Erythropoietin inhibits eryptosis induced by reactive oxygen species

AIM: To observe the influence of erythropoietin (EPO) on eryptosis and production of reactive oxygen species (ROS) in erythrocytes under stimulation of hydrogen peroxide (H₂O₂),and to explore its related mechanism. METHODS: The erythrocyte suspension (1%) was cultured in vitro and divided into 3 groups:control group (C group, the culture medium was PBS), H₂O₂ group (H group, the culture medium was PBS containing H₂O₂ at final concentration of 100 μmol/L) and EPO group (E group, the culture medium was PBS containing H₂O₂ at final concentration of 100 μmol/L and EPO at final concentration of 2×10⁴ U/L). The erythrocytes were collected at 24 h and 60 h. The eryptosis was detected by flow cytometry with Annexin V staining. The production of ROS and intracellular calcium ion concentration ([Ca²⁺]_i) were also analyzed by flow cytometry. RESULTS: The eryptosis in C group was increased as the incubating time extended. The eryptosis in H group was higher than that in C group (P<0.01), while that in E group was lower than that in H group (P<0.01). Meanwhile, ROS production and[Ca²⁺]_i were higher in H group than those in C group (P<0.01), but those were lower in E group than those in H group (P<0.05 or P<0.01). CONCLUSION: EPO inhibits eryptosis induced by H₂O₂ and its mechanism may be related to antioxidant effect and change of[Ca²⁺]_i.     

Effects of uremic serum on the proliferation and trans-differentiation of human renal tubular epithelial cells

AIM: To explore the effects of uremic serum on proliferation and trans-differentiation of human renal tubular epithelial cells. METHODS: Human renal tubular epithelial cell line (HK-2) was cultured in RPMI-1640 medium. The proliferation effects of uremic serum at different concentrations were evaluated by methylene blue assay (MTT method) and flow cytometry. The positive cells percentage of α-smooth muscle actin(α-SMA)in different concentration uremic serum medium was also measured by flow cytometry in vitro. RESULTS: Absorbance 490 (A₄₉₀) was increased in 5%-20% uremic serum groups compared with that in normal controls with the use of MTT. Cells in G₁ phase were decreased, but proliferation index (PI) was increased in 10%-20% uremic serum groups compared with that in normal controls with the use of flow cytometry. No significant difference of cell proliferation index was found among uremic serum groups. The positive percentage of α-SMA cells was increased significantly in uremic serum groups compared with that in normal controls, and increased in parallel with the increasing of uremic serum concentration. CONCLUSION:These data suggest that AGEs could induce a high expression of MIP-1αm RNA and protein in cultured HUVECs in a dose-dependent and time-dependent manner.     

L-Carnitine inhibits H2O2-mediated NFATc3 nuclear translocation

AIM:To explore the effect of L-carnitine on nuclear factor of activated T-cells,cytoplasmic 3 (NFATc3) in cardiomyocytes under H2O2 stimulation. METHODS:Primary cultured neonatal rat myocardial cells were stimulated by H2O2 at concentration of 200 μmol/L for 12 h to induce oxidative stress injury. In treatment group, L-carnitine and cyclosporin A (CsA), a specific inhibitor of calcineurin (CaN), were administered 30 min prior to H2O2 stimulation. After treatment, total, cytoplasmic and nuclear NFATc3 protein levels were determined by Western blotting. The method of immunofluoresence was used to evaluate the distribution of NFATc3. RESULTS:H2O2 treatment produced no effect on the expression of total NFATc3, but caused its translocation from the cytosolic to nuclear compartment, which was greatly blunted by L-carnitine pretreatment. CONCLUSION: L-carnitine antagonized oxidative stress injury via alleviating NFATc3 nuclear translocation.     

