Characteristics of morphology and left ventricular function in the mouse with myocarditis

AIM:To determine the relationship between microhistology and cardiac contractility in myocarditis animal model. METHODS: Setting up myocarditis animal model by injecting Coxsackivevirus B₃ (CVB₃) into mice, then observed myocardial morphological changes and measured left ventricular function of mice at the time of first three days and two weeks after injecting CVB₃. RESULTS:Subcellular structure (mitochondria) changed at the first three days after injecting CVB₃. The left ventricular pressure (LVP) and the rate of intraventricular pressure development (dp/dt) which is the index of reflecting cardiac contractility depressed in this stage (14.2±0.8) kPa and (273.1±10.0)kPa/s, respectively. There were (17.1±0.7)kPa and (359.8±9.3)kPa/s in normal mice, respectively (P＜0.01). Myocardial lesions were more severe during immune response stage-two weeks after injecting CVB₃, including myocardial inflammation and necrosis. LVP was (11.8±0.2)kPa and dp/dt was (209.5±6.1)kPa/s in immune response stage. There was significant difference between mice with myocarditis at early stage and at immune response stage (P＜0.01).CONCLUSIONS:The factor of causing the depression of cardiac contractility in early stage (virus-induced damage) is mainly change of subcellular structure. Mitochondria cannot provide energy as normal. There were more severe myocardial lesions in later stage (cell-mediated autoimmune response)than in early stage. The depression of cardiac contractility is a consequence of multifactor.     

Role of PERK pathway in morphine-induced myocardial protection of H9c2 cells

AIM: To explore whether morphine protects oxidative stress-damaged myocardial cells by inhibiting the PERK pathway to reduce endoplasmic reticulum stress and prevent mitochondrial permeability transition pore (mPTP) opening. METHODS: Rat myocardial H9c2 cells were cultured to establish an oxidative stress model, and then randomly divided into control group, H₂O₂ group, H₂O₂+morphine group, H₂O₂+morphine+PERK pathway inhibitor GSK2656157 group, morphine group and GSK2656157 group. Immunohistochemical method was used to detect the effects of morphine on expression of glucose-regulated protein (GRP) 78 and GRP94 induced by oxidative stress. The protein levels of PERK signaling pathway-related molecules were determined by Western blot. Confocal microscopy was used to observe the effects of morphine on mPTP opening and endoplasmic reticulum induced by oxidative stress. Cellular toxicity was detected by lactate dehydrogenase (LDH) kit and cell viability was measured by MTT assay. RESULTS: Compared with control group, GRP78 and GRP94 proteins in H₂O₂ group were strongly expressed, and the brown-yellow particles were significantly increased, but morphine significantly inhibited this process. Compared with control group, the phosphorylation of PERK was significantly reduced with GSK2656157 treatment at different concentrations, among which 2 μmol/L had the most significant effect (P < 0.05). Oxidative stress significantly increased the protein levels of GRP78, GRP94, p-PERK and CHOP, but significantly decreased p-GSK-3β level. These changes were inhibited by morphine, and the effects of morphine were further enhanced by GSK2656157 (P < 0.05). Compared with control group, oxidative stress significantly reduced the fluorescence intensity of mitochondrial TMRE and ER-Tracker Red. Morphine significantly inhibited this effect even when mitochondrial membrane potential was reduced, mPTP was open, and endoplasmic reticulum was damaged, while GSK2656157 further enhanced the effect of morphine (P < 0.05). Compared with control group, H₂O₂ significantly increased cellular toxicity and decreased the cell viability. Morphine inhibited this effect and GSK2656157 significantly enhanced the effect of morphine (P < 0.05). CONCLUSION: Morphine protects cardiac H9c2 cells under oxidative condition by inhibiting endoplasmic reticulum stress through PERK pathway and preventing the mPTP opening via GSK-3β inactivation.     

