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ObjectiveTo investigate the protective effect of Guiqi Baizhu prescription combined with oxaliplatin on the intestinal barrier of tumor-bearing mice with gastric cancer by regulating downstream aquaporin 3 （AQP3） and aquaporin 4 （AQP4） through the vasoactive intestinal peptide （VIP）/cyclic adenosine monophosphate （cAMP）/protein kinase A （PKA） signaling pathway. MethodThe gastric cancer cell lines MFC with a density of 1×107/mL were prepared into cell suspension. The tumor-bearing mouse model of gastric cancer was established by inoculating 0.2 mL cell suspension under the right axilla of mice. After successful modeling， mice were randomly divided into 5 groups， namely， model group， oxaliplatin group （10 mg·kg-1）， and high， medium， and low-dose oxaliplatin + Guiqi Baizhu prescription groups （17.68， 8.84， 4.42 g·kg-1）， with 10 mice in each group， and the remaining 10 mice were set as a blank group. Mice in each group were treated with Chinese medicine， oxaliplatin， or normal saline by gavage or intraperitoneal injection for 14 d. The next day after the last dose， blood was taken from the eyeball to separate serum and take colonic samples. Hematoxylin-eosin （HE） staining was used to observe the changes in tissue morphology. The content of D-lactate acid （D-LA） and diamine oxidase （DAO） in the serum was determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expressions of VIP， cAMP， PKA， AQP3， and AQP4 were detected by Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the blank group， the model group showed edema in the colonic submucosa， disordered arrangement of intestinal glands in the mucosal layer， loss of goblet cells， infiltration of inflammatory cells， and villus shedding. However， there were different degrees of improvement in each administration group. As compared with the blank group， the serum levels of DAO and D-LA in the model group were significantly increased （P<0.01）. As compared with the model group， the levels of DAO and D-LA in the high-dose oxaliplatin + Guiqi Baizhu prescription group and the level of D-LA in the medium-dose oxaliplatin + Guiqi Baizhu prescription group were decreased （P<0.05， P<0.01）. As compared with the oxaliplatin group， the levels of D-LA in the high and medium-dose oxaliplatin + Guiqi Baizhu prescription groups were decreased （P<0.05）， and the levels of DAO and D-LA in other administration groups were decreased as well， but the difference had no statistical significance. As compared with the blank group， the mRNA and protein expression levels of VIP， cAMP， PKA， AQP3， and AQP4 in the model group were significantly decreased （P<0.05， P<0.01）. As compared with the model group， the mRNA and protein expression levels of VIP， cAMP， PKA， AQP3， and AQP4 in each administration group were increased， and those in the high-dose oxaliplatin + Guiqi Baizhu prescription group were significantly increased （P<0.05， P<0.01）， while the protein expression level of cAMP in the medium-dose oxaliplatin + Guiqi Baizhu prescription group were increased （P<0.05）. As compared with the oxaliplatin group， the protein expression levels of cAMP in the high-dose oxaliplatin + Guiqi Baizhu prescription group were increased （P<0.05）， and the mRNA and protein expressions of these indexes in the other groups were also increased but the differences were not statistically significant. ConclusionGuiqi Baizhu prescription combined with oxaliplatin can regulate AQP3 and AQP4 through the VIP/cAMP/PKA signaling pathway to protect the intestinal barrier of tumor-bearing mice with gastric cancer.
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Objective:To investigate the expression difference of miR-3189-3p in renal cancer tissue and adjacent tissue and its effect on the biological function of renal cancer cells.Methods:quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression level of miR-3189-3p in renal cancer tissues and adjacent tissues, renal cancer cell lines (Caki-1, ACHN, A498, OS-RC-2) and normal renal tubular epithelial cells HK-2. miR-NC or miR-3189-3p mimics were transfected into renal cancer cells with the lowest expression of miR-3189-3p, respectively, named miR-NC group and miR-3189-3p group. The effects of miR-3189-3p on the proliferation and invasion of renal cancer cells were detected by CCK-8 method and Transwell migration experiment. miRanda and miRTarBase software was used to predict the downstream gene of miR-3189-3p. The dual luciferase reporter gene experiment was used to verify the downstream gene of miR-3189-3p. qRT-PCR and Western blotting were used to detect the expression of miR-3189-3p downstream gene. Measurement data were expressed as mean ± standard deviation ( ± s), and t-test was used for comparison between groups. Results:The relative expression of miR-3189-3p in renal cancer tissue and paracancerous tissue was 1.97±0.61 and 6.19±0.73, respectively, and the relative expression of miR-3189-3p in renal cancer tissue was lower than that in paracancerous tissue ( P<0.01). The relative expression of miR-3189-3p in renal cancer cell lines was lower than that in HK-2 cells ( P<0.05). The relative expression of miR-3189-3p in OS-RC-2 cells was the lowest ( P<0.01). The relative expression levels of miR-3189-3p in OS-RC-2 cells in the miR-NC group and miR-3189-3p group were 1.01±0.11 and 9.27±1.43, respectively, and the relative expression levels of miR-3189-3p in the miR-NC group significantly lower than the miR-3189-3p group ( P<0.01). Compared with the miR-NC group, the proliferation ability of OS-RC-2 cells with high expression of miR-3189-3p was significantly reduced ( P<0.05). The numbers of penetrating cells in the miR-NC group and miR-3189-3p group were 165.40±17.02 and 41.07±6.36, respectively, and the invasive ability of OS-RC-2 cells in the miR-3189-3p group was significantly reduced ( P<0.01). The target gene of miR-3189-3p is Aquaporin 3 ( AQP3) and miR-3189-3p can target AQP3 mRNA ( P<0.01). Compared with the miR-NC group, the expression of AQP3 gene in the high-expressing miR-3189-3p cells was significantly reduced at both the mRNA level and the protein level ( P<0.01). Conclusions:The expression of miR-3189-3p is down-regulated in renal cell carcinoma. High expression of miR-3189-3p can significantly inhibit the proliferation and invasion of renal cell carcinoma OS-RC-2 cells. The molecular mechanism is that miR-3189-5p targets and inhibits the expression of AQP3 gene.
