Acutely Inhibiting AQP4 With TGN-020 Improves Functional Outcome by Attenuating Edema and Peri-Infarct Astrogliosis After Cerebral Ischemia.

Background: Ischemic stroke is one of the leading causes of human death and disability. Brain edema and peri-infarct astrocyte reactivity are crucial pathological changes, both involving aquaporin-4 (AQP4). Studies revealed that acute inhibition of AQP4 after stroke diminishes brain edema, however, its effect on peri-infarct astrocyte reactivity and the subacute outcome is unclear. And if diffusion-weighted imaging (DWI) could reflect the AQP4 expression patterns is uncertain. Methods: Rats were subjected to middle cerebral artery occlusion (MCAO) and allocated randomly to TGN 020-treated and control groups. One day after stroke, brain swelling and lesion volumes of the rats were checked using T2-weighted imaging (T2-WI). Fourteen days after stroke, the rats successively underwent neurological examination, T2-WI and DWI with standard b-values and ultra-high b-values, apparent diffusion coefficient (ADC) was calculated correspondingly. Finally, the rats' brains were acquired and used for glial fibrillary acidic protein (GFAP) and AQP4 immunoreactive analysis. Results: At 1 day after stroke, the TGN-020-treated animals exhibited reduced brain swelling and lesion volumes compared with those in the control group. At 14 days after stroke, the TGN-020-treated animals showed fewer neurological function deficits and smaller lesion volumes. In the peri-infarct region, the control group showed evident astrogliosis and AQP4 depolarization, which were reduced significantly in the TGN-020 group. In addition, the ultra-high b-values of ADC (ADCuh) in the peri-infarct region of the TGN-020 group was higher than that of the control group. Furthermore, correlation analysis revealed that peri-infarct AQP4 polarization correlated negatively with astrogliosis extent, and ADCuh correlated positively with AQP4 polarization. Conclusion: We found that acutely inhibiting AQP4 using TGN-020 promoted neurological recovery by diminishing brain edema at the early stage and attenuating peri-infarct astrogliosis and AQP4 depolarization at the subacute stage after stroke. Moreover, ADCuh could reflect the AQP4 polarization.

Inhibition of CAMK II modulates water permeability by reducing AQP4 expression in astrocytes after oxygen-glucose deprivation.

The predominant form of edema that occurs during the early stage of ischemic stroke is cytotoxic, resulting in neuronal injury during brain ischemia and reperfusion. Intracellular calcium (Ca2+) is elevated following brain ischemia leading to increased cell membrane permeability. Ca2+/calmodulin-dependent protein kinase II (CaMK II), the downstream molecular signal of N-methyl-d-aspartate receptors (NMDARs), is sensitive to elevations in intracellular Ca2+. Aquaporin-4 (AQP4), which is expressed primarily in the brain, is a water-transport protein. However, it is unclear whether CaMK II regulates AQP4 expression to modulate cellular water permeability. We exposed cultured astrocytes to a hypoxic and glucose-free environment to mimic an ischemic environment in vitro. We investigated the effects of oxygen-glucose deprivation (OGD) on astrocytic viability and swelling, as well as CaMK II and AQP4 expression. We also studied the effects of CaMK II inhibition on cell swelling, viability and AQP4 expression. OGD increased astrocytic swelling and expression of CaMK II and AQP4, and it decreased astrocyte viability. Inhibition of CaMK II resulted in reduced astrocyte water permeability and AQP4 expression. We concluded that the upregulation of CaMK II promoted astrocyte swelling by increasing the expression of AQP4 after OGD.

Adipose Tissue-Derived Mesenchymal Stem Cell Concentrated Conditioned Medium Alters the Expression Pattern of Glutamate Regulatory Proteins and Aquaporin-4 in the Retina after Mild Traumatic Brain Injury.