Astragalus membranaceus inhibits bleomycin-induced pulmonary fibrosis in mice by antioxidation

AIM:To investigate the effect of Astragalus membranaceus on the balance of oxidation and antioxidation in bleomycin-induced pulmonary fibrosis in mice and the possible anti-fibrosis mechanism of Astragalus membranaceus.METHODS:Female KM mice (n=36) were randomly divided into 3 groups.The mice in control group were administered with saline aerosol intratracheally.The mice in fibrosis group were administered with bleomycin at dose of 3 mg/kg aerosol intratracheally.The mice in Astragalus membranaceus group were administered with bleomycin at dose of 3 mg/kg aerosol intratracheally and then intraperitoneal injected with Astragalus membranaceus parenteral solution at daily dose of 1.7 g/kg.All mice were sacrificed 14 d after the treatment,and the lungs and serum were collected for detection.Hematoxylin-eosin staining was performed in the lung tissue.The mRNA expression of superoxide dismutase 1/2/3(SOD1/2/3),catalase (CAT),NADPH oxidase 2/4(NOX2/4) and α-smooth muscle actin (α-SMA) was detected by RT-PCR,and the protein expression of α-SMA and NOX2/4 was determined by Western blot.The concentration of malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in the serum were measured by a colorimetric method.RESULTS:The pathological injury was obviously observed in bleomycin group compared with control group,but was attenuated in Astragalus membranaceus group.The α-SMA mRNA and protein expression,MDA/T-AOC,NOX2,NOX4 and SOD3 mRNA expression,and NOX2 protein expression in bleomycin group were significantly higher than those in control group,while those in Astragalus membranaceus group were significantly lower than those in bleomycin group.The protein expression of NOX4 in bleomycin group was significantly lower than that in control group,while that in Astragalus membranaceus group was higher than that in bleomycin group.The mRNA expression of SOD1 and CAT in Astragalus membranaceus group and bleomycin group were decreased compared with control group.No significant difference of SOD2 mRNA expression among the 3 groups was observed.CONCLUSION:Astragalus membranaceus inhibits bleomycin-induced pulmonary fibrosis in mice by maintaining the balance of oxidation and antioxidation.     

Mechanism of hepatic insulin resistance induced by high-fat diet

AIM: To observed the relationship between oxidative stress and development of insulin resistance in hepatic tissues of Sprague dawley(SD) rats by analyzing reactive oxygen species(ROS) level and NADPH oxidase 3(NOX3) expression in livers. METHODS: Four-week-old male SD rats were fed with high-fat diet containing 20% fat and 20% sucrose for 12 weeks to induce insulin resistance. Plasma insulin level was detected by radioimmunoassay. The content of liver intracellular glycogen was measured using a glycogen assay kit. ROS generation in the liver tissues was assessed by dihydroethidium(DHE) fluorescence. The expression of NOX3 was determined by Western blotting.RESULTS: After 12 weeks of high-fat diet feeding, the content of blood glucose was increased but still maintained in normal level in the rats. However, the index of insulin sensitivity obviously decreased. Hepatic glycogen content in the rats fed with high-fat diet was significantly decreased, indicating that insulin resistance developed. Enhanced ROS production in hepatic tissues of the rats fed with high-fat diet was observed. Importantly, the expression of NOX3 in the liver was up-regulated in response to high-fat diet in vivo.CONCLUSION: High-fat diet feeding decreases insulin sensitivity, enhances ROS level and NOX3 expression, and reduces glycogen content in the livers.     

Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human umbilical vein endothelial cells