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OBJECTIVE@#To observe the effect of acupuncture and moxibustion on the water content of stratum corneum (WCSC), expression of serum inflammatory factors and aquaporin 3 (AQP3) in skin, lung and rectum in guinea pigs with eczema of skin damp-heat accumulation, and to explore the possible mechanism of acupuncture and moxibustion for regulating skin barrier function.@*METHODS@#A total of 24 male albino guinea pigs were randomly divided into a blank group (n=6) and a modeling group (n=18). The guinea pigs in the modeling group were induced by 2,4-dinitrochlorobenzene (DNCB) to establish the eczema model of skin damp-heat accumulation. The guinea pigs with successful modeling were further randomly divided into a model group, a medication group and an acupuncture-moxibustion group, 6 guinea pigs in each group. The guinea pigs in the medication group were treated with loratadine tablets (0.8 mg/kg) by gavage, once a day for 7 days; the guinea pigs in the acupuncture-moxibustion group were treated with acupuncture at "Feishu" (BL 13), "Pishu" (BL 20), "Quchi" (LI 11), "Zusanli" (ST 36) and "Xuehai" (SP 10); at the same time, moxibustion was applied at "Feishu" (BL 13) and "Zusanli" (ST 36), moxibustion intervention for 10 min and needle retaining for 15 min at each acupoint, once a day for 7 days. The eczema area and severity index (EASI) score was evaluated before and After intervention, and WCSC and trans-epidermal water loss (TEWL) were measured by skin tester. After intervention, The HE staining was used to observe the changes of skin histomorphology in each group; ELISA was used to measure the contents of serum immunoglobulin E (IgE), interleukin (IL)-4 and IL-17; Western blot was used to measure the protein expression of AQP3 in skin, lung and rectum.@*RESULTS@#Before the intervention, compared with the blank group, the EASI scores and TEWL were increased in the remaining groups (P<0.01), and the WCSC was decreased (P<0.01). After the intervention, compared with the model group, the EASI scores and TEWL were decreased (P<0.05, P<0.01), and WCSC was increased (P<0.01) in the medication group and the acupuncture-moxibustion group. The epidermal structure in the blank group was complete and the fibers in the dermis were arranged orderly; in the model group, epidermal hyperkeratosis, proliferation of granular layer, spinous cell layer and basal layer, and disordered arrangement of dermal fibers and infiltration of inflammatory cells were observed. The morphological performance in the medication group and the acupuncture-moxibustion group was better than that in the model group. Compared with the blank group, the contents of serum IgE and IL-17 were increased (P<0.01), and the content of serum IL-4 and the protein expression of AQP3 in skin, lung and rectum were decreased in the model group (P<0.01, P<0.05). Compared with the model group, the contents of serum IgE and IL-17 were decreased and the contents of serum IL-4 were increased in the medication group and the acupuncture-moxibustion group (P<0.01), and the protein expression of AQP3 in skin, lung and rectum in the acupuncture- moxibustion group were increased (P<0.05). Compared with the medication group, the contents of serum IgE and IL-17 were increased (P<0.01), and the content of serum IL-4 was decreased (P<0.01) in the acupuncture-moxibustion group.@*CONCLUSION@#Acupuncture and moxibustion could improve the epidermal water metabolism and skin tissue morphology in guinea pigs with eczema of skin damp-heat accumulation. Its mechanism may be related to regulating inflammatory factors, up-regulating the expression of AQP3, and then repairing the skin barrier function.
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Humans , Male , Acupuncture Therapy , Eczema/therapy , Hot Temperature , Immunoglobulin E , Interleukin-17 , Interleukin-4 , Moxibustion , WaterABSTRACT
Mannosylerythritol lipids (MELs) are glycolipids and have several pharmacological efficacies. MELs also show skin-moisturizing efficacy through a yet-unknown underlying mechanism. Aquaporin-3 (AQP3) is a membrane protein that contributes to the water homeostasis of the epidermis, and decreased AQP3 expression following ultraviolet (UV)-irradiation of the skin is associated with reduced skin moisture. No previous study has examined whether the skin-moisturizing effect of MELs might act through the modulation of AQP3 expression. Here, we report for the first time that MELs ameliorate the UVA-induced downregulation of AQP3 in cultured human epidermal keratinocytes (HaCaT keratinocytes). Our results revealed that UVA irradiation decreases AQP3 expression at the protein and messenger RNA (mRNA) levels, but that MEL treatment significantly ameliorated these effects. Our mitogen-activated protein kinase inhibitor analysis revealed that phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38, mediates UVA-induced AQP3 downregulation, and that MEL treatment significantly suppressed the UVA-induced phosphorylation of JNK. To explore a possible mechanism, we tested whether MELs could regulate the expression of peroxidase proliferator-activated receptor gamma (PPAR-γ), which acts as a potent transcription factor for AQP3 expression. Interestingly, UVA irradiation significantly inhibited the mRNA expression of PPAR-γ in HaCaT keratinocytes, whereas a JNK inhibitor and MELs significantly rescued this effect. Taken together, these findings suggest that MELs ameliorate UVA-induced AQP3 downregulation in HaCaT keratinocytes by suppressing JNK activation to block the decrease of PPAR-γ. Collectively, our findings suggest that MELs can be used as a potential ingredient that modulates AQP3 expression to improve skin moisturization following UVA irradiation-induced damage.
