Effects of MDL 72527, a Specific Inhibitor of Polyamine Oxidase, on Brain Edema, Ischemic Injury Volume, and Tissue Polyamine Levels in Rats After Temporary Middle Cerebral After Occlusion

Abstract The possible effects of the polyamine interconversion pathway on tissue polyamine levels, brain edema formation, and ischemic injury volume were studied by using a selective irreversible inhibitor, MDL 72527, of the interconversion pathway enzyme, polyamine oxidase. In an intraluminal suture occlusion model of middle coerebral artery in spontaneously hypertensive rats, 100 mg/kg MDL 72527 changed the brain edema formation from 85.7 ± 0.3 to 84.5 ± 0.9% in cortex ( P < 0.05) and from 79.9 ± 1.7 to 78.4 ± 2.0% in subcortex (difference not significant). Ischemic injury volume was reduced by 22% in the cortex ( P < 0.05) and 17% in the subcortex ( P < 0.05) after inhibition of polyamine oxidase by MDL 72527. There was an increase in tissue putrescine levels together with a decrease in spermine and spermidine levels at the ischemic site compared with the nonischemic site compared with the nonischemic site after ischemia-reperfusion injury. The increase in putrescine levels at the ischemic cortical and subcortical region was reduced by a mean of 45% with MDL 72527 treatment. These results suggest that the polyamine interconversion pathway has an important role in the postischemic increase ini putrescine levels and that blocking of this pathway can be neuroprotective against neuronal cell damage after temporary focal cerebral ischemia.     

The anti-inflammatory and anti-apoptotic effects of nesfatin-1 in the traumatic rat brain

Nesfatin-1 has been demonstrated to possess anti-inflammatory and anti-apoptotic effects in the rat brain with subarachnoid hemorrhage. The study was designed to investigate the influence of nesfatin-1 on inflammatory responses and neuronal cell apoptosis after traumatic brain injury. Wistar rats were subjected to 5, 10 or 20 μg/kg of nesfatin-1 at designed time points (0.5, 2, 4 or 8h after head trauma) intraperitoneally. Rats were sacrificed at hours 2, 6 and 12, as well as day 1, 2, 3 and 5 after head trauma. The administration of 10 or 20 μg/kg of nesfatin-1 at hour 0.5 after head trauma could significantly suppress gene expressions of nuclear factor kappa-B, lessen concentrations of tumor necrosis factor-alpha, interleukin-1beta and interleukin-6, diminish caspase-3 activity as well as reduce number of apoptotic neuronal cells in traumatic rat brain tissues (P<0.05), but the administration of 5 μg/kg of nesfatin-1 not (P>0.05). Moreover, 20 μg/kg nesfatin-1 also significantly suppressed the inflammation and neuronal cell apoptosis when applied 2, 4 or 8h after head trauma. However, a clear concentration-response or time-response relationship was not found. These findings suggest that nesfatin-1 may inhibit nuclear factor kappa-B-dependent inflammatory responses, and lessen caspase-3-mediated neuronal cell apoptosis after traumatic brain injury in rats.     

NRG-1/ErbB4通路与脑缺血再灌注损伤细胞凋亡关系及电针干预作用

神经通路在电针治疗脑缺血再灌注损伤（CIRI）领域研究日渐深入。NRG-1/ErbB4通路是神经调节素（NRG）与其ErbB受体组成的一条在细胞的增殖分化以及神经系统发育等生命过程中起着关键作用的神经信号传导通路,前期研究发现该通路与神经发育异常疾病、心力衰竭、心肌梗死以及癌症等疾病密切相关。近年,国内外研究发现CIRI后细胞凋亡与该通路以及Caspase-3、NF-κB、Bcl-2/Bax等因子的调控有关。本文综述NRG-1/ErbB4通路以及相关凋亡因子在CIRI治疗中的研究进展。     

Suppression of REDD1 attenuates oxygen glucose deprivation/reoxygenation-evoked ischemic injury in neuron by suppressing mTOR-mediated excessive autophagy

