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

OBJECTIVE: To observe the effect of methyl eugenol on the expression of aquaporin (AQP) 5 in nasal mucosa of rats with allergic rhinitis and to explore its significance. METHODS: In the study, 128 Wistar rats were randomly divided into normal control group, AR model control group, budesonide positive control group, 80 mg/kg group, 40 mg/kg group, 20 mg/kg group and 10 mg/kg group, and ovalbumin (OVA) was used to establish the model of allergic rhinitis. After successful modeling, castor oil, budesonide and corresponding doses of methyl eugenol were given respectively. After 1, 2 and 4 weeks of administration, the distribution of AQP5 in nasal mucosa was observed by immunohistochemistry. The expression of AQP5 in nasal mucosa of each group was compared by Western blotting. The expression of AQP5 mRNA was compared with real-time PCR. RESULTS: AQP5 was mainly located in the glandular epithelium and ductal epithelial cell membrane and cytoplasm. The expression of AQP5 and AQP5 mRNA in nasal mucosa of the rats in the model control group was lower than that in the normal control group (P<0.05). AQP5 and AQP5 mRNA in nasal mucosa of the rats in each treatment group were higher than those in the model control group in varying degrees. The expression of AQP5 in the budesonide group was not significantly different from that in the normal control group 1, 2 and 4 weeks after drug intervention (P>0.05), but there was significant difference between the budesonide group and the model control group (P<0.05). The expression of AQP5 mRNA in the budesonide group was significantly different from that in the normal control group and the model control group (P<0.05).After 2 weeks of intervention, the expression of AQP5 in each dose group of methyleugenol was not significantly different from that in the budesonide group (P>0.05). After 1 week of intervention, there was no significant difference in AQP5 mRNA between the 20 mg/kg group and the normal control group (P>0.05), but there was significant difference between the 20 mg/kg group and the model control group (P<0.05). CONCLUSION Methyl eugenol may increase the degree of edema of the nasal mucosa by reducing the expression of AQP5 and reduce the secretion of glands, thus alleviating the symptoms of allergic rhinitis, sneezing and runny nose.
Animals ; Aquaporin 5 ; Eugenol/analogs & derivatives* ; Nasal Mucosa ; Rats ; Rats, Wistar ; Rhinitis, Allergic

Xylitol is well-known to have an anti-caries effect by inhibiting the replication of cariogenic bacteria. In addition, xylitol enhances saliva secretion. However, the precise molecular mechanism of xylitol on saliva secretion is yet to be elucidated. Thus, in this study, we aimed to investigate the stimulatory effect of xylitol on saliva secretion and to further evaluate the involvement of xylitol in muscarinic type 3 receptor (M3R) signaling. For determining these effects, we measured the saliva flow rate following xylitol treatment in healthy individuals and patients with dry mouth. We further tested the effects of xylitol on M3R signaling in human salivary gland (HSG) cells using real-time quantitative reverse-transcriptase polymerase chain reaction, immunoblotting, and immunostaining. Xylitol candy significantly increased the salivary flow rate and intracellular calcium release in HSG cells via the M3R signaling pathway. In addition, the expressions of M3R and aquaporin 5 were induced by xylitol treatment. Lastly, we investigated the distribution of M3R and aquaporin 5 in HSG cells. Xylitol was found to activate M3R, thereby inducing increases in Ca²⁺ concentration. Stimulation of the muscarinic receptor induced by xylitol activated the internalization of M3R and subsequent trafficking of aquaporin 5. Taken together, these findings suggest a molecular mechanism for secretory effects of xylitol on salivary epithelial cells.
Aquaporin 5 ; Bacteria ; Calcium ; Calcium Signaling ; Candy ; Epithelial Cells ; Humans ; Immunoblotting ; Mouth ; Polymerase Chain Reaction ; Receptors, Muscarinic ; Saliva ; Salivary Glands ; Xylitol

