The Influence of Aerial Exposure on Sea Anemones Aulactinia veratra Mucin Genes Expression Using the RNA Sequencing

Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air during the low tide and produces large quantities of mucus as an external covering. The relation between low tide and mucus secretion is still unclear, and what is the role of mucin during arial exposure is not yet investigated. This study hypothesised that the mucin genes in A. veratra would have significantly high expression in response to aerial exposure. Therefore, the aim of current study was to examine and analyses the response of A. veratra mucins in response to an experiment involving three hours of aerial exposure. To achieve this, aim the RNA-sequencing and bioinformatics analyses were used to examine the expression profile of A. veratra mucin genes in response to aerial exposure. The generated results have shown that, Mucin4-like and mucin5B-like were up-regulated in response to the three hours of aerial exposure in A. veratra. This finding shows a significant role of mucin5B-like and mucin4-like genes in response to air stress at low tide. The data generated from this study could be used in conjunction with future mucin gene studies of sea anemones and other cnidarians to compare A. veratra mucin gene expression results across time, and to extend our understanding of mucin stress response in this phylum.

中华蜜蜂肠粘蛋白AcMucin5AC-1的鉴定和定位分析

【目的】本研究旨在鉴定中华蜜蜂Apis cerana cerana肠粘蛋白AcMucin5AC-1的结构、分布及对中肠的影响,为解析蜜蜂中肠的生理功能提供理论依据。【方法】利用生物信息学比较分析中华蜜蜂AcMucin5AC蛋白的序列特征;利用RT-qPCR检测AcMucin 5 AC-1在中华蜜蜂4日龄幼虫中肠和表皮以及2日龄幼虫感染中华蜜蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)后0,12,24,48和72 h时中肠中的表达量。通过原核表达系统对中华蜜蜂AcMucin5AC-1进行表达,亲和层析柱纯化重组蛋白并制备相应多克隆抗体;Western blot检测AcMucin5AC-1在中华蜜蜂健康3-6日龄幼虫中以及4日龄幼虫中肠、表皮和围食膜中的表达,应用间接免疫荧光试验分析AcMucin5AC-1在中华蜜蜂4日龄幼虫中的定位。通过饲喂法利用RNAi沉默中华蜜蜂2日龄幼虫AcMucin 5 AC-1后12,24,48和72 h时分析RNAi干扰效率,利用苏木精-伊红(hematoxylin-eosin,HE)染色法探究RNAi干扰2日幼虫AcMucin 5 AC-1后24,48和72 h时对中华蜜蜂幼虫整体组织形态的影响。【结果】中华蜜蜂基因组中有8个AcMucin5AC基因,其氨基酸序列均含有粘蛋白结构域。RT-qPCR检测结果表明,AcMucin 5 AC-1在中华蜜蜂4日龄幼虫中肠中表达量较表皮中的高,且在感染CSBV病毒的12和72 h时的2日龄幼虫中肠中比对照组显著下调。成功获得约50 kD的重组蛋白AcMucin5AC-1和多克隆抗体,Western blot结果显示在中华蜜蜂3-6日龄幼虫以及4日龄幼虫中肠、表皮和围食膜的总蛋白中均能检测到AcMucin5AC-1。间接免疫荧光试验结果表明AcMucin5AC-1主要定位在4日龄幼虫中肠和围食膜。RNAi效率检测结果表明,2.0μg/头ds AcMucin 5 AC-1干扰24 h时中华蜜蜂AcMucin 5 AC-1较ds EGFP对照组表达量下调92%。HE染色检测结果表明,在AcMucin 5 AC-1的RNAi后72 h时中华蜜蜂幼虫整个肠腔的细胞间的致密程度减弱,形态结构紊乱。【结论】中华蜜蜂AcMucin5AC-1是位于幼虫的中肠和围食膜的粘蛋白,AcMucin 5 AC-1的下调影响中华蜜蜂幼虫中肠的形态,暗示该基因在幼虫中肠发育过程中可能发挥重要作用。