Cerebral ischemia/reperfusion (I/R) typically occurs after mechanical thrombectomy to treat ischemic stroke, generation of reactive oxygen species (ROS) after reperfusion may result in neuronal insult, ultimately leading to disability and death. Regulated in development and DNA damage responses 1 (REDD1) is a conserved stress response protein under various pathogenic conditions. Recent research confirms the controversial role of REDD1 in injury processes. Nevertheless, the role of REDD1 in cerebral I/R remains poorly defined. In the current study, increased expression of REDD1 was observed in neurons exposed to simulated I/R via oxygen glucose deprivation/reoxygenation (OGD/R) treatment. Knockdown of REDD1 enhanced OGD/R-inhibited cell viability, but suppressed lactate dehydrogenase (LDH) release in neurons upon OGD/R. Simultaneously, suppression of REDD1 also antagonized OGD/R-evoked cell apoptosis, Bax expression, and caspase-3 activity. Intriguingly, REDD1 depression abrogated neuronal oxidative stress under OGD/R condition by suppressing ROS, MDA generation, and increasing antioxidant SOD levels. Further mechanism analysis corroborated the excessive activation of autophagy in neurons upon OGD/R with increased expression of autophagy-related LC3 and Beclin-1, but decreased autophagy substrate p62 expression. Notably, REDD1 inhibition reversed OGD/R-triggered excessive neuronal autophagy. More importantly, depression of REDD1 also elevated the expression of p-mTOR. Preconditioning with mTOR inhibitor rapamycin engendered not only a reduction in mTOR activation, but also a reactivation of autophagy in REDD1 knockdown-neurons upon OGD/R. In addition, blocking the mTOR pathway muted the protective roles of REDD1 inhibition against OGD/R-induced neuron injury and oxidative stress. Together these data suggested that REDD1 may regulate I/R-induced oxidative stress injury in neurons by mediating mTOR-autophagy signaling, supporting a promising therapeutic strategy against brain ischemic diseases.     

早期跑步运动对大鼠脑缺血/再灌注损伤后神经行为与神经元凋亡的影响

目的: 探讨早期跑步运动对大鼠脑缺血后神经行为与神经元凋亡的影响。方法:雄性SD大鼠随机分为4组:假手术+安静组(Sham-St、假手术+运动组(Sham-Ex)、缺血(大脑中动脉闭塞(MCAO) +运动组((MCAO -Ex)和缺血+安静组(MCAO-St),每组15只。MCAO-Ex 和 MCAO-St 组大鼠行MCAO 60 min,再灌注2 d后,MCAO-Ex 和Sham-Ex大鼠在跑步机上进行5 d的30 min/d跑步运动(15 m/min),之后进行神经行为学评价,最后大鼠断头取脑进行TTC方法染色,评估各组大鼠梗死体积以及缺血半影Caspase-3和TUNEL阳性细胞表达水平。结果:与Sham-St相比,MCAO-St和MCAO-Ex大鼠缺血半影区Caspase-3表达均显著升高 (P＜0.05);与MCAO-St 组大鼠相比,MCAO-Ex组大鼠脑梗死体积明显减少,大鼠神经功能评分明显改善,大鼠缺血半影区Caspase-3和TUNEL阳性细胞表达水平显著降低 (P＜0.05)。结论: 早期运动可能通过抑制大鼠脑缺血后神经元凋亡发挥神经保护作用。     

Neuroglobin expression and oxidant/antioxidant balance after graded traumatic brain injury in the rat

Neuroglobin is a neuron-specific hexacoordinated globin capable of binding various ligands, including O₂, NO, and CO, the biological function of which is still uncertain. Various studies seem to indicate that neuroglobin is a neuroprotective agent when overexpressed, acting as a potent inhibitor of oxidative and nitrosative stress. In this study, we evaluated the pathophysiological response of the neuroglobin gene and protein expression in the cerebral tissue of rats sustaining traumatic brain injury of differing severity, while simultaneously measuring the oxidant/antioxidant balance. Two levels of trauma (mild and severe) were induced in anesthetized animals using the weight-drop model of diffuse axonal injury. Rats were then sacrificed at 6, 12, 24, 48, and 120 h after traumatic brain injury, and the gene and protein expression of neuroglobin and the concentrations of malondialdehyde (as a parameter representative of reactive oxygen species-mediated damage), nitrite + nitrate (indicative of NO metabolism), ascorbate, and glutathione (GSH) were determined in the brain tissue. Results indicated that mild traumatic brain injury, although causing a reversible increase in oxidative/nitrosative stress (increase in malondialdehyde and nitrite + nitrate) and an imbalance in antioxidants (decrease in ascorbate and GSH), did not induce any change in neuroglobin. Conversely, severe traumatic brain injury caused an over nine- and a fivefold increase in neuroglobin gene and protein expression, respectively, as well as a remarkable increase in oxidative/nitrosative stress and depletion of antioxidants. The results of this study, showing a lack of effect in mild traumatic brain injury as well as asynchronous time course changes in neuroglobin expression, oxidative/nitrosative stress, and antioxidants in severe traumatic brain injury, do not seem to support the role of neuroglobin as an endogenous neuroprotective antioxidant agent, at least under pathophysiological conditions.     

