CDKN1B Mediates Apoptosis of Neuronal Cells and Inflammation Induced by Oxyhemoglobin via miR-502-5p After Subarachnoid Hemorrhage.

Subarachnoid hemorrhage is a common disease in the neural system, which causes high fatality rate. Therefore, it is necessary to figure out inner mechanisms of factors related to this disease. RT-qPCR was applied for measuring expressions of CDKN1B and miR-502-5p and other factors of apoptosis and inflammation. Cell viabilities were detected through CCK-8. Binding conditions between miR-502-5p and CDKN1B were detected through luciferase report assay. Western blot was used for measuring levels of proteins in PPARγ/NF-κB signaling pathway. Apoptosis and inflammation of HT22 cell line, a kind of nerve cell line, were enhanced and viabilities were suppressed by oxyhemoglobin. CDKN1B expressed lower in induced HT22 cell line and overexpressed CDKN1B could promote viabilities and suppress apoptosis as well as inflammation. MiR-502-5p was the target gene of CDKN1B and enhanced apoptosis and inflammation of cells in HT22 cell line. Furthermore, miR-502-5p reversed functions of CKDN1B in induced cells through regulating proteins in PPARγ/NF-κB signaling pathway. CDKN1B was the gene that could inhibit SAH caused by apoptosis in nerve cells and inflammation by sponging miR-502-5p and regulating factors in PPARγ/NF-κB signaling pathway, suggesting it could be a factor for protecting functions of nerve cells after SAH.

Evidence for the protective effects of curcumin against oxyhemoglobin-induced injury in rat cortical neurons.

Curcumin (CCM) is a natural polyphenolic compound in Curcuma longa that has been reported to exhibit neuroprotective effects. Subarachnoid hemorrhage (SAH) is a severe neurological disorder with an unsatisfactory prognosis. Oxyhemoglobin (OxyHb) plays an important role in mediating the neurological deficits following SAH. The present study, therefore, aimed to investigate the effect of CCM on primary cortical neurons exposed to OxyHb neurotoxicity. Cortical neurons were exposed to OxyHb at a concentration of 10 µM in the presence or absence of 5 µM (low dose) or 10 µM (high dose) CCM for 24 h. Morphological changes in the neurons were observed. Cell viability and lactate dehydrogenase (LDH) release were assayed to determine the extent of cell injury. Additionally, levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and reactive oxygen species (ROS) were measured. Neuronal apoptosis was assayed via TUNEL staining and protein levels of cleaved caspase-3, Bax, and Bcl-2 were measured by Western blot. Levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 were measured using ELISA kits. Our results suggested that CCM at both low and high doses markedly improved cell viability and decreased LDH release. CCM treatment decreased neuronal apoptosis. Additionally, oxidative stress and inflammation induced by OxyHb were alleviated by CCM treatment. In conclusion, CCM inhibits neuronal apoptosis, and alleviates oxidative stress and inflammation in neurons subjected to OxyHb, suggesting that it may be beneficial in the treatment of brain damage following SAH.

Absence of developmental toxicity in a canine model after infusion of a hemoglobin-based oxygen carrier: Implications for risk assessment.

Bovine-derived hemoglobin-based oxygen carriers (HBOCs) have been investigated for use in humans (HBOC-201) and approved for veterinary medicine (HBOC-301). We infused pregnant beagles with HBOC-201 to test whether HBOC-induced developmental toxicity previously observed in rats would occur in a species devoid of an inverted visceral yolk sac (invVYS). Phase 1 assessed developmental toxicity of 6g/kg HBOC-201 on gestational day (GD) 21. Phase 2 investigated single infusions of 6g/kg HBOC-201 on one of GDs 21, 25, 29 or 33. Phase 3 studied multiple sequential infusions on GDs 21, 23,25,27,29, 31, and 33 at 0.52g/kg/day (3.6g/kg total dose). Mild to moderate maternal toxicity occurred in all phases. There was an unequivocal absence of developmental toxicity in all phases. Overall, our hypothesis that HBOC, which interferes with the function of the invVYS, would not affect the offspring in dogs was supported. The implications relative to human risk are discussed.

Depletion of Nrf2 enhances inflammation induced by oxyhemoglobin in cultured mice astrocytes.

Nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element pathway has been proved to be the key regulator in reducing inflammatory damage, which is involved in subarachnoid hemorrhage (SAH). Here, in a traditional in vitro SAH model, we investigated the effect of Nrf2 depletion on pro-inflammatory cytokines production. Primary cultured astrocytes from Nrf2 wild type (WT) or knockout (KO) mouse were exposed or not exposed to oxyhemoglobin (OxyHb). Then the DNA-binding activity of transcription factor nuclear factor-κB (NF-κB) was detected by EMSA. The expression of TNF-α, IL-1ß, IL-6 and MMP9 were evaluated. The activity of MMP9 was measured by Gelatin zymography. After exposure to OxyHb, NF-κB was activated and the expression of downstream pro-inflammatory cytokines was up-regulated in astrocytes. And such up-regulation was much higher in KO astrocytes than in WT astrocytes, which means more aggravated inflammation in Nrf2 deficient astrocytes. These results suggest that astrocytes participate in inflammatory process after SAH and the absence of Nrf2 may induce more aggressive inflammation through activation of NF-κB pathway.

All hemoglobin-based oxygen carriers are not created equally.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) also known as "blood substitutes" have been under active clinical development over the last two decades. Cell-free Hb outside its natural protective red blood cell environment, as is the case with all HBOCs, has been shown to be vasoactive in part due to the scavenging of vascular endothelial nitric oxide (NO) and may in some instances induce heme-mediated oxidative stress. Chemical modification intended to stabilize HBOCs in the tetrameric or polymeric forms introduces conformational constraints that result in proteins with diverse allosteric responses as well as oxidative and nitrosative redox side reactions. Intra and inter-molecular cross-linking may in some instances also determine the interactions between HBOCs and normal oxidative inactivation and clearance mechanisms. Oxygen and oxidative reactions of normal and several cross-linked Hbs as well as their interactions with endogenous plasma protein (haptoglobin) and cellular receptor pathways (macrophage CD163) differ significantly. Therefore, safety and efficacy may be addressed by designing HBOCs with modifications that limit hypertension, minimize heme destabilization and take into account endogenous Hb removal mechanisms to optimize exposure times for a given indication.

Caspase inhibitors attenuate oxyhemoglobin-induced apoptosis in endothelial cells.

BACKGROUND AND PURPOSE: Our recent study showed that oxyhemoglobin (OxyHb) induces apoptosis in cultured endothelial cells. Apoptosis requires the action of various classes of proteases, including a family of cysteine proteases known collectively as the caspases. This study was undertaken to investigate the effect of 2 caspase inhibitors, Z-VDVAD-FMK and Z-DEVD-FMK, in the protection of endothelial cells from OxyHb-induced apoptosis. METHODS: Cultured bovine brain microvascular endothelial cells (passages 5 to 9) were exposed to OxyHb (10 micromol/L) for 24 to 72 hours with and without caspase inhibitors. Cell attachment, DNA ladder, Western blotting of poly(ADP-ribose) polymerase (PARP), and caspase activities were measured to confirm the cytotoxic effect of OxyHb and the protective effect of the caspase inhibitors. RESULTS: (1) OxyHb produced cell detachment in a time-dependent manner. (2) OxyHb increased caspase-2 and -3 activities, produced DNA ladders, and cleaved PARP in endothelial cells. (3) Z-VDVAD-FMK and Z-DEVD-FMK (100 micromol/L) attenuated OxyHb-induced cell detachment, reduced caspase-2 and -3 activities, abolished OxyHb-induced DNA ladders, and prevented OxyHb-induced cleavage of PARP. CONCLUSIONS: OxyHb activates caspase-2 and -3 in cultured brain microvessel endothelial cells. Caspase inhibitors attenuated the cytotoxic effect of OxyHb.

Oxyhemoglobin produces necrosis in cultured smooth muscle cells.

OBJECT: Myonecrosis in the tunica media, which is defined morphologically, is one of the most striking alterations in the cerebral arterial wall following subarachnoid hemorrhage (SAH). In this study, oxyhemoglobin (OxyHb) was added to cultured rat aortic smooth muscle cells to determine the pattern of cell death by morphological and biochemical techniques. METHODS: Confluent rat aortic smooth muscle cells were treated with OxyHb in a concentration- and time-dependent manner. Cell density was assayed by counting the number of cells that attached to the culture dishes after exposed to OxyHb. To identify cell death pattern, DNA analysis, electron microscopy, and Western blotting using poly (ADP-ribose) polymerase (PARP) antibody were performed. CONCLUSIONS: OxyHb decreased cell density in a concentration- and time-dependent manner. DNA analysis showed a smear pattern characteristic of cell necrosis. Transmission electron microscopy demonstrated disintegration of cell membrane and destruction of cell organelles. No apoptotic changes, such as condensation of chromatin or apoptotic bodies were observed. Western blotting using PARP antibody revealed that 116 kDa PARP was not cleaved to 85 kDa, an apoptosis-related fragment. These results demonstrated morphologically and biochemically that OxyHb induced necrosis, not apoptosis, in cultured smooth muscle cells.

