Activation of adenosine A1 receptor attenuates tumor necrosis factor-α induced hypertrophy of cardiomyocytes

Tumor necrosis factor (TNF)-α has been implicated in the pathogenesis of cardiac hypertrophy, while the activation of adenosine receptors has been shown to exert antihypertrophic effect on the heart. However, it remains unknown whether adenosine can attenuate hypertrophy induced by TNF-α. This study was aimed to address this issue using transverse aortic constriction (TAC) mouse models and cultured neonatal rat cardiomyocytes. Plasma TNF-α was significantly increased in hypertrophied hearts (Sham vs TAC group: 46.8 ± 2.5 vs 67.0 ± 1.6 pg/ml, P = 0.021), while myocardial TNF-α level, expression of TNF receptor 1 and TNF-α-converting enzyme were positively correlated with heart weight to body weight ratio (r = 0.930, 0.676 and 0.891, respectively, P < 0.01-0.05). Myocardial adenosine levels were increased significantly at 4 weeks (Sham vs TAC group: 16.15 ± 1.59 vs 86.54 ± 13.49 nmol/mg protein, P < 0.01) and decreased from 6 to 11 weeks after TAC. N6-cyclopentyladenosine, an adenosine A1 receptor agonist inhibited protein synthesis of cardiomyocytes induced by TNF-α in a dose-dependent manner. This antihypertrophic effect could not be mimicked by agonists of A2a, A2b and A3 adenosine receptors. These findings indicate that TNF-α signal system plays important role in the process of cardiac hypertrophy, and activation of adenosine receptor 1 inhibits hypertrophy of cardiomyocytes induced by TNF-α.     

Minocycline attenuates brain tissue levels of TNF-α produced by neurons after prolonged hypothermic cardiac arrest in rats

Neuro-cognitive disabilities are a well-recognized complication of hypothermic circulatory arrest. We and others have reported that prolonged cardiac arrest (CA) produces neuronal death and microglial proliferation and activation that are only partially mitigated by hypothermia. Microglia, and possibly other cells, are suggested to elaborate tumor necrosis factor alpha (TNF-α), which can trigger neuronal death cascades and exacerbate edema after CNS insults. Minocycline is neuroprotective in some brain ischemia models in part by blunting the microglial response. We tested the hypothesis that minocycline would attenuate neuroinflammation as reflected by brain tissue levels of TNF-α after hypothermic CA in rats. Rats were subjected to rapid exsanguination, followed by a 6 min normothermic CA. Hypothermia (30 °C) was then induced by an aortic saline flush. After a total of 20 min CA, resuscitation was achieved via cardiopulmonary bypass (CPB). After 5 min reperfusion, minocycline (90 mg kg⁻¹; n = 6) or vehicle (PBS; n = 6) was given. Hypothermia (34 °C) was maintained for 6 h. Rats were sacrificed at 6 or 24 h. TNF-α was quantified (ELISA) in four brain regions (cerebellum, CEREB; cortex, CTX; hippocampus, HIP; striatum, STRI). Naïve rats (n = 6) and rats subjected to the same anesthesia and CPB but no CA served as controls (n = 6). Immunocytochemistry was used to localize TNF-α. Naïve rats and CPB controls had no detectable TNF-α in any brain region. CA markedly increased brain TNF-α. Regional differences were seen, with the highest TNF-α levels in striatum in CA groups (10-fold higher, P < 0.05 vs. all other brain regions). TNF-α was undetectable at 24 h. Minocycline attenuated TNF-α levels in CTX, HIP and STRI (P < 0.05). TNF-α showed unique co-localization with neurons. In conclusion, we report region-dependent early increases in brain TNF-α levels after prolonged hypothermic CA, with maximal increases in striatum. Surprisingly, TNF-α co-localized in neurons and not microglia. Minocycline attenuated TNF-α by approximately 50% but did not totally ablate its production. That minocycline decreased brain TNF-α levels suggests that it may represent a therapeutic adjunct to hypothermia in CA neuroprotection.     