黏液-纤毛标志物Mucin 5AC-acetylated alpha-tubulin在过敏性鼻炎患者鼻黏膜中的表达

目的探究过敏性鼻炎(AR)患者鼻腔黏液-纤毛清除功能在鼻黏膜分子水平上的损伤表现和严重程度。方法选取2021年3月至2022年3月期间在我院进行鼻中隔偏曲手术的40例患者的下鼻甲黏膜组织,其中AR患者21例(AR组),非AR患者(对照组)19例。通过免疫荧光染色方法在蛋白水平上观察及评估黏液分泌标志物Mucin 5AC(MUC5AC)和纤毛标志物acetylated alpha-tubulin(acet.α-tubulin)表达情况。结果与对照组相比,AR组黏液分泌的总免疫荧光强度增加32.365%,纤毛长度缩短将近1/2,纤毛脱落评分上升将近3倍,纤毛总免疫荧光强度下降24.555%。结论AR患者黏液分泌量显著增加,纤毛排列不齐、变短、稀疏以及脱落增加可能是导致AR患者鼻腔黏膜黏液-纤毛清除功能减弱,继而加重AR症状严重程度的分子依据。

IRF5、MUC1水平与IBS患者肠道菌群、胃肠症状严重程度的相关性分析

目的探究血清干扰素调节因子5(IRF5)、黏蛋白1(MUC1)水平与肠易激综合征(IBS)患者肠道菌群、胃肠症状严重程度的相关性,为IBS临床研究提供新的生物学指标。方法选取2022年1月至2023年1月期间本院收治的IBS患者120例,根据IBS诊断标准分为IBS-C组64例与IBS-D组56例。选择同期本院健康体检者60例为对照组。采用Pearson相关性分析探究血清中IRF5、MUC1水平与肠道菌群的相关性;Spearman相关性分析评估血清IRF5、MUC1表达水平与肠易激综合征症状严重程度量表(IBS-SSS)评分的关系。结果IBS-D组患者血清IRF5表达水平、普氏菌、双歧杆菌数量明显低于IBS-C组和对照组,IBS-C组明显低于对照组;IBS-D组患者MUC1表达水平、肠球菌、大肠杆菌数量明显高于IBS-C组和对照组,IBS-C组明显高于对照组(P<0.05)。与IBS-C组相比,IBS-D组IBS-SSS评分明显升高(P<0.05)。结论IBS患者血清IRF5呈低表达,MUC1呈高表达,二者血清水平与肠道菌群及胃肠症状严重程度具有一定相关性。

Comparison between colorectal low- and high-grade mucinous adenocarcinoma with MUC1 and MUC5AC

AIM:To explore useful prognostic factors for mucinous adenocarcinoma(MAC) in the colon and rectum.METHODS:MAC was divided into low-and high-grade types based on the degree of structural differentiation;low-grade MAC arisen from well to moderately differentiated adenocarcinoma and papillary carcinoma,and high-grade MAC from poorly differentiated adenocarcinoma and signet ring cell carcinoma.Immunohistochemically,the expression of 2 types of MUC1(MUC1/DF and MUC1/CORE),MUC2,2 types of MUC5AC(MUC5AC/CHL2 and HGM),MUC6,CDX2,and CD10 was examined in 16 cases of MAC consisting of 6 low-and 10 high-grade types.RESULTS:MUC1/DF3 was expressed in 3 of 6 low-grade MAC(50) and 10 of 10 high-grade MAC(100).MUC1/CORE was expressed in 1 of 6 lowgrade MAC(16.7) and 7 of 10 high-grade MAC(70).MUC2 was expressed in all MAC regardless of the grade.MUC5AC was expressed in 6 of 6 low-grade MAC(100) and 4 of 10 high-grade MAC(40).HGM was expressed in 5 of 6 low-grade MAC(83.3) and 6 of 10 high-grade MAC(60).Expression of MUC6 and CD10 was undetected in all MAC regardless of the grade.CDX2 was expressed in 5 of 6 low-grade MAC(83.3) and 7 of 10 high-grade MAC(70).Taken together,MUC1/DF3 was expressed significantly more frequently in high-grade MAC than in low-grade,and MUC5AC/CHL2 was expressed significantly more frequently in low-grade MAC than in high-grade.CONCLUSION:It is proposed that MUC1/DF3 and MUC5AC/CHL2 immunostaining is useful to discriminate high-grade MAC from low-grade MAC.

