Mucin Protein Production Research Articles

인간 기도 상피세포에서 IKK/IκBα/NF-κB p65 신호전달 경로를 경유한 호흡기 MUC5AC 뮤신 유전자의 발현에 미치는 올레아놀산의 영향

In the present study, we investigated whether oleanolic acid, an anti-inflammatory natural product, significantly affects the phorbol 12-myristate 13-acetate (PMA)-induced gene expression of airway MUC5AC mucin. Confluent NCIH292 cells were pretreated with oleanolic acid for 30 min and then stimulated with PMA for 24 h or the indicated periods. The MUC5AC mucin mRNA expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA. To elucidate the action mechanism of oleanolic acid, effect of oleanolic acid on PMA-induced NF-B signaling pathway was investigated by western blot analysis. The results were as follows: (1) Oleanolic acid significantly inhibited the production of MUC5AC mucin protein and down-regulated the expression of MUC5AC mucin mRNA; (2) Oleanolic acid inhibited PMA-induced activation (phosphorylation) of inhibitory kappa B kinase (IKK), and thus phosphorylation and degradation of inhibitory kappa Ba (IB); (3) Oleanolic acid inhibited PMA-induced nuclear translocation of nuclear factor kappa B (NF-B) p65, led to the down-regulation of MUC5AC mucin gene expression in NCI-H292 cells. These results suggest that oleanolic acid can regulate the gene expression of mucin by acting on airway epithelial cells through regulation of NF-B signaling pathway.

배양된 인간 기도 상피세포에서 포볼 에스터로 유발된 뮤신 유전자 발현과 세포 내 NF-κB 신호전달 경로에 대한 하이퍼로사이드의 작용

We investigated whether hyperoside, an anti-inflammatory natural product, significantly affects phorbol 12- myristate 13-acetate (PMA)-induced airway expression of MUC5AC encoded mucin. Confluent NCI-H292 cells were pretreated with hyperoside for 30 min, then stimulated with PMA for 24 h or the indicated periods. MUC5AC mucin mRNA expression was measured by RT-PCR. MUC5AC mucin protein expression was quantitated by ELISA. To elucidate the mechanism of hyperoside action, we investigated the effect of hyperoside on PMA-induced NF-κB signaling by western blot analysis. The results were as follows: (1) Hyperoside significantly inhibited MUC5AC mucin protein production and down-regulated MUC5AC mucin mRNA expression; (2) Hyperoside inhibited PMA-induced activation (phosphorylation) of inhibitory kappa B kinase (IKK), thereby inhibiting phosphorylation and degradation of inhibitory kappa Bα (IκBα); (3) Hyperoside inhibited PMA-induced nuclear translocation of nuclear factor kappa B (NF-κB) p65, thereby down-regulating MUC5AC mucin gene expression in NCI-H292 cells. These results suggest that hyperoside can regulate mucin gene expression by airway epithelial cells through regulation of the NF-κB signaling pathway.

Effect of Paeoniae Radix Rubra (Paeonia lactiflora Pall.) extract on mucin secretion, gene expression in human airway epithelial cells

Ethnopharmacological relevancePaeoniae Radix Rubra (PRR), the root of Paeonia lactiflora Pall., is a traditional Chinese medicine which has the effects of regulating various inflammatory diseases, treating blood stasis, and enhancing blood circulation. Aim of the studyThis study examined whether Paeoniae Radix rubra extract (PRRE) and Paeoniflorin (PF) affect mucin production, gene expression including MUC5AC, and protein expression related to the ERK pathway induced by TNF-α from human airway epithelial cells. Materials and methodsNCI–H292 cells induced by TNF-α were treated with each agent. MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription polymerase chain reaction, staining, and enzyme-linked immunosorbent assay. Western blot was used to investigate the cell signaling pathways. ResultsPRRE and PF inhibited the production of MUC5AC mucin protein and gene expression in TNF-α-induced H292 cells. In Western blot, PRRE was involved in protein expression related to the ERK pathway. ConclusionsOverall, PRRE effectively inhibited the MUC5AC, and inflammatory cytokines expression caused by TNF-α, which was closely involved in the ERK pathway. PRRE may have the potential for treating mucus producing respiratory inflammation.