AIM:To explore the effect of atorvastatin on high glucose-induced oxidative stress and underlying mechanisms in human endothelial cells. METHODS:Human umbilical vein endothelial cells(HUVECs) were cultured in medium 199 containing normal concentration of glucose(5.5 mmol/L). For high glucose treatment, glucose solution was added to the final concentration of 25 mmol/L. Reactive oxygen species(ROS) were detected by flow cytometry and confocal microscopy. The activity of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase was measured by lucigenin assay. Phosphorylated protein kinase C(PKC) and the expression levels of NADPH oxidase subunits Nox4 and Nox2/gp91^phox were determined by quantitative real-time PCR and immunoblotting. RESULTS:High glucose increased ROS production, NADPH oxidase activity and the expression of Nox4 and Nox2/gp91^phox subunits. Treatment of endothelial cells with atorvastatin resulted in significant inhibition(in a concentration-dependent manner) of high glucose-induced ROS production, NADPH oxidase activation and the expression of Nox4 and Nox2/gp91^phox subunits. PKC inhibitor showed a similar effect to that of atorvastatin on high glucose-induced oxidative stress. Furthermore, atorvastatin rapidly inhibited high glucose-induced activation of protein kinase C, an upstream activator of NADPH oxidase. CONCLUSION:PKC is involved in high glucose-induced oxidative stress in HUVECs. Atorvastatin inhibits high glucose-induced oxidative stress by depressing PKC activity in human endothelial cells.     

Effect of Chaihushugansan on pancreatic fibrosis in mice with chronic pancreatitis induced by DBTC plus ethanol and its anti-oxidation mechanism

AIM:To explore the role of superoxide dismutase (SOD) and malondialdehyde (MDA) in chronic pancreatitis (CP) induced by dibutyltin dichloride (DBTC) combined with ethanol, and the mechanisms for prevention and treatment of pancreatic fibrosis by Chaihushugansan. METHODS:The KM mice were randomly divided into control group, CP group (DBTC combined with ethanol) and Chaihushugansan group (CP+Chaihushugansan). Except for control group, the mice in other groups were intravenously injected in tail with DBTC (8 mg/kg) and drank 10% ethanol. The mice in Chaihushugansan group were administered intragastrically with Chaihushugansan (6 g·kg-1·d-1) at the following experimenal period. Before modeling and 1 week, 2 weeks, 4 weeks and 8 weeks after modeling, the mice were anesthetized and sacrificed. The activity of amylase and the content of hyaluronic acid in the serum were measured. The morphology and the degree of fibrosis in the pancreas were observed by HE staining. The activity of SOD and the level of MDA in the pancreas homogenate were analyzed. The protein of pancreas was extracted to detect the expression of type I collagen by Western blotting. RESULTS:DBTC combined with ethanol induced CP with increased serum amylase and hyaluronic acid levels， while the serum amylase and hyaluronic acid levels in Chaihushugansan group were significantly lowered (P<0.05). In 1 week, 2 weeks, 4 weeks and 8 weeks, the pancreas were obviously injured and appeared different degrees of fibrosis. The content of MDA and the expression of type I collagen in the increased significantly, but the SOD was decreased. In Chaihushugansan group, the pathological damage and the degree of fibrosis of the pancreas were improved. The level of MDA and type I collagen expression in the pancreas were significantly reduced, but the SOD was increased. CONCLUSION: The oxidative stress may take part in the development of CP. Inhibition of oxidative stress in the pancreas is one of the mechanisms that Chaihushugansan attenuates the development of CP.     

Effect of taurine on kidney injury induced by paraquat poisoning in rats

AIM:To study the effects of taurine at different doses on renal oxidative stress and inflammation induced by paraquat in rats.METHODS:Male SD rats (n=48) were randomly divided into 4 groups:negative control group,paraquat group,paraquat+low-dose taurine group,and paraquat+high-dose taurine group.The serum levels of creatinine and urea nitrogen were detected by a biochemical analyzer.The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured by colorimetry.The plasma concentrations of IL-6 and intercellular adhesion molecule (ICAM)-1 were detected by ELISA.Renal reactive oxygen species (ROS) was checked by fluorescence probe dihydroethidium (DHE).The protein levels of renal p-P38 MAPK,p-ERK1/2 and p-JNK were determined by Western blot.The mRNA expression of TNF-α,IL-6 and TGF-β1 was detected by real-time PCR.RESULTS:Serum creatinine and urea nitrogen increased after paraquat poisoning,and decreased after feeding with taurine in poisoned rats,with better result in high-dose taurine group.Taurine reduced the oxidative stress and inflammation in the renal tissue,and also reduced the protein levels of p-JNK,p-ERK1/2 and p-P38 in the kidney of paraquat-poisoned rats.CONCLUSION:Taurine attenuates renal injury induced by paraquat poisoning in rats.The mechanism may be related to reducing renal MAPK activity,oxidative stress and inflammatory response.     