Regulation of MUC5AC mucin production by the cell attachment dependent pathway involving integrin <i>β</i>1 in NCI-H292 human lung epithelial cells

Mucus hypersecretion in airways is a common pathological change observed in chronic inflammatory diseases and asthma. We investigated the new role of cell attachment to the extracellular matrix (ECM) on the production of the airway mucus protein, MUC5AC mucin, in human airway epithelial cells, NCI-H292. MUC5AC levels of cells cultured on low adhesion plates were 10-fold higher than those of cells cultured on adhesion plates. Cells cultured on bovine serum albumin (BSA) coated plates, which produce low adhesion conditions, also induced the up-regulation of MUC5AC. Mucin staining by PAS and MUC5AC immunodetection confirmed that mucin proteins were overproduced under low adhesion conditions. The major adhesion molecule between cells and the ECM was integrins. A time-course experiment showed that the expression patterns of integrin β1 and MUC5AC protein were inversely proportional. The inhibition of integrin β1 induced an increase in MUC5AC production in cells cultured under adhesion conditions, but not under low adhesion conditions. These results suggested that cell attachment regulates MUC5AC production, which is up-regulated by low adhesion to the ECM, and MUC5AC production is inversely proportional to the function of integrin β1.

<i>Citrus jabara</i>Extracts Suppress MUC5AC Mucin Production in Human Lung Epithelial Cells

In the human airway, the overproduction of MUC5AC mucin is a key feature of allergic asthma, and it induces airway narrowing and obstruction. The production of MUC5AC is regulated by several signals, but the mechanism is not completely understood. We investigated the effect of jabara, a citrus containing abundant flavonoids, on the regulation of MUC5AC production. When NCI-H292 human airway epithelial cells were cultured with jabara extracts, we found that the expression of Periodic acid-schiff stained mucin was suppressed with downregulated MUC5AC production. In human primary airway cells derived from asthmatic patients, MUC5AC production was also suppressed by jabara extracts. The treatment of cells with jabara extracts decreased ERK activation in NCI-H292 and in primary cells. These results show that jabara extracts contain some factors that suppress MUC5AC production and ERK activity and suggest that it will be useful for relieving asthma.

哮喘小鼠肺组织水通道蛋白5的表达及地塞米松对其的调节

目的:探讨支气管哮喘(哮喘)小鼠肺组织中水通道蛋白5(aquaporin 5,AQP5)和黏蛋白5AC(MUC5AC)的变化及地塞米松对其的调节作用。方法:雌性C57BL/6小鼠48只,随机分为对照组、哮喘组和地塞米松干预组,每组16只。测定各组小鼠肺组织病理变化及湿/干质量比值,应用实时定量PCR法测定其肺组织中AQP5和MUC5AC mRNA的表达,应用免疫组化法测定各组AQP5蛋白在肺组织中的分布,免疫印迹法测定AQP5蛋白在肺组织中的表达。ELISA法测定各组支气管肺泡灌洗液中MUC5AC的含量。结果:哮喘组小鼠肺组织中AQP5表达较对照组明显降低[mRNA减少(42.5±3.6)%,蛋白质减少(64.3±8.2)%]。而MUC5AC表达显著升高[mRNA升高(93.3±8.1)%;蛋白质水平升高(98.3±7.2)%]。地塞米松分别负调节其表达(与模型组比较P<0.05)。结论:哮喘小鼠肺组织中AQP5表达明显降低可能与气道MUC5AC表达的升高有关,地塞米松对其显著负调节从而改善气道黏液高分泌、炎性浸润和肺水渗出的病理生理过程。

