Expression Of AQP1 Research Articles

Hypoxia reduces mouse urine output via HIF1α–mediated up–regulation of renal AQP1

Introduction: Patients with acute mountain sickness (AMS) due to hypoxia at high altitudes often exhibit abnormal water metabolism. Hypoxia-inducible factors (HIFs) are major regulators of adaptive responses to hypoxia. As transcription factors, HIFs are involved in the regulation of erythropoiesis, iron metabolism, angiogenesis, energy metabolism, and cell survival by promoting the transcriptional expression of hundreds of target genes. Roxadustat, a novel drug for the treatment of anemia associated with chronic kidney disease, acts by inhibiting the degradation of HIFs to increase their protein levels. However, the clinical use of roxadustat is frequently associated with peripheral edema, suggesting the involvement of HIFs in regulating the body's water balance possibly by modulating water reabsorption in the kidney. Methods: we first evaluated the effect of hypoxia (8% O2) on mouse urine output. We then performed in vitro experiments using hypoxia (1% O2) and roxadustat on mouse primary proximal tubular cells (mPTCs). The qPCR, western blot, and immunofluorescence were used to assess AQP1 mRNA and protein expression levels. Luciferase, CHIP, and EMSA were used to investigate the transcriptional regulation of AQP1 by HIF1α. Results: We found that mice exposed to hypoxia (8% O2) had significantly reduced urine volume compared to mice exposed to normoxia (21% O2). Hypoxia significantly elevated AQP1 expression at both mRNA and protein levels. In vitro experiments using mouse primary cultured proximal tubular cells (mPTCs) revealed that both hypoxia and roxadustat increased AQP1 expression. Mechanistically, overexpression of HIF1α, but not HIF2α, markedly increased AQP1 protein expression. Furthermore, the up-regulation of AQP1 by hypoxia and roxadustat can be blocked by the HIF1α inhibitor PX-478 in mPTCs. Finally, we found that the AQP1 gene promoter contains a putative hypoxia response element (HRE) and confirmed that AQP1 is a target gene of HIF1α using Luciferase reporter, CHIP, and EMSA assays. Conclusion: This study demonstrates that hypoxia can reduce the urine volume of mice via upregulating AQP1 expression by HIF1α in the proximal tubular epithelial cells. Our findings also suggest a potential mechanism involved in water metabolism disorders in patients with AMS and in patients with chronic kidney disease (CKD) receiving roxadustat treatment.

Involvement of aquaporins in Shiga toxin-induced swelling and water transport dysfunction in human renal microvascular endothelial cells

One of the hallmarks of Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is kidney damage. Our previous research demonstrated that Shiga toxin type 2 (Stx2a) decreases cell viability and induces swelling of human glomerular endothelial cells (HGEC). However, Stx2a can disrupt net water transport across HGEC monolayers without affecting cell viability. This work aimed to elucidate the possible mechanisms involved in the water transport disruption caused by Stx2a across HGEC monolayers. We investigated paracellular and transcellular water transfer across HGEC by analyzing the passage of FITC-Dextran and the hydrostatic pressure (Phydr) and measuring the osmotic pressure (Posm), respectively. Stx2a selectively affected the transcellular pathway without impacting the paracellular route. Furthermore, Stx2a cell swelling was prevented by pretreatment with aquaporin inhibitors tetraethylammonium chloride (TEA), Mercury (II) chloride (HgCl2) or TGN-020, suggesting aquaporin involvement in this process. Confocal microscopy revealed that Stx2a increased HGEC total volume, which TEA and TGN-020 counteracted. Additionally, we identified in HGEC not only the expression of aquaporin-1 (AQP1) but also the expression of aquaporin-4 (AQP4). Surprisingly, we observed a decrease in the expression of both AQPs after Stx2a exposure. Our findings suggest that Stx2a may induce water movement into HGEC via AQP1 and AQP4, increasing total cell volume. Subsequently, decreased AQP1 and AQP4 expression could inhibit transcellular water transfer, potentially as a protective mechanism against excessive water entry and cell lysis.