Concentrated conditioned media from adipose tissue-derived mesenchymal stem cells (ASC-CCM) show promise for retinal degenerative diseases. In this study, we hypothesized that ASC-CCM could rescue retinal damage and thereby improve visual function by acting through Müller glia in mild traumatic brain injury (mTBI). Adult C57Bl/6 mice were subjected to a 50-psi air pulse on the left side of the head, resulting in an mTBI. After blast injury, 1 µL (∼100 ng total protein) of human ASC-CCM was delivered intravitreally and followed up after 4 weeks for visual function assessed by electroretinogram and histopathological markers for Müller cell-related markers. Blast mice that received ASC-CCM, compared with blast mice that received saline, demonstrated a significant improvement in a- and b-wave response correlated with a 1.3-fold decrease in extracellular glutamate levels and a concomitant increase in glutamine synthetase (GS), as well as the glutamate transporter (GLAST) in Müller cells. Additionally, an increase in aquaporin-4 (AQP4) in Müller cells in blast mice received saline restored to normal levels in blast mice that received ASC-CCM. In vitro studies on rMC-1 Müller glia exposed to 100 ng/mL glutamate or RNA interference knockdown of GLAST expression mimicked the increased Müller cell glial fibrillary acidic protein (a marker of gliosis) seen with mTBI, and suggested that an increase in glutamate and/or a decrease in GLAST might contribute to the Müller cell activation in vivo. Taken together, our data suggest a novel neuroprotective role for ASC-CCM in the rescue of the visual deficits and pathologies of mTBI via restoration of Müller cell health.

Inhibition of LncRNA MALAT1 Attenuates Cerebral Ischemic Reperfusion Injury via Regulating AQP4 Expression.

Stroke is one of the leading causes of mortality and disability worldwide. Long noncoding RNAs (lncRNAs) including MALAT1 have been shown to have critical roles in cerebral ischemia reperfusion injury (CIRI). However, the underlying mechanism of MALAT1 in CIRI has not been elucidated. The present study aimed to investigate the function and potential regulatory mechanism of MALAT1 in cerebral ischemic reperfusion injury. We established the middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation/reoxygenation (OGD/RX) model in vivo and in vitro, and then Cell Counting Kit-8 (CCK-8), RT-qPCR, flow cytometry analysis, lactate dehydrogenase (LDH) analysis, and 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to examine cell viability, MALAT1, aquaporin-4 (AQP4) expression, LDH release, and infarct volume, respectively. The level of AQP4 was remarkably upregulated in CIRI 24 h/48 h or OGD/RX 24 h/48 h compared with the sham group. Knockdown of AQP4 could alleviate OGD/RX-induced injury through enhancing cell viability and reducing LDH release and the rate of apoptotic cells. Furthermore, we found that MALAT1 was also increased in OGD/RX 24 h/48 h and silencing of MALAT1 could decrease AQP4. Inhibition of MALAT1 could also protect OGD/RX-induced injury, while the protective effect of MALAT1 siRNA on cerebral ischemic reperfusion was disappeared after transfection with AQP4 plasmid, indicating that MALAT1 may play a protective role in brain stroke through regulating AQP4. Taken together, our study provides evidence that MALAT1 is involved in ischemic stroke by inhibiting AQP4. Therefore, MALAT1 may serve as a potential target for therapeutic intervention in ischemic brain injury.

Activated microglia-derived macrophage-like cells exacerbate brain edema after ischemic stroke correlate with astrocytic expression of aquaporin-4 and interleukin-1 alpha release.

Brain edema following brain infarction affects mobility and mortality. The mechanisms underlying this process remain to be elucidated. Animal studies have shown that aquaporin-4 (AQP4) expression in astrocytes increases after stroke, and its deletion significantly reduces brain swelling. Recently, two kinds of cells, resident microglia-derived macrophage-like cells (MG-MΦ) and bone marrow-derived macrophages (BM-MΦ), have been reported to accumulate in the ischemic core and stimulate adjacent astrocytes. Therefore, we hypothesized that these cells play crucial roles in the expression of AQP4 and ultimately lead to exacerbated brain edema. To verify this hypothesis, we investigated the role of MG- or BM-MΦ in brain edema using a rat model of transient middle cerebral artery occlusion and rat astrocyte primary cultures. AQP4 expression significantly increased in the peri-infarct tissue at 3-7 days post-reperfusion (dpr) and in the core tissue at 5 and 7 dpr, which synchronized with the expression of Iba1, Il1a, Tnf, and C1qa mRNA. Interleukin (IL)-1α treatment or coculture with MG- and BM-MΦ increased AQP4 expression in astrocytes, while an IL-1 receptor type I antagonist reduced these effects. Furthermore, aggravated animals exhibited high expression of Aqp4 and Il1a mRNA in the ischemic core at 7 dpr, which led to the exacerbation of brain edema. MG-MΦ signature genes were highly expressed in the ischemic core in aggravated rats, while BM-MΦ signature genes were weakly expressed. These findings suggest that IL-1α produced by MG-MΦ induces astrocytic AQP4 expression in the peri-infarct and ischemic core tissues, thereby exacerbating brain edema. Therefore, the regulation of MG-MΦ may prevent the exacerbation of brain edema.

