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。

MUC1 and MUC5AC mucin expression in liver fluke-associated intrahepatic cholangiocarcinoma

AIM： To investigate the expressions of MUC1 and MUC5AC in intrahepatic cholangiocarcinoma （ICC）. Association of expressions of mucins MUC1 and MUC5AC with clinical findings, metastasis, and survival of the liver fluke-associated ICC patients was determined. METHODS： The expressions of MUC1 and MUC5AC mucins were examined by immunohistochemical staining in 87 cases of histologically-proven ICC. The expressions of mucins in relationship between clinicopathological significance and prognosis of the patients were evaluated. RESULTS： Fifty-two patients （60%） exhibited both MUC1 and MUCSAC expressions, whereas 31% expressed either MUC1 or MUC5AC, and 9% expressed neither. High MUC1 immunoreactivity displayed a significant correlation wibh tumor progression as reflected by vascular invasion （P〈0.001）, whereas high expression of MUC5AC significantly correlated with neural invasion （P = 0.022） and advanced ICC stage （P = 0.008）. Patients with high expression of MUC1 had a significantly shorter survival （P = 0.0002）. According to multivariate analyses, MUC1 reactivity （P = 0.026）, histological grading and stage of tumor represented the least probability of survival. CONCLUSION： MUC1 is overexpressed in liver flukeassociated cholangiocarcinoma and relates to vascular invasion and poor prognosis, whereas MUC5AC mucin is neoexpressed and relates to neural invasion and advanced ICC stage. High MUC1 expression in tumor may be useful for predicting the poor outcome of ICC patients.

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.

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。

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.

Gastric mucin expression in Helicobacter pylori-related, nonsteroidal anti-inflammatory drug-related and idiopathic ulcers

AIM: To determine the pattern of secreted mucin expression in Helicobacter pylori (H. pylori)-related, nonsteroidal anti-inflammatory drug (NSAID)-related and idiopathic gastric ulcers. METHODS: We randomly selected 92 patients with H. pylori-associated (n = 30), NSAID-associated (n = 18), combined H. pylori and NSAID-associated gastric ulcers (n = 24), and patients with idiopathic gastric ulcers (n = 20). Immunohistochemistry for T-cell CD4/CD8, andfor mucin 5AC (MUC5AC) and mucin 6 (MUC6), was performed on sections of the mucosa from the ulcer margin. Inflammation score was assessed according to the Sydney system. RESULTS: MUC5AC was expressed on the surface epithelium (98.9%) and neck glands (98.9%) with minimal expression in the deep glands (6.5%). MUC6 was strongly expressed in the deep glands (97.8%), variable in the neck glands (19.6%) and absent in the surface epithelium (0%). The pattern of mucin expression in idiopathic ulcer margins was not different from the expression in ulcers associated with H. pylori, NSAIDs, or combined H. pylori and NSAIDs. CD4/CD8 ratio was higher in H. pylori-positive patients (P = 0.009). Idiopathic ulcers are associated with hospitalized patients and have higher bleeding and mortality rates. CONCLUSION: Idiopathic ulcers have a unique clinical profile. Gastric mucin expression in idiopathic gastric ulcers is unchanged compared with H. pylori and/or NSAID-associated ulcers.

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.

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。

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.

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.

Curcumin Inhibits Lipopolysaccharide-lnduced Mucin 5AC Hypersecretion and Airway Inflammation via Nuclear Factor Erythroid 2-Related Factor 2