Myeloid-derived growth factor promotes M2 macrophage polarization and attenuates Sjögren’s syndrome via suppression of the CX3CL1/CX3CR1 axis

IntroductionPrimary Sjögren syndrome (pSS) is a systemic autoimmune disease that is characterized by the infiltration of immune cells into the salivary glands. The re-establishment of salivary glands (SGs) function in pSS remains a clinical challenge. Myeloid-derived growth factor (MYDGF) has anti-inflammatory, immunomodulatory, and tissue-functional restorative abilities. However, its potential to restore SGs function during pSS has not yet been investigated.MethodsNonobese diabetic (NOD)/LtJ mice (pSS model) were intravenously administered with adeno-associated viruses carrying MYDGF at 11 weeks of age. Salivary flow rates were determined before and after treatment. Mice were killed 5 weeks after MYDGF treatment, and submandibular glands were collected for analyses of histological disease scores, inflammatory cell infiltration, PCR determination of genes, and Western blotting of functional proteins. Furthermore, mRNA sequencing and bioinformatics were used to predict the mechanism underlying the therapeutic effect of MYDGF.ResultsTreatment of NOD/LtJ mice with MYDGF alleviated pSS, as indicated by increased salivary flow rate, reduced lymphocyte infiltration, attenuated glandular inflammation, and enhanced AQP5 and NKCC1 expression. The gene expression levels of cytokines and chemokines, including Ccl12, Ccl3, Il1r1, Ccr2, Cx3cr1, Il7, Mmp2, Mmp14, Il1b, and Il7, significantly decreased after treatment with MYDGF, as determined by RNA sequencing. Meanwhile, MYDGF inhibits infiltration of macrophages (Mϕ) in SGs, induces polarization of M2ϕ, and suppresses C-X3C motif ligand 1 (CX3CL1)/C-X3C motif receptor 1 (CX3CR1) axis.ConclusionsOur findings showed that MYDGF could revitalize the SGs function of pSS, inhibit infiltration of Mϕ, and promote M2ϕ polarization via suppression of the CX3CL1/CX3CR1 axis, which has implications for potential therapy for pSS.

Therapeutic Potential of Intermittent Hypoxia in Atrial Fibrillation

Intermittent hypoxia (IH) has been extensively studied in recent years, demonstrating adverse and beneficial effects on several physiological systems. However, the precise mechanism underlying its cardiac effects on the heart remains unclear. This study aims to explore the effect of treatment on atrial fibrillation under IH conditions, providing data that can potentially be used in the treatment of heart disease. An atrial fibrillation (AF) model was induced by injecting monocrotaline (MCT, 60 mg/kg) into rats. The study included 32 rats divided into four groups: Control, Control + IH, AF, and AF + IH. We evaluated molecular changes associated with AF using ELISA and Western blot and performed electrophysiological experiments to evaluate AF. Arrhythmia-related calcium and fibrosis markers were investigated. Phosphorylation levels of CaMKII, Phospholamban, and RyR2 all increased in the AF group but decreased in the IH-exposed group. Additionally, fibrosis marker expressions such as SMA, MMP2, MMP9, and TGF-β increased in the AF group but were significantly downregulated with IH treatment. Connexin 43 and AQP4 expression were restored in the IH-treated group. These findings suggest that IH may prevent AF by downregulating the expression of calcium-handling proteins and fibrosis-associated proteins in an AF-induced rat model.

Orthosilicic acid inhibits human osteoclast differentiation and bone resorption.

Silicon (Si), which is present in the diet in the bioavailable form of orthosilicic acid (OSA) and is detected as a dissolution product of certain bone-substitute materials, is suggested to promote bone health, and enhance bone healing, respectively. Silicon has been shown to stimulate osteoblastic cell differentiation and function, although the effect of Si on human osteoclasts is unclear. The present study investigated the direct effects of Si on human osteoclast differentiation, gene expression, and bone resorption. Human CD14+ monocytes were isolated from buffy coats and cultured with M-CSF and RANKL in medium without or with Si (50 μg/ml; constituting 75% OSA). The effects of Si on osteoclast differentiation were evaluated by TRAP-staining and the expression levels of CtsK, CalcR, TRAP, and DC-STAMP measured by RT-qPCR. The effect of Si on the expression level of AQP9, which encodes a potential Si transporter, was also analyzed. Bone resorption was determined based on the number of resorption pits formed when the RANKL-stimulated monocytes were cultured on bone slices, and by the levels of type I collagen fragments released into the cell culture medium. Silicon significantly inhibited the number of TRAP+ multinucleated cells and the expression of osteoclast related genes but increased the late expression of AQP9. Furthermore, Si significantly inhibited the number of resorption pits and the amount of collagen fragments in the medium when cells were cultured on bone slices. Our results demonstrate that OSA inhibits RANKL-stimulated human osteoclast differentiation, gene expression of osteoclast phenotypic markers, and bone resorption.

