Astaxanthin suppresses LPS-induced myocardial apoptosis by regulating PTP1B/JNK pathway in vitro.

PURPOSE: Myocardial injury induced by sepsis can increase the patient's mortality, which is an important complication of sepsis. Myocardial apoptosis plays a key role in septic myocardial injury. Here we explored the potential mechanism of astaxanthin (ATX) inhibiting myocardial apoptosis induced by lipopolysaccharide (LPS) in vitro. METHODS: The H9C2 cell experiment was conducted in three parts. In the first part, we set up three groups: control group, LPS group (10 µg/ml), a model of septic myocardial injury, and LPS + ATX (5, 10, 30 µM); In the second part, we set up four groups: control group, LPS group, LPS + PTP1B-IN-1, a protein tyrosine phosphatase 1B (PTP1B) inhibitor, and LPS + PTP1B-IN-1 + ATX; In the third part, we set up four groups: control group, LPS group, LPS + Anisomycin, a c-Jun N-terminal kinase (JNK) activator, and LPS + Anisomycin + ATX. We assessed H9C2 cell viability using the Cell Counting Kit-8 (CCK-8) assay. We observed cell apoptosis using flow cytometry analysis. We tested the mitochondrial membrane potential (ΔΨm) using JC-1 staining. To identify the molecular targets of ATX, Astaxanthin targets were predicted through the SwissTargetPrediction database. We verified the binding affinity of ATX and its targets using microscale thermophoresis (MST). We investigated the p-JNK expression using immunofluorescence staining. Finally, Western blot was used to evaluate PTP1B, JNK, p-JNK and the mitochondrial apoptosis-associated protein expression. RESULTS: LPS inhibited H9C2 cell viability in a time-dependent manner and ATX treatment enhances H9C2 cell viability in a concentration dependent manner after LPS administration. ATX inhibited the LPS-induced apoptosis and loss of mitochondrial membrane potential in H9C2 cells. As predicted by the SwissTargetPrediction database, PTP1B was a potential target of ATX, and the interaction between ATX and PTP1B was further verified by MST. ATX attenuated the LPS-induced protein expression of PTP1B and p-JNK, regardless of PTP1B inhibition. Both immunofluorescence staining and Western blotting showed that ATX suppressed the LPS-induced p-JNK expression in H9C2 cells, regardless of Anisomycin administration. In addition, by adding Anisomycin to overexpress JNK, ATX inhibited the LPS-induced apoptosis, loss of mitochondrial membrane potential and upregulation of mitochondrial apoptosis-associated proteins in H9C2 cells via JNK signaling. CONCLUSION: ATX inhibited LPS-induced mitochondrial apoptosis of H9C2 cells by PTP1B/JNK pathway and PTP1B was the target of ATX.

The immune response of T cells and therapeutic targets related to regulating the levels of T helper cells after ischaemic stroke.

Through considerable effort in research and clinical studies, the immune system has been identified as a participant in the onset and progression of brain injury after ischaemic stroke. Due to the involvement of all types of immune cells, the roles of the immune system in stroke pathology and associated effects are complicated. Past research concentrated on the functions of monocytes and neutrophils in the pathogenesis of ischaemic stroke and tried to demonstrate the mechanisms of tissue injury and protection involving these immune cells. Within the past several years, an increasing number of studies have elucidated the vital functions of T cells in the innate and adaptive immune responses in both the acute and chronic phases of ischaemic stroke. Recently, the phenotypes of T cells with proinflammatory or anti-inflammatory function have been demonstrated in detail. T cells with distinctive phenotypes can also influence cerebral inflammation through various pathways, such as regulating the immune response, interacting with brain-resident immune cells and modulating neurogenesis and angiogenesis during different phases following stroke. In view of the limited treatment options available following stroke other than tissue plasminogen activator therapy, understanding the function of immune responses, especially T cell responses, in the post-stroke recovery period can provide a new therapeutic direction. Here, we discuss the different functions and temporal evolution of T cells with different phenotypes during the acute and chronic phases of ischaemic stroke. We suggest that modulating the balance between the proinflammatory and anti-inflammatory functions of T cells with distinct phenotypes may become a potential therapeutic approach that reduces the mortality and improves the functional outcomes and prognosis of patients suffering from ischaemic stroke.

Astaxanthin suppresses lipopolysaccharide­induced myocardial injury by regulating MAPK and PI3K/AKT/mTOR/GSK3ß signaling.

