PI3K/Akt调节线粒体Cx43蛋白表达在H_2S后处理离体大鼠心肌中的保护作用

目的探讨PI3K/Akt信号通路是否通过调节线粒体缝隙连接蛋白Cx43而在硫化氢(H2S)后处理中减轻离体大鼠心脏缺血/再灌注(I/R)损伤。方法 56只♂SpragueDawley(SD)大鼠随机分为4组(n=14):缺血/再灌注组(I/R组),PI3K/Akt信号通路抑制剂LY294002组(LY组),硫化氢后处理组(NP组),硫化氢后处理+PI3K/Akt信号通路抑制剂LY294002组(N+L组)。采用离体心脏Langen-dorff灌注模型,平衡灌注20 min后停灌30 min复灌60 min。记录平衡末及灌注结束时的心率(HR)、左室舒张末期压(LVEDP)、左室发展压(LVDP)、左室内压上升最大速率(+dp/dtmax)、左室内压下降最大速率(-dp/dtmax);灌注结束时,TTC法染色心肌切片并计算心肌梗死面积百分比;TUNEL法检测心肌细胞凋亡,计算凋亡指数(AI);Westernblot半定量线粒体总的Cx43(total connexin 43,tCx43)和磷酸化Cx43(phosphorylated connexin 43,pCx43)表达水平。结果平衡灌注末各组间心功能指标差异无统计学意义。再灌注后,与I/R组比较,NP组心功能的各项指标明显改善(P<0.05);心肌梗死面积减少(26.5±4.2)%vs(44.5±5.3)%(P<0.05);凋亡指数降低(25.9±3.0)%vs(43.1±1.9)%(P<0.05);线粒体tCx43和pCx43蛋白表达水平明显升高。LY294002逆转了H2S后处理产生的心肌保护效应,使N+L组心功能指标及线粒体中tCx43和pCx43的表达水平降低(P<0.05),心肌梗死面积及凋亡指数均增加(P<0.05)。结论 PI3K/Akt信号通路通过上调线粒体缝隙连接蛋白Cx43蛋白的表达而在硫化氢(H2S)后处理中减轻离体大鼠I/R损伤。     

Rosiglitazone‐induced myocardial protection against ischaemia–reperfusion injury is mediated via a phosphatidylinositol 3‐kinase/Akt‐dependent pathway

1. Rosiglitazone is widely used in the treatment of Type 2 diabetes. However, in recent years it has become evident that the therapeutic effects of peroxisome proliferator‐activated receptor γ ligands reach far beyond their use as insulin sensitizers. Recently, the ability of rosiglitazone pretreatment to induce cardioprotection following ischaemia–reperfusion (I/R) has been well documented; however, the protective mechanisms have not been elucidated. In the present study, examined the role of the phosphatidylinositol 3‐kinase (PI3‐K)/Akt signalling pathway in rosiglitazone cardioprotection following I/R injury. 2. Mice were pretreated with 3 mg/kg per day rosiglitazone for 14 days before hearts were subjected to ischaemia (30 min) and reperfusion (2 h). Wortmannin (1.4 mg/kg, i.p.), an inhibitor of PI3‐K, was administered 10 min prior to myocardial I/R. Then, activation of the PI3‐K/Akt/glycogen synthase kinase (GSK)‐3α signalling pathway was examined. The effects of PI3‐K inhibition on rosiglitazone‐induced cardioprotection were also evaluated. 3. Compared with control rats, the ratio of infarct size to ischaemic area (area at risk) and the occurrence of sustained ventricular fibrillation in rosiglitazone‐pretreated rats was significantly reduced (P < 0.05). Rosiglitazone pretreatment attenuated cardiac apoptosis, as assessed by ELISA to determine cardiomyocyte DNA fragmentation. Rosiglitazone pretreatment significantly increased levels of phosphorylated (p‐) Akt and p‐GSK‐3α in the rat myocardium. Pharmacological inhibition of PI3‐K by wortmannin markedly abolished the cardioprotection induced by rosiglitazone. 4. These results indicate that rosiglitazone‐induced cardioprotection in I/R injury is mediated via a PI3‐K/Akt/GSK‐3α‐dependent pathway. The data also suggest that modulation of PI3‐K/Akt/GSK‐3α‐dependent signalling pathways may be a viable strategy to reduce myocardial I/R injury.     