POU2F2 activates the Akt/mTOR signalling pathway and enhances B lymphocyte function during diabetic kidney disease by promoting PIK3CD transcription.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD) is the predominant isoform of the catalytic subunit of PI3K in lymphocytes. Based on comprehensive bioinformatics, this study explores the functions of PIK3CD in diabetic kidney disease (DKD) progression in mice and B lymphocyte activity. Non-obese diabetic (NOD) mice that spontaneously develop DKD were applied as animal models. Lentiviral vector-mediated PIK3CD silencing was introduced in mice, followed by histological staining and biomarker assessments to evaluate DKD-associated symptoms. The activity of the Akt/mTOR signalling pathway was determined by western blot analysis. Following bioinformatics analyses, the interaction between POU class 2 homeobox 2 (POU2F2) and PIK3CD in B lymphocytes was determined by chromatin immunoprecipitation and luciferase reporter assays. Their functions in B cell function were identified by MTT, flow cytometry and IgG deposition assessment. PIK3CD was found to be highly expressed in the kidney of DKD mice. Knockdown of PIK3CD inactivated the Akt/mTOR signalling, thus ameliorating tissue injury and fibrosis, enhancing the expression of AQP1 and decreasing urinary NGAL contents, UACR, BUN and β-NAG/creatinine ratio. These effects were negated by the Akt-specific activator SC79. POU2F2 was found to promote PIK3CD transcription by binding to its promoter, thus activating the Akt/mTOR pathway. Knockdown of POU2F2 suppressed B cell proliferation in vitro and decreased B cell population and IgG deposition in the mouse kidney. However, these trends were reversed upon additional PIK3CD overexpression. This study demonstrates that POU2F2 mediates PIK3CD-dependent Akt/mTOR signalling activation, thus enhancing B lymphocyte function and promoting DKD progression.

Gene expression analysis of Aquaporin genes in ruminants during growth phase in response to heat stress

Aquaporins (AQPs) are trans-membrane protein involved in water transport and different cellular functions such as cell adhesion, signalling and proliferation. These membrane proteins are essential for key physiological functions such as organ development, osmoregulation, tissue regeneration and metabolism. The regulation of AQP5 gene expression in ruminants during growth phase has not been analysed in-vivo. Therefore, the gene expression pattern was analysed in Jamunapari goats during 3 months to 12 month of age and adult age group in response to heat stress. The genotyping of the AQP5 gene was carried out by High-Resolution Melting (HRM) in four different goat breeds, which indicated four distinct genotypes in the population. The nucleotide diversity for the AQP5 gene ranged from 0.315 and 0.524 across the breeds. Additionally, a close evolutionary relationship between AQP5 and the HSP70 gene was observed, indicating a shared pathway for heat stress regulation. The m-RNA expression level of AQP5 at 3, 9, 12 month and adult age group exhibited 47.24, 1140, 43.17 and 12.55-fold higher expression than control. The m-RNA expression level of the AQP5 gene was up-regulated and significantly higher (P < 0.05) at 9-month age as compared to the other age groups. Heat stress phenotypes were classified based on respiration rate and heart rate, and the m-RNA expression of AQP5 was higher in heat stress-susceptible (HSS) individuals than heat stress-tolerant (HST) individuals at 3, 9, and 12 months of age. The AQP5 plays a significant role in thermoregulation during growth phases in response to heat stress in goats, however, it is required to understand the role of aquaporins at cellular level as well as to establish the association with production performance in ruminant system in-vivo.

Maternal alcohol consumption up to mouse organogenesis disrupts fetal-placental interface at mid-gestation associated with dysregulation of AQP3 immunoexpression

Adequate trophoblast development during placentation involves the AQP3 regulation. The link between potential placental fetal-maternal interface abnormalities and AQP3 expression after perigestational alcohol intake was not explored yet. Female mice were treated (TF) with 10 % ethanol in drinking water before and up to day 10 of gestation, and control females (CF) with ethanol-free water. At gestational day 13, TFs showed increased fetal/placental weight ratio and reduced histological placental thickness compared to CFs. TF-placentas had disorganized fetal face layers, increased junctional zone (JZ), and decreased labyrinth (Lab). Concomitantly, immunoexpression of cleaved caspase-3 significantly increased in TF-JZ and Lab vs controls. Consistent with placental changes, AQP3 expression was higher in junctional trophoblast giant cells (TGCs), glycogen cells (GCs), spongiotrophoblasts (spg), and lab-syncytiotrophoblasts compared to CF-placentas. This study reveals, for the first time, that perigestational alcohol consumption up to organogenesis causes abnormal placental development associated with dysregulation of AQP3 expression.

