This study aims to explore the role and mechanism of berberine in promoting the expression of aquaporin 4(AQP4) in astrocytes by regulating the expression of miR-383-5p in HepG2 cell-derived exosomes under insulin resistance(IR). The IR-HepG2 cell model was established with 1×10~(-6) mol·L~(-1) insulin. With metformin as the positive control, the safe concentrations of berberine and metformin were screened by cell counting kit-8(CCK-8) and lactate dehydrogenase(LDH) leakage assays, and the effect of berberine on the IR of HepG2 cells was evaluated by glucose consumption. NanoSight was used to measure the particle size and concentration of exosomes secreted by HepG2 cells in each group. HepG2 cell-derived exosomes in each group were incubated with astrocytes for 24 h, and the protein and mRNA levels of AQP4 in HA1800 cells were determined by Western blot and qRT-PCR, respectively. qRT-PCR was performed to determine the expression of miR-383-5p in HepG2 cell-derived exosomes and HA1800 cells after co-incubation. Western blotting was employed to determine the expression levels of miRNAs and proteins associated with exosome production and release in HepG2 cells. The results showed that 10 μmol·L~(-1) berberine and 1 mmol·L~(-1) metformin significantly alleviated the IR of HepG2 cells and reduced the concentration of exosomes in HepG2 cells. The exosomes of HepG2 cells treated with berberine and metformin significantly up-regulated the protein and mRNA levels of AQP4 in HA1800 cells. The mRNA level of miR-383-5p in HepG2 cell exosomes and HA1800 cells co-incubated with berberine and metformin decreased significantly. The intervention with berberine and metformin significantly down-regulated the expression of proteins associated with the production of miRNAs(Dicer, Drosha) as well as the production(Alix, Vps4A) and release(Rab35, VAMP3) of exosomes in IR-HepG2 cells. In conclusion, berberine can promote the expression of AQP4 in astrocytes by inhibiting the production and release of miR-383-5p in HepG2-derived exosomes under IR.
Humans ; MicroRNAs/metabolism* ; Berberine/pharmacology* ; Hep G2 Cells ; Exosomes/genetics* ; Aquaporin 4/metabolism* ; Insulin Resistance ; Astrocytes/drug effects*

This study investigated the therapeutic effects and mechanisms of Modified Yiyi Fuzi Baijiang Powder on ulcerative colitis(UC) in rats from the perspective of dampness. SD rats were randomly allocated into six groups(n=10): control, model, mesalazine, and Modified Yiyi Fuzi Baijiang Powder at low(3.96 g·kg~(-1)·d~(-1)), medium(7.92 g·kg~(-1)·d~(-1)), and high(15.84 g·kg~(-1)·d~(-1)) doses. UC was induced in all groups except the control by administration with 3% dextran sulfate sodium(DSS) solution for 7 days. The disease activity index(DAI) was recorded, and the colon tissue was collected for analysis. Histopathological changes were assessed by hematoxylin-eosin staining. Serum levels of D-lactic acid(D-LA) and diamine oxidase(DAO) were measured by ELISA. Immunohistochemistry and PCR were employed to evaluate the expression of aquaporins(AQP3, AQP4) and tight junction proteins [zonula occludens-1(ZO-1) and occludin] at both protein and mRNA levels. Compared with the control group, the model group showed an increased DAI scores(P<0.05), intestinal mucosal damage, elevated serum levels of DAO and D-LA(P<0.05), and decreased expression of AQP3, AQP4, ZO-1, and occludin(P<0.05). Treatment with Modified Yiyi Fuzi Baijiang Powder reduced the DAI scores(P<0.05), lowered the serum levels of D-LA and DAO(P<0.05), and upregulated the expression of AQP3, AQP4, ZO-1, and occludin at both protein and mRNA levels compared with the model group. These findings suggest that Modified Yiyi Fuzi Baijiang Powder exerts therapeutic effects on UC by reducing the intestinal mucosal permeability, promoting colonic mucosal repair, and regulating abnormal intestinal water metabolism, which may involve the upregulation of AQP3 and AQP4 expression.
Animals ; Colitis, Ulcerative/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Rats ; Intestinal Mucosa/metabolism* ; Male ; Aquaporin 3/metabolism* ; Aquaporin 4/metabolism* ; Permeability/drug effects* ; Humans ; Powders ; Intestinal Barrier Function