C1q/TNF-related protein 6 (CTRP6) attenuates renal ischaemia-reperfusion injury through the activation of PI3K/Akt signalling pathway

C1q/TNF-related protein 6 (CTRP6) is a member of the CTRP family that has been reported to exhibit a nephroprotective effect. However, the role of CTRP6 in renal ischaemia/reperfusion (I/R) injury (IRI) remains unclear. In the present study, we aimed to explore the protective effect of CTRP6 in renal IRI and the potential mechanism. We found that CTRP6 expression was markedly decreased in the kidneys of mice subjected to I/R and HK-2 cells in response to hypoxia/reoxygenation (H/R) stimulation. Recombinant CTRP6 protein protected against renal I/R injury by the reduction of blood urea nitrogen (BUN) and creatinine levels. The increased production of ROS and malondialdehyde (MDA), as well the decreased activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) caused by H/R induction were mitigated by CTRP6 in HK-2 cells. The caspase-3 activity and apoptotic rate were both decreased in CTRP6-overexpressing HK-2 cells. In addition, we also found that knockdown of CTRP6 aggravated H/R-caused oxidative stress and cell apoptosis in HK-2 cells. Moreover, CTRP6 overexpression enhanced the H/R-stimulated activation of PI3K/Akt pathway in HK-2 cells. Inhibition of PI3K reversed the nephroprotective effects of CTRP6 in HK-2 cells. Taken together, CTRP6 exerted protective effects against H/R-caused oxidative injury in HK-2 cells via activating the PI3K/Akt pathway.     

bFGF inhibits ER stress induced by ischemic oxidative injury via activation of the PI3K/Akt and ERK1/2 pathways

Extensive research has focused on finding effective strategies to prevent or improve recovery from brain ischemia and reperfusion (I/R) injury. The basic fibroblast growth factor (bFGF) has been shown to have therapeutic potential in some central nervous system (CNS) disorders, including ischemic injury. In this study, we demonstrate that bFGF administration can improve locomotor activity and inhibit the ER stress induced in the CA1 region of the hippocampus in a mouse model of I/R injury. In vitro, bFGF exerts a protective effect by inhibiting the ER stress response proteins CHOP, XBP-1, ATF-6 and caspase-12 that are induced by H(2)O(2) treatment. Both of these in vivo and in vitro effects are related to the activation of two downstream signaling pathways, PI3K/Akt and ERK1/2. Inhibition of the PI3K/Akt and ERK1/2 pathways by specific inhibitors, LY294002 and U0126, respectively, partially reduce the protective effect of bFGF. Taken together, our results indicate that the neuroprotective role of bFGF involves the suppression of ER stress in the ischemic oxidative damage models and oxidative stress-induced PC12 cell injury, and these effects is underlying the activation of the PI3K/Akt and ERK1/2 signal pathway.     

PI3K/Akt信号通路在外源性硫化氢后处理大鼠离体心肌中的作用

目的探讨PI3K/Akt信号通路是否参与硫化氢后处理减轻离体大鼠心肌缺血/再灌注损伤。方法 70只♂Sprague Dawley(SD)大鼠随机分为5组(n=14):缺血/再灌注组(I/R组),硫化氢后处理组(N组),溶媒组(D组),LY294002组(L组),硫化氢后处理+LY294002组(N+L组)。采用离体心脏Langendorff灌注模型,平衡灌注20min后停灌40min复灌60min。记录平衡末及灌注结束时的左室舒张末期压(LVEDP)、左室发展压(LVDP)、左室内压上升最大速率(+dp/dt)、左室内压下降最大速率(-dp/dt)、心率(HR)、冠脉血流量(CF);灌注结束时,TTC法染色心肌切片并计算心肌梗死面积百分比,TUNEL法检测心肌细胞凋亡计算凋亡指数(AI),Western blot半定量p-Akt和总的Akt表达水平。结果平衡灌注末各组间心功能指标(基础值)差异未见统计学意义(P>0.05)。灌注结束时,与I/R组比较,N组可改善再灌注损伤心功能的各项指标(P<0.05),使心肌梗死面积缩小和凋亡指数降低(P<0.05),p-Akt表达水平升高(P<0.05)。LY294002逆转了硫化氢后处理的心功能指标、心肌梗死面积、凋亡指数及p-Akt表达水平(P<0.05),使L组和N+L组p-Akt蛋白表达明显低于N组(P<0.05)。结论外源性硫化氢后处理通过PI3K/Akt信号通路减轻离体大鼠心肌缺血/再灌注损伤。     

