Potential molecular mechanism of Guiqi Baizhu Decoction in radiation-induced intestinal edema by regulating HIF-1a, AQP4 and Na+/K+-ATPase.

BACKGROUND: Guiqi Baizhu Decoction (GQBZD) has a good protective effect on radiation-induced intestinal edema (RIIE). However, the underlying molecular mechanisms need further elucidation. PURPOSE: To reveal the potential mechanism of RIIE and GQBZD treatment. METHODS: SD rats were irradiated with 6Gy X-ray to establish RIIE model. The general condition of the rats was observed; the dry/wet weight ratio of colon tissue was detected; the morphological changes of colon tissue were observed by HE staining; the expressions of ROS, HIF-1α and AQP4 in colon tissue were detected by confocal laser scanning; the expression of edema-related proteins was detected by Western blot. In addition, human colon epithelial cells (NCM460) was irradiated with 2Gy X-ray, and HIF-1α expression in NCM460 was knocked down by small interfering RNA (siRNA) transfection, and the activity of Na+/K+-ATPase was detected by enzyme activity kit; the ROS expression was detected by flow cytometer; the AQP4 expression was detected by laser confocal microscopy; and the expression of edema-related proteins were detected by Western blot. RESULTS: We found that after irradiation, the colon tissue of rats was significantly edema, mainly manifested as mucosal and submucosal edema, and the ultrastructure was reflected in the structural damage of nucleus and mitochondria. ROS, HIF-1α and AQP4 were significantly expressed, and Na+/K+-ATPase expression/activity was decreased. After the intervention of GQBZD, the edema of the colon tissue of the rats was improved, the expressions of ROS, HIF-1α and AQP4 were decreased, and the expression/activity of Na+/K+-ATPase was increased. CONCLUSION: Ionizing radiation (IR) can cause significant intestinal edema. AQP4 and Na+/K+-ATPase are the key factors of RIIE, which are regulated by ROS and HIF-1α. GQBZD can improve hypoxia and oxidative stress, regulate the expression of AQP4 and Na+/K+-ATPase, and achieve a protective effect on RIIE. This study is the first to reveal the mechanism of RIIE.

Xinshuitong Capsule extract attenuates doxorubicin-induced myocardial edema via regulation of cardiac aquaporins in the chronic heart failure rats.

Doxorubicin (Dox), an effective antineoplastic drug, was limited use for cardiotoxicity. Xinshuitong Capsule (XST), a patented herbal formula, showed desirable beneficial effects in the treatment of chronic heart failure (CHF) patients. However, the drug on Dox-induced cardiotoxicity remains unclear. Ninety male Sprague-Dawley rats were randomized into two groups: 15 rats were selected as the normal group and 75 rats were injected intraperitoneally with Dox to establish CHF rat models, the success ones were randomly divided into five groups: low XST (LXST), medium XST (MXST) or high XST (HXST) (4.9, 9.8, or 19.6 g/kg d) administrated intragastrically twice a day for 4 weeks, with the captopril-treated group and the model group as comparison. The model group showed the cardiac functions generally impaired, and CHF mortality rate higher (47%) than those in the XST-treated groups (averaged 24%, P < 0.05). Compared with XST-treated groups, myocardial remodeling, inflammation and desarcomerization, and higher water content more severe in the cardiac tissue in the model group (P < 0.05), which was associated with higher expressions of mRNA or protein levels of AQP1, 4 and 7. Dox-impaired cardiac functions, cardiac remodeling and myocardial edema could be dose-dependently reverted by XST treatment. XST could inhibit AQP1, 4 and 7 at mRNA levels or at protein levels, which was associated with the attenuation of myocardial edema and cardiac remodeling, decreasing the ventricular stiffness and improving the cardiac functions and rats' survival. AQPs is involved in cardiac edema composed one of the mechanisms of Dox-induced cardiotoxicity, XSTvia inhibition of AQPs relieved the Dox-induced side effects.

Effects of cannabinoid (CBD) on blood brain barrier permeability after brain injury in rats.