Subject(s)
Humans , Down-Regulation , Epidermis , Glycolipids , Homeostasis , JNK Mitogen-Activated Protein Kinases , Keratinocytes , Membrane Proteins , Peroxidase , Phosphorylation , Phosphotransferases , PPAR gamma , Protein Kinases , RNA, Messenger , Skin , Transcription Factors , WaterABSTRACT
Objective To investigate the effect and significance of Jiangu granule on the expression of aquaporin 3 (AQP3) in articular cartilage of knee osteoarthritis (OA) rats.Methods Sprague-Dawley (SD) rats were randomly divided into sham operation group, model group, and Jiangu granule treatment group.The treatment group was divided into Jiangu granule high dose, medium dose, and low dose groups, with 12 rats in each group.The osteoarthritis model was made by injecting 4％ papain and 0.03 mol/L Lcysteine mixture into the knee joint.Sham-operated group and model group received normal feeding every day.Treatment group (high, medium, and low dose groups), the next day after the completion of the mod el, were given different dose Jiangu granule by gavage twice a day for 4 weeks.After treatment, the ratswere executed uniformly, using general optical microscope and the pathological section of histology to observe the articular cartilage damage.The expression of AQP3 in articular cartilage of rats was detected by real time polymerase chain reaction (RT-PCR) and Western blot.Results The articular cartilage destruction of Jiangu granule treatment group was significantly less than that of the model group (P ＜ 0.05).The mRNA and protein expression levels of AQP3 in model group was significantly higher than that in the sham operation group (P ＜ 0.05).OA rats with Jiangu granule after 4 weeks treatment, the mRNA and protein expression levels of AQP3 in articular cartilage were decreased significantly.Jiangu granule in each sub group relative to the model group had statistical significance (P ＜ 0.05), and the high dose group was decreased significantly.Conclusions Jiangu granule can reduce the expressions of AQP3 in OA articular cartilage of rats and alleviate the swelling of joints, which may have protective effects on articular cartilage.
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Objective To observe the effect of Yang-warming and Qi-tonifying Recipe (YQP)on aquaporin 3(AQP3) and AQP8 in rats with slow transit constipation,and to explore its therapeutic mechanism. Methods Forty rats were randomly divided into modeling group(N=30)and normal control group(N = 10). After successful modeling by gastric gavage of loperamide,the modeled rats were randomly divided into model group,YQP group and Mosapride group,10 rats in each group,and were separately treated with corresponding medicine for 2 weeks. After treatment, the colonic transit function was measured by carbon propelling test. The protein levels of AQP3 and AQP8 were detected by immunohistochemistry and their mRNA expression levels were detected with real-time fluorescence polymerase chain reaction (qPCR). Results Compared with the normal control group,the propelling rate of carbon particle in the model group was decreased,and the protein and mRNA expression levels of AQP3 and AQP8 were significantly increased(P<0.05 or P<0.01). Compared with the model group,the propelling rate of carbon particle of YQP group and mosapride group was significantly increased, and the protein and mRNA expression levels of AQP3 and AQP8 were significantly decreased (P<0.05),but there was no significant difference between YQP group and mosapride group (P >0.05). Conclusion YQP had therapeutic effects on loperamide-induced constipation through decreasing the expression of AQP3 and AQP8 in the intestine,reducing the reabsorption of intestinal fluid, and increasing the fecal water content.