Cardiac dysfunction is a significant manifestation of sepsis and it is associated with the prognosis of the disease. Astaxanthin (ATX) has been discovered to serve a variety of pharmacological effects, including anti­inflammatory, antioxidant and antiapoptotic properties. The present study aimed to investigate the role and mechanisms of ATX in sepsis­induced myocardial injury. Male C57BL/6 mice were divided into three groups (15 mice per group): Control group, lipopolysaccharide (LPS) group and LPS + ATX group. The cardiac dysfunction model was induced through an intraperitoneal injection of LPS (10 mg/kg) and ATX (40 mg/kg) was administered to the LPS + ATX group by intraperitoneal injection 30 min following the administration of LPS. All animals were sacrificed after 24 h. Inflammatory cytokine levels in the serum were detected using ELISAs, and cardiac B­type natriuretic peptide (BNP) levels were analyzed using western blot analysis and reverse transcription­quantitative PCR. Furthermore, the extent of myocardial injury was evaluated using pathological analysis, and cardiomyocyte apoptosis was analyzed using a TUNEL assay, in addition to determining the expression levels of Bcl­2 and Bax. The expression levels of proteins involved in the mitogen activated protein kinase (MAPK) and PI3K/AKT signaling pathways were also analyzed using western blot analysis. ATX significantly suppressed the LPS­induced increased production of TNF­α and IL­6 and suppressed the protein expression levels of BNP, Bax and Bcl­2 to normal levels. ATX also prevented the histopathological changes to the myocardial tissue and reduced the extent of necrosis. Furthermore, the treatment with ATX suppressed the LPS­activated MAPK and PI3K/AKT signaling. ATX additionally exerted a protective effect on cardiac dysfunction caused by sepsis by inhibiting MAPK and PI3K/AKT signaling.

Prognostic Implications of Immune-Related Genes' (IRGs) Signature Models in Cervical Cancer and Endometrial Cancer.

Cervical cancer and endometrial cancer remain serious threats to women's health. Even though some patients can be treated with surgery plus chemoradiotherapy as a conventional option, the overall efficacy is deemed unsatisfactory. As such, the development for new treatment approaches is truly necessary. In recent years, immunotherapy has been widely used in clinical practice and it is an area of great interest that researchers are keeping attention on. However, a thorough immune-related genes (IRGs) study for cervical cancer and endometrial cancer is still lacking. We therefore aim to make a comprehensive evaluation of IRGs through bioinformatics and large databases, and also investigate the relationship between the two types of cancer. We reviewed the transcriptome RNAs of IRGs and clinical data based on the TCGA database. Survival-associated IRGs in cervical/endometrial cancer were identified using univariable and multivariable Cox proportional-hazard regression analysis for developing an IRG signature model to evaluate the risk of patients. In the end, this model was validated based on the enrichment analyses through GO, KEGG, and GSEA pathways, Kaplan-Meier survival curve, ROC curves, and immune cell infiltration. Our results showed that out of 25/23 survival-associated IRGs for cervical/endometrial cancer, 13/12 warranted further examination by multivariate Cox proportional-hazard regression analysis and were selected to develop an IRGs signature model. As a result, enrichment analyses for high-risk groups indicated main enriched pathways were associated with tumor development and progression, and statistical differences were found between high-risk and low-risk groups as shown by Kaplan-Meier survival curve. This model could be used as an independent measure for risk assessment and was considered relevant to immune cell infiltration, but it had nothing to do with clinicopathological characteristics. In summary, based on comprehensive analysis, we obtained the IRGs signature model in cervical cancer (LTA, TFRC, TYK2, DLL4, CSK, JUND, NFATC4, SBDS, FLT1, IL17RD, IL3RA, SDC1, PLAU) and endometrial cancer (LTA, PSMC4, KAL1, TNF, SBDS, HDGF, LTB, HTR3E, NR2F1, NR3C1, PGR, CBLC), which can effectively evaluate the prognosis and risk of patients and provide justification in immunology for further researches.

The New Biomarker for Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC) Based on Public Database Mining.

