversed by pretreatment with ZAL or E₂, and there was no significant difference between their effects in the same dose of ZAL and E₂. CONCLUSION:These results suggest that phytoestrogen ZAL protects HUVECs from H/R injury by inhibiting the oxidative stress, which was similar to E₂.     

Anti-aging Klotho protein reduce the hypoxia/reoxygenation injury of neonatal rat myocardial cells

AIM: To study the effects of anti-aging Klotho protein on neonatal rat myocardial cells with hypo-xia/reoxygenation (H/R) injury. METHODS: The cardiomyocytes of neonatal SD rats were cultured to establish hypoxia/reoxygenation model. The myocardial cells were divided into normal control group, H/R model group, different concentrations of Klotho protein (0.1 μmol/L, 1 μmol/L and 10 μmol/L) pretreatment groups. The myocardial cells pulse frequency was observed before and after H/R. The cell viability was measured by MTT assay. The leakages of LDH, CK and AST, the content of MDA and the activity of SOD were detected. The apoptotic rate of the myocardial cells was analyzed by flow cytometry. The mRNA expression of endoplasmic reticulum stress markers and apoptosis-related molecules GRP78, CRT, CHOP and caspase-12 was measured by real-time PCR. The protein levels of CHOP, caspase-12 and phosphorylated Akt in the myocardial cells were determined by Western blot. RESULTS: Compared with normal control group, the pulse frequency, cell viability rate and SOD activity of myocardial cells were significantly decreased, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were increased in H/R model group. The mRNA expressions of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and caspase-12 were increased, whereas p-Akt level was decreased obviously. Compared with H/R model group, the pulse frequency, cell viability rate and SOD activity were increased significantly, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were decreased in Klotho pretreated group. The mRNA expression of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and caspase-12 were decreased, while p-Akt level increased significantly. CONCLUSION: Anti-aging Klotho protein improves the myocardial cell survival and inhibits the apoptosis by increasing the resistance of the cells to oxidative stress and excessive endoplasmic reticulum stress response, which is related with the activation of Akt phosphorylation in H/R-injured mycardial cells.     

Effect of L-carnitine on apoptosis and oxidant damage of cardiomyocytes induced by hypoxia/reoxygenation in vitro

AIM: To examine the effects of L-carnitine on apoptosis and oxidant injury in cultured neonatal rat cardiomyocytes induced by hypoxia/reoxygenation and its possible mechanism. METHODS: The cultured cardiomyocytes were divided into three groups， control， A/R group （anoxia for 120 min, reoxygenation for 240 min） and L-carnitine treatment group, in which cells were exposed to 20 mg/L, 50 mg/L, 100 mg/L, 200 mg/L L-carnitine respectively at 2 h before anoxia. The superoxide dismutase (SOD), succinate dehydrogenase (SDH) activities and malondialdehyde (MDA) content were examined, and the apoptosis was determined by flow of cytometry (FCM). In addition, the ultrastructure was observed by transmission electron microscopy. RESULTS: In A/R group, SOD and SDH activities were lower, the apoptosis rate and MDA content were higher than those in control group (P<0.05). In L-carnitine treatment group, SOD and SDH activities were higher, the apoptosis rate and MDA content were lower than those in A/R group, a L-carnitine concentration-dependent effect was found. Moreover, impairment of myocardial ultrastructure was more severe in A/R group than L-carnitine treatment group. CONCLUSION: L-carnitine can protect cardiomyocytes against hypoxia/reoxygenation-induced injury in a dose-dependent manner.     

Effect of Auricularia auricular polysaccharide on ischemia myocardial injury

AIM: To investigate the protective effect of auricularia auricular polysaccharide (AAP) on the myocardial injury induced by ischemia and its underlying mechanism. METHODS: AAP was orally administrated to rats at the does of 50, 100 or 200 mg·kg-1·d-1 for 20 d. Myocardial injury was induced in anesthetized Sprague-Dawley rats by left anterior descending coronary artery ligation. Myocardial infarct size, the level of lactate dehydrogenase (LDH), the activity of superoxide dismutase (SOD), the production of lipid peroxidation malondialdehyde (MDA) and protein level of myocardial collagen of the heart were measured. RESULTS: The average myocardial infarct size in AAP groups was significantly smaller than that in ischemia group. The level of serum LDH induced by regional myocardial ischemia was significantly decreased in AAP group compared to ischemia group (P<0.01). AAP inhibited the production of MDA and increased the activity of SOD. Furthermore, AAP reduced the protein level of myocardial collagen after ischemia (P<0.01). CONCLUSION: AAP prevents myocardium from ischemia injury as an antioxidant.     

