Retroductal dexamethasone administration promotes the recovery from obstructive and inflammatory salivary gland dysfunction.

Introduction: Salivary gland dysfunction, often resulting from salivary gland obstruction-induced inflammation, is a prevalent condition. Corticosteroid, known for its anti-inflammatory and immunomodulatory properties, is commonly prescribed in clinics. This study investigates the therapeutic implications and potential side effects of dexamethasone on obstructive sialadenitis recovery using duct ligation mice and salivary gland organoid models. Methods: Functional and pathological changes were assessed after administering dexamethasone to the duct following deligation 2 weeks after maintaining ligation of the mouse submandibular duct. Additionally, lipopolysaccharide- and tumor necrosis factor-induced salivary gland organoid inflammation models were established to investigate the effects and underlying mechanisms of action of dexamethasone. Results: Dexamethasone administration facilitated SG function restoration, by increasing salivary gland weight and saliva volume while reducing saliva lag time. Histological evaluation revealed, reduced acinar cell atrophy and fibrosis with dexamethasone treatment. Additionally, dexamethasone suppressed pro-inflammatory cytokines IL-1ß and TNF expression. In a model of inflammation in salivary gland organoids induced by inflammatory substances, dexamethasone restored acinar markers such as AQP5 gene expression levels, while inhibiting pro-inflammatory cytokines TNF and IL6, as well as chemokines CCL2, CXCL5, and CXCL12 induction. Macrophages cultured in inflammatory substance-treated media from salivary gland organoid cultures exhibited pro-inflammatory polarization. However, treatment with dexamethasone shifted them towards an anti-inflammatory phenotype by reducing M1 markers (Tnf, Il6, Il1b, and Cd86) and elevating M2 markers (Ym1, Il10, Cd163, and Klf4). However, high-dose or prolonged dexamethasone treatment induced acino-ductal metaplasia and had side effects in both in vivo and in vitro models. Conclusions: Our findings suggest the effectiveness of corticosteroids in treating obstructive sialadenitis-induced salivary gland dysfunction by regulating pro-inflammatory cytokines.

Reticulated Retinoic Acid Synthesis is Implicated in the Pathogenesis of Dry Eye in Aqp5 Deficiency Mice.

Purpose: Abnormalities in aquaporins are implicated in the pathological progression of dry eye syndrome. Retinoic acid (RA) regulates cellular proliferation, differentiation, and apoptosis in the cornea, thereby being associated with dry eye disease (DED). The objective of this study is to explore the underlying mechanisms responsible for RA metabolic abnormalities in corneas lacking aquaporin 5 (AQP5). Methods: Dry eye (DE) models were induced via subcutaneous scopolamine hydrobromide. Aqp5 knockout (Aqp5-/-) mice and DE mice were utilized to assess corneal epithelial alterations. Tear secretion, goblet cell counts, and corneal punctate defects were evaluated. The impact of Aqp5 on RA-related enzymes and receptors was investigated using pharmacological RA or SR (A JunB inhibitor), a transcription factor JunB inhibitor, treatment in mouse corneal epithelial cells (CECs), or human corneal epithelial cells (HCECs). The HCECs and NaCl-treated HCECs underwent quantitative real-time PCR (qRT-PCR), immunofluorescent, Western blot, and TUNEL assays. The regulation of transcription factor JunB on Aldh1a1 was explored via ChIP-PCR. Results: Aqp5 and Aldh1a1 were reduced in both CECs of DE mice and NaCl-induced HCECs. Aqp5-/- mice exhibited DE phenotype and reduced Aldh1a1. RA treatment reduced apoptosis, promoted proliferation, and improved the DE phenotype in Aqp5-/- mice. JunB enrichment in the Aldh1a1 promoter was identified by ChIP-PCR. SR significantly increased Aldh1a1 expression, Ki67, and ΔNp63-positive cells, and decreased TUNEL-positive cells in CECs and HCECs. Conclusions: Our findings demonstrated the downregulation of Aqp5 expression and aberrant RA metabolism in DE conditions. Knockout of Aqp5 resulted in reduced production of RA through activation of JunB, subsequently leading to the manifestation of DE symptoms.

Efficient generation of a stable CHO-K1 cell line overexpressing the human water channel aquaporin-5 as tool to generate therapeutic antibodies.

