瞬时受体电位香草酸亚型1激活释放的P物质在小鼠急性心肌梗死后凋亡中的保护作用

瞬时受体电位香草酸亚型1 (transient receptor potential vanilloid 1, TRPV1)在心肌缺血激活后可传导心绞痛信号和释放P物质(substance P, SP).SP是速激肽家族成员之一,主要通过结合并激活神经激肽1 (neurokinin 1,NK1)受体发挥作用. TRPV1和SP在缺血性心脏病中对心功能的恢复和重塑有一定保护作用,但对心肌梗死后凋亡的作用及具体机制尚不明确.本研究用TRPV1基因敲除(TRPV1-/- )小鼠和野生型(wide type, WT)小鼠建立心肌梗死模型,并外源性给予SP和NK1受体拮抗剂RP67580,用TTC染色法观察梗死的面积,TUNEL法检测心肌细胞凋亡指数,Western印迹方法检测caspase-3、Bcl-2、Bax、p53的蛋白表达.结果发现,心肌梗死24 h后,TRPV1-/-小鼠比WT小鼠梗死面积更大,凋亡指数和caspase-3活性更高,Bcl-2/Bax和p53蛋白表达更低. SP预处理可以明显缩小TRPV1-/-小鼠梗死面积,降低凋亡指数、caspase-3活性和升高Bcl-2/Bax比值,而在WT小鼠中改善不明显.外源性给予RP67580,阻断SP与NK1受体结合后,与相应对照组相比,WT小鼠梗死面积和凋亡指数更大,caspase-3蛋白表达更高,Bcl-2/Bax比值更低;TRPV1-/-小鼠与相应对照组比较,凋亡指数和caspase-3表达升高,Bcl-2/Bax比值降低.研究结果表明,SP可能介导了TRPV1在急性心肌梗死后凋亡中的保护作用.     

Expression of dominant-negative Fas-associated death domain blocks human keratinocyte apoptosis and vesication induced by sulfur mustard

DNA damaging agents up-regulate levels of the Fas receptor or its ligand, resulting in recruitment of Fas-associated death domain (FADD) and autocatalytic activation of caspase-8, consequently activating the executioner caspases-3, -6, and -7. We found that human epidermal keratinocytes exposed to a vesicating dose (300 microm) of sulfur mustard (SM) exhibit a dose-dependent increase in the levels of Fas receptor and Fas ligand. Immunoblot analysis revealed that the upstream caspases-8 and -9 are both activated in a time-dependent fashion, and caspase-8 is cleaved prior to caspase-9. These results are consistent with the activation of both death receptor (caspase-8) and mitochondrial (caspase-9) pathways by SM. Pretreatment of keratinocytes with a peptide inhibitor of caspase-3 (Ac-DEVD-CHO) suppressed SM-induced downstream markers of apoptosis. To further analyze the importance of the death receptor pathway in SM toxicity, we utilized Fas- or tumor necrosis factor receptor-neutralizing antibodies or constructs expressing a dominant-negative FADD (FADD-DN) to inhibit the recruitment of FADD to the death receptor complex and block the Fas/tumor necrosis factor receptor pathway following SM exposure. Keratinocytes pretreated with Fas-blocking antibody or stably expressing FADD-DN and exhibiting reduced levels of FADD signaling demonstrated markedly decreased caspase-3 activity when treated with SM. In addition, the processing of procaspases-3, -7, and -8 into their active forms was observed in SM-treated control keratinocytes, but not in FADD-DN cells. Blocking the death receptor complex by expression of FADD-DN additionally inhibited SM-induced internucleosomal DNA cleavage and caspase-6-mediated nuclear lamin cleavage. Significantly, we further found that altering the death receptor pathway by expressing FADD-DN in human skin grafted onto nude mice reduces vesication and tissue injury in response to SM. These results indicate that the death receptor pathway plays a pivotal role in SM-induced apoptosis and is therefore a target for therapeutic intervention to reduce SM injury.     

