Metformin protects against hyperglycemia-induced cardiomyocytes injury by inhibiting the expressions of receptor for advanced glycation end products and high mobility group box 1 protein

Metformin (MET), an anti-diabetic oral drug with antioxidant properties, has been proved to provide cardioprotective effects in patients with diabetic disease. However, the mechanism is unclear. This study aimd to investigate the effects of MET on the expressions of receptor for advanced glycation end products (RAGE) and high mobility group box 1 protein (HMGB1) in hyperglycemia-treated neonatal rat ventricular myocytes. Cardiocytes were prepared and cultured with high glucose and different concentrations of MET. The expressions of RAGE and HMGB1 were evaluated by Western blot analysis. The superoxide dismutase (SOD), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), lactate dehydrogenase (LDH) and creatine kinase (CK) were measured. After 12 h-incubation, MET significantly inhibited the increase of MDA, TNF-α, LDH and CK levels induced by high glucose, especially at the 5 × 10^?5 to 10^?4 mol/L concentrations while inhibiting the decrease of SOD level. Meanwhile, RAGE and HMGB1 expression were significantly increased induced by hyperglycaemia for 24 h (P < 0.05). MET inhibited the expressions of RAGE and HMGB1 in a dose-dependent manner, especially at the 5 × 10^?5 to 10^?4 mol/L concentrations (P < 0.05). In conclusion, our study suggested that MET could reduce hyperglycemia-induced cardiocytes injury by inhibiting the expressions of RAGE and HMGB1.     

B型钠尿肽对急性心肌缺血损伤发挥保护作用的机制

本研究旨在研究B型钠尿肽(brain natriuretic peptide, BNP)对异丙肾上腺素(isoproterenol, ISO)诱导的急性心肌缺血(acute myocardial ischemia, AMI)相关损伤的保护作用。将大鼠随机分作5组:对照组、ISO处理组、ISO+普萘洛尔(5 mg/kg)处理组、ISO+BNP (5 mg/kg)和ISO+BNP (10 mg/kg)处理组。再将4个给药组中的大鼠连续2 d皮下注射ISO。通过心电图(ECG)记录ST段数据;ELISA检测血清中肌酸激酶(CK)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、丙二醛(MDA)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6 (IL-6)和IL-1β的表达水平;接着使用Western blotting对Bax-2、Bcl-2、PI3K、Akt、GSK-3β、MDA5和SOD1蛋白表达水平进行检测,并测定PI3K、Akt和GSK-3β的磷酸化。实验结果显示,BNP (5 mg/kg, 10 mg/kg)可降低血清中MDA和CK的表达水平以及SOD和LDH的蛋白活性;经BNP预处理显著降低了促炎因子IL-1β、IL-6和TNF-α的表达水平;同时BNP也降低了ST段抬高。此外,BNP改善了ISO诱导的大鼠中SOD1、MDA5、Bax-2、Bcl-2、p-PI3K、p-Akt和p-GSK-3β的表达。综上所述,通过调节PI3K/Akt/GSK-3β信号传导途径,BNP保护大鼠免受AMI损伤。     

Ethyl pyruvate reduces myocardial ischemia and reperfusion injury by inhibiting high mobility group box 1 protein in rats

High mobility group box 1 protein (HMGB1) plays an important role in myocardial ischemia and reperfusion (I/R) injury. Ethyl pyruvate (EP), a potent reactive oxygen species scavenger, has been reported to inhibit myocardial apoptosis and reduce myocardial I/R injury. The aim of this study was to investigate the mechanism by which EP reduces myocardial I/R injury in rats. Anesthetized male rats were once treated with EP (50 mg/kg, i.p.) before ischemia, and then subjected to ischemia for 30 min followed by reperfusion for 4 h. Lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD) activity and infarct size were measured. HMGB1 expression was assessed by immunoblotting. The results showed that pretreatment of EP (50 mg/kg) could significantly reduce the infarct size and the levels of LDH and CK after 4 h reperfusion (all P < 0.05). EP could also significantly inhibit the increase of the MDA level, the decrease of the SOD level (both P < 0.05). Meanwhile, EP could significantly inhibit the expression of HMGB1 induced by I/R. The present study suggested that ethyl pyruvate could attenuate myocardial I/R injury by inhibiting HMGB1 expression.     