OBJECTIVE: To investigate the effect of aquaporin 5 (AQP5) on Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor κB (NF-κB) signaling pathway in Sjögren syndrome (SS) rats. METHODS: The SS gene expression data sets GSE406611 and GSE84844 were extracted from the Gene Expression Omnibus (GEO), and the AQP5 mRNA expression was analyzed by R software. The rat SS model was constructed. The successfully modeled rats were divided into SS group, SS+NC group, and SS+pc group, 10 rats in each group; and 10 rats were set as Normal group. The rats in the SS+NC group were injected with 10 μg of rno-pcDNA3.1-AQP5-NC at the submandibular gland, subcutaneously every day for 28 days. The rats in the SS+pc group were injected with 10 μg of rno-pcDNA3.1-AQP5 at the submandibular gland, subcutaneously every day for 28 days. The enzyme-linked immunosorbent assay (ELISA) kit was used to detect the content of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the serum. High-throughput sequencing was used to identify the target genes. Quantitative real-time PCR (qPCR) and Western blot were used to detect the mRNA and protein expressions of AQP5, TLR4, MyD88, and NF-κB in the rat submandibular gland tissue. RESULTS: In the SS dataset GSE406611 and GSE84844, the mRNA expression of AQP5 in SS was significantly reduced. Compared with the Normal group, the content of TNF-α and IL-1β in the serum, the mRNA and protein expressions of TLR4, MyD88, and NF-κB in the SS group were significantly increased, the mRNA and protein expressions of AQP5 were significantly decreased. After overexpression of AQP5, the content of TNF-α and IL-1β in the serum, the mRNA and protein expressions of TLR4, MyD88, and NF-κB in the SS+pc group were significantly decreased, the mRNA and protein expressions of AQP5 were significantly increased. The differences were statistically significant (all P < 0.05). CONCLUSION The expression of AQP5 is involved in the progression of SS. Increasing the expression of AQP5 can significantly inhibit inflammatory stress and reduce the pathological damage of submandibular gland tissue. This may be related to the inhibition of TLR4/MyD88/NF-κB conduction.
Animals ; Toll-Like Receptor 4/genetics* ; Myeloid Differentiation Factor 88/genetics* ; Aquaporin 5/metabolism* ; Sjogren's Syndrome/genetics* ; Signal Transduction ; NF-kappa B/metabolism* ; Rats ; Rats, Sprague-Dawley ; Interleukin-1beta/metabolism* ; Female

Cerebral edema is characterized by fluid accumulation, and the glymphatic system (GS) plays a pivotal role in regulating fluid transport. Using the Tenecteplase system, magnesium salt of salvianolic acid B/ginsenoside Rg1 (SalB/Rg1) was injected intravenously into mice 4.5 h after middle cerebral artery occlusion and once every 24 h for the following 72 h. GS function was assessed by Evans blue imaging, near-infrared fluorescence region II (NIR-II) imaging, and magnetic resonance imaging (MRI). SalB/Rg1 had significant effects on reducing the infarct volume and hemorrhagic transformation score, improving neurobehavioral function, and protecting tissue structure, especially inhibiting cerebral edema. Meanwhile, the influx/efflux drainage of GS was enhanced by SalB/Rg1 according to NIR-II imaging and MRI. SalB/Rg1 inhibited matrix metalloproteinase-9 (MMP-9) activity, reduced cleaved β-dystroglycan (β-DG), and stabilized aquaporin-4 (AQP4) polarity, which was verified by colocalization with CD31. Our findings indicated that SalB/Rg1 treatment enhances GS function and attenuates cerebral edema, accompanying the regulation of the MMP9/β-DG/AQP4 pathway.
Animals ; Ginsenosides/administration & dosage* ; Brain Edema/etiology* ; Male ; Benzofurans/administration & dosage* ; Glymphatic System/diagnostic imaging* ; Mice ; Infarction, Middle Cerebral Artery/drug therapy* ; Aquaporin 4/metabolism* ; Disease Models, Animal ; Mice, Inbred C57BL ; Matrix Metalloproteinase 9/metabolism* ; Neuroprotective Agents/pharmacology* ; Depsides