Muscarinic acetylcholine receptors (mAChRs), including M1-M5 subtypes, are classic receptors in regulating water, ion, and solute transport in salivary gland. Our work focuses on the studies on the expression pattern and function of mAChR in the submandibular gland (SMG), and the underlying mechanism involved in the mAChR-regulated secretion, together with the effect of parasympathectomy on the salivary secretion. Microvascular autotransplantation of SMG into the temporal fossa provides a continuous and endogenous source of fluids, and is currently an effective method for treating severe keratoconjunctivitis sicca. By using RT-PCR, Western blotting, and immunofluorescence, our data demonstrated that the expression of M1 and M3 subtypes were decreased in latent period in rabbit SMG autotransplantation model, whereas carbachol stimulation promoted the salivary secretion, as well as M1 and M3 expressions. By contrast, mAChRs were hypersensitive in epiphora SMGs, whereas atropine gel and botulinum toxin A application significantly inhibited the hypersecretion in both animal models and patients. Furthermore, the possible intracellular signal molecules involved in the mAChR-modulated salivary secretion were explored. Activation of mAChR upregulated the expression of aquaporin 5 (AQP5), the main transporter that mediated water secretion through transcellular pathway, and led to AQP5 trafficking from lipid rafts to non-lipid microdomain. Extracellular signal-regulated kinase 1/2 (ERK1/2) was involved in the mAChR-regulated AQP5 content. mAChR activation also modulated the expression, distribution, and function of tight junction proteins, and increased paracellular permeability. ERK1/2/β-arrestin2/clathrin/ubiquitin signaling pathway was responsible for the mAChR-regulated downregulation of tight junction molecule claudin-4. Cytoskeleton filamentous actin (F-actin) was also involved in the distribution and barrier function of epithelial tight junctions. Besides, endothelial tight junctions were opened by mAChR agonist-evoked salivation in the mice. Furthermore, parasympathetic denervation increased resting salivary secretion in the long terminrats and minipigs. Taken together, our work demonstrated that mAChR regulated saliva secretion via transcellular and paracellular pathways in SMG epithelium as well as tight junction opening in SMG endothelium. Modulation of mAChR might be a promising strategy to ameliorate SMG dysfunction.
Animals ; Aquaporin 5 ; Carbachol ; Humans ; Mice ; Rabbits ; Receptors, Muscarinic ; Salivation ; Submandibular Gland

BACKGROUND AND OBJECTIVES: Salivary hypofunction is one of the common side effects after radioiodine therapy, and its pathophysiology is salivary ductal stenosis resulting from ductal cell injury. This study aimed to develop the functional culture environment of human parotid gland ductal cells in in vitro three-dimensional perfusion culture system. MATERIALS AND METHODS: We compared plastic dish culture method and three-dimensional culture system containing Matrigel and nanofiber. Morphogenesis of reconstituted salivary structures was assessed by histomorphometry. Functional characteristics were assessed by immunohistochemistry and reverse transcription polymerase chain reaction (aquaporin 5, CK7, CK18, connexin 43, and p21). In addition, we designed the media perfusion culture system and identified higher rate of cell proliferation and expression of connexin 43 in perfusion system comparing to dish. RESULTS: Human parotid ductal cells were well proliferated with the ductal cell characters under environment with Matrigel. In the presence of Matrigel, aquaporin 5, CK18 and connexin 43 were more expressed than 2D dish and 3D nanofiber setting. In the media perfusion culture system, ductal cells in 3D culture media showed higher cells count and connexin 43 expression compared to 2D dish. CONCLUSION: This in vitro ductal cell perfusion culture system using Matrigel could be used to study for radioiodine induced sialadenitis model in vivo.
Aquaporin 5 ; Cell Proliferation ; Connexin 43 ; Constriction, Pathologic ; Culture Media ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Methods ; Morphogenesis ; Nanofibers ; Parotid Gland ; Perfusion ; Plastics ; Polymerase Chain Reaction ; Reverse Transcription ; Salivary Ducts ; Salivary Glands ; Sialadenitis ; Thyroid Neoplasms

Alcohol intake is known to affect various organs in the human body, causing reduction of salivation in the oral cavity. Hypo-salivation effect of alcohol is a common feature, but the mechanism in salivary glands is still poorly studied. Therefore, in this study, the changes in salivary secretion and water channel protein (aquaporin5, AQP5) in salivary glands of mice were investigated after ethanol administration. Animals were divided in to 4 groups with the control, 4 g/kg ethanol, 8 g/kg ethanol and 16 g/kg ethanol administration groups. One hour after ethanol administration, saliva was collected from the oral cavity, and the animals were killed and parotid and submandibular glands were extracted to analyze the histopathology, AQP5 immunihistochemistry and AQP5 protein level. According to the results, the salivation rate decreased irrespective of the ethanol dose in mice, and viscosities increased with increase in ethanol dose. However, there were no pathological changes in parotid and submandibular glands due to ethanol administration. Expression of AQP5 in parotid and submandibular glands decreased with increase ethanol administration These results indicate that the reduction of salivary secretion due to acute alcohol intake is closely related to decrease of the water channel protein such as AQP5 in parotid glands and submandibular glands, rather than the damage of salivary glands.
Animals ; Aquaporin 5 ; Eating ; Ethanol ; Human Body ; Mice ; Mouth ; Parotid Gland ; Saliva ; Salivary Glands ; Salivation ; Submandibular Gland ; Viscosity ; Water