Comparative Antiapoptotic Effects of KB-R7943 and Ischemic Postconditioning During Myocardial Ischemia Reperfusion

We examined whether KB-R7943 reduced infarct size by attenuating apoptosis during reperfusion and also compared antiapoptotic effects of KB-R7943 and IPost. For this purpose, isolated rat hearts underwent 30-min global ischemia and 120-min reperfusion. Ischemic postconditioning (IPost) (n?=?15; three cycles of 10-s reperfusion/10-s ischemia or three cycles of 30-s reperfusion/30-s ischemia) and KB-R7943 (n?=?15; 1???M KB-R at the onset of reperfusion or before ischemia) were compared with controls (n?=?12; ischemia?Creperfusion only). Myocardial injury was determined by TTC staining, TUNEL assay and caspase-3 activity. AKT and eNOS phosphorylation were measured by immunoblotting. We found that IPost (10?s), Pre KB-R, and Reperf KB-R reduced infarct size (29?±?4.1, 35?±?5.0, 28.6?±?3.4?%, respectively, vs. controls 46?±?8.7?%; P?<?0.05) and attenuated cell apoptosis (TUNEL-positive cardiomyocyte nuclei) in the myocardium (P?<?0.01). Moreover, IPost (10?s), Pre KB-R and Reperf KB-R significantly decreased caspase-3 activation caused by myocardial ischemia?Creperfusion. However, IPost (30?s) did not show any effect on necrosis and apoptosis. Akt, eNOS phosphorylation, at 30?min of reperfusion/IPost-10?s was significantly higher than other groups. In conclusion, KB-R7943 was as effective as IPost in reducing necrosis and inhibiting apoptosis and it might be an ideal pharmacological agent to provide a more amenable approach to cardioprotection.     

Darbepoetin alfa, a long-acting erythropoietin analog, offers novel and delayed cardioprotection for the ischemic heart

Recent studies from our lab and others have shown that the hematopoietic cytokine erythropoietin (EPO) can protect the heart from ischemic damage in a red blood cell-independent manner. Here we examined any protective effects of the long-acting EPO analog darbepoetin alfa (DA) in a rat model of ischemia-reperfusion (I/R) injury. Rats were subjected to 30-min ischemia followed by 72-h reperfusion. In a dose-response study, DA (2, 7, 11, and 30 mug/kg) or vehicle was administered as a single bolus at the start of ischemia. To determine the time window of potential cardioprotection, a single high dose of DA (30 mug/kg) was given at either the initiation or the end of ischemia or at 1 or 24 h after reperfusion. After 3 days, cardiac function and infarct size were assessed. Acute myocyte apoptosis was quantified by TUNEL staining on myocardial sections and by caspase-3 activity assays. DA significantly reduced infarct size from 32.8 +/- 3.5% (vehicle) to 11.0 +/- 3.3% in a dose-dependent manner, while there was no difference in ischemic area between groups. Treatment with DA as late as 24 h after the beginning of reperfusion still demonstrated a significant reduction in infarct size (17.0 +/- 1.6%). Consistent with infarction data, DA improved in vivo cardiac reserve compared with vehicle. Finally, DA significantly decreased myocyte apoptosis and caspase-3 activity after I/R. These data indicate that DA protects the heart against I/R injury and improves cardiac function, apparently through a reduction of myocyte apoptosis. Of clinical importance pointing toward a relevant therapeutic utility, we report that even if given 24 h after I/R injury, DA can significantly protect the myocardium.     

Anti-apoptotic effect of magnolol in myocardial ischemia and reperfusion injury requires extracellular signal-regulated kinase1/2 pathways in rat in vivo