Aquaporins (AQPs) are a family of water permeable channels expressed on the plasma membrane with AQP5 being the major channel expressed in several human tissues including salivary and lacrimal glands. Anti-AQP5 autoantibodies have been observed in patients with Sjögren's syndrome who are characterised by dryness of both salivary and lacrimal glands, and they have been implicated in the underlying mechanisms of glandular dysfunction. AQP5 is formed by six transmembrane helices linked with three extracellular and two intracellular loops. Develop antibodies against membrane protein extracellular loops can be a challenge due to the difficulty in maintaining these proteins as recombinant in their native form. Therefore, in this work we aimed to generate an efficient stable-transfected cell line overexpressing human AQP5 (CHO-K1/AQP5) to perform primarily cell-based phage display biopanning experiments to develop new potential recombinant antibodies targeting AQP5. We also showed that the new CHO-K1/AQP5 cell line can be used to study molecular mechanisms of AQP5 sub-cellular trafficking making these cells a useful tool for functional studies.

Structural Basis for the Interaction between the Ezrin FERM-Domain and Human Aquaporins.

The Ezrin/Radixin/Moesin (ERM) family of proteins act as cross-linkers between the plasma membrane and the actin cytoskeleton. This mechanism plays an essential role in processes related to membrane remodeling and organization, such as cell polarization, morphogenesis and adhesion, as well as in membrane protein trafficking and signaling pathways. For several human aquaporin (AQP) isoforms, an interaction between the ezrin band Four-point-one, Ezrin, Radixin, Moesin (FERM)-domain and the AQP C-terminus has been demonstrated, and this is believed to be important for AQP localization in the plasma membrane. Here, we investigate the structural basis for the interaction between ezrin and two human AQPs: AQP2 and AQP5. Using microscale thermophoresis, we show that full-length AQP2 and AQP5 as well as peptides corresponding to their C-termini interact with the ezrin FERM-domain with affinities in the low micromolar range. Modelling of the AQP2 and AQP5 FERM complexes using ColabFold reveals a common mode of binding in which the proximal and distal parts of the AQP C-termini bind simultaneously to distinct binding sites of FERM. While the interaction at each site closely resembles other FERM-complexes, the concurrent interaction with both sites has only been observed in the complex between moesin and its C-terminus which causes auto-inhibition. The proposed interaction between AQP2/AQP5 and FERM thus represents a novel binding mode for extrinsic ERM-interacting partners.

Application of anti-vascular endothelial growth factor antibody restores the function of saliva secretion in a type 2 diabetes mouse model.

OBJECTIVES: Xerostomia, a common complication of type 2 diabetes, leads to an increased risk of caries, dysphagia, and dysgeusia. Although anti-vascular endothelial growth factor (VEGF) antibodies, such as ranibizumab (RBZ), have been used to treat diabetic retinopathy, their effects on the salivary glands are unknown. This study evaluated the effects of RBZ on salivary glands to reduce inflammation and restore salivary function in a mouse model of type 2 diabetes. METHODS: Male KK-Ay mice with type 2 diabetes (10-12 weeks old) were used. The diabetes mellitus (DM) group received phosphate-buffered saline, while the DM + RBZ group received an intraperitoneal administration of RBZ (100 µg/kg) 24 h before the experiment. RESULTS: Ex vivo perfusion experiments showed a substantial increase in salivary secretion from the submandibular gland (SMG) in the DM + RBZ group. In addition, the mRNA expression levels of TNF-α and IL-1ß were considerably lower in this group. In contrast, those of aquaporin 5 were substantially higher in the DM + RBZ group, as revealed by quantitative reverse transcription PCR. Furthermore, the number of lymphocyte infiltration spots in the SMG was notably lower in the DM + RBZ group. Finally, intracellular Ca2+ signaling in acinar cells was considerably higher in the DM + RBZ group than that in the DM group. CONCLUSION: Treating a type 2 diabetic mouse model with RBZ restored salivary secretion through its anti-inflammatory effects.

Topical naltrexone increases aquaporin 5 production in the lacrimal gland and restores tear production in diabetic rats.