To reconstruct the ceRNA biological network of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and to select an appropriate mRNA as a biomarker that could be used for CESC early diagnosis and prognosis evaluation. We downloaded CESC data from the TCGA public database, and statistical analysis was conducted with the R software to find out differential expressed genes encoding for lncRNAs, miRNAs, and mRNAs. The differentially expressed mRNAs (DEmRNAs) screened in the ceRNA network were analyzed for survival to find the mRNAs with significantly linked to the survival prognosis. These mRNAs were searched in the Pathological Atlas to identify the final appropriate mRNAs. Differential expression analysis revealed 773 lncRNAs, 94 miRNAs, and 2466 mRNAs. Survival analysis of DEmRNAs in the ceRNA network indicated that ADGRF4, ANXA8L1, HCAR3, IRF6, and PDE2A (P < 0.05) were negatively correlated with survival time. Verification of these six DEmRNAs in the Pathology Atlas indicated that PDE2A was a possible biomarker for CESC patients. PDE2A might be a biomarker for early diagnosis and prognosis evaluation of CESC patients, but due to the lack of available data, further studies may be needed for confirmation.

The Akt/glycogen synthase kinase-3ß pathway participates in the neuroprotective effect of interleukin-4 against cerebral ischemia/reperfusion injury.

Interleukin-4 (IL-4) has a protective effect against cerebral ischemia/reperfusion injury. Animal experiments have shown that IL-4 improves the short- and long-term prognosis of neurological function. The Akt (also called protein kinase B, PKB)/glycogen synthase kinase-3ß (Akt/GSK-3ß) signaling pathway is involved in oxidative stress, the inflammatory response, apoptosis, and autophagy. However, it is not yet clear whether the Akt/GSK-3ß pathway participates in the neuroprotective effect of IL-4 against cerebral ischemia/reperfusion injury. In the present study, we established a cerebral ischemia/reperfusion mouse model by middle cerebral artery occlusion for 60 minutes followed by a 24-hour reperfusion. An IL-4/anti-IL-4 complex (10 µg) was intraperitoneally administered 30 minutes before surgery. We found that administration of IL-4 significantly alleviated the neurological deficits, oxidative stress, cell apoptosis, and autophagy and reduced infarct volume of the mice with cerebral ischemia/reperfusion injury 24 hours after reperfusion. Simultaneously, IL-4 activated Akt/GSK-3ß signaling pathway. However, an Akt inhibitor LY294002, which was injected at 15 nmol/kg via the tail vein, attenuated the protective effects of IL-4. These findings indicate that IL-4 has a protective effect on cerebral ischemia/reperfusion injury by mitigating oxidative stress, reducing apoptosis, and inhibiting excessive autophagy, and that this mechanism may be related to activation of the Akt/GSK-3ß pathway. This animal study was approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University, China (approval No. WDRY2017-K037) on March 9, 2017.

NLRP3 inflammasome as a potential treatment in ischemic stroke concomitant with diabetes.

The NLRP3 (nucleotide-binding oligomerization domain-like receptor [NLR] family pyrin domain-containing 3) inflammasome is a member of the NLR family of innate immune cell sensors. These are crucial regulators of cytokine secretions, which promote ischemic cell death and insulin resistance. This review summarizes recent progress regarding the NLRP3 inflammasome as a potential treatment for ischemic stroke in patients with diabetes, two complicated diseases that often occur together. Stroke worsens glucose metabolism abnormalities, and the outcomes after stroke are more serious for diabetic patients compared with those without diabetes. Inflammation contributes to organ injury after ischemic stroke and diabetes. Recent research has focused on inhibiting the activation of inflammasomes and thus reducing the maturation of proinflammatory cytokines such as interleukin (IL)-1ß and IL-18. Studies suggest that inhibition of NLRP3 prevents or alleviates both ischemic stroke and diabetes. Targeting against the assembly and activity of the NLRP3 inflammasome is a potential and novel therapy for inflammasome-associated diseases, including ischemic stroke concomitant with diabetes.

Serum prealbumin as an effective prognostic indicator for determining clinical status and prognosis in patients with hemorrhagic stroke.

Serum prealbumin is a recognized marker of malnutrition, but its prognostic role in patients with hemorrhagic stroke remains unclear. In this study, we retrospectively reviewed the records of 105 patients with hemorrhagic stroke admitted to Renmin Hospital of Wuhan University, China, from January to December 2015. We collected demographic and radiological data, and recorded serum prealbumin levels at admission and on days 1, 3, 6, 9, and 14-21. The existence of infections and gastrointestinal hemorrhage, and clinical condition at discharge were also recorded. Serum prealbumin levels during hospitalization were significantly lower in patients with infections compared with those without infections, and also significantly lower in patients with gastrointestinal hemorrhage compared with those without. Serum prealbumin levels at discharge were significantly higher in patients with good recovery than in those with poor recovery. We conclude that regular serum prealbumin measurements in patients with hemorrhagic stroke may be a useful indicator for determining clinical status and prognosis, which may therefore help to guide clinical decision-making.