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Objective To evaluate the effects of extracts of Semen Coicis (ESC) on a BALB/c mouse model of atopic dermatitis (AD),and to explore its potential mechanism.Methods Forty specific pathogen-free (SPF) female BABL/c mice were randomly divided into blank group (8 mice,receiving no treatment) and AD model group (32 mice).The mice in the model group were topically treated with 2,4-dinitrochlorobenzene (DNCB) in acetone/olive oil to establish the mouse model of AD.After modeling,8 mice in the blank group and 8 in the model group were sacrificed immediately.The other 24 mice in the model group were randomly and equally divided into 3 groups:model control group receiving no treatment,ESC group and ESC vehicle group topically treated with ESC and ESC vehicle respectively once every day on the back and aural region of the mice for 28 consecutive days.Changes in skin lesions were observed by naked eyes every day.A thickness tester was used to measure the thickness of skin lesions on the left ear before modeling,at completion of modeling and 12 hours after the final treatment.At 12 hours after the final treatment,the mice in the above 3 groups were sacrificed,and the eyeballs were removed for collecting blood.Then,the sera were isolated,and skin tissue specimens were obtained from the skin lesions on the back.These tissue sections were subjected to hematoxylin and eosin (HE) staining and toluidine blue staining for observing the infiltration of inflammatory cells in skin lesions.An immunohistochemical study was performed to determine the expression of aquaporin 3 (AQP3),Toll-like receptor 2 (TLR2) and TLR4,and enzyme-linked immunosorbent assay (ELISA) to detect the serum levels of IgE,interleukin-4 (IL-4) and interferon-/ (IFN-γ).Results After 28-day treatment,skin lesions were improved in the ESC group.Compared with the model control group,the ESC group showed a significantly lower clinical symptom score (1.50 ± 0.58 vs.2.50 ± 0.58,P ＜ 0.05),decreased lesional thickness on the left ear ([0.31 ± 0.01] mm vs.[0.33 ± 0.01] mm,P ＜ 0.05),and lower number of infiltrating mast cells per high-power field (15.18 ± 1.64 vs.28.94 ± 1.28,P ＜ 0.05).Immunohistochemical findings indicated that the ESC group showed significantly lower expression of AQP3,TLR2 and TLR4 compared with the model control group,and decreased AQP3 expression in the spinous layer.Compared with the model control group,the ESC group showed significantly lower total serum IgE and IL-4 levels,but higher IFN-γ levels (all P ＜ 0.05).Conclusion Topical ESC is effective for the treatment of skin lesions in mouse models of AD,likely by regulating serum levels of IgE,IL-4 and IFN-γ and affecting the expression of AQP3,TLR2 and TLR4.
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To expand the clinical application of gamboges, it is necessary to study crude gamboges' toxicity after oral administration and attenuation mechanism during processing. In this study, crude gamboges' toxicity was judged by multiple assays, including inflammatory mediums [such as nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6)] released by macrophage RAW264.7, and pathological manifestations of rat stomach and duodenal tissues after oral administration with crude and processed gamboges. The attenuation mechanism during processing was studied by detecting AQP3, AQP4 protein and mRNA expression in rat gastric and duodenal tissues using immunohistochemical assay and real-time fluorescent quantitative PCR technique. According to the results, crude gamboges group showed promotion in release of NO, TNF-α and IL-6 by macrophage RAW264.7 in a dose-dependent manner; Compared with crude gamboges group, processed gamboges group showed reduction in release of NO and IL-6, with increase in TNF-α. Crude gamboges could cause rat diarrhea, white blood cells increase, lymphocytes decrease, hyperemia and edema in rat gastric mucosa, duodenal mucosal necrosis and inflammatory cells infiltration. All of these results proved that gamboges had the inflammatory toxicity in gastric and duodenal tissues after oral administration in a dose-dependent manner, which however reduced after processing. In addition to the inflammatory toxicity, the mRNA and protein expressions of aquaporin 3 (AQP3), aquaporin 4 (AQP4) in gastric and duodenal tissues of high-dose crude gamboges group were increased significantly (P<0.05), while the protein and mRNA expressions of AQP3, AQP4 were weakened in processed gamboges group. The results showed that AQP3, AQP4 protein and mRNA expressions were positively correlated with the inflammatory toxicity. In conclusion, down-regulation of AQP3, AQP4 protein and mRNA expressions may be one of attenuation mechanisms in processing gamboges.
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Objective To analyze the roles and mechanisms of lactitol and Bifidobacterium infantis in the treatment of rat constipation and to investigate their effects on aquaporin3 (AQP3) and interstitial cells of Cajal (ICC) in colon tissues.Methods Thirty SD male rats were recruited in this study,6 of which were randomly selected as the control and the rest were given 4 mg/kg.d of loperamide for 5 consecutive days to construct the rat model of constipation.The rats with constipation were randomly divided into four groups including model group,lactitol treatment group,Bifidobacterium infantis treatment group and lactitol in combination with Bifidobacterium infantis treatment group.General indexes including food intake,water intake,body weight,fecal water content and intestinal transit rate of each rat were measured after receiving corresponding treatments for 7 consecutive days.The levels of substance P (SP) and vasoactive intestinal peptide (VIP) in serums samples were detected by ELISA.The expression of protein kinase A (PKA) and neurokinin-1 receptor (NK-1) at mRNA level in colon tissues were detected by real-time polymerase chain reaction (real-time PCR).Western blot assay and real-time PCR analysis were used to detect the expression of AQP3 and c-kit at protein and mRNA levels,respectively.Results Compared with the rats in model group,the levels of fecal water content and intestinal transit rate,the concentrations of SP and VIP in serums samples,the expression of PKA and NK-1 at mRNA level and the expression of AQP3 and c-kit at mRNA and protein levels were significantly increased in rats from the three treatment groups (P＜0.05).The most effective treatment was lactitol in combination with Bifidobacterium infantis,followed by the lactitol treatment and then the Bifidobacterium infantis treatment.Conclusion The combination therapy with lactitol and Bifidobacterium infantis increased the serum levels of SP and VIP in rats with constipation.SP could enhance the contraction of gastrointestinal smooth muscles and improve the intestinal motility by binding to the NK-1 receptor on the membrane of ICC.VIP could promote the absorption of water in intestinal tracts,soften stools and alleviate constipation by upregulating the expression of AQP3 at both protein and mRNA levels via the cyclic adenosine monophosphate-PKA (cAMP-PKA) signaling pathway.