Background： Excess mucus production is an important pathophysiological feature of chronic inflammatory airway diseases. Effective therapies are currently lacking. The aim of the study was to evaluate the effects ofcurcumin （CUR） on lipopolysaccharide （LPS）-induced mucus secretion and inflammation, and explored the underlying mechanism in vivo and in vitro. Methods： For the in vitro study, human bronchial epithelial （NCI-H292） cells were pretreated with CUR or vehicle for 30 min, and then exposed to LPS for 24 h. Next, nuclear factor erythroid 2-related factor 2 （Nrf2） was knocked down with Nrf2 small interfering RNA （siRNA） to confirm the specific role of Nrf2 in mucin regulation of CUR in NCI-H292 cells. In vivo, C57BL/6 mice were randomly assigned to three groups （n = 7 for each group）： control group, LPS group, and LPS ＋ CUR group. Mice in LPS and LPS ＋ CUR group were injected with saline or CUR （50 mg/kg） intraperitoneally 2 h before intratracheal instillation with LPS （ 100 μg/ml） for 7 days. Cell lysate and lung tissue were obtained to measured Mucin 5AC （MUC5AC） and Nrf2 mRNA and protein expression by a real-time polymerase chain reaction and Western blotting. Bronchoalveolar lavage fluid （BALF） was collected to enumerate total cells and neutrophils. HistopathologicaI changes of the lung were observed. Data were analyzed by one-way analysis of variance. Student＇s t-test was used when two groups were compared. Results： CUR significantly decreased the expression ofMUC5AC mRNA and protein in NCI-H292 cells exposed to LPS. This effect was dose dependent （2.424 ± 0.318 vs. 7.169 ± 1.785, t = 4.534, and 1.060 ± 0.197 vs. 2.340 ± 0.209, t = 7.716; both P 〈 0.05, respectively） and accompanied by increased mRNA and protein expression of Nrf2 （1.952 ± 0.340 vs. 1.142 ± 0.176, t = -3.661, and 2.010 ± 0.209 vs. 1.089 ±0. 132, t = -6.453; both P 〈 0.05, respectively）. Furthermore, knockdown of Nrf2 with siRNA increased MUC5A C mRN A expression by 47.7%, compared with levels observed in the siRNA-negative group （6.845 ± 1.478 vs. 3.391 ± 0.517, t = -3.821, P 〈 0.05）. Knockdown of Nrf2 with siRNA also markedly increased MUC5A C protein expression in NCI-H292 cells. CUR also significantly decreased LPS-induced mRNA and protein expression of MUC5A C in mouse lung （ 1.672 ± 0.721 vs. 5.961 ± 2.452, t = 2.906, and 0.480 ± 0.191 vs. 2.290 ± 0.834, t = 3.665, respectively; both P 〈 0.05）. Alcian blue/periodic acid-Schiff staining also showed that CUR suppressed mucin production. Compared with the LPS group, the numbers of inflammatory cells （247 ± 30 vs. 334 ± 24, t = 3.901, P 〈 0.05） and neutrophils （185 ± 22 vs. 246 ± 20, t = 3.566, P 〈 0.05） in BALF decreased in the LPS ＋ CUR group, as well as reduced inflammatory cell infiltration in lung tissue. Conclusion： CUR inhibits LPS-induced airway mucus hypersecretion and inflammation through activation of Nrf2 possibly.

Effect of electroacupuncture on expressions of acetylcholine and mucin 5AC in the lungs of rats with chronic obstructive pulmonary disease

Objective： To observe the effect of electroacupuncture （EA） on the expressions of acetylcholine （ACh） and mucin 5AC （MUC5AC） in the lungs of rats with chronic obstructive pulmonary disease （COPD）, and explore the mechanism of EA in treating COPD. Methods： Thirty Sprague-Dawley （SD） rats were randomly divided into a control group, a COPD group, and an EA group, with 10 rats in each group. The control group was a group of normal rats. The COPD rat model was induced by cigarette smoke combined with lipopolysaccharide （LPS）. The COPD rats were treated with EA at bilateral Feishu （BL 13） and Zusanli （ST 36） in the EA group, 30 rain each time, once a day, successively for 14 d. The lung function was tested. The contents of ACh and MUC5AC in lungs and bronchoalveolar lavage fluid （BALF） were detected by enzyme-linked immunosorbent assay （ELISA）. Pearson method was used to analyze the correlation between pulmonary function and the content of MUC5AC in lungs. The mRNA and protein expressions of MUC5AC in lung tissues were detected by real-time polymerase chain reaction （RT-PCR） and Western blot （WB）, respectively. The immune response of MUC5AC was observed by immunohistochemistry. Results： Eight rats were left in each group, and the other two died. Compared with the control group, the total airway resistance （Raw） increased significantly and dynamic compliance （Cdyn） decreased significantly in the COPD group （P〈0.01）; compared with the COPD group, the Raw level declined significantly and Cdyn increased significantly in the EA group （P〈0.01）. The contents of ACh and MUC5AC in the lungs and BALF were remarkably higher in the COPD group compared with those in the control group （P〈0.01, P〈0.001）; compared with the COPD group, the contents of ACh and MUC5AC were significantly lower in the EA group （P〈0.05, P〈0.001）. There was a negative correlation between MUC5AC content and lung function （P〈0.001）. The mRNA and protein expressions of MUC5AC in the lungs were significantly higher in the COPD group than in the control group （P〈0.001）; compared with the COPD group, the expressions were significantly lower in the EA group （P〈0.01）. Compared with the control group, the immune response of MUC5AC in the airway epithelium significantly increased in the COPD group （P〈0.001）; the immune response of MUC5AC was significantly lower in the EA group compared with that in the COPD group （P〈0.001）. Conclusion： EA treatment can improve the lung function of COPD rats, which may be related to its effect in the down-regulation of ACh and MUC5AC contents in the lungs as well as the inhibition of mucus hypersecretion.