Propofol exerts anti-hepatocellular carcinoma by microvesicle-mediated transfer of miR-142-3p from macrophage to cancer cells.

OBJECTIVE: We previously confirmed that propofol directly inhibited the viability, proliferation, and invasiveness of hepatocellular carcinoma cells in vitro. In this study, we investigated the mechanism underlying the anti-HCC effects of propofol. METHODS: In vivo antitumor activity was investigated in tumor-bearing mice following an intraperitoneal injection of propofol, with or without clodrolip. The co-culture system was used to verify that miR-142-3p was transported from macrophages to HCC cells. A miR-142-3p inhibitor was used to down-regulate the expression of miR-142-3p. RESULTS: Propofol drastically inhibited tumor growth in tomor-bearing mice through macrophage activation, and stimulated tumor-associated macrophages (TAMs) to secrete microvesicles (MVs), which delivered miR-142-3p to HCC cells, resulting in the inhibition of HCC cell invasion. In addition, MVs collected from the plasma of the tumor-bearing mice injected with propofol suppressed tumor growth. More importantly, down-regulation of the expression miR-142-3p reversed the effect of propofol on HCC cell migration. CONCLUSIONS: This study reveals a novel role for propofol in the inhibition of HCC through MV-mediated transfer of miR-142-3p from macrophages to cancer cells in vivo.

Buyang Huanwu decoction increases angiopoietin-1 expression and promotes angiogenesis and functional outcome after focal cerebral ischemia.

Buyang Huanwu decoction (BYHWD), a traditional Chinese herbal prescription, has been widely used clinically to treat stroke in China for hundreds of years; however, the mechanisms of this drug for stroke treatment are still unclear. This study aims to observe the cerebral angiogenesis effects of BYHWD on chronic brain injury after focal cerebral ischemia in rats and to explore its possible mechanisms. The ischemia was induced by occlusion of the right middle cerebral artery for 90 min. BYHWD (12.5 and 25.0 g/(kg ∙ d), equivalent to the dry weight of the raw materials) was orally administered twice a day beginning 2 h after surgery. BYHWD significantly attenuated the neurological dysfunction, infarct volume, and brain atrophy after ischemia. There was a significant increase in the microvessel density, as assessed by immunofluorescence CD31, and a significant increase in angiopoietin-1 (Ang-1) in the penumbra areas of the rats was shown by immunohistochemical staining and Western blotting. The results indicate that the neurorestorative effects of BYHWD are associated with angiogenesis and the enhancement of the expressions of Ang-1 on chronic brain injury after focal cerebral ischemia.

Probenecid protects against transient focal cerebral ischemic injury by inhibiting HMGB1 release and attenuating AQP4 expression in mice.

Stroke results in inflammation, brain edema, and neuronal death. However, effective neuroprotectants are not available. Recent studies have shown that high mobility group box-1 (HMGB1), a proinflammatory cytokine, contributes to ischemic brain injury. Aquaporin 4 (AQP4), a water channel protein, is considered to play a pivotal role in ischemia-induced brain edema. More recently, studies have shown that pannexin 1 channels are involved in cerebral ischemic injury and the cellular inflammatory response. Here, we examined whether the pannexin 1 channel inhibitor probenecid could reduce focal ischemic brain injury by inhibiting cerebral inflammation and edema. Transient focal ischemia was induced in C57BL/6J mice by middle cerebral artery occlusion (MCAO) for 1 h. Infarct volume, neurological score and cerebral water content were evaluated 48 h after MCAO. Immunostaining, western blot analysis and ELISA were used to assess the effects of probenecid on the cellular inflammatory response, HMGB1 release and AQP4 expression. Administration of probenecid reduced infarct size, decreased cerebral water content, inhibited neuronal death, and reduced inflammation in the brain 48 h after stroke. In addition, HMGB1 release from neurons was significantly diminished and serum HMGB1 levels were substantially reduced following probenecid treatment. Moreover, AQP4 protein expression was downregulated in the cortical penumbra following post-stroke treatment with probenecid. These results suggest that probenecid, a powerful pannexin 1 channel inhibitor, protects against ischemic brain injury by inhibiting cerebral inflammation and edema.