Isosteviol Sodium Exerts Anti-Colitic Effects on BALB/c Mice with Dextran Sodium Sulfate-Induced Colitis Through Metabolic Reprogramming and Immune Response Modulation.

PurposeInflammatory bowel diseases (IBDs) are global health problems that are associated with immune regulation, but clinical IBDs treatment is currently inadequate. Effective preventive or therapeutic methods for immune disorders rely on controlling the function of immune cells. Isosteviol sodium (STV-Na) has antioxidant activity, but the therapeutic effect of STV-Na against IBD remain undocumented. Herein, we investigated the therapeutic effect of STV-Na in mice models with IBDs.MethodsMice received 3.5% DSS for 7 days to establish IBD models. Intraperitoneal STV-Na was given 2 days before DSS and lasted for 9 days. Commercially available drugs used in treating IBDs (5-aminosalicylic acid, dexamethasone, and infliximab) were used as positive controls. Samples were collected 7 days after colitis induction. Histopathological score, biochemical parameters, molecular biology methods, and metabolomics were used for evaluating the therapeutic effect of STV-Na.ResultsOur data revealed that STV-Na could significantly alleviate colon inflammation in mice with colitis. Specifically, STV-Na treatment improved body weight loss, increased colon length, decreased histology scores, and restored the hematological parameters of mice with colitis. The untargeted metabolomics analysis revealed that metabolic profiles were restored by STV-Na treatment. Furthermore, STV-Na therapy suppressed the number of CD68 macrophages and F4/80 cell infiltration. And STV-Na suppressed M1 and M2 macrophage numbers along with the mRNA expressions of proinflammatory cytokines. Moreover, STV-Na administration increased the number of regulatory T (Treg) cells while decreasing Th1/Th2/Th17 cell counts in the spleen. Additionally, STV-Na treatment restored intestinal barrier disruption in DSS-triggered colitis tissues by ameliorating the TJ proteins, increasing goblet cell proportions, and mucin protein production, and decreasing the permeability to FITC-dextran, which was accompanied by decreased plasma LPS and DAO contents.ConclusionThese results indicate that STV-Na can ameliorate colitis by modulating immune responses along with metabolic reprogramming, and could therefore be a promising therapeutic strategy for IBDs.

Effects of laminarin zwitterionic carboxylate and sulfonate on the intestinal barrier function and gut microbiota

Ulcerative colitis (UC) has become a global chronic disease that keeps increasing. This study was to explore the treatment effectiveness of two functional zwitterionic laminarins, zwitterionic sulfonate (LZS) and zwitterionic carboxylate (LZC), in dextran sulfate sodium (DSS) induced mouse model. FT-IR and NMR techniques were used to characterize the aforementioned functional zwitterion. Compared to UC mice, the composition and diversity of gut microbiota were significantly increased in the treated mice. Specifically, the composition of Bacteroidetes increased and the level of Firmicutes decreased. Moreover, we demonstrated the alleviation of colitis by LZS and LZC reflected by the improved integrity of intestinal mucosa, which includes increased number of goblet cells, mucin protein production, maintenance of collagens, as well as the lower extent of intestinal fibrosis. These findings indicated the potentials of LZC and LZS as promising agents to prevent colitis via adjusting gut microbiota and maintaining intestinal barrier integrity.