人羊膜间充质干细胞移植对哮喘小鼠气道杯状细胞增生及黏蛋白5ac表达的影响

背景:前期实验研究表明,人羊膜间充质干细胞移植可减轻哮喘小鼠气道炎症反应,但对哮喘小鼠气道黏液高分泌的影响尚不明确。目的:探讨人羊膜间充质干细胞移植对哮喘小鼠气道杯状细胞增生及黏蛋白5ac表达的影响。方法:28只BALB/C小鼠随机分为4组:假手术组(第1,13天给予生理盐水致敏,第19-23天给予生理盐水雾化激发),模型组(给予卵清白蛋白致敏和雾化激发构建哮喘小鼠模型),实验组(第18天给予哮喘模型小鼠尾静脉移植人羊膜间充质干细胞进行干预),空白组(给予生理盐水致敏和雾化激发,第18天尾静脉移植人羊膜间充质干细胞进行干预),每组7只小鼠。各组小鼠均于第24天处死,观察气道组织病理学变化、上皮杯状细胞增生情况,检测支气管肺泡灌洗液中白细胞介素5水平、肺组织中气道黏蛋白5ac蛋白和m RNA的表达水平。结果与结论:①模型组支气管、血管周围及肺间质可见大量炎症细胞浸润,气道上皮杯状细胞增生明显,管壁不同程度增厚,部分管腔内见大量黏液分泌,管腔狭窄甚至闭塞;实验组气道炎症细胞浸润、杯状细胞增生情况较模型组减轻;②与假手术组和空白组比较,模型组气道上皮杯状细胞占总细胞的面积比例、杯状细胞数量、肺泡灌洗液中白细胞介素5水平、气道黏蛋白5ac蛋白和m RNA的表达量明显增高(P <0.01);与模型组比较,实验组上述指标明显降低(P <0.01);③结果表明,人羊膜间充质干细胞移植可以抑制哮喘小鼠气道炎症反应和上皮杯状细胞增生,下调气道黏蛋白5ac的表达,减轻气道黏液高分泌。

脂多糖刺激黏液下腺细胞后黏蛋白5AC和水通道蛋白5的变化

目的:探索脂多糖(lipopolysaccharide,LPS)刺激下水通道蛋白5(aquaporin-5,AQP5)和黏蛋白5AC(mucin 5AC,MUC5AC)的变化。方法:采用不同的时间点、不同浓度LPS干预SPC-A1细胞株,荧光定量聚合酶链反应(polymerase chainreaction,PCR)检测AQP5和MUC5AC的mRNA水平,Western杂交、酶联免疫吸附试验(enzyme-linked immunosorbent as-say,ELISA)法检测AQP5和MUC5AC蛋白的变化。结果:给予SPC-A1细胞LPS 0μg/mL、5μg/mL、10μg/mL、20μg/mL、40μg/mL浓度刺激后,AQP5mRNA和AQP5蛋白表达呈LPS浓度依赖性降低,而MUC5AC mRNA和MUC5AC蛋白表达呈LPS浓度依赖性升高。LPS干预0、12、24、48h后,AQP5mRNA和AQP5蛋白呈时间依赖性降低,MUC5AC mRNA和MUC5AC蛋白升高,两者呈负相关。结论:LPS刺激后AQP5和MUC5AC的变化呈负相关。

Differential mucin phenotypes and their significance in a variation of colorectal carcinoma