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OBJECTIVE To investigate the protective effect of collagen peptides from walleye pollock skin(CPWPS)in the hydrogen peroxide(H2O2)-induced oxidative stress damage model of the human keratinocyte cell line (HaCaT). METHODS HaCaT cells were pretreated with CPWPS 50,100 and 200 mg · L-1 for 12 h. Then H2O2 was added to the final concentration of 300μmol · L-1. After 12 h,the activity of catalase(CAT),glutathion peroxidase(GSH-Px),total superoxide dismutase(T-SOD)and total antioxidant capacity (T- AOC) were detected by enzymes and biochemical method. The expression of aquaporin3 (AQP3) in HaCaT cells was measured by Western blotting. RESULTS Compared with control group,CPWPS group did not exhibit significant cytotoxicity at a concentration up to 300 mg·L-1. The viability of cells treated with H2O2 300μmol·L-1 for 12 h decreased to 54.0%(P<0.01). The viability of cells pretreated with CPWPS 50,100 and 200 mg · L-1 for 12 h increased to 69.4%,79.4%and 89.1%(P<0.05,P<0.01),respectively. Compared with the model group,the activity of CAT,GSH-Px,T-SOD and T-AOC in HaCaT cells pretreated with CPWPS 50,100 and 200 mg·L-1 for 12 h increased. Compared with model group,the activity of CAT,GSH-Px,T-SOD and T-AOC of CPWPS 200 mg · L-1 pretreatment group increased by 56.90%,48.68%,48.24% and 71.43%(P<0.01),respectively. Western blotting revealed that the expression of AQP3 in H2O2 model group was in?creased relative to that in control group,while the expression of AQP3 in CPWPS pretreatment groups was decreased. The expression of AQP3 in CPWPS 200 mg · L-1 pretreatment group decreased to 67.1%(P<0.01) compared to the H2O2 model group. CONCLUSION CPWPS can protect HaCaT cells against H2O2- induced oxidative stress damage. The cytoprotective effects are associated with enhanced antioxidant capacity and decreased expression of AQP3.
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Objective To investigate the expression of aquaporin-3 (AQP3) in tongue tissues of rats with experimental severe acute pancreatitis (SAP) and regulating effects of emodin. Methods Rat models with SAP were established by injecting 5% sodium taurocholate retrogradely into gallbladder and pancreas. SD male rats were randomly divided into sham-operation group, model group, and emodin group. After model establishment, rats in the emodin group received gavage with emodin 20 mg/kg each day, while rats in the model group and sham-operation group received gavage with normal saline. The mental state, thick greasy tongue fur and mortality of rats were observed every day after model establishment, and 5 days later, protein and genetic expression of AQP3 were detected by Western blot and RT-PCR. Results Compared with the model group, the mortality and the thick greasy tongue fur significantly decreased, the protein and genetic expression of AQP3 significantly decreased in the emodin group (P<0.05). On the 5th day, 11 rats in the model group survived, and 5 rats had thick greasy tongue fur. Compared with the sham-operation group, the protein and genetic expression of AQP3 in the model group were higher (P<0.05). Conclusion Emodin can improve the severity of SAP and decrease the incidence of thick greasy tongue fur significantly by reducing the protein and genetic expression of AQP3.
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Objective To investigate the effects of aquaporin 3 (AQP3) and phospholipase D2 (PLD2) on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431.Methods Three small interfering RNAs (siRNAs) were constructed targeting the AQP3 and PLD2 genes separately,and transfected into A431 cells using liposomes.Then,fluorescence quantitative PCR was performed to find the most efficient siRNAs.Western blot was conducted to detect the protein expression levels of AQP3 and PLD2 in A431 cells after transfection with the selected AQP3-siRNA and PLD2-siRNA.Some A431 cells were divided into five groups:normal control group without any treatment,transfection reagent group treated with the oligofectamine reagent only,negative control group transfected with the negative control siRNA,AQP3-siRNA group transfected with the selected AQP3-siRNA,PLD2-siRNA group transfected with the selected PLD2-siRNA.After additional culture,cell counting kit-8 assay was performed to evaluate the proliferation of A431 cells,flow cytometry to detect the apoptosis of A431 cells after annexin V-fluorescein isocyanate/propidium iodide double-staining.Statistical analysis was carried out by the paired t test.Results The transfection with AQP3-siRNA and PLD2-siRNA induced a significant decrease in the mRNA and protein expressions of AQP3 and PLD2 respectively in A431 cells when compared with the untransfected cells.Compared with the negative control group,the proliferation of A431 cells was significantly decelerated at 24,48 and 72 hours after transfection in the AQP3-siRNA group (t =24.10,11.00,9.54,respectively,all P ＜ 0.01) and PLD2-siRNA group (t =30.47,7.02,8.73,respectively,all P ＜ 0.01).A significant increase was observed in the apoptosis of A431 cells at 48 and 72 hours after transfection with AQP3-siRNA (t =11.36,20.91,respectively,both P ＜ 0.01),and at 72 hours after transfection with PLD2-siRNA (t =4.86,P ＜ 0.05) compared with the negative control group.Conclusion The down-regulation of AQP3 and PLD2 expressions by siRNA can inhibit the proliferation,but induce the apoptosis,of A431 cells.