Diabetes mellitus is a prevalent disease that is often accompanied by ocular surface abnormalities including delayed epithelial wound healing and decreased corneal sensitivity. The impact of diabetes on the lacrimal functional unit (LFU) and the structures responsible for maintaining tear homeostasis, is not completely known. It has been shown that the Opioid Growth Factor Receptor (OGFr), and its ligand, Opioid Growth Factor (OGF), is dysregulated in the ocular surface of diabetic rats leading to overproduction of the inhibitory growth peptide OGF. The opioid antagonist naltrexone hydrochloride (NTX) blocks the OGF-OGFr pathway, and complete blockade following systemic or topical treatment with NTX restores the rate of re-epithelialization of corneal epithelial wounds, normalizes corneal sensitivity, and reverses dry eye in diabetic animal models. These effects occur rapidly and within days of initiating treatment. The present study was designed to understand mechanisms related to the fast reversal (<5 days) of dry eye by NTX in type 1 diabetes (T1D) by investigating dysregulation of the LFU. The approach involved examination of the morphology of the LFU before and after NTX treatment. Male and female adult Sprague-Dawley rats were rendered hyperglycemic with streptozotocin, and after 6 weeks rats were considered to be a T1D model. Rats received topical NTX twice daily to one eye for 10 days. During the period of treatment, tear production and corneal sensitivity were recorded. On day 11, animals were euthanized and orbital tissues including conjunctiva, eyelids, and lacrimal glands, were removed and processed for histologic examination including immunohistochemistry. Male and female T1D rats had significantly decreased tear production and corneal insensitivity, significantly decreased number and size of lacrimal gland acini, decreased expression of aquaporin-5 (AQP5) protein and decreased goblet cell size. Thus, 10 days of NTX treatment restored tear production and corneal sensitivity to normal values, increased AQP5 expression, and restored the surface area of goblet cells to normal. NTX had no effect on the number of lacrimal gland acini or the number of conjunctival goblet cells. In summary, blockade of the OGF-OGFr pathway with NTX reversed corneal and lacrimal gland complications and restored some components of tear homeostasis confirming the efficacy of topical NTX as a treatment for ocular defects in diabetes.

Downregulated GPX4 in salivary gland epithelial cells contributes to salivary secretion dysfunction in Sjogren's syndrome via lipid ROS/pSTAT4/AQP5 axis.

Sjogren's syndrome (SS) is an autoimmune disease characterized by dysfunction of exocrine glands, such as salivary glands. However, the molecular mechanism of salivary secretion dysfunction in SS is still unclear. Given the significance of glutathione peroxidase 4 (GPX4) in cellular redox homeostasis, we hypothesized that dysregulation of GPX4 may play a pivotal role in the pathogenesis of salivary secretion dysfunction observed in SS. The salivary gland of SS patients and the SS mouse model exhibited reduced expression of the ferroptosis inhibitor GPX4 and the important protein aquaporin 5 (AQP5), which is involved in salivary secretion. GPX4 overexpression upregulated and GPX4 knockdown downregulated AQP5 expression in salivary gland epithelial cells (SGECs) and salivary secretion. Bioinformatics analysis of GSE databases from SS patients' salivary glands revealed STAT4 as a key intermediary regulator between GPX4 and AQP5. A higher level of nuclear pSTAT4 was observed in the salivary gland of the SS mouse model. GPX4 overexpression inhibited and GPX4 knockdown promoted STAT4 phosphorylation and nuclear translocation in SGECs. CHIP assay confirmed the binding of pSTAT4 within the promoter of AQP5 inhibiting AQP5 transcription. GPX4 downregulation accumulates intracellular lipid ROS in SGECs. Lipid ROS inhibitor ferrostatin-1 treatment during in vitro and in vivo studies confirmed that lipid ROS activates STAT4 phosphorylation and nuclear translocation in SGECs. In summary, the downregulated GPX4 in SGECs contributes to salivary secretion dysfunction in SS via the lipid ROS/pSTAT4/AQP5 axis. This study unraveled novel targets to revitalize the salivary secretion function in SS patients.

Targeting Aquaporin-5 by Phosphodiesterase 4 Inhibition Offers New Therapeutic Opportunities for Ovarian Ischemia Reperfusion Injury in Rats.