Effects of simvastatin on expression of Bcl-2 and Bax in myocardium of rats with renal ischemia-reperfusion injury

AIM: To observe the effect of simvastatin on myocardial tissue after renal ischemia-reperfusion injury and its mechanism. METHODS: A rat model of renal ischemia-reperfusion injury was prepared by clamping the bilateral renal arteries for 45 min. The rats (n=36) were randomly divided into sham operation group, renal ischemia-reperfusion (I/R) group and simvastatin group with 12 rats in each group. The content of serum creatinine (SCr), blood urea nitrogen (BUN) and myocardial tissue malondialdehyde (MDA), the myocardial activity of lactate dehydrogenase (LDH), creatine kinase (CK) and superoxide dismutase (SOD), and the myocardial protein expression of Bcl-2 and Bax were detected. RESULTS: Compared with sham operation group, the content of SCr, BUN and myocardial MDA, and the myocardial activity of LDH and CK in I/R group were significantly increased (P<0.05), and the activity of SOD was significantly decreased (P<0.05). Compared with I/R group, the content of SCr, BUN and myocardial MDA, and the myocardial activity of LDH and CK in simvastatin group were significantly decreased (P<0.05), while SOD activity was enhanced (P<0.05). The protein expression of Bcl-2 and Bax in sham operation group was less than that in I/R group (P<0.05), and the protein level of Bax in simvastatin group was significantly lower than that in I/R group (P<0.05), while the protein level of Bcl-2 was increased (P<0.05). CONCLUSION: Simvastatin has a protective effect on the myocardium of the rats with renal ischemia-reperfusion injury, and the protective mechanism may be related to the elimination of free radicals by simvastatin, increase in the protein expression of Bcl-2 and decrease in the protein expression of Bax.     

Effects of PTEN on apoptosis,oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation

AIM: To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on the apoptosis, oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation (A/R). METHODS: The H9c2 cells were used to establish a model of A/R. The H9c2 cells were transfected with PTEN small interfering RNA (siRNA) and negative control. After A/R, the expression of PTEN at mRNA and protein levels was determined by RT-PCR and Western blot, respectively. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Xanthine oxidase method was used to determine superoxide dismutase (SOD) activity. The content of malondialdehyde (MDA) was detected by thiobarbituric acid method. The lactate dehydrogenase (LDH) activity in the supernatant was evaluated by 4-dinitrophenylhydrazine method. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in culture supernatant were examined by ELISA. The protein levels of cleaved caspase-3, Bax and FasL in the cells were determined by Western blot. RESULTS: After A/R, the expression of PTEN at mRNA and protein levels was significantly increased in the H9c2 cells (P<0.05). The mRNA and protein levels of PTEN were decreased significantly after transfection with PTEN siRNA (P<0.05). The viability of H9c2 cells was decreased after A/R, while the apoptotic rate was increased. The protein levels of cleaved caspase-3, Bax and FasL were increased in the cells. The MDA level was elevated, the activity of SOD was decreased, and the levels of LDH, TNF-α, IL-1β and IL-6 in the culture supernatant were increased (P<0.05). Down-regulation of PTEN partly antagonized the effects of A/R on the viability, apoptotic rate, MDA content, SOD activity, and the levels of LDH, TNF-α, IL-1β and IL-6 in culture supernatant. CONCLUSION: Down-regulation of PTEN attenuates oxidative damage induced by A/R, reduces apoptosis and secretion levels of TNF-α, IL-1β and IL-6 in the H9c2 cells.     