BACKGROUND: Aquaporins are water channel proteins that play a major role in the movement of water in various human tissues. Recently, it has been found that aquaporins have influence in the carcinogenesis of human malignancies. We analyzed the prognostic impact of aquaporin 5 (AQP5) in non-small lung cancer (NSCLC). METHODS: Seventy-six cases of NSCLC were studied, including 44 cases of adenocarcinoma (ADC) and 32 cases of squamous cell carcinoma (SQCC). Tissue microarray was constructed and immunohistochemical staining for AQP5 was performed. RESULTS: AQP5 was positive in 59.2% of the total enrolled NSCLCs (63.7% in ADC and 53.1% in SQCC). The difference in expression of AQP5 according to the histologic grade of the tumor was significant (p<.047), but not in a serial order. When ADC and SQCC were separately evaluated, no significant difference was observed according to the histologic grade of the tumor (p=.076 in ADC and p=.631 in SQCC). No difference was observed between AQP5 expression and other demographic data and tumor characteristics. Disease-free survival (DFS) was higher in AQP5 negative cases than positive cases in ADC (p=.047), but no significance was found in SQCC (p=.068). We were unable to find a significance between AQP5 overexpression and overall survival in either ADC (p=.210) or SQCC (p=.533). CONCLUSIONS: AQP5 expression is associated with DFS in ADC of the lung and tumor grade of NSCLC. The present study suggests that AQP5 can be a prognostic factor of NSCLC.
Adenocarcinoma ; Aquaporin 5* ; Aquaporins ; Carcinogenesis ; Carcinoma, Non-Small-Cell Lung* ; Carcinoma, Squamous Cell ; Disease-Free Survival ; Humans ; Lung ; Lung Neoplasms ; Water

OBJECTIVETo observe the effect of Yangfei Ziyin Decoction (YZD) on symptoms, serum levels of TNF-alpha, IL-6, and aquaporin-5 (AQP-5), and pathology of Sj6gren's syndrome (SS) model mice.

METHODSTotally 60 mice were divided into 6 groups according to random digit table, i.e., the model group, the normal control group, the high, medium, low dose YZD groups (administered with YZD at 36.7, 18.4, 9.2 g/kg, 0.2 mL/10 g), the Chinese patent medicine group [CPM, administered with total glucosides of paeony at 0.6 g/kg], 10 mice in each group. All intervention was performed for six successive days in a week, with an interval of one day, a total of 50 days. Body weight, salivary secretion, food and water intake were measured at day10, 20, 30, 40, and 50, respectively. At day 50 blood was collected. Submandibular gland, thymus, and spleen were weighed. Serum levels of TNF-alpha, IL-6, and AQP-5 were detected by ELISA. Pathological changes of submandibular gland were observed. Results Compared with the normal control group, there was no change in water intake of mice in the model group, but with reduced salivary secretion (P < 0.01, P < 0.05). Thymus/spleen/submandibular gland weight and index increased in the model group (P < 0.01, P < 0.05). Compared with the model group at the same time point, salivary secretion increased in the CPM group and 3 YZD groups (P < 0.01 , P < 0.05). Compared with the model group, thymus/spleen/submandibular gland weight and index decreased in the CPM group (P < 0.01, P < 0.05). Thymus/submandibular gland weight and index decreased in the low dose YZD group (P < 0.01, P < 0.05). Thymus/submandibular gland weight and index, and spleen index decreased in high and medium dose YZD groups (P < 0.01 , P < 0.05). Levels of TNF-alpha and IL-6 decreased, but AQP-5 level increased in the CPM group (P < 0.05). AQP-5 level increased in high and medium dose YZD groups (P < 0.01 , P < 0.05). In the model group alveoli and duct of salivary gland were destroyed, alveoli and duct were irregular, epithelial cells were degenerated, necrotic, and desquamated. Mild-to-moderate lymphocytic infiltration occurred around submandibular gland. Pathological changes were alleviated in the CPM group and 3 YZD groups.

