The endoplasmic reticulum stress inhibitor salubrinal inhibits the activation of autophagy and neuroprotection induced by brain ischemic preconditioning

Aim:

To investigate whether endoplasmic reticulum (ER) stress participates in the neuroprotective effects of ischemic preconditioning (IPC)-induced neuroprotection and autophagy activation in rat brains.

Methods:

The right middle cerebral artery in SD rats was occluded for 10 min to induce focal cerebral IPC, and was occluded permanently 24 h later to induce permanent focal ischemia (PFI). ER stress inhibitor salubrinal (SAL) was injected via intracerebral ventricle infusion 10 min before the onset of IPC. Infarct volume and motor behavior deficits were examined after the ischemic insult. The protein levels of LC3, p62, HSP70, glucose-regulated protein 78 (GRP 78), p-eIF2α and caspase-12 in the ipsilateral cortex were analyzed using immunoblotting. LC3 expression pattern in the sections of ipsilateral cortex was observed with immunofluorescence.

Results:

Pretreatment with SAL (150 pmol) abolished the neuroprotective effects of IPC, as evidenced by the significant increases in mortality, infarct volume and motor deficits after PFI. At the molecular levels, pretreatment with SAL (150 pmol) significantly increased p-eIF2α level, and decreased GRP78 level after PFI, suggesting that SAL effectively inhibited ER stress in the cortex. Furthermore, the pretreatment with SAL blocked the IPC-induced upregulation of LC3-II and downregulation of p62 in the cortex, thus inhibiting the activation of autophagy. Moreover,SAL blocked the upregulation of HSP70, but significantly increased the cleaved caspase-12 level, thus promoting ER stress-dependent apoptotic signaling in the cortex.

Conclusion:

ER stress-induced autophagy might contribute to the neuroprotective effect of brain ischemic preconditioning.     

A novel quinone-based derivative (DTNQ-Pro) induces apoptotic death via modulation of heat shock protein expression in Caco-2 cells

Background and purpose:

The resistance of human colon adenocarcinoma cells to antineoplastic agents may be related to the high endogenous expression of stress proteins, including the family of heat shock proteins (HSPs). Recently, a quinone-based pentacyclic derivative, DTNQ-Pro, showed high cytotoxic activity in human colon carcinoma cell lines. The aim of the present study was to determine the precise cellular mechanisms of this cytotoxic action of DTNQ-Pro.

Experimental approach:

Using human colorectal carcinoma-derived Caco-2 cells as a model, we studied the effects of DTNQ-Pro on cellular viability and oxidative stress; HSP70 and HSP27 accumulation; and cell cycle, differentiation and apoptosis.

Key results:

Incubation of Caco-2 cells with DTNQ-Pro reduced cell growth and increased the levels of reactive oxygen species in mitochondria. After 48 h of treatment, cells surviving showed an increased expression of Mn-superoxide dismutase (SOD), nitric oxide production and membrane lipid peroxidation. Treatment with DTNQ-Pro decreased HSP70 expression, and redistributed HSP27 and vimentin within the cell. DTNQ-Pro down-regulated the expression of A and B cyclins with arrest of the cell cycle in S phase and increased cellular differentiation. A second treatment of Caco-2 cells with DTNQ-Pro induced cellular death by activation of the apoptotic pathway.

Conclusions and implications:

DTNQ-Pro causes Caco-2 cell death by induction of apoptosis via inhibition of HSP70 accumulation and the intracellular redistribution of HSP27. These findings suggest the potential use of DTNQ-Pro in combination chemotherapy for colon cancer.     

The edaravone and 3-n-butylphthalide ring-opening derivative 10b effectively attenuates cerebral ischemia injury in rats

Aim:

Compound 10b is a hybrid molecule of edaravone and a ring-opening derivative of 3-n-butylphthalide (NBP). The aim of this study was to examine the effects of compound 10b on brain damage in rats after focal cerebral ischemia.