Sulforaphane inhibits diethylnitrosamine-induced hepatocellular carcinoma tumorigenesis in rats

AIM: To investigate the inhibitory effect of sulforaphane (SFN) on the hepatocellular carcinoma (HCC) induced by diethylnitrosamine (DEN) and its possible mechanism. METHODS: DEN was repeatedly injected into the SD rats to induce HCC model, and different doses (0.19 mg/kg, 0.38 mg/kg and 0.57 mg/kg) of SFN were given at the initial symptoms of fibrosis or cirrhosis. The morphological changes of liver specimens and the number of cancerous nodules were observed, and the degree of hepatocyte injury and hepatocellular carcinogenesis were evaluated by HE and Masson staining. The levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (TBIL), alanine aminotransferase (ALT), interleukin (IL)-1α, IL-6, IL-10, IL-1β, tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) in liver tissues were measured by ELISA. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and content of mdlondialdehyde (MDA) in liver tissues were detected by spectrophotometry method. RESULTS: Macroscopic observation showed that the number of cancerous nodules in SFN intervention groups was lower than that in DEN group, and the dosage of SFN was negatively correlated with the degree of liver canceration. HE staining and Masson staining showed that SFN inhibited the liver canceration and inflammatory cell infiltration induced by DEN, and the degree of alleviation was positively correlated with the dosage of SFN. The data of ELISA showed that SFN attenuated the hepatocyte injury induced by DEN, and the higher the concentration of SFN was used, the lower the levels of AST, ALT, TBIL and ALP in liver tissues were detected. The levels of inflammatory factors IL-1α, IL-6, IL-1β and TNF-α in liver tissues were decreased after administration of SFN, and the degree of reduction was positively correlated with the concentration of administration, while the levels of inflammatory factors IL-10 and TGF-β were positively correlated with the concentrations of SFN. The activity of SOD, CAT and GPx was decreased with the increase in SFN concentration. CONCLUSION: SFN has a certain inhibitory effect on the liver cancer development induced by DEN, which may be related to the anti-inflammatory, antioxidant and liver injury-reducing effects of SFN.     

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.     

Dependence of adrenoceptor regulation on oxidative stress in cardiac injury induced by high sympathetic activity in rats

AIM：To investigate the dependence of the adrenoceptor regulation on oxidative stress in the rats with cardiac injury induced by high sympathetic activity. METHODS：Healthy SD rats were randomly divided into 7 groups: control, model, propranolol (Pro), prazosin (Praz), Pro+Praz, vitamin E (VE) and Pro+Praz+VE. The rats were intraperitoneally injected with norepinephrine (NE) for continuous 16 d to reproduce cardiac injury, and treated with the respective drugs. During the experimental process, the body weight was recorded. At the end of the experiments, the following parameters were measured: the ventricular remodeling indexes (cardiac index and hydroxyproline of the left ventricle), histopathologic examination, oxidative/antioxidative indexes ［MDA, SOD, catalase (CAT), GSH-Px and total antioxidant capacity (T-AOC)］, and energy metabolism (Na+-K+ ATPase and Ca2+-Mg2+ATPase). RESULTS：The increase of body weight in model group was significantly slower than that in control group after 9 d of treatment (P<0.05). The cardiac index and left ventricular hypertrophy were significantly increased. Oxidation/antioxidation and energy metabolism were disturbed. In Pro, Praz, Pro+Praz and VE groups, the body weight, cardiac index, left ventricular fibrosis and oxidative/antioxidative dysfunction were ameliorated. Pro, Praz and Pro+Praz increased the activity of Na+-K+ ATPase and Ca2+-Mg2+ATPase. Treatment with Pro+Praz showed the best result in all of the indexes (P<0.05). CONCLUSION：The dependence of adrenoceptor regulation plays an important role in the formation of oxidative stress in the process of rat cardiac injury induced by high sympathetic activity.     