Study of hemodynamics during cardiac arrest and after restoration of spontaneous circulation in a porcine cardiac arrest model induced by acute pulmonary embolism北大核心CSCD

Objective: To observe the hemodynamic change during cardiac arrest (CA) and after restoration of spontaneous circulation (ROSC) in a porcine acute pulmonary embolism model. Methods: A total of 14 inbred Beijing Landraces were used to estalish the model of CA and ROSC induced by acute pulmonary embolism through injection of thrombus followed by cardiopulmonary resuscitation and thrombolytic therapy (urokinase, 15 000 U/kg, iv). Five resuscitated pigs restored spontaneous circulation. Hemodynamic changes were determined at baseline, CA, ROSC, and 0.5, 1, 1.5, 2, 2.5, 4, and 6 h after ROSC. Results: Compared with the baseline, mean arterial pressure was decreased significantly, mean pulmonary arterial pressure and right ventricular pressure were increased significantly, and the heart rate had no change during CA induced by acute pulmonary embolism. The mean arterial pressure restored normal level gradually after ROSC, but was decreased at 4 h after ROSC compared with the baseline (P<0.05). The heart rate was faster at ROSC and 0.5-2 h after ROSC than the baseline (P<0.05). The mean pulmonary arterial pressure restored the baseline level after ROSC; The right ventricular pressure were decreased at 2.5 h (26.5±11.4) mmHg and 4 h (24.8±9.3) mmHg after ROSC compared with the level during CA (46.2± 13.01) mmHg (P<0.05). The systemic vascular resistance peaked at 4 h after ROSC. The pulmonary vascular resistance level at ROSC was higher than the baseline [(96.5±24.8)DS/cm5 vs. (26.5±13.4)DS/cm5, P<0.05], and was decreased at 1 h and 2 h after ROSC, but was increased at 4 h and 6 h after ROSC [(98.5±0.7)DS/cm5 and (98.0±1.4)DS/cm5]. The changes of heart function: compared with the baseline, the left ventricular function at ROSC and 1-6 h after ROSC were declined significantly (all P<0.05), and right cardiac output declined at ROSC and 4 h and 6 h after ROSC (all P<0.05), and the level of cardiac function index was dropped at 1 h and 2 h after ROSC (P<0.05). Conclusions: The mean arterial pressure was declined, mean pulmonary arterial pressure, right ventricular pressure and pulmonary vascular resistance were increased, cardiac function was decreased during CA induced by acute pulmonary embolism; After ROSC, hemodynamic changes were described as compensated in the early stage (1-2 h after ROSC) and decompensated (4 h after ROSC) with time. © 2018 Chinese Medical Association. All rights reserved.     

Liposome‐mediated transfection with extract from neonatal rat cardiomyocytes induces transdifferentiation of human adipose‐derived stem cells into cardiomyocytes

Objective. Recent studies indicate that direct cell‐to‐cell interaction is involved in transdifferentiation of adult stem cells into cardiomyocytes. We investigated whether transdifferentiation of human adipose‐tissue‐derived stem cells could be achieved by transfecting the cells with a nuclear neonatal cardiomyocyte extract using a liposome‐based transfection system. Material and methods. In this study, we isolated stem cells derived from human subcutaneous adipose tissue. These cells were transfected with nuclear protein extracts from either isolated cardiomyocytes or whole hearts of neonatal rats. Results. We found that transfection induced expression of the cardiac markers α‐sarcomeric actin, Nk×2.5, troponin I and troponin T after 1–3 weeks. Whole‐heart protein extracts showed the additional capacity to induce differentiation into endothelial‐like and smooth muscle‐like cells. Conclusion. We demonstrate that transfection with nuclear protein extracts from neonatal rat cardiomyocytes can induce a cardiomyogenic differentiation pathway in human stem cells.     