Moderate hypothermia inhibits brain inflammation and attenuates stroke-induced immunodepression in rats.

AIMS: Stroke causes both brain inflammation and immunodepression. Mild-to-moderate hypothermia is known to attenuate brain inflammation, but its role in stroke-induced immunodepression (SIID) of the peripheral immune system remains unknown. This study investigated the effects in rats of moderate intra-ischemic hypothermia on SIID and brain inflammation. METHODS: Stroke was induced in rats by permanent distal middle cerebral artery occlusion combined with transient bilateral common carotid artery occlusion, while body temperature was reduced to 30°C. Real-time PCR, flow cytometry, in vitro T-cell proliferation assays, in vivo delayed-type hypersensitivity (DTH) reaction and confocal microscopy were used to study SIID and brain inflammation. RESULTS: Brief intra-ischemic hypothermia helped maintain certain leukocytes in the peripheral blood and spleen and enhanced T-cell proliferation in vitro and delayed-type hypersensitivity in vivo, suggesting that hypothermia reduces SIID. In contrast, in the brain, brief intra-Ischemic hypothermia inhibited mRNA expression of anti-inflammatory cytokine IL-10 and proinflammatory mediators INF-γ, TNF-α, IL-2, IL-1ß and MIP-2. Brief intra-Ischemic hypothermia also attenuated the infiltration of lymphocytes, neutrophils (MPO(+) cells) and macrophages (CD68(+) cells) into the ischemic brain, suggesting that hypothermia inhibited brain inflammation. CONCLUSIONS: Brief intra-ischemic hypothermia attenuated SIID and protected against acute brain inflammation.

Astrocyte-enriched miR-29a targets PUMA and reduces neuronal vulnerability to forebrain ischemia.

Following transient forebrain ischemia, astrocytes play a key role in determining whether or not neurons in the hippocampal CA1 sector go on to die in a delayed fashion. MicroRNAs (miRNAs) are a novel class of RNAs that control gene expression at the post-transcriptional level and the miR-29 family is highly expressed in astrocytes. In this study we assessed levels of miR-29 in hippocampus following forebrain ischemia and found that after transient forebrain ischemia and short periods of reperfusion, miR-29a significantly increased in the resistant dentate gyrus, but decreased in the vulnerable CA1 region of the hippocampus. We demonstrate that miR-29a targets BH3-only proapoptotic BCL2 family member PUMA by luciferase reporter assay and by Western blot. Comparing primary neuron and astrocyte cultures, and postnatal brain, we verified the strongly astrocytic expression of miR-29a. We further found that miR-29a mimic protects and miR-29a inhibitor aggravates cell injury and mitochondrial function after ischemia-like stresses in vitro. Lastly, by overexpressing and reducing miR-29a we demonstrate the protective effect of miR-29a on CA1 delayed neuronal death after forebrain ischemia. Our data suggest that by targeting a pro-apoptotic BCL2 family member, increasing levels of miR-29a might emerge as a strategy for protection against ischemia-reperfusion injury.

Dissociation of vasospasm-related morbidity and outcomes in patients with aneurysmal subarachnoid hemorrhage treated with clazosentan: a meta-analysis of randomized controlled trials.

OBJECT: Clazosentan therapy after aneurysmal subarachnoid hemorrhage (SAH) has been found to be effective in reducing the incidence of vasospasm in randomized controlled trials. However, while vasospasm-related morbidity, including delayed ischemic neurological deficits (DINDs) and delayed cerebral infarctions, was consistently decreased, statistical significance was not demonstrated and outcomes were not affected by clazosentan treatment. The objective of this meta-analysis was to determine whether clazosentan treatment after aneurysmal SAH significantly reduced the incidence of DINDs and delayed cerebral infarctions and improved outcomes. METHODS: All randomized controlled trials investigating the effect of clazosentan were retrieved via searches with sensitive and specific terms. Six variables were abstracted after the assessment of the methodological quality of the trials. Analyses were performed following the method guidelines of the Cochrane Back Review Group. RESULTS: Four randomized, placebo-controlled trials met eligibility criteria, enrolling a total of 2181 patients. The meta-analysis demonstrated a significant decrease in the incidence of DINDs (relative risk [RR] 0.76 [95% CI 0.62-0.92]) and delayed cerebral infarction (RR 0.79 [95% CI 0.63-1.00]) in patients treated with clazosentan after aneurysmal SAH. However, this treatment regimen was not shown to outcomes including functional outcomes measured by Glasgow Outcome Scale-Extended (RR 1.12 [95% CI 0.96-1.30]) or mortality (RR 1.02 [95%CI 0.70-1.49]). Adverse events, including pulmonary complications, anemia, and hypotension, were all significantly increased in patients who received clazosentan therapy. CONCLUSIONS: The results of the present meta-analysis show that treatment with clazosentan after aneurysmal SAH significantly reduced the incidence of the vasospasm-related DINDs and delayed cerebral infarctions, but did not improve poor neurological outcomes in patients with aneurysmal SAH. Further study is required to elucidate the dissociation between vasospasm-related morbidity and outcomes.