Up-regulation of CD36 induced by casein-induced inflammation promotes renal injury in mice

AIM: To investigate the role of CD36 in casein-induced mouse renal injury.METHODS: Eight-week-old male C57BL/6J mice and CD36 knockout (CD36KO) mice were randomly divided into C57BL/6J saline injection group, C57BL/6J casein injection group and CD36KO casein injection group (n=8 in each group). After 14 weeks of treatment with high-fat diet, the mouse serum, 24 h urine and kidney tissue samples were collected for analysis. The serum content of tumor necrosis factor-α (TNF-α) was measured by ELISA. The renal function markers in the serum and urine were determined by an automatic biochemical analyzer. The pathological changes of the kidney were observed by HE staining and Masson staining. The expression of CD36 and cytokines/chemokines (TNF-α, IL-6 and MCP-1) at mRNA and protein levels in the renal tissues were determined by real-time PCR and Western blot. The content of tissue hydrogen peroxide (H₂O₂) was measured by a commercial kit. The protein levels of Nrf2 and TGF-β1 in the renal tissues were measured by immunohistochemical staining.RESULTS: Compared with saline injection group, casein injection increased the level of TNF-α in the serum and in the kidney tissues of C57BL/6J mice (P<0.05), suggesting that casein injection successfully induced chronic inflammation in C57BL/6J mice. Casein injection also promoted the protein expression of CD36 and TGF-β1 in the renal tissues of the C57BL/6J mice, accompanied with glomerular sclerosis, proteinuria, increased serum creatinine content, increased H₂O₂ content, and decreased Nrf2 protein level and the ability of antioxidant in the kidneys (P<0.05). Furthermore, CD36 deficiency protected the mice from casein-induced renal injury, as evidenced by improved kidney pathological changes and decreased proteinuria. The content of H₂O₂ in the kidneys of casein-treated CD36 knockout mice was also lower than that in casein-treated C57BL/6J mice.CONCLUSION: Inflammatory responses promote the oxidative stress and renal injury in a CD36-dependent manner.     

PPARγ agonist inhibits high glucose-induced production of reactive oxygen species by UCP2 up-regulation

AIM: To explore the effects of PPARγ on the elevated level of reactive oxygen species (ROS) induced by high glucose and its mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured with DMEM containing high glucose (33 mmol/L D-glucose), and DMEM containing lower glucose (5.5 mmol/L D-glucose) was used as control. Superoxide anion and nitric oxide fluorescence probes were used to observe the effects of PPARγ agonist on ROS and NO productions in the HUVECs. The uncoupling protein 2 (UCP2) protein level in the HUVECs was detected by Western blotting. RESULTS: PPARγ agonist pioglitazone inhibited the ROS generation and prevented the decrease in NO level under high glucose condition, and these effects were reversed by pretreatment with PPARγ antagonist GW9662. The results of Western blotting indicated that PPARγ agonist pioglitazone up-regulated the UCP2 expression under high glucose condition, and this effect was also blocked by GW9662. Inhibition of UCP2 by genipin attenuated the effect of pioglotazone on the ROS production. CONCLUSION: Activation of PPARγ inhibits ROS generation under high glucose condition, and this effect may mediate by up-regulation of UCP2.     