Magnolol, an active component extracted from Magnolia officinalis, has been reported to have protective effect on ischemia and reperfusion (I/R)-induced injury in experimental animals. The aim of the present investigation was to further evaluate the mechanism(s) by which magnolol reduces I/R-induced myocardial injury in rats in vivo. Under anesthesia, left anterior descending (LAD) coronary artery was occluded for 30 min followed by reperfusion for 24 h (for infarct size and cardiac function analysis). In some experiments, reperfusion was limited to 1 h or 6 h for analysis of biochemical and molecular events. Magnolol and DMSO solution (vehicle) were injected intra-peritoneally 1 h prior to I/R insult. The infarct size was measured by TTC technique and heart function was monitored by Millar Catheter. Apoptosis related events such as p-ERK, p-Bad, Bcl-xl and cytochrome c expression were evaluated by Western blot analysis and myocardial caspase-3 activity was also measured. Magnolol (10 mg/kg) reduced infarct size by 50% (P < 0.01 versus vehicle), and also improved I/R-induced myocardial dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in magnolol-treated rats. Magnolol increased the expression of phosphor ERK and Bad which resulted in inhibition of myocardial apoptosis as evidenced by TUNEL analysis and DNA laddering experiments. Application of PD 98059, a selective MEK1/2 inhibitor, strongly antagonized the effect of magnolol. Taken together, we concluded that magnolol inhibits apoptosis through enhancing the activation of ERK1/2 and modulation of the Bcl-xl proteins which brings about reduction of infarct size and improvement of cardiac function in I/R-induced injury.     

A20 inhibits oxidized low-density lipoprotein-induced apoptosis through negative Fas/Fas ligand-dependent activation of caspase-8 and mitochondrial pathways in murine RAW264.7 macrophages

A20 was originally characterized as a TNF-inducible gene in human umbilical vein endothelial cells. As an NF-kappaB target gene, A20 is also induced in many other cell types by a wide range of stimuli. Expression of A20 has been shown to protect from TNF-induced apoptosis and also functions via a negative-feedback loop to block NF-kappaB activation induced by TNF and other stimuli. To date, there are no reports on whether A20 can protect OxLDL-induced apoptosis in macrophages. For the first time we report that A20 expression blocks OxLDL-mediated cell toxicity and apoptosis. OxLDL induced the expression of Fas and FasL, and the subsequent caspase-8 cleavage and treatment with a neutralizing ZB4 anti-Fas antibody blocked apoptosis induced by OxLDL. Expression of dominant negative FADD efficiently prevented OxLDL-induced apoptosis and caspase-8 activation. A20 expression significantly attenuated the increased expression of Fas and FasL, and Fas-mediated apoptosis. These findings suggest that A20-mediated protection from OxLDL may occur at the level of Fas/FADD-caspase-8 and be FasL dependent. Treatment of RAW264.7 cells with OxLDL induces a series of time-dependent events, including the release of cytochrome c, Smac and Omi from the mitochondria to the cytosol, activation of caspase-9, -6, -2, and -3, which are blocked by A20 expression. No cleaved form of Bid was detected, even treatment with OxLDL for 48 h. Expression of dominant negative FADD also efficiently prevented OxLDL-induced the above apoptotic events. The release of cyto c, Smac and Omi from mitochondria to cytosol, activated by OxLDL treatment, and the activation of caspase-9 may not be a downstream event of caspase-8-mediated Bid cleavage. Therefore, the protective effect of A20 on mitochondrial apoptotic pathway activated by OxLDL may be dependent on FADD. A20 expression reversed OxLDL-mediated G(0)/G(1) stage arrest by maintaining the expression of cyclin B1, cyclin D1, and cyclin E, and p21 and p73. Thus, A20 expression blocks OxLDL-mediated apoptosis in murine RAW264.7 macrophages through disrupting Fas/FasL-dependent activation of caspase-8 and the mitochondria pathway.     

BAX contributes to apoptotic-like death following neonatal hypoxia-ischemia: evidence for distinct apoptosis pathways.

