Background. Excessive autophagy is a major mechanism of myocardial ischemia reperfusion injury (I/RI) in diabetes with enhanced oxidative stress. Antioxidant N-acetylcysteine (NAC) reduces myocardial I/RI. It is unknown if inhibition of autophagy may represent a mechanism whereby NAC confers cardioprotection in diabetes. Methods and Results. Diabetes was induced in Sprague-Dawley rats with streptozotocin and they were treated without or with NAC (1.5 g/kg/day) for four weeks before being subjected to 30-minute coronary occlusion and 2-hour reperfusion. The results showed that cardiac levels of 15-F2t-Isoprostane were increased and that autophagy was evidenced as increases in ratio of LC3 II/I and protein P62 and AMPK and mTOR expressions were significantly increased in diabetic compared to nondiabetic rats, concomitant with increased postischemic myocardial infarct size and CK-MB release but decreased Akt and eNOS activation. Diabetes was also associated with increased postischemic apoptotic cell death manifested as increases in TUNEL positive cells, cleaved-caspase-3, and ratio of Bax/Bcl-2 protein expression. NAC significantly attenuated I/RI-induced increases in oxidative stress and cardiac apoptosis, prevented postischemic autophagy formation in diabetes, and reduced postischemic myocardial infarction (all p < 0.05). Conclusions. NAC confers cardioprotection against diabetic heart I/RI primarily through inhibiting excessive autophagy which might be a major mechanism why diabetic hearts are less tolerant to I/RI.
BACKGROUND: Postoperative acute lung injury (ALI) is a severe complication after liver transplantation, which severely affects postoperative patients' survival. The underlying mechanism is largely unknown and effective treatment limited. We explored the role of serpin protease inhibitor B1 (SERPINB1), a potent inhibitor of neutrophil serine proteases, in ALI in liver transplantation and its interplay with signal transducer and activator of transcription 3 (STAT3) and heme oxygenase-1 (HO-1). METHODS: Sprague-Dawley rats underwent orthotopic autologous liver transplantation (OALT) were treated with recombinant SB1 (rSB1) in the absence or presence of STAT3 specific inhibitor, WP1066. Then SB1-siRNA was used to knockdown endogenous SERPINB1. Also, alveolar epithelial cells RLE-6TN and BEAS-2B were exposed to TNF-alpha without or with SERPINB1 and the roles of STAT3 and HO-1 were examined by respective gene knockdown. Finally, rats were treated with ERK1/2 inhibitor U0126, p38 MAPK inhibitor SB20358, or JNK inhibitor SP600125 after rSB1 pretreatment and then subjected to OALT. RESULTS: OALT resulted in increased pulmonary inflammation and oxidative stress, accompanied by severe lung injury that was coincident with increased pulmonary SERPINB1, HO-1, and STAT3. SERPINB1 gene knockdown increased post-OALT lung injury and pulmonary inflammation. rSB1 administration dose-dependently reduced post-OALT lung injury and decreased pulmonary inflammation and oxidative stress with concomitant enhanced HO-1 and STAT3 protein expression. These protective effects of SERPINB1 were abolished by STAT3 inhibition. Similarly, in RLE-6TN cells and BEAS-2B cells, TNF-alpha induced cell injury and increased HO-1 and STAT3. SERPINB1 further increased HO-1 and STAT3 protein expression and attenuated TNF-alpha-induced cellular oxidative stress, apoptotic cells, and mitochondria damage, which were cancelled by STAT3 or HO-1 gene knockdown. Furthermore, these SERPINB1-mediated STAT3/HO-1 activation and pulmonary protective effects were abolished by inhibition of ERK1/2 but not p38 MAPK or JNK. CONCLUSIONS: SERPINB1 decreased inflammation, ameliorated oxidative stress in the lung, and attenuated ALI in rats with OALT by activating HO-1 and it does so through STAT3 and it does so by activating ERK1/2.