AIM: To investigate mucin expression profiles in colorectal carcinoma (CRC) histological subtypes with regard to clinicopathologic variables and prognosis. METHODS: Mucin (MUC)2 and MUC5AC expressions were assessed by immunohistochemistry for a total of 250 CRC cases that underwent surgical resection. CRCs included 63 well-to-moderately differentiated adenocar-cinomas (WMDAs), 91 poorly differentiated adenocarcinomas (PDAs), 81 mucinous adenocarcinoma (MUAs), and 15 signet-ring cell carcinomas (SRCCs). MUC2 and MUC5AC were scored as positive when ≥ 25% and ≥ 1% of cancer cells were stained positive, respectively. The human mutL homolog 1 and human mutS homolog 2 expressions were assessed by immunohistochemistry in PDAs to investigate mismatch-repair (MMR) status.Tumors that did not express either of these two were considered MMR-deficient. Results were analyzed for associations with clinicopathologic variables and the prognosis in individual histological CRC subtypes. RESULTS: MUC2-positive and MUC5AC-positive WMDA percentages were 49.2% and 30.2%, respectively. In contrast, MUC2-positive and MUC5AC-positive PDA percentages were 9.5% and 51.6%, respectively. MUC2 levels tended to decrease and MUC5AC levels tended to increase from WMDA to PDA. In 21 tumors comprising both adenoma and adenocarcinoma components in a single tumor (4 WMDAs, 7 PDAs, and 10 MUAs), MUC2 was significantly downregulated in PDA and MUC5AC was downregulated in PDA and MUA in the adenoma-carcinoma sequence. These results suggested that MUC2 levels might be associated with malignant potential and that MUC5AC expression was an early event in tumorigenesis. Despite worse prognoses than WMDA, high MUC2 expression levels were maintained in MUA (95.1%) and SRCC (71.5%), which suggested a pathogenesis for these subtypes distinct from that of WMDA. No significant associations were found between MUC2 expression and any clinicopathologic variables in any histological subtype. MUC5AC expression in PDA was closely associated with right-sided location (P = 0.017), absence of nodal metastasis (P = 0.010), low tumor node metastasis stage (P = 0.010), and MMR deficiency (P = 0.003). MUC2 expression in WMDA was a marginal prognostic factor for recurrence/metastasis-free survival (RFS) by univariate Cox analysis (P = 0.077) but not by multivariate Cox analysis (P = 0.161). MUC5AC expression in PDA was a significant prognostic factor for RFS by univariate Cox analysis (P = 0.007) but not by multivariate Cox analysis (P = 0.104). Kaplan-Meier curves and log-rank tests revealed that MUC2 expression was marginally associated with a better WMDA prognosis [P = 0.064 for RFS and P = 0.172 for overall survival (OS)] but not for PDA. In contrast, MUC5AC expression was significantly and marginally associated with a better PDA prognosis in terms of RFS and OS, respectively(P = 0.004 for RFS and P = 0.100 for OS), but not for WMDA and MUA. CONCLUSION: Mucin core protein expression profiles and clinical significance differ according to histological CRC subtypes. This may reflect different pathogeneses for these tumors.

18β-甘草次酸对变应性鼻炎大鼠鼻黏膜中水通道蛋白5及黏蛋白5AC的影响

目的观察18β-甘草次酸(18β-glycyrrhetinic acid,18β-GA)对变应性鼻炎(AR)鼻黏膜中水通道蛋白5(aquaporin 5,AQP5)与黏蛋白5AC(mucin 5AC,MUC5AC)表达的影响并探讨其意义。方法将30只大鼠随机分为正常对照组、AR模型组、18β-GA组,每组10只,采用OVA致敏法建立大鼠AR模型,18β-GA组大鼠灌胃给予18β-GA(23.4 mg/kg),模型组灌胃给予同体积的生理盐水;给药期间每周末次给药后,对大鼠进行行为学评分测定;随后麻醉大鼠取鼻黏膜组织光镜下观察病理形态学变化;免疫组织化学法观察AQP5及MUC5AC的蛋白表达情况;同时测定小鼠血清中白细胞介素-4(IL-4)、特异性IgE(sIgE)、干扰素-γ(IFN-γ)的含量及鼻黏膜组织中AQP5及MUC5AC的表达情况。结果大鼠AR模型建立成功,与AR模型组比较,18β-GA组大鼠的行为学评分明显降低(4.63±1.02 vs 8.74±1.88,t=7.567,P<0.01);已损伤的黏膜上皮出现好转,腺体和扩张的腺管趋于正常,肥大的杯状细胞明显减少,黏膜固有层浸润的炎性细胞减少。与AR模型组比较,18β-GA组大鼠的血清IL-4、sIgE的含量显著降低[(5.63±1.08)ng/ml vs(6.98±1.24)ng/ml,(0.772±0.185)ng/ml vs(1.886±0.394)ng/ml,t=2.608、8.096,P<0.01],IFN-γ含量显著升高[(65.42±8.02)pg/ml vs(35.14±4.83)pg/ml,t=10.237,P<0.01];AQP5蛋白表达量升高(0.84±0.13 vs 0.57±0.03,t=9.234,P<0.01),而MUC5AC蛋白表达量显著降低(1.46±0.34 vs 1.82±0.31,t=6.318,P<0.01)。结论18β-GA可能通过改变AR小鼠体内的炎症因子的含量,同时升高鼻黏膜组织中AQP5的表达及降低MUC5AC表达的方式来达到治疗AR的效果。