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Objective To investigate the expression of aquaporins-3 (AQP3) in amniotic epithelial cells regulated by cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signal pathway and to explore the mechanisms of its expression.Methods The amniotic epithelial cells were collected from 30 patients who underwent elective caesarean sections at term with normal amniotic fluid volume and primarily cultured.The cultured cells were treated with (1) forskolin groups: different concentration (0,2.5,5,50 or 100 μmol/L) of forskolin treated cells for 2 hours,and the optimal concentration of forskolin treated cells with different time (0,1,2,10 or 20 hours) ; (2)SP-cAMP groups: different concentration (0,2.5,5,50 or 100 μmol/L) of SP-cAMP treated cells for 2 hours,and the optimal concentration of SP-cAMP treated cells with different time (0,1,2,10 or 20 hours); (3)H-89 groups: different concentration (0,5,10,50 or 100 μmol/L) of H-89 treated cells for 2 hours,and the optimal concentration of H-89 treated cells with different time (0,1,2,10 or 20 hours).The level of intracellular cAMP and activity of PKA were detected by using ELISA,and immunohistochemistry was used to detect the localization of AQP3,the protein expression of total cAMP-response element binding protein (CREB) and phospho-CREB (p-CREB) and AQP3 were assessed by western blot analysis.Cell proliferation was assessed by cell counting kit-8 (CCK-8)assay.Results (1) The brown staining of AQP3 was detected in both cell membrane and cytoplasm in each group.(2) There was no significant change of the cell proliferation rate among groups with different concentration of forskolin,SP-cAMP and H-89 treatment (P ＞ 0.05).(3) After different concentration of forskolin treated 2 hours,the expression of total CREB had no significant difference among them(P ＞ 0.05).While the expression of cAMP level,PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 2.5 μmol/L,5 μmol/L,50 μmol/L forskolin group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 5 μmol/L forskolin group were higher than that in 2.5 μmol/L and 50 μmol/L (P ＜ 0.05).The optimal forskolin concentration was 5 μmol/L.(4) After different concentration of SP-cAMP treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 5 μμmol/L,50 μmol/L SP-cAMP group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 50 μmol/L SP-cAMP group were higher than that in 5 μmol/L (P ＜0.05).The optimal SP-cAMP concentration was 50 μmol/L (5) After different concentration of H-89 treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were lower in 10 μmol/L,50 μmol/L and 100 μmol/L H-89 group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 10 μmol/L H-89 group were lower than that in 50 μmol/L,100 μmol/L (P ＜ 0.05).The optimal H-89 concentration was 10 μmol/L.(6) p-CREB and AQP3 protein expression were significantly lower in 5 μmol/L forskolin combined 10 μmol/L H-89 incubating 2 hours group when compared with 5 μmol/L forskolin,but higher than that in 10 μmol/L H-89 treated group (P ＜ 0.05).Total CREB was no significant difference among the three groups (P ＞ 0.05).Conclusion cAMP-PKA signal transduction pathway may regulate AQP3 protein expression in human amniotic epithelial cells.
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Objective To investigate the effect of compound catechu anti-diarrhea ointment on the expression of aquaporin3 (AQP3) in the colonic tissues of diarrhea rat model, and the mechanism thereof. Methods Forty SD rats were randomly divided into four groups:blank control group, model control group, positive control group and compound catechu anti-diarrhea ointment group. Rats were given 6 g blank matrix cream in model control group, 2 mL suspension of berberine in positive control group and compound catechu anti-diarrhea ointment in compound catechu anti-diarrhea ointment group, two times/d for 7 d. The rat model of diarrhea was established by using senna intragastric administration. The water content of feces was measured. The expression of AQP3 in colonic tissues was detected by immunohistochemistry assay. Results The water contents of feces were significantly higher in model control group (64.09±0.41)%than those of other three groups (F=53.879,P＜0.05). There was no significant difference in the water content of feces between compound catechu anti-diar-rhea ointment group (48.83 ± 1.08)%and positive control group (46.87 ± 2.19)%. The AQP3-positive cells were mainly ex-pressed in the intestinal mucosa. The dyeing index was significantly lower in model control group (0.85±0.18) than that of other three groups (F=14.971,P＜0.05). There was no significant difference in the dyeing index between compound catechu anti-diarrhea ointment group (1.30±0.18) and positive control group (1.37±0.14). Conclusion Compound catechu anti-di-arrhea ointment can significantly reduce the water contents of feces, which may be related to the increased AQP 3 expression in colonic tissues.
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Objective To investigate the expression and distribution of aquaporin 3 (AQP3) in mouse early embryos at different stages.Methods Controlled ovarian hyperstimulation model of Kunming mouse was used to collect four-cell embryos,eight-cell embryos,morula stage,and early blastocysts.Immunofluorescence microscopy and laser confocal microscopy were used to detect expression and distribution of AQP3 channels in these stages.Results Fluorescence signal of AQP3 was found in four embryonic stages of mice.Distribution within embryo was different at different embryonic stages.AQP3 was mainly expressed on the karyotheca of blastomeres at four-cell and eight-cell stage.In morula stage,AQP3 was mainly expressed on cell membrane of each blastomere.In early blastocysts,AQP3 was predominantly expressed on the cell membrane and cytoplasm of trophoblastic cell.Conclusions AQP3 trans-membrane channel might have potential regulation function on mouse embryonic development.