Isoflurane postconditioning (IsoPostC) attenuates myocardial ischaemia/reperfusion injury (IRI). Signal transducer and activator of transcription-3 (STAT3) is critical in ischaemic postconditioning cardioprotection, which can be regulated by the Brahma-related gene (Brg1) and nuclear factor-erythroid 2-related factor 2 (Nrf2), although they are both reduced in diabetic hearts. We hypothesized that reduced Brg1/Nrf2 and STAT3 activation may jeopardize IsoPostC-mediated cardioprotection in diabetic hearts. In the present study, Langendorff-perfused, non-diabetic (control) and 8-week-old streptozotocin-induced Type 1 diabetic rat hearts were subjected to 30 min of global ischaemia and 120 min of reperfusion without or with IsoPostC, which was achieved by administering emulsified isoflurane (2.0%, v/v) in Krebs-Henseleit (KH) solution immediately at the onset of reperfusion for 10 min and switching to KH solution perfusion alone thereafter. Cultured H9C2 cells were exposed to normal glucose (NG, 5.5 mM) or high glucose (HG, 30 mM) and subjected to hypoxia/reoxygenation (HR) in the presence or absence of IsoPostC. Diabetic rats displayed larger post-ischaemic myocardial infarction and more severe haemodynamic dysfunction, associated with increased myocardial oxidative stress and reduced cardiac Brg1, Nrf2 and STAT3 phosphorylation/activation (p-STAT3), compared with controls. These changes were reversed/prevented by IsoPostC in control but not in diabetic rats. In H9C2 cells exposed to NG but not HG, IsoPostC significantly attenuated HR-induced cellular injury and superoxide anion production with increased Brg1, Nrf2 and p-STAT3. These beneficial effects of IsoPostC were abolished by Brg1, Nrf2 or STAT3 gene knockdown. Brg1 or Nrf2 gene knockdown abolished IsoPostC-induced STAT3 activation. N-acetylcysteine restored Brg1, Nrf2 and p-STAT3, and IsoPostC-induced protection in H9C2 cells exposed to HG and HR. In conclusion, IsoPostC confers cardioprotection through Brg1/Nrf2/STAT3 signalling, and impairment of this pathway may be responsible for the loss of IsoPostC cardioprotection in diabetes.
The present study explored the potential causal link between ischemia-driven cyclooxygenase-2 (COX-2) expression and enhanced apoptosis during myocardial ischemia/reperfusion (I/R) by using H9C2 cardiomyocytes and primary rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R). The results showed that H/R resulted in higher COX-2 expression than that of controls, which was prevented by pretreatment with Helenalin (NFkappaB specific inhibitor). Furthermore, pretreatment with NS398 (COX-2 specific inhibitor) significantly attenuated H/R-induced cell injury [lower lactate dehydrogenase (LDH) leakage and enhanced cell viability] and apoptosis (higher Bcl2 expression and lower level of cleaved caspases-3 and TUNEL-positive cells) in cardiomyocytes. The amelioration of posthypoxic apoptotic cell death was paralleled by significant attenuation of H/R-induced increases in proinflammatory cytokines [interleukin 6 (IL6) and tumor necrosis factor (TNFalpha)] and reactive oxygen species (ROS) production and by higher protein expression of phosphorylated Akt and inducible nitric oxide synthase (iNOS) and enhanced nitric oxide production. Moreover, the application of LY294002 (Akt-specific inhibitor) or 1400W (iNOS-selective inhibitor) cancelled the cellular protective effects of NS398. Findings from the current study suggest that activation of NFkappaB during cardiomyocyte H/R induces the expression of COX-2 and that higher COX-2 expression during H/R exacerbates cardiomyocyte H/R injury via mechanisms that involve cross talks among inflammation, ROS, and Akt/iNOS/NO signaling.
Sevofluane postconditioning (SPostC) protects heart against ischemia/reperfusion injury. However, SPostC cardioprotection is lost in diabetes whose cardiac heme oxygenase-1 (HO-1) is reduced. Brahma-related gene 1 (Brg1) facilitates nuclear factor-erythroid-2-related factor-2 (Nrf2) to activate HO-1 to increase myocardial antioxidant capacity in response to oxidative stress. However, cardiac Brg1 is reduced in diabetes. We hypothesized that SPostC confers cardioprotection by activating HO-1 through Nrf2/Brg1 and that impaired Nrf2/Brg1/HO-1 in diabetes is responsible for the loss of SPostC. Control and streptozotocin-induced diabetic mice were subjected to 45min coronary artery occlusion followed by 2h reperfusion with or without SPostC achieved by exposing the mice to 2% sevoflurane for 15min at the onset of reperfusion. In invitro study, H9c2 cells were exposed to normal or high glucose and subjected to 3h hypoxia followed by 6h reoxygenation. Diabetic mice displayed larger post-ischemic infarct size, severer cardiomyocytes apoptosis, and increased oxidative stress concomitant with reduced HO-1, nuclear Nrf2 and Brg1 protein expression. These changes were prevented/reversed by SPostC in control but not in diabetic mice, and these beneficial effects of SPostC were abolished by HO-1 inhibition. In H9c2 cells exposed to normal glucose but not high glucose, SPostC significantly attenuated hypoxia/reoxygenation-induced cellular injury and oxidative stress with increased HO-1 and nuclear Nrf2. These SPostC beneficial effects were canceled by HO-1 inhibition. In conclusion, SPostC protects against myocardial ischemia/reperfusion injury through activation of Nrf2/Brg1/HO-1 signaling and impairment of this signaling may be responsible for the loss of SPostC cardioprotection in diabetes.