Effects of Glucocorticoid on IL-13-induced Muc5ac Expression in Airways of Mice

Summary: To study the effects of glucocorticoid on the IL-13-induced Muc5ac expression in airways of mice, and investigate its role in mucus secretion of airways, 24 pathogen-free BALB/c mice were randomly divided into 3 groups. IL-13 group received an nasal instillation of 100 μg of recombinant murine IL-13 solution on days 1, 3 and 5. In dexamethasone group, dexamethasone (0.5 mg/kg) was administered intraperitoneally 24 h before and 1 h before the first instillation of IL-13 and on 4 consecutive days (day 0 to day 5, 6 consecutive days in total), while control group was not treated with IL-13 or dexamethasone. Bronchoalveolar lavage fluid (BALF) was collected and eosinophils were counted, and expression of Muc5ac mRNA and protein in lungs were detected by reverse transcription-polymerase chain reaction (RT-PCR) technology and immunohistochemical assay respectively. Our results showed that the number of mice, with positve Muc5ac protein expression, expression of Muc5ac mRNA and eosinophils in BALF after IL-13 treatment were all significantly higher than that of control group ( all P<0.01). Despite eosinophils reduced (P<0.01), the number of mice with positive Muc5ac protein expression, expression of Muc5ac mRNA after dexamethasone treatment didn't decreas significantly as compared with that of IL-13 group. It is concluded that IL-13 can up-regulate the expression of Muc5ac mRNA and protein, which may play a pivotal role in the mucus overproduction of airways. Dexamethasone can suppress IL-13-induced eosinophilic infiltration in lung but can't inhibit the mucus overproduction.

MUC5B Production Is Unaffected by Akt Inhibition in Human Lung Epithelial NCI-H292 Cells

In the human airway, the gelforming mucin subtypes MUC5B and MUC5AC play important roles in biophylaxis. However, the regulation of MUC5B production is less clear than that of MUC5AC. Therefore, the regulation of MUC5B production by cell attachment and Akt was investigated in human lung epithelial NCI-H292 cells. We found that low cell attachment to culture plates induced the upregulated production of both MUC5B and MUC5AC. Cell attachment induces the activation of Akt, a serine/threonine kinase. Cell treatment with Akt inhibitor I decreased Akt phosphorylation and activation. However, MUC5B production was unaffected by Akt inhibition, whereas MUC5AC production was upregulated. MUC5B production was also unaffected by Akt inhibition in cells cultured on type IV collagen or fibronectin. These results suggest that the production of both MUC5B and MUC5AC is regulated by cell attachment. However, the regulation of MUC5B is unaffected by Akt inhibition, in contrast to that of MUC5AC.