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Objective To evaluate the effect of green tea polyphenol epigallocatechin-3-gallate (EGCG)and ultraviolet B (UVB) on the expression of aquaporin 3 and epidermal growth factor receptor (EGFR)/extracellular signal-regulated protein kinase (ERK) signaling pathway in keratinocytes.Methods Twenty healthy human subjects were enrolled in this study.Both legs of each subjects were separated into 4 areas to remain untreated (control area),be topically treated with 3％ and 1％ EGCG cream and the vehicle of EGCG cream respectively once a day for 2 weeks followed by the measurement of skin moisture content and transepidermal water loss (TEWL).Cultured keratinocytes were classified into various groups to be irradiated with different doses (10,20 and 30 mJ/cm2) of UVB,or be pretreated with different concentrations of EGCG (10-7,10-6,10-5 mol/L) or EGFR/ERK phosphorylation inhibitors for 1 hour followed by irradiation with UVB of 30 mJ/cm2.After various durations of additional culture,Western blot was conducted to quantify the expression of AQP3 and phosphorylated-EGFR (p-EGFR) and-ERK (p-ERK) of keratinocytes.Data were processed by SPSS 10.0 software,and statistical analysis was carried out by t test.Results Skin moisture content was significantly increased,while TEWL was decreased in healthy skin after treatment with 1％ and 3％ EGCG cream compared with vehicle-treated skin areas and untreated skin areas.Increased AQP3 expression was observed in keratinocytes pretreated with EGCG of 10-7,10-6,10-5 mol/L (172.36 ± 12.42,320.66 ± 15.51 and 368.10 ± 11.39 vs.100.00,t =12.16,26.75 and 38.62 respectively,all P ＜ 0.05) and in those pretreated with the EGFR inhibitor PD153035 of 1.0 μmol/L and ERK inhibitor U0126 of 10 μmol/L (413.85 ± 25.27 and 268.85 ± 16.33 vs.100.00,t =35.16,19.25 respectively,both P ＜ 0.05)compared with those irradiated with UVB of 30 mJ/cm2 alone.UVB irradiation stimulated the phosphorylation of EGFR/ERK in keratinocytes,and the stimulation was markedly inhibited by the pre-treatment with EGCG of 10-7,10-6 and 10-5 mol/L (all P ＜ 0.05).Conclusions EGCG can enhance skin barrier function.AQP3 expression is down-regulated by UVB irradiation in keratinoctyes,while EGCG can inhibit the downregulation likely by suppressing the UVB-induced activation of EGFR and ERK.
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Objective To investigate the role of mitogen-activated protein kinases (MAPK)/extracellular signal regulated kinase1/2 (ERK1/2) signal transduction pathway in regulating the expression of aquaporin 3 (AQP3) in human amnion epithelial cells.Methods Primary cell culture of human amnion epithelial cells deriving from amnion of term pregnancy with normal amniotic fluid volume (AFV) or isolated oligohydramnios was conducted in the Second Affiliated Hospital of Wenzhou Medical College from January to November 2011.Either group included 10 elective cesarean cases.The primarily cultured cells were treated with different concentrations (0,5,10,20 and 40 mol/L) of ERK inhibitors (U0126) for 12 h,and then the optimal concentration of U0126 which resulted in the lowest expression of phospho~ERK1/2 (p-ERK1/2) was added for different durations(0,2,6,12 and 24 h).Immunocytochemistry was used to detect the localization of AQP3 and Western blot analysis was used to examine the expression of total ERK1/2,p-ERK1/2 and AQP3 in human amnion epithelial cells.Statistical analysis was performed by t-test and one-way ANOVA.Results (1) Compared with those in normal AFV group,p-ERK1/2 and AQP3 expression in human amnion epithelium cells decreased in oligohydramnios group,respectively (p-ERK1/2:3.46 ± 0.33 and 2.46±0.25;AQP3:2.34 ± 0.18 and 1.56±0.10,t=9.243 and 13.292,P＜0.01).(2) In oligohydramnios groups,after treated with different concentrations of U0126,the expressions of total ERK1/2 did not change (F=0.365,P＞0.05).The expression of p-ERK1/2 and AQP3 in 5,10,20,40 μmol/L U0126 (p-ERK1/2:0.96±0.16,1.12±0.13,0.98±0.17 and 1.02±0.26; AQP3:1.10±0.09,1.12±0.08,1.13±0.06 and 1.11±0.06) were all significantly lower than those in 0 μmol/LU0126 group (p-ERK1/2:2.46±0.25; AQP3:1.56±0.10,P＜0.05).However,the expression of p-ERK1/2 and AQP3 showed no significant difference among 5,10,20,and 40μmol/L U0126 groups (P＞0.05).The optimal concentration of U0126 was 5 μmol/L.After treated with 5 μmol/L U0126 for 2 h,the expressions of p-ERK1/2 and AQP3 (1.27±0.29 and 1.44±0.12)were lower than those after treated for 0 h (2.55±0.12 and 2.15±0.09,P＜0.05).However,there was no significant difference among groups treated for 2,6,12 and 24 h.Therefore,the optimal treatment time was 2 h.(3) The expression of AQP3 was distributed in both cell membrane and cytoplasm in amnion epithelial cells with normal amniotic fluid volume or isolated oligohydramnios,but mainly in cytoplasm.U0126 did not change the localization of AQP3 expression.Conclusions U0126 inhibits the phosphorylation of ERK1/2 and expression of AQP3 of women with oligohydramnios,indicating that the MAPK/ERK1/2 signal transduction pathway might regulate the expression of AQP3 in human amnion epithelial cells,and therefore affect the balance of amniotic fluid volume.