Yangxue qingnao-containing serum inhibits rat vascular smooth muscle cell proliferation induced by lysophosphatidic acid

AIM: To observe the effects of Yangxue qingnao-containing serum on rat vascular smooth muscle cell (VSMC) proliferation induced by lysophosphatidic acid (LPA). METHODS: The [³H]-TdR incorporation and mitogen-activated protein kinasc (MAPK) activity were measured in cultured VSMC. End product of lipid peroxidation-MDA levels were also detected. RESULTS: 1×10^－9,1×10^－8 and 1×10^－7 mol/L LPA enhanced the cultured VSMC [³H]-TdR incorporation, increased MAPK activity and MDA content in a concentration-dependent manner. 5％, 10％ and 15％ Yangxue qingnao-containing serum concentration-dependently inhibited the increase in VSMC [³H]-TdR incorporation, MAPK activity and MDA content induced by LPA. CONCLUSIONS: LPA has a stimulating effect on VSMC proliferation. The LPA-induced intracellular signal transduction may be related to MAPK activity. Yangxue-qingnao can efficiently inhibit LPA -induced VSMC proliferation,MAPK activity and lipid peroxidation.     

Establishment of pancreatic β cell iron overload model and β cell injury induced by iron overload

AIM: To establish rat insulinoma INS-1 cell iron overload model, and to study the effect of iron overload on the viability, insulin secretion, mitochondrial defect and oxidative stress change in the INS-1 cells. METHODS:INS-1 cells were cultured with ferric ammonium citrate (FAC) at different concentrations (0, 5, 10, 20, 40, 80, 160 and 320 μmol/L). Labile iron pool (LIP) were calculated by detecting calcein-AM fluorescence in 24 h, 48 h and 72 h. The cell viability was measured by CCK8 assay. Iron overload model was established by screening for the best combination to ensure both high LIP level and cell viability. Reactive oxygen species (ROS) level was further analyzed by flow cytometry after fluorescent probe staining. The function of insulin secretion was measured by ELISA. The mitochondrial membrane potential was detected by JC-1 kit, and the mitochondrial changes were observed by transmission electron microscopy. RESULTS: Intracellular LIP levels were significantly increased in FAC groups in a concentration-dependent manner (P<0.05). The viability of INS-1 cells was suppressed with increasing FAC concentration or culture time (P<0.05). The highest LIP level and cell viability over 50% were observed with the condition of exposure to FAC for 48 h, indicating that INS-1 cell iron overload model was established. With the increase in the FAC concentrations, the insulin secretion was also increased and then decreased, and that in 160 and 320 mol/L groups showed statistical difference compared with control group (P<0.05). The ROS level was significantly increased by FAC exposure as compared with control group (P<0.05). Mitochondrial membrane potential was decreased with the increase in the iron concentration (P<0.05). After iron overload, the mitochondria of INS-1 cells were swollen, the internal cristae were expanded, and the normal structure was lost. With the increase in the FAC concentration, the mitochondrial structure was destroyed more obviously. CONCLUSION: Co-culture of INS-1 cells with FAC for 48 h successfully establish the iron overload model. Iron overload significantly damages mitochondrial structure and increases intracellular ROS level. The viability of pancreatic β cells is sensitive to iron, even lower doses of iron can damage β cells. The insulin secretion is reduced when the number of β cells is decreased to a certain extent.     

Genipin inhibits oxidative stress and apoptotic damage in high glucose-induced H9c2 cardiomyocytes