Oxytocin Reduces Brain Injury and Maintains Blood-Brain Barrier Integrity After Ischemic Stroke in Mice.

The present study was designed to determine the effect of different doses of oxytocin (OXT) on neuronal injury, spatial memory, blood-brain barrier (BBB) integrity and to explore possible underlying molecular mechanisms in the early stage of stroke in mice. Stroke model was generated by middle cerebral artery occlusion (MCAO) for 60 min and 24 h reperfusion in mice. OXT at doses of 1, 2, 4 and 8 IU/per mouse was administrated intranasally at the beginning of brain ischemia. Brain injury, BBB integrity, and spatial memory were evaluated by standard methods. Changes in the expression of nuclear factor-kappa B (NF-κB), and TUNEL positive cell were detected by immunohistochemistry. The levels of vascular endothelial growth factor (VEGF), aquaporin-4 (AQP4) and brain-derived neurotrophic factor (BDNF) proteins were determined by western blotting and ELISA methods. OXT at doses of 4 and 8 IU/per mouse reduced the infarct size by 42% and 52%, respectively, and improved spatial memory function (p < 0.001). OXT (8 IU/per mouse) significantly reduced brain edema, BBB disruption and upregulated the AQP4 expression (p < 0.001). Finally, OXT significantly diminished the number of apoptotic, NF-κB positive cells and enhanced the expression of BDNF and VEGF proteins in the brain tissue (p < 0.001). These findings provide important evidences that OXT significantly suppresses neuronal damage in the early stage of stroke by inhibiting apoptotic and NF-κB signaling pathway, increasing the expression of VEGF, AQP4 and BDNF proteins and reducing the BBB leakage.

Poldip2 mediates blood-brain barrier disruption and cerebral edema by inducing AQP4 polarity loss in mouse bacterial meningitis model.

BACKGROUND: Specific highly polarized aquaporin-4 (AQP4) expression is reported to play a crucial role in blood-brain barrier (BBB) integrity and brain water transport balance. The upregulation of polymerase δ-interacting protein 2 (Poldip2) was involved in aggravating BBB disruption following ischemic stroke. This study aimed to investigate whether Poldip2-mediated BBB disruption and cerebral edema formation in mouse bacterial meningitis (BM) model occur via induction of AQP4 polarity loss. METHODS AND RESULTS: Mouse BM model was induced by injecting mice with group B hemolytic streptococci via posterior cistern. Recombinant human Poldip2 (rh-Poldip2) was administered intranasally at 1 hour after BM induction. Small interfering ribonucleic acid (siRNA) targeting Poldip2 was administered by intracerebroventricular (i.c.v) injection at 48 hours before BM induction. A specific inhibitor of matrix metalloproteinases (MMPs), UK383367, was administered intravenously at 0.5 hour before BM induction. Western blotting, immunofluorescence staining, quantitative real-time PCR, neurobehavioral test, brain water content test, Evans blue (EB) permeability assay, transmission electron microscopy (TEM), and gelatin zymography were carried out. The results showed that Poldip2 was upregulated and AQP4 polarity was lost in mouse BM model. Both Poldip2 siRNA and UK383367 improved neurobehavioral outcomes, alleviated brain edema, preserved the integrity of BBB, and relieved the loss of AQP4 polarity in BM model. Rh-Poldip2 upregulated the expression of MMPs and glial fibrillary acidic protein (GFAP) and downregulated the expression of ß-dystroglycan (ß-DG), zonula occludens-1 (ZO-1), occludin, and claudin-5; whereas Poldip2 siRNA downregulated the expression of MMPs and GFAP, and upregulated ß-DG, ZO-1, occludin, and claudin-5. Similarly, UK383367 downregulated the expression of GFAP and upregulated the expression of ß-DG, ZO-1, occludin, and claudin-5. CONCLUSION: Poldip2 inhibition alleviated brain edema and preserved the integrity of BBB partially by relieving the loss of AQP4 polarity via MMPs/ß-DG pathway.

Dexamethasone Upregulates the Expression of Aquaporin4 by Increasing SUMOylation in A549 Cells.