Nanosecond pulsed electric fields impair viability and mucin expression in mucinous colorectal carcinoma cell

Nanosecond pulsed electric fields (nsPEFs) are a non-thermal technology that can induce a myriad of biological responses and changes in cellular physiology. nsPEFs have gained significant attention as a novel cancer therapy. However, studies investigating the application of nsPEF in mucinous carcinomas are scarce. In this study, we explored several biological responses in two mucinous colorectal adenocarcinoma cell lines, LS 174T and HT-29, to nsPEF treatment. We determined the overall cell survival and viability rates following nsPEF treatment using CCK-8 and colony formation assays. We measured the intracellular effects of nsPEF treatment by analyzing cell cycle distribution, cell apoptosis and mitochondrial potential. We also analyzed mucin production at both mRNA and protein levels. Our results showed that nsPEF treatment significantly reduced mucinous cell viability in a dose-dependent manner. nsPEF treatment increased cell cycles arrest at G0/G1 while the proportion of G2/M cells gradually decreased. Cell apoptosis increased following nsPEF treatment with a clear loss in mitochondrial membrane potential. Furthermore, the protein expression of functional mucin family members decreased after nsPEF treatment. In conclusion, nsPEF treatment reduced MCRC cell viability, cell proliferation, and mucin protein production while promoted apoptosis. Our work is a pilot study that projects some insights into the potential clinical applications of nsPEFs in treating mucinous colorectal carcinoma.

Effect of Korean Red Ginseng and Rg3 on Asian Sand Dust-Induced MUC5AC, MUC5B, and MUC8 Expression in Bronchial Epithelial Cells.

Korean Red ginseng (KRG), commonly used in traditional medicine, has anti-inflammatory, anti- oxidative, and anti-tumorigenic properties. Asian sand dust (ASD) is known to aggravate upper and lower airway inflammatory responses. BEAS-2B cells were exposed to ASD with or without KRG or ginsenoside Rg3. Mucin 5AC (MUC5AC), MUC5B, and MUC8 mRNA and protein expression levels were determined using quantitative RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor kappa B (NF-κB), activator protein 1, and mitogen-activated protein kinase expression and activity were determined using western blot analysis. ASD induced MUC5AC, MUC5B, and MUC8 mRNA and protein expression in BEAS-2B cells, which was significantly inhibited by KRG and Rg3. Although ASD-induced mucin expression was associated with NF-κB and p38 mitogen-activated protein kinase (MAPK) activity, KRG and Rg3 significantly suppressed only ASD-induced NF-κB expression and activity. KRG and Rg3 inhibited ASD-induced mucin gene expression and protein production from bronchial epithelial cells. These results suggest that KRG and Rg3 have potential for treating mucus-producing airway inflammatory diseases.

Modulation of Mucin (MUC2, MUC5AC and MUC5B) mRNA Expression and Protein Production and Secretion in Caco-2/HT29-MTX Co-Cultures Following Exposure to Individual and Combined Aflatoxin M1 and Ochratoxin A.

Aflatoxin M1 (AFM1) and ochratoxin A (OTA), which widely coexist in milk, may pose a serious threat to human health. Mucin is a major component of the intestinal mucus layer, which plays an important role in maintaining intestinal mucosal homeostasis. However, the effect of mycotoxins AFM1 and OTA on intestinal mucin production is still not clear. This study aimed to investigate individual and interactive effects of mycotoxins AFM1 and OTA on the intestinal barrier and the mRNA expression of intestinal mucin (MUC2, MUC5AC and MUC5B) and on protein production in Caco-2/HT29-MTX cultures after 48 h of exposure. Our results show that individual mycotoxins and their mixtures significantly reduced intestinal cell viability and transepithelial electrical resistance (TEER) values, as well as significantly altered intestinal mucin mRNA expression and protein abundance. Moreover, OTA showed toxicity similar to AFM1 in cell viability and TEER value at the same concentration. When the two mycotoxins acted in combination, the synergistic effects observed in the assessment of cell viability and protein abundance in all mono- and co-cultures. In general, this study provides evidence that AFM1 and OTA can damage the intestine, and it contributes to optimized maximum permissible limits of mycotoxins in milk.

Obtusifolin isolated from the seeds of Cassia obtusifolia regulates the gene expression and production of MUC5AC mucin in airway epithelial cells via affecting NF-κB pathway.

