AQP1在人类胎盘、胎膜中的分布及表达

目的本文通过研究水通道蛋白1(aquaporin 1,AQP1)在人类胎盘、胎膜中的分布及表达,初步探讨AQP1在人类羊水循环中的作用。方法采取免疫组织化学SABC法检测15例羊水量正常孕妇,9例羊水过少孕妇,11例羊水过多孕妇胎盘、胎膜组织中AQP1的分布及表达强度。结果AQP1主要表达在人类胎盘及胎膜的羊膜上皮细胞的细胞膜上,AQP1在羊水量正常、羊水过少及羊水过多的分布及表达强度差异无显著性(P>0.05)。结论提示AQP1在人类胎盘、胎膜中的表达表明AQP1可能参与介导胎儿和母体的体液交换,推测羊水量异常的发病机制中,AQP1的结构改变可能比其表达量的改变更为重要,具体机制尚需进一步研究。

Aquaporin1～9 mRNA在成人大肠黏膜中的表达

明确Aquaporin基因各亚型(aqps)mRNA在成人大肠黏膜的表达,探讨aqps在正常大肠生理中的作用.以RT-PCR方法分别检测左半结肠、右半结肠黏膜各10例标本中1～9共9个亚型mRNA的表达情况.结果示aqp6在左、右半结肠均未见有表达,1、2、3、4、5、7、8、9 mRNA在左右半结肠黏膜均有表达,aqp9在左半结肠表达更丰富.结论:成人大肠黏膜中多种水通道蛋白的表达提示aqps尤其aqp9与大肠的水分吸收、黏液分泌等有密切关系.

Comment on: Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in the gastrointestinal system

TO THE EDITOR Sir, I read with great interest the recently published article in the World Journal of Gastroenterology by Jin and co-workers on the cloning and characterization of porcine aquaporin 1 water channel from the pig liver and studies on its expression in the porcine gastrointestinal system. The authors should be congratulated for making this important and valuable contribution to the field of aquaporin biology and porcine gastrointestinal physiology. However, there are a number of unresolved issues and controversies concerning the expression of aquaporins （especially aquaporin 1） in the gastrointestinal system that are worthy of additional comment and discussion by Jin and co-workers.

GST Fusion Protein Based Specific Polyclonal Antibody Preparation of Mouse Aquaporin 1

Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.

Aquaporin 1 Facilitated Hepatocellular Carcinoma SMMC7221 Cell Migration Associated with Water Permeability

The authors investigated the regulation of human aquaporin I（hAQP1） and the involvement of aquaporin 1 （AQP 1） in the migration of human hepatocellular carcinoma SMMC-7221 cells using RNA intereference technology Firstly, two short hairpin RNA（shRNA） constructs in PBSU6 vector were reconstructed and their knockdown effects were identified in SMMC-7221 cells. Next, the involvement of endogenous hAQP1 in regulating the migration of SMMC-7221 cells was investigated via siRNA technology. HAQPI-shRNA can specifically inhibit AQP1 dependent osmotic water permeability. Meanwhile the migration of SMMC-7221 cells was inhibited remarkably after silencing AQP1 by performing transwell cell migration assay and in vitro wound healing assay. Furthermore, in the presence of an inhibitor HgCl2, the water permeability of the cell membrane was remarkably decreased, the expression of AQP1 was upregulated after HgCla treatment and the cell movement was decreased at the moment. Increased AQP1 cannot attenuate cell migration ability when cell membrane loses its water permeability function. This demonstrates that the cell migration was remarkably related to the transporting water function of cell membrane.

Expression and role of AQP1 in cervical squamous carcinoma and its precancerous lesions

Objective： To investigate the expression of aquaporin 1 in cervical squamous carcinomas （CSC） and cervical precancerous lesions, and the relationship between the tumor clinicopathological parameters, prognosis and the expression of AQP1. Methods： Immunohistochemical method （EliVision） was used to detect the expression of AQP1 in samples from 106 patients [20 with normal cervical tissue, 30 with cervical intraepithelial neoplasia （stage Ⅰ and Ⅱ） and 56 with CSC]. Survival analysis was performed by Kaplan-Meier method. Results： AQP1 protein was expressed in vascular endothelia of all samples. It showed upregulation of AQP1 expression in CSC. There was a significant difference between CSC and normal cervical tissues （P〈0.05）. AQP1 was expressed in some tumor cells and unexpressed in normal squamous epithelial cells. And APQl-expressing tumor cells were positively related to lymph node metastasis. Patients with APQl-expressing tumor cells had the lower survival rate than the ones without. Conclusion： Abnormal expression of AQP1 plays an important role in the development of CSC. Positive expression of AQP1 in tumor cells maybe enhances tumor metastasis and could be used as a marker for tumor prognosis.