Diabetic cardiomyopathy (DCM) is a disorder of the heart muscle in people with diabetes that can occur independent of hypertension or vascular disease. The underlying mechanism of DCM is incompletely understood. Some transcription factors have been suggested to regulate the gene program intricate in the pathogenesis of diabetes prompted cardiac injury. Forkhead box transcription factor 1 is a pleiotropic transcription factor that plays a pivotal role in a variety of physiological processes. Altered FOXO1 expression and function have been associated with cardiovascular diseases, and the important role of FOXO1 in DCM has begun to attract attention. In this review, we focus on the FOXO1 pathway and its role in various processes that have been related to DCM, such as metabolism, oxidative stress, endothelial dysfunction, inflammation and apoptosis.
Signal transducer and activator of transcription 3 (STAT3) activation is key for ischemic postconditioning (IPo) to attenuate myocardial ischemia-reperfusion injury (MIRI), but IPo loses cardioprotection in diabetes in which cardiac STAT3 activation is impaired and adiponectin (APN) reduced. We found that IPo increased postischemic cardiomyocyte-derived APN, activated mitochondrial STAT3 (mitoSTAT3), improved mitochondrial function, and attenuated MIRI in wild-type but not in APN knockout (Adipo(-/-)) mice subjected to 30 min coronary occlusion, followed by 2 or 24 h of reperfusion. Hypoxic postconditioning-induced protection against hypoxia/reoxygenation injury was lost in Adipo(-/-) cardiomyocytes but restored by recombinant APN, but this APN beneficial effect was abolished by specific STAT3 or APN receptor 1 (AdipoR1) gene knockdown, or caveolin-3 (Cav3) disruption. APN activated cardiac STAT3 and restored IPo cardioprotection in 4-week diabetic rats where AdipoR1 and Cav3 were functionally interactive but not in 8-week diabetic rats whose cardiac Cav3 was severely reduced and AdipoR1/Cav3 signaling impaired. We concluded that IPo activates mitoSTAT3 through APN/AdipoR1/Cav3 pathway to confer cardioprotection, whereas in diabetes, IPo loses cardioprotection due to impaired APN/AdipoR1/Cav3 signaling. Therefore, effective means that may concomitantly activate APN and repair APN signaling (i.e., AdipoR1/Cav3) in diabetes may represent promising avenues in the treatment of MIRI in diabetes.
Bupivacaine, a commonly used local anesthetic, has potential neurotoxicity through diverse signaling pathways. However, the key mechanism of bupivacaine-induced neurotoxicity remains unclear. Cultured human SH-SY5Y neuroblastoma cells were treated (bupivacaine) or untreated (control) with bupivacaine for 24 h. Compared to the control group, bupivacaine significantly increased cyto-inhibition, cellular reactive oxygen species, DNA damage, mitochondrial injury, apoptosis (increased TUNEL-positive cells, cleaved caspase 3, and Bcl-2/Bax), and activated autophagy (enhanced LC3II/LC3I ratio). To explore changes in protein expression and intercommunication among the pathways involved in bupivacaine-induced neurotoxicity, an 8-plex iTRAQ proteomic technique and bioinformatics analysis were performed. Compared to the control group, 241 differentially expressed proteins were identified, of which, 145 were up-regulated and 96 were down-regulated. Bioinformatics analysis of the cross-talk between the significant proteins with altered expression in bupivacaine-induced neurotoxicity indicated that phosphatidyl-3-kinase (PI3K) was the most frequently targeted protein in each of the interactions. We further confirmed these results by determining the downstream targets of the identified signaling pathways (PI3K, Akt, FoxO1, Erk, and JNK). In conclusion, our study demonstrated that PI3K may play a central role in contacting and regulating the signaling pathways that contribute to bupivacaine-induced neurotoxicity.