Role of glycine receptor in protection of glycine against anoxia/reoxygenation injury in cardiomyocytes

AIM:To investigate whether glycine receptor is involved in the protection of glycine against anoxia/reoxygenation injury in cardiomyocytes by detecting oxygen free radical metabolism, apoptosis and intracellular calcium overload. METHODS:The neonatal rat cardiomyocytes were cultured and exposed to anoxia and reoxygenation (A/R) in the presence of glycine receptor antagonist, glycine or in free chloride buffer. The superoxide dismutase (SOD) activity, the contents of malondialdehyde (MDA) and nitric oxide (NO), the intracellular free calcium concentration and the apoptotic rate in the cardiomyocytes were determined. RESULTS:SOD activity and NO content in cardiomyocytes were lower, but MDA content, intracellular free calcium concentration and apoptotic rate in cardiomyocytes were higher in A/R group than those in control. Pretreatment with glycine inhibited the above changes caused by A/R, which was reversed by strychnine treatment and in the free chloride medium. CONCLUSIONS:Glycine inhibits free radical production, attenuates calcium overload, decreases apoptotic rate and increases SOD activity and NO release in cardiomyocytes exposed to A/R. These findings suggest that glycine exerts a protective effect against A/R injury via glycine receptor and glycine protects the neonatal rat cardiomycytes from A/R-induced injury in a chloride-dependent manner.     

Nitric oxide-mediated the cardioprotection of tumor necrosis factor-alpha on cultured neonatal rat cardiomyocytes during hypoxia/reoxygenation

AIM: To investigate the role of nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and protein kinase C (PKC) signaling in tumor necrosis factor-α (TNF-α)-induced cardioprotection against hypoxia/reoxygenation (H/R) injury. METHODS: Neonatal rat ventricular myocytes were pretreated with TNF-α or sodium nitroprusside (SNP) or L-arginine (L-Arg), respectively, for 12 h and then subjected to continuous hypoxia for 12 h, followed by reoxygenation for 6 h. The manganese superoxide dismutase (Mn-SOD) activity of the cells was measured after H/R. Myocyte injury was determined by the release of lactic dehydrogenase (LDH). RESULTS: TNF-α (10⁵ U/L) significantly increased the Mn-SOD activity and decreased release of LDH from ventricular myocytes. The cardioprotection against H/R injury was induced by the pretreatment with SNP (5 μmol/L) or L-Arg (5 mmol/L), which was blocked by ODQ (10 μmol/L), the specific sGC inhibitor, and Chel (5 μmol/L), the specific PKC inhibitor. Pretreatment with L-NAME (100 μmol/L), ODQ, Chel, antoxidant 2-MPG (400 μmol/L) or tyrosine kinase inhibitor genistein (50 μmol/L) attenuated the increased Mn-SOD activity and reduced LDH level induced by TNF-α. CONCLUSION: The results suggest that NO may play a role in TNF-α-induced cardioprotection, which is mediated by sGC and PKC.     

Sevoflurane preconditioning inhibits brain injury in hypoxic mice

AIM: To observe the effects of sevoflurane preconditioning on brain injury in hypoxic mice and its possible mechanism. METHODS: Male C57BL/6J mice were randomly divided into control (C) group, hypoxia (H) group, 2% sevoflurane preconditioning for 30 min + hypoxia (S1+H) group, 2% sevoflurane preconditioning for 60 min+hypoxia (S2+H) group and 4% sevoflurane preconditioning for 30 min + hypoxia (S3+H) group. The hypoxia model was established by continuous inhalation of (6.5±0.1)% O₂ for 24 h. The sevoflurane preconditioning treatments, S1, S2 and S3, were conducted by inhalation of 2% sevoflurane for 30 min, 2% sevoflurane for 60 min and 4% sevoflurane for 30 min, respectively, with the carrier of (21.0±0.5)% O₂, followed by washout for 15 min and then hypoxia treatment. The histological changes of the hippocampal CA1 area were observed under light microscope and transmission electron microscope (TEM), and serum lactate dehydrogenase (LDH) activity was measured by colorimetric method. Furthermore, the protein levels of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in brain tissue homogenate were examined by ELISA, and the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured by microplate reader. RESULTS: After hypoxia for 24 h, cell edema or pyknosis in the hippocampal CA1 area was observed in H group. Sevoflurane preconditioning reduced hypoxic injury, and the cell ultrastructure under TEM was significantly improved in S2+H group. Compared with C group, the serum LDH activity and the levels of EPO, VEGF and MDA in brain tissues were significantly increased in H group, while the activity of SOD and GPx decreased. After sevoflurane pretreatment, the serum LDH activity and the levels of EPO and VEGF in brain tissues were lower than those in H group, and the most significant difference was observed in S2+H group. Moreover, the MDA content and SOD activity decreased, and the GPx activity increased in the sevoflurane preconditioning groups. CONCLUSION: Sevoflurane preconditioning attenuates brain injury in hypoxic mice by regulating antihypoxic protein synthesis and reducing oxidative stress.     