AIM:To explore the effects of genipin (GEN) on high glucose (HG)-induced oxidative stress injury and apoptosis in H9c2 cardiomyocytes.METHODS:H9c2 cells were cultured in vitro and HG-induced injury model was established. H9c2 cells were divided into 4 groups:normal control (NC) group (glucose at 5.6 mmol/L), HG group (glucose at 50 mmol/L), NG+GEN group and HG+GEN group. The concentration of genipin was used at 10 μmol/L. The viability of the H9c2 cells was measured by CCK-8 assay. The intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined by enzyme labeling and WST-1 methods, respectively. The activity of lactate dehydrogenase (LDH) in the cell culture supernatant was detected by microplate method. Fluorescent probe DCF was used to detect intracellular levels of reactive oxygen species (ROS). Nucleosome fragments was measured to evaluate cell apoptosis by ELISA. The intracellular mitochondrial membrane potential was detected by JC-1 method. The protein levels of Mn-SOD, cytochrome C (Cyt C), Bax and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with HG group, the cell viability in HG+GEN group was increased significantly (P<0.05), the levels of MDA and LDH were decreased (P<0.05), SOD activity was increased (P<0.05), the levels of ROS and nucleosome fragments in HG+GEN group were decreased (P<0.05), and the mitochondrial membranes potential was notably increased (P<0.05). Compared with NG group, the activation of Mn-SOD was decreased, but the protein levels of Cyt C, Bax and cleaved caspase-3 were increased in HG group (P<0.05). Compared with HG group, the activation of Mn-SOD was increased, and the protein levels of Cyt C, Bax and cleaved caspase-3 were decreased in HG+GEN group (P<0.05).CONCLUSION:Genipin protects HG-induced H9c2 cardiomyocytes against oxidative stress injury and apoptosis.     

Role of CRIF1 in cardiomyocyte apoptosis induced by palmitic acid

AIM: To investigate the effects of CR6-interacting factor 1 (CRIF1) on the apoptosis of mouse cardiomyocytes (MCMs) induced by palmitic acid. METHODS: The MCMs cultured with medium containing palmitic acid at 0 and 300 μmol/L for 24 h were divided into control group and palmitic acid group, respectively. In order to explore the effects of CRIF1 on MCMs injuries induced by high fat, MCM exposed to palmitic acid at 300 μmol/L for 24 h were divided into vehicle group, scrambled (Scra) siRNA group and CRIF1 siRNA group. The cells in vehicle group were only treated with transfection reagent, the cells in Scra siRNA group were given a treatment with transfection reagent and scrambled RNA, and the cells in CRIF1 siRNA group were given a treatment with transfection reagent and CRIF1 specific siRNA. In order to further confirm the specific mechanism of CRIF1 in high fat-induced MCM injuries, MCMs in CRIF1 siRNA group were divided into DMSO group and N-acetyl cysteine (NAC) group, and were given the same intervention of palmitic acid. RT-qPCR, Western blot and immunofluorescence staining were used to detect the mRNA and protein expression of CRIF1. The apoptotic rate was analyzed by flow cytometry, and the level of intracellular reactive oxygen species (ROS) was tested by DHE staining and ELISA. RESULTS: The expression of CRIF1 was significantly increased after exposure to palmitic acid (P<0.05). Compared with control group, the apoptotic rate was increased significantly in vehicle group and Scra siRNA group (P<0.05). The apoptotic rate was significantly increased in CRIF1 siRNA group (P<0.05). Compared with control group, the intracellular ROS content was significantly increased in vehicle group and Scra siRNA group (P<0.05). Compared with vehicle group and Scra siRNA group, the intracellular ROS content was significantly increased in CRIF1 siRNA group (P<0.05). Compared with DMSO group, the intracellular ROS content and the apoptotic rate were remarkably decreased in NAC group (P<0.05). CONCLUSION: With the inhibition of oxidative stress, CRIF1 may reduce the apoptosis of MCMs induced by high fat.     

Potassium antagonizes damage of coronary artery induced by high salt intake

AIM：To investigate the effect of potassium treatment on coronary arterial impairment induced by high salt intake. METHODS：Sprague-Dawley rats (4-week-old, n=10 in each group) received distilled water (NS), water containing 1.5% NaCl (HS), or 1.5% NaCl and 0.5% KCl (HS+HP) for 16 weeks. Systolic blood pressure (SBP) was determined by tail plethysmography every 2 weeks. After 16 weeks of treatment, vascular remodeling, superoxide production, malondialdehyde (MDA) content, and endothelial nitric oxide synthase (eNOS) and gp91 expression in the coronary arteries were detected. RESULTS：After 16 weeks of salt loading, the rats in HS group was divided into salt sensitive subgroup and salt resistance subgroup according to the tail-cuff blood pressure. In this experiment, the salt-sensitive rats were selected as HS group. In HS group, salt loading significantly increased SBP, serum MDA and gp91 expression, decreased serum NO and eNOS expression in the coronary arteries, and induced the coronary artery remodeling compared with NS group. In salt-loaded SD rats, 16-week potassium treatment abrogated the effects induced by salt loading. CONCLUSION：High salt may affect structural and functional changes in coronary arteries by activating oxidative stress. Potassium treatment antagonizes the effect of high salt intake.     