Oxidative stress plays a critical role in the pathogenesis of intestinal ischemia reperfusion (IIR) injury. Enhancement in endogenous Lipoxin A4 (LXA4), a potent antioxidant and mediator, is associated with attenuation of IIR. However, the effects of LXA4 on IIR injury and the potential mechanisms are unknown. In a rat IIR (ischemia 45 minutes and subsequent reperfusion 6 hours) model, IIR caused intestinal injury, evidenced by increased serum diamine oxidase, D-lactic acid, intestinal-type fatty acid-binding protein, and the oxidative stress marker 15-F2t-Isoprostane. LXA4 treatment significantly attenuated IIR injury by reducing mucosal 15-F2t-Isoprostane and elevating endogenous antioxidant superoxide dismutase activity, accompanied with Keap1/Nrf2 pathway activation. Meanwhile, LXA4 receptor antagonist Boc-2 reversed the protective effects of LXA4 on intestinal injury but failed to affect the oxidative stress and the related Nrf2 pathway. Furthermore, Nrf2 antagonist brusatol reversed the antioxidant effects conferred by LXA4 and led to exacerbation of intestinal epithelium cells oxidative stress and apoptosis, finally resulting in a decrease of survival rate of rat. Meanwhile, LXA4 pretreatment upregulated nuclear Nrf2 level and reduced hypoxia/reoxygenation-induced IEC-6 cell damage and Nrf2 siRNA reversed this protective effect of LXA4 in vitro. In conclusion, these findings suggest that LXA4 ameliorates IIR injury by activating Keap1/Nrf2 pathway in a LXA4 receptor independent manner.
The effect of sevoflurane postconditioning (sevo-postC) cardioprotection is compromised in diabetes which is associated with increased oxidative stress. We hypothesized that antioxidant N-Acetylcysteine may enhance or restore sevo-postC cardioprotection in diabetes. Control or streptozotocin-induced Type 1 diabetic rats were either untreated or treated with N-Acetylcysteine for four weeks starting at five weeks after streptozotocin injection and were subjected to myocardial ischemia-reperfusion injury (IRI), in the absence or presence of sevo-postC. Diabetes showed reduction of cardiac STAT3 activation (p-STAT3) and adiponectin with concomitantly increase of FoxO1 and CD36, which associated with reduced sevo-postC cardioprotection. N-Acetylcysteine and sevo-postC synergistically reduced the infarct size in diabetic groups. N-Acetylcysteine remarkably increased cardiac p-STAT3 which was further enhanced by sevo-postC. N-Acetylcysteine but not sevo-postC decreased myocardial FoxO1 while sevo-postC but not N-Acetylcysteine significantly increased myocardiac adiponectin in diabetic rats. It is concluded that late stage diabetic rats displayed reduction of cardiac p-STAT3, adiponectin deficiency, and increase of FoxO1 and CD36 expression, which may be responsible for the loss of myocardial responsiveness to sevo-postC cardioprotection. N-Acetylcysteine restored Sevo-postC cardioprotection in diabetes possibly through enhancing cardiac p-STAT3 and adiponectin and reducing Fox1 and CD36.