Dexamethasone can alleviate the severity of bronchial and alveolar edema and therefore is widely applied in the treatment of various exudative diseases including pulmonary edema. However, the effectiveness of dexamethasone is still being questioned and its mechanism is not fully understood. Aquaporins (AQPs) are mainly responsible for the transmembrane transport of water, which is tightly associated with pulmonary edema. Small ubiquitin-like modifiers (SUMOs) are considered to play a protective role in some pathological conditions. In this study, we demonstrated that dexamethasone can upregulate the expression of AQPs in A549 cells by inducing SUMOylation. We found that a low dose of dexamethasone significantly upregulated the levels of SUMOylation and AQP expression in A549 cells, accompanied by a translocation of SUMOs from the cytoplasm to the nucleus. We also explored the possible relation between SUMOylation and AQPs. Knockdown of SUMO2/3 by RNA interference decreased the level of AQP4 in A549 cells after dexamethasone stimulation. Together, our findings demonstrated that AQP4 expression was upregulated in A549 cells exposed to dexamethasone, and SUMOylation may participate in the regulation of AQP4.

Concomitant enlargement of perivascular spaces and decrease in glymphatic transport in an animal model of cerebral small vessel disease.

OBJECTIVES: To observe glymphatic transport and evaluate enlarged perivascular spaces (PVSs) in spontaneously hypertensive rats (SHRs). METHODS: SHRs were used as an animal model of cerebral small vessel disease, and Wistar Kyoto (WKY) rats were used as the control group. Histopathology was used to evaluate the enlargement of PVSs. A fluorescent tracer was infused into the cisterna magna of rats, and the proportion of the brain parenchyma area exposed to the fluorescent tracer was later quantified to evaluate the influx and efflux function of the glymphatic system. The global and polarized expression of aquaporin protein 4 (AQP4) was analyzed by immunofluorescence. RESULTS: Compared with WKY rats, SHRs exhibited obviously enlarged PVSs and significantly decreased influx and efflux function of the glymphatic system. The results showed a significant decrease in AQP4 polarity in SHRs, but a difference in global AQP4 expression was not observed between SHRs and WKY rats. CONCLUSIONS: Impaired glymphatic transport may be involved in the pathogenesis of arteriolosclerotic cerebral small vessel disease, and enlarged PVSs may be a manifestation of the impaired glymphatic system.

Time course changes in AQP4 expression patterns in progressive skeletal muscle atrophy during the early stage of denervation.

OBJECTIVES: In the skeletal muscles, water metabolism is mainly regulated by water channel aquaporin 4 (AQP4). Although the expression level of AQP4 was reduced by long-term denervation, during denervation the relationship between muscle atrophy initiation and AQP4 expression decrease initiation remains unknown. The present study examined the relationship between the timing of muscle atrophy initiation and that of AQP4 expression decrease initiation, during the early stage of denervation. METHODS: Female 344 rats (8 weeks of age) were randomly assigned to control (C), day 1 post-sciatic denervation (D1), day 4 post- sciatic denervation (D4) and day 7 post- sciatic denervation (D7) groups (n=6 per group). In the tibialis anterior (TA) muscles of each group, the expression levels of some target proteins were quantified by Western blot analysis. RESULTS: The expression level of AQP4 significantly decreased on day 4 post-denervation (p<0.05). Moreover, the beginning of the decrease in AQP4 expression level was concurrent with the timing of muscle atrophy in the skeletal muscles during the early stage of denervation. CONCLUSIONS: The present study suggested that the progression of the decrease in the AQP4 expression level is partly related to the progression of muscle atrophy during the early stage of denervation.

Reduced sarcolemmal aquaporin 4 expression can support the differential diagnosis of neuromyelitis optica spectrum disorders.

This study aimed to investigate the underlying pathological muscle damage in neuromyelitis optica spectrum disorder (NMOSD) patients without muscular symptoms. We prospectively enrolled 15 patients with aquaporin 4 (AQP4) antibody seropositive NMOSD and 16 patients with non-NMOSD diseases as a control group. Biceps biopsy samples from 18 patients were examined. Six NMOSD patients exhibited inflammatory lesions/edema in lower muscles on muscle MRI. On histopathological examination, NMOSD samples showed significantly decreased IgG-targeting AQP4 expression on sarcolemma compared with non-NMOSD samples in terms of the area of positive staining and integrated optical density. Muscle biopsy can support the differential diagnosis of NMOSD.