Importance of mitochondrial calcium uniporter in high glucose-induced cardiac myocyte apoptosis

AIM: To investigate the role of mitochondrial calcium uniporter (MCU) in high glucose(HG)-induced apoptosis of cardiac myocytes. METHODS: Cardiac myocytes were exposed to normal glucose (5.5 mmol/L glucose+ 19.5 mmol/L mannitol), HG (25 mmol/L glucose), or HG combined with 5 μmol/L spermine for 72 h. Mitochondrial free Ca²⁺ concentration ([Ca²⁺]_m), MCU at mRNA and protein levels, pyruvate dehydrogenase (PDH) activity, mitochondrial membrane potential (Δψ_m), the levels of ATP and reactive oxygen species (ROS), and apoptosis were determined. RESULTS: The [Ca²⁺]_m, the mRNA and protein levels of MCU, PDH activity, ATP levels, and Δψ_m were reduced (P<0.05), while ROS content and the protein levels of caspase-9 and caspase-3 were increased in HG group (P<0.05). Adding 5 μmol/L spermine returned these parameters toward control levels (P<0.05). Moreover, apoptosis was reduced by adding spermine and HG treatment (P<0.05). CONCLUSION: HG-induced cardiac myocyte apoptosis may be associated with the decreased MCU expression and activity, abnormal mitochondrial Ca²⁺ handling, deviant mitochon-drial respiratory chain, and mitochondrial dysfunction.     

Effects of urocortin-I preconditioning on myocardial mitochondrial respiratory function and enzyme activity in rats

ATM: To investigate the influence of urocortin-I (Ucn I) preconditioning on the myocardial mitochondrial respiratory function and enzyme activity in the rats with ischemia reperfusion, and to observe the changes of ATP content in the myocardial cells. METHODS: (1) The healthy male Sprague-Dawley rats were randomly divided into 4 groups:normal group (Nor group), ischemia reperfusion group (IR group), Ucn I preconditioning group (Ucn I group), 5-hydroxy acid (5-HD)+Ucn I group. Langendorff perfusion was used to establish the in vitro model of cardiac ischemia reperfusion. At the end of the balance (T₁), before ischemia (T₂) and at the end of the reperfusion (T₃) respectively, the myocardial mitochondria was extracted, the mitochondrial respiratory function and respiratory enzyme activity in each group were determined. (2) The method of MPA isolated heart perfusion was used to isolate myocardial cells of the adult rats. After cultured for 24 h, myocardial cells were divided into 4 groups:Nor group, hypoxia/reoxygenation group (I/R group), Ucn I group, 5-HD+Ucn I group. Hypoxia/reoxygenation model of myocardial cells was established. At the end of reoxygenation, the changes of myocardial ATP content were measured by high performance liquid chromatography.RESULTS: (1) Compared with T₁, T₂ time points, the respiratory function (state 3 respiratory rate, respiratory control rate) and NADH oxidase, succinate oxidase and cytochrome C oxidase activities at T₃ time point were significantly decreased (P<0.05) in all groups except Nor group. At T₃ time point, the myocardial mitochondrial respiratory function and respiratory enzyme activity in Ucn I group were superior to 5-HD+Ucn I group and IR group (P<0.05), but was inferior to Nor group (P<0.05). At T₃ time point, the respiratory function of myocardial mitochondria and respiratory enzyme activities (NADH oxidase, succinate oxidase) in 5-HD+Ucn I group were better than those in IR group (P<0.05), but no statistical difference of the cytochrome C oxidase activity between the 2 groups was observed. The respiratory function and 3 kinds of respiratory enzyme activities at T₁, T₂ time points had no statistical change. (2) At the end of the reoxygenation, the myocardial ATP content in Nor group was higher than that in other groups (P<0.01). The myocardial ATP contents in I/R group and 5-HD+Ucn I group were lower than that in Ucn I group (P<0.05). In additon, 5-HD+Ucn I group was higher ATP content compared with I/R group (P<0.05). CONCLUSION: Ucn I preconditioning attenuates the ischemia/reperfusion induced damages of myocardial mitochondrial respiratory function and respiratory enzyme activity, thus ensuring the myocardial ATP contents under the condition of hypoxia/reoxygenation.     

Effect of transmyocardial laser revascularization on myocardial lactatic metabolism and mitochondria of myocardial cells