Necrosis amplifies inflammation and plays important roles in acute respiratory distress syndrome (ARDS). Necroptosis is a newly identified programmed necrosis that is mediated by receptor interacting protein 3 (RIP3). However, the potential involvement and impact of necroptosis in lipopolysaccharide (LPS)-induced ARDS remains unknown. We therefore explored the role and mechanism of RIP3-mediated necroptosis in LPS-induced ARDS. Mice were instilled with increasing doses of LPS intratracheally to induce different degrees of ARDS. Lung tissues were harvested for histological and TUNEL staining and western blot for RIP3, p-RIP3, X-linked inhibitor of apoptosis protein (XIAP), mixed lineage kinase domain-like protein (MLKL), total and cleaved caspases-3/8. Then, wild-type and RIP3 knock-out mice were induced ARDS with 30 mg/kg LPS. Pulmonary cellular necrosis was labeled by the propidium Iodide (PI) staining. Levels of TNF-a, Interleukin (IL)-1beta, IL-6, IL-1alpha, IL-10 and HMGB1, tissue myeloperoxidase (MPO) activity, neutrophil counts and total protein concentration were measured. Results showed that in high dose LPS (30mg/kg and 40mg/kg) -induced severe ARDS, RIP3 protein was increased significantly, accompanied by increases of p-RIP3 and MLKL, while in low dose LPS (10mg/kg and 20mg/kg) -induced mild ARDS, apoptosis was remarkably increased. In LPS-induced severe ARDS, RIP3 knock-out alleviated the hypothermia symptom, increased survival rate and ameliorated the lung tissue injury RIP3 depletion also attenuated LPS-induced increase in IL-1alpha/beta, IL-6 and HMGB1 release, decreased tissue MPO activity, and reduced neutrophil influx and total protein concentration in BALF in severe ARDS. Further, RIP3 depletion reduced the necrotic cells in the lung and decreased the expression of MLKL, but had no impact on cleaved caspase-3 in LPS-induced ARDS. It is concluded that RIP3-mediated necroptosis is a major mechanism of enhanced inflammation and lung tissue injury in high dose LPS- induced severe ARDS in mice.
BACKGROUND: Recent clinical and animal studies suggested that remote limb ischemic postconditioning (RIPostC) can invoke potent cardioprotection or neuroprotection. However, the effect and mechanism of RIPostC against renal ischemia/reperfusion injury (IRI) are poorly understood. T-LAK-cell-originated protein kinase (TOPK) is crucial for the proliferation and migration of tumor cells. However, the function of TOPK and the molecular mechanism underlying renal protection remain unknown. Therefore, this study aimed to evaluate the role of TOPK in renoprotection induced by RIPostC. MATERIALS AND METHODS: The renal IRI model was induced by left renal pedicle clamping for 45min followed by 24h reperfusion and right nephrectomy. All mice were intraperitoneally injected with vehicle, TOPK inhibitor HI-TOPK-032 or Akt inhibitor LY294002. After 24h reperfusion, renal histology, function, and inflammatory cytokines and oxidative stress were assessed. The proteins were measured by Western blotting. RESULTS: The results showed that RIPostC significantly protected the kidneys against IRI. The protective effects were accompanied by the attenuation of renal dysfunction, tubular damage, inflammation and oxidative stress. In addition, RIPostC increased the phosphorylation of TOPK, PTEN, Akt, GSK3beta and the nuclear translocation of Nrf2 and decreased the nuclear translocation of NF-kappaB. However, all of the above renoprotective effects of RIPostC were eliminated either by the inhibition of TOPK or Akt with HI-TOPK-032 or LY294002. CONCLUSIONS: The current data reveal that RIPostC protects against renal IRI via activation of TOPK/PTEN/Akt signaling pathway mediated anti-oxidation and anti-inflammation.
Hyperglycemia-induced oxidative stress is implicated in the development of cardiomyopathy in diabetes that is associated with reduced adiponectin (APN) and heme oxygenase-1 (HO-1). Brahma-related gene 1 (Brg1) assists nuclear factor-erythroid-2-related factor-2 (Nrf2) to activate HO-1 to increase myocardial antioxidant capacity in response to oxidative stress. We hypothesized that reduced adiponectin (APN) impairs HO-1 induction which contributes to the development of diabetic cardiomyopathy, and that supplementation of APN may ameliorate diabetic cardiomyopathy by activating HO-1 through Nrf2 and Brg1 in diabetes. Control (C) and streptozotocin-induced diabetic (D) rats were untreated or treated with APN adenovirus (1x10(9) pfu) 3 weeks after diabetes induction and examined and terminated 1 week afterward. Rat left ventricular functions were assessed by a pressure-volume conductance system, before the rat hearts were removed to perform histological and biochemical assays. Four weeks after diabetes induction, D rats developed cardiac hypertrophy evidenced as increased ratio of heart weight to body weight, elevated myocardial collagen I content, and larger cardiomyocyte cross-sectional area (all P<0.05 vs C). Diabetes elevated cardiac oxidative stress (increased 15-F2t-isoprostane, 4-hydroxynonenal generation, 8-hydroxy-2'-deoxyguanosine, and superoxide anion generation), increased myocardial apoptosis, and impaired cardiac function (all P<0.05 vs C). In D rats, myocardial HO-1 mRNA and protein expression were reduced which was associated with reduced Brg1 and nuclear Nrf2 protein expression. All these changes were either attenuated or prevented by APN. In primarily cultured cardiomyocytes (CMs) isolated from D rats or in the embryonic rat cardiomyocytes cell line H9C2 cells incubated with high glucose (HG, 25 mM), supplementation of recombined globular APN (gAd, 2mug/mL) reversed HG-induced reductions of HO-1, Brg1, and nuclear Nrf2 protein expression and attenuated cellular oxidative stress, myocyte size, and apoptotic cells. Inhibition of HO-1 by ZnPP (10muM) or small interfering RNA (siRNA) canceled all the above gAd beneficial effects. Moreover, inhibition of Nrf2 (either by the Nrf2 inhibitor luteolin or siRNA) or Brg1 (by siRNA) canceled gAd-induced HO-1 induction and cellular protection in CMs and in H9C2 cells incubated with HG. In summary, our present study demonstrated that APN reduced cardiac oxidative stress, ameliorated cardiomyocyte hypertrophy, and prevented left ventricular dysfunction in diabetes by concomitantly activating Nrf2 and Brg1 to facilitate HO-1 induction.