Effects of spermine on myocardial ischemia/ reperfusion injury in rats

AIM: To observe the effect of exogenous spermine (low concentration) on myocardial ischemia/reperfusion injury in rats.METHODS: 40 Wistar rats were randomly divided into 4 groups: sham- operation group (Sham), ischemic reperfusion group (I/R), spermine group (Sp) and natural saline group (NS). The model of ischemic/reperfusion injury was established by ligating rat coronary artery. In Sp group, spermine (0.5 mmol/L, 2 mL/kg) was injected slowly into rat vein. During the process, we recorded the electrocardiogram and the LV functional parameters, assayed the levels of SOD, LDH, NO and MDA in serum, and examined the ultrastructure of the myocardium. RESULTS: In I/R group, the incidence of arrhythmia was 90%, myocardial ultrastructure was injured seriously, values of LVSP and ±dp/dtmax decreased, levels of LDH, NO and MDA increased while SOD activity decreased (P<0.05 or P<0.01, compared with Sham group). Compared with I/R and NS group, all those indexes in Sp group changed significantly (P<0.05 or P<0.01). CONCLUSION: Exogenous spermine alleviates myocardial ischemia/ reperfusion injury in rats. The mechanism may be related to its antioxidant effect and relieving the injury caused by oxygen free radical.     

Role of ROS/PKC/p38 MAPK pathway in protective effects of polysaccharide from Fructus cornion rat cardiomyocytes against hypoxia-reoxygenation injury

AIM: To investigate the effect of polysaccharide from Fructus corni(PFC) on cardiomyocytes against hypoxia/reoxygenation (H/R) injury and its possible relationship with ROS/PKC/p38 MAPK pathway.METHODS: Primary cardiomyocytes were isolated from neonatal SD rats and randomly divided into normal group, H/R group, PFC (20 mg/L, 100 mg/L and 200 mg/L) preconditioning+H/R groups, chelerythrine+PFC (100 mg/L)+H/R group and SB203580+PFC (100 mg/L)+H/R group. The cell viability was measured by inverted microscopic observation. Apoptosis in the cardiomyocytes was detected by Hoechst 33258 staining and fluorescence microscopy. The levels of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the cell culture supernatants, and the reactive oxygen species (ROS) in the cells were also measured by microplate reader. The protein levels of PKC, p-p38 MAPK and HSP70 in the cells were detected by Western blotting.RESULTS: Compared with normal group, the cell viability and beating frequency were decreased in H/R group. LDH and ROS contents, apoptotic rate and p-p38 MAPK level increased significantly (P<0.01). Compared with H/R group, PFC preconditioning increased beating frequency, SOD activity and the protein level of PKC and HSP70, and decreased ROS production, the protein level of p-p38 MAPK and cell apoptotic rate. However, the effect of PFC was inhibited by chelerythrine or SB203580.CONCLUSION: PFC may protect cardiomyocytes from hypoxia/reoxygenation injury. Its mechanism is possibly involved in the inhibition of ROS via increasing the activity of SOD and the activation of PKC, and suppression of excessive activation of p38 MAPK.     

CaMKII aggravates hypoxia/reoxygenation injury in H9c2 cells by activating apoptosis

AIM: To investigate the effect of Ca²⁺/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) on hypoxia/reoxygenation (H/R) injury in H9c2 cells. METHODS: H9c2 cells were randomized into 4 groups:control group, KN-93 (an inhibitor of CaMKⅡ; 1 μmol/L) treatment group, H/R group and H/R+KN-93 (1 μmol/L) treatment group. The cells in KN-93 group and KN-93+H/R group were pretreated with KN-93 for 2 h before the other treatment was performed. The viability of H9c2 cells in each group was measured by CCK-8 assay. Lactate dehydrogenase (LDH) activity in the culture medium was detected. The protein levels of phosphorylated CaMKⅡ (p-CaMKⅡ), phosphorylated phospholamban (p-PLN) and cleaved caspase-3 were determined by Western blot. The apoptosis was analyzed by TUNEL staining and the flow cytometry. RESULTS: No significant difference of all indexes tested between control group and KN-93 group was observed. H/R treatment significantly reduced the cell viability, and increased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). KN-93 (1 μmol/L) significantly increased the cell viability, and decreased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). CONCLUSION: CaMKⅡ aggravates hypoxia/reoxygenation injury in the H9c2 cells by activating apoptosis.     