miR-181 regulates GRP78 and influences outcome from cerebral ischemia in vitro and in vivo.

MicroRNAs (miRNA) are short (~22nt) single stranded RNAs that downregulate gene expression. Although recent studies indicate extensive miRNA changes in response to ischemic brain injury, there is currently little information on the roles of specific miRNAs in this setting. Heat shock proteins (HSP) of the HSP70 family have been extensively studied for their multiple roles in cellular protection, but there is little information on their regulation by miRNAs. We used bioinformatics to identify miR-181 as a possible regulator of several HSP70 family members. We validated GRP78/BIP as a target by dual luciferase assay. In response to stroke in the mouse we find that miR-181 increases in the core, where cells die, but decreases in the penumbra, where cells survive. Increased levels of miR-181a are associated with decreased GRP78 protein levels, but increased levels of mRNA, implicating translational arrest. We manipulated levels of miR-181a using plasmid overexpression of pri-miR-181ab or mimic to increase, and antagomir or inhibitor to reduce levels. Increased miR-181a exacerbated injury both in vitro and in the mouse stroke model. Conversely, reduced levels were associated with reduced injury and increased GRP78 protein levels. Studies in C6 cells show that if GRP78 levels are maintained miR-181a no longer exerts a toxic effect. These data demonstrate that miR-181 levels change in response to stroke and inversely correlate with levels of GRP78. Importantly, reducing or blocking miR-181a protects the brain from stroke.

Propofol inhibits aquaporin 4 expression through a protein kinase C-dependent pathway in an astrocyte model of cerebral ischemia/reoxygenation.

BACKGROUND: Aquaporin 4 (AQP4) plays a key role in maintaining water balance in the central nervous system, and its dysfunction may lead to brain edema. Previous studies have suggested that propofol may be involved in neuroprotection by preventing brain edema. In this study, we examined the effects of propofol on edema and assessed its neuroprotective actions in an oxygen and glucose deprivation (OGD) model of cultured rat astrocytes. We assessed the effects of propofol on AQP4 expression and the possible role of the protein kinase C (PKC) pathway on this effect. METHODS: Neocortical astrocytes were exposed to OGD in an anaerobic chamber. After 6 h of OGD exposure, astrocytes were subsequently subjected to 24 h of reoxygenation. Propofol was added during the OGD phase of the model. Cell morphology was assessed by light microscopy. Astrocyte viability was assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide absorbency (optical density value) and the percentage of lactate dehydrogenase released by injured astrocytes. AQP4 expression was evaluated with Western blot analysis. To investigate the possible mechanism of propofol's effects on AQP4 expression, cultured astrocytes were pretreated for 24 h with the PKC activator, 12-O-tetradecanoylphorbol 13-acetate, before the propofol treatment/OGD 6 h/reoxygenation 24 h. RESULTS: We found by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide testing that astrocyte viability began to decrease after about 4 h of OGD exposure and decreased to 60% after 6 h of OGD. When 6 h of OGD was followed by 24 h of reoxygenation, cell viability was further decreased. AQP4 expression was attenuated after 6 h of OGD exposure but was reversed and exceeded baseline levels after 24 h of reoxygenation. Propofol dose-dependently reduced cell death assessed by lactate dehydrogenase test (P < 0.05), and 10 muM propofol significantly down-regulated AQP4 expression in astrocytes after 6 h of OGD followed by 24 h of reoxygenation (P < 0.01). Prolonged (24 h) pretreatment with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate before OGD significantly reversed the effect of propofol on AQP4 expression (P < 0.01). CONCLUSION: Propofol, administered during OGD, provided neuroprotective effects and down-regulated AQP4 expression in the OGD/reoxygenation model of cultured rat astrocytes. Activation of the PKC pathway may block the effects of propofol.