Methods:

SD rats were subjected to 2-h-middle cerebral artery occlusion (MCAO). At the onset of reperfusion, the rats were orally treated with NBP (60 mg/kg), edaravone (3 mg/kg), NBP (60 mg/kg)+edaravone (3 mg/kg), or compound 10b (70, 140 mg/kg). The infarct volume, motor behavior deficits, brain water content, histopathological alterations, and activity of GSH, SOD, and MDA were analyzed 24 h after reperfusion. The levels of relevant proteins in the ipsilateral striatum were examined using immunoblotting.

Results:

Administration of compound 10b (70 or 140 mg/kg) significantly reduced the infarct volume and neurological deficits in MCAO rats. The neuroprotective effects of compound 10b were more pronounced compared to NBP, edaravone or NBP+edaravone. Furthermore, compound 10b significantly upregulated the protein levels of the cytoprotective molecules Bcl-2, HO-1, Nrf2, Trx, P-NF-κB p65, and IκB-α, while decreasing the expression of Bax, caspase 3, caspase 9, Txnip, NF-κB p65, and P-IκB-α.

Conclusion:

Oral administration of compound 10b effectively attenuates rat cerebral ischemia injury.     

RNA interference-mediated downregulation of Beclin1 attenuates cerebral ischemic injury in rats

Aim:

To test the role of the Beclin 1-dependent autophagy pathway in brain damage during cerebral ischemia.

Methods:

Focal cerebral ischemia was established in rats using a middle cerebral artery occlusion (MCAO) model. A lentiviral vector-associated RNA interference (RNAi) system was stereotaxically injected into the ipsilateral lateral ventricle to reduce Beclin1 expression. We measured the ipsilateral infarct volume, autophagosome formation, neurogenesis and apoptosis, all of which could be modulated by Beclin1 RNAi.

Results:

On the 14th day after MCAO, Beclin1 downregulation by RNAi increased the population of neural progenitor cells (BrdU⁺-DCX⁺), newborn immature cells (BrdU⁺-Tuj-1⁺) and mature neurons (BrdU⁺-MAP-2⁺), and reduced the apoptosis of immature neurons (caspase-3⁺-DCX⁺ and caspase-3⁺-Tuj-1⁺) surrounding the ischemic core of the ipsilateral hemisphere. Furthermore, RNAi-mediated downregulation of Beclin1 decreased infarct volume and inhibited histological injury and neurological deficits.

Conclusion:

RNAi-mediated downregulation of Beclin1 improves outcomes after transient MCAO.     

Mesenchymal stem cells transplantation suppresses inflammatory responses in global cerebral ischemia: contribution of TNF-α-induced protein 6

Aim:

To investigate the effects of mesenchymal stem cells (MSCs) transplantation on rat global cerebral ischemia and the underlying mechanisms.

Methods:

Adult male SD rats underwent asphxial cardiac arrest to induce global cerebral ischemia, then received intravenous injection of 5×10⁶ cultured MSCs of SD rats at 2 h after resuscitation. In another group of cardiac arrest rats, tumor necrosis factor-α-induced protein 6 (TSG-6, 6 μg) was injected into the right lateral ventricle. Functional outcome was assessed at 1, 3, and 7 d after resuscitation. Donor MSCs in the brains were detected at 3 d after resuscitation. The level of serum S-100B and proinflammatory cytokines in cerebral cortex were assayed using ELISA. The expression of TSG-6 and proinflammatory cytokines in cerebral cortex was assayed using RT-PCR. Western blot was performed to determine the levels of TSG-6 and neutrophil elastase in cerebral cortex.

Results:

MSCs transplantation significantly reduced serum S-100B level, and improved neurological function after global cerebral ischemia compared to the PBS-treated group. The MSCs injected migrated into the ischemic brains, and were observed mainly in the cerebral cortex. Furthermore, MSCs transplantation significantly increased the expression of TSG-6, and reduced the expression of neutrophil elastase and proinflammatory cytokines in the cerebral cortex. Intracerebroventricular injection of TSG-6 reproduced the beneficial effects of MSCs transplantation in rats with global cerebral ischemia.

Conclusion:

MSCs transplantation improves functional recovery and reduces inflammatory responses in rats with global cerebral ischemia, maybe via upregulation of TSG-6 expression.     