BACKGROUND: Hypoxic-ischemic (H-I) injury to the neonatal brain has been shown to result in rapid cell death with features of acute excitotoxicity/necrosis as well as prominent delayed cell death with features of apoptosis such as marked caspase-3 activation. BAX, a pro-apoptotic molecule, has been shown to be required for apoptotic neuronal cell death during normal development but the contribution of endogenous BAX in cell death pathways following H-I injury to the developing or adult brain has not been studied. MATERIALS AND METHODS: Bax +/+, +/-, and -/- mice at post-natal day 7 were subjected to unilateral carotid ligation followed by exposure to 45 minutes of 8% oxygen. At different timepoints following H-I, brain tissue was studied by conventional histology, immunohistochemistry, immunofluorescence, Western blotting, and enzymatic assay to determine the extent and type of cell injury as well as the amount of caspase activation. RESULTS: We found that bax -/- mice had significantly less (38%) hippocampal tissue loss than mice expressing bax. Some of the remaining cell death in bax -/- mice, however, still had features of apoptosis including evidence of nuclear shrinkage and caspase-3 activation. Though bax -/- mice had significantly decreased caspase-3 activation as compared to bax expressing mice following H-I, the density of cells with activated caspase-8 in the CA3 region of the hippocampus did not differ between bax +/- and bax -/- mice. CONCLUSIONS: These findings demonstrate that endogenous BAX plays a role in regulating cell death in the central nervous system (CNS) following neonatal H-I, a model of cerebral palsy. In addition, while BAX appears to modulate the caspase-3 activation following neonatal H-I, caspase-8 which is linked to death receptor activation, may contribute to apoptotic-like neuronal death in a BAX-independent manner.     

Bid is cleaved upstream of caspase-8 activation during TRAIL-mediated apoptosis in human osteosarcoma cells

TRAIL induces apoptosis in many malignant cell types. In this study, we used the human papilloma virus (HPV) 16 E6 protein as a molecular tool to probe the TRAIL pathway in HCT116 colon carcinoma cells and U2OS osteosarcoma cells. Intriguingly, we found that while E6 protected HCT116 cells from TRAIL, U2OS cells expressing E6 remained sensitive to TRAIL. Furthermore, silencing FADD and procaspase-8 expression with siRNA did not prevent TRAIL-induced apoptosis in U2OS cells. However, siBid provided significant protection from TRAIL, and the cleavage kinetics of Bid and caspase-8 revealed that Bid was cleaved prior to the activation of caspase-8. Cathepsin B activity in U2OS cells was significantly activated shortly after exposure to TRAIL, and the cathepsin B inhibitor, CA074Me, inhibited both TRAIL- and anti-DR5-mediated apoptosis and delayed the cleavage of Bid. These findings suggest that TRAIL activates a pathway dependent on Bid, but largely independent of FADD and caspase-8, in U2OS cells.     

FADD is required for multiple signaling events downstream of the receptor Fas.

To identify essential components of the Fas-induced apoptotic signaling pathway, Jurkat T lymphocytes were chemically mutagenized and selected for clones that were resistant to Fas-induced apoptosis. We obtained five cell lines that contain mutations in the adaptor FADD. All five cell lines did not express FADD by immunoblot analysis and were completely resistant to Fas-induced death. Complementation of the FADD mutant cell lines with wild-type FADD restored Fas-mediated apoptosis. Fas activation of caspase-2, caspase-3, caspase-7, and caspase-8 and the proteolytic cleavage of substrates such as BID, protein kinase Cdelta, and poly(ADP-ribose) polymerase were completely defective in the FADD mutant cell lines. In addition, Fas activation of the stress kinases p38 and c-Jun NH2 kinase and the generation of ceramide in response to Fas ligation were blocked in the FADD mutant cell lines. These data indicate that FADD is essential for multiple signaling events downstream of Fas.     

Myrtucommulone from <Emphasis Type="Italic">Myrtus communis</Emphasis> induces apoptosis in cancer cells via the mitochondrial pathway involving caspase-9