Atorvastatin inhibits atherogenesis by RXRα-mediated depressing oxidative stress in STZ-induced diabetic ApoE-/- mice with fat-rich diet

AIM: To explore the effects of atorvastatin (Atorv) on atherosclerosis in streptozotocin (STZ)-induced diabetic apolipoprotein E knockout (ApoE-/-) mice with fat-rich diet and the possible mechanism. METHODS：C57 mice served as control. ApoE-/- mice (n=34) fed with high-fat diet were randomly divided into ApoE-/- group, STZ-ApoE-/- group and STZ-ApoE-/-+Atorv group. Intraperitoneal injection of streptozotocin was performed to create diabetic animal model. Blood glucose was determined by glucose oxidase method. Blood lipid levels were detected by enzymic method or selective homogeneous method. The plaque area in the thoracic aorta was measured by HE staining. The protein level of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase subunit gp91phox in the thoracic aorta was determined by Western blotting. The levels of reactive oxygen species (ROS) in blood and thoracic aorta homogenates were detected by Fenton reaction and Griess reagent. Human umbilical vein endothelial cells (HUVECs) were isolated from healthy umbilical cords by collagenase I and cultured. ROS production was detected by flow cytometry. NADPH oxidase activity was measured using lucigenin assay.Effects of retinoid X receptor α (RXRα) on inhibition of oxidative stress by atorvastatin were evaluated by RNA interference and plasmid transfection. RESULTS：(1) Compared with C57 group, the plaque areas of the thoracic aorta in ApoE-/- group were increased. No difference of the fasting glucose between the 2 groups was observed. The levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), thoracic aorta gp91phox protein and ROS in blood and thoracic aorta homogenates were higher in ApoE-/- group than those in C57 group. (2) Compared with ApoE-/- group, the plaque areas of the thoracic aorta in STZ-ApoE-/- group were further enlarged ［(314.13±35.72) μm2 vs (215.88±34.19) μm2, P<0.05］. The levels of blood glucose, TG, TC and LDL-C, thoracic aorta gp91phox protein and ROS in blood and thoracic aorta homogenates were higher in STZ-ApoE-/- group than those in ApoE-/- group (P<0.05). (3) Compared with STZ-ApoE-/- group, the plaque areas of the thoracic aorta in STZ-ApoE-/- +Atorv group were reduced ［(217.47±24.56） μm2 vs （314.13±35.72） μm2, P<0.05］. The levels of blood glucose, LDL-C, TC, HDL-C and TG showed no significant difference between the 2 groups. Thoracic aorta gp91phox protein level and ROS production in blood and thoracic aorta homogenates were lower in STZ-ApoE-/- +Atorv group than those in STZ-ApoE-/- group (P<0.05). (4) High glucose-induced increases in NADPH oxidase activity and gp91phox expression were significantly inhibited by atorvastatin (10-6 mol/L) in HUVECs. The inhibitory effects of atorvastatin on high glucose-induced ROS production and NADPH oxidase activation were largely impaired when the cells were transfected with RXRα siRNA. However, the effect of atorvastatin was significantly strengthened when RXRα was over-expressed in the HUVECs transfected with RXRα plasmid. CONCLUSION：Atorvastatin inhibits atherogenesis by depressing high glucose-induced oxidative stress in diabetic ApoE-/- mice with fat-rich diet. The anti-oxidative stress effect of atorvastatin is mediated by RXRα.