Angiotensin II type 1 receptor antagonists prevent glucose‐induced increases in islet blood flow in rats

Objective. Renin‐angiotensin system inhibitors are reported to be beneficial in delaying the onset of diabetes mellitus. Since islet blood hyperperfusion during hyperglycaemia may be detrimental to endothelium in pancreatic islets and eventually lead to β‐cell dysfunction, we studied acute and chronic effects of angiotensin II type 1 receptor blockers (ARB) on islet blood flow before and after glucose load. Material and methods. Islet blood flow was measured using the colour microsphere method in anaesthetized Sprague‐Dawley rats before and 3?min after glucose injection. Results. Olmesartan significantly reduced blood pressure, but did not affect islet blood flow 10?min after its injection. However, pretreatment with olmesartan blunted the glucose‐induced increase in islet blood flow (62?% of control). In rats treated with olmesartan or candesartan for 4 weeks, islet blood flow was not different from untreated control, whereas the glucose‐induced increase in islet blood flow was significantly suppressed in chronically ARB‐treated rats (olmesartan 59?% of control, candesartan 64?% of control, respectively). Acute or chronic treatment with ARB did not change insulin secretion before and in response to glucose load. Pancreatic or duodenal blood flow was not affected by ARB treatment, although acute olmesartan administration reduced pancreatic blood flow after glucose load. Conclusion. ARB appears to suppress the hyperglycaemia‐induced islet hyperperfusion, which may ameliorate haemodynamic stress in pancreatic islets.     

Apoptosis of neuronal cells induced by serum of patients with acute brain injury: a new in vitro prognostic model

Objective To investigate whether serum draining from the jugular bulb of patients with traumatic or haemorrhagic brain injury induced apoptosis of neuronal PC12 cells in vitro and whether the apoptotic rate correlated with patients' outcome at 6 months. Design and setting Prospective clinical investigation in a 21-bed intensive care unit (ICU) in a university hospital. Patients Seventy patients who had suffered from acute brain injury requiring intensive care. Interventions Jugular bulb vein and systemic samples were obtained on admission to the ICU and after 48 h. PC12 cells were incubated in the presence of 10% of heat-inactivated patient's sera and apoptotic rate was determined by flow cytometry using annexin V and 7-aminoactinomycin D. Results Regional serum draining from the lesions induced higher early apoptosis of PC12 cells than systemic serum. Early apoptotic rate, Glasgow coma score, APACHE II score and the presence of pupil abnormalities were associated with mortality at 6 months in univariate statistical analyses. In logistic regression analysis only early apoptotic rate was an independent factor associated with mortality at 6 months (odds ratio: 1.502, 95% CI 1.2–1.9; p < 0.001). The final model has a sensitivity of 82.4% and a specificity of 84.8% for predicting death within 6 months. Conclusions We developed a simple and reproducible in vitro model for predicting outcome in patients with traumatic or haemorrhagic brain injury that survived in the early phase. Our in vitro model combined with clinical and radiological measurements might improve the value of prognostic models to predict acute brain injury patients' outcome.     

Paeoniflorin attenuates cardiac dysfunction in endotoxemic mice via the inhibition of nuclear factor-κB

The impaired cardiac function caused by reduced myocardial contractility is a typical manifestation of sepsis/septic shock. Paeoniflorin (Pae) has reportedly exhibited anti-inflammatory effect and protection against LPS-induced cardiac dysfunction in mice, but the molecular mechanism is still not fully understood. This study was designed to investigate the protective effects of Pae on lipopolysaccharide (LPS)-induced septic cardiac dysfunction and inflammation response in mice. Mice were intraperitoneal injection with Pae (15 mg/kg) for 3d before the LPS challenge (10 mg/kg, i.p.). Pae significantly protected against LPS-induced cardiac dysfunction and damage. Pae decreased production of inflammatory cytokines, e.g., TNF-α, IL-1β, IL-6, IL-12, MCP-1, IFN-γ, and inducible nitric oxide synthase (iNOS), in the heart of LPS-treated mice. Furthermore, Pae prevented NF-κB activation in endotoxemic mice. Pae pretreatment preserved the level of phospho-Akt. Pae effectively improved cardiac function during endotoxemia in mice. This action is attributed to Pae-induced reduction of inflammatory cytokine release and NF-κB activation, which possibly occurred via the activation PI3K/Akt signaling.     