Cannabidiol is a natural herbal medicine known to protect the brain from traumatic brain injury (TBI). Here, a TBI rat model was established, with cannabidiol administered intraperitoneally at doses of 5, 10, or 20 mg/kg, 30 min before surgery and 6 h after surgery until sacrifice. Brain water content, body weight, and modified neurological severity scores were determined, and enzyme-linked immunosorbent assay, immunofluorescence staining, hematoxylin and eosin staining, Nissl staining, Evans-blue dye extravasation, and western blotting were performed. Results showed that cannabidiol decreased the number of aquaporin-4-positive and glial fibrillary acidic protein-positive cells. Cannabidiol also significantly reduced the protein levels of proinflammatory cytokines (TNF-α and IL-1ß) and significantly increased the expression of tight junction proteins (claudin-5 and occludin). Moreover, cannabidiol administration significantly mitigated water content in the brain after TBI and blood-brain barrier disruption and ameliorated the neurological deficit score after TBI. Cannabidiol administration improved the integrity and permeability of the blood-brain barrier and reduced edema in the brain after TBI.

The protective effects of yellow tea extract against loperamide-induced constipation in mice.

Yellow tea, a rare type tea from China, has a rich breadth of functional ingredients and benefits the gastrointestinal tract. However, it is not clear whether the yellow tea extract can alleviate constipation. Therefore, we used loperamide-induced constipation in mice to evaluate the effects of yellow tea extract. Fifty Kunming mice were randomly divided into five groups: normal, model, low-dose yellow tea extract, low-dose yellow tea extract prevention group, and high-dose yellow tea extract prevention group. Mice were administered yellow tea extract for 5 weeks followed by loperamide-induced constipation for the final 2 weeks. The results showed that yellow tea extract alleviated constipation symptoms by improving the fecal water content, defecation weight, and gastrointestinal transit rate. Yellow tea extract intervention also protected colon tissue, regulated serum neurotransmitters, and decreased the vasoactive intestinal peptide level. Furthermore, qRT-PCR indicated that yellow tea extract regulated genes associated with the constipation state, raised 5-HT3 and 5-HT4 and reduced AQP3 and AQP4 mRNA expression. Moreover, we found that yellow tea extract changed the gut microbiota composition. Community diversity and richness were increased and principal co-ordinate analysis demonstrated that the yellow tea extract prophylaxis groups differed from the model group. Difference analysis indicated that yellow tea extract increased Roseburia, Lachnospiraceae_UCG-006, and Bifidobacterium and decreased norank_f_Clostridiales_vadinBB60_group, unclassified_o_Bacteroidales, and Bacteroides, which are correlated with constipation. Based on these results, we believe that regular yellow tea consumption can effectively alleviate constipation.

Acorus tatarinowii Schott extract reduces cerebral edema caused by ischemia-reperfusion injury in rats: involvement in regulation of astrocytic NKCC1/AQP4 and JNK/iNOS-mediated signaling.

BACKGROUND: This study aimed to evaluate the effects of the Acorus tatarinowii Schott [Shi Chang Pu (SCP)] extract administered at the start of 2 h of middle cerebral artery occlusion (MCAo), followed by 3 d of reperfusion, and to determine mechanisms involved in anti-edema effects in the penumbra of the cerebral cortex. METHOD: Rats were intraperitoneally administered the SCP extract at a dose of 0.25 g/kg (SCP-0.25 g), 0.5 g/kg (SCP-0.5 g), or 1 g/kg (SCP-1 g) at the start of MCAo. RESULT: SCP-0.5 g and SCP-1 g treatments effectively reduced the cerebral infarct size, ameliorated cerebral edema, reduced blood-brain barrier permeability, and restored neurological function. SCP-0.5 g and SCP-1 g treatments markedly downregulated the levels of glial fibrillary acidic protein, Na+-K+-2Cl- cotransporter type 1 (NKCC1), aquaporin 4 (AQP4), phospho-c-Jun N-terminal kinase (p-JNK)/JNK, inducible nitric oxide synthase (iNOS), 3-nitrotyrosine, intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor-A (VEGF-A), and zonula occluden-1 (ZO-1) and upregulated ZO-3 expression in the penumbra of the cerebral cortex 3 d after reperfusion. CONCLUSIONS: SCP-0.5 g and SCP-1 g treatments exert neuroprotective effects against cerebral infarction and cerebral edema partially by mitigating astrocytic swelling and blood-brain barrier disruption. Moreover, the anti-cerebral edema effects of SCP extract treatments are possibly associated with the downregulation of astrocytic NKCC1/AQP4 and JNK/iNOS-mediated ICAM-1/MMP-9 signaling in the penumbra of the cerebral cortex 3 d after reperfusion.