Myrtucommulone (MC) is a unique, nonprenylated acylphloroglucinol contained in the leaves of myrtle (Myrtus communis). Here, we addressed the potential of MC to induce apoptosis of cancer cells. MC potently induced cell death of different cancer cell lines (EC₅₀ 3–8 μM) with characteristics of apoptosis, visualized by the activation of caspase-3, -8 and -9, cleavage of poly(ADP-ribose)polymerase (PARP), release of nucleosomes into the cytosol, and DNA fragmentation. MC was much less cytotoxic for non-transformed human peripheral blood mononuclear cells (PBMC) or foreskin fibroblasts (EC₅₀ cell death = 20–50 μM), and MC up to 30 μM hardly caused processing of PARP, caspase-3, -8 and -9 in human PBMC. MC-induced apoptosis was mediated by the intrinsic rather than the extrinsic death pathway. Thus, MC caused loss of the mitochondrial membrane potential in MM6 cells and evoked release of cytochrome c from mitochondria. Interestingly, Jurkat cells deficient in caspase-9 were resistant to MC-induced cell death and no processing of PARP or caspase-8 was evident. In cell lines deficient in either CD95 (Fas, APO-1) signalling, FADD or caspase-8, MC was still able to potently induce cell death and PARP cleavage. Conclusively, MC induces apoptosis in cancer cell lines, with marginal cytotoxicity for non-transformed cells, via the mitochondrial cytochrome c/Apaf-1/caspase-9 pathway. I. Tretiakova and D. Blaesius contributed equally to this work.     

A caspase-8-independent component in TRAIL/Apo-2L-induced cell death in human rhabdomyosarcoma cells

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) belongs to the Tumor necrosis factor (TNF) family of death-inducing ligands, and signaling downstream of TRAIL ligation to its receptor(s) remains to be fully elucidated. Components of the death-inducing signaling complex (DISC) and TRAIL signaling downstream of receptor activation were examined in TRAIL - sensitive and -resistant models of human rhabdomyosarcoma (RMS). TRAIL ligation induced DISC formation in TRAIL-sensitive (RD, Rh18, Rh30) and TRAIL-resistant RMS (Rh28, Rh36, Rh41), with recruitment of FADD and procaspase-8. In RD cells, overexpression of dominant-negative FADD (DNFADD) completely abolished TRAIL-induced cell death in contrast to dominant-negative caspase- 8 (DNC8), which only partially inhibited TRAIL-induced apoptosis, growth inhibition, or loss in clonogenic survival. DNC8 did not inhibit the cleavage of Bid or the activation of Bax. Overexpression of Bcl-2 or Bcl-xL inhibited TRAIL-induced apoptosis, growth inhibition, and loss in clonogenic survival. Bcl-2 and Bcl-xL, but not DNC8, inhibited TRAIL-induced Bax activation. Bcl-xL did not inhibit the early activation of caspase-8 (<4 h) but inhibited cleavage of Bid, suggesting that Bid is cleaved downstream of the mitochondria, independent of caspase-8. Exogenous addition of sphingosine also induced activation of Bax via a caspase-8-and Bid-independent mechanism. Further, inhibition of sphingosine kinase completely protected cells from TRAIL-induced apoptosis. Data demonstrate that in RMS cells, the TRAIL signaling pathway circumvents caspase-8 activation of Bid upstream of the mitochondria and that TRAIL acts at the level of the mitochondria via a mechanism that may involve components of the sphingomyelin cycle.     

FADD deficiency sensitises Jurkat T cells to TNF-alpha-dependent necrosis during activation-induced cell death

Activation-induced cell death (AICD) in activated T lymphocytes is largely mediated by Fas/Fas ligand (FasL) interaction. The cytoplasmic adaptor molecule Fas-associated death domain protein (FADD) plays an essential role in the apoptotic signalling of the Fas death pathway. In the present study, we observed that FADD deficient (FADD(-/-)) Jurkat T cells undergo AICD to a similar extent as wild-type cells. AICD in wild-type Jurkat T cells is via apoptosis, whereas it is non-apoptotic in FADD(-/-) cells. The latter took up propidium iodide, exhibit a loss in mitochondrial membrane potential and have no detectable cleavage products of caspase-8 or -3 activation, suggesting that these cells die by necrosis. Wild-type Jurkat T cells undergo apoptosis when incubated with recombinant FasL and Trail but not with TNF-alpha. In contrast, FADD(-/-) Jurkat T cells are resistant to FasL and Trail but die of necrosis when incubated with TNF-alpha. We showed that neutralising anti-TNF-alpha blocked AICD as well as TNF-alpha-induced necrosis in FADD(-/-) Jurkat T cells. Furthermore, down regulating the receptor interacting protein, RIP, with geldanamycin treatment, which is essential for TNF-alpha signalling, markedly inhibited AICD in FADD(-/-) Jurkat T cells. In addition, caspase-8-deficient Jurkat T cells are resistant to Fas- and TNF-alpha-induced cell death. Taken together, our results suggest that a deficiency in FADD and not caspase-8 or the inhibition of the Fas signalling pathway sensitises Jurkat T cells to TNF-alpha-dependent necrosis during AICD.     