This study aims to explore the pharmacodynamic effect of baicalin on rat brain edema induced by cerebral ischemia reperfusion injury and discuss the mechanism from the perspective of inhibiting astrocyte swelling, which is expected to serve as a refe-rence for the treatment of cerebral ischemia with Chinese medicine. To be specific, middle cerebral artery occlusion(suture method) was used to induce cerebral ischemia in rats. Rats were randomized into normal group, model group, high-dose baicalin(20 mg·kg~(-1)) group, and low-dose baicalin(10 mg·kg~(-1)) group. The neurobehavior, brain index, brain water content, and cerebral infarction area of rats were measured 6 h and 24 h after cerebral ischemia. Brain slices were stained with hematoxylin and eosin(HE) for the observation of pathological morphology of cerebral cortex after baicalin treatment. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the content of total L-glutathione(GSH) and glutamic acid(Glu) in brain tissue, Western blot to measure the content of glial fibrillary acidic protein(GFAP), aquaporin-4(AQP4), and transient receptor potential vanilloid type 4(TRPV4), and immunohistochemical staining to observe the expression of GFAP. The low-dose baicalin was used for exploring the mechanism. The experimental results showed that the neurobehavioral scores(6 h and 24 h of cerebral ischemia), brain water content, and cerebral infarction area of the model group were increased, and both high-dose and low-dose baicalin can lower the above three indexes. The content of GSH dropped but the content of Glu raised in brain tissue of rats in the model group. Low-dose baicalin can elevate the content of GSH and lower the content of Glu. According to the immunohistochemical staining result, the model group demonstrated the increase in GFAP expression, and swelling and proliferation of astrocytes, and the low-dose baicalin can significantly improve this situation. The results of Western blot showed that the expression of GFAP, TRPV4, and AQP4 in the cerebral cortex of the model group increased, and the low-dose baicalin reduce their expression. The cerebral cortex of rats in the model group was severely damaged, and the low-dose baicalin can significantly alleviate the damage. The above results indicate that baicalin can effectively relieve the brain edema caused by cerebral ischemia reperfusion injury in rats, possibly by suppressing astrocyte swelling and TRPV4 and AQP4.
Animals ; Aquaporin 4/genetics* ; Astrocytes ; Brain Edema/drug therapy* ; Brain Ischemia/metabolism* ; Flavonoids ; Infarction, Middle Cerebral Artery/drug therapy* ; Rats ; Rats, Sprague-Dawley ; Reperfusion ; TRPV Cation Channels/therapeutic use*

OBJECTIVES: To observe the changes of expression of aquaporin-1（AQP-1） and AQP-4 in drowned and postmortem immersed rats' lungs. METHODS: Thirty healthy male Wistar rats were randomly divided into drowning group, postmortem immersion group and cervical dislocation group. The morphological changes of rats' lungs were observed using HE staining. The mRNA and protein expressions of AQP-1 and AQP-4 in rats' lungs were detected by real-time PCR, immunohistochemistry and Western blotting, respectively. RESULTS: The results of immunohistochemistry and the Western blotting showed that the protein expression of AQP-1 of the drowning group was higher than the postmortem immersion group and the cervical dislocation group （P<0.05）. The result of immunohistochemistry showed that the protein expression of AQP-4 of the drowning group was higher than the postmortem immersion group and the cervical dislocation group （P<0.05） while no difference were detected among the three of them by Western blotting （P>0.05）. The mRNA expressions of AQP-1 and AQP-4 in rats' lungs of the drowning group was significantly higher than the postmortem immersion group （P<0.05）. CONCLUSIONS The increase of mRNA and protein expressions of AQP-1 and AQP-4 in lungs of rats with cute lung injury of the drowning group would be useful for differentiating vital drowning from postmortem immersion.
Animals ; Aquaporin 1/metabolism* ; Aquaporin 4/metabolism* ; Autopsy ; Blotting, Western ; Drowning ; Immunohistochemistry ; Lung/metabolism* ; Male ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Real-Time Polymerase Chain Reaction

OBJECTIVE: To observe the expression of aquaporin 4 (AQP4) in diffuse brain injury (DBI) of rats and to explore the corresponding effect of AQP4 for brain edema. METHODS: The rat model of DBI was established using Marmarou's impact-compression trauma model. Brain water content was measured by dry-wet weight method. Blood-brain barrier permeability was evaluated by Evans blue (EB) staining. Immunohistochemical method was used to observe the expression of AQP4. RESULTS: Brain water content increased after 3 h and peaked at 24 h after DBI. Brain EB content significantly increased and peaked at 12 h after DBI. The expression of AQP4 significantly increased after 3 h and peaked at 24 h after DBI, and the number of AQP4 positive astrocytes increased. CONCLUSION The increment of the permeability of blood-brain barrier and the expression of AQP4 may contribute to the development of brain edema in rat DBI. The change of AQP4 expression in astrocytes may also contribute to determine DBI.
Animals ; Aquaporin 4/metabolism* ; Astrocytes ; Blood-Brain Barrier/metabolism* ; Brain ; Brain Edema/metabolism* ; Brain Injuries/metabolism* ; Cell Membrane Permeability/genetics* ; Disease Models, Animal ; Permeability ; Rats ; Water

OBJECTIVETo investigate the role of extracellular signal-regulated kinase1/2 (ERK1/2) pathway in the regulation of aquaporin 4 (AQP4) expression in cultured astrocytes after scratch-injury.

METHODSThe scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase (LDH) leakages were observed to assess the degree of scratch-injury. AQP4 expression was detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2 (p-ERK1/2) expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 µmol/L U0126 (ERK1/2 inhibitor) was incubated in the medium at 30 min before the scratch-injury in some groups.

RESULTSIncreases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expression was associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury.