Bcl-2-dependent upregulation of autophagy by sequestosome 1/p62 in vitro

Aim:

To investigate whether sequestosome 1/p62 (p62), a key cargo adaptor protein involved in both the ubiquitin-proteasome system and the autophagy-lysosome system, could directly regulate autophagy in vitro.

Methods:

HEK 293 cells or HeLa cells were transfected with p62-expressing plasmids or siRNA targeting p62. The cells or the cell lysates were subsequently subjected to immunofluorescence assay, immunoprecipitation assay, or immunoblot analysis. In vitro pulldown assay was used to study the interaction of p62 with Bcl-2.

Results:

Overexpression of p62 significantly increased the basal level of autophagy in both HEK 293 cells and HeLa cells, whereas knockdown of p62 significantly decreased the basal level of autophagy. In vitro pulldown assay showed that p62 directly interacted with Bcl-2. It was observed in HeLa cells that p62 co-localized with Bcl-2. Furthermore, knockdown of p62 in HEK 293 cells significantly increased the amount of Beclin 1 that co-immunoprecipitated with Bcl-2.

Conclusion:

p62 induces autophagy by disrupting the association between Bcl-2 and Beclin 1.     

Human umbilical cord blood mesenchymal stem cell transplantation suppresses inflammatory responses and neuronal apoptosis during early stage of focal cerebral ischemia in rabbits

Aim： Human umbilical cord blood mesenchymal stem cells （hUCB-MSCs） have been shown to ameliorate cerebral ischemia in animal models. In this study we investigated the effects of hUCB-MSCs on inflammatory responses and neuronal apoptosis during the early stage of focal cerebral ischemia in rabbits. Methods： Focal cerebral ischemia was induced in male New Zealand rabbits by occlusion of MCA for 2 h. The blood samples were collected at different time points prior and during MCAO-reperfusion. The animals were euthanized 3 d after MCAO, and the protein levels of IL-113, IL-6, IL-IO, and TNF-a in the serum and peri-ischemic brain tissues were detected using Western blot and ELISA, respectively. Inflammatory cell infiltration, neuronal apoptosis and neuronal density were measured morphologically, hUCB-MSCs （5x106） were iv injected a few minutes after MCAO. Results： The serum levels of IL-113, IL-6, and TNF-~ were rapidly increased, and peaked at 2 h after the start of MCAO. hUCB-MSC transplantation markedly and progressively suppressed the ischemia-induced increases of serum IL-113, IL-6, and TNF-a levels within 6 h MCAO-reperfusion. Focal cerebral ischemia decreased the serum level of IL-IO, which was prevented by hUCB-MSC transplantation The expression of IL-113, IL-6, IL-IO, and TNF-a in the peri-ischemic brain tissues showed similar changes as in the serum, hUCB-MSC transplantation markedly suppressed the infiltration of inflammatory cells, and increased the neuronal density around the ischemic region. Furthermore, hUCB-MSC transplantation significantly decreased the percentage of apoptosis around the ischemic region. Conclusion： hUCB-MSCs transplantation suppresses inflammatory responses and neuronal apoptosis during the early stage focal cerebral ischemia in rabbits.     

The effects of a single nucleotide polymorphism in SLCO1B1 on the pharmacodynamics of pravastatin

AIM

To determine whether the SNP rs4149056 in SLCO1B1 alters the pharmacodynamics of pravastatin.

METHODS

rs4149056 was genotyped in 626 pravastatin-treated participants in the WOSCOPS trial and the response after 1 year of treatment was compared between the different genotypes.

RESULTS

Pravastatin reduced serum LDL cholesterol by 22.2% in TT homozygotes, by 22.2% in TC heterozygotes and by 17.7% in CC homozygotes (TT + TC vs. CC P value 0.33). There were no significant differences in the response of total cholesterol, LDL, HDL, triglycerides or CRP to pravastatin between the genotypes.

CONCLUSION

The rs4149056 SNP did not significantly affect the pharmacodynamics of pravastatin.     