This study aimed to examine the effect of Phosphodiesterase 4 (PDE4) inhibition on Aquaporin-5 (AQP5) and its potential cell signaling pathway in the ovarian ischemia reperfusion (OIR) model. Thirty adult female rats were divided into five groups: Group 1; Control: Sham operation, Group 2; OIR that 3 hour ischemia followed by 3 hour reperfusion, Group 3; OIR + Rolipram 1 mg/kg, Group 4; OIR + Rolipram 3 mg/kg, Group 5; OIR + Rolipram 5 mg/kg. Rolipram was administered intraperitoneally to the rats in groups 3-4 and 5 at determined doses 30 minutes before reperfusion. From ovary tissue; Tumor necrosis factor-a (TNF-α), Cyclic adenosine monophosphate (cAMP), Nuclear factor kappa (NF-κB), Interleukin-6 (IL-6), Phosphodiesterase 4D (PDE4D), Mitogen-activated protein kinase (MAPK) and AQP5 levels were measured by ELISA. We also measured the level of AQP5 in ovary tissue by real-time reverse-transcription polymerase chain reaction (RT-PCR). In the OIR groups; TNF-α, NF-κB, IL-6, MAPK inflammatory levels increased, and cAMP and AQP5 levels decreased, which improved with the administration of rolipram doses. Also histopathological results showed damaged ovarian tissue after OIR, while rolipram administration decrased tissue damage in a dose dependent manner. We propose that the protective effect of PDE4 inhibition in OIR may be regulated by AQP5 and its potential cell signaling pathway and may be a new target in OIR therapy. However, clinical studies are needed to appraise these data in humans.

Botulinum toxin type a blocks aquaporin 5 trafficking by decreasing synaptosomal-associated protein 23 in submandibular acinar cells.

The trafficking of aquaporin 5 (AQP5) is critical for salivary secretion. Synaptosomal-associated protein 23 (SNAP23) is an important regulator in the process of membrane fusion. However, the role of SNAP23 on AQP5 trafficking has not been explored. Botulinum toxin type A (BoNT/A) is a bacterial toxin that effectively treats sialorrhea. We previously reported that BoNT/A induced AQP5 redistribution in cultured acinar cells, but the mechanism remained unclear. In this study, SNAP23 was predominantly localized to the plasma membrane of acinar cells in the rat submandibular gland (SMG) and colocalized with AQP5 at the apical membrane of acinar cells. In stable GFP-AQP5-transfected SMG-C6 cells, the acetylcholine receptor agonist carbachol (CCh) induced trafficking of AQP5 from intracellular vesicles to the apical membrane. Furthermore, SNAP23 knockdown by siRNA significantly inhibited CCh-induced AQP5 trafficking, whereas this inhibitory effect was reversed by SNAP23 re-expression, indicating that SNAP23 was essential in AQP5 trafficking. More importantly, BoNT/A inhibited salivary secretion from SMGs, and the underlying mechanism involved that BoNT/A blocked CCh-triggered AQP5 trafficking by decreasing SNAP23 in acinar cells. Taken together, these results identified a crucial role for SNAP23 in AQP5 trafficking and provided new insights into the mechanism of BoNT/A in treating sialorrhea and thereby a theoretical basis for clinical applications.

Distribution of alpha-synuclein in rat salivary glands.

Expression of alpha-synuclein (Syn), a presynaptic neuronal protein, was immunohistochemically examined in intact rat submandibular, sublingual, and lingual glands. The submandibular gland contained abundant periductal Syn-immunoreactive (-ir) nerve fibers. Abundant Syn-ir varicosities were present in acini of the sublingual and serous lingual glands. By confocal laser scanning microscopy, Syn-ir nerve fibers around smooth muscle actin (SMA)-ir cells alone were infrequent; however, those around aquaporin-5 (AQP5)-ir cells alone and both SMA- and AQP5-ir cells were abundant in the sublingual and serous lingual glands. SMA-ir cells were occasionally immunoreactive for toll-like receptor 4, a Syn receptor. Syn-ir nerve fibers contained tyrosine hydroxylase (TH) in the submandibular gland and choline acetyltransferase (ChAT) in all examined salivary glands. In the superior cervical (SCG), submandibular, and intralingual ganglia, sympathetic and parasympathetic neurons co-expressed Syn with TH and ChAT, respectively. SCG neurons innervating the submandibular gland contained mostly Syn. In the thoracic spinal cord, 14.7% of ChAT-ir preganglionic sympathetic neurons co-expressed Syn. In the superior salivatory nucleus, preganglionic parasympathetic neurons projecting to the lingual nerve co-expressed Syn and ChAT. The present findings indicate that released Syn acts on myoepithelial cells. Syn in pre- and post-ganglionic neurons may regulate neurotransmitter release and salivary volume and composition.