Photobiomodulation reduces spinal cord edema by decreasing the expression of AQP4 in the astrocytes of male spinal cord injury rats via the JAK2/STAT3 signaling pathway.

BackgroundSpinal cord swelling commonly occurs following SCI. Previous studies suggest that PBM may reduce inflammation and scar formation after SCI. However, whether PBM can alleviate post-spinal cord injury edema and its underlying mechanisms have not yet been reported. This study aims to investigate the effects of PBM on spinal cord swelling in rats following SCI and explore the underlying mechanisms. MethodsA rat model of SCI was established, and the rats received continuous PBM therapy for two weeks. Tissue hydration, motor function, AQP4 expression, and pathological changes in the spinal cord were evaluated at different time points. In vitro, astrocytes were subjected to PBM and treated with either cucurbitacin I or TGN020 following OGD. ResultsThe results indicate that PBM reduces tissue swelling in rats with SCI, improves motor function recovery, and inhibits the upregulation of AQP4 and GFAP associated with SCI. In vitro, PBM reduces abnormal activation of the JAK2/STAT3 signaling pathway in astrocytes, leading to decreased AQP4 synthesis and astrocyte activation. ConclusionsThese findings suggest that PBM reduces spinal cord swelling in rats after injury. This effect is associated with the inhibition of JAK2/STAT3 signaling pathway activation in astrocytes and the reduction in AQP4 expression.

Improvement effect and mechanism of XuanFuDaiZhe tang on rats with diarrheal irritable bowel syndrome induced by colorectal dilation

Ethnopharmacological relevanceXuanFuDaiZhe Tang (XFDZT) is used in traditional Chinese medicine (TCM) to treat diarrhoea-predominant irritable bowel syndrome (IBS-D). Our laboratory has demonstrated that XFDZT remarkably improves various gastrointestinal motility disorders in animal models. However, previous studies have only focused on one or several protein targets without systematically investigating dynamic changes and protein interrelations. Aim of the studyTo explore the mechanisms underlying the therapeutic action of XFDZT in IBS-D using a network pharmacology approach and in vivo experiments. Materials and methodsThe active compounds of XFDZT were selected from TCM Systems Pharmacology and TCM Integrated databases, and potential targets were identified using the Swiss Target Prediction databases. Targets related to IBS-D were mined from the DisGeNet, Drug Bank, and Therapeutic Target databases. The intersecting protein–protein interactions (PPIs) of the drug–disease crossover genes were analysed, and a central PPI network was constructed using the STRING database and Cytoscape 3.7.2. Following Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, a gene pathway network was constructed to identify key target genes and pathways. Using haematoxylin and eosin staining and western blotting, we validated how XFDZT controls water expression in the body to treat IBS-D infection. ResultsFirst, the results showed that XFDZT contained 1037 active ingredients and 1458 corresponding targets. After intersecting the 252 IBS targets, 108 targets were identified. The main targets of XFDZT were albumin, aquaporins such as AQP1 and AQP3, calmodulin, and the cellular enzyme CYP2C9. GO and KEGG enrichment predicted that the action pathways were the neuroactive ligand–receptor interaction, calcium signalling pathway, serotonergic synapse signalling pathway, cGMP–PKG signalling pathway, cAMP signalling pathway, and MLCK–MLC signalling pathway. Second, an IBS-D rat model was constructed using colorectal dilation (CRD). CRD can significantly induce IBS-D symptoms such as diarrhoea, abdominal pain, and anxiety and depression-like behaviour in rats. XFDZT (10, 20, and 40 g/kg) administered for 14, 21, and 28 days significantly reversed these changes in IBS-D rats in a time- and dose-dependent manner, suggesting that XFDZT significantly improved IBS-D. Finally, the mechanism by which XFDZT improves IBS-D was explored from the perspective of AQPs, tight junction proteins, and motility-related proteins in colon tissue. Compared with the control group, the protein expression of AQP1, AQP3, and AQP8 in the colon tissue of IBS-D rats was significantly downregulated, whereas the protein expression of AQP7 was significantly upregulated. The expression of tight junction-related proteins claudin-1, occludin, and ZO-1 in colon tissue was significantly downregulated, whereas the expression of motility-related proteins p-MLC, MLC, MLCK, and CaM in colon tissue was significantly upregulated, suggesting that IBS-D rats had AQP disorders, epithelial intercellular connections, and motility in colon tissue. The above changes were significantly reversed by XFDZT administration (5, 10, and 20 g/kg) for 14 days. ConclusionXFDZT significantly improved diarrhoea, abdominal pain, anxiety, and depression in IBS-D rats, and its mechanism of action may be related to the regulation of AQPs, tight junction proteins, and the MLCK–MLC pathway. This study provided a pharmacological experimental basis for the development of XFDZT as a novel drug for the treatment of IBS-D.