Melatonin Induces Akt Phosphorylation through Melatonin Receptor- and PI3K-Dependent Pathways in Primary Astrocytes

Melatonin has been reported to protect neurons from a variety of neurotoxicity. However, the underlying mechanism by which melatonin exerts its neuroprotective property has not yet been clearly understood. We previously demonstrated that melatonin protected kainic acid-induced neuronal cell death in mouse hippocampus, accompanied by sustained activation of Akt, a critical mediator of neuronal survival. To further elucidate the neuroprotective action of melatonin, we examined in the present study the causal mechanism how Akt signaling pathway is regulated by melatonin in a rat primary astrocyte culture model. Melatonin resulted in increased astrocytic Akt phosphorylation, which was significantly decreased with wortmannin, a specific inhibitor of PI3K, suggesting that activation of Akt by melatonin is mediated through the PI3K-Akt signaling pathway. Furthermore, increased Akt activation was also significantly decreased with luzindole, a non-selective melatonin receptor antagonist. As downstream signaling pathway of Akt activation, increased levels of CREB phoshorylation and GDNF expression were observed, which were also attenuated with wortmannin and luzindole. These results strongly suggest that melatonin exerts its neuroprotective property in astrocytes through the activation of plasma membrane receptors and then PI3K-Akt signaling pathway.     

七氟烷预处理对成年及幼年大鼠心肌缺血/再灌注损伤保护作用的对比研究

目的观察比较七氟烷预处理对成年及幼年大鼠心肌缺血/再灌注损伤保护作用及可能机制。方法 36只成年♂SD大鼠随机分为3组:对照组(sham 1组),缺血/再灌注组(I/R 1组),七氟烷预处理组(S 1组);36只幼年♂SD大鼠随机分为3组:对照组(sham 2组),缺血/再灌注组(I/R 2组),七氟烷预处理组(S 2组)。采用Langendorff离体大鼠心肌灌注模型。对照组,自然灌流120 min;缺血/再灌注组,平衡灌注30 min,缺血30 min,复灌60 min;七氟烷组,平衡灌注15 min,含七氟烷的K-H液10 min,洗出5 min,缺血30min,再灌注60 min。记录各组心脏在平衡末及复灌15 min的左室舒张末压(LVEDP)、左室发展压(LVDP)、左室压力升高或降低最大速率(±dp/dtmax)、心率(HR)。复灌15 min时,TUNEL法检测凋亡细胞,免疫印迹法(Western blot)半定量检测p-Akt和总Akt的含量;复灌末TTC法计算心肌梗死面积百分比。结果平衡灌注末各组间心功能指标(基础值)差异未见统计学意义(P>0.05)。灌注结束时,S 1组较I/R 1组以及S 2组较I/R 2组心功能明显改善,心肌梗死面积减少和凋亡指数降低(P<0.05),同时p-Akt表达水平升高(P<0.05)。结论 1 MAC的七氟烷预处理可能通过增强Akt的磷酸化来减轻成年及幼年大鼠的心肌缺血/再灌注损伤。     

紫草素通过PI3K/Akt通路促进人乳腺癌MCF-7细胞自噬

目的研究紫草素(Shikonin)对人乳腺癌MCF-7细胞自噬的影响及其作用机制。方法 CCK-8法检测紫草素处理人乳腺癌MCF-7细胞24、48 h细胞的存活率,Western blot方法检测紫草素处理24 h和48 h时LC3、p62、PI3K、Akt、p-PI3K、p-Akt蛋白水平。结果 1μmol.L-1紫草素处理细胞48 h以及2.5、5μmol.L-1紫草素处理MCF-7细胞24 h和48 h时,MCF-7细胞的活力受到明显抑制,LC3-Ⅱ/LC3-Ⅰ值增加,p62表达减少,总PI3K、Akt、p-PI3K、p-Akt均减少。结论紫草素促进乳腺癌MCF-7细胞自噬,其作用机制可能与PI3K/Akt通路受到抑制有关。     

PTEN, the Achilles' heel of myocardial ischaemia/reperfusion injury?