Methazolamide Reduces the AQP5 mRNA Expression and Immune Cell Migration-A New Potential Drug in Sepsis Therapy?

Sepsis is a life-threatening condition caused by the dysregulated host response to infection. Novel therapeutic options are urgently needed and aquaporin inhibitors could suffice as aquaporin 5 (Aqp5) knockdown provided enhanced sepsis survival in a murine sepsis model. Potential AQP5 inhibitors provide sulfonamides and their derivatives. In this study, we tested the hypothesis that sulfonamides reduce AQP5 expression in different conditions. The impact of sulfonamides on AQP5 expression and immune cell migration was examined in cell lines REH and RAW 264.7 by qPCR, Western blot and migration assay. Subsequently, whether furosemide and methazolamide are capable of reducing AQP5 expression after LPS incubation was investigated in whole blood samples of healthy volunteers. Incubation with methazolamide (10-5 M) and furosemide (10-6 M) reduced AQP5 mRNA and protein expression by about 30% in REH cells. Pre-incubation of the cells with methazolamide reduced cell migration towards SDF1-α compared to non-preincubated cells to control level. Pre-incubation with methazolamide in PBMCs led to a reduction in LPS-induced AQP5 expression compared to control levels, while furosemide failed to reduce it. Methazolamide appears to reduce AQP5 expression and migration of immune cells. However, after LPS administration, the reduction in AQP5 expression by methazolamide is no longer possible. Hence, our study indicates that methazolamide is capable of reducing AQP5 expression and has the potential to be used in sepsis prophylaxis.

Goji Berry Juice Prevents Tumor Necrosis Factor Alpha-Induced Xerostomia in Human Salivary Gland Cells.

Sjögren's syndrome (SS) is an autoimmune disorder characterized by oral dryness that is primarily attributed to tumor necrosis factor alpha (TNF-α)-mediated reduction in saliva production. In traditional Chinese medicine, goji berries are recognized for their hydrating effect and are considered suitable to address oral dryness associated with Yin deficiency. In the present study, we used goji berry juice (GBJ) to investigate the potential preventive effect of goji berries on oral dryness caused by SS. Pretreatment of human salivary gland cells with GBJ effectively prevented the decrease in aquaporin-5 (AQP-5) mRNA and protein levels induced by TNF-α. GBJ also inhibited histone H4 deacetylation and suppressed the generation of intracellular reactive oxygen species (ROS). Furthermore, GBJ pretreatment reserved mitochondrial membrane potential and suppressed the upregulation of Bax and caspase-3, indicating that GBJ exerted an antiapoptotic effect. These findings suggest that GBJ provides protection against TNF-α in human salivary gland cells and prevents the reduction of AQP-5 expression on the cell membrane. Altogether, these results highlight the potential role of GBJ in preventing oral dryness caused by SS.

Distributions and expressions of Aquaporin-5 and 7 in the testes of developing male chicks.

Aquaporins (AQPs) are integral membrane proteins that act as water channels for which a total of 13 orthologs of AQP genes in birds have been reported. Tissue expression and cellular or subcellular localization of AQPs have been poorly investigated in the male reproductive system of birds. We aimed to determine the distribution and localization of AQP5 and AQP7 proteins by immunocytochemistry in testicular tissues obtained from developing chicks (14, 21, 28, 35 and 42 days old). Totally 175 male chicks (Ross 308) were used in the study from which testicular tissue was removed, fixed in 10% formaldehyde solution, then embedded in paraffin blocks. Five µm sections were cut, mounted on poly-L-lysine slides, dried in an oven, then dehydrated using standard immunohistochemistry staining protocol. The sections were imaged with a Nikon Eclipse 50i trinocular light microscope. Immunohistochemical evaluation of the immune reactivity of AQP5 revealed a positive immune reaction in spermatocytes and interstitial areas of the testes in 14-day-old chicks. Testicular tissue AQP5 immune reactivity was observed in the tubule and the interstitial regions of 21-, 28-, 35- and 42-day-old chicks. AQP7 immune reactions were determined in the tubule and interstitial areas testes of developing chicks' testis tissue, with increasing positivity corresponding to older age. The expression of AQP5 and AQP7 appears to be species-specific due to differences in localization and expression in male chicks compared with studies of other mammals, which is likely to play an important role in regulating fluid and sperm volume. This research can serve as a base for future studies that will contribute to the understanding of the male genital system of AQPs.