Amelioration of streptozotocin‐induced diabetes mellitus,oxidative stress and dyslipidemia in rats by tomato extract lycopene

The effects of various doses of lycopene were studied in streptozotocin (STZ)‐induced hyperglycaemic rats to evaluate its possible hypoglycaemic, hypolipidaemic and antioxidant activity in diabetes. Compared to the normoglycaemic group, the treatment of rats with a single dose of STZ (65?mg/kg body weight) revealed a significant increase (p<0.05) only in plasma hydrogen peroxide (H₂O₂), i.e. by 230?%; it increased the thiobarbituric acid reactive substances (TBARS) as index of the lipid peroxidation level by 69?%, while total antioxidant activity was decreased by 36?%, with a consistently significant decrease (p<0.05) in the activity of erythrocytes antioxidative enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx). The levels of total lipid, triglycerides and total cholesterol in serum of hyperglycaemic rats were increased by 14?%, 65?% and 36?%, respectively, while HDL‐C decreased by 22?% compared to the normoglycaemic group. Exogenous administration of individual gradual doses of lycopene to hyperglycaemic rats causes a dose‐dependent decrease in glucose level, an increase of insulin concentration, a decrease of H₂O₂ and TBARS levels, as well as increased total antioxidant status with increased antioxidant enzyme activities (CAT, SOD and GPx) with improvement in serum lipid profile. It is obvious from this study that lycopene acts as an antidiabetic agent through lowering the free radical and has an improving effect on serum that reaches the normal level; the greatest effect of lycopene was observed at 90?mg/kg body weight.     

Study on adenovirus mediated antisense p38α fragment gene in suppressing apoptosis of the myocardium of neonatal rat by burn serum and hypoxia

Objective To investigate the relationship between the activation of p38 mitogen-activated protein kinase (p38MAPK) in the myocardium and the apoptosis in the presence of burn serum and hypoxia. Methods Ventricular myocardium isolated from neonatal rats were employed in this study, and they were divided into three groups as the normal control group, with the myocardium grew naturally; burn serum+ hypoxia group, in which the myocardium was stimulated by the serum collected from the rat 6 hours after burn injury involving 40% of total body surface area (TBSA), and at the same time exposed to 1%O2, 5% CO2, and 94 %N2; antisense blocking group, in which rats were pretreated by AD-antisense (AS) p38α, then exposed to the same conditions as burn serum+hypoxia group.The phosphorylation of p38 in the myocardium was determined by Western blotting.The level of myocardium apoptosis was determined by DNA ladder and flow cytometry.Results Compared with normal control group, the level of phosphorylation of p38 (gray value) was markedly increased (8.68±0.14 vs.3.21±0.05, P＜0.01＝ after being exposed to burn serum and hypoxia, and at the same time myocardium apoptosis was strikingly increased[(50.367±0.451)% vs.(2.063±0.111)%, P＜0.01＝.When the myocardium was transfected by AD-ASp38α, the phosphorylation of p38 (gray level) was decreased remarkably (5.46±0.16 vs.8.68±0.14, P＜0.01＝, the rate of the apoptosis was lowered remarkably[(13.200 ± 0.121 ) % vs.(50.367 ± 0.451)%, P ＜ 0.01]. Conclusion Burn serum combined with hypoxia can induce apoptosis of the myocardium by activating p38MAPK;blockage of p38MAPK signal transduction pathway may lessen the damage of the myocardium in early period of severe burn.     

A novel role for the cyclin-dependent kinase inhibitor p27Kip1 in angiotensin II–stimulated vascular smooth muscle cell hypertrophy

Angiotensin II (Ang II) has been shown to stimulate either hypertrophy or hyperplasia. We postulated that the differential response of vascular smooth muscle cells (VSMCs) to Ang II is mediated by the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1), which is abundant in quiescent cells and drops after serum stimulation. Ang II treatment (100 nM) of quiescent VSMCs led to upregulation of the cell-cycle regulatory proteins cyclin D1, Cdk2, proliferating cell nuclear antigen, and Cdk1. p27(Kip1) levels, however, remained high, and the activation of the G1-phase Cdk2 was inhibited as the cells underwent hypertrophy. Overexpression of p27(Kip1) cDNA inhibited serum-stimulated [(3)H]thymidine incorporation compared with control-transfected cells. This cell-cycle inhibition was associated with cellular hypertrophy, as reflected by an increase in the [(3)H]leucine/[(3)H]thymidine incorporation ratio and by an increase in forward-angle light scatter during flow cytometry at 48 hours after transfection. The role of p27(Kip1) in modulating the hypertrophic response of VSMCs to Ang II was further tested by antisense oligodeoxynucleotide (ODN) inhibition of p27(Kip1) expression. Ang II stimulated an increase in [(3)H]thymidine incorporation and the percentage of S-phase cells in antisense ODN-transfected cells but not in control ODN-transfected cells. We conclude that p27(Kip1) plays a role in mediating VSMC hypertrophy. Ang II stimulation of quiescent cells in which p27(Kip1) levels are high results in hypertrophy but promotes hyperplasia when levels of p27(Kip1) are low, as in the presence of other growth factors.     