Neuroprotective activities of catalpol against CaMKII-dependent apoptosis induced by LPS in PC12 cells

Background and Purpose

Neurodegenerative diseases present progressive neurological disorder induced by cell death or apoptosis. Catalpol, an iridoid glucoside isolated from the root of Rehmannia glutinosa Libosch, is present in a wide range of plant families. Although catalpol is an effective anti-apoptotic agent in LPS-induced neurodegeneration, the underlying mechanism has not been established. Here we have identified some of the mechanisms involved the prevention by catalpol of apoptosis induced by LPS in an experimental model of neurodegeneration in vitro.

Experimental Approach

Apoptosis was induced by adding LPS (80 ng·mL⁻¹) to pheochromocytoma (PC12) cells, pretreated with catalpol for 12 h. We measured intracellular reactive oxygen species (ROS), apoptosis and intracellular calcium concentration ( [Ca²⁺]i) by flow cytometry or laser confocal scanning microscopy. We also analysed the protein expression of Bcl-2, Bax and Ca²⁺-calmodulin-dependent protein kinase II (CaMKII)-dependent apoptosis signal-regulating kinase-1 (ASK-1)/JNK/p38 signalling pathway in PC12 cells by Western blot.

Key Results

Catalpol stimulated expression of Bcl-2 and inhibited the expression of Bax. Catalpol also attenuated the increase in Ca²⁺ concentration induced by LPS in PC12 cells and down-regulated CaMK phosphorylation. The CaMKII-dependent ASK-1/JNK/p38 signalling cascade was blocked by catalpol. All these changes were accompanied by a decrease of apoptosis induced by LPS in PC12 cells.

Conclusions and Implications

The data presented here provide new mechanistic insights into the links between the CaMKII-dependent ASK-1/JNK/p38 signalling pathway and the protective effect of catalpol on apoptosis induced by LPS in PC12 cells.     

Tribulosin protects rat hearts from ischemia/ reperfusion injury

Aim:

To investigate the protective effect of tribulosin, a monomer of the gross saponins from Tribulus terrestris, against cardiac ischemia/reperfusion injury and the underlying mechanism in rats.

Methods:

Isolated rat hearts were subjected to 30 min of ischemia followed by 120 min of reperfusion using Langendorff''s technique. The hearts were assigned to seven groups: control, ischemia/reperfusion (I/R), treatment with gross saponins from Tribulus terrestris (GSTT) 100 mg/L, treatment with tribulosin (100, 10, and 1 nmol/L) and treatment with a PKC inhibitor (chelerythrine) (1 μmol/L). Infarct size was assessed by triphenyltetrazolium chloride staining. Malondialdehyde (MDA), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) contents as well as superoxide dismutase (SOD) and creatine kinase (CK) activities were determined after the treatment. Histopathological changes in the myocardium were observed using hematoxylin-eosin (H&E) staining. Apoptosis was detected with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay. Bcl-2, Bax, caspase-3, and PKCɛ protein expression were examined using Western blotting.

Results:

Tribulosin treatment significantly reduced MDA, AST, CK and LDH contents, and increased the activity of SOD. The infarct size of I/R group was 40.21% of the total area. GSTT and various concentrations of tribulosin treatment decreased the infarct size to 24.33%, 20.24%, 23.19%, and 30.32% (P<0.01). Tribulosin treatment reduced the myocardial apoptosis rate in a concentration-dependent manner. Bcl-2 and PKCɛ protein expression was increased after tribulosin preconditioning, whereas Bax and caspase-3 expression was decreased. In the chelerythrine group, Bcl-2 and PKCɛ expression was decreased, whereas Bax and caspase-3 expression was increased.

Conclusion:

Tribulosin protects myocardium against ischemia/reperfusion injury through PKCɛ activation.     

PGE(2) -EP(2) signalling in endothelium is activated by haemodynamic stress and induces cerebral aneurysm through an amplifying loop via NF-κB

BACKGROUND AND PURPOSE

Cerebral aneurysm is a frequent cerebrovascular event and a major cause of fatal subarachnoid haemorrhage, but there is no medical treatment for this condition. Haemodynamic stress and, recently, chronic inflammation have been proposed as major causes of cerebral aneurysm. Nevertheless, links between haemodynamic stress and chronic inflammation remain ill-defined, and to clarify such links, we evaluated the effects of prostaglandin E₂ (PGE₂), a mediator of inflammation, on the formation of cerebral aneurysms.