MicroRNA-449b-5p targets HMGB1 to attenuate hepatocyte injury in liver ischemia and reperfusion

MicroRNAs (miRNAs) participate in the pathological process of liver ischemia/reperfusion (I/R) injury. MiR-449b-5p is the target miRNA of high mobility group box 1 (HMGB1). Its role and molecular mechanism in liver I/R injury remain unidentified. In this study, we found a protective effect of miR-449b-5p against hepatic I/R injury. HMGB1 expression significantly increased, whereas miR-449b-5p dramatically decreased in patients after liver transplant and in L02 cells exposed to hypoxia/reoxygenation (H/R). A dual-luciferase reporter assay confirmed the direct interaction between miR-449b-5p and the 3′ untranslated region of HMGB1 messenger RNA. We also found that overexpression of miR-449b-5p significantly promoted cell viability and inhibited cell apoptosis of L02 cells exposed to H/R. Moreover, miR-449b-5p repressed HMGB1 protein expression and nuclear factor-κB (NF-κB) pathway activation in these L02 cells. In an in vivo rat model of hepatic I/R injury, overexpression of miR-449b-5p significantly decreased alanine aminotransferase and aspartate aminotransferase and inhibited the HMGB1/NF-κB pathway. Our study thus suggests that miR-449b-5p alleviated hepatic I/R injury by targeting HMGB1 and deactivating the NF-κB pathway, which may provide a novel and promising therapeutic target for hepatic I/R injury.     

高铁血红素对心肌缺血／再灌注损伤的防护作用及其机制

目的：在大鼠急性心肌缺血／再灌ii（I／R）模型上,观察高铁血红素在钙激活中性蛋白酶（calpain）介导的心肌I／R损伤中的作用。并初步探讨其可能的机制。方法：64只雄性SD大鼠随机8组（n：8）：假手术组（sham组）、（I／R）组、MDL28170＋I／R组、单纯MDL28170组、高铁血红素＋I／R组、单纯高铁血红素组、锌原卟啉Ⅸ＋高铁血红素＋I／R组、单纯锌原卟啉Ⅸ组。采用大鼠离体心脏Langendorff灌流技术,心脏I／R后,测定左室发展压（LVDP）、心肌梗死面积、冠脉流出液中的乳酸脱氢酶（LDH）释放量。检测calpain、血红素氧化酶（HO）、和半胱氨酸天冬氨酸蛋白酶3（caspase3）活性。Westernblot观察心肌钙蛋白酶抑制蛋白（calpastatin）蛋白表达。结果：①心肌I／R后,calpain、caspase3活性明显增高。calpain抑制剂MDL28170可抑制I／R诱导的LDH释放量增加,增高LVDP,缩小心肌梗死面积。②与单纯I／R组相比,大鼠预先给予高铁血红素后,心脏HO-1活性增加,calpain和caspase3活性下降。同时,LDH释放量减少,LVDP明显增高,心肌梗死面积缩小。③I／R组心肌calpastatin表达量明显低于对照组,高铁血红素组大鼠calpastatin表达量增高。HO-1的抑制剂锌原卟啉Ⅸ可取消高铁血红素对calpastain表达量的影响,并取消其心肌保护作用。结论：高铁血红素预处理可通过抑制calpain的激活,减轻大鼠心肌I／R损伤,其机制可能与增加calpastatin蛋白表达有关。     