Aquaporin 4 regulation by ginsenoside Rb1 intervenes with oxygen-glucose deprivation/reoxygenation-induced astrocyte injury.

BACKGROUND: Spinal cord ischemia-reperfusion injury (SCII) is a common complication of spinal surgery as well as thoracic and abdominal surgery. Acute cytotoxic edema is the key pathogenic alteration. Therefore, avoiding or decreasing cellular edema has become the major target for SCII treatment. METHODS: The antiedema activity of ginsenoside Rb1 on aquaporin (AQP) 4, nerve growth factor (NGF), and brain-derived neurotrophic factor expression was detected by western blot and real-time polymerase chain reaction under conditions of oxygen-glucose deprivation/reoxygenation (OGD/R) in a rat astrocyte model in vitro. In addition, the cellular membrane permeability of AQP4 overexpressing cells or AQP4 small interfering RNA-transfected cells was detected. RESULTS: Ginsenoside Rb1 significantly prevented OGD/R-induced AQP4 downregulation in rat astrocytes. In addition, ginsenoside Rb1 treatment or AQP4 overexpression in rat astrocytes significantly attenuated the OGD/R-induced increase of cellular membrane permeability. Moreover, ginsenoside Rb1 obviously prevented the OGD/R-induced decrease of NGF and BDNT expression in rat astrocytes. CONCLUSION: These findings demonstrate that ginsenoside Rb1 can relieve spinal cord edema and improve neurological function by increasing AQP4 expression.

Regulation of aquaporin 4 expression by lipoxin A4 in astrocytes stimulated by lipopolysaccharide.

Aquaporin (AQP4) could be associated with inflammation, common in central nervous system diseases. We investigated the effect of lipoxin A4 (LXA4) on the activation of astrocytes, AQP4 expression, and inflammatory response induced by lipopolysaccharide (LPS). Astrocytes were cultured in vitro and changes in transcript and protein levels of AQP4, interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), and cyclooxygenase-2 (COX-2), and protein levels of P38 and phospho-P38 were determined. The LPS group showed increased AQP4, IL-1ß, TNF-α, and COX-2 levels, whereas they decreased in the LPS + LXA4 group, suggesting that LXA4 inhibits AQP4 expression. Furthermore, levels of phospho-P38 increased in the LPS group, but decreased in the LPS + LXA4 group. In conclusion, LXA4 alleviated the LPS-induced increase in AQP4 expression and inflammatory cytokine secretion by astrocytes, possibly by inhibiting P38 phosphorylation. For the first time, we found that LXA4 may inhibit the expression of inflammatory factors by regulating the expression of AQP4. AQP4 on astrocytes is likely to be the target of anti-inflammatory effect of LXA4.

Cerebral aquaporin-4 expression is independent of seizures in tuberous sclerosis complex.

Astrocytes serve many functions in the human brain, many of which focus on maintenance of homeostasis. Astrocyte dysfunction in Tuberous Sclerosis Complex (TSC) has long been appreciated with activation of the mTORC1 signaling pathway resulting in gliosis and possibly contributing to the very frequent phenotype of epilepsy. We hypothesized that aberrant expression of the astrocyte protein aquaporin-4 (AQP4) may be present in TSC and contribute to disease pathology. Characterization of AQP4 expression in epileptic cortex from TSC patients demonstrated a diffuse increase in AQP4. To determine if this was due to exposure to seizures, we examined Aqp4 expression in mouse models of TSC in which Tsc1 or Tsc2 inactivation was targeted to astrocytes or glial progenitors, respectively. Loss of either Tsc1 or Tsc2 from astrocytes resulted in a marked increase in Aqp4 expression which was sensitive to mTORC1 inhibition with rapamycin. Our findings in both TSC epileptogenic cortex and in a variety of astrocyte culture models demonstrate for the first time that AQP4 expression is dysregulated in TSC. The extent to which AQP4 contributes to epilepsy in TSC is not known, though the similarities in AQP4 expression between TSC and temporal lobe epilepsy supports further studies targeting AQP4 in TSC.

Aquaporin-4 expression dynamically varies after acute spinal cord injury-induced disruption of blood spinal cord barrier in rats.