AIM: We studied the therapeutic effect and mechanism of transmyocardial laser revascularization (TMLR) for the acute myocardial ischemia (AMI). METHODS: 18 dogs were divided randomly and evenly into the control group, the AMI group and the TMLR group. A continuous wave Nd: YAG laser was used for TMLR. Concentration of lactate in artery and coronary sinus (A.Lat and CS.Lat), myocardial metabolic rate of lactate acid (MLR) and myocardial lactate extraction (MLE) were measured before the left anterior descending coronary artery (LAD) ligation and 60 min after the LAD ligation. Myocardial biopsy was made 4 h after the LAD ligation to quantitatively observe the shape and number of mitochondria in myocardial cells by a electric microscope. RESULTS: 60 min after the LAD ligation, CS.Lat were (7.63±4.27) mmol/L in the AMI and (5.78±3.98) mmol/L in the TMLR, respectively (P<0.05); MLR were (0.03±0.01) mmol·100 g^-1 myocardium·min^-1 in the AMI and (0.06±0.02) mmol·100 g^-1 myocardium·min^-1 in the TMLR, respectively (P<0.05); MLE were (12.04±3.04) in the AMI and (21.84±8.49)% in the TMLR, respectively (P<0.05). The volume density of mitochondria were (27.51±7.93)% in the AMI and (31.26±3.85)% in the TMLR, respectively (P>0.05). The area density of mitochondria were (1.25±0.18) μm^-1 in the AMI and (1.64±0.28) μm^-1 in the TMLR, respectively (P<0.01). The number density of mitochondria were (0.10±0.03) μm^-3 in the AMI and (0.18±0.05) μm^-3 in the TMLR, respectively (P<0.01). The average volume of mitochondria were (5.27±2.85) μm³ in the AMI and (2.80±0.54) μm³ in the TMLR, respectively (P<0.05). The average diameter of mitochondria were (2.06±0.36) μm in the AMI and (1.78±0.12)μm in the TMLR, respectively (P＜0.05). CONCLUSION: The study suggests that TMLR may effectively improve myocardial lactatic metabolism and protect the myocardial cells from ischemic injury in dogs with the AMI.     

Salvianolic acid B reduces apoptosis of bone marrow stem cells induced by hydrogen peroxide

AIM: To investigate the effect of salvianolic acid B (Sal B) on apoptosis of rat bone mesenchymal stem cells(BMSCs) induced by hydrogen peroxide(H₂O₂). METHODS: BMSCs were incubated with Sal B at the concentration of 1, 10 or 100 μmol/L while treated with lethal concentration of H₂O₂ (500 μmol/L). The effect of Sal B at different concentrations on the viability of BMSCs was detected by MTT. Flow cytometry were used to determine the protective role of Sal B in apoptosis of BMSCs. The changes of chromatin distribution in BMSCs were observed by Hoechst 33342 staining. The expression of p-ERK1/2 was detected by Western blotting. RESULTS: Sal B protected the BMSCs against H₂O₂ as the cell viability was increased from (53.60±4.21)% to (85.33±9.08)% or (75.78±6.28)% in a dose-dependent manner. After exposed to H₂O₂, about 50%-65% BMSCs displayed apoptotic morphology. Treatment with Sal B at the concentrations of 10 and 100 μmol/L reduced the cytotoxic effect of H₂O₂ on BMSCs to about 32% and 47%, respectively. The results of flow cytometric analysis confirmed the cytoprotective effect of Sal B. This protective effect was concomitant with significant reduction of ROS generation. Moreover, H₂O₂ time-dependently induced a pronounced increase in ERK1/2 phosphorylation,which was effectively inhibited by Sal B.CONCLUSION: Sal B protects BMSCs against H₂O₂-induced apoptosis. Sal B may exert its protective effect on BMSCs by triggering intracellular anti-apoptosis mechanism as well as reducing the oxidative stress.     

MSC-conditioned medium activates Nrf2/ARE pathway to protect H9c2 cells against oxidative stress

AIM: To investigate the protective effect of mesenchymal stem cell (MSC)-conditioned medium (MSCCM) on myocardial cell line H9c2 and its mechanism. METHODS: Verification of MSC was performed by flow cytometry analysis, followed by MTT assay to determine the optimal incubation time of MSCCM with myocardial cells. The cells were divided into 4 groups: normal (N) group, model (M) group, M+MSCCM group and MSCCM group. The cells in M+MSCCM group and MSCCM group were pre-incubated with MSCCM for 24 h. The cells in M group and M+MSCCM group were treated with 300 μmol/L H₂O₂ for 4 h to imitate oxidative injury of myocardial cells. Mitochondrial membrane potential and apoptotic rate of injured myocardial cells were detected by flow cytometry. The ROS production was measured by fluorescence microscopy. The nuclear translocation of Nrf2 and expression of HO-1 was examined by Western blot. RESULTS: No difference of mitochondrial membrane potential, apoptotic rate or ROS production between MSCCM group and N group was observed (P>0.05). The mitochondrial membrane potential depolarization, apoptotic rate and ROS production in M+MSCCM group were significantly lower than those in M group (P<0.01). The nuclear translocation of Nrf2 and expression of HO-1 in the myocardial cells were increased with MSCCM incubation time prolonged. CONCLUSION: MSCCM protects the myocardial cells against oxidative injury induced by H₂O₂. The anti-oxidative mechanism would be associated with the activation of Nrf2/ARE pathway.     