The Attenuation of Traumatic Brain Injury via Inhibition of Oxidative Stress and Apoptosis by Tanshinone IIA.

Traumatic brain injury (TBI) is a major source of mortality and long-term disability worldwide. The mechanisms associated with TBI development are poorly understood, and little progress has been made in the treatment of TBI. Tanshinone IIA is an effective agent to treat a variety of disorders; however, the mechanisms of Tanshinone IIA on TBI remain unclear. The aim of the present study was to investigate the therapeutic potential of Tanshinone IIA on TBI and its underlying molecular mechanisms. Changes in microvascular permeability were examined to determine the extent of TBI with Evans blue dye. Brain edema was assessed by measuring the wet weight to dry weight ratio. The expression levels of CD11, interleukin- (IL-) 1ß, and tumor necrosis factor- (TNF-) α mRNA were determined by reverse transcription-quantitative PCR. Aquaporin-4 (AQP4), glial fibrillary acidic protein (GFAP), and p47phox protein expression levels were detected by western blotting. Superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-PX) activities, and malondialdehyde (MDA) content were determined using commercial kits. Cell apoptosis was detected by western blotting and TUNEL staining. Tanshinone IIA (10 mg/kg/day, intraperitoneal administration) significantly reduced brain water content and vascular permeability at 12, 24, 48, and 72 h after TBI. Tanshinone IIA downregulated the mRNA expression levels of various factors induced by TBI, including CD11, IL-1ß, and TNF-α. Notably, CD11 mRNA downregulation suggested that Tanshinone IIA inhibited microglia activation. Further results showed that Tanshinone IIA treatment significantly downregulated AQP4 and GFAP expression. TBI-induced oxidative stress and apoptosis were markedly reversed by Tanshinone IIA, with an increase in SOD and GSH-PX activities and a decrease in the MDA content. Moreover, Tanshinone IIA decreased TBI-induced NADPH oxidase activation via the inhibition of p47phox. Tanshinone IIA attenuated TBI, and its mechanism of action may involve the inhibition of oxidative stress and apoptosis.

Oryeongsan (Goreisan) Ameliorates Experimental Autoimmune Encephalomyelitis.

Objective Oryeongsan (Goreisan), a formula composed of five herbal medicines, has long been used to treat impairments of the regulation of body fluid homeostasis. Goreisan has been revealed to have anti-inflammatory actions and inhibit a water channel, the aquaporin (AQP). We herein report the therapeutic effect of Goreisan on experimental autoimmune encephalomyelitis (EAE in, an animal model of inflammatory demyelinating diseases. Materials and Methods EAE mice immunized with MOG35-55 peptide were divided into Goreisan- and sham-treated groups. The clinical EAE score and histopathological finding of the central nervous system (CNS) were analyzed. For the proliferation assay, prepared spleen cells from immunized mice were cultured and analyzed for the [3H]-thymidine uptake and cytokine concentrations of the culture supernatant. The relative quantification of AQP4 mRNA in the CNS of EAE mice was analyzed quantitatively. Results The EAE score of the Goreisan-treated mice was significantly lower than that of the sham-treated mice. The CD4-positive cell number in the CNS of Goreisan-treated mice was lower than that of sham-treated mice. In the recall response to MOG35-55 peptide, the cell proliferation did not differ markedly between the spleen cells from Goreisan- and sham-treated mice. Furthermore, Goreisan decreased the mRNA level of AQP4 in the spinal cord during EAE. Conclusion Goreisan prevented the disease activity of EAE by inhibiting the migration of pathogenic cells into the CNS by suppressing the AQP4 expression in the CNS. Goreisan may have a therapeutic effect on inflammatory demyelinating diseases.