Adrenomedullin gene delivery attenuates myocardial infarction and apoptosis after ischemia and reperfusion

Adrenomedullin (AM) has been shown to protect against cardiac remodeling. In this study, we investigated the potential role of AM in myocardial ischemia-reperfusion (I/R) injury through adenovirus-mediated gene delivery. One week after AM gene delivery, rats were subjected to 30-min coronary occlusion, followed by 2-h reperfusion. AM gene transfer significantly reduced the ratio of infarct size to ischemic area at risk and the occurrence of sustained ventricular fibrillation compared with control rats. AM gene delivery also attenuated apoptosis, assessed by both terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and DNA laddering. The effect of AM gene transfer on infarct size, arrhythmia, and apoptosis was abolished by an AM antagonist, calcitonin gene-related peptide [CGRP(8-37)]. Expression of human AM significantly increased cardiac cGMP levels and reduced superoxide production, superoxide density, NAD(P)H oxidase activity, p38 MAPK activation, and Bax levels. Moreover, AM increased Akt and Bad phosphorylation and Bcl-2 levels, but decreased caspase-3 activation. These results indicate that AM protects against myocardial infarction, arrhythmia, and apoptosis in I/R injury via suppression of oxidative stress-induced Bax and p38 MAPK phosphorylation and activation of the Akt-Bad-Bcl-2 signaling pathway. Successful application of this technology may have a protective effect in coronary artery diseases.     

Human cell-death-inducing DFF45-like effector C induces apoptosis via caspase-8

Human cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector C (CIDEC) is a potent apoptotic inducer. Previous studies have indicated that the Fat-specific protein 27 (Fsp27), a mouse homolog of CIDEC, induces apoptosis via caspase-3, -7, and -9 and triggers the release of cytochrome c from mitochondria, which implies that the mitochondrial pathway is involved in Fsp27-induced apoptosis. In the current study, we found that CIDEC-induced apoptosis was mediated by caspase-8. The caspase inhibitor assay showed that CIDEC-induced apoptosis was dramatically reduced in the presence of the general caspase inhibitor, the caspase-3 inhibitor, and the caspase-8 inhibitor, whereas the caspase-9 inhibitor only weakly inhibited CIDEC-induced apoptosis. These results confirmed that the activation of caspase-3 and caspase-8 were involved in CIDEC-induced apoptosis. Moreover, in caspase-3- or caspase-8-deficient cells, CIDEC-induced apoptosis were dramatically decreased, which demonstrated that CIDEC-induced apoptosis might require the activation of caspase-3 and caspase-8. Because caspase-8 in general is a key effecter of death-receptor pathway and activated by Fas-Associated protein with Death Domain (FADD), we examined whether FADD was involved in CIDEC-induced apoptosis. Our results demonstrated that CIDEC-induced apoptosis was independent of FADD, suggesting that CIDEC-induced apoptosis might be in a death-receptor-independent, caspase-8-dependent manner. It was also found that the region of amino acid 168-200 in carboxyl domain of CIDEC was critical for its crucial pro-apoptotic function.     

The prevalence of TNFα-induced necrosis over apoptosis is determined by TAK1-RIP1 interplay