We investigated whether obtusin, obtusifolin, and cassiaside isolated from the seeds of Cassia obtusifolia inhibit the gene expression and production of airway mucin 5AC (MUC5AC). Confluent NCI-H292 cells were pretreated with obtusin, obtusifolin, or cassiaside for 30min and then stimulated with epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA), or tumor necrosis factor-α (TNF-α) for 24hr. The MUC5AC mucin gene expression was measured by reverse transcription-polymerase chain reaction. Production of MUC5AC mucin protein was measured by enzyme-linked immunosorbent assay. To elucidate the action mechanism of obtusifolin, effect of obtusifolin on PMA-induced nuclear factor kappa B (NF-κB) signaling pathway was investigated by western blot analysis. Obtusin, obtusifolin, or cassiaside inhibited the expression of MUC5AC mucin gene and the production of MUC5AC mucin protein, induced by EGF, PMA, or TNF-α. Obtusifolin inhibited PMA-induced activation (phosphorylation) of inhibitory kappa B kinase, and thus phosphorylation and degradation of inhibitory kappa B alpha. Obtusifolin inhibited PMA-induced nuclear translocation of NF-κB p65. These results suggest that obtusifolin can regulate the production and gene expression of mucin by acting on airway epithelial cells through regulation of NF-κB signaling pathway.

Effects of Natural Products Derived from Persicae Semen including Daucosterol on gene Expression and Production of Respiratory Mucin induced by Growth Factor and Phorbol Ester

Persicae semen was used as folk remedy for regulating pulmonary inflammatory diseases in traditional oriental medicine. In the prersent study, we investigated whether prunasin, erucic acid or daucosterol derived from Persicae semen affects airway MUC5AC mucin gene expression and production, induced by phorbol 12-myristate 13-acetate (PMA) or epi-dermal growth factor (EGF) from NCI-H292 cells. Confluent cells were pretreated with each agent for 30 min and then stim-ulated with PMA or EGF for 24h. The MUC5AC mucin gene expression was measured by RT-PCR (Reverse Transcription-Polymerase Chain Reaction). Production of MUC5AC mucin protein was measured by enzyme-linked immunosorbent assay (ELISA). The results were as follows: (1) Erucic acid inhibited the gene expression and production of MUC5AC mucin from NCI-H292 cells; (2) Daucosterol also inhibited the gene expression and production of MUC5AC mucin from NCI-H292 cells. This result suggests that erucic acid or daucosterol can inhibit the production and gene expression of mucin induced by PMA or EGF, through directly acting on airway epithelial cells.

Tussilagone suppressed the production and gene expression of MUC5AC mucin via regulating nuclear factor-kappa B signaling pathway in airway epithelial cells

In the present study, we investigated whether tussilagone, a natural product derived from Tussilago farfara, significantly affects the production and gene expression of airway MUC5AC mucin. Confluent NCI-H292 cells were pretreated with tussilagone for 30 min and then stimulated with EGF (epidermal growth factor) or PMA (phorbol 12-myristate 13-acetate) for 24 h or the indicated periods. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA. To elucidate the action mechanism of tussilagone, effect of tussilagone on PMA-induced NF-κB signaling pathway was investigated by western blot analysis. Tussilagone significantly inhibited the production of MUC5AC mucin protein and down-regulated the expression of MUC5AC mucin gene, induced by EGF or PMA. Tussilagone inhibited PMA-induced activation (phosphorylation) of inhibitory kappa B kinase (IKK), and thus phosphorylation and degradation of inhibitory kappa Ba (IκBα). Tussilagone inhibited PMA-induced phosphorylation and nuclear translocation of nuclear factor kappa B (NF-κB) p65. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. These results suggest that tussilagone can regulate the production and gene expression of mucin by acting on airway epithelial cells through regulation of NF-κB signaling pathway.