Gamabufotalin induces a negative feedback loop connecting ATP1A3 expression and the AQP4 pathway to promote temozolomide sensitivity in glioblastoma cells by targeting the amino acid Thr794.

OBJECTIVES: Temozolomide (TMZ) is one of the most commonly used clinical drugs for glioblastoma (GBM) treatment, but its drug sensitivity needs to be improved. Gamabufotalin (CS-6), the primary component of the traditional Chinese medicine "ChanSu," was shown to have strong anti-cancer activity. However, more efforts should be directed towards reducing its toxicity or effective treatment doses. METHODS: Target fishing experiment, Western blotting, PCR, confocal immunofluorescence and molecular cloning techniques were performed to search for possible downstream signalling pathways. In addition, GBM xenografts were used to further determine the potential molecular mechanisms of the synergistic effects of CS-6 and TMZ in vivo. RESULTS: Mechanistic research revealed a negative feedback loop between ATP1A3 and AQP4 through which CS-6 inhibited GBM growth and mediated the synergistic treatment effect of CS-6 and TMZ. In addition, by mutating potential amino acid residues of ATP1A3, which were predicted by modelling and docking to interact with CS-6, we demonstrated that abrogating hydrogen bonding of the amino acid Thr794 interferes with the activation of ATP1A3 by CS-6 and that the Thr794Ala mutation directly affects the synergistic treatment efficacy of CS-6 and TMZ. CONCLUSIONS: As the main potential target of CS-6, ATP1A3 activation critically depends on the hydrogen bonding of Thr794 with CS-6. The combination of CS-6 and TMZ could significantly reduce the therapeutic doses and promote the anti-cancer efficacy of CS-6/TMZ monotherapy.

Antibacterial, antidiarrheal, anti-inflammatory and analgesic activities of compound Shikuqin powder.

The MIC and MBC values of Shikuqin (SKQ) against 5 bacteria that readily cause diarrhea were measured by the broth micro dilution method. The castor oil-induced diarrhea method was used to evaluate the antidiarrheal activity. Intestinal transit and gastric emptying were also evaluated with normal and neostigmine-induced intestinal transit in rodents. In addition, the antidiarrheal activity of SKQ was assessed in vivo with isolated rabbit ileum. Xylene-induced ear edema was used to evaluate the anti-inflammatory activities in mice, while hot plate and writhing tests were performed to assess the analgesic effects. Senna decoction (0.3g/mL) was administered intragastrically to induce a rat model of diarrhea. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect AQP4 mRNA, and Western blot was performed to quantify the protein level of AQP4 in the colon. SKQ exhibits remarkable antidiarrheal, anti-inflammatory and analgesic effects in the gastrointestinal tract disorders, and can therefore be developed as a promising antidiarrheal agent.

Ligustilide Ameliorates the Permeability of the Blood-Brain Barrier Model In Vitro During Oxygen-Glucose Deprivation Injury Through HIF/VEGF Pathway.

Chuanxiong rhizome has been widely used for the treatment of cerebral vascular disease in traditional Chinese medicine. The integrity of blood-brain barrier (BBB) is closely linked to the cerebral vascular disease. The protective effects of ligustilide, the major bioactive component in Chuanxiong rhizome, on cerebral blood vessels have been reported previously, but its effects and potential mechanism on BBB have not been entirely clarified. In the current work, the effects of ligustilide on BBB permeability and the underlying molecular mechanisms had been investigated using the model of BBB established by coculturing astrocytes and brain microvascular endothelial cells isolated from the rat brain. The ischemia-damaged model of BBB has been established with oxygen and glucose deprivation (OGD). Our results indicated that OGD significantly increased the permeability in the coculture BBB model. This OGD-induced increase in permeability could suppress by ligustilide in a concentration-dependent manner. Also, ligustilide promoted both gene and protein expression of tight junction proteins. Ligustilide suppressed the upregulation of HIF-1α, vascular endothelial growth factor, and AQP-4 in the BBB model induced by OGD. Collectively, all results have demonstrated that ligustilide is capable of reducing the permeability of BBB in vitro model induced by OGD through HIF-1α/vascular endothelial growth factor pathway and AQP-4, which provide a new target for the clinical application of ligustilide on BBB after stroke in future.

