Cytochrome P450: major player in reperfusion injury

While attention has historically focused on mitochondria as the primary source of ROS in myocardial ischemia/reperfusion injury, recent evidence has implicated cytochrome P450 monooxygenases (CYPs) as a significant factor. CYPs represent a large family of enzymes that catalyze the oxidation of endogenous and exogenous compounds. They catalyze arachidonic acid oxidation to a variety of biologically active eicosanoids that regulate ion channels and protein kinases, with effects on vasomotor tone and cardiac inotropy. They also represent a significant source of reactive oxygen species that may target cellular homeostatic mechanisms and mitochondria. In this review, we will consider the contribution of cytochrome P450 enzymes to reperfusion injury and will speculate on whether the mechanism of injury is due to CYP-mediated ROS production or arachidonic acid metabolites.     

N—acetylserotonin对肝缺血再灌注小鼠氧化应激损伤的保护作用

目的探讨NAS对肝缺血再灌注所诱导的脂质过氧化损伤产生的保护作用。方法采用夹闭肝蒂法30min、再灌注6h制作肝缺血再灌注模型,冰冻切片,HE染色,光学显微镜下观察肝细胞形态结构的变化;比色法检测损伤后血清中谷丙转氨酶（ALT）水平及肝组织中超氧化物歧化酶（SOD）、丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH—Px）的含量。结果夹闭肝蒂30min、再灌注6h后,肝小叶结构紊乱、肝血窦淤血,其间有白细胞浸润、肝细胞出现变性、坏死;血清中ALT水平升高,肝组织中s0D和GSH—Px的含量降低,MDA升高;NAS可减少缺血再灌注后血清ALT的释放,使肝组织中SOD和GSHPx的含量升高,MDA的含量降低;NAS＋Luz可逆转NAS的这一作用。结论NAS对肝缺血再灌注小鼠的氧化应激损伤具有保护作用。     

Postconditioning induces an anti-apoptotic effect and preserves mitochondrial integrity in isolated rat hearts

Postconditioning (PostC) may limit mitochondrial damage and apoptotic signaling. We studied markers of apoptosis and mitochondrial protection in isolated rat hearts, which underwent a) perfusion without ischemia (Sham), b) 30-min ischemia (I) plus 2-hour reperfusion (R), or c) PostC protocol (5 intermittent cycles of 10-s reperfusion and 10-s ischemia immediately after the 30-min ischemia). Markers were studied in cytosolic (CF) and/or mitochondrial (MF) fractions. In CF, while pro-apoptotic factors (cytochrome c and caspase-3) were reduced, the anti-apoptotic markers (Bcl-2 and Pim-1) were increased by PostC, compared to the I/R group. Accordingly, phospho-GSK-3β and Bcl-2 levels increased in mitochondria of PostC group. Moreover, I/R reduced the level of mitochondrial structural protein (HSP-60) in MF and increased in CF, thus suggesting mitochondrial damage and HSP-60 release in cytosol, which were prevented by PostC. Electron microscopy confirmed that I/R markedly damaged cristae and mitochondrial membranes; damage was markedly reduced by PostC. Finally, total connexin-43 (Cx43) levels were reduced in the CF of the I/R group, whereas phospho-Cx43 level resulted in higher levels in the MF of the I/R group than the Sham group. PostC limited the I/R-induced increase of mitochondrial phospho-Cx43. Data suggest that PostC i) increases the levels of anti-apoptotic markers, including the cardioprotective kinase Pim-1, ii) decreases the pro-apoptotic markers, e.g. cytochrome c, iii) preserves the mitochondrial structure, and iv) limits the migration of phospho-Cx43 to mitochondria.     

Promoting osteoblast differentiation by the flavanes from Huangshan Maofeng tea is linked to a reduction of oxidative stress

Epidemiological evidence has shown an association between tea consumption and the prevention of bone loss in the elderly. Previous studies indicated that green tea exerted osteoprotective effect in vivo. This study aims to investigate the constituents in Huangshan Maofeng tea and systemically evaluate their antioxidative and osteogenic effects in vitro. Five flavanes, isolated from Huangshan Maofeng tea, showed effects on proliferation of osteoblastic cells and ameliorated H₂O₂-induced C2C12 mouse myoblast cell apoptosis at 3.125–50 μg/ml. (−)-Epicatechin observably increased alkaline phosphatase (ALP) activity and hydroxyproline content. (−)-Epiafzelechin at 25 μg/ml significantly increased the area of mineralized bone nodules. The activities of flavanes in promoting osteblastic proliferation and differentiation are positively correlated with activities in protecting against apoptosis in C2C12 cells. It indicates that anti-osteoporosis effect of these flavanes may be linked to their antioxidative activity. The observed effects of these flavanes suggest that these flavanes may have beneficial effects on bone health.     