CONCLUSIONThese results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

Animals ; Aquaporin 4 ; metabolism ; Astrocytes ; enzymology ; metabolism ; Butadienes ; administration & dosage ; Cells, Cultured ; Down-Regulation ; Enzyme Activation ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; MAP Kinase Signaling System ; Nitriles ; administration & dosage ; Rats ; Rats, Wistar ; Skin ; injuries

The deficiency of aquaporin-4 (AQP4) has been reported to alter release of neurotransmitters in the mouse brain. However, the functional relevance of AQP4 in mediating essential components of the general anaesthetic state is unknown. The aim of the present study was to investigate the role of AQP4 in general anaesthesia in mice lacking AQP4. The hypnotic effects of propofol, ketamine, and pentobarbital in AQP4 knockout (KO) and CD1 control mice were evaluated using the behavioural endpoint of loss of righting reflex (LORR). The effects of propofol on extracellular levels of amino acids in prefrontal cortex of freely moving mice were investigated using microdialysis coupled to high performance liquid chromatography with fluorescent detection. The result showed that, after receiving ketamine or pentobarbital, LORR occurred at earlier time in KO mice than that in control animals. Intraperitoneal injection of ketamine or pentobarbital increased the duration of LORR. After the administration of propofol, the duration of LORR was significantly reduced in KO mice compared with that in controls. Propofol increased the extracellular levels of aspartate, glutamate, and GABA, but not taurine, in prefrontal cortex. There were significant differences of increase patterns of the three kinds of neurotransmitters between KO and WT mice. Notably, the duration of GABA level increase correlated with the duration of LORR in two genotypes of mice. These results provide in vivo evidence of different responses in time-dependent release of excitatory and inhibitory neurotransmitters in prefrontal cortex of the two genotypes of mice. It is suggested that changes in anaesthetic reactions in mice with AQP4 loss may be related to neurotransmitter regulation, and that normal functioning of AQP4 plays an important role in the maintenance of anaesthetic hypnosis.
Anesthetics, Intravenous ; pharmacology ; Animals ; Aquaporin 4 ; deficiency ; genetics ; Hypnotics and Sedatives ; pharmacology ; Ketamine ; pharmacology ; Mice ; Mice, Knockout ; Neurotransmitter Agents ; metabolism ; Pentobarbital ; pharmacology ; Prefrontal Cortex ; drug effects ; metabolism ; Propofol ; pharmacology

BACKGROUNDAlthough some studies have reported that aquaporin-4 (AQP4) plays an important role in the brain edema after traumatic brain injury (TBI), little is known about the AQP4 expression in the early stage of TBI, or about the correlation between the structural damage to the blood-brain barrier (BBB) and angioedema. The aim of this project was to investigate the relationship between AQP4 expression and damage to the BBB at early stages of TBI.

METHODSOne hundred and twenty healthy adult Wistar rats were randomly divided into two groups: sham operation group (SO) and TBI group. The TBI group was divided into five sub-groups according to the different time intervals: 1, 3, 6, 12, and 24 hours. The brains of the animals were taken out at different time points after TBI to measure brain water content. The cerebral edema and BBB changes in structure were examined with an optical microscopy (OM) and transmission electron microscopy (TEM), and the IgG content and AQP4 protein expression in traumatic brain tissue were determined by means of immunohistochemistry and Western blotting. The data were analyzed with SPSS 13.0 statistical software.

RESULTSIn the SO group, tissue was negative for IgG, and there were no abnormalities in brain water content or AQP4 expression. In the TBI group, brain water content significantly increased at 6 hours and peaked at 24 hours following injury. IgG expression significantly increased from 1 to 6 hours following injury, and remained at a high level at 24 hours. Pathological observation revealed BBB damage at 1 hour following injury. Angioedema appeared at 1 hour, was gradually aggravated, and became obvious at 6 hours. Intracellular edema occurred at 3 hours, with the presence of large glial cell bodies and mitochondrial swelling. These phenomena were aggravated with time and became obvious at 12 hours. In addition, microglial proliferation was visible at 24 hours. AQP4 protein expression were reduced at 1 hour, lowest at 6 hours, and began to increase at 12 hours, showing a V-shaped curve.

CONCLUSIONSThe angioedema characterized by BBB damage was the primary type of early traumatic brain edema. It was followed by mixed cerebral edema that consisted of angioedema and cellular edema and was aggravated with time. AQP4 expression was down-regulated during the angioedema attack, but AQP4 expression was upregulated during intracellular edema.

Animals ; Aquaporin 4 ; metabolism ; Blood-Brain Barrier ; metabolism ; Blotting, Western ; Brain Edema ; metabolism ; Brain Injuries ; metabolism ; Immunohistochemistry ; Rats ; Rats, Wistar