Involvement of aquaporin 5 and Na-K-2Cl cotransporter 1 in the pathogenesis of primary focal hyperhidrosis: evidence from the primary sweat gland cell culture.

People with primary focal hyperhidrosis (PFH) usually have an overactive sympathetic nervous system, which can activate the sweat glands through the chemical messenger of acetylcholine. The role of aquaporin 5 (AQP5) and Na-K-2Cl cotransporter 1 (NKCC1) in PFH is still unknown. The relative mRNA and protein levels of AQP5 and NKCC1 in the sweat gland tissues of three subtypes of patients with PFH (primary palmar hyperhidrosis, PPH; primary axillary hyperhidrosis, PAH; and primary craniofacial hyperhidrosis, PCH) were detected with real-time PCR (qPCR) and Western blot. Primary sweat gland cells from healthy controls (NPFH-SG) were incubated with different concentrations of acetylcholine, and the relative mRNA and protein expression of AQP5 and NKCC1 were also detected. NPFH-SG cells were also transfected with si-AQP5 or shNKCC1, and acetylcholine stimulation-induced calcium transients were assayed with Fluo-3 AM calcium assay. Upregulated AQP5 and NKCC1 expression were observed in sweat gland tissues, and AQP5 demonstrated a positive Pearson correlation with NKCC1 in patients with PPH (r = 0.66, P < 0.001), patients with PAH (r = 0.71, P < 0.001), and patients with PCH (r = 0.62, P < 0.001). Upregulated AQP5 and NKCC1 expression were also detected in primary sweat gland cells derived from three subtypes of patients with PFH when compared with primary sweat gland cells derived from healthy control. Acetylcholine stimulation could induce the upregulated AQP5 and NKCC1 expression in NPFH-SG cells, and AQP5 or NKCC1 inhibitions attenuated the calcium transients induced by acetylcholine stimulation in NPFH-SG cells. The dependence of ACh-stimulated calcium transients on AQP5 and NKCC1 expression may be involved in the development of PFH.NEW & NOTEWORTHY The dependence of ACh-stimulated calcium transients on AQP5 and Na-K-2Cl cotransporter 1 (NKCC1) expression may be involved in the development of primary focal hyperhidrosis (PFH).

Effect of Infliximab on Radiation-Induced Submandibular Gland Dysfunction in Rats.

Inflammatory response is one of the essential parts of various pathogenic mechanisms of radiation-induced salivary dysfunction. The effect of decreasing the levels of inflammatory cytokines on alleviating submandibular gland injuries after irradiation is unclear. This study aimed to explore the effect of the antibody against tumor necrosis factor-alpha, infliximab, on radiation-induced submandibular gland dysfunction in rats. Male Wistar rats received a single 20 Gy dose to the right submandibular gland region or sham irradiated. Meanwhile, the irradiated group was divided into infliximab treatment groups or untreated groups. Animals were euthanized at 1, 6, and 12 weeks postirradiation, and the irradiated submandibular gland was dissected for subsequent detection. Submandibular gland exposure caused obvious pathological changes. The increased levels of inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1ß, and interleukin-6, represent an aggravated inflammatory response. The results of the western blot, reverse transcription-quantitative polymerase chain reaction, and immunofluorescence staining showed upregulated levels of claudin-1, claudin-3, and aquaporin 5 and downregulated levels of claudin-4. Moreover, nuclear factor kappa-B phosphorylation levels were also up-regulated. In subsequent experiments, we found that infliximab alleviated inflammatory response, up-regulated tumor necrosis factor-alpha, interleukin-1ß, and interleukin-6 levels, and improved claudin-1, claudin-3, claudin-4, and aquaporin 5 expression. Our results indicate that infliximab might improve the para-cellular pathway and trans-cellular pathway destruction by reducing the inflammatory.