黄芪甲苷抑制缺血再灌注诱导的大鼠心肌细胞凋亡

摘要 目的：本研究旨在探究黄芪甲苷（Astragaloside IV,Ast-IV）在缺血再灌注（ischemia-reperfusion,I/R）大鼠模型中的保护作用,并讨论黄芪甲苷在抑制I/R诱导心肌细胞凋亡过程中的作用。方法：通过左冠状动脉前降支结扎构建I/R大鼠模型;将40只SD大鼠分为4组：假手术组（Sham组）、模型组（I/R组）、黄芪甲苷干预组（Ast组）和黄芪甲苷+LY294002干预组（Ast+LY组）。使用试剂盒测定血清中心脏功能障碍标记物CPK、ALT、LDH和tropornin-T的表达水平;通过HE染色和TUNEL分别检测心肌组织病理学变化和心肌细胞凋亡情况;通过超氧化物荧光探针染色检测细胞内ROS水平;通过ELISA试剂盒测定心肌组织MDA、GSH和GSH-PX含量;免疫组织化学检测SOD2和HO-1蛋白表达水平,分析心肌氧化应激状态;通过Western blot检测PI3K、p-Akt、Akt、p-eNOS、eNOS、caspase-3、Bax和Bcl-2蛋白表达水平变化。结果：Ast组大鼠血浆CPK、ALT、LDH和tropornin-T酶活性均明显低于I/R组（P<0.05）。Ast组大鼠心肌纤维断裂,心肌细胞坏死和炎性细胞浸润等病变程度均低于I/R组。Ast组大鼠TUNEL阳性细胞数低于I/R组（P<0.05)。相较于I/R组,Ast组大鼠Caspase3和Bax表达水平均明显下调,Bcl-2和PI3K蛋白表达水平上调,p-Akt/Akt和p-eNOS/eNOS比值均显著上调（P<0.05）。Ast组大鼠DHE荧光强度显著低于Ast+LY组（P<0.05）。与I/R组相比,Ast组大鼠心肌组织中MDA含量降低,GSH、GSH-PX、SOD2和HO-1表达水平升高（P<0.05）;与Ast组相比,Ast+LY组大鼠心肌组织中MDA含量升高,GSH、GSH-PX、SOD2和HO-1表达水平降低（P<0.05）。结论：黄芪甲苷通过激活PI3K/Akt信号通路,抑制心肌细胞氧化应激反应,从而减少I/R诱导大鼠心肌细胞凋亡,缓解I/R后大鼠心肌损伤。     

Protective effect of docosahexaenoic acid against brain injury in ischemic rats

Evidence suggests that inactivation of cell-damaging mechanisms and/or activation of cell-survival mechanisms may provide effective preventive or therapeutic interventions to reduce cerebral ischemia/reperfusion (I/R) injuries. Docosahexaenoic acid (DHA) is an essential polyunsaturated fatty acid in the central nervous system that has been shown to possess neuroprotective effects. We examined whether different preadministrative protocols of DHA have effects on brain injury after focal cerebral I/R and investigated the potential neuroactive mechanisms involved. Sprague–Dawley rats were intraperitoneally pretreated with DHA once 1 h or 3 days being subjected to focal cerebral I/R or daily for 6 weeks before being subjected to focal cerebral I/R. Reduction of brain infarction was found in all three DHA-pretreated groups. The beneficial effect of DHA on the treatment groups was accompanied by decreases in blood–brain barrier disruption, brain edema, malondialdehyde (MDA) production, inflammatory cell infiltration, interleukin-6 (IL-6) expression and caspase-3 activity. Elevation of antioxidative capacity, as evidenced by decreased MDA level and increased superoxide dismutase activity and glutathione level, was detected only in the chronic daily-administration group. The two single-administration groups showed increased phosphorylation of extracellular-signal-regulated kinase (ERK). Elevation of Bcl-2 expression was detected in the chronic daily-administration and 3-day-administration groups. In vitro study demonstrated that DHA attenuated IL-6 production from stimulated glial cells involving nuclear factor κB inactivation. Therefore, the data suggest that the neuroprotective mechanisms of DHA pretreatment are, in part, mediated by attenuating damaging mechanisms through reduction of cytotoxic factor production and by strengthening survival mechanisms through ERK-mediated and/or Bcl-2-mediated prosurvival cascade.     