Characteristics of energy metabolism in brain mitochondria of rats exposed to hypoxia

AIM:To explore the characteristics of energy metabolism in brain mitochondria of rats exposed to acute and chronic hypoxia. METHODS: Animal grouping: Wistar rats were randomized into acute hypoxic group (AH), chronic hypoxic group (CH) and the control. Respiratory function, F₀F₁-ATPase activity, mitochondrial ATP, ADP and AMP contents and ATP production rate were measured respectively. RESULTS: In AH, brain mitochondrial respiratory state IV (ST₄) was increased, while respiratory control rate (RCR), mitochondrial ATP content, ATP production rate and F₀F₁-ATPase activity were decreased respectively. In CH, ST₄, RCR, mitochondrial ATP content and F₀F₁-ATPase activity were reversed partially.CONCLUSION: Acute hypoxia may impair brain mitochondria energy metabolism by way of depressing mitochondrial oxidative phosphorylation and ATP production and these parameters gain partial reablement during chronic hypoxia.     

Effects of human urotensin II on ischemia/reperfusion injury in isolated perfused rat hearts

AIM: To investigate the effects of human urotensin II (hUII) on ischemia/reperfusion (I/R) injury in isolated rat hearts. METHODS: In the ischemia/reperfusion (I/R) model of isolated perfused rat hearts, the effects of hUII pretreatment on cardiac function was monitored with cardiac function software of MFL Lab200. ATP, total calcium, and malondialdehyde (MDA) content in myocardium were detected. The coronary perfusion flow (CPF) and lactate dehydrogenase (LDH) activity in coronary effluent were measured during reperfusion. RESULTS: In the hUII pretreated group, the release of LDH from myocardium was lower [(78.3±18.1)U/L] than I/R group [(109.3±23.9) U/L, P< 0.05], with decreased contents of MDA and calcium in myocardium (decreased by 24% and 27%, respectively, P< 0.05) and an increased myocardial ATP content [(3.8±0.4)μmol/g dw vs (2.2±0.4)μmol/g dw, P< 0.05)]. At the same time, hUII pretreatment increased CPF [(5.4±0.7) mL/min vs (3.8±0.8) mL/min in I/R group, P< 0.05], reduced left ventricular end-diastolic pressure (LVEDP) by 20% ( P< 0.05) with increased±d p /d t max [(217±38) kPa/s and (119±18) kPa/s vs (173±29) kPa/s and (82±25) kPa/s in I/R groups, respectively, P< 0.05]. hUII pretreatment also increased natrite/natrate (NO₂^-/NO₃^-) content in coronary effluent [(52.2±12.0)μmol/L vs (32.1±10.2)μmol/L in I/R group, P< 0.05)]. CONCLUSION: hUII pretreatment attenuated I/R injury in isolated perfused rat hearts. The protective mechanism might be associated with NO-mediated coronary vasodilation.     

Metallothionein protects rat hepatic nuclear nucleoside triphosphatase from hydroxyl radical-induced suppression

AIM:Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotection during metal exposure and oxidative stress. The present study was designed to investigate whether MT can directly protect NTPase on nuclear envelope from damage induced by hydroxyl radical.METHODS:Isolated hepatic nuclei from rat liver were exposed to Fe²⁺/H₂O₂ with or without MT, and the NTPase activity on nuclei was assayed using ATP and GTP as substrate, respectively.RESULTS:Incubation of rat hepatic nuclei with the Fe²⁺/H₂O₂ (in μmol·L^-1/μmol·L^-1 : 0.1/0.5, 0.5/2.5, 1/5, 5/25) resulted in a concentration-dependent decrease in nuclear NTPase activities (P＜0.01). Incubation of hepatic nuclei with different concentrations of MT (10^-9-10^-4mol·L^-1)and Fe²⁺/H₂O₂ (1 μmol·L^-1/5 μmol·L^-1) for 10 min, nuclear NTPase activities were increased in a MT concentration-dependent fashion as compared with that of incubation with Fe²⁺/H₂O₂(1 μmol·L^-1/5 μmol·L^-1) alone. When MT was at 10^-4 mol·L^-1, TNPase activities reversed to (102±10) nmol·mg^-1 protein·min^-1(for ATP as substrated) and (131±12) μmol·g^-1 protein·min^-1(for GTP as substrate), which had no significant defferences from that of the controls (112±8 and 142±10 μmol·g^-1 protein·min^-1, respectively) (P＞0.05). In addition, incubation of hepatic nuclei with only MT had no effect on nuclear NTPase activity. CONCLUSION:These data demonstrate that hydroxyl radical generated from Fe²⁺/H₂O₂ might attack nuclear NTPase. MT antagonistically reduces toxicity of Fe²⁺/H₂O₂ system to the NTPase.     