Identification of a contractile deficit in adult cardiac myocytes expressing hypertrophic cardiomyopathy–associated mutant troponin T proteins

The direct effects of expressing hypertrophic cardiomyopathy-associated (HCM-associated) mutant troponin T (TnT) proteins on the force generation of single adult cardiac myocytes have not been established. Replication-defective recombinant adenovirus vectors were generated for gene transfer of HCM-associated I79N and R92Q mutant cardiac TnT cDNAs into fully differentiated adult cardiac myocytes in primary culture. We tested the hypothesis that the mutant TnT proteins would be expressed and incorporated into the cardiac sarcomere and would behave as dominant-negative proteins to directly alter calcium-activated force generation at the level of the single cardiac myocyte. Interestingly, under identical experimental conditions, the ectopic expression of the mutant TnTs was significantly less ( approximately 8% of total) than that obtained with expression of wild-type TnT ( approximately 35%) in the myocytes. Confocal imaging of immunolabeled TnT showed a regular periodic pattern of localization of ectopic mutant TnT that was not different than that in normal controls, suggesting that mutant TnT incorporation had no deleterious effects on sarcomeric architecture. Direct measurements of isometric force production in single cardiac myocytes demonstrated marked desensitization of submaximal calcium-activated tension, with unchanged maximum tension generation in mutant TnT-expressing myocytes compared with control myocytes. Collectively, these results demonstrate an impaired expression of the mutant protein and a disabling of cardiac contraction in the submaximal range of myoplasmic calcium concentrations. Our functional results suggest that development of new pharmacological, chemical, or genetic approaches to sensitize the thin-filament regulatory protein system could ameliorate force deficits associated with expression of I79N and R92Q in adult cardiac myocytes.     

Study of the relationship between brain injury and glucose metabolism in rat model of cardiac arrest北大核心CSCD

Objective To determine the relationship between brain injury and cerebral glucose metabolism in rat model of cardiac arrest. Methods Asphyxia-induced cardiac arrest model was established. Forty-two male Wistar rats were randomly assigned to sham or experimental groups. Rats in the CA4,CA6 and CA8 group were treated with cardiopulmonary resuscitation(CPR) 4 min, 6 min and 8 min after cardiac arrest, respectively. The maximum standardized uptake value (SUV^J of glucose was detected by PET, and neural deficit score (NDS) were evaluated at 24 h and 72 h after ROSC. The numbers of injured neurons and apoptotic cells and the protein level of hexokmase I (HXK I) were measured at 72 h after ROSC. Results SUV, NDS and the level of HXK I were all decreased after ROSC, and interestingly, this declination of these markers was conelated with the prolongation of the duration of CA, the longer duration of CA the more declination of these biomarkers. Accordingly, the number of injured neurons and apoptotic cells increased were correlated with duration of CA, and thus CA8 group had greater numbers of those cells than CA6 group and CA4 group (P<0.05),and CA6 group had greater numbers of those cells than CA4 group(P<0.05). In addition, the SUV, was positively correlated with NDS (P<0.05), and negatively correlated with the numbers of injured neurons and apoptotic index(P<0.05). Conclusions The degree of brain injury is associated with cerebral glucose metabolism, and PET may become a novel method to assess the severity of brain damage after CA. © 2018 Chinese Medical Association. All rights reserved.     