Evaluation of the Choroid Plexus Epithelium Inflammation TLR4/NF-κB/NKCC1 Signal Pathway Activation in the Development of Hydrocephalus.

Hydrocephalus is characterized by secretion, circulation, and absorption disorder of cerebrospinal fluid (CSF) with high morbidity and complication rate. The relationship between inflammation and abnormal secretion of CSF by choroid plexus epithelium (CPE) had received more attention. In this study, we aim to detect the role of Toll-like receptor 4/nuclear factor-kappa B/Na+/K+/2Cl-cotransporter 1(TLR4/NF-κB/NKCC1) signal pathway in the development of hydrocephalus. Hydrocephalus was induced in adult rats (8 weeks) by intracisternal kaolin injection, then pyrrolidinedithiocarbamate (PDTC) and bumetanide were administrated to the rats mode. Then the rat model was evaluated, and ventricular volume was calculated at different time points. Then CPE, cortex, preventricular tissue, and CSF were obtained. Protein expressions of TLR-4, NKCC/serine-threonine STE20/SPS1-related, proline-alanine-rich kinase (SPAK), pNKCC1, pSPAK, GFAP, AQP1, and AQP4 were measured by RT-PCR, western blot, and immunofluorescence (IF) stains in CPE, respectively. Our data showed that inflammation factors tumor necrosis factor-(TNF-α), interleukin 18(IL-18), and glial fibrillary acidic protein (GFAP) concentrations were significantly higher in the model group than in controls. The TLR4/NF-κB/NKCC1 signal pathway were actived by NF-κB-p65, NKCC1, pNKCC1- pSPAK complex, and Aquaporin1 (AQP1) high expression. PDTC and bumetanide use can help regular TLR4/NF-κB/NKCC1 expression and reduced AQP1 expression by down-regulate NF-B-p65 and inhibiting NKCC1, respectively. As a result, the treatment groups alleviated CPE abnormal secretion and ventricle enlargement. These results confirmed that the inflammatory reaction contributes TLR4/NF-κB/NKCC1 mediated CPE abnormal secretion and consequent hydrocephalus. Regulation of TLR4/NF-κB/NKCC1 and AQP1 can prevent this process. Our study provides a strong rationale for further exploring alleviating CPE abnormal secretion as a therapeutic perspective of hydrocephalus.

Lentivirus-mediated RNA interference targeting HMGB1 modulates AQP1 to reduce pain induced by chronic compression of the dorsal root ganglia.

Neuropathic pain (NP) is a kind of chronic pain that has attracted much attention in clinical practice, characterized by high morbidity, complex mechanisms, and difficulties in clinical treatment, with which the activation of High mobility group box 1 (HMGB1) is closely related. The aim of this study was to investigate the effects of lentivirus-mediated RNA interference gene therapy targeting HMGB1 on neuropathic pain in rats with chronic dorsal root ganglion compression (CCD) and its specific mechanisms, so as to explore new pharmacological targets. Adult male Wistar rats were surgically subjected to chronic compression of the dorsal root ganglia (CCD). Behavioral tests were performed by calculating the paw withdrawal mechanical threshold (PWMT) and the thermal paw withdrawal latency (TPWL). Co-immunoprecipitation (CO-IP) was used to clarify protein interactions. Gene silencing was induced by injecting lentivirus expressing HMGB1 short hairpin RNA (shRNA) into rats. An LPS-inflammation-stimulated rat astrocyte model was established to validate the animal experiment results further. Western blot analysis and real-time quantitative PCR were used to detect pathway protein expression. After first establishing the rat CCD model, both PWMT and PTWL were significantly reduced in rats, indicating that the model construction was successful. After lentiviral silencing of HMGB1 expression, NP was significantly alleviated in CCD rats. CO-IP experiments showed a link between HMGB1 and AQP1; After silencing HMGB1 expression, the expression of AQP1 was significantly reduced, and HMGB1 was able to modulate the effect of AQP1 on NP. Further use of an inhibitor of the HMGB1 receptor showed that after inhibition of RAGE, AQP1 was significantly reduced; HMGB1 may regulate AQP1 through its receptor RAGE to affect NP. Silencing of HMGB1 resulted in a significant decrease in NF-κB, and HMGB1 affects the inflammatory pathways it mediates. After silencing AQP1, NF-κB also decreased significantly, indicating that AQP1 is an upstream regulator of NF-κB. Lentivirus-mediated RNA interference (RNAi) silencing targeting HMGB1 may play a key role in the development of neuropathic pain in rats by regulating AQP1 expression via RAGE and ultimately activating NF-κB.