Myocardial ischaemia/reperfusion injury leading to myocardial infarction is one of the most frequent causes of debilitation and death in man. Considerable research has been undertaken to investigate the possibility of reducing myocardial infarction and increasing cell survival by activating certain endogenous prosurvival signaling pathways. Thus, it has been established that the activation of the PI3K (Phosphoinositide-3 kinase)/Akt (Protein kinase B, PKB) signaling pathway is essential for protection against ischaemia/reperfusion injury. This pathway has been shown to be activated by mechanical procedures (e.g. pre and post conditioning) as well as by a number of pharmacological agents. Although the activation of this prosurvival signaling pathway induces the phosphorylation of a large number of substrates implicated in increased cell survival, when activated over a prolonged period this pathway can have detrimental consequences by facilitating unwanted growth and malignancies. Importantly PTEN (phosphatase and tensin homolog deleted on chromosome ten), is the main phosphatase which negatively regulates the PI3K/Akt pathway. In this review we discuss: a) the significance and the limitations of inhibiting PTEN in myocardial ischaemia/reperfusion injury; b) PTEN and its relationship to ischaemic preconditioning, c) the role of PTEN in the development of tolerance to chronic administration of drugs known to limit infarction by activating PI3K/Akt pathway when given acutely, and d) the possible role of PTEN in the ischaemic/reperfused diabetic heart. The experimental evidence discussed in this review illustrates the importance of PTEN inhibition in the protection of the heart against ischaemia/reperfusion injury.     

Orientin-induced cardioprotection against reperfusion is associated with attenuation of mitochondrial permeability transition

In this study, we provide new evidence that orientin from bamboo leaves (Phyllostachys nigra) protect H9c2 cardiomyocytes against ischemia/reperfusion (I/R) injury through the mitochondrial apoptotic pathway. A previous work has identified that orientin could protect myocardium against ischemia/reperfusion injury. Mitochondria are both critical determinants of cardioprotection and crucial targets of cardioprotective signaling. Their role during reperfusion is conspicuously critical because the conditions promote apoptosis through the mitochondrial pathway and necrosis though irreversible damage to mitochondria, which is in association with mitochondrial permeability transition (MPT). After myocardial ischemia, opening of the mPTP is a critical determinant of cell death. The relationship of orientin and mPTP in mediating reperfusion-induced cardiomyocytes injury is still elusive. Here, our results indicate that the protective effect of orientin in H9c2 cells subjected to I/R injury is associated with depression of the mPTP opening, resultant mitochondrial dysfunction, and apoptosis. Further investigation of cellular mechanisms revealed that these effects were associated with inhibition of reactive oxygen species (ROS) generation, repolarization of mitochondrial membrane potential (Δψ(m)), suppression of mitochondrial cytochrome C release, enhancement of the Bcl-2 level, and inhibition of Bax and Smac/DIABLO levels. Furthermore, these beneficial effects of orientin were blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, and orientin could enhance Akt phosphorylation. In summary, we demonstrate that orientin protects H9c2 cardiomytocytes against I/R-induced apoptosis by modulating the mPTP opening, and this role of orientin may involve the PI3K/Akt signaling pathway.     