Oxyhemoglobin-induced cytotoxicity and arachidonic acid release in cultured bovine endothelial cells.

BACKGROUND AND PURPOSE: An impairment of endothelial function is associated with vasospasm after subarachnoid hemorrhage. Oxyhemoglobin is considered to be a critical trigger in the pathogenesis of vasospasm. The present studies examined the direct effects of oxyhemoglobin on cultured endothelial cells from bovine carotid artery. METHODS: Confluent endothelial cells were treated with oxyhemoglobin, and the following were studied: 1) cell morphology, 2) cell density, and 3) the release of radiolabel from [3H]arachidonic acid-treated cells. RESULTS: Endothelial cells exposed to oxyhemoglobin exhibited detachment vacuoles, and cell density was significantly decreased in time- and dose-dependent manners. Superoxide dismutase, a free radical scavenger, provided partial protection against the cytotoxic effects of oxyhemoglobin. The release of radiolabel from [3H]arachidonic acid-treated cells was increased by oxyhemoglobin in time- and dose-dependent manners. Treatment with an inhibitor of phospholipase A2 or a calcium chelator inhibited the effects of oxyhemoglobin on arachidonic acid release and cellular viability. CONCLUSIONS: Oxyhemoglobin exerts a direct cytotoxic effect on cultured endothelial cells, and this effect is associated with increased release from [3H]arachidonic acid-labeled cells. Phospholipase A2 and free radicals appear to participate in the pathogenesis of endothelial cell damage. Oxyhemoglobin-induced compromise of endothelial cells may contribute to cerebrovascular pathology.

Effect of intrathecal superoxide dismutase and catalase on oxyhemoglobin-induced vasospasm in monkeys.

A gel consisting of agarose and oxyhemoglobin (OxyHb) was developed so that, when placed in the subarachnoid space, OxyHb would be slowly released, simulating lysis of erythocytes after subarachnoid hemorrhage. The system was used to investigate the importance of reactions mediated by free radicals in the genesis of OxyHb-induced vasospasm in monkeys. Seventeen monkeys were randomly assigned to have subarachnoid placement, on Day 0, of one of the following: 1) agarose gel alone (n = 2); 2) agarose plus OxyHb (n = 3); 3) agarose plus OxyHb plus intrathecal administration of superoxide dismutase and catalase (n = 6); and 4) agarose plus OxyHb plus intrathecal administration of placebo (n = 6). Vasospasm was assessed by comparison of angiograms performed on Day 0 and 7 days after subarachnoid placement of compounds, and by electron microscopy. OxyHb alone caused significant reduction in the diameter of the middle cerebral artery (40 +/- 8%, P less than 0.005, paired t test), which was associated with ultrastructural damage to smooth muscle. Treatment with superoxide dismutase plus catalase or with placebo attenuated vasospasm of the middle cerebral artery, although significant narrowing persisted in both groups (27 +/- 12% and 26 +/- 13%, respectively, P less than 0.05, paired t test). Analysis of variance showed no difference in the degree of vasospasm between groups exposed to subarachnoid placement of OxyHb. Cerebrospinal fluid aspirated from the cisterna magna on Day 7 contained elevated activity of superoxide dismutase in animals that received treatment. Malondialdehyde was undetectable in cerebrospinal fluid after subarachnoid placement of agarose alone, although it was present in similar amounts in all groups that received subarachnoid placement of OxyHb. Since intrathecal superoxide dismutase and catalase failed to protect against OxyHb-induced vasospasm, mechanisms mediated by free radicals may not be important in its genesis. As only one combination of doses of superoxide dismutase and catalase was administered, however, it may be that other dosage schedules might be efficacious.

A potential blood substitute from carboxylic dextran and oxyhemoglobin. II. Physicochemical and physiological assessments. Preliminary results on guinea pig.