Changes of potassium currents in rabbit ventricle with healed myocardial infarction

AIM: To elucidate the mechanism of arrhythmia in healed myocardial infarction (HMI), and to investigate the changes of action potential duration (APD),transient outward potassium current (I_to), delayed rectifier potassium current (I_K) and inward rectifier potassium current (I_K1) of left ventricular myocytes in noninfarcted zone of HMI. METHODS: 12 rabbits were randomly assigned in two groups: HMI group (thoracotomy and ligation of the circumflex coronary); sham-operated group (thoracotomy but no conorary ligation). 3 months after operation, whole cell patch clamp technique was used to record APD, I_to, I_K and I_K1 of ventricular myocytes in non-infarcted zone. RESULTS: Membrane capacitance was larger in HMI group than that in sham-operated group. Action potential duration was lengthened significantly in HMI group and early after depolarization (EAD) appeared in HMI group. The densities of I_to, I_K,tail and I_K1 were reduced significantly in HMI group (P<0.01), from (6.72±0.42) pA/pF, (1.54±0.13) pA/pF and (25.6±2.6) pA/pF in Sham-operated group to (4.03±0.33) pA/pF, (1.14±0.11) pA/pF and (17.6±2.3) pA/pF, respectively. CONCLUSION: The reduced densities of I_to, I_K,tail and I_K1 in ventricular myocytes of non-infarcted zone in HMI are responsible for the prolongation of APD and the presentation of EAD, which play important roles in the malignant arrhythmia of HMI.     

Effect of inhaled nitric oxide on adhesion molecule CD11b expression in lung neutrophils of meconium aspiration rabbits

AIM:To evaluate effects of inhaled nitric oxide(iNO) on adhesion molecule CD11b expression on lung neutrophils in experimental meconium aspiration syndrome(MAS) rabbits treated with conventional mechanical ventilation under room air or 100%O₂. METHODS:Animals were randomly allocated to 8 groups(n=48) of 6 each: two MAS model groups(under room air or 100%O₂ without iNO treatment), 6 treatment groups were treated with continuous NO inhalation at a dose of 0.2×10^-6mol/L, 0.33×10^-6mol/L or 0.67×10^-6mol/L respectively for 12 hours under room air or 100%O₂. Mean systemic arterial pressure(SAP) and methemoglobin (MeHb) were performed at basement time, 0, 2, 4, 12 hours. Expression of CD11b on neutrophils in the bronchoalveolar lavage fluid(BALF) was detected with flow cytometry. RESULTS:SAP, MeHb at different time among different groups were within the normal scale. CD11b expression on the neutrophils in the BALF significantly decreased in groups of inhalation 0.33×10^-6 mol/L or 0.67×10^-6 mol/L NO, compared with the two MAS model groups. (x±s: under 21%O₂, 0.33×10^-6 mol/L NO, 121±20 υs 392±204; 0.67×10^-6 mol/L NO, 112±30 υs 392±204;under 100%O₂, 0.33×10^-6 mol/L NO, 113±24υs293±65; 0.67×10^-6 mol/L 102±114 υs293±65, P<0.05). 0.2×10^-6mol/L NO inhalation did no effect on CD11b expression. (x±s:21%O₂, 190±101 υs 392±204; 100%O₂, 222±85 υs 293±65; P>0.05). No statistic difference was observed between groups inhaled 0.33×10^-6 mol/L NO and 0.67×10^-6mol/L NO. CONCLUSION:0.33×10^-6 mol/L or 0.67×10^-6 mol/L NO inhalation down-regulated the CD11b expression on the neutrophils in BALF to reduce the sequestration of neutrophils in rabbit lung.     