The importance of the nine-amino acid C-terminal sequence of exendin-4 for binding to the GLP-1 receptor and for biological activity

Exendin-4, a 39-amino acid (AA) peptide, is a long-acting agonist at the glucagon-like peptide-1 (GLP-1) receptor. Consequently, it may be preferable to GLP-1 as a long-term treatment for type 2 diabetes mellitus. Exendin-4 (Ex-4), unlike GLP-1, is not degraded by dipeptidyl peptidase IV (DPP IV), is less susceptible to degradation by neutral endopeptidase, and possesses a nine-AA C-terminal sequence absent from GLP-1. Here we examine the importance of these nine AAs for biological activity of Ex-4, a sequence of truncated Ex-4 analogs, and native GLP-1 and GLP-1 analogs to which all or parts of the C-terminal sequence have been added. We found that removing these AAs from Ex-4 to produce Ex (1-30) reduced the affinity for the GLP-1 receptor (GLP-1R) relative to Ex-4 (IC50: Ex-4, 3.22+/-0.9 nM; Ex (1-30), 32+/-5.8 nM) but made it comparable to that of GLP-1 (IC50: 44.9+/-3.2 nM). The addition of this nine-AA sequence to GLP-1 improved the affinity of both GLP-1 and the DPP IV resistant analog GLP-1 8-glycine for the GLP-1 receptor (IC50: GLP-1 Gly8 [GG], 220+/-23 nM; GLP-1 Gly8 Ex (31-39), 74+/-11 nM). Observations of the cAMP response in an insulinoma cell line show a similar trend for biological activity.     

HMGB1 regulates ferroptosis through Nrf2 pathway in mesangial cells in response to high glucose

Ferroptosis, a novel type of programmed cell death, is involved in inflammation and oxidation of various human diseases, including diabetic kidney disease. The present study explored the role of high-mobility group box-1 (HMGB1) on the regulation of ferroptosis in mesangial cells in response to high glucose. Compared with healthy control, levels of serum ferritin, lactate dehydrogenase (LDH), reactive oxygen species (ROS), malonaldehyde (MDA), and HMGB1 were significantly elevated in diabetic nephropathy (DN) patients, accompanied with deregulated ferroptosis-related molecules, including long-chain acyl-CoA synthetase 4 (ACSL4), prostaglandin-endoperoxide synthase 2 (PTGS2), NADPH oxidase 1 (NOX1), and glutathione peroxidase 4 (GPX4). In vitro assay revealed that erastin and high glucose both induced ferroptosis in mesangial cells. Suppression of HMGB1 restored cellular proliferation, prevented ROS and LDH generation, decreased ACSL4, PTGS2, and NOX1, and increased GPX4 levels in mesangial cells. Furthermore, nuclear factor E2-related factor 2 (Nrf2) was decreased in DN patients and high glucose-mediated translocation of HMGB1 in mesangial cells. Knockdown of HMGB1 suppressed high glucose-induced activation of TLR4/NF-κB axis and promoted Nrf2 expression as well as its downstream targets including HO-1, NQO-1, GCLC, and GCLM. Collectively, these findings suggest that HMGB1 regulates glucose-induced ferroptosis via Nrf2 pathway in mesangial cells.     

Effect of GLP-1 on glucose transport and its cell signalling in human myocytes

Glucagon-like peptide-1 (GLP-1) controls glucose metabolism in extrapancreatic tissues participating in glucose homeostasis, through receptors not associated to cAMP. In rat hepatocytes, activation of PI3K/PKB, PKC and PP-1 mediates the GLP-1-induced stimulation of glycogen synthase. We have investigated the effect of GLP-1 in normal human myocytes, and that of its structurally related peptides exendin-4 (Ex-4) and its truncated form 9-39 (Ex-9) upon glucose uptake, and the participation of cellular enzymes proposed to mediate insulin actions. GLP-1 and both exendins activated, like insulin, PI3K/PKB and p42/44 MAPK enzymes, but p70s6k was activated only by GLP-1 and insulin. GLP-1, Ex-4 and Ex-9, like insulin, stimulated glucose uptake; wortmannin blocked the action of GLP-1, insulin and Ex-9, and reduced that of Ex-4; PD98059 abolished the effect of all peptides/hormones, while rapamycin blocked that of insulin and partially prevented that of GLP-1. H-7 abolished the action of GLP-1, insulin and Ex-4, while Ro 31-8220 prevented only the Ex-4 and Ex-9 effect. In conclusion, GLP-1, like insulin, stimulates glucose uptake, and this involves activation of PI3K/PKB, p44/42 MAPKs, partially p70s6k, and possibly PKC; Ex-4 and Ex-9 both have GLP-1-like effect upon glucose transport, in which both share with GLP-1 an activation of PI3K/PKB--partially in the case of Ex-4--and p44/42 MAPKs but not p70s6k.     