The hemoglobin solution covalently linked to dextran benzene tetracarboxylate (dex-BTC) described in Part I, offered the following characteristics: [Hb] = 70 g/l, non modified Hb less than 15%, metHb less than 5%, P50 close to blood value, viscosity and oncotic pressure near to physiological values (37 degrees C). It was biocompatible, stable in plasma, non hypotensive in rat model, non pyrogenic and did not present abnormal toxicity (on mice, according to the french Pharmacopea). Nevertheless, on account of the presence of dextran in the conjugate and due to the literature, we observed an anaphylactoïd reaction in rats consecutive to the injections: absence of hypotension but presence of a massive oedema resulting from an important increase in the capillary permeability with harmful consequences to the vascular retention and survival time. As guinea pigs are insensitive to dextran, we are beginning to assess this conjugate solution in this animal. The first results are very promising: a lack of mortality after large injection, an almost complete vascular retention, a weak urinary loss (only non conjugated Hb), and a plasmatic half-disappearance time close to 7h. Results from haemorrhagic shocks to a final hematocrit less than 0.10 l/l and total and isovolemic exchange transfusions seem to prove a real oxygen-carrying capacity of this new Hb solution.

Identification of 2-azelaoylphosphatidylcholine as one of the cytotoxic products generated during oxyhemoglobin-induced peroxidation of phosphatidylcholine.

Cytotoxic product(s), which are responsible for inducing the release of acetylcholinesterase-enriched vesicles from human erythrocytes and cell lysis, are generated when 1-saturated-2-polyunsaturated glycerophosphocholine was incubated with oxyhemoglobin (Itabe, H., Kobayashi, T. and Inoue, K. (1988) Biochim. Biophys. Acta 961, 13-21). To identify the products, a model compound, 1-O-octadecyl-2-linoleoylglycerophosphocholine was incubated with oxyhemoglobin. The oxidation products were isolated by both straight-phase and reverse-phase HPLC. The products, which were responsible for inducing erythrocyte membrane damage, were analyzed by secondary ion mass spectrometry and 1H-NMR. One of the cytotoxic products isolated was identified as 1-O-octadecyl-2-azelaoylglycerophosphocholine. Methyl esterification of the product confirmed the proposed structure.

[On the mechanism underlying spasmogenic actions of oxy-hemoglobin on the cerebral artery, analyzed from the inhibitory effects of nicardipine, procaine and indomethacin].

The present study was to analyze the basic mechanism underlying spasmogenic actions of Oxy-hemoglobin (Oxy-Hb) on the bovine cerebral arteries. Using helical strips of the middle cerebral arteries (M2), the changes in muscular tension during an isometric contraction induced by either Oxy-Hb, hydrogen peroxide (H2O2), high potassium-ion-Tyrode (30 mM K+), prostaglandin F2 alpha (PGF2 alpha), or carbocyclic thromboxane A2 (cTXA2) were recorded on the polygraph. Blocking effects of nicardipine, procaine indomethacin were compared on the contractions produced by each reagent described above. The results obtained are summarized as follows. 1) H2O2 dissolved in different concentrations produced arterial contractions similar to those by equimolar Oxy-Hb, showing similar dose-response curves. In contrast, equimolar met-hemoglobin (Met-Hb) always produced much weaker contractions. When the equimolar H2O2 was applied during an Oxy-Hb-induced contraction, the response to H2O2 was completely occluded. When H2O2 was applied during a Met-Hb-induced contraction, the response to H2O2 was not occluded and always additive to the response to Met-Hb. Indomethacin blocked both responses to Oxy-Hb and H2O2, showing similar dose dependence. 2) The above results suggested that Oxy-Hb induced contraction consisted of two components; a strong contraction by active oxygen within Hb molecule, and a weak contraction by Hb molecule itself. The former may be mediated by some PG's which are produced inside the muscle cells as the results of arachidonic acid release and subsequent cyclooxygenase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

[The effect of prazosin hydrochloride on cerebral vasospasm--an experimental study].

The effect of Prazosin, a new alpha-receptor blocking agent, to release and prevent experimental cerebral vasospasm induced by oxyhemoglobin was investigated. In the first group of cats Prazosin was administered one hour before the induction of vasospasm by oxyhemoglobin, and thirty minutes after the application of oxyhemoglobin in the second group. Doses of 0.5 mg/kg and 0.1 mg/kg were subjected to both groups respectively. In the third group, the divided administration of Prazosin was done; 0.05 mg/kg was given one hour before and two hours after the application of oxyhemoglobin. In the first group, prevention of vasospasm was observed. In the second group, induced vasospasm was released from three hours after the administration of Prazosin. These effects were seemed to be dependent on doses of Prazosin. In the third group, more constant prevention of vasospasm was observed than the former groups. The possible mechanism of preventing and releasing effect of Prazosin on cerebral vasospasm was discussed.