Acupuncture promotes tear secretion by up-regulating VIP/cAMP/PKA/AQP5 signaling in guinea pigs with aqueous tear deficiency dry eye. / 基于VIP/cAMP/PKA/AQP5信号通路探讨针刺治疗水液缺乏型干眼的作用机制.

OBJECTIVES: To observe the effect of acupuncture on the ocular surface symptoms and the protein expression of vasoactive intestinal peptide (VIP) / cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) / aquaporin 5(AQP5) signaling pathway in lacrimal gland tissue of aqueous tear deficiency (ATD) type dry eye model, so as to investigate its mechanism underlying improvement of ATD. METHODS: British shorthair guinea pigs were randomly divided into blank control, model, acupuncture, sham-acupuncture and medication group, with 8 guinea pigs in each group. The ATD model was established by subcutaneous injection of scopolamine hydrobromide (0.6 mg/dose, 4 times/d for 10 days). For guinea pigs of the acupuncture group, filiform needles were inserted into bilateral "Jingming"(BL1), "Cuanzhu"(BL2), "Sizhukong"(TE23), "Taiyang"(EX-HN5), and "Tongziliao"(GB1) for 15 min. For guinea pigs of the sham-acupuncture group, a blunt filiform needle was used to repeatedly prick (not pierce) the skin of the same acupoints mentioned above. The treatment in the above two groups was conducted once daily for 14 days. The guinea pigs in the medication group received administration of sodium hyaluronate eye drops in both eyes, three times a day for 14 days. The objective tests of tear film break-up time (BUT), corneal fluorescein staining score (FLS) and phenol red thread (PRT) test were conducted before and after modeling and after the intervention. After the intervention, the lacrimal index (weight of lacrimal gland/body weight) was calculated. Histopathological changes of the lacrimal gland were observed after H.E. staining. The expression of AQP5 in the lacrimal gland were detected by immunofluorescence, and the contents of VIP and AQP5 in the lacrimal gland were measured by ELISA, the protein expression levels of VIP, cAMP, PKA, p-PKA and AQP5 in the lacrimal gland were detected by Western blot. RESULTS: In comparison with the blank control group, the PRT, BUT, lacrimal index, AQP5 immunoactivity, contents of VIP and AQP5, and protein expression levels of VIP, cAMP, PKA, p-PKA and AQP5 were significantly decreased(P<0.01, P<0.05), and FLS was obviously increased (P<0.01) in the model group . Compared to the model group, the PRT, BUT, lacrimal index, AQP5 immunoactivity, contents of VIP and AQP5, and expression levels of VIP and AQP5 in both acupuncture and medication groups, and the expression levels of cAMP, PKA, p-PKA in the acupuncture group were considerably increased (P<0.01, P<0.05), while the FLS was markedly decreased in both acupuncture and medication groups (P<0.01, P<0.05). Compared with the medication group, the acupuncture group had increased PRT (P<0.05). CONCLUSIONS: Acupuncture intervention is effective in reducing ocular surface damage and promoting tear secretion in guinea pigs with ATD, which may be related to its function in activating VIP/cAMP/PKA signaling, and promoting the expression of AQP5 in the lacrimal gland.

Regulation of Axon Guidance by Slit2 and Netrin-1 Signaling in the Lacrimal Gland of Aqp5 Knockout Mice.