BACKGROUND: Pretreatment with the angiotensin-converting inhibitor captopril or volatile anesthetic isoflurane has, respectively, been shown to attenuate myocardial ischemia reperfusion (MI/R) injury in rodents and in patients. It is unknown whether or not captopril pretreatment and isoflurane preconditioning (Iso) may additively or synergistically attenuate MI/R injury. METHODS AND RESULTS: Patients selected for heart valve replacement surgery were randomly assigned to five groups: untreated control (Control), captopril pretreatment for 3 days (Cap3d), or single dose captopril (Cap1hr, 1 hour) before surgery with or without Iso (Cap3d+Iso and Cap1hr+Iso). Rabbit MI/R model was induced by occluding coronary artery for 30 min followed by 2-hour reperfusion. Rabbits were randomized to receive sham operation (Sham), MI/R (I/R), captopril (Cap, 24 hours before MI/R), Iso, or the combination of captopril and Iso (Iso+Cap). In patients, Cap3d+Iso but not Cap1hr+Iso additively reduced postischemic myocardial injury and attenuated postischemic myocardial inflammation. In rabbits, Cap or Iso significantly reduced postischemic myocardial infarction. Iso+Cap additively reduced cellular injury that was associated with improved postischemic myocardial functional recovery and reduced myocardial apoptosis and attenuated oxidative stress. CONCLUSION: A joint use of 3-day captopril treatment and isoflurane preconditioning additively attenuated MI/R by reducing oxidative stress and inflammation.
Clinical evidence shows that circulating levels of adipocyte fatty-acid-binding protein (A-FABP) are elevated in patients with diabetes and closely associated with ischaemic heart disease. Patients with diabetes are more susceptible to myocardial ischaemia/reperfusion (MI/R) injury. The experiments in the present study investigated the role of A-FABP in MI/R injury with or without diabetes. Non-diabetic and diabetic (streptozotocin-induced) A-FABP knockout and wild-type mice were subjected to MI/R or sham intervention. After MI/R, A-FABP knockout mice exhibited reductions in myocardial infarct size, apoptotic index, oxidative and nitrative stress, and inflammation. These reductions were accompanied by an improved left ventricular function compared with the relative controls under non-diabetic or diabetic conditions. After diabetes induction, A-FABP knockout mice exhibited a preserved cardiac function compared with that in wild-type mice. Endothelial cells, but not cardiomyocytes, were identified as the most likely source of cardiac A-FABP. Cardiac and circulating A-FABP levels were significantly increased in mice with diabetes or MI/R. Diabetes-induced superoxide anion production was significantly elevated in wild-type mice, but diminished in A-FABP knockout mice, and this elevation contributed to the exaggeration of MI/R-induced cardiac injury. Phosphorylation of endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) were enhanced in both diabetic and non-diabetic A-FABP knockout mice after MI/R injury, but diminished in wild-type mice. The beneficial effects of A-FABP deficiency on MI/R injury were abolished by the NOS inhibitor N(G)-nitro-L-arginine methyl ester. Thus, A-FABP deficiency protects mice against MI/R-induced and/or diabetes-induced cardiac injury at least partially through activation of the eNOS/NO pathway and reduction in superoxide anion production.