Long-term aerobic exercise protects the heart against ischemia/reperfusion injury via PI3 kinase-dependent and Akt-mediated mechanism

Objective Physical activity has been shown to improve cardiovascular function and to be beneficial to type 2 diabetic patients. However, the effects of aerobic exercise (AE) on myocardial ischemia/reperfusion (MI/R) are largely unclear. Therefore, the aims of the present study were to determine whether long-term AE can protect the heart against I/R injury, and if so, to investigate the underlying mechanism. Methods Adult male Sprague–Dawley rats were randomly subjected to 8 weeks of either sedentary or free-loading swimming exercise (3 h/day, 5 d/week). Then the animals were subjected to 30 min MI followed by 4 h R. Arterial blood pressure and left ventricular pressure (LVP) were monitored throughout the whole MI/R procedure. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured spectrophotometrically. Myocardial infarction and myocardial apoptosis (TUNEL analysis) were determined in a blinded manner. Results MI/R caused significant cardiac dysfunction and myocardial apoptosis (strong TUNEL-positive staining). Compared with sedentary group, rats subjected to 8 weeks of AE showed protection against MI/R as evidenced by reduced myocardial infarction (26.8 ± 1.5% vs. 35.3 ± 2.4%, n = 8, P < 0.05), inhibited cardiomyocyte apoptosis (decreased apoptotic index (12.4 ± 1.1% vs. 21.0 ± 1.7%, n = 8, P < 0.01) and decreased myocardial caspase-3 activity), decreased plasma CK and LDH activities and improved recovery of cardiac systolic/diastolic function (including LVSP and ±LVdP/dt) at the end of R. Moreover, exercise resulted in 1.7-fold, 2.5-fold and 2.5-fold increases in Akt expression, Akt phosphorylation and glycogen synthase kinase-3β phosphorylation in I/R myocardium, respectively (n = 3, all P < 0.05). More importantly, treatment with wortmannin, a PI3 kinase inhibitor, 15 min before R not only significantly blocked Akt phosphorylation (P < 0.05) in exercise rats, but also abolished long-term AE-induced cardioprotection for the I/R heart as manifested by increased apoptosis and myocardial infarction, and reduced cardiac function. Conclusion Long-term AE exerts cardioprotective effect against MI/R injury, including anti-cardiomyocyte apoptosis, which is at least partly via PI3 kinase-dependent and Akt-mediated mechanism.     

高压氧对肾脏缺血再灌注损伤的保护作用及其机制研究

目的:观察高压氧(hyperbaric oxygen,HBO)对肾脏缺血再灌注损伤的保护作用并探讨其作用机制.方法:56只SD大鼠被随机分为三组,假手术组(n=8);I/R组(n=24),夹闭双肾动脉45分钟后恢复血流灌注;I/R+HBO组(n=24),夹闭双肾动脉45分钟并在恢复血流后1h、24 h、48 h行HBO治疗,每次HBO后采血并取双肾,比色法测定血浆尿素氮(BUN)、肌酐(Cr)值,原位末端标记(TUNEL)法检测肾小管上皮细胞凋亡情况,实时定量PCR法检测促凋亡基因Bax的mRNA含量.结果:与sham组(BUN值为9.563± 1.384 mmol/L;Cr值为45.912±2.685 mmo1/L,TUNEL值为2.088％)比较,I/R组大鼠再灌注1小时尿素氮(12.5±1.487 mmol/L)和血肌酐水平(51.388±3.092 mmol/L)升高,但差异无统计学意义,而TUNEL阳性细胞数(9.775％)和Bax的mR-NA(3.219± 0.427)表达水平均显著升高(P＜0.05),再灌注24小时及48小时后尿素氮(28.087± 2.012 mmol/L、41.225± 1.397mmol/L)和血肌酐(241.75± 11.853 mmol/L、278.75± 12.578 mmol/L)水平、TUNEL阳性细胞数(12.512％、14.413％)和Bax的mRNA(5.541±0.227、6.407± 0.291)表达水平均显著升高(P＜0.05);而HBO治疗可显著降低再灌注24小时及48小时的大鼠尿素氮(14.15±1.397 mmol/L、25.962± 2.497 mmol/L)和血肌酐(146.375± 8.782 mmolL、210.125± 11.519 mmol/L)水平(P＜0.05),但仍显著高于假手术组(P＜0.05).结论:HBO治疗可以改善I/R后肾功能,其作用机制可能与在早期明显降低Bax的mRNA表达,减轻肾小管上皮细胞凋亡有关.     