Baicalin mitigated IL-1β-Induced osteoarthritis chondrocytes damage through activating mitophagy

Mitophagy is related to chondrocyte homeostasis and plays a key role in the progress of osteoarthritis (OA). Baicalin has a protective effect on OA chondrocytes, the aim of this study was to explore whether the effect of Baicalin on IL-1β-induced chondrocyte injury is related to the regulation of mitophagy. The expression of collagen II in chondrocytes was detected to identify chondrocytes. The effects of different concentrations of Baicalin (10, 20 and 40 μM), autophagy inhibitor (3-Methyladenine), autophagy activator (rapamycin) and Baicalin combined with PI3K agonist (740Y-P) on the viability (cell counting kit 8), apoptosis (flow cytometry), autophagy activation (Monodansylcadaverine staining) and mitochondrial membrane potential (JC-1 kit) of IL-1β-induced chondrocytes were evaluated. The co-localization of autophagosome and mitochondria was determined by immunofluorescence. Apoptosis-, autophagy-, PI3K/AKT/mTOR pathway- and mitophagy-related proteins were detected by western blot. Our result revealed that Baicalin and rapamycin facilitated cell viability, autophagy and mitophagy, elevated mitochondrial membrane potential and suppressed apoptosis of IL-1β-induced rat chondrocytes. In addition, Baicalin and rapamycin upregulated the levels of Bcl-2, Beclin 1, LC3-II/LC3-I, p-Drp1, PINK1 and Parkin as well as downregulated the levels of Bax, cleaved caspase-3, P62, p-PI3K/PI3K, p-mTOR/mTOR and Drp1 in IL-1β-induced rat chondrocytes. However, 3-Methyladenine did the opposite effects of Baicalin and 740Y-P reversed the effects of Baicalin on IL-1β-induced rat chondrocytes. In conclusion, Baicalin activated mitophagy in IL-1β-induced chondrocytes by inhibiting PI3K/AKT/mTOR pathway and activating PINK1/Parkin and PINK1/Drp-1 pathway, thereby reducing the chondrocyte injury.     

Cornin protects astrocytes against autophagy induced by cerebral ischemia-reperfusion via PI3K/Akt/mTOR pathway

OBJECTIVE The purpose of this study was to investigate the effect of cornin against astrocytes autophagy induced by cerebral ischemia-reperfusion and its mechanism. METHODS In vitro, U87 cells were used to establish oxygen glucose deprivation/reperfusion(OGD/R) as a cerebral ischemia model. In vivo, a SpragueDawley(SD) rat middle cerebral artery occlusion(MCAO) model was used to evaluated the effects of cornin against autophagy in astrocytes. RESULTS The results demonstrated that cornin was able to increase the ratio of apoptosis regulator Bcl-2/Bax and decrease the level of cleaved-caspase-3 protein. Similarly, cornin reduce the rate of apoptotic cells by flow cytometry. In addition, cornin(100 nmol·L~(-1)) decreased the expression of microtubule-associated proteins 1A/1B light chain 3 B(LC3-Ⅱ) and Beclin-1 protein, increased the level of p62,and upregulated phosphorylated protein kinase m TOR(m TOR), phosphorylated(p) RAC α serine/threonine protein kinase(Akt) in OGD treated U87 cells. However,following PI3 K inhibitor LY294002 and m TOR si RNA reversed this result. Cornin(10 mg · kg~(-1)) significantly reduced the cerebral infarction volume and the leakage rate of blood-brain barrier(BBB). The expression of autophagy proteins(LC3-Ⅱ, Beclin-1, P62) and apoptosis-associated proteins(Bcl-2/Bax, cleaved caspase-3) in brain tissue showed the similar results as in cells. Similarly, PI3 K inhibitor LY294002 was able to reversed the protective effect of cornin and upregulate autophagy-associated proteins. In addition, immunohistochemistry staining was applied for Neu N, GFAP, LC3 B and Beclin-1, and cornin could alleviated pathological injury 24 h after ischemiareperfusion. CONCLUSION These results indicated that cornin against astrocytes autophagy induced by cerebral ischemia-reperfusion through PI3 K/Akt/m TOR signaling pathway.     