Effects of Homogentisic Acid and Natural Products Derived from Pinellia ternata on Secretion, Production and Gene Expression of MUC5AC Mucin from Cultured Airway Epithelial Cells

In this study, we investigated whether adenosine, adenine, uridine and homogentisic acid derived from Pinellia ternata affect the secretion, production and gene expression of MUC5AC mucin from airway epithelial cells. Confluent NCI-H292 cells were pretreated with adenosine, adenine, uridine or homogentisic acid for 30 min and then stimulated with PMA (phorbol 12-myristate 13-acetate) for 24 h. The MUC5AC mucin gene expression, mucin protein production and secretion were measured by RT-PCR and ELISA, respectively. The results were as follows: (1) Adenine and homogentisic acid decreased PMA-induced MUC5AC mucin gene expression, although adenosine and uridine did not affect the mucin gene expression; (2) Adenosine, adenine, uridine and homogentisic acid inhibited PMA-induced MUC5AC mucin production; (3) Homogentisic acid inhibited the secretion of MUC5AC mucin from NCI-H292 cells. These results suggest that, among the four compounds examined, homogentisic acid showed the regulatory effect on the steps of gene expression, production and secretion of mucin, by directly acting on airway epithelial cells.

Verticine, ebeiedine and suchengbeisine isolated from the bulbs of Fritillaria thunbergii Miq. inhibited the gene expression and production of MUC5AC mucin from human airway epithelial cells

BackgroundThe bulb of Fritillaria thunbergii has been utilised as mucoregulators and expectorants for controlling the airway inflammatory diseases in folk medicine. Hypothesis/PurposeWe investigated whether verticine, ebeiedine and suchengbeisine isolated from the bulbs of Fritillaria thunbergii inhibit the gene expression and production of MUC5AC mucin from human airway epithelial cells. Study designConfluent NCI-H292 cells were pretreated with verticine, ebeiedine or suchengbeisine for 30min and then stimulated with EGF, PMA or TNF-α for 24h. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA. Results(1) Verticine, ebeiedine or suchengbeisine inhibited the expression of MUC5AC mucin gene induced by EGF, PMA or TNF-α; (2) The production of MUC5AC mucin protein induced by EGF, PMA or TNF-α were also inhibited by treatment of verticine, ebeiedine or suchengbeisine. ConclusionThese results suggest that verticine, ebeiedine and suchengbeisine isolated from the bulbs of Fritillaria thunbergii inhibit the gene expression and production of MUC5AC mucin, by directly acting on airway epithelial cells, and the results are consistent with the traditional use of Fritillaria thunbergii as remedy for diverse inflammatory pulmonary diseases.

Dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppressed the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor

BackgroundThe root of Asparagus cochinchinensis (Lour.) Merr. has been utilized as mucoregulators and expectorants for controlling the airway inflammatory diseases in folk medicine. Hypothesis/purposeWe investigated whether dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis (Lour.) Merr. suppress the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor. Study designConfluent NCI-H292 cells were pretreated with dioscin or methylprotodioscin for 30 min and then stimulated with EGF or PMA for 24 h. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA. Results(1) Dioscin and methylprotodioscin suppressed the expression of MUC5AC mucin gene induced by EGF or PMA; (2) dioscin suppressed the production of MUC5AC mucin induced by either EGF at 10−5 M (p < 0.05) and 10−6 M (p < 0.05) or PMA at 10−4 M (p < 0.05), 10−5 M (p < 0.05) and 10−6 M (p < 0.05); (3) methylprotodioscin also suppressed the production of MUC5AC mucin induced by either EGF at 10−4 M (p < 0.05) or PMA at 10−4 M (p < 0.05). ConclusionThese results suggest that dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppress the gene expression and production of MUC5AC mucin, by directly acting on airway epithelial cells, and the results are consistent with the traditional use of Asparagus cochinchinensis as remedy for diverse inflammatory pulmonary diseases.