Exendin-4 ameliorates motor neuron degeneration in cellular and animal models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by a progressive loss of lower motor neurons in the spinal cord. The incretin hormone, glucagon-like peptide-1 (GLP-1), facilitates insulin signaling, and the long acting GLP-1 receptor agonist exendin-4 (Ex-4) is currently used as an anti-diabetic drug. GLP-1 receptors are widely expressed in the brain and spinal cord, and our prior studies have shown that Ex-4 is neuroprotective in several neurodegenerative disease rodent models, including stroke, Parkinson's disease and Alzheimer's disease. Here we hypothesized that Ex-4 may provide neuroprotective activity in ALS, and hence characterized Ex-4 actions in both cell culture (NSC-19 neuroblastoma cells) and in vivo (SOD1 G93A mutant mice) models of ALS. Ex-4 proved to be neurotrophic in NSC-19 cells, elevating choline acetyltransferase (ChAT) activity, as well as neuroprotective, protecting cells from hydrogen peroxide-induced oxidative stress and staurosporine-induced apoptosis. Additionally, in both wild-type SOD1 and mutant SOD1 (G37R) stably transfected NSC-19 cell lines, Ex-4 protected against trophic factor withdrawal-induced toxicity. To assess in vivo translation, SOD1 mutant mice were administered vehicle or Ex-4 at 6-weeks of age onwards to end-stage disease via subcutaneous osmotic pump to provide steady-state infusion. ALS mice treated with Ex-4 showed improved glucose tolerance and normalization of behavior, as assessed by running wheel, compared to control ALS mice. Furthermore, Ex-4 treatment attenuated neuronal cell death in the lumbar spinal cord; immunohistochemical analysis demonstrated the rescue of neuronal markers, such as ChAT, associated with motor neurons. Together, our results suggest that GLP-1 receptor agonists warrant further evaluation to assess whether their neuroprotective potential is of therapeutic relevance in ALS.     

Divergent effects of GLP-1 analogs exendin-4 and exendin-9 on the expression of myosin heavy chain isoforms in C2C12 myotubes

Exendin 1-39 amide (Ex-4) and its truncated form exendin 9-39 amide (Ex-9) are peptides of non-mammalian nature, which act as an agonist and antagonist, respectively, of the glucagon-like peptide-1 (GLP-1) receptor in mammals. GLP-1 is an intestinal peptide that plays an important role in the regulation of glucose metabolism and glucose uptake in skeletal muscle; however, the effects of its two analogs (Ex-4 and Ex-9) on myofiber properties are still unclear. Here, we report the effects of Ex-4 and Ex-9 alone or in combination on the myosin heavy chain (MyHC) type composition and the glucose uptake capacity in differentiated C2C12 myotubes. Neither Ex-4 nor Ex-9 altered basal glucose uptake, whereas Ex-9 significantly increased insulin-stimulated glucose uptake, suggesting enhanced insulin sensitivity. The mRNA expression of MyHC I and 2A as well as the percentage of MyHC I protein was remarkably increased in Ex-9-treated myotubes. In contrast, Ex-4, alone or in combination with Ex-9, caused a significant reduction in MyHC 2A mRNA expression and the percentage of MyHC I protein. Consistent with the MyHC type switching peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α expression in myotubes was remarkably increased by Ex-9 yet was significantly inhibited by Ex-4. In addition, intracellular concentrations of free Ca²⁺ were increased in all treatment groups, but only Ex-9-treated myotubes showed higher calcineurin A protein content. Taken together, our data suggest that Ex-9 promotes oxidative differentiation in myotubes to improve cell insulin sensitivity, probably through calcineurin and PGC-1α mediated pathways.     