Role of ATP-sensitive potassium channels in inhibitory effect of hydrogen sulfide on high glucose-induced injury in H9c2 cardiac cells

AIM: To investigate the roles of ATP-sensitive potassium (K_ATP) channels in high glucose-induced cardiac injury and the inhibitory effect of hydrogen sulfide (H₂S) on the cardiomyocyte injury. METHODS: The expression level of K_ATP channel protein was tested by Western blot. The cell viability was measured by CCK-8 assay. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining. Mitochondrial membrane potential (MMP) was examined by JC-1 staining. RESULTS: After the H9c2 cells were treated with 35 mmol/L glucose (high glucose, HG) for 1~24 h, the protein level of K_ATP channel was significantly reduced at 6 h, 9 h, 12 h and 24 h, reaching the minimum level at 12 h and 24 h. Pretreatment of the cells with 400 μmol/L NaHS (a donor of H₂S) prior to exposure to HG for 12 h considerably blocked the down-regulation of K_ATP channels induced by HG. Pretreatment of the cells with 100 μmol/L mitochondrial K_ATP channel opener diazoxide, 50 μmol/L non-selective K_ATP channel opener pinacidil or NaHS obviously inhibited HG-induced injuries, leading to an increase in the cell viability, and decreases in the number of apoptotic cells and the MMP loss. Pretreatment with 100 μmol/L mitochondrial K_ATP channel antagonist 5-hydroxydecanoic acid or 1 mmol/L non-selective K_ATP channel antagonist glibenclamide attenuated the above cardioprotective effects of NaHS. CONCLUSION: K_ATP channels mediate the inhibitory effect of H₂S on HG-induced cardiac injury.     

Expression and functional role of p38 MAPK in the kidney after unilateral ureteral obstruction in rats

AIM: To investigate the expression and functional role of p38MAPK in the kidney after unilateral ureteral obstruction in rats. METHODS: Unilateral ureteral obstruction (UUO) models were induced by ligating the left ureter. Rats were sacrificed at 1 h, 3 h, 6 h, 12 h, 1, 3, 5, 7, 14, 21, and 28 days after UUO was initiated. p38MAPK activity was assayed by immunohistochemical staining and specific substrate phosphorylation with immunoprecipitation and Western blotting. TGFβ mRNA and protein expression were analyzed with in situ hybridization and immunohistochemical stainning. RESULTS: A basic p38MAPK activity was detectable in the normal kidney(0.22±0.06).p38MAPK pathway was rapidly act ivated at 1 hour(0.45±0.14 vs control,P<0.05)and this was steadily in creased by 12 hours(0.91±0.07 vs control,P<0.01)after UUO.Afterwards,the activity of p38MAPK reduced grad ually,then increased again from 3 days and this was steadily increased by 7 days(0.93±0.06 vs control,P<0.01). Upregulation of TGFβ₁ was markedly tested at 3 days(13.55±6.33 vs control,P<0.05)and this was steadily in creased by 7 days(26.78 8.77 vs control,P<0.01).The activation of p38MAPK preceded markedly the expression of TGF 1.The early activity of p38 MAPK was positively related to the amount of TGFβ₁ expression.The amount of TGFβ₁ expressed in obstructed kidney also related significant ly to the late activity of p38MAPK(r=0.84,P<0.01). CONCLUSION: The activity of p38MAPK is increased significantly in the obstructed kidney, which may cause renal fibrosis via inducing the expression of TGFβ₁.     

Protective effect of ecdysterone on H9c2 cells against oxidative stress

AIM: To investigate the effect of ecdysterone (EDS) on H9c2 cardiomyocytes after oxidative stress. METHODS: H9c2 cells were cultured in vitro and divided into control group, high dose (2 μmol/L) of EDS group, middle dose (1.5 μmol/L) of EDS group, low dose (1 μmol/L) of EDS group, and H₂O₂ group. H9c2 cardiomyocytes in H₂O₂ group and high, middle and low doses of EDS groups were exposed to H₂O₂ for 6 h to establish the model of oxidative stress. The viability of the H9c2 cells was detected by CCK-8 assay. The apoptosis of H9c2 cells was analyzed by flow cytometry. The levels of lactate dehydogenase (LDH) and creatine kinase-MB (CK-MB) in the culture medium, and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the H9c2 cells were measured by colorimetry. The generation of reactive oxygen species (ROS) and the mitochondrial membrane potential were evaluated by flow cytometry and confocal laser scanning microscopy. The protein levels of Bax, Bcl-2 and cleaved caspase-3 in the H9c2 cells were determined by Western blot. RESULTS: Ecdysterone at the selected concentrations had no effect on the viability of H9c2 cells. Compared with control group, the levels of LDH, CK-MB, ROS and MDA, and the apoptotic rates of the H9c2 cells were significantly increased after treated with H₂O₂, but were decreased by EDS treatment in a dose-dependent manner. The levels of SOD and mitochondrial membrane potential of the H9c2 cells in H₂O₂ group were reduced significantly compared with control group, but high, middle and low doses of EDS treatments up-regulated the levels of SOD and mitochondrial membrane potential in H₂O₂-treated H9c2 cells. The protein levels of Bax and cleaved caspase-3 in the H9c2 cells in H₂O₂ group showed significant elevation in comparison with control group, and the protein expression of Bcl-2 declined in H₂O₂ group compared with control group, but high, middle and low doses of ecdysterone treatments down-regulated the protein levels of Bax, cleaved caspase-3 and up-regulated the expression of Bcl-2 in H₂O₂-treated H9c2 cells. CONCLUSION: Ecdysterone attenuates the effect of H₂O₂-induced oxidative stress on H9c2 cardiomyocytes. The mechanism may be involved in scavenging oxidative stress products, increasing antioxidant enzyme activity and improving mitochondrial function.     