Pretreatment with Korean red ginseng or dimethyl fumarate attenuates reactive gliosis and confers sustained neuroprotection against cerebral hypoxic-ischemic damage by an Nrf2-dependent mechanism.

The transcriptional factor Nrf2, a master regulator of oxidative stress and inflammation that are tightly linked to the development and progression of cerebral ischemia pathology, plays a vital role in inducing the endogenous neuroprotective process. Here, hypoxic-ischemia (HI) was performed in adult Nrf2 knockout and wildtype mice that were orally pretreated either with standardized Korean red ginseng extract (Ginseng) or dimethyl fumarate (DMF), two candidate Nrf2 inducers, to determine whether the putative protection was through an Nrf2-dependent mechanism involving the attenuation of reactive gliosis. Results show that Nrf2 target cytoprotective genes were distinctly elevated following HI. Pretreatment with Ginseng or DMF elicited robust neuroprotection against the deterioration of acute cerebral ischemia damage in an Nrf2-dependent manner as revealed by the reductions of neurological deficits score, infarct volume and brain edema, as well as enhanced expression levels of Nrf2 target antioxidant proteins and anti-inflammation mediators. In both ischemic striatum and cortex, the dynamic pattern of attenuated reactive gliosis in astrocytes and microglia, including affected astrocytic dysfunction in glutamate metabolism and water homeostasis, correlated well with the Nrf2-dependent neuroprotection by Ginseng or DMF. Furthermore, such neuroprotective benefits extended to the late phase of ischemic brain damage after HI, as evidenced by improvements in neurobehavioral outcomes, infarct volume and brain edema. Overall, pretreatment with Ginseng or DMF identically attenuates reactive gliosis and confers long-lasting neuroprotective efficacy against ischemic brain damage through an Nrf2-dependent mechanism. This study also provides new insight into the profitable contribution of reactive gliosis in the Nrf2-dependent neuroprotection in acute brain injury.

Changes of Spinal Edema and Expression of Aquaporin 4 in Methylprednisolone-treated Rats with Spinal Cord Injury.

The aim of the current study was to investigate the impact of methylprednisolone (MP) on the expression of Aquaporin 4 (AQP4) and spinal edema in rats with acute spinal cord injury (ASCI). Sprague-Dawley (SD) Wistar rats were randomly divided into the following groups: control (C), sham operation (S), ASCI, and MP-treated. After the ASCI model was induced and prepared, 30 mg/kg MP was injected via the tail vein, on postoperative days 1, 3, and 7. The Tarlov scoring method was used to evaluate the neurological outcome; spinal water content was measured using the wet/dry weight method; spinal pathological changes were evaluated by hematoxylin-eosin (HE) staining, and the expression of AQP4 was detected using Western blot. Compared to the S group, the motor nerve functional score (MNFS) was significantly lower in the ASCI group. Furthermore, their spinal water content and AQP4 expression was also significantly higher (P<0.05). Moreover, compared with the ASCI group, the MNFS, spinal water content, and AQP4 expression of the MP group was significantly lower (P<0.05). The ASCI groups showed severe, perivascular and neuronal edema, a reduction in the neuron number, shrinkage of the nuclei, necrosis of some spinal tissue white matter degeneration, and the formation of numerous cysts and vacuoles. In the MP group, the spinal gray matter and white matter had much clearer borders, with reduced bleeding range and karyopyknosis. Furthermore, this group showed improved perivascular and neuronal edema and an even distribution of intranuclear chromatins. MP inhibited spinal edema in rats with ASCI, which might be related to the reduced AQP4 expression in spinal tissues.

Protective role of astragalus injection in spinal cord ischemia-reperfusion injury in rats.