LncRNA OIP5-AS1 facilitates ox-LDL-induced endothelial cell injury through the miR-98-5p/HMGB1 axis

Cerebrovascular diseases have a high mortality and disability rate in developed countries. Endothelial cell injury is the main cause of atherosclerosis and cerebrovascular disease. Long non-coding RNA (lncRNA) has been proved to participate in the progression of endothelial cell. Our study aimed to develop the function of lncRNA opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell injury. The expression of OIP5-AS1, miR-98-5p and High-mobility group protein box-1 (HMGB1) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry were used to detect the cell proliferation and apoptosis. The levels of cyclinD1, Bcl-2 Associated X Protein (Bax), Cleaved-caspase-3, Toll like receptors 4 (TLR4), phosphorylation of p65 (p-P65), phosphorylation of nuclear factor-kappa B inhibitor α (p-IκB-α) and HMGB1 were measured by Western blot. The concentrations of Interleukin-6 (IL-6), Interleukin-1β (IL-1β) and Tumor necrosis factor-α (TNF-α) were detected by Enzyme-linked immunosorbent assay (ELISA). The production of Reactive oxygen species (ROS), Superoxide Dismutase (SOD) and malondialdehyde (MDA) was detected by the corresponding kit. The targets of OIP5-AS and miR-98-5p were predicted by starBase 3.0 and TargetScan and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The expression of OIP5-AS1 was upregulated, while miR-98-5p was downregulated in ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Functionally, knockdown of OIP5-AS1 induced proliferation and inhibited apoptosis, inflammatory injury and oxidative stress injury in ox-LDL-induced HUVEC cells. Interestingly, miR-98-5p was a target of OIP5-AS1 and miR-98-5p inhibition abolished the effects of OIP5-AS1 downregulation on ox-LDL-induced HUVECs injury. More importantly, miR-98-5p directly targeted HMGB1, and OIP5-AS1 regulated the expression of HMGB1 by sponging miR-98-5p. Finally, OIP5-AS1 regulated the TLR4/nuclear factor-kappa B (NF-κB) signaling pathway through miR-98-5p/HMGB1 axis. LncRNA OIP5-AS1 accelerates ox-LDL-induced endothelial cell injury through regulating HMGB1 mediated by miR-98-5p via the TLR4/NF-κB signaling pathway.

     

间歇运动对心梗大鼠心肌氧化应激和炎症的影响及其机制

目的:探讨间歇运动激活心肌梗死(MI)大鼠SIRT1-Nox4-ROS通路抑制心脏氧化应激和炎症的作用.方法:选取雄性SD大鼠30只,随机分为假手术对照(C)组,心肌梗死(MI)组和心梗+间歇运动(ME)组,每组10只.MI组采用心脏左冠状动脉前降支(LAD)结扎法,建立MI模型.C组大鼠实施假手术,ME组大鼠在MI手...     

Characteristic of GLP-1 effects on glucose metabolism in human skeletal muscle from obese patients

Direct effects of GLP-1, kinase-mediated, on glucose and lipid metabolism in rat and human extrapancreatic tissues, are amply documented and also changes in type-2 diabetic (T2D) patients. Here, we explored the characteristics of the GLP-1 action and those of its analogs Ex-4 and Ex-9, on muscle glucose transport (GT) and metabolism in human morbid obesity (OB), as compared with normal and T2D subjects. In primary cultured myocytes from OB, GT and glycogen synthase a (GSa) activity values were lower than normal, and comparable to those reported in T2D patients; GT was increased by either GLP-1 or Ex-9 in a more efficient manner than in normal or T2D, up to normal levels; the Ex-4 increasing effect on GSa activity was two times that in normal cells, while Ex-9 failed to modify the enzyme activity. In OB, the control value of all kinases analyzed - PI3K, PKB, MAPKs, and p70s6K - although lower than that in normal or T2D subjects, the cells maintained their response capability to GLP-1, Ex-4, Ex-9 and insulin, with some exceptions. GLP-1 and exendins showed a direct normalizing action in the altered glucose uptake and metabolism in the muscle of obese subjects, which in the case of GLP-1 could account, at least in part, for the reported restoration of the metabolic conditions of these patients after restrictive surgery.     