泼尼松对支气管上皮细胞黏蛋白5AC、细胞间黏附分子-1、白细胞介素-6分泌的影响

目的:探讨泼尼松对脂多糖(LPS)诱导的支气管上皮细胞黏蛋白5AC(MUC5AC)、细胞间黏附分子-1(ICAM-1)、白细胞介素-6(IL-6)分泌的影响。方法:体外分离鉴定原代支气管上皮细胞,LPS以1μg/m L剂量诱导细胞炎症反应,泼尼松5、10、20、40、50、100μmol/L干预治疗,酶联免疫法(ELISA)检测细胞上清液MUC5AC、ICAM-1、IL-6水平变化,采用定量逆转录PCR(RT-PCR)、免疫印迹(Western-blot)检测MUC5AC、ICAM-1、IL-6 m RNA和蛋白表达情况,免疫荧光法检测核因子κB(NF-κB)核定位,Western-blot检测泼尼松干预治疗前后表皮生长因子受体(EGFR)、细胞外调节蛋白激酶1/2(ERK1/2)活化情况。结果:泼尼松以剂量依赖性降低LPS诱导的支气管上皮细胞内MUC5AC、ICAM-1、IL-6 m RNA水平和蛋白水平,抑制NF-κB、EGFR、ERK1/2活化。结论:泼尼松在体外能抑制MUC5AC、ICAM-1、IL-6的产生,其机制可能与抑制NF-κB、EGFR、ERK1/2的激活有关。

半夏提取物对气道黏液高分泌的影响

目的探讨半夏提取物(EP)对脂多糖诱导的大鼠气道黏液高分泌模型的干预作用。方法30只Wistar大鼠随机分为5组(n=6),分别为空白组、模型组、低剂量EP组、中剂量EP组及高剂量EP组。模型组和EP组经气道注入脂多糖(LPS)复制大鼠气道黏液高分泌模型。低、中、高剂量EP组连续4 d分别给予管饲EP 10、30及60 g/kg;采用免疫组化法检测肺内气道的黏蛋白5AC(MUC5AC)蛋白表达,RT-PCR法检测肺组织MUC5AC mRNA、水通道蛋白5(AQP-5)mRNA;ELISA法检测BALF中的TNF-α浓度。结果大鼠气道上皮MUC5AC蛋白和肺组织MUC5AC mRNA的表达在模型组中均明显高于空白组(P<0.05);在低剂量及中剂量EP组较模型组稍降低(P>0.05),仍显著高于空白组(P<0.05);在高剂量EP组显著低于模型组(P<0.05)。大鼠AQP-5 mRNA表达在模型组显著低于空白组(P<0.05);在低剂量及中剂量EP组较模型组稍升高(P>0.05),仍较空白组显著降低(P<0.05);在高剂量EP组显著高于模型组(P<0.01)。BALF中TNF-α浓度与肺组织MUC5AC mRNA的表达呈正相关(r=0.948,P<0.05),肺组织AQP-5 mRNA与MUC5AC mRNA及TNF-α的表达呈负相关(r=-0.955,P<0.05;r=-0.909,P<0.05)。结论高剂量的EP能明显抑制大鼠气道上黏液高分泌状态。

二陈汤对慢性支气管炎气道黏液高分泌的影响

目的观察二陈汤对慢性支气管炎大鼠气道黏液高分泌的影响及其机制。方法采用香烟烟熏加内毒素脂多糖制备慢性支气管炎大鼠模型,大鼠随机分为正常组,模型组,二陈汤低、中、高剂量组,急支糖浆组。正常组和模型组灌胃生理盐水6 ml/d,急支糖浆组灌胃给予太极急支糖浆12 g/kg,二陈汤低、中、高剂量组分别灌胃2.45、4.90、9.80 g/kg,连续14 d。免疫组化检测黏蛋白(MUC)5AC和水通道蛋白(AQP)5在支气管组织中的表达。结果与正常组相比,模型组MUC5AC蛋白表达显著增强,AQP5蛋白表达显著减弱(均P<0.01),MUC5AC与AQP5蛋白表达呈负相关(r=-0.55,P<0.01);与模型组相比,二陈汤中剂量组MUC5AC蛋白表达显著减弱,AQP5蛋白表达增强(均P<0.01),MUC5AC与AQP5蛋白表达呈负相关(r=-0.75,P<0.01)。结论二陈汤对慢性支气管炎气道黏液高分泌有调节作用,其机制可能与抑制MUC5AC、上调AQP5蛋白表达有关。