Downregulation of Aquaporin 4 Expression through Extracellular Signal-regulated Kinases1/2 Activation in Cultured Astrocytes Following Scratch-injury

Objective To investigate the role of extracellular signal-regulated kinase1/2（ERK1/2） pathway in the regulation of aquaporin 4（AQP4） expression in cultured astrocytes after scratch-injury. Methods The scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase（LDH） leakages were observed to assess the degree of scratch-injury. AQP4 expression was detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2（p-ERK1/2） expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 μmol/L U0126（ERK1/2 inhibitor） was incubated in the medium at 30 min before the scratch-injury in some groups. Results Increases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expression was associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury. Conclusion These results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

Correlation between the Expression of Aquaporin 1 and Hypoxia-inducible Factor 1 in Breast Cancer Tissues

The correlation between aquaporin 1 （AQP1） and hypoxia-inducible factor 1 （HIF 1） in breast cancer tissues was preliminarily studied. In 155 cases of breast cancer, the expression levels of AQP1 were detected by immunohistochemisty in HIFl-positive group or HIFl-negative group, and the correlation between AQP1 and HIF1 was analyzed. The overexpression of AQP1 and HIF1 were observed in 155 cases of breast cancer tissues. The expression level of AQP1 in HIFl-positive group was significantly higher than that in HIFl-negative group. The positive expression rate of AQP1 was 296.55±24.67 and 168.37±37.53 in HIFl-positive group and HIFl-negative group respectively with the difference being very significant between them （P〈0.001）. It was concluded that AQP1 was overexpressed in the HIFl-positive group and there were some correlations between AQP1 and HIF1, suggesting they interact each other and regulate the oncogenesis of breast cancer.

6-苄氨基嘌呤防治急性高原低氧大鼠心肌水肿的实验研究

目的探讨6-苄氨基嘌呤(6-BA)防治高原低氧大鼠心肌水肿的效果及可能机制。方法雄性SD大鼠54只,使用随机数字表法分为常压常氧对照组、高原低氧组、6-BA干预组,每组各18只。应用低氧实验舱模拟海拔7000 m高原环境。6-BA组应用100(mg·d)/kg溶液灌胃,连续灌胃1周。对照组、低氧组应用同体积去离子水灌胃。观察各组大鼠心肌组织病理变化,测定心肌组织含水量,检测心肌组织水通道蛋白1(AQP1)mRNA和miR-144-3p表达。结果与对照组比较,低氧组和6-BA组大鼠心肌组织含水量显著升高(P<0.05)。病理分析显示,低氧组大鼠心肌细胞肿胀、肌浆凝聚、横纹不清。6-BA组大鼠可见个别心肌细胞肿胀。与对照组比较,低氧组大鼠心肌组织AQP1 mRNA和miR-144-3p表达显著升高(P<0.05),6-BA组大鼠心肌组织AQP1mRNA和miR-144-3p表达无显著变化(P>0.05)。与低氧组比较,6-BA组大鼠心肌组织AQP1mRNA和miR-144-3p表达显著降低(P<0.01)。结论6-BA通过下调高原低氧大鼠心肌组织AQP1和miR-144-3p表达,减轻心肌水肿,具有心脏保护作用。

Aquaporin 1 overexpression may enhance glioma tumorigenesis by interacting with the transcriptional regulation networks of Foxo4,Maz,and E2F families