Effects of diammonium glycyrrhizinate on the expression of Toll-like receptor 4 in rat ' s kidney with acute kidney injury induced by paraquat北大核心CSCD

Objective To investigate the effects of diammonium glycyrrhizinate (DG) on the expression of Toll-like receptor 4 in rat's kidney poisoned by paraquat (PQ). Methods The PQ (100 mg/kg) were administered via a gastric tube to make an animal model of acute kidney injury in rats. Then DG (50 mg/kg per 24 h) were administered immediately by intraperitoneal injection in the DG group. The ELISA assay was applied to measuring the IL-6 and IL-10 in kidneys and in blood. And the protein expressions of TLR-4, Myd88 and NF-κB P65 were examined by Immunohistochemistry. The gene expressions of TLR-4, Myd88 and NF-κB on kidneys were detected by RT-PCR. Results The levels of serum Cr, BUN, IL-6 and renal IL-6 in PQ group were significantly higher than those in NS group (F = 266.014, 168. 160, 63.279, 192.997, respectively, P<0.01). While, the levels of serum Cr, BUN, IL-6 and renal IL-6 in DG group were significantly lower than those in PQ group (F = 266. 014, 168. 160, 63.279, 192.997, respectively, P<0.01). And the levels of serum and renal IL-10 in PQ group were higher than those in NS group (F = 87. 511, 79. 473, respectively, P < 0.01). But the levels of serum and renal IL-10 in DG group were lower than those in PQ group (F = 87. 511, 79.473, respectively, P < 0.01). The results of PCR confirmed that the expressions of TLR-4 mRNA, myd88 mRNA and NF-κB mRNA in PQ group were higher than those in NS group (F = 84. 408, 60. 683, 86. 272, respectively, P < 0.01), while, the expressions of TLR4 mRNA, myd88 mRNA and NF-κB mRNA in DG group were lower than those in PQ group (F = 84. 408, 60.683, 86.272, respectively, P<0.01). The protein levels of TLR4, Myd88 and NF-κB P65 in PQ group were higher than those in NS group (F = 79. 139, 61.799, 112. 740, respectively, P < 0.01). While the protein levels of TLR-4, Myd88 and NF-κB P65 in DG group were lower than those in PQ group (F = 79. 139, 61.799, 112.740, respectively, P < 0.01). Conclusions Diammonium Glycyrrhizinate can attenuate rat ' s acute kidney injury caused by paraquat poisoning and decrease the expressions of TLR-4, Myd88 and NF-κB.     

Expression of Toll‐like receptors 2 and 4 on innate immunity cells modulated by cardiac surgical operation

Objective. This study aimed to follow‐up on the changes in the expression of Toll‐like receptors (TLRs) on monocytes and granulocytes in venous blood of patients undergoing cardiac surgical operation. Material and methods. TLR2 and TLR4 expression on blood cells was determined by flow cytometry in 40 patients undergoing cardiac surgery performed either with cardiopulmonary bypass (CPB) (“on‐pump”) or without it (“off‐pump”). Results. Intensity of the expression of TLR2 on both monocytes and granulocytes, expressed as median fluorescence intensity, is significantly reduced during CPB, being lower in both groups at the finish of surgery. These changes are not so remarkable in the case of TLR4 expression. Compared to “on‐pump” patients, there is a higher relative number of TLR2⁺ granulocytes in “off‐pump” patients at the finish of surgery and of TLR4⁺ granulocytes on the first postoperative day. Conclusions. We found characteristic patterns in the expression of TLR2 and TLR4 on monocytes and granulocytes in venous blood of patients undergoing cardiac surgery with or without CPB.     

The acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl– channels

Bradykinin (BK) is an inflammatory mediator and one of the most potent endogenous pain-inducing substances. When released at sites of tissue damage or inflammation, or applied exogenously, BK produces acute spontaneous pain and causes hyperalgesia (increased sensitivity to potentially painful stimuli). The mechanisms underlying spontaneous pain induced by BK are poorly understood. Here we report that in small nociceptive neurons from rat dorsal root ganglia, BK, acting through its B₂ receptors, PLC, and release of calcium from intracellular stores, robustly inhibits M-type K⁺ channels and opens Ca²⁺-activated Cl^– channels (CaCCs) encoded by Tmem16a (also known as Ano1). Summation of these two effects accounted for the depolarization and increase in AP firing induced by BK in DRG neurons. Local injection of inhibitors of CaCC and specific M-channel openers both strongly attenuated the nociceptive effect of local injections of BK in rats. These results provide a framework for understanding spontaneous inflammatory pain and may suggest new drug targets for treatment of such pain.