17β‐Oestradiol Protects Primary‐Cultured Rat Cortical Neurons from Ketamine‐Induced Apoptosis by Activating PI3K/Akt/Bcl‐2 Signalling

Numerous studies in rodents have indicated that exposure to ketamine during the period when synaptogenesis is highly active induces neurodegeneration. Thus, there is a growing need to develop strategies to prevent ketamine‐induced brain injury in the developing brain. Oestradiol is a neuroactive steroid that prevents neuronal cell death in different experimental models by activating cell survival signals and inhibiting apoptotic signals. The main goal of this study was to investigate the neuroprotective effects of 17β‐oestradiol against ketamine‐induced apoptotic neurodegeneration in primary‐cultured cortical neurons. The data revealed that 17β‐oestradiol (0.1 μM) in combination with ketamine (100 μM) increased cell viability in the MTT assay and reduced the number of apoptotic cells detected by TUNEL and Hoechst 33258 staining. To elucidate a possible mechanism by which 17β‐oestradiol exerts its neuroprotective effect, we investigated the PI3K pathway using an inhibitor of PI3K, LY294002. The protective effects of 17β‐oestradiol were abrogated by LY294002. Furthermore, we found that 17β‐oestradiol not only induced phosphorylation of the PI3K substrate Akt, but also increased the expression of Bcl‐2, which down‐regulated ketamine‐induced caspase‐3 activity and inhibited neuronal apoptosis. These data demonstrate that 17β‐oestradiol exerts a neuroprotective effect against ketamine‐induced neuronal apoptosis by activating the PI3K/Akt/Bcl‐2 signalling pathway. Therefore, 17β‐oestradiol appears to be a promising agent in preventing or reversing ketamine's toxic effects on neurons at an early developmental stage.     

Fraxetin suppresses reactive oxygen species-dependent autophagy by the PI3K/Akt pathway to inhibit isoflurane-induced neurotoxicity in hippocampal neuronal cells

Isoflurane, a common volatile anesthetic, has been widely used to provide general anesthesia in operations. However, exposure to isoflurane may cause widespread neurotoxicity in the developing animal brain. Fraxetin, a natural coumarin derivative extracted from the bark of Fraxinus rhynchophylla, possesses versatile pharmacological properties including anti-oxidative, anti-inflammatory, and neuroprotective effects. However, the effect and action mechanism of fraxetin on neurotoxicity induced by isoflurane are unknown. Reactive oxygen species (ROS) generation, cell viability, lactate dehydrogenase (LDH) release, and apoptosis were estimated by 2′,7′-dichlorofluorescin-diacetate (DCFH-DA) staining, MTT, LDH release, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining assays, respectively. The protein levels of light chain 3 (LC3)-I, LC3-II, p62, protein kinase B (Akt), and phosphorylated Akt (p-Akt) were detected by western blot analysis. Isoflurane induced ROS, LDH release, apoptosis, and autophagy, but inhibited the viability in HT22 cells, which were overturned by fraxetin or ROS scavenger N-acetyl-L-cysteine. Fraxetin suppressed isoflurane-induced PI3K/Akt inactivation in HT22 cells. PI3K/Akt inactivation by LY294002 resisted the effects of fraxetin on isoflurane-induced autophagy and autophagy-modulated neurotoxicity in HT22 cells. In conclusion, fraxetin suppressed ROS-dependent autophagy by activating the PI3K/Akt pathway to inhibit isoflurane-induced neurotoxicity in hippocampal neuronal cells.     

Erythropoietin protects the human myocardium against hypoxia/reoxygenation injury via phosphatidylinositol-3 kinase and ERK1/2 activation

BACKGROUND AND PURPOSES: Erythropoietin (EPO) has been shown to protect against myocardial infarction in animal studies by activating phosphatidylinositol-3 kinase (PI3K)/Akt and ERK1/2. However these pro-survival pathways are impaired in the diabetic heart. We investigated the ability of EPO to protect human atrial trabeculae from non-diabetic and diabetic patients undergoing coronary artery bypass surgery, against hypoxia-reoxygenation injury. EXPERIMENTAL APPROACH: Human atrial trabeculae were exposed to 90min hypoxia and 120min reoxygenation. EPO was administered throughout reoxygenation. The developed force of contraction, calculated as a percentage of baseline force of contraction, was continuously monitored. The involvement of PI3K and ERK1/2 and the levels of activated caspase 3(AC3) were assessed. KEY RESULTS: EPO improved the force of contraction in tissue from non-diabetic patients (46.7+/-1.7% vs. 30.2+/-2.2% in control, p<0.001). These beneficial effects were prevented by the PI3K inhibitor, LY294002 and the ERK1/2 inhibitor, U0126. EPO also significantly improved the force of contraction in the diabetic tissue, although to a lesser degree. The levels of activated caspase 3 were significantly reduced in EPO treated trabeculae from both non-diabetic and diabetic patients, relative to their respective untreated controls. CONCLUSIONS AND IMPLICATIONS: EPO administered at reoxygenation protected human myocardial muscle by activating PI3K and ERK1/2 and reducing the level of activated caspase 3. This cardioprotection was also observed in the diabetic group. This data supports the potential of EPO being used as a novel cardioprotective strategy either alone or as an adjunct in the clinical setting alongside existing reperfusion therapies.     