Remobilization with whole-body vibration improves functionality, histomorphometric parameters, and AQP1 expression in the soleus muscle of Wistar rats.

Whole-body vibration (WBV) is used to enhance physical performance in sports and rehabilitation. The present study analyzed the effects of remobilization with WBV on the soleus muscle of Wistar rats. Twenty-eight animals were separated into four experimental groups (n = 7): CON (control); IM (immobilized); FR (immobilization and free remobilization); and WBV (immobilization and remobilization with WBV). The immobilization of the pelvic limb was carried out according to the standard protocol using a plaster cast for 15 days. For remobilization with WBV, a Frequency of 60 Hz was applied for 10 min, five days a week, for two weeks. After the remobilization period, the animals were euthanized, and the right soleus muscle was dissected followed by processing for histomorphometric analysis and immunolocalization of Aquaporin 1 (AQP1). We observed a reduced larger diameter in IM compared to CON, with restored values in WBV. For the estimation of connective tissue, a significant increase was observed in the immobilized groups, while a reduction was noted in the remobilized groups. AQP1 expression decreased significantly in IM and increased in WBV. Immobilization caused morphofunctional damage to the soleus muscle, and remobilization with WBV is efficient and offers advantages over free remobilization.

Extract of Nanhaia speciosa J. Compton & Schrire alleviates LPS-induced acute lung injury via the NF-κB/Nrf2/AQPs pathway

Ethnopharmacological relevanceNanhaia speciosas J. Compton & Schrire (the name Nanhaia speciosas J. Compton & Schrire has been accepted by the World Checklist of Vascular Plants https://www.worldfloraonline.org/taxon/wfo-0001444004) is a traditional Zhuang medicine that have been widely used for centuries. It has been used in the treatment of lung inflammation, tuberculosis, rheumatic pain, lumbar muscle strain, and various other ailments, such as chronic hepatitis, menoxenia, leukorrhea, and injuries. In addition, N. speciosa has also been used to treat acute lung injury (ALI). Aim of the studyThe objective of this study was to conduct a comparative analysis of the effects of various constituents present in N. speciosas extract (NSE) on ALI and the related mechanisms while also elucidating the potential active monomeric components. Materials and methodsNSE was extracted using an AB-8 macroporous resin column, and five fractions (Fr. 0%, 25%, 50%, 75% and 95%) were obtained. The anti-inflammatory and antioxidant capacities of the five fractions were evaluated in an A549 cell-based in vitro model, with the aim of evaluating their potential therapeutic effects. The anti-inflammatory and antioxidant capacities of NSE were assessed in a murine model of ALI induced by intratracheal injection of LPS. We utilized an in vitro model to analyse the critical molecular mechanisms through which NSE ameliorates ALI. The chemical composition of the optimal fraction was analysed and confirmed using UHPLC/MS. ResultsDifferent fractions (especially Fr. 75%) significantly reduced inflammation and oxidative stress in A549 cells. Fr.75% abrogated LPS-induced pathological alterations and decreased the lung W/D ratio, total protein concentration in BALF, and the levels of the proinflammatory factors TNF-α, IL-6, and IL-1β. Moreover, Fr.75% reduced MPO and MDA concentrations and elevated SOD and GSH concentrations in pulmonary tissues. Additionally, it decreased the pulmonary tissue inflammation caused by LPS by downregulating the expression of p-NF-κB p65 and upregulating the expression of Nrf2, AQP1 and AQP5. Fr. 75% decreased p-NF-κB p65 protein levels; increased Keap1, Nrf2, HO-1, NQO1, AQP1 and AQP5 protein levels; and promoted the entry of Nrf2 into the nucleus. After UHPLC/MS analysis was conducted, the flavonoid Maackiain was determined to potentially play a pivotal role in this process. ConclusionFr.75% alleviates ALI by regulating the NF-κB/Nrf2/AQPs signalling pathway. The flavonoid Maackiain may also play an important role in this process. Overall, N. speciosas may be a potential therapeutic agent for the prevention and treatment of ALI.