CONCLUSIONYZD could improve clinical symptoms, serum levels of TNF-alpha, IL-6, AQP-5, and pathological changes of SS model mice.

Animals ; Aquaporin 5 ; Disease Models, Animal ; Drugs, Chinese Herbal ; therapeutic use ; Glucosides ; Interleukin-6 ; Mice ; Paeonia ; Salivation ; Sjogren's Syndrome ; drug therapy ; Submandibular Gland ; Tumor Necrosis Factor-alpha

OBJECTIVETo explore the molecular mechanism of exocrine immune inflammatory injury of Sjögren's Syndrome and the intervention of Banxia Qinlian Decoction (BQD).

METHODSTotally 18 female NOD mice were randomly divided into the model group, the positive drug group, and the BQD group, 6 in each group. Six female BALB/c mice were recruited as a blank control group. Mice in the blank control group and the model group were gavaged with deionized water at the daily dose of 0.1 mL/10 g body weight. Tripterygium Tablet was administered by gastrogavage to mice in the positive group at the daily dose of 10 mg/kg. BQD was administered by gastrogavage to mice in the BQD group at the daily dose of 60 g crude drugs/kg. After 12 weeks of medication, mice were sacrificed. Their eyeballs were excised and blood collected. Tissues of bilateral parotids and submandibular glands were kept. mRNA transcriptional levels of IL-17, IL-6, type 3 muscarinic acetylcholine receptors (M3R), aquaporin protein-5 (AQP5) were detected by RT-PCR. Expression levels of M3R and AQP5 protein were detected by Western blot. Protein expression levels of IL-17 and IL-6 were detected by ELISA.

RESULTSCompared with the normal group, mRNA transcriptional levels and protein expression levels of IL-17, IL-6, M3R, and AQP5 were significantly up-regulated in the model group (P < 0.01). Compared with the model group, mRNA transcriptional levels and protein expression levels of IL-17, IL-6, M3R, and AQP5 were significantly down-regulated in the positive drug group and the BQD group with statistical difference (P < 0.01, P < 0.05). Compared with the BQD group, mRNA-transcriptional levels of IL-17, IL-6, and M3R, as well as M3R and AQP5 protein expression levels were significantly down-regulated in the positive drug group (all P < 0.01).

CONCLUSIONThe molecular mechanism of BQD in inhibiting SS exocrine neurotoxic injury might be possibly related to regulating Th17/IL-17 immune inflammatory way.

Animals ; Aquaporin 5 ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Interleukin-17 ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred NOD ; Sjogren's Syndrome ; drug therapy ; immunology ; Submandibular Gland ; Th17 Cells ; Up-Regulation

Ischemic stroke results in the diverse phathophysiologies including blood brain barrier (BBB) disruption, brain edema, neuronal cell death, and synaptic loss in brain. Vitamin C has known as the potent anti-oxidant having multiple functions in various organs, as well as in brain. Dehydroascorbic acid (DHA) as the oxidized form of ascorbic acid (AA) acts as a cellular protector against oxidative stress and easily enters into the brain compared to AA. To determine the role of DHA on edema formation, neuronal cell death, and synaptic dysfunction following cerebral ischemia, we investigated the infarct size of ischemic brain tissue and measured the expression of aquaporin 1 (AQP-1) as the water channel protein. We also examined the expression of claudin 5 for confirming the BBB breakdown, and the expression of bcl 2 associated X protein (Bax), caspase-3, inducible nitric oxide synthase (iNOS) for checking the effect of DHA on the neurotoxicity. Finally, we examined postsynaptic density protein-95 (PSD-95) expression to confirm the effect of DHA on synaptic dysfunction following ischemic stroke. Based on our findings, we propose that DHA might alleviate the pathogenesis of ischemic brain injury by attenuating edema, neuronal loss, and by improving synaptic connection.
Aquaporins ; Aquaporin 1 ; Ascorbic Acid ; bcl-2-Associated X Protein ; Blood-Brain Barrier ; Brain ; Brain Edema* ; Brain Injuries ; Brain Ischemia* ; Caspase 3 ; Cell Death ; Claudin-5 ; Dehydroascorbic Acid* ; Edema ; Neurons ; Nitric Oxide Synthase Type II ; Oxidative Stress ; Post-Synaptic Density ; Stroke