Astragalus polysaccharides attenuate endothelial cell injury caused by homocysteine via AMPK pathway

AIM:To investigate the protective effect of Astragalus polysaccharides (APS) on human umbilical vein endothelial cells (HUVECs) injured by homocysteine (Hcy) and its mechanism. METHODS:HUVECs cultured in vitro were divided into 4 groups:control group, APS group[APS (200 mg/L) treatment for 24 h], Hcy group[Hcy (1 mmol/L) treatment for 24 h], and Hcy+APS group[Hcy (1 mmol/L) and APS (200 mg/L) co-treatment for 24 h]. The cell viability were measured by MTT assay. The activity of lactate dehydrogenase (LDH) and superoxidase dismutase (SOD), and the content of malondialdehyde (MDA) in HUVECs were detected by the commercial kits. The mRNA expression of SOD1, catalase (CAT) and NADPH oxidase 2 (NOX2) was detected by RT-qPCR. The protein levels of AMP-activated protein kinase α (AMPKα) and phosphorylated AMPKα (p-AMPKα) were determined by Western blot. RESULTS:Compared with control group, the cell viability, the activity of SOD, and the mRNA expression of SOD1 and CAT in the HUVECs were decreased, but the activity of LDH, the content of MDA, and the mRNA expression of NOX2 were increased significantly in Hcy group(P<0.05). APS inhibited the decrease in cell viability, and the increases in LDH acti-vity and MDA content induced by Hcy. APS increased SOD activity and the mRNA expression of SOD1 and CAT, but reduced the mRNA expression of NOX2. Compound C, an AMPK inhibitor, reduced the protective effect of APS on HUVECs injured by Hcy. CONCLUSION:APS protects HUVECs from Hcy-induced injury via AMPK signaling pathway to regulate intracellular oxidative stress.     

Expression of calcium sensing receptor during ischemia/reperfusion myocardial damage and relationship between CaSR and injury of myocardium

AIM: To investigate the expression of calcium sensing receptor(CaSR) during myocardial injury induced by ischemia/reperfusion and disclose the relationship between CaSR and myocardial ischemia/reperfusion. METHODS: The experimental model was established by the 30 min ligating and 1 h, 2 h, 4 h, 6 h reperfusing the left descending coronary artery (LAD) in rats. Wistar rats were randomly divided into 5 groups: sham group, ischemia/reperfusion 1 h, 2 h, 4 h, 6 h groups (I/R 1 h, 2 h, 4 h, 6 h group). CaSR mRNA expression was detected by RT-PCR. Left ventricular function was recorded. The levels of plasma lactate dehydrogenase (LDH), alondialdehyde (MDA) and superoxide dismutase (SOD) were measured. The change of ultrastructure in the ischemia/reperfusion myocardium of rats was observed by electron microscopy. RESULTS: LVSP,±dp/dtmax and SOD activity decreased gradually with the reperfusion time prolonged. LDH and MDA peaked at 2 h. The ultramicro-structural injury at the 1 h and 2 h was more serious than that at 4 h and 6 h. The expression of CaSR increased significantly after reperfusion of 1 h and 2 h, and decreased after 4 h and 6 h. CONCLUSION: The increased expression of CaSR mRNA and serious injure of myocardium were observed. CaSR may be associated with the myocardial ischemia/reperfusion injury.     