Exacerbation of Hepatic Damage in Endothelial Aquaporin 1 Transgenic Mice after Experimental Heatstroke.

Heatstroke induces fluid loss and electrolyte abnormalities owing to high ambient temperature (AT) and relative humidity (RH). Aquaporin 1 (AQP1) is a key protein for water homeostasis; however, its role in heatstroke remains unclear. This study examines endothelial AQP1 in Tie2-Cre/LNL-AQP1 double transgenic (dTG) mice with upregulated Aqp1 in endothelial cells. For experimental heatstroke, mice were exposed to 41 °C AT and >99% RH. Blood, brain, kidney, and liver samples were collected 24 h later. Blood was analyzed for electrolytes and tissue damage markers, and organs were examined using morphological and immunohistological staining for 3-nitrotyrosine (3-NT), AQP1, and Iba-1. No difference in Aqp1 expression was observed in the whole brain; however, it was detected in dTG mice after capillary deprivation. AQP1 immunostaining revealed immunoreaction in blood vessels. After heat exposure, wild-type and dTG mice showed electrolyte abnormalities compared with non-heatstroke wild-type mice. Hepatic damage markers were significantly higher in dTG mice than in wild-type mice. Hematoxylin-eosin staining and 3-NT immunoreactivity in the liver indicated hepatic damage. The number of Iba-1-positive cells adherent to hepatic vasculature was significantly higher in dTG mice than in wild-type mice. This study is the first to suggest that endothelial AQP1 contributes to hepatic damage after heatstroke.

Comprehensive analysis of an mRNA co-expression network and a ceRNA network reveals potential prognostic biomarkers in oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC) is a prevalent and aggressive oral cancer with a poor prognosis. Its polygenic risk is likely influenced by complex transcriptional disorders involving networks of co-expressed and functionally related genes, though such investigations are limited in OSCC.MethodsWe analyzed the GSE37991 dataset, comprising 40 OSCC and 40 normal oral tissue samples from the Gene Expression Omnibus. Tumor-specific modules were identified using weighted correlation network analysis (WGCNA), leading to the selection of hub mRNAs and lncRNAs. These lncRNAs were used to construct lncRNA–mRNA and competing endogenous RNA networks. We further examined the expression profiles and survival data of these genes from the Cancer Genome Atlas. Prognostic markers were identified and validated through 5-year survival analysis and Cox proportional hazards modeling. RT-qPCR was used to validate the expression levels in clinical OSCC tissues.ResultsWe identified 1847 differentially expressed genes in OSCC tissues. WGCNA revealed four OSCC-specific modules, screening 120 hub mRNAs and five hub lncRNAs. Two prognostic markers (AQP5, IL-26) from hub mRNAs and three (FRMD5, INHBB, GUCY1A3) from the lncRNA–mRNA network were associated with survival. Validation showed lower expression of AQP5 and GUCY1A3, and higher expression of FRMD5 and INHBB in OSCC compared to normal tissues.ConclusionThis study enhances our understanding of transcriptional dysregulation in OSCC and may highlights AQP5, IL-26, FRMD5, INHBB, and GUCY1A3 as promising prognostic biomarkers.

Patterns of VEGF/KDR and AQP3 expression in the materno-fetal interface and vascularization of mouse placenta after perigestational alcohol consumption up to early organogenesis

Patterns of VEGF/KDR and AQP3 expression in the materno-fetal interface and vascularization of mouse placenta after perigestational alcohol consumption up to early organogenesis

Hepatocyte aquaporin 8-mediated water transport facilitates bile dilution and prevents gallstone formation in mice