OBJECTIVE: To investigate the neuroprotective effect of Astragalus injection in a spinal cord ischemia-reperfusion (I/R) injury model. METHODS: A total of 27 Sprague Dawley rats were randomly divided into 3 groups: control group (n=3), I/R group (n=12), and Astragalus injection group (Ast group, n=12). Spinal cord ischemia was induced by occlusion of the abdominal aorta above the right renal artery for 32 min. Animals in the Ast group were administered Astragalus injection (6.42 mL/kg) at 30 min before the induction of ischemia. After reperfusion for 8, 12, 24, or 48 hours, the serum neuron-specific enolase (NSE) concentration was measured by enzyme-linked immunosorbent assay (ELISA) and the aquaporin-4 (AQP4) protein level was detected by western blotting. RESULTS: The pathological changes, as assessed by hematoxylin and eosin (HE) staining, were milder in the spinal cords of the Ast group compared to the I/R group. Enzyme-linked immunosorbent assay demonstrated that the NSE concentration of the Ast group was significantly lower than that of the I/R group (p<0.05). However, the NSE concentrations of the I/R and Ast groups were significantly higher than that of the control group (p=0.05). Additionally, the expression of AQP4 in the Ast group was lower than that of the I/R group at each time point. CONCLUSION: These findings indicate that Astragalus injection has a neuroprotective effect in spinal cord I/R injury by decreasing the AQP4 expression.

Er Shen Wan extract reduces diarrhea and regulates AQP 4 and NHE 3 in a rat model of spleen-kidney Yang deficiency-induced diarrhea.

INTRODUCTION: Er Shen Wan (ESW), a traditional Chinese medicinal formula comprised of Psoraleae Fructus (Babchi seeds, from Psoralea corylifolia Linn.) and Myristicae Semen (Nutmeg, from Myristica fragrans Houtt.), is widely used to treat spleen-kidney Yang deficiency (SKYD)-induced diarrhea. Previous studies have demonstrated preliminarily that the petroleum ether extract of ESW (ESWP) exhibits significant anti-diarrheal activity. The present study aimed to evaluate the anti-diarrhea activity of ESWP and to explore the underlying mechanisms with respect to fluid metabolism in a rat model of SKYD-induced diarrhea. MATERIALS AND METHODS: A high-performance liquid chromatography-diode array detector (HPLC-DAD) approach was developed and validated for qualitative and quantitative analyses of the main constituents of ESWP. SKYD model rats were established and treated with an effective dose (3.5?g/kg) of the extract for two weeks. Anti-diarrheal activity and stool properties were observed. After the experiment, the appearance and histology of the intestines were evaluated. Serum levels of neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were also determined. Furthermore, to characterize the regulation of aquaporin-4 (AQP 4) and Na+/H+ exchanger isoform 3 (NHE 3) in the colon, quantitative real-time RT-PCR (qRT-PCR), immunohistochemistry (IHC) and Western blotting (WB) were employed to detect mRNA and protein expression levels. RESULTS: In the rat models, oral ESWP administration significantly reduced the diarrhea score and the number and weight of wet stools. Jejunal and ileac histological damage was impeded, and the histology score decreased. Serum VIP levels were significantly decreased, in contrast to NPY levels. In addition, AQP 4 and NHE 3 expression levels increased significantly. CONCLUSIONS: These results showed that ESWP's anti-diarrheal effect might at least partially involve the regulation of hormones intimately involved in maintaining fluid and electrolyte levels, as well as by increasing AQP 4 and NHE 3 expression levels and enhancing the absorption of Na+ and water.

Endothelin-2 Injures the Blood-Retinal Barrier and Macroglial Müller Cells: Interactions with Angiotensin II, Aldosterone, and NADPH Oxidase.