Exendin-4, a GLP-1 receptor agonist, directly induces adiponectin expression through protein kinase A pathway and prevents inflammatory adipokine expression

Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist that has been used as a drug injected subcutaneously for treatment of type 2 diabetes. Many studies have revealed molecular targets of Ex-4, but its influence on adipokines has not been determined. Our study showed that Ex-4 induced secretion of adiponectin into the culture medium of 3T3-L1 adipocytes. This effect of Ex-4 is due to increased adiponectin mRNA level through the GLP-1R. Both forskolin and 3-isobutyl-1-methylxanthine (IBMX), which may finally elevate cyclic adenosine monophosphate (cAMP) concentration, prevented the induction of adiponectin expression by Ex-4. Moreover, H89, a protein kinase A inhibitor, blocked the effect of Ex-4 on adiponectin. On the other hand, Ex-4 decreased the mRNA levels of inflammatory adipokines. The results indicate that Ex-4 directly promotes adiponectin secretion via the protein kinase A pathway in 3T3-L1 adipocytes and may ameliorate insulin resistance.     

蒙药绍沙-7味丸防治大鼠心肌缺血/再灌注损伤的作用

目的:探讨蒙药绍沙-7味丸对心肌缺血/再灌注损伤大鼠的防治作用及机制.方法:60只大鼠随机分成6组:假手术组、模型组、蒙药绍沙-7味丸低、中、高剂量组以及阳性药对照组,每组10只;蒙药绍沙-7味丸低、中、高剂量组分别灌胃0.4 g/kg、0.8 g/kg、1.6 g/kg蒙药绍沙-7味丸,阳性药对照组灌胃0.3 g/k...     

Exogenous 3-Deoxyglucosone-Induced Carbonyl and Oxidative Stress Causes β-Cells Dysfunction by Impairing Gut Permeability in Rats

3-Deoxyglucosone (3DG) is a highly reactive dicarbonyl species, and its accumulation evokes carbonyl and oxidative stress. Our recent data reveal the role of 3DG as an independent factor for the development of prediabetes and suggest that intestine could be its novel target tissue. The present study investigated whether exogenous 3DG increases intestinal permeability by triggering carbonyl and oxidative stress, thus contributing to β-cell dysfunction. Rats were administered 3DG for two weeks by gastric gavage. Then levels of insulin, ROS, MDA, SOD, NLRP3, TNF-α and IL-1β in blood plasma as well as the ROS level and content of TNF-α and IL-1β in pancreas were assessed. Also, the expression of E-cadherin and ZO-1 as well as levels of 3DG, protein carbonylation, ROS, TNF-α and IL-1β in colon were determined. The 3DG-treated rats showed an elevation in systemic oxidative stress (ROS, MDA and SOD) and in inflammation (TNF-α and IL-1β), decreased plasma insulin level 15 min after the glucose load, and increased levels of TNF-α, IL-1β and ROS in pancreatic tissue. In colon tissues of the 3DG-treated rats, decreased E-cadherin expression and increased ROS production as well as an elevation of TNF-α and IL-1β levels were observed. Interestingly, elevation of colon protein carbonylation was observed in the 3DG-treated rats that displayed 3DG deposition in colon tissues. We revealed for the first time that 3DG deposition in colon triggers carbonyl and oxidative stress and, as a consequence, impairs gut permeability. The enhanced intestinal permeability caused by 3DG deposition in colon results in systemic and pancreatic oxidative stress and inflammatory process, contributing to the development of β-cell dysfunction.     

Effects of Selenium-Enriched Agaricus blazei Murill on Liver Metabolic Dysfunction in Mice,a Comparison with Selenium-Deficient Agaricus blazei Murill and Sodium Selenite