EXPERIMENTAL APPROACH

Expression of COX and prostaglandin E synthase (PGES) and PGE receptors were examined in human and rodent cerebral aneurysm. The incidence, size and inflammation of cerebral aneurysms were evaluated in rats treated with COX-2 inhibitors and mice lacking each prostaglandin receptor. Effects of shear stress and PGE receptor signalling on expression of pro-inflammatory molecules were studied in primary cultures of human endothelial cells (ECs).

KEY RESULTS

COX-2, microsomal PGES-1 and prostaglandin E receptor 2 (EP₂) were induced in ECs in the walls of cerebral aneurysms. Shear stress applied to primary ECs induced COX-2 and EP₂. Inhibition or loss of COX-2 or EP₂in vivo attenuated each other''s expression, suppressed nuclear factor κB (NF-κB)-mediated chronic inflammation and reduced incidence of cerebral aneurysm. EP₂ stimulation in primary ECs induced NF-κB activation and expression of the chemokine (C-C motif) ligand 2, essential for cerebral aneurysm.

CONCLUSIONS AND IMPLICATIONS

These results suggest that shear stress activated PGE₂-EP₂ pathway in ECs and amplified chronic inflammation via NF-κB. We propose EP₂ as a therapeutic target in cerebral aneurysm.     

WIN55,212-2 protects oligodendrocyte precursor cells in stroke penumbra following permanent focal cerebral ischemia in rats

Aim:

To explore whether the synthetic cannabinoid receptor agonist WIN55,212-2 could protect oligodendrocyte precursor cells (OPCs) in stroke penumbra, thereby providing neuroprotection following permanent focal cerebral ischemia in rats.

Methods:

Adult male SD rats were subjected to permanent middle cerebral artery occlusion (p-MCAO). The animals were administered WIN55,212-2 at 2 h, and sacrificed at 24 h after the ischemic insult. The infarct volumes and brain swelling were assessed. The expression of cannabinoid receptor type 1 (CB1) in the stroke penumbra was examined using Western blot assay. The pathological changes and proliferation of neural glial antigen 2-positive OPCs (NG2⁺ cells) in the stroke penumbra were studied using immunohistochemistry staining.

Results:

p-MCAO significantly increased the expression of CB1 within the stroke penumbra with the highest level appearing at 2 h following the ischemic insult. Administration of WIN55,212-2 (9 mg/kg, iv) significantly attenuated the brain swelling, and reduced the infarct volume as well as the number of tau-immunoreactive NG2⁺ cells (tau-1⁺/NG2⁺ cells) in the stroke penumbra. Moreover, WIN55,212-2 significantly promoted the proliferation of NG2⁺ cells in the stroke penumbra and in the ipsilateral subventricular zone at 24 h following the ischemic insult. Administration of the selective CB1 antagonist rimonabant (1 mg/kg, iv) partially blocked the effects caused by WIN55,212-2.

Conclusion:

Tau-1 is expressed in NG2⁺ cells following permanent focal cerebral ischemic injury. Treatment with WIN55,212-2 reduces the number of tau-1⁺/NG2⁺ cells and promotes NG2⁺ cell proliferation in the stroke penumbra, which are mediated partially via CB1 and may contribute to its neuroprotective effects.     

Ferulic acid inhibits nitric oxide-induced apoptosis by enhancing GABA81 receptor expression in transient focal cerebral ischemia in rats

Aim:

Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) provides neuroprotection against apoptosis in a transient middle cerebral artery occlusion (MCAo) model. This study was to further investigate the anti-apoptotic effect of FA during reperfusion after cerebral ischemia.

Methods:

Rats were subjected to 90 min of cerebral ischemia followed by 3 or 24 h of reperfusion after which they were sacrificed.

