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Lentiviral shRNA against KCa3.1 inhibits allergic response in allergic rhinitis and suppresses mast cell activity via PI3K/AKT signaling pathway.

Lin H, Zheng C, Li J, Yang C, Hu L - Sci Rep (2015)

Bottom Line: KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction.LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels.LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Otorhinolaryngology, Shanghai Jiao Tong University Affiliated Sixth People's hospital, Shanghai, China [2] Department of Otorhinolaryngology, Eye and ENT Hospital of Fudan University, Shanghai, China.

ABSTRACT
Calcium-activated potassium ion channel-3.1 (KCa3.1) plays a pivotal role in the potassium-calcium exchange involved in atopy. This study aimed to explore the impact of lentiviral-mediated shRNA silencing KCa3.1 on allergic response in a murine allergic rhinitis (AR) model. The BALB/c mice were divided into four groups: untreated AR group, negative control AR group, lentiviral KCa3.1-shRNA treated AR group and normal control group. Concentrations of ovalbumin (OVA)-specific IgE, histamine and leukotrienes C4 (LTC4) in serum, and IL-4, IL-9 and IL-17 in nasal lavage fluid (NLF) were analyzed. Goblet cells and mast cells were counted. KCa3.1 positive cells were counted after immunolabelling by immunofluorescence method. KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction. Furthermore, P815 cell line was used to explore the role and mechanism of lentiviral KCa3.1-shRNA on mast cells. The results showed that LV-KCa3.1-shRNA intervention effectively attenuated allergic responses in LV-KCa3.1-shRNA treated mice. LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels. LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway.

No MeSH data available.


Related in: MedlinePlus

KCa3.1 immunolabelling assayed by immunofluorescence in UnT-AR group, NeC-AR group, Ksh-AR and normal control group.Arrows indicate KCa3.1 positive mucosal epithelial cells. Empty triangles indicate KCa3.1 positive epithelial cells surrounding basal lamina. Filled triangles indicate KCa3.1 positive sub-mucosal inflammatory cells. Scale bar = 50 μm.
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f4: KCa3.1 immunolabelling assayed by immunofluorescence in UnT-AR group, NeC-AR group, Ksh-AR and normal control group.Arrows indicate KCa3.1 positive mucosal epithelial cells. Empty triangles indicate KCa3.1 positive epithelial cells surrounding basal lamina. Filled triangles indicate KCa