TMEM16A mediates the hypersecretion of mucus induced by Interleukin-13

Previous studies showed that the Ca2+-activated Cl− channel (CaCC) was involved in the pathogenesis of mucus hypersecretion induced by Interleukin-13 (IL-13). However, the mechanisms underlying the process were unknown. Recently, transmembrane protein 16A (TMEM16A) was identified as the channel underlying the CaCC current. The aim of the current study was to investigate whether the TMEM16A channel is part of the mechanism underlying IL-13-induced mucus hypersecretion. We observed that both TMEM16A mRNA and protein expression were significantly up-regulated after treatment with IL-13 in human bronchial epithelial 16 (HBE 16) cells, which correlated with an increase in mucus production. Additionally, mucus hypersecretion in rat airways was induced by intratracheal instillation of IL-13 and similar increases were observed in the expression of TMEM16A mRNA and protein in the bronchial epithelium. Niflumic acid (NA), a selective antagonist of CaCC, markedly blocked IL-13-induced mucin (MUC) 5AC mRNA and protein production in vivo and in vitro. Further investigation with HBE16 cells revealed that TMEM16A overexpression clearly promoted mucus production, IκBα phosphorylation, and p65 accumulation in the nucleus. The loss of TMEM16A resulted in inhibition of mucus production, and the TMEM16A-mediated production of MUC5AC was significantly blocked by a nuclear factor-kappa B (NF-κB) inhibitor. Therefore, the TMEM16A channel acts upstream of NF-κB in the regulation of mucus production. This is the first demonstration that the TMEM16A-NF-κB pathway is positively involved in IL-13-induced mucus production, which provides novel insight into the molecular mechanism of mucin overproduction.

Impact of Staphylococcus Epidermidis Lysates on Middle Ear Epithelial Proinflammatory and Mucogenic Response

BackgroundChronic otitis media with effusion (COME) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Staphylococcus epidermidis, typically considered a commensal...

Butyrate modulates bacterial adherence on LS174T human colorectal cells by stimulating mucin secretion and MAPK signaling pathway.

BACKGROUND/OBJECTIVESFermentation of dietary fiber results in production of various short chain fatty acids in the colon. In particular, butyrate is reported to regulate the physical and functional integrity of the normal colonic mucosa by altering mucin gene expression or the number of goblet cells. The objective of this study was to investigate whether butyrate modulates mucin secretion in LS174T human colorectal cells, thereby influencing the adhesion of probiotics such as Lactobacillus and Bifidobacterium strains and subsequently inhibiting pathogenic bacteria such as E. coli. In addition, possible signaling pathways involved in mucin gene regulation induced by butyrate treatment were also investigated.MATERIALS/METHODSMucin protein content assay and periodic acid-Schiff (PAS) staining were performed in LS174T cells treated with butyrate at various concentrations. Effects of butyrate on the ability of probiotics to adhere to LS174T cells and their competition with E. coli strains were examined. Real time polymerase chain reaction for mucin gene expression and Taqman array 96-well fast plate-based pathway analysis were performed on butyrate-treated LS174T cells.RESULTSTreatment with butyrate resulted in a dose-dependent increase in mucin protein contents in LS174T cells with peak effects at 6 or 9 mM, which was further confirmed by PAS staining. Increase in mucin protein contents resulted in elevated adherence of probiotics, which subsequently reduced the adherent ability of E. coli. Treatment with butyrate also increased transcriptional levels of MUC3, MUC4, and MUC12, which was accompanied by higher gene expressions of signaling kinases and transcription factors involved in mitogen-activated protein kinase (MAPK) signaling pathways.CONCLUSIONSBased on our results, butyrate is an effective regulator of modulation of mucin protein production at the transcriptional and translational levels, resulting in changes in the adherence of gut microflora. Butyrate potentially stimulates the MAPK signaling pathway in intestinal cells, which is positively correlated with gut defense.

Apigenin Inhibits Tumor Necrosis Factor-α-Induced Production and Gene Expression of Mucin through Regulating Nuclear Factor-Kappa B Signaling Pathway in Airway Epithelial Cells.