Death receptor-induced programmed necrosis is regarded as a secondary death mechanism dominating only in cells that cannot properly induce caspase-dependent apoptosis. Here, we show that in cells lacking TGFβ-activated Kinase-1 (TAK1) expression, catalytically active Receptor Interacting Protein 1 (RIP1)-dependent programmed necrosis overrides apoptotic processes following Tumor Necrosis Factor-α (TNFα) stimulation and results in rapid cell death. Importantly, the activation of the caspase cascade and caspase-8-mediated RIP1 cleavage in TNFα-stimulated TAK1 deficient cells is not sufficient to prevent RIP1-dependent necrosome formation and subsequent programmed necrosis. Our results demonstrate that TAK1 acts independently of its kinase activity to prevent the premature dissociation of ubiquitinated-RIP1 from TNFα-stimulated TNF-receptor I and also to inhibit the formation of TNFα-induced necrosome complex consisting of RIP1, RIP3, FADD, caspase-8 and cFLIP(L). The surprising prevalence of catalytically active RIP1-dependent programmed necrosis over apoptosis despite ongoing caspase activity implicates a complex regulatory mechanism governing the decision between both cell death pathways following death receptor stimulation.     

Ginsenoside Rd Attenuates Myocardial Ischemia/Reperfusion Injury via Akt/GSK-3β Signaling and Inhibition of the Mitochondria-Dependent Apoptotic Pathway

Evidence suggests Ginsenoside Rd (GSRd), a biologically active extract from the medical plant Panax Ginseng, exerts antioxidant effect, decreasing reactive oxygen species (ROS) formation. Current study determined the effect of GSRd on myocardial ischemia/reperfusion (MI/R) injury (a pathological condition where ROS production is significantly increased) and investigated the underlying mechanisms. The current study utilized an in vivo rat model of MI/R injury and an in vitro neonatal rat cardiomyocyte (NRC) model of simulated ischemia/reperfusion (SI/R) injury. Infarct size was measured by Evans blue/TTC double staining. NRC injury was determined by MTT and lactate dehydrogenase (LDH) leakage assay. ROS accumulation and apoptosis were assessed by flow cytometry. Mitochondrial membrane potential (MMP) was determined by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetrathylbenzimidazol carbocyanine iodide (JC-1). Cytosolic translocation of mitochondrial cytochrome c and expression of caspase-9, caspase-3, Bcl-2 family proteins, and phosphorylated Akt and GSK-3β were determined by western blot. Pretreatment with GSRd (50 mg/kg) significantly augmented rat cardiac function, as evidenced by increased left ventricular ejection fraction (LVEF) and ±dP/dt. GSRd reduced myocardial infarct size, apoptotic cell death, and blood creatine kinase/lactate dehydrogenase levels after MI/R. In NRCs, GSRd (10 µM) inhibited SI/R-induced ROS generation (P<0.01), decreased cellular apoptosis, stabilized the mitochondrial membrane potential (MMP), and attenuated cytosolic translocation of mitochondrial cytochrome c. GSRd inhibited activation of caspase-9 and caspase-3, increased the phosphorylated Akt and GSK-3β, and increased the Bcl-2/Bax ratio. Together, these data demonstrate GSRd mediated cardioprotective effect against MI/R–induced apoptosis via a mitochondrial-dependent apoptotic pathway.     

Disassembly of the JIP1/JNK molecular scaffold by caspase-3-mediated cleavage of JIP1 during apoptosis

We report here the cleavage of the c-Jun N-terminal Kinase (JNK) pathway scaffold protein, JNK Interacting Protein-1 (JIP1), by caspases during both Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and staurosporine-induced apoptosis in HeLa cells. During the initiation of apoptosis, maximal JNK activation is observed when JIP1 is intact, whereas cleavage of JIP1 correlates with JNK inactivation and progression of apoptosis. JIP1 is cleaved by caspase-3 at two sites, leading to disassembly of the JIP1/JNK complex. Inhibition of JIP1 cleavage by the caspase-3 inhibitor DEVD.fmk inhibits this disassembly, and is accompanied by sustained JNK activation. These data suggest that TRAIL and staurosporine induce JNK activation in a caspase-3-independent manner and that caspase-3-mediated JIP1 cleavage plays a role in JNK inactivation via scaffold disassembly during the execution phase of apoptosis. Caspase-mediated cleavage of JIP scaffold proteins may therefore represent an important mechanism for modulation of JNK signalling during apoptotic cell death.     