Activation of PKCbeta (protein kinase Cbeta) plays a critical role in myocardial I/R (ischaemia/reperfusion) injury in non-diabetic rodents. In the myocardium of diabetes, PKCbeta2 overexpression is associated with increased vulnerability to post-ischaemic I/R injury with concomitantly impaired cardiomyocyte Cav (caveolin)-3 and Akt signalling compared with non-diabetic rats. We hypothesized that myocardial PKCbeta overexpression in diabetes exacerbates myocardial I/R injury through impairing Cav-3/Akt signalling. Streptozotocin-induced diabetic rats were treated with the selective PKCbeta inhibitor ruboxistaurin (RBX, 1 mg/kg per day) for 4 weeks, starting from 1 week after diabetes induction, before inducing myocardial I/R achieved by occluding the left descending coronary artery followed by reperfusion. Cardiac function was measured using a pressure-volume conductance system. In an in vitro study, cardiac H9C2 cells were exposed to high glucose (30 mmol/l) and subjected to hypoxia followed by reoxygenation (H/R) in the presence or absence of the selective PKCbeta2 inhibitor CGP53353 (1 mumol/l), siRNAs of PKCbeta2 or Cav-3 or Akt. Cell apoptosis and mitochondrial membrane potential were assessed by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) and JC-1 staining respectively. RBX significantly decreased post-ischaemic myocardial infarct size (35+/-5% compared with 49+/-3% in control, P<0.05) and attenuated cardiac dysfunction, and prevented the reduction in cardiac Cav-3 and enhanced phosphorylated/activated Akt (p-Akt) in diabetic rats (P<0.05). H/R increased cardiomyocyte injury under high glucose conditions as was evident by increased TUNEL-positive and increased JC-1 monomeric cells (P<0.05 compared with control), accompanied with increased PKCbeta2 phosphorylation/activation and decreased Cav-3 expression. Either CGP53353 or PKCbeta2 siRNA significantly attenuated all of these changes and enhanced p-Akt. Cav-3 gene knockdown significantly reduced p-Akt and increased post-hypoxic cellular and mitochondrial injury despite a concomitant reduction in PKCbeta2 phosphorylation. PKCbeta2 inhibition with RBX protects diabetic hearts from myocardial I/R injury through Cav-3-dependent activation of Akt.
Inhalation anesthetic isoflurane inhibits hypoxia pulmonary vasoconstriction (HPV), while dexmedetomidine (Dex) could reduce the dose of isoflurane inhalation and potentiate HPV, but the mechanism is unclear. Inhibition of reactive oxygen species (ROS) production can favor HPV during one-lung ventilation (OLV). Similarly, nitric oxide (NO), an important endothelium-derived vasodilator in lung circulation, can decrease the regional pulmonary vascular resistance of ventilated lung and reduce intrapulmonary shunting. We hypothesized that Dex may augment HPV and improve oxygenation during OLV through inhibiting oxidative stress and increasing NO release. Patients undergoing OLV during elective thoracic surgery were randomly allocated to either isoflurane + saline (NISO, n = 24) or isoflurane + dexmedetomidine (DISO, n = 25) group. Anesthesia was maintained with intravenous remifentanil and inhalational isoflurane (1.0-2.0%), with concomitant infusion of dexmedetomidine 0.7 mugkg(-1)h(-1) in DISO and saline 0.25 mL kg(-1)h(-1) in NISO group. Hemodynamic variables or depth of anesthesia did not significantly differ between groups. Administration of Dex significantly reduced Qs/Qt and increased PaO2 after OLV, accompanied with reduced lipid peroxidation product malondialdehyde and higher levels of SOD activity as well as serum NO (all P < 0.05 DISO versus NISO). In conclusion, reducing oxidative stress and increasing NO release during OLV may represent a mechanism whereby Dex potentiates HPV.