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。

TLR4在慢性支气管炎大鼠气道粘液高分泌中的作用及多粘菌素B干预的影响

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及多粘菌素B(PMB)干预的影响,并探讨其可能的机制。方法 SPF级雄性Wistar大鼠40只,随机分为正常对照组(NS组)、慢性支气管炎组(LPS组)、多粘菌素B干预组(LPS+PMB组)和多粘菌素B自身对照组(PMB组)各10只。用气管内注入LPS 200μg/200μl及每日LPS溶液雾化吸入1小时的方法建立慢性支气管炎动物模型,3周后收集支气管肺泡灌洗液(BALF)进行细胞学计数和白细胞分类检测,酶联免疫吸附实验(ELISA)测定BALF中肿瘤坏死因子-α(TNF-α)含量,病理学检测大鼠肺组织阿先蓝-过碘酸雪夫(AB-PAS)阳染面积,用免疫组化法检测粘蛋白Muc5AC、TLR4在支气管肺内的表达及荧光定量RT-PCR法检测Muc5AC mRNA、TLR4mRNA在肺内的表达。结果 LPS组在BALF中细胞总数、中性粒细胞、TNF-α含量及AB-PAS阳染面积、粘蛋白Muc5AC、TLR4蛋白及Muc5AC mRNA、TLR4mRNA表达与LPS+PMB组比较,差异均有显著性(均P<0.05),NS组和PMB组上述指标间差异无显著性(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。PMB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。

健脾清热化瘀方治疗胃癌前病变的疗效及对胃黏膜黏蛋白5AC表达的影响

目的:观察健脾清热化瘀方治疗脾虚湿热瘀血型胃癌前病变的临床疗效并探讨其可能的作用机制。方法:60例患者按入院前后顺序分为治疗组和对照组各30例。治疗组予健脾清热化瘀方,1剂/d,水煎分两次2次口服;对照组予胃复春片,4片/次,3次/d,饭前30 min口服。两组疗程均为24周。观察两组患者各临床症状评分、病理评分的变化、幽门螺旋杆菌(HP)根除率及胃黏膜黏蛋白5 AC(MUC5 AC)的表达变化。结果:治疗组综合临床疗效、胃镜疗效及病理疗效均优于对照组(P<0.05);治疗组HP根除率为65.0%,优于对照组的26.3%(P<0.05);治疗后治疗组脾虚湿热瘀血证各症状评分均低于对照组(P<0.01)治疗后治疗组肠上皮化生和异型增生评分均低于对照组(P<0.01)治疗后治疗组MUC5 AC阳性表达评分高于对照组(P<0.05)。结论:健脾清热化瘀方可以明显改善胃癌前病变患者症状,减轻腺体的肠化生和异型增生,对胃癌前病变具有肯定的疗效,其上调胃黏膜MUC5 AC蛋白的表达,增强胃黏膜的保护可能是其作用机制之一。

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。

Exocyst复合物关键亚基Sec3蛋白在气道黏液高分泌中的作用研究

目的探讨Exocyst复合物关键亚基Sec3蛋白在气道黏液高分泌形成过程中的作用。方法将60只大鼠随机分成3组:正常对照组,烟雾暴露组、烟雾暴露+人中性粒细胞弹性蛋白酶(HNE)组。烟雾暴露组大鼠采用烟雾暴露法建立慢性支气管炎症病理性改变,HNE攻击形成气道黏液高分泌大鼠模型。取肺组织支气管上皮,观察气道上皮杯状细胞的增生及黏液分泌情况,测定Sec3和气道黏蛋白5AC(MUC5AC)表达。HNE处理人支气管上皮细胞(16HBE),分析HNE对16HBE的Sec3、p-Sec3和MUC5AC蛋白表达的影响。sh-Sec3转染HNE处理的16HBE,分析转染效率,以及Sec3对HNE诱导的16HBE中MUC5AC分泌情况的影响。免疫共沉淀鉴定Sec3与MUC5AC的相互作用。结果与正常对照组比较,烟雾暴露组和烟雾暴露+HNE组大鼠气道上皮杯状细胞增生及黏液分泌量增加,Sec3和MUC5AC蛋白表达水平升高,且烟雾暴露+HNE组变化更明显(P<0.05);与16HBE组比较,16HBE+HNE组的Sec3、p-Sec3和MUC5AC蛋白相对表达水平升高(P<0.05),且呈时间依赖性;与16HBE+HNE组和空质粒转染组比较,sh-Sec3组Sec3蛋白和mRNA及MUC5AC蛋白相对表达水平降低(P<0.05);Sec3蛋白可被MUC5AC抗体共沉淀,MUC5AC蛋白亦可被Sec3抗体共沉淀,且在HNE刺激下,这种相互作用可加强。结论Sec3参与气道黏液高分泌过程,且与MUC5AC在体内外环境中均具有相互作用,介导了HNE诱导的MUC5AC高分泌。