In the present study, we investigated whether apigenin significantly affects tumor necrosis factor-α (TNF-α)-induced production and gene expression of MUC5AC mucin in airway epithelial cells. Confluent NCI-H292 cells were pretreated with apigenin for 30 min and then stimulated with TNF-α for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription - polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Apigenin significantly inhibited MUC5AC mucin production and down-regulated MUC5AC gene expression induced by TNF-α in NCI-H292 cells. To elucidate the action mechanism of apigenin, effect of apigenin on TNF-α-induced nuclear factor kappa B (NF-κB) signaling pathway was also investigated by western blot analysis. Apigenin inhibited NF-κB activation induced by TNF-α. Inhibition of inhibitory kappa B kinase (IKK) by apigenin led to the suppression of inhibitory kappa B alpha (IκBα) phosphorylation and degradation, p65 nuclear translocation. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Apigenin also has an influence on upstream signaling of IKK because it inhibited the expression of adaptor protein, receptor interacting protein 1 (RIP1). These results suggest that apigenin can regulate the production and gene expression of mucin through regulating NF-κB signaling pathway in airway epithelial cells.

Luteolin inhibited the gene expression, production and secretion of MUC5AC mucin via regulation of nuclear factor kappa B signaling pathway in human airway epithelial cells

Luteolin, a flavonoidal compound derived from Lonicera japonica Thunb. and Chrysanthemum indicum L., has been reported to show anti-inflammatory, anti-oxidative and anti-carcinogenic effects. In this study, we investigated whether luteolin significantly affects the secretion, production and gene expression of airway mucin. Confluent NCI-H292 cells were pretreated with luteolin for 30 min and then stimulated with EGF (epidermal growth factor) or PMA (phorbol 12-myristate 13-acetate) for 24 h or the indicated periods. The MUC5AC mucin gene expression was measured by RT-PCR. Production and secretion of MUC5AC mucin protein were measured by ELISA. To elucidate the action mechanism of luteolin, effect of luteolin on PMA-induced NF-κB signaling pathway was investigated by western blot analysis. The results were as follows: (1) Luteolin inhibited the secretion of MUC5AC mucin protein induced by EGF or PMA; (2) Luteolin inhibited the production of MUC5AC mucin protein and the expression of MUC5AC mucin gene induced by EGF or PMA; (3) Luteolin inhibited PMA-induced phosphorylation and degradation of inhibitory kappa Bα (IκBα); (4) Luteolin inhibited PMA-induced phosphorylation and nuclear translocation of nuclear factor kappa B (NF-κB) p65. This result suggests that luteolin can regulate the secretion, production and gene expression of mucin by acting on airway epithelial cells via regulation of NF-kB signaling pathway.

DA-6034-induced mucin secretion via Ca2+-dependent pathways through P2Y receptor stimulation.

We evaluated whether DA-6034 is involved in mucin secretion via P2Y receptor activation and/or intracellular Ca2+ concentration ([Ca2+]i) change. Also, we investigated the effect of P2Y receptor inhibitors or Ca2+ chelators on the DA-6034-induced mucin secretion and [Ca2+]i increases. Effects of DA-6034 on mucin expression in primary, cultured, conjunctival epithelial cells was studied using RT-PCR, Western blot analysis, and periodic acid-schiff (PAS) staining. To evaluate thin film layer thickness generated by mucin and fluid secretion, cells were incubated in DA-6034 with/without P2Y antagonists or extracellular/intracellular Ca2+ chelators, and were imaged with confocal microscope using Texas Red-dextran dye. In addition, DA-6034-induced Ca2+-dependent Cl- channels opening was evaluated using perforated patch clamp. Fluo-4/AM was used to measure changes in [Ca2+]i induced by DA-6034 in Ca2+-free or Ca2+-containing buffered condition, as well as P2Y antagonists. DA-6034 induced the expression of mucin genes, production of mucin protein, and increase of number of mucin-secreting cells. P2Y antagonists inhibited DA-6034-induced mucin and fluid secretion, which was also affected by extracellular/intracellular Ca2+ chelators. DA-6034 stimulated Cl- channel opening and [Ca2+]i elevation. Further, [Ca2+]i increases induced by DA-6034 were lacking in either P2Y antagonists or Ca2+-free buffered condition, and diminished when endoplasmic reticulum Ca2+ was depleted by cyclopiazonic acid in Ca2+-free buffered condition. This study demonstrated that DA-6034 has a potential to induce mucin secretion via Ca2+-dependent pathways through P2Y receptors in multilayer, cultured, human conjunctival epithelial cells.