Preparation of glycine liposomes and it's effect on cardiomyocyte injury induced by hypoxia/reoxygenation

AIM: To prepare glycine liposome microparticle and observe the effect of glycine liposomes on cardiomyocyte injury induced by hypoxia/reoxygenation. METHODS: （1） Reverse-phase evaporation method was used to produce glycine liposomes, the effects of different organic solvents: aether, chloroform and two mixtures of aether/chloroform on entrapment efficiency were evaluated, transmission electron microscope was used to detect the particle diameter of glycine liposomes. （2） A cardiomyocyte injury model was established by using hypoxia/reoxygenation, the activities of lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase isoenzyme (CK-MB) of each group were detected. RESULTS: The entrapment efficiency of glycine liposomes prepared with the mixtures of aether/chloroform is highest compared with other organic solvents （64.8％, P<0.01）; all of glycine, glycine liposomes and blank liposomes inhibited the release of LDH, CK and CK-MB induced by hypoxia/reoxygenation in cultured cardiomyocytes, and the inhibition by glycine liposomes was the most obvious （P<0.05，P<0.01）. CONCLUSIONS: The mixtures of aether/chloroform can be used to prepare glycine liposomes, which can get higher entrapment efficiency; glycine liposomes protects cultured cardiomyocytes against hypoxia/reoxygenation-induced injury, and the protective effect of glycine liposomes is better than that of glycine.     

Protective effect of anti-aging Klotho protein on human umbilical vein endothelial cells treated with high glucose

AIM: To study the protective effect of anti-aging Klotho protein on human umbilical vein endothelial cells (HUVECs) treated with high glucose (HG).METHODS: HUVECs were cultured in vitro, and divided into PBS control group, 5.5 mmol/L glucose group, 33.3 mmol/L glucose group, 0.1 μmol/L Klotho+33.3 mmol/L glucose group, 1 μmol/L Klotho+33.3 mmol/L glucose group, and 10 μmol/L Klotho+33.3 mmol/L glucose group. The viability of the HUVECs was measured by MTT assay. The content of malondialdehyde (MDA), and the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione (GSH) in cell culture supernatants were observed. The production of reactive oxygen species (ROS) in HUVECs was analyzed by flow cytometry. The levels of nitric oxide (NO), endothelin (ET-1), intercellular adhesion molecule-1 (ICAM-1) in HUVEC culture medium were detected by ELISA. The protein expression of nuclear factor-kappa B (NF-κB) in the HUVECs was determined by Western blot. RESULTS: Compared with PBS control group, 33.3 mmol/L glucose significantly decreased the HUVEC viability, increased ROS, LDH and MDA levels, reduced the activities of SOD and GSH, decreased the NO secretion, and induced the ET-1 and ICAM-1 secretion and the protein expression of NF-κB in HUVECs. When HUVECs were treated with Klotho protein at different concentrations combined with 33.3 mmol/L glucose, the cell viability was increased significantly, the ROS, LDH and MDA levels were decreased significantly, the antioxidant SOD and GSH activities were significantly increased, the secretion of NO was increased, but ET-1 and ICAM-1 releases and protein expression of NF-κB were significantly reduced.CONCLUSION: Anti-aging Klotho protein promotes the viability of HUVECs treated with HG, reduces the oxidative damage and ROS production, and restores the normal secretory function of HUVECs, thus playing a protective role in vascular endothelial cells through reducing the protein expression of NF-κB.     

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The cytotoxicity of nitric oxide induced by inflammatory cytokine in combination with LPS in endothelial cells

AIM: To explore the mechanism underlying inducible nitric oxide (NO) caused injury of endothelial cells during inflammation. METHODS:The activity of iso-enzymes of NO synthase (NOS), NO level and iNOS expression were examined using NADPH method, Griess reaction and RT-PCR, respectively. Furthermore, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content were also measured. RESULTS:Co-administration of cytokines (TNF-α 5×10⁵ U/L, IL-1β 2×10⁵ U/L, INF-γ 2×10⁵ U/L) and LPS (10 mg/L) caused an obvious increase in NOS activity, NO levels (about two-fold) and a significant injury of the cells. At the same time, a significant increase in iNOS mRNA was also detected. Wheareas, treatment of the cells separately with cytokines or LPS for 24 h had no significant effect on NOS activity and NO level in cell lysates, however, it caused a significant increase in LDH release and MDA content. Also, the effect of cytokines and LPS on cell viability was concentration-and time-dependent. L-NMMA, a inhibitor of NOS, can suppress inducible NO production and protect cells against NO induced injury. CONCLUSION:Co-administration of cytokines (TNF-α, IL-1β and INF-γ) and LPS significant activated iNOS and NO production which, in turn, induced oxidative reaction in endothelial cells.