In the present study, we investigated the effects of Se-enriched Agaricus blazei Murill (Se-AbM) on liver injury in mice induced by acute alcohol administration. Mice received ethanol (5 g/kg body weight (BW)) by gavage every 12 h for a total of 3 doses. Se-AbM was administrated before ethanol administration. Subsequent serum alanine aminotransferase (ALT) level, aspartate aminotransaminase (AST) level, maleic dialdehyde (MDA) level, hepatic total antioxidant status (TAOS), nuclear factor kappa B (NF-κB) level, polymorphonuclear cells (PMN) level, interleukin-1β (IL-1β) level, inducible nitric oxide synthase (iNOS) level, tumor necrosis factor-α (TNF-α) level, intercellular adhesion molecule 1 (ICAM-1), and cyclooxygenase-2 (COX-2) were determined by ELISA and immunohistochemistry, respectively. Se-AbM administration markedly (p?<?005) decreased serum ALT, AST, and MDA levels, hepatic IL-1β and TNF-α levels, as well as PMN infiltration and the expression of ICAM-1, COX-2, iNOS, and NF-κB compared with alcohol administration. In conclusion, we observed that Se-AbM supplementation could restrain the hepatic damage caused by acute alcohol exposure.     

Activation of glucagon-like peptide-1 receptor in microglia attenuates neuroinflammation-induced glial scarring via rescuing Arf and Rho GAP adapter protein 3 expressions after nerve injury

Rationale: The neuroinflammation is necessary for glial group initiation and clearance of damaged cell debris after nerve injury. However, the proinflammatory polarization of excessive microglia amplifies secondary injury via enhancing cross-talk with astrocytes and exacerbating neurological destruction after spinal cord injury (SCI). The glucagon-like peptide-1 receptor (GLP-1R) agonist has been previously shown to have a neuroprotective effect in neurodegeneration, whereas its potency in microglial inflammation after SCI is still unknown.Methods: The effect and mechanism of GLP-1R activation by exendin-4 (Ex-4) were investigated in in vitro cultured glial groups and in vivo in SCI mice. Alterations in the gene expression after GLP-1R activation in inflammatory microglia were measured using mRNA sequencing. The microglial polarization, neuroinflammatory level, and astrocyte reaction were detected by using western blotting, flow cytometry, and immunofluorescence. The recoveries of neurological histology and function were also observed using imaging and ethological examinations.Results: GLP-1R activation attenuated microglia-induced neuroinflammation by reversing M1 subtypes to M2 subtypes in vitro and in vivo. In addition, activation of GLP-1R in microglia blocked production of reactive astrocytes. We also found less neuroinflammation, reactive astrocytes, corrected myelin integrity, ameliorated histology, and improved locomotor function in SCI mice treated with Ex-4. Mechanistically, we found that Ex-4 rescued the RNA expression of Arf and Rho GAP adapter protein 3 (ARAP3). Knockdown of ARAP3 in microglia reversed activation of RhoA and the pharmacological effect of Ex-4 on anti-inflammation in vitro.Conclusion: Ex-4 exhibited a previously unidentified role in reducing reactive astrocyte activation by mediation of the PI3K/ARAP3/RhoA signaling pathway, by neuroinflammation targeting microglia, and exerted a neuroprotective effect post-SCI, implying that activation of GLP-1R in microglia was a therapeutical option for treatment of neurological injury.     

胰高血糖素样肽-1抑制高糖诱导的新生大鼠心室肌细胞氧化应激效应

本文旨在观察胰高血糖素样肽-1(glucagon like peptide-1,GLP-1)对高糖所致新生大鼠心室肌细胞氧化应激的影响,并探讨PI3K-Akt通路在其中所起的作用。将酶消化法分离的经α-肌动蛋白免疫荧光法鉴定的原代培养72~96h的新生大鼠心室肌细胞分为5组:正常对照组、高糖组、高糖+GLP-1组、高糖+GLP-1+LY294002(PI3K-Akt通路的抑制剂)组、高渗对照组。采用硫代巴比妥酸显色法测定细胞上清液中丙二醛(malonaldehyde,MDA)含量,用黄嘌呤氧化酶法测定细胞上清液中超氧化物歧化酶(superoxide dismutase,SOD)的活性,运用PCR凝胶电泳检测NADPH P47phox亚基mRNA的变化,运用荧光显微镜及流式细胞术检测心室肌细胞内的ROS含量,运用流式细胞术及DNAladder法检测心室肌细胞的凋亡,Western blot检测各组心肌细胞Akt磷酸化水平。结果显示:(1)与正常对照组相比,高糖组细胞生长状态较差,搏动频率减慢甚至消失,细胞凋亡率显著增加,DNA ladder呈现凋亡独有的梯状图谱,胞浆MDA水平增高,上清液中SOD活性下降,心肌...