甘草查尔酮A对局灶性脑缺血再灌注小鼠Nrf2/HO-1信号通路和神经炎症反应的影响

目的:探讨甘草查尔酮A对局灶性脑缺血再灌注小鼠Nrf2/HO-1信号通路和神经炎症反应的影响。方法:体重23~25 g的雄性C57BL/6小鼠总计96只,随机分为4组(n=24):假手术对照组(Sham组)、脑缺血再灌注组(MCAO组)、溶剂组(Vehicle组)、甘草查尔酮A组(LA组)。采用大脑中动脉栓塞(MCAO)模型致大鼠脑缺血损伤。72 h后行神经功能学评分,2,3,5-三苯基氯化铵(TTC)染色检测脑梗死体积,Western blot法检测脑Nrf2、HO-1、TNF-α、IL-6蛋白表达水平,TUNEL染色法检测凋亡细胞数。结果:与Sham组比较,MCAO组和Vehicle组小鼠神经功能评分明显降低(P0.05),脑梗死体积显著增加(P0.05),而核蛋白Nrf2和胞浆蛋白HO-1蛋白表达水平较低(P0.05),炎症因子IL-6和TNF-α表达水平明显增加(P0.05),脑实质炎性细胞浸润显著增多(P0.05);与MCAO组和Vehicle组比较,LA组小鼠神经功能评分明显增加(P0.05),脑梗死体积显著减少(P0.05),而核蛋白Nrf2和胞浆蛋白HO-1蛋白表达水平更高(P0.05),炎症因子IL-6和TNF-α表达水平明显减少(P0.05),脑实质炎性细胞浸润明显减少(P0.05)。结论:甘草查尔酮A可缓解脑缺血再灌注损伤后出现的神经炎症反应及细胞凋亡,进而减轻神经功能障碍和脑梗死体积,其机制可能与激活Nrf2/HO-1信号通路相关。     

Role of Keap1-Nrf2/ARE signal transduction pathway in protection of dexmedetomidine preconditioning against myocardial ischemia/reperfusion injury

Objective: To explore the role and mechanism of the Kelch sample related protein-1-nuclear factor erythroid-2 related factor 2/antioxidant response element (Keap1-Nrf2/ARE) signaling pathway in protection of dexmedetomidine (DEX) preconditioning against myocardial ischemia/reperfusion injury (MIRI). Methods: A total of 70 male SD rats were randomly divided into seven equal groups (n=10): blank control (S group), ischemia/reperfusion injury (C group), DEX preconditioning (DEX group), tertiary butylhydroquinone (tBHQ) control (tBHQ group), combined tBHQ and DEX preconditioning (tBHQ+DEX group), all-trans retinoic acid (ATRA) control (ATRA group), and combined ATRA and DEX preconditioning (ATRA+DEX group). Serum creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) concentrations were measured by ELISA kits, and the infarct size (IS) was assessed by Evan’s blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Oxidative stress was assessed through Western blotting for expression of Keap1-Nrf2/ARE pathway members and oxidative stress markers. Results: Cardioprotection of DEX, tBHQ, and tBHQ+DEX preconditioning treatments were shown as lower concentrations of serum CK-MB and cTnI and a smaller IS following MIRI in rats compared with those of MIRI rats without pre-treatment. In addition, tBHQ+DEX preconditioning exhibited stronger myocardial protection compared with DEX preconditioning. Mechanistically, the cardioprotection offered by DEX, tBHQ, and tBHQ+DEX preconditioning treatments was mediated via exerting antioxidant stress through activation of the Keap1-Nrf2/ARE signal transduction pathway. Conversely, the protective effects of DEX were diminished by blocking the Keap1-Nrf2/ARE pathway with inhibitor ATRA. Conclusion: DEX preconditioning protects against MIRI by exerting antioxidant stress through activation of the Keap1-Nrf2/ARE signal transduction pathway, while inhibition of the Keap1-Nrf2/ARE signal transduction pathway reverses the protective effect of DEX preconditioning on MIRI.     