Although increasing evidence indicates that endothelin-2 (Edn2) has distinct roles in tissue pathology, including inflammation, glial cell dysfunction, and angiogenesis, its role in the retina and the factors that regulate its actions are not fully understood. We hypothesized that Edn2 damages the blood-retinal barrier (BRB) and that this is mediated by interactions with the renin-angiotensin-aldosterone system and reactive oxygen species derived from NADPH oxidase (Nox). C57BL/6J mice received an intravitreal injection of Edn2 or control vehicle to examine the blood pressure-independent effects of Edn2. Mice administered Edn2 were randomized to receive by intraperitoneal injection treatments that inhibited the Edn type a receptor, Edn type b receptor, angiotensin type 1 receptor, mineralocorticoid receptor, or Nox isoforms 1 to 4. One month later, mice administered Edn2 exhibited breakdown of the BRB with increased vascular leakage, vascular endothelial growth factor expression, and infiltrating macrophages (Ly6C+CD45highCD11b+). Further, macroglial Müller cells, which influence the integrity of the BRB and prevent retinal edema, became gliotic and expressed increased levels of water (aquaporin-4) and ion (Kir4.1) channels. This Edn2-mediated retinopathy was reduced by all treatments. Complementary in vitro studies in cultured Müller cells supported these findings and demonstrated the importance of reactive oxygen species in mediating these events. In conclusion, Edn2 has detrimental effects on the BRB and Müller cells that involve interactions with the renin-angiotensin aldosterone system and Nox1/4.

Goreisan Prevents Brain Edema after Cerebral Ischemic Stroke by Inhibiting Aquaporin 4 Upregulation in Mice.

BACKGROUND: Aquaporin 4 (AQP4) is a water-selective transport protein expressed in astrocytes throughout the central nervous system. AQP4 level increases after cerebral ischemia and results in ischemic brain edema. Brain edema markedly influences mortality and motor function by elevating intracranial pressure that leads to secondary brain damage. Therefore, AQP4 is an important target to improve brain edema after cerebral ischemia. The Japanese herbal Kampo medicine, goreisan, is known to inhibit AQP4 activity. Here, we investigated whether goreisan prevents induction of brain edema by cerebral ischemia via AQP4 using 4-hour middle cerebral artery occlusion (4h MCAO) mice. METHODS: Goreisan was orally administered at a dose of 500 mg/kg twice a day for 5 days before MCAO. AQP4 expression and motor coordination were measured by Western blotting and rotarod test, respectively. RESULTS: Brain water content of 4h MCAO mice was significantly increased at 24 hours after MCAO. Treatment with goreisan significantly decreased both brain water content and AQP4 expression in the ischemic brain at 24 hours after MCAO. In addition, treatment with goreisan alleviated motor coordination deficits at 24 hours after MCAO. CONCLUSIONS: The results of this study suggested that goreisan may be a useful new therapeutic option for ischemic brain edema.

Effects of erythropoietin and methylprednisolone on AQP4 expression in astrocytes.

Methylprednisolone sodium succinate (MPSS) has been suggested as a treatment for spinal cord injury (SCI), but its use has been limited due to its adverse effects. Erythropoietin (EPO) has been suggested as a promising candidate for limiting SCI in mammals. The aim of the present study was to investigate the effects of EPO in combination with MPSS on astrocytes following ischemic injury in vitro. Astrocytes were isolated from the cerebral cortex of postnatal day 3 Sprague­Dawley rats and cultured in vitro. Astrocyte ischemic injury was induced by oxygen and glucose deprivation for 4 h, and reperfusion was simulated by subsequent culture under normoxic conditions. The effects of EPO and MPSS on the expression of aquaporin­4 (AQP4) were investigated. Ischemic astrocytes were treated with EPO (10 U/ml), MPSS (10 µg/ml), or EPO (10 U/ml) in combination with MPSS (10 µg/ml) during reperfusion. The cell viability of astrocytes was assessed using an MTT assay. The mRNA and protein expression levels of AQP4 were determined using reverse transcription­quantitative polymerase chain reaction and western blot analysis, respectively. The role of the protein kinase C (PKC) signaling pathway in the molecular mechanisms underlying the effects of EPO and MPSS was also investigated. The present results demonstrated that following treatment with EPO and MPSS, the mRNA expression levels of AQP4 were upregulated and cell viability was enhanced. EPO and MPSS effectively inhibited the oxygen and glucose deprivation­mediated downregulation of AQP4 following reperfusion. In addition, the combined treatment with EPO and MPSS exhibited higher AQP4 expression levels and cell viability compared with each treatment alone. Finally, the effects of EPO and MPSS on AQP4 expression were partially reversed by pretreatment with the PKC inhibitor Ro 31­8220. The present study indicated that EPO and MPSS had a synergistic effect on AQP4 expression following reperfusion, and suggest that they may be combined in the treatment of SCI.