Adaptation characteristics of mitochondria in plateau pikas at different altitudes

AIM: To investigate the adaptive characteristics of mitochondria in plateau pikas at different altitudes. METHODS: According to the altitudes of the capture area, plateau pikas were divided into 4 300 m group (Mado Star Sea, n=6) and 2 900 m group (Laoji Mountain South, n=6). The red blood cells and hemoglobin of the animals in 2 groups were measured. The levels of mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) in the liver and skeletal muscle, and mitochondrial H₂O₂ production level in the skeletal muscle were determined by high-resolution mitochondrial respirometer. RESULTS: The hemoglobin of the plateau pika in 4 300 m group was significantly lower than those in 2 900 m group. The liver state 4 respiration rate of mitochondrial respiratory chain complex I (CI-LEAK), mitochondrial respiratory chain complex I state 3 respiration rate (CI-OXPHOS) and respiratory chain complex I+Ⅱ state 3 respiration rate(CI+Ⅱ-OXPHOS), electron transfer capacity of respiratory chain complex I+Ⅱ (CI+Ⅱ-ETS), the electron transport capacity of the respiratory chain complex Ⅱ (CⅡ-ETS) in 2 900 m group were increased, and the coupling efficiency was decreased compared with 4 300 m group (P<0.05). The skeletal muscle CI-LEAK,CI+Ⅱ-OXPHOS and CI+Ⅱ-ETS were higher than those in 4 300 m group. Mitochondrial state 4 respiration rate with palmitic acid carnitine and malate as substrates (FAO-LEAK) of liver and skeletal muscle in 2 900 m group were increased compared with 4 300 m group. The skeletal muscle with palmitic acid carnitine as a substrate for fatty acid metabolism state 3 respiratory rate (FAO-OXPHOS) was increased in 2 900 m group compared with 4 300 m group. The H₂O₂/O₂ flow ratio (ΔH₂O₂) in 2 900 m group was lower in state 4 respiration rate of mitochondrial respiratory chain complex I (LEAK), state 3 of mitochondrial respiratory chain complex I (OXPHOS), state 3 of mitochondrial respiratory chain complex I+Ⅱ (OXPHOS*) and electron transfer capacity of respiratory chain complex I+Ⅱ (ETS) than 4 300 m group. CONCLUSION: Plateau pikas with different altitudes have different mitochondrial adaptation characteristics.     

Effects of schisandrin B on apoptosis of lens epithelial cells treated with H2O2

AIM: To investigate the effects of Schisandrin B (Sch B) on apoptosis of lens epithelial cells (LEC) treated with H₂O₂. METHODS: Eyes in SDrats were excised and lenses were separated under operating microscope in sterilized condition. Lenses were divided randomly into four groups with different treatment: control group, hydrogen peroxide group (H₂O₂), pirenoxine sodium group (PS) and schisandrin Bgroup (Sch B). Lenses were incubated in CO₂ incubator for 24 h with 300 μmol·L^-1 H₂O₂ and with or without 0.5 mmol·L^-1 Sch B. LECaoptosis and apoptosis rate were measured by TUNELmethod. Ultrastructure changes and apoptosis bodies of LECwere observed via transmitted electron microscope. RESULTS: (1) Apoptosis rate in H₂O₂ group (92.0±2.6) was significantly higher than that in control group (3.5±1.8). Apoptosis rate in Sch Bgroup (13.8±3.27) was remarkably lower than that in H₂O₂ group and PSgroup. (2) Ultrastructure observation indicated that apoptosis cells occurred in most LEC in H₂O₂ group and the changes were severe presenting different stages. While a few apoptosis cells were observed in Sch Bgroup, the changes were slight and most of them were in early and middle stages. CONCLUSION: These data indicated that Sch Bsignificantly inhibited apoptosis of LECduring experimental oxidative injury, the effects were stronger than PS.     