Results:

Intravenous FA (100 mg/kg) administered immediately after middle cerebral artery occlusion (MCAo) or 2 h after reperfusion effectively abrogated the elevation of postsynaptic density-95 (PSD-95), neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), nitrotyrosine, and cleaved caspase-3 levels as well as apoptosis in the ischemic cortex at 24 h of reperfusion. FA further inhibited Bax translocation, cytochrome c release, and p38 mitogen-activated protein (MAP) kinase phosphorylation. Moreover, FA enhanced the expression of gamma-aminobutyric acid type B receptor subunit 1 (GABA_B1) in the ischemic cortex at 3 and 24 h of reperfusion. In addition, nitrotyrosine-positive cells colocalized with cleaved caspase-3-positive cells, and phospho-p38 MAP kinase-positive cells colocalized with nitrotyrosine- and Bax-positive cells, indicating a positive relationship among the expression of nitrotyrosine, phospho-p38 MAP kinase, Bax, and cleaved caspase-3. The mutually exclusive expression of GABA_B1 and nitrotyrosine revealed that there is a negative correlation between GABA_B1 and nitrotyrosine expression profiles. Additionally, pretreatment with saclofen, a GABA_B receptor antagonist, abolished the neuroprotection of FA against nitric oxide (NO)-induced apoptosis.

Conclusion:

FA significantly enhances GABA_B1 receptor expression at early reperfusion and thereby provides neuroprotection against p38 MAP kinase-mediated NO-induced apoptosis at 24 h of reperfusion.     

Homoharringtonine acts synergistically with SG235TRAIL, a conditionally replicating adenovirus, in human leukemia cell lines

Aim:

To investigate the synergistic effects of SG235-TRAIL, a novel oncolytic adenovirus expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and homoharringtonine (HHT) in human leukemia cell lines.

Methods:

The combined effect of SG235-TRAIL and HHT was assessed using a crystal violet assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, followed by combination index analysis. Cell apoptosis was measured using flow cytometry combined with fluorescein-isothiocyanate-Annexin V staining. The activation of caspase pathway and the expression of Bcl-2 family proteins, TRAIL, and E1A were examined using Western blotting.

Results:

HHT synergized the cytotoxicity of SG235-TRAIL against leukemia cell lines Kasumi-1, KG-1, HL-60, and U937, concomitantly with increased apoptosis and enhanced activity of caspase-3 and -9. The combination therapy resulted in significantly lower levels of Bcl-2, Mcl-1, and Bid compared to treatment of cells with either HHT or SG235-TRAIL alone, suggesting that HHT sensitizes leukemia cells to SG235-TRAIL virus through alteration of anti-apoptotic signaling elements. Importantly, HHT combined with SG235-TRAIL did not show significant cytotoxicity to normal human mononuclear cells and mesenchymal stem cells.

Conclusion:

Combining oncolytic adenovirus SG235-TRAIL and HHT synergistically enhances cytotoxicity in leukemia cells in vitro, suggesting that the combination therapy could represent a rational approach for the treatment of leukemia.     

Sevoflurane post-conditioning protects isolated rat hearts against ischemia-reperfusion injury via activation of the ERK1/2 pathway

Aim:

To investigate the role of extracellular signal-regulated kinases (ERKs) in sevoflurane post-conditioning induced cardioprotection in vitro.

Methods:

Isolated rat hearts were subjected to 30 min ischemia followed by 120 min reperfusion (I/R). Sevoflurane post-conditioning was carried out by administration of O₂-enriched gas mixture with 3% sevoflurane (SEVO) for 15 min from the onset of reperfusion. Cardiac functions, myocardial infarct size, myocardial ATP and NAD⁺ contents, mitochondrial ultrastructure, and anti-apototic and anti-oncosis protein levels were measured.

Results:

Sevoflurane post-conditioning significantly improved the heart function, decreased infarct size and mitochondria damage, and increased myocardial ATP and NAD⁺ content in the I/R hearts. Furthermore, sevoflurane post-conditioning significantly increased the levels of p-ERK and p-p70S6K, decreased the levels of porimin, caspase-8, cleaved caspase-3, and cytosolic cytochrome c in the I/R hearts. Co-administration of the ERK1/2 inhibitor PD98059 (20 μmol/L) abolished the sevoflurane-induced protective effects against myocardial I/R.

Conclusion:

Sevoflurane post-conditioning protects isolated rat hearts against myocardial I/R injury and inhibits cell oncosis and apoptosis via activation of the ERK1/2 pathway.