Protein Kinase RNA/FADD/Caspase-8 Pathway Mediates the Proapoptotic Activity of the RNA-binding Protein Human Antigen R (HuR)

The RNA-binding protein human antigen R (HuR) has been implicated in apoptosis in multiple ways. Several studies have shown that in response to a variety of stresses HuR promotes the expression of proapoptotic mRNAs, whereas others reported its regulatory effect on antiapoptotic messages. We recently showed that in response to severe stress, HuR is cleaved to generate two cleavage products (CPs), HuR-CP1 (24 kDa) and HuR-CP2 (8 kDa), by which it promotes apoptotic cell death. Here, we show that this cleavage event is dependent on protein kinase RNA (PKR). Surprisingly, although in response to the apoptotic inducer staurosporine PKR itself is not phosphorylated, PKR triggers the cleavage of HuR via its downstream effector FADD that in turn activates the caspase-8/caspase-3 pathway. This effect, however, does not require the phosphorylation of the eukaryotic translation initiation factor 2α. Additionally, we observed that these HuR-CPs are sufficient to trigger cell death in the absence of activation of the PKR pathway. Therefore, our results support a model whereby in response to lethal stress, PKR, without being phosphorylated, activates the FADD/caspase-8/caspase-3 pathway to trigger HuR cleavage, and the HuR-CPs are then capable of promoting apoptosis.     

Neuroprotective Effect of Baicalin in a Rat Model of Permanent Focal Cerebral Ischemia

This investigation was performed to determine the neuroprotective effect of baicalin on permanent cerebral ischemia injury in rats and the potential mechanisms in this process. Adult male Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (pMCAO). The rats were then received intraperitoneal injection with baicalin (10, 30 and 100 mg/kg) or vehicle. Morphological characteristic, neurological deficit scores, cerebral infarct volume and the enzymatic activity of myeloperoxidase (MPO) were measured 24 h after pMCAO. The mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were determined by RT-PCR. Neuronal apoptosis was determined by TUNEL staining and Western blot. Baicalin (30 and 100 mg/kg) reduced neurological deficit scores and cerebral infarct volume 24 h after pMCAO. Baicalin significantly decreased the enzymatic activity of MPO and the expression of iNOS mRNA and COX-2 mRNA in rat brain, it also significantly inhibited neuronal apoptosis and the expression of cleaved caspase-3 protein after pMCAO. Our results suggested that baicalin possesses potent anti-inflammatory and anti-apoptotic properties and attenuates cerebral ischemia injury. This protection might be associated with the downregulated expression of iNOS mRNA, COX-2 mRNA, and cleaved caspase-3 protein.     

Effects of captopril and angiotensin II receptor blockers (AT1, AT2) on myocardial ischemia-reperfusion induced infarct size

The renin-angiotensin system (RAS) plays a major role in regulating the cardiovascular system, and disorders of the RAS contribute largely to the cardiac pathophysiology, including myocardial ischemia-reperfusion (MI/R) injury. Two subtypes of angiotensin II (Ang II) receptors have been defined on the basis of their differential pharmacological properties. The current study was undertaken to address the question as to whether the inhibition of the angiotensin converting enzyme (ACE) by captopril and the AT1 and AT2 receptor blockers losartan and PD123319 modulate MI/R-induced infarct size in an in vivo rat model. To produce necrosis, a branch of the descending left coronary artery was occluded for 30 min followed by two hours of reperfusion. ECG changes, blood pressure, and heart rate were measured during the experiment. Captopril (3 mg/kg), losartan (2 mg/kg), and PD123319 (20 μg/kg/min) were given in an IV 10 min before ischemia and were continued during the ischemic period. The infarcted area was measured by TTC staining. The volume of infarct and the risk zone was determined by planimetry. Compared to the control group (55.62±4.00%) both captopril and losartan significantly reduced the myocardial infarct size (30.50±3.26% and 37.75±4.44%), whereas neither PD123319 nor PD123319+losartan affected the infarct size volume (46.50±3.72% and 54.62±2.43%). Our data indicates that captopril and losartan exert cardioprotective activity after an MI/R injury. Also, infarct size reduction by losartan was halted by a blockade of the AT2 receptor. Therefore, the activation of AT2 receptors may be potentially protective and appear to oppose the effects mediated by the AT1 receptors.