Background The glioblastoma has served as a valuable experimental model system for investigating the growth and invasive properties of glioblastoma.Aquaporin-1(AQP1)in facilitating cell migration and potentially contributing to tumor progression.In this study,we analyzed the role of AQP1 overexpression in glioblastoma and elucidated the main mechanisms involved.Methods AQP1 overexpression recombinant vector was introduced into C6 rat glioma cells to construct an AQP1 overexpression C6 cell line,and its effect on cell viability and migration ability was detected by MTT and Transwell.RNA was extracted by Trizol method for gene sequencing and transcriptomics analysis,and the differentially expressed genes(DEGs)were enriched for up-and downregulated genes by Principal component analysis(PCA),and the molecular mechanism of AQP1 overexpression was analyzed in comparison with the control group using the NCBI GEO database.Statistical analysis was performed using Mann-Whitney paired two tailed t test.Results The cell viability of AQP1-transfected cell lines increased by 23%and the mean distance traveled increased by 67%compared with the control group.Quantitative analysis of gene expression showed that there were 12,121 genes with an average transcripts per million(TPM)value greater than 1.DEGs accounted for 13%of the genes expressed,with the highest correlation with upregulated genes being FOXO4 and MAZ,and the highest with down-regulated genes being E2F TFs.Conclusions AQP1 may be implicated in glioma formation by interacting with the transcriptional regulation networks involving the FOXO4,MAZ,and E2F1/2.These findings shed light on the potential significance of AQP1 in glioma pathogenesis and warrant further investigations to unravel the underlying molecular mechanisms.

Shen Qi Wan attenuates renal interstitial fibrosis through upregulating AQP1

Renal interstitial fibrosis(RIF)is the crucial pathway in chronic kidney disease(CKD)leading to the end-stage renal failure.However,the underlying mechanism of Shen Qi Wan(SQW)on RIF is not fully understood.In the current study,we investigated the role of Aquaporin 1(AQP1)in SQW on tubular epithelial-to-mesenchymal transition(EMT).A RIF mouse model induced by adenine and a TGF-β1-stimulated HK-2 cell model were etablished to explore the involvement of AQP 1 in the protective effect of SQW on EMT in vitro and in vivo.Subsequently,the molecular mechanism of SQW on EMT was explored in HK-2 cells with AQP1 knockdown.The results indicated that SQW alleviated kidney injury and renal collagen deposition in the kidneys of mice induced by adenine,increased the protein expression of E-cadherin and AQP1 expression,and decreased the expression of vimentin andα-smooth muscle actin(α-SMA).Similarly,treatmement with SQW-containing serum significantly halted EMT process in TGF-β1 stimulated HK-2 cells.The expression of snail and slug was significantly upregulated in HK-2 cells after knockdown of AQP1.AQP1 knockdown also increased the mRNA expression of vimentin andα-SMA,and decreased the expression of E-cadherin.The protein expression of vimentin increased,while the expression of E-cadherin and CK-18 significantly decreased after AQP1 knockdown in HK-2 cells.These results revealed that AQP1 knockdown promoted EMT.Furthermore,AQP1 knockdown abolished the protective effect of SQW-containing serum on EMT in HK-2 cells.In sum,SQW attentuates EMT process in RIF through upregulation of the expression of AQP1.

Decreased expression of AQP1 and AQP5 in acute injured lungs in rats

OBJECTIVE: To determine if aquaporin1 (AQP1) and aquaporin5 (AQP5) are expressed in the alveolar capillary membrane in rats. Moreover, to investigate the alteration of AQP1 and AQP5 in acute injured lungs. METHODS: The distribution of AQP1 and AQP5 in alveolar capillary membrane were investigated by immunohistochemistry and immunoelectron microscopy with affinity-purified antibodies to human AQP1 and AQP5. To study the possibility that alveolar capillary membrane AQP1 and AQP5 undergo altered regulation, we established a rat model using alveolar instillation of lipopolysaccharide (LPS). RESULTS: Immunolabelling showed AQP1 was stained primarily in the microvascular endotheli a of normal lungs, while AQP5 was expressed in type I pneumocytes. Immunohisto chemical analysis showed a significant decrease in the expression of AQP1 and AQP5 in injured lungs at 4h-48h after LPS instillation. AQP1 protein was resumed partly at 24h after LPS instillation and steroid administration, whereas AQP5 was unchanged. CONCLUSION: The decreased expressions of AQP1 and AQP5 in injured lungs suggest that both of them may play a role in abnormal fluid transportation.