BACKGROUND: Postliver transplantation acute kidney injury (AKI) severely affects patient survival, whereas the mechanism is unclear and effective therapy is lacking. The authors postulated that reperfusion induced enhancement of connexin32 (Cx32) gap junction plays a critical role in mediating postliver transplantation AKI and that pretreatment/precondition with the anesthetic propofol, known to inhibit gap junction, can confer effective protection. METHODS: Male Sprague-Dawley rats underwent autologous orthotopic liver transplantation (AOLT) in the absence or presence of treatments with the selective Cx32 inhibitor, 2-aminoethoxydiphenyl borate or propofol (50 mg/kg) (n = 8 per group). Also, kidney tubular epithelial (NRK-52E) cells were subjected to hypoxia-reoxygenation and the function of Cx32 was manipulated by three distinct mechanisms: cell culture in different density; pretreatment with Cx32 inhibitors or enhancer; Cx32 gene knock-down (n = 4 to 5). RESULTS: AOLT resulted in significant increases of renal Cx32 protein expression and gap junction, which were coincident with increases in oxidative stress and impairment in renal function and tissue injury as compared to sham group. Similarly, hypoxia-reoxygenation resulted in significant cellular injury manifested as reduced cell growth and increased lactate dehydrogenase release, which was significantly attenuated by Cx32 gene knock-down but exacerbated by Cx32 enhancement. Propofol inhibited Cx32 function and attenuated post-AOLT AKI. In NRK-52E cells, propofol reduced posthypoxic reactive oxygen species production and attenuated cellular injury, and the cellular protective effects of propofol were reinforced by Cx32 inhibition but cancelled by Cx32 enhancement. CONCLUSION: Cx32 plays a critical role in AOLT-induced AKI and that inhibition of Cx32 function may represent a new and major mechanism whereby propofol reduces oxidative stress and subsequently attenuates post-AOLT AKI.
Both oxidative stress and mast cell (MC) degranulation participate in the process of small intestinal ischemia reperfusion (IIR) injury, and oxidative stress induces MC degranulation. Propofol, an anesthetic with antioxidant property, can attenuate IIR injury. We postulated that propofol can protect against IIR injury by inhibiting oxidative stress subsequent from NADPH oxidase mediated MC activation. Cultured RBL-2H3 cells were pretreated with antioxidant N-acetylcysteine (NAC) or propofol and subjected to hydrogen peroxide (H2O2) stimulation without or with MC degranulator compound 48/80 (CP). H2O2 significantly increased cells degranulation, which was abolished by NAC or propofol. MC degranulation by CP further aggravated H2O2 induced cell degranulation of small intestinal epithelial cell, IEC-6 cells, stimulated by tryptase. Rats subjected to IIR showed significant increases in cellular injury and elevations of NADPH oxidase subunits p47(phox) and gp91(phox) protein expression, increases of the specific lipid peroxidation product 15-F2t-Isoprostane and interleukin-6, and reductions in superoxide dismutase activity with concomitant enhancements in tryptase and beta-hexosaminidase. MC degranulation by CP further aggravated IIR injury. And all these changes were attenuated by NAC or propofol pretreatment, which also abrogated CP-mediated exacerbation of IIR injury. It is concluded that pretreatment of propofol confers protection against IIR injury by suppressing NADPH oxidase mediated MC activation.
Endothelial dysfunction induced by oxidative stress and inflammation plays a critical role in the pathogenesis of cardiovascular diseases. The anesthetic sevoflurane confers cytoprotective effects through its anti-inflammatory properties in various pathologies such as systemic inflammatory response syndrome and ischemic-reperfusion injury but mechanism is unclear. We hypothesized that sevoflurane can protect against tumor necrosis factor (TNF)-alpha-induced endothelial dysfunction through promoting the production of endothelium-dependent nitric oxide (NO). Primary cultured human umbilical vein endothelial cells (HUVECs) were pretreated with different concentrations (0.5, 1.5 and 2.5 minimum alveolar concentration, MAC) of sevoflurane for 30 min before TNF-alpha (10 ng/mL) stimulation for 4 h. Sevoflurane pretreatment significantly reduced TNF-alpha-induced VCAM-1, ICAM-1, IkappaBalpha, and NF-kappaB activation, and blocked leukocytes adhesion to HUVECs. Meanwhile, sevoflurane (1.5 and 2.5 MAC) significantly induced endothelial nitric oxide synthase (eNOS) phosphorylation and enhanced NO levels both intracellularly and in the cell culture medium. All these cytoprotective effects of sevoflurane were abrogated by NG-nitro-l-arginine methyl ester (l-NAME), a non-specific nitric oxide synthase inhibitor. Collectively, these data indicate that sevoflurane protects against TNF-alpha -induced vascular endothelium dysfunction through activation of eNOS/NO pathway and inhibition of NF-kappaB.