The blood-spinal cord barrier (BSCB) changes badly after spinal cord injury (SCI), and it is an important pathophysiological basis of SCI secondary damage. Aquaporin-4 (AQP4), one of the transmembrane proteins in spinal cord, has been shown to be closely related to the development of the BSCB and edema. We established a SCI model in rats using a free-falling weight drop device to subsequently investigate AQP4 expression. AQP4 messenger RNA (mRNA) and protein expression and immunoreactivity were detected in spinal cord tissue using reverse transcription-real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry and Western blot analysis. We found the water content and edema of the spinal cord were significantly higher than the control group after SCI, which was related to the growth of BSCB permeability; both reached their peak on the third day after injury. One, 3, 5, 7 days after injury, the immune response and protein expression in the model group increased from 1 to 3 days, with a plateau period from 3 to 5 days and a decline from 5 to 7 days, showing a significant difference compared with the sham group at each time point (P < 0.05), while the RT-qPCR results showed a decline of mRNA just after 3 days. In conclusion, after SCI, the water content of the spinal cord and the BSCB permeability increases, together with the excessive expression of AQP4, which reached a peak on the third day. AQP4 expression is closely relative to the permeability of BSCB and the water content of the spinal cord.

Isoflurane exposure in infant rats acutely increases aquaporin 4 and does not cause neurocognitive impairment.

Isoflurane is commonly used in pediatric population, but its mechanism of action in cognition is unclear. Aquaporin 4 (AQP4) regulates water content in blood, brain, and cerebrospinal fluid. Various studies have provided evidence for the role of AQP4 in synaptic plasticity and neurocognition. In this study, we aimed to determine whether a prolonged exposure to isoflurane in infant rats is associated with cognition and what effect this exposure has on AQP4 expression. Ten-day-old [postnatal day (P) 10] Wistar albino rats were randomly allocated to isoflurane group (n = 32; 1.5% isoflurane in 50% oxygen for 6 hours) or control group (n = 32; only 50% oxygen for 6 hours). Acute (P11) and long-term (P33) effects of 6-hour anesthetic isoflurane exposure on AQP4 expression were analyzed in whole brains of P11 and P33 rats by RT-qPCR and Western blot. Spatial learning and memory were assessed on P28 to P33 days by Morris Water Maze (MWM) test. The analysis revealed that isoflurane increased acutely both mRNA (~4.5 fold) and protein (~90%) levels of AQP4 in P11 rats compared with control group. The increasing levels of AQP4 in P11 were not observed in P33 rats. Also, no statistically significant change between isoflurane and control groups was observed in the latency to find the platform during MWM training and probe trial. Our results indicate that a single exposure to isoflurane anesthesia does not influence cognition in infant rats. In this case, acutely increased AQP4 after isoflurane anesthesia may have a protective role in neurocognition.

Expression patterns of aquaporins 1 and 4 in stroke.

Ischemic stroke occurs through embolic or thrombotic obliteration of an artery from cerebral circulation and represents over 80% of all stroke cases. One of the fiercest complications after stroke is edema, which results from imbalanced water diffusion around the blood vessels walls. Water diffusion around blood vessel walls occurs physiologically mainly through two protein-formed pores, namely aquaporins (AQPs) 1 and 4. Here, we compare for the first time the expression patterns and colocalization degrees of the two AQPs in control brain tissue and in peri-ischemic regions, on tissue obtained from eight patients with confirmed ischemic pathology and from five control cases. Our analysis showed that AQP4 is more abundant that AQP1, especially in the cortex and in the organized scar areas. The colocalization of the two markers was high, both located on the astrocytes membranes, but the colocalization degree decreased in the scar peri-ischemic regions. Colocalization with basement membranes was also lower for AQP1 compared to AQP4, in all regions analyzed.

Expression of AQP2, AQP4 and AQP 8 in mouse intestine induced by unprocessed and processed Euphorbia lathyris.