半滑舌鳎两种Aquaporin1同源基因的克隆与表达分析

克隆了半滑舌鳎(Cynoglossus semilaevis)AQP1o和AQP1基因的全长cDNA序列,并对其序列和表达模式进行了分析.结果表明,AQP1ocDNA全长为993 bp,包括120 bp的5′非翻译区,789 bp的开放阅读框,84bp的3′非翻译区,编码262个氨基酸的蛋白质,分子质量为28.3×103.AQP1的cDNA全长为1 335 bp,包括63bp的5′非翻译区,786 bp的开发阅读框,486 bp的3′非翻译区,编码261个氨基酸的蛋白质,分子质量为27.4×103.聚类分析表明半滑舌鳎AQP1o蛋白与金头鲷和塞内加尔鳎等海洋鱼类在卵巢中特异表达的AQP1o聚为一类,而AQP1与非卵巢特异表达的AQP1聚为一类,表明该两类基因为不同类型的AQP1同源基因.RT-PCR分析表明两个基因具有不同的表达模式,AQP1o主要在卵巢中表达,提示其在卵巢中具有特定的生理功能、而AQP1则在雌雄鱼的多种组织中表达.

Effects of shenmai injection on pulmonary aquaporin 1 in rats following traumatic brain injury

Background Aquaporin-1 （AQP1） has involved in fluid transport in diverse pulmonary edema diseases. Our study aimed to explore the dynamic changes of AQP1 in pulmonary water metabolism in rats following traumatic brain injury （TBI） and the protective effect provided by shenmai injection.Methods Sixty male Sprague Dawley rats weighting 280-300 g were randomly divided into three groups： the normal control group, the model group and the shenrnai injection （SMI） group. One piece skull was taken away without injuring cerebral tissue in normal control group, while rats in model group and SMI group were subject to free fall injury in the cerebral hemisphere. Rats in model group received intraperitoneal normal sodium （15 mi/kg） at one hour post-injury and the same dose of shenmai injection instead in SMI group, respectively. The expression of AQP1 was detected by immunohistochemical analysis and semi-quantitative RT-PCR at 0 hour, 10 hours, 72 hours and 120 hours after TBI.Arterial blood gas analysis and lung wet to dry were also measured.Results AQP1 was mainly presented in the capillary endothelium and slightly alveolar epithelial cells in three groups, but the expression of AQP1 in the normal control group was positive and tenuous, weakly positive in the model and SMI groups,respectively. Compared with normal control group, AQP1 mRNA levels were down regulated in the model and SMI groups at 10 hours, 72 hours and 120 hours （P ￡1/40.05）. While AQP1 mRNA levels in the SMI group was up-regulated than that in the model group （P￡1/40.05）. Lung wet to dry weight ratio （W/D） in the model and SMI groups at 10 hours were higher than that in normal control group （P ￡1/40.05）. Compared with normal control group, PaO2 was markedly lower in the model and SMI groups （P ￡1/40.05）, but there were no statistically significant differences between model and SMI groups （P ￡3/40.05）.Conclusions The decreased AQP1 expression may be involved in the increased lung water content and dysfunction of pulmonary water metabolism following TBI. The treatment with SMI could improve water metabolism by promoting AQP1 expression.

Expression of Phytoestrogens in pGL2/AQPI Promoter Reporter System

In this study, we amplified aqnaporin I（AQP1） promoter sequence with polymerase chain reaction（PCR）, then AQP1 promoter fragment and pGL2 basic vector were linked to create an artificial pGL2/AQP1 promoter re- porter system. A certain concentration of 17β-estradiol（E2） activated pGL2/AQPlp, which demonstrated the pGL2/AQPlp transcriptional system effective. The pGL2/AQP1 promoter reporter system was applied to evaluate the activate effect on AQP1 of different kinds of phytoestrogens. Dual hiciferase reporter gene activity assay showed that a certain concentration phytoestrogens including daidzein and genistein can increase AQP1 promoter transcription activity. In addition, E2, daidzein and genistein can make AQP1 protein endogenous expression level increase and promote the function of water scretion. The result can guide the clinical application to treat the Sjogren＇s syndrome and provide a necessary molecular tool for the subsequent drug screening.