异丙酚对离体大鼠心肌缺血、再灌注损伤后细胞凋亡及其机制研究

目的：观察异丙酚对离体大鼠心肌缺血/再灌注损伤的影响并从氧化应激和线粒体介导的凋亡方面探讨其作用机制。方法：应用Langendorff离体心脏灌注系统建立心肌缺血/再灌注损伤模型。40只SD大鼠随机分为正常对照组、缺血/再灌注模型（I/R）组、异丙酚15、30、60μmol.L-1组。除正常对照组外,各组分别平衡灌注20 min后,常温全心停灌25 min,再灌注30 min。Powerlab/8s仪记录各组平衡末、缺血前及再灌30 min时的各项心功能指标并测定冠脉流出液中乳酸脱氢酶（LDH）、肌酸激酶（CK）活性;检测心肌线粒体活力、膜肿胀度、锰超氧化物岐化酶（Mn-SOD）活性和丙二醛（MDA）含量;流式细胞仪检测心肌细胞凋亡;流式细胞术检测Bcl-2和Bax的表达,免疫组化法测定天冬氨酸特异的半胱氨酸蛋白酶（caspase）-3,9,8蛋白的表达。结果：与I/R组相比,异丙酚30、60μmol.L-1能明显改善缺血/再灌注后的心功能,减弱冠脉流出液中LDH、CK的活性（P〈0.05）;心肌线粒体活力有所恢复,膜肿胀度减轻,Mn-SOD活性升高,MDA生成明显减少（P〈0.05）,心肌细胞凋亡明显减少,Bcl-2表达上调,Bax表达下调,caspase-3,9阳性表达细胞数明显减少（P〈0.05）。结论：异丙酚明显减轻缺血/再灌注所致的心肌线粒体的过氧化损伤,抑制线粒体途径的凋亡,可能是其心肌保护作用机制之一。     

针刺对缺氧缺血性脑损伤大鼠脑神经功能及5-HTR1A/cAMP/PKA信号通路的影响机制研究

摘要 目的：探讨与研究针刺对缺氧缺血性脑损伤大鼠脑神经功能及5-HTR1A/cAMP/PKA信号通路的影响机制。方法：研究时间为2022年6月到2022年12月,SPF级健康雄性SD大鼠36只平分为空白组、模型组、针刺组,每组各12只大鼠。空白组不进行造模,针刺组在造模完成1周后进行针刺治疗,空白组、模型组不进行治疗。结果：所有大鼠都顺利完成实验,无死亡大鼠出现。针刺组、模型组治疗后2周与4周的神经功能评分都显著高于空白组（P<0.05）,针刺组的神经功能评分与模型组相比显著降低(P<0.05)。针刺组、模型组治疗后2周与4周的脑缺氧缺血组织体积都高于空白组(P<0.05),针刺组的脑缺氧缺血组织体积与模型组相比显著降低（P<0.05）。针刺组、模型组治疗后2周与4周的血清超氧化物歧化酶活力低于空白组（P<0.05）,血清丙二醛含量高于空白组（P<0.05）,针刺组与模型组对比也有显著差异（P<0.05）。针刺组、模型组治疗后2周与4周的大脑组织5-HTR1A蛋白、cAMP蛋白、PKA蛋白相对表达水平明显低于空白组（P<0.05）,针刺与模型组相比显著提高（P<0.05）。结论：针刺在缺氧缺血性脑损伤大鼠的应用能激活5-HTR1A/cAMP/PKA信号通路,能提高超氧化物歧化酶活力,降低血清丙二醛含量,能改善大鼠的脑神经功能,降低脑缺氧缺血组织体积。     

High levels of endogenous nitric oxide produced after burn injury in rats arrest activated T lymphocytes in the first G1 phase of the cell cycle and then induce their apoptosis

Major physical traumas provoke a systemic inflammatory response and immune dysfunction. In a model of thermal injury in rats, we previously showed that an overproduction of nitric oxide (NO) was responsible for the collapse of lymphoproliferative responses. In the present work, we performed a time-course analysis of cell proliferation and cell death parameters in order to establish the sequence of events triggered by the high NO output in Wistar/Han rat splenocytes activated with Con A, 10 days after burn injury. We demonstrate that activated T cells from burned rats never divided whereas normal T cells underwent four division cycles. However, T cells from both burned and normal rat entered the G1 phase as shown by increase of cell size, mitochondria hyperpolarization, and expression of cyclin D1. Burned rat T cells progressed to the late G1 phase as shown by expression of the nuclear Ki-67 antigen, but they never entered the S phase. They underwent apoptosis as shown by morphological parameters, disruption of transmembrane mitochondrial potential, and DNA fragmentation. Persistent accumulation of the p53 protein accompanied these phenomena. NO synthase inhibitors antagonize alterations of cell proliferation and cell death parameters in burned rat T cells and accelerated p53 turnover.