The present research was designed to study expression of AQP2, AQP4 and AQP8 in mouse intestines induced by unprocessed and processed Euphorbia lathyris. KM mice were given by different dose lavage of unprocessed and processed Euphorbia lathyris, Euphorbia factor L1, Euphorbia factor L2, Euphorbia factor L3. Samples of mouse intestine were collected for protein levels of AQP2, AQP 4 and AQP 8 which were assessed by immunohistochemical staining and mRNA expression of AQP2, AQP 4 and AQP 8 which were quantified by Real Time-PCR. Comparing to the normal control group, the protein levels of AQP2, AQP 4 and AQP 8 were significantly decreased (P<0.05)by Semen Euphorbiae group and Semen Euphorbiae Pulveratum group (unprocessed and processed Euphorbia lathyris) induced. Protein expression of AQP2, AQP 4 and AQP 8 in the Euphorbia factor L1, Euphorbia factor L2 and Euphorbia factor L3 group were not significantly lower than normal control group. There had no differences on the levels of AQP2 and AQP 8 mRNA expressions between the high-dose group of semen Euphorbiae group, semen Euphorbiae Pulveratum group and positive control group, while significantly lower than normal control group (P<0.05). Expression of AQP4 mRNA in the Semen Euphorbiae group and Semen Euphorbiae Pulveratum group has not significantly decreased. But levels of AQP2, AQP 4 and AQP 8 mRNA in the Euphorbia factor L1 group had no significant differences in normal control group and positive control group. These findings suggest that semen Euphorbiae could regulate expression of AQP2, AQP 4 and AQP 8 protein and mRNA, which may be the possible one reason of semen Euphorbiae induces diarrhea. The semen Euphorbiae group has more significant effects on the levels of AQP2, AQP 4 and AQP 8 protein and mRNA than semen Euphorbiae Pulveratum group, which may be one of the mechanisms of processing attenuation.

Hydrogen­rich solution against myocardial injury and aquaporin expression via the PI3K/Akt signaling pathway during cardiopulmonary bypass in rats.

Myocardial ischemia, hypoxia and reperfusion injury are induced by aortic occlusion, cardiac arrest and resuscitation during cardiopulmonary bypass (CPB), which can severely affect cardiac function. The aim of the present study was to investigate the effects of hydrogen­rich solution (HRS) and aquaporin (AQP) on cardiopulmonary bypass (CPB)­induced myocardial injury, and determine the mechanism of the phosphatidylinositol 3­kinase (PI3K)/protein kinase B (Akt) signaling pathway. Sprague Dawley rats were divided into a sham operation group, a CPB surgery group and a HRS group. A CPB model was established, and the hemodynamic parameters were determined at the termination of CPB. The myocardial tissues were observed by hematoxylin and eosin, and Masson staining. The levels of myocardial injury markers [adult cardiac troponin I (cTnI), lactate dehydrogenase (LDH), creatine kinase MB (CK­MB) and brain natriuretic peptide (BNP)], inflammatory factors [interleukin (IL)­1ß, IL­6 and tumor necrosis factor­α (TNF­α)] and oxidative stress indicators [superoxide dismutase (SOD), malondialdehyde (MDA) and myeloperoxidase (MPO)] were determined by ELISA. Furthermore, H9C2 cells were treated with HRS following hypoxia/reoxygenation. Cell viability and cell apoptosis were investigated. The expression of apoptosis regulator Bcl­2 (Bcl­2), apoptosis regulator Bax (Bax), caspase 3, AQP­1, AQP­4, phosphorylated (p)­Akt, heme oxygenase 1 (HO­1) and nuclear factor erythroid 2­related factor 2 (Nrf2) were investigated using western blotting and quantitative­polymerase chain reaction of tissues and cells. Following CPB, myocardial cell arrangement was disordered, myocardial injury markers (cTnI, LDH, CK­MB and BNP), inflammatory cytokines (IL­1ß, IL­6 and TNF­α) and MDA levels were significantly increased compared with the sham group; whereas the SOD levels were significantly downregulated following CPB compared with the sham group. HRS attenuated myocardial injury, reduced the expression levels of cTnI, LDH, CK­MB, BNP, IL­1ß, IL­6, TNF­α, MDA and MPO, and increased SOD release. Levels of Bcl­2, AQP­1, AQP­4, p­Akt, HO­1 and Nrf2 were significantly increased following HRS; whereas Bax and caspase­3 expression levels were significantly reduced following CPB. HRS treatment significantly increased the viability of myocardial cells, reduced the rate of myocardial cell apoptosis and the release of MDA and LDH compared with the CPB group. A PI3K inhibitor (LY294002) was revealed to reverse the protective effect of HRS treatment. HRS was demonstrated to attenuate CPB­induced myocardial injury, suppress AQP­1 and AQP­4 expression following CPB treatment and protect myocardial cells via the PI3K/Akt signaling pathway.