Neuroprotection of selenite against ischemic brain injury through negatively regulating early activation of ASK1/JNK cascade via activation of PI3K/AKT pathway

AIM: To investigate whether selenite, a known antioxidant, could decrease the activation of apoptosis signal regulating kinase 1/c-jun N-terminal kinase (ASK1/ JNK) signaling cascade in cerebral ischemia/reperfusion (I/R) by activating the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in rat hippocampi, and the neuroprotective effect of selenite against ischemic injury after 15 min of transient brain ischemia. METHODS: Transient global brain ischemia was induced by 4-vessel occlusion into adult male Sprague-Dawley rats weighing 250-300 g. The rats were pretreated only with selenite (0.3 mg/kg dissolved in 0.9% saline) every 24 h for 7 d by means of intravenous injection of the tail or combined with LY294002 from d 5 by left cerebral ventricle injection before surgery. RESULTS: Selenite significantly increased AKT1 activation and decreased the activation of ASK1/ JNK cascade via phosphorylating ASK1 at Ser-83 residue by AKT1 during early reperfusion after 15 min transient global brain ischemia. On the contrary, combined pretreatment of the rats with LY294002 (a specific PI3K inhibitor) and selenite significantly inhibited the effects solely with selenite. CONCLUSION: The activation of the pro-apoptotic ASK1/JNK cascade, which is closely associated with oxidative stress, could be suppressed by selenite through activating the antiapoptotic PI3K/AKT pathway during early reperfusion after cerebral ischemia in rat hippocampi.     

瑞芬太尼预处理对心力衰竭大鼠心肌的保护作用

目的探讨瑞芬太尼预处理对阿霉素心衰大鼠离体心肌缺血/再灌注损伤的作用。方法 60只成年♂SD大鼠(250±20)g,尾静脉注射阿霉素2μg·g-1,每周1次,共6周,制成阿霉素心衰大鼠模型。随机将阿霉素心衰大鼠分为6组:对照组(Sham组)、缺血/再灌注组(I/R组)、缺血预处理组(IPC组)、10μg·L-1瑞芬太尼预处理组(RPC 1组)、30μg·L-1瑞芬太尼预处理组(RPC 2组)、60μg·L-1瑞芬太尼预处理组(RPC 3组)。采用Langendorff离体大鼠心肌灌注模型。除Sham组为持续灌注165 min外,所有心脏予以30 min缺血,90 min再灌注。IPC组在缺血前结扎左冠状动脉5 min,松开5 min,共3个循环。RPC组在缺血前给予含浓度分别为10、30、60μg·L-1的瑞芬太尼的K-H液灌注5 min后改用不含瑞芬太尼的K-H液灌注5 min,共3个循环。记录各组心脏在平衡末、再灌5 min、再灌30 min、再灌90 min时的心率(HR)、左室发展压(LVDP)和左室压力升高或降低最大速率(±dp/dtmax)、冠脉流量(CF)并测定冠脉流出液中乳酸脱氢酶(LDH)的活性。再灌末TTC法计算心肌缺血梗死区(IS/AAR)。Western blot半定量检测p-Akt和总Akt的含量。结果平衡灌注末各组间心功能指标(基础值)差异未见统计学意义(P>0.05)。再灌5、30、90 min时,RPC 2组和RPC 3组的LVDP、±dp/dtmax、CF较I/R组高,LDH值较I/R组低(P<0.05)。灌注结束后,见RPC 2组、RPC 3组的IS/AAR较I/R小,p-Akt表达水平升高(P<0.05),而IPC组和RPC 1组的各项指标较I/R组无差异。结论 RPC在一定程度上减轻阿霉素心衰大鼠心肌缺血/再灌注损伤,而缺血预处理对阿霉素心衰大鼠心肌缺血/再灌注损伤无明显保护作用。     