孟鲁斯特对人支气管上皮细胞黏液蛋白分泌的影响

目的探讨孟鲁斯特对脂多糖(LPS)诱导的人支气管上皮细胞黏液蛋白分泌的影响。方法体外分离鉴定人原代支气管上皮细胞,LPS以1μg/ml剂量诱导细胞炎症反应,孟鲁斯特(50、20、10μmol/L)进行干预治疗,酶联免疫法(ELISA)检测分泌细胞上清黏蛋白5AC(MUC5AC)水平变化,并采用定量逆转录PCR(RT-PCR)、免疫印迹(Western-blot)检测MUC5AC m RNA和蛋白表达情况;2’,7’-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针检测活性氧(ROS)变化;Western-blot检测孟鲁斯特干预治疗前后磷酸化细胞核转录因子κB(p-NF-κB)、磷酸化NF-κB抑制蛋白(p-IκBα)、磷酸化细胞外调节激酶1,2(ERK1/2)活化情况。结果孟鲁斯特以剂量依赖性降低LPS诱导的人支气管上皮细胞内MUC5AC mRNA水平和蛋白水平,抑制ROS产生,抑制NF-κB/p-IκBα、ERK活化。结论孟鲁斯特在体外能抑制MUC5AC的产生,其机制可能与抑制NF-κB、EKR的激活有关。

TLR4在慢性支气管炎大鼠气道粘液高分泌中的作用

目的观察TLR4在细菌内毒素(LPS)致慢性支气管炎大鼠气道粘液高分泌中的作用。方法 SPF级雄性Wistar大鼠40只,随机分为4组,正常对照组(NS组)10只,慢性支气管炎组(LPS组)10只,多粘菌素B干预组(LPS+PMB组)10只,多粘菌素B自身对照组(PMB组)10只。用气管内注入LPS 200μg/200μL及每日LPS溶液雾化吸入1h的方法建立慢性支气管炎动物模型,3周后对大鼠肺脏进行病理学检查,肺组织阿先蓝-过碘酸雪夫(AB-PAS)阳染面积,用免疫组化法观察粘蛋白Muc5AC、TLR4在支气管肺内的表达及荧光定量RT-PCR Muc5AC mRNA、TLR4 mRNA在肺内的表达。结果 LPS组在AB-PAS阳染面积、粘蛋白Muc5AC、TLR4蛋白及Muc5AC mRNA、TLR4 mRNA表达与LPS+PMB组比较差异有统计学意义(P<0.05),NS组和PMB组在上述指标则组间差异无统计学意义(P>0.05)。结论 TLR4介导LPS诱发的慢性呼吸道炎症反应可能导致气道粘蛋白基因Muc5AC mRNA的高表达和粘蛋白Muc5AC的高分泌。多粘菌素B可能通过抑制肺组织TLR4 mRNA及其蛋白的表达而抑制气道粘液高分泌。

SO_2衍生物对人BEP2D细胞哮喘基因表达影响

目的研究二氧化硫(SO2)衍生物染毒后人支气管上皮细胞(BEP2D)哮喘相关基因黏蛋白5AC(MUC5AC)和白介素-13(IL-13)表达的变化。方法以不同浓度SO2衍生物(0.000 1,0.001,0.01,0.1,1 mmol/L)对BEP2D细胞染毒,采用荧光实时定量RT-PCR、免疫细胞化学和酶联免疫吸附法(ELISA)等方法测定基因的表达。结果不同浓度SO2衍生物处理的BEP2D细胞中MUC5AC和IL-13 mRNA和蛋白表达比对照组明显增加;经0.1 mmol/L SO2衍生物处理后换新鲜培养基再孵育不同的时间,MUC5AC和IL-13 mRNA水平均显著增高,在更换新鲜培养基孵育4 h时达到峰值,24 h时虽然有所降低,但仍比对照组显著增高。结论SO2衍生物能增加EBP2D细胞MUC5AC和IL-13在转录和翻译水平上的表达,提示这可能是SO2加重哮喘疾病的原因之一。