Long noncoding RNA FTX regulates cardiomyocyte apoptosis by targeting miR-29b-1-5p and Bcl2l2

Cardiomyocyte apoptosis correlates with the pathogenesis of heart disease. Long noncoding RNA (LncRNA) emerges as a class of noncoding RNAs that regulate gene expression and participate in various cellular processes. However, the role of lncRNAs in cardiomyocyte apoptosis remains to be elucidated. In our study, we found that lncRNA FTX is significantly down-regulated upon ischemia/reperfusion injury and hydrogen peroxide treatment. Enhanced expression of FTX inhibits cardiomyocyte apoptosis induced by hydrogen peroxide. miR-29b-1-5p was found to interact with FTX and regulate the expression of Bcl2l2. Inhibition of miR-29b-1-5p attenuated cardiomyocyte apoptosis upon hydrogen peroxide treatment. We then found that FTX functions as endogenous sponge for miR-29b-1-5p and regulates the activity of miR-29b-1-5p. The results demonstrate that FTX regulates cardiomyocyte apoptosis through modulating the expression of Bcl2l2 which is mediated by miR-29b-1-5p. Our findings reveal a novel regulatory model which is composed of FTX, miR-29b-1-5p and Bcl2l2. Manipulating of their levels may become a new approach to tackling cardiomyocyte apoptosis related heart diseases.     

Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes

The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca2+]i. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl3 was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl3 during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a Deltapsim, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl3. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl3 but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl3 in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.     

Hypothermia attenuates ischemia/reperfusion-induced endothelial cell apoptosis via alterations in apoptotic pathways and JNK signaling

Hypothermia is the most effective means of protecting the brain, heart and other organs during ischemia/reperfusion (I/R) injury. However, the precise mechanisms for hypothermia to inhibit I/R-induced endothelial cell apoptosis are not fully understood. In the present study, human umbilical endothelial cells (HUVECs) were exposed to ischemia followed by reperfusion under normothermia (37 °C) or hypothermia (33 °C). Our results showed that hypothermia markedly reduced I/R-induced endothelial cell apoptosis, the expression of cleaved caspase-3 and PARP. Moreover, hypothermia markedly reversed I/R-induced activation of Fas/caspase-8, the increase of Bax and decrease of Bcl-2. Furthermore, hypothermia inhibited JNK1/2 activation via MKP-1 induction. Together, these data demonstrate that hypothermia represses I/R-induced endothelial cell apoptosis by inhibiting both extrinsic- and intrinsic-dependent apoptotic pathways and activation of JNK1/2.     

The hepatoprotective effects of Hypericum perforatum L. on hepatic ischemia/reperfusion injury in rats

Little is known about the effective role of Hypericum perforatum on hepatic ischemia–reperfusion (I/R) injury in rats. Hence, albino rats were subjected to 45 min of hepatic ischemia followed by 60 min of reperfusion period. Hypericum perforatum extract (HPE) at the dose of 50 mg/kg body weight (HPE₅₀) was intraperitonally injected as a single dose, 15 min prior to ischemia. Rats were sacrificed at the end of reperfusion period and then, biochemical investigations were made in serum and liver tissue. Liver tissue homogenates were used for the measurement of malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GPx) levels. At the same time alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were assayed in serum samples and compared statistically. While the ALT, AST, LDH activities and MDA levels were significantly increased, CAT and GPx activities significantly decreased in only I/R-induced control rats compared to normal control rats (p < 0.05). Treatment with HPE₅₀ significantly decreased the ALT, AST, LDH activities and MDA levels, and markedly increased activities of CAT and GPx in tissue homogenates compared to I/R-induced rats without treatment–control group (p < 0.05). In oxidative stress generated by hepatic ischemia–reperfusion, H. perforatum L. as an antioxidant agent contributes an alteration in the delicate balance between the scavenging capacity of antioxidant defence systems and free radicals in favour of the antioxidant defence systems in the body.     

Rooibos (Aspalathus linearis) beyond the farm gate: From herbal tea to potential phytopharmaceutical

Aspalathus linearis (Burm.f.) Dahlg. (Fabaceae, Tribe Crotalarieae), an endemic South African fynbos species, is cultivated to produce the well-known herbal tea, rooibos. It is currently sold in more than 37 countries with Germany, the Netherlands, the United Kingdom, Japan and the United States of America representing 86% of the export market in 2010. Its caffeine-free and comparatively low tannin status, combined with its potential health-promoting properties, most notably antioxidant activity, contributes to its popularity. First marketed in 1904 in its fermented (oxidised) form, green rooibos is a new product recently on the market. The utilisation of rooibos has also moved beyond a herbal tea to intermediate value-added products such as extracts for the beverage, food, nutraceutical and cosmetic markets. Its potential as a phytopharmaceutical, shown in recent scientific studies, has not yet been exploited. This review focuses on past and current research aimed at enhancing the value of rooibos herbal tea as a specialised, niche product and expanding its value-adding potential against the background of its traditional use and the current market. The focus falls specifically on aspects such as composition, processing, quality and rooibos as food and potential medicine.