Although water channel aquaporin-8 (AQP8) has been implicated in hepatic bile formation and liver diseases associated with abnormal bile flow in human and animal studies, direct evidence of its involvement in bile secretion is still lacking. This study aimed to determine the role of AQP8 in bile secretion and gallstone formation. We generated various transgenic knock-in and knockout mouse models and assessed liver AQP8 expression by immunostaining and immunoblotting, hepatic bile secretion by cannulation of the common bile duct, cholesterol gallstone formation by feeding a high-fat lithogenic diet, and identified regulatory small molecules by screening the organic fractions of cholagogic Chinese herbs and biochemical characterization. We identified a novel expression pattern of AQP8 protein in the canalicular membrane of approximately 50% of the liver lobules. AQP8-deficient mice exhibited impaired hepatic bile formation, characterized by the secretion of concentrated bile with a lower flow rate and higher levels of bile lipids than that of wild-type littermates. AQP8-/- mice showed accelerated gallstone formation, which was rescued by AAV-mediated hepatic expression of AQP8 or AQP1. Moreover, we identified a small molecule, scutellarin, that upregulates hepatocyte AQP8 expression in vitro and in vivo. In AQP8+/+ mice, scutellarin significantly increased bile flow, decreased bile lipid concentrations, and prevented gallstone formation compared to AQP8-/- mice. Molecular studies revealed that scutellarin promoted the ubiquitination and degradation of HIF-1α, a transcriptional negative regulator of AQP8, by disrupting its interactions with HSP90. AQP8 plays a crucial role in facilitating water transport and bile dilution during hepatic bile formation, thereby mitigating gallstone formation in mice. Small-molecule intervention validated hepatocyte AQP8 as a promising drug target for gallstone therapy. The incidence of gallstone disease is high, and current drug treatments for gallstones are very limited, necessitating the identification of novel drug targets for developing new drugs with universal applicability. To our knowledge, this is the first study to provide direct evidence that hepatic water channel AQP8 plays a key role in bile dilution and gallstone formation. Modulation of hepatic water transport may provide a universal therapeutic strategy for all types of gallstone diseases.

The Impact of High-Dose Fish Oil Supplementation on Mfsd2a, Aqp4, and Amyloid-β Expression in Retinal Blood Vessels of 5xFAD Alzheimer's Mouse Model.

In patients with Alzheimer's disease (AD) and in animal models, the increased accumulation of amyloid β (Aβ) in retinal blood vessels strongly correlates with brain amyloid deposits and cognitive decline. The accumulation of Aβ in blood vessels may result from impaired transcytosis and a dysfunctional ocular glymphatic system in AD. High-dose fish oil (FO) supplementation has been shown to significantly change the expression of major facilitator superfamily domain-containing protein 2a (Mfsd2a), a key regulator of transcytosis, and Aquaporin 4 (Aqp4), an essential component of the glymphatic system in the retinas of WT mice. We examined the expression of Mfsd2a and Aqp4 in the retinas of 4-month-old 5xFAD female mice supplemented with high-dose FO for three weeks. There was a significant increase in Mfsd2a expression in 5xFAD retinas supplemented with FO compared to control 5xFAD mice. Additionally, the increase in Aqp4 expression observed in 4-month-old 5xFAD retinas, indicative of an impaired glymphatic system, was significantly decreased. Simultaneously, Aβ accumulation in 5xFAD retinal blood vessels was reduced following FO supplementation. These findings suggest that high-dose FO supplementation could serve as an adjunct in developing new treatments aimed at improving the regulation of transcytosis or the function of the glymphatic system in the AD retina.

Distinctive small molecules blend: Promotes lacrimal gland epithelial cell proliferation in vitro and accelerates lacrimal gland injury repair in vivo

PurposeThis study aims to develop a novel serum-free culture strategy containing only two small molecules, Y27632 and SB431542 (2C), for in vitro expansion of mouse lacrimal gland epithelial cells (LGECs) and investigate an innovative therapeutic approach for lacrimal gland (LG) injury. MethodsLGECs proliferative capacity was assessed by cell counting, crystal violet staining, qRT-PCR and immunofluorescence. Cell differentiation was achieved by manipulating culture conditions and assessed by qRT-PCR and AQP5 immunofluorescence. LGECs were seeded in Matrigel for three-dimensional culture and assessed by qRT-PCR and immunofluorescence. Secretory function of the cultures was assayed by ELISA. In vivo, 2C injection verified its reparative capacity in a mouse LG injury model. Corneal fluorescein staining, phenol red cotton thread, H&E, immunofluorescence and Western blot were used to assess LG injury repair. ResultsLGECs cultured with 2C exhibited high expression of stemness/proliferation markers and maintained morphology and proliferative capacity even after the tenth passage. Removal of 2C was efficacious in achieving LGECs differentiation, characterized by the increased AQP5 expression and LTF secretion. 3D spheroids cultured with 2C demonstrated differentiation potential, forming microglandular structures containing multiple LG cell types with secretory functions after 2C removal. In vivo, 2C improved the structural integrity and function of the injured LG. ConclusionsWe present a small molecule combination, 2C, that promotes LGECs expansion and differentiation in vitro and accelerates LG injury repair in vivo. This approach has potential applications for providing a stable source of seed cells for tissue engineering applications, providing new sights for LG-related diseases treatment.