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PURPOSE: Aquaporin (AQP), a protein located in the cellular membrane, allows rapid passage of water across the cell membrane. Various AQP subtypes have been associated with ureteral obstruction. In particular, AQP3 has two functions: water and glycerol transport. The aim of this study was to investigate the expression of AQP3 in the ipsilateral rat kidney in unilateral partial ureteral obstruction (UPUO). MATERIALS AND METHODS: Sprague-Dawley rats (n=30, 200-250 g) were divided into two groups. A sham operation was performed in the control group (n=10) and UPUO of the left upper ureter with a silicone tube was induced in the UPUO group (n=20). The left kidney was obtained from both groups 7 days after the operations. The kidney specimens underwent immunofluorescent staining with AQP3 monoclonal antibody, and the density of AQP3 in the tissue was measured with an image analyzer. RESULTS: In the UPUO group, thinning of the epithelial layer and infiltration of inflammatory cells was seen along with the localized expression of AQP3 in the basolateral aspect of the principal collecting duct cells. The mean optical density of AQP3 was significantly lower in the UPUO group than in the control group (100.9+/-17.5 compared with 131.7+/-16.9; p<0.001). CONCLUSIONS: These results suggest that a decrease in the expression of AQP3 may be the result of a urinary stasis reaction caused by UPUO in response to local and intrarenal factors. These changes suggest that AQP3 may have a pathophysiological role in UPUO.
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Animals , Rats , Aquaporin 3 , Cell Membrane , Glycerol , Kidney , Membranes , Rats, Sprague-Dawley , Salicylamides , Silicones , Ureter , Ureteral ObstructionABSTRACT
ObjectiveTo study the roles of aquaporin 3(AQP3),Caspase 14 and bleomycin hydrolase(BH) in skin barrier in patients with chronic eczema and to tackle the mechanisms underlying the therapeutic effect of narrow-band ultraviolet B(NB-UVB) on eczema.MethodsTransepidermal water loss (TEWL)and water content of skin were detected by a muhi-functional skin tester in lesions and non-lesional skin of 30 patients with chronic eczema.The protein expressions of AQP3,Caspase 14 and BH were detected by immunohistochemistry in tissue specimens from lesioual and non-lesional skin of these patients.Cultured keratinocytes were irradiated with 3 different doses(400,800,1200 mJ/cm2) of UVB,and Western blot was performed to quantify the protein expressions of AQP3,Caspase 14 and BH at 24 hours after the irradiation.ResultsThere was a significant decrease in water content of skin(11.32 ± 5.25 vs.49.68 ± 8.62) but an increase in TEWL (86.28 ± 16.35 vs.14.62 ± 6.68) in lesional skin compared with non-lesional skin from the patients with chronic eczema(both P ＜ 0.05).The protein expression levels of AQP3,Caspase 14 and BH in lesional skin were 5.3 times,4.2 times and one-third of,those in non-lesional skin from the patients,respectively,and significant differences existed between the lesional and non-lesional skin in the 3 parameters (all P ＜ 0.05 ).After irradiation with NB-UVB at 400,800 and 1200 mJ/cm2,the expression level of AQP3 in keratinocytes was decreased by 52％,77％ and 84％,respectively,and that of Caspase 14 was decreased by 18％,22％ and 78％,respectively,compared with unirradiated keratinocytes (all P ＜ 0.05).A marked decrease(by 66％) was also observed in the expression of Caspase 14 in keratinocytes after irradiation with NB-UVB at 1200 mJ/cm2(P ＜ 0.05).ConclusionsThe impairment of skin barrier function in chronic eczema may be related to the downregulated expression of BH and upregulated expression of Caspase 14 and AQP3.The therapeutic effect of NB-UVB on chronic eczema may be partly attributed to the diminished expression of AQP3,Caspase 14 and BH.
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Vitiligo is an acquired depigmentary disorder of the skin that results from the loss of functioning epidermal melanocytes. Most studies on vitiligo have concentrated on the abnormality of melanocytes rather than the abnormality of keratinocytes; however, epidermal melanocytes form a functional and structural unit with neighboring keratinocytes. In fact, direct cell-to cell contact stimulates in vitro proliferation of melanocytes, and growth factors produced by adjacent keratinocytes regulate the proliferation and differentiation of melanocytes. The potential role of keratinocyte-derived cytokines has also been presented. We focused on the structural changes in vitiliginous keratinocytes, which may result in loss of melanocytes, to examine the pathomechanism of vitiligo. The results of a comparison between depigmented and normally pigmented epidermis in patients with vitiligo showed that the keratinocytes in the depigmented epidermis were more vulnerable to apoptosis. Impaired Phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase (Akt) activation followed by reduced nuclear factor-kappaB activation under increased tumor necrosis factor-alpha levels was demonstrated as a mechanism for keratinocyte apoptosis. The role of aquaporin 3 in keratinocyte apoptosis was addressed based on the relationship between the PI3K/AKT pathway and the E-cadherin-catenin complex. Apoptotic keratinocytes induced a lower expression of keratinocyte-derived factors, including stem cell factor, in depigmented epidermis, resulting in passive melanocyte death.