Purpose: Dry eye disease (DED) is multifactorial and associated with nerve abnormalities. We explored an Aquaporin 5 (AQP5)-deficiency-induced JunB activation mechanism, which causes abnormal lacrimal gland (LG) nerve distribution through Slit2 upregulation and Netrin-1 repression. Methods: Aqp5 knockout (Aqp5-/-) and wild-type (Aqp5+/+) mice were studied. LGs were permeabilized and stained with neuronal class III ß-tubulin, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). Whole-mount images were acquired through tissue clearing and 3D fluorescence imaging. Mouse primary trigeminal ganglion (TG) neurons were treated with LG extracts and Netrin-1/Slit2 neutralizing antibody. Transcription factor (TF) prediction and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) experiments verified the JunB binding and regulatory effect on Netrin-1 and Slit2. Results: Three-dimensional tissue and section immunofluorescence showed reduced LG nerves in Aqp5-/- mice, with sympathetic and sensory nerves significantly decreased. Netrin-1 was reduced and Slit2 increased in Aqp5-/- mice LGs. Aqp5+/+ mice LG tissue extracts (TEs) promoted Aqp5-/- TG neurons axon growth, but Netrin-1 neutralizing antibody (NAb) could inhibit that promotion. Aqp5-/- mice LG TEs inhibited Aqp5+/+ TG axon growth, but Slit2 NAb alleviated that inhibition. Furthermore, JunB, a Netrin-1 and Slit2 TF, could bind them and regulate their expression. SR11302, meanwhile, reversed the Netrin-1 and Slit2 shifts caused by AQP5 deficiency. Conclusions: AQP5 deficiency causes LG nerve abnormalities. Persistent JunB activation, the common denominator for Netrin-1 suppression and Slit2 induction, was found in Aqp5-/- mice LG epithelial cells. This affected sensory and sympathetic nerve fibers' distribution in LGs. Our findings provide insights into preventing, reversing, and treating DED.

Ablation of AQP5 gene in mice leads to olfactory dysfunction caused by hyposecretion of Bowman's gland.

Smell detection depends on nasal airflow, which can make absorption of odors to the olfactory epithelium by diffusion through the mucus layer. The odors then act on the chemo-sensitive epithelium of olfactory sensory neurons (OSNs). Therefore, any pathological changes in the olfactory area, for instance, dry nose caused by Sjögren's Syndrome (SS) may interfere with olfactory function. SS is an autoimmune disease in which aquaporin (AQP) 5 autoantibodies have been detected in the serum. However, the expression of AQP5 in olfactory mucosa and its function in olfaction is still unknown. Based on the study of the expression characteristics of AQP5 protein in the nasal mucosa, the olfaction dysfunction in AQP5 knockout (KO) mice was found by olfactory behavior analysis, which was accompanied by reduced secretion volume of Bowman's gland by using in vitro secretion measure system, and the change of acid mucin in nasal mucus layer was identified. By excluding the possibility that olfactory disturbance was caused by changes in OSNs, the result indicated that AQP5 contributes to olfactory functions by regulating the volume and composition of OE mucus layer, which is the medium for the dissolution of odor molecules. Our results indicate that AQP5 can affect the olfactory functions by regulating the water supply of BGs and the mucus layer upper the OE that can explain the olfactory loss in the patients of SS, and AQP5 KO mice might be used as an ideal model to study the olfactory dysfunction.

TGF-ß1 Triggers Salivary Hypofunction via Attenuating Protein Secretion and AQP5 Expression in Human Submandibular Gland Cells.

Aging-related salivary gland degeneration usually causes poor oral health. Periductal fibrosis frequently occurs in the submandibular gland of the elderly. Transforming growth factor ß1 (TGF-ß1) is the primary driving factor for fibrosis, which exhibits an increase in the fibrotic submandibular gland tissue. This study aimed to investigate the effects of TGF-ß1 on the human submandibular gland (HSG) cell secretory function and its influences on aquaporin 5 (AQP5) expressions and distribution. We found that TGF-ß1 reduces the protein secretion amount of HSG and leads to the abundance alteration of 151 secretory proteins. Data are available via ProteomeXchange with the identifier PXD043185. The majority of HSG secretory proteins (84.11%) could be matched to the human saliva proteome. Meanwhile, TGF-ß1 enhances the expression of COL4A2, COL5A1, COL7A1, COL1A1, COL2A1, and α-SMA, hinting that TGF-ß1 possesses the potential to drive HSG fibrosis-related events. Besides, TGF-ß1 also attenuates the AQP5 expression and its membrane distribution in HSGs. The percentage for TGF-ß1-induced AQP5 reduction (52.28%) is much greater than that of the TGF-ß1-induced secretory protein concentration reduction (16.53%). Taken together, we concluded that TGF-ß1 triggers salivary hypofunction via attenuating protein secretion and AQP5 expression in HSGs, which may be associated with TGF-ß1-driven fibrosis events in HSGs.