Reduction of Ischemic Brain Edema by Combined use of Paeoniflorin and Astragaloside IV via Down-Regulating Connexin 43.

Paeoniflorin (PF) and astragaloside IV (AS-IV) have protective effects on cerebral ischemia. We aimed to test the effects of combined use of PF and AS-IV on ischemic brain edema and investigate whether the effects were dependent on connexin43 (Cx43). We detected the expression of Cx43 induced by PF and AS-IV after cerebral ischemia. We also examined the effects of combined use of PF and AS-IV on ischemic edema and further investigated the related pathways. We demonstrated PF and AS-IV decreased Cx43 and aquaporin4 (AQP4) associating with reduction of brain edema by dry-wet weight and brain-specific gravity methods after cerebral ischemia. Administration of PF and AS-IV displayed a further attenuation of brain edema with lower Cx43 levels. Meanwhile, Cx43 blockade inhibited AQP4 down-regulation by the two drugs. Moreover, phosphorylation of C-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) were increased by PF and AS-IV, respectively. The effects of PF and AS-IV to down-regulate Cx43 were suppressed by JNK and ERK inhibitors, respectively. Our data indicate that PF and AS-IV alleviate ischemic brain edema, which has close relation to Cx43 down-regulation causing decrease of AQP4 via JNK and ERK pathways activation, respectively. Combined administration elicits synergistic effects on brain edema reduction. Copyright © 2017 John Wiley & Sons, Ltd.

Combine effect of Chondroitinase ABC and low level laser (660nm) on spinal cord injury model in adult male rats.

After spinal cord injury (SCI) there are many recoveries inhibiting factors such as chondroitin sulfate proteoglycan (CSPG) and inflammation. The present study investigated the combinational effect of low level laser therapy (LLLT) as anti-inflammatory agent and Chondroitinase ABC (ChABC) enzyme as CSPG digesting factor on spinal cord after injury. This study performed on 44 male Wistar rats, spinal cord injury induced by a clip compression injury. Animals received two-weeks treatment of 660nm low level laser (LLL) and intraspinal injection of 1µg ChABC. Functional recovery, cavity size, myelination, axonal projections around the cavity, fibroblast invasion and expression of glycogen synthase kinase-3ß (GSk 3ß), CSPG and aquaporin 4 (AQP4) expression were evaluated. In statistical evaluation p<0.05 considered significant. Result showed the combination of LLLT and ChABC have more effect on reduction of cavity size, improvement of myelination and number of axons around the cavity and decreasing the expression of GSK3ß, CSPG and AQP4 expression compared to LLLT and ChABC alone. In the laser and laser+enzyme groups AQP4 expression decreased significantly after SCI. Functional recovery, improved in LLLT and ChABC treated animals, but higher recovery belonged to the combination therapy group. The current study showed combination therapy by LLLT and ChABC is more efficient than a single therapy with each of them.

Proton Magnetic Resonance Spectroscopy Indicates Preserved Cerebral Biochemical Composition in Duchenne Muscular Dystrophy Patients.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin. DMD is associated with specific learning and behavioural disabilities. In the brain, dystrophin is associated with GABAA receptors and aquaporin-4 in neurons and astrocytes, respectively, but little is known about its function. OBJECTIVE AND METHODS: In this study we aimed to compare the biochemical composition between patients and healthy controls in brain regions that are naturally rich in dystrophin using magnetic resonance spectroscopy. Given previous conflicting results obtained at clinical field strengths, we obtained data using a 7 Tesla system with associated higher signal-to-noise ratio and spectral resolution. RESULTS: Results indicated unchanged biochemical composition in all regions investigated, and increased variance in glutamate in the frontal cortex.