Rutaecarpine inhibits hypoxia/reoxygenation-induced apoptosis in rat hippocampal neurons

Our previous studies showed that rutaecarpine (Rut) protected against myocardial ischemia/reperfusion (I/R) injury, which was associated with activation of transient receptor potential vanilloid subtype 1 (TRPV1). Recently, TRPV1 activation was also reported to exert neuroprotective effects. The present study was to investigate the effect of Rut on hypoxia/reoxygenation (H/R)-induced apoptosis in primary rat hippocampal neurons. Three-hour hypoxia (1% O2) and consequent 24-h reoxygenation significantly increased the apoptotic death of hippocampal neurons as evidenced by increases in both TUNEL-positive cell number and caspase-3 activity. However, pretreatment with Rut (1-10microM) or caspase-3 specific inhibitor DEVD-CHO could markedly attenuate H/R-induced apoptosis in neurons. Rut markedly induced the phosphorylation of Akt and PI3K inhibitor LY294002 prevented the survival effect of Rut on neurons. Intracellular oxidative stress was significantly induced after H/R, which was inhibited by Rut and LY294002 as well as antioxidant PDTC. TRPV1 antagonist capsazepine or intracellular Ca2+ chelator BAPTA/AM could abolish these effects of Rut mentioned above. In summary, the present data suggest that Rut inhibits H/R-induced apoptosis of hippocampal neurons via TRPV1-[Ca2+]i-dependent and PI3K/Akt signaling pathway, which is related to inhibiting oxidative stress and caspase-3 activation.     

异鼠李素激活PI3K/Akt/GSK-3β/CREB信号通路减轻鱼藤酮诱导的PC12细胞损伤

目的探讨异鼠李素(isorhamnetin,ISO)是否能够通过激活PI3K/Akt/GSK-3β/CREB通路减轻鱼藤酮对PC12细胞的损伤作用。方法采用MTT法检测细胞活力,LDH检测乳酸脱氢酶释放,Western blot法测定p-Akt、Akt、p-GSK-3β、GSK-3β、p-CREB和CREB蛋白表达。结果与对照组相比,鱼藤酮损伤后PC12细胞活力明显降低,CREB的磷酸化程度显著降低。异鼠李素预处理组细胞存活率和磷酸化CREB的表达均高于鱼藤酮损伤模型组。此外,异鼠李素预处理增强了鱼藤酮损伤后PC12细胞中Akt和GSK-3β的磷酸化程度。加入PI3K抑制剂LY294002可以抑制Akt、GSK-3β和CREB的磷酸化水平,从而部分消除异鼠李素对鱼藤酮损伤PC12细胞的神经保护作用。结论异鼠李素可能通过PI3K/Akt/GSK-3β/CREB信号通路发挥PC12细胞保护作用。     

Cyanidin-3-rutinoside increases glucose uptake by activating the PI3K/Akt pathway in 3T3-L1 adipocytes

In this study, the effect of cyanidin-3-rutinoside (C3R) on glucose uptake by 3T3-L1 adipocytes was studied. C3R significantly increased glucose uptake, which was associated with enhanced plasma membrane glucose transporter type 4 (PM-GLUT4) expression in 3T3-L1 adipocytes. The potentiating effect of C3R on glucose uptake and PM-GLUT4 expression was related to enhanced phosphorylation of insulin receptor substrate 1 (IRS-1) and Akt, as well as augmented activation of phosphatidylinositol-3-kinase (PI3K) in the insulin signaling pathway. C3R induced glucose uptake was inhibited only by the PI3K inhibitor, but not by an AMPK inhibitor in 3T3-L1 adipocytes. Therefore, C3R likely up-regulates glucose uptake and PM-GLUT4 expression in 3T3-L1 adipocytes by activating the PI3K/Akt pathways.