Effects of several harmful factors on expression of glycine receptor α1 subunit in neonatal rat myocardial cells

AIM: To study the expression of glycine receptor α₁ subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells. METHODS: Neonatal rat myocardial cells were cultured in vitro. The expression of glycine receptor α₁ subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell viability was measured by CCK-8 assay, and the expression of glycine receptor α₁ subunit was determined by Western blotting. RESULTS: The expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells was positively detectable by Western blotting. Compared with control group, no significant difference of the cell viability (P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed. The expression of glycine receptor α₁ subunit was increased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION: Glycine receptor α₁ subunit exists in the neonatal rat myocardial cells. A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptor α₁ subunit, but HG downregulates the expression of glycine receptor α₁ subunit in cultured neonatal rat myocardial cells.     

Effects of hemorrhagic shock on BKCa channel tyrosine phosphorylation level in mesenteric artery in rats

AIM: To study the effects of hemorrhagic shock on BK_Ca channel tyrosine phosphorylation in rat superior mesenteric artery and the role of nitric oxide (NO) in BK_Ca channel tyrosine phosphorylation. METHODS: The hemorrhagic shock model [(35±5)mmHg] was established in rats and the whole lysate of superior mesenteric artery were extracted and analyzed by immune precipition (IP) and immunoblotting. The tyrosine phosphorylation levels of BK_Ca channel alpha-subunit in mesenteric artery in hemorrhagic shock rats were investigated, and the modulation of BK_Ca channel alpha-subunit tyrosine phosphorylation by NO and its dose-and time-dependended relationships were observed. RESULTS: The tyrosine phosphorylation level of BK_Ca channel alpha-subunit in mesenteric artery in rats increased significantly after hemorrhagic shock 2 h and 4 h (P<0.01 ), and L-arginine (5×10^-5-5×10^-4 mol/L) up-regulated BK_Ca channel alpha-subunit tyrosine phosphorylation in primary cultured VSMC in a 30 min incubation and without significant decrease after 2 h; L-arginine induced BK_Ca channel alpha-subunit tyrosine phosphorylation in a dose-dependent manner. CONCLUSION: Hemorrhagic shock enhances BK_Ca channel tyrosine phosphorylation in resistant artery in rats, and NO is involved in this process.     

Identification of ATTM as a novel H2S donor and investigation of its protective effect on HaCaT skin cells

AIM: To investigate the ability of a metal complex ammonium tetrathiomolybdate (ATTM) to release H₂S and its cytoprotective effect on an oxidative injury model. METHODS: Released H₂S was absorbed in a reaction flask from ATTM dissolved in the cell medium. Staining with dichlorodihydrofluorescein diacetate or rhodamine 123 followed by photofluorography was conducted for the observation of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ_m) levels, respectively. Cell viability and release of lactate dehydrogenase (LDH) from the cells were measured with commercial kits. RESULTS: Similar to another H₂S donor GYY4137, ATTM had an ability to release H₂S in the cell medium in a dose-dependent manner. Treatment of human skin HaCaT cells with ATTM at concentrations of 25~400 μmol/L didn't significantly alter cell viability. Exposure of the cells to ultraviolet rays or a ROS donor H₂O₂ increased the intracellular ROS levels. Treatment with 400 μmol/L H₂O₂ significantly reduced the viability of HaCaT cells (P<0.01). However, before the treatment with H₂O₂, pretreatment with ATTM at 100 and 200 μmol/L markedly prevented the H₂O₂-induced cell injury (P<0.01). In addition, the treatment with H₂O₂ triggered ΔΨ_m loss (P<0.01) and LDH release from the cells (P<0.01). Prior to suffering from H₂O₂ injury, the preconditioning with 200 μmol/L ATTM significantly improved ΔΨ_m levels (P<0.05) and attenuated LDH release from the cells (P<0.01).CONCLUSION: ATTM is capable of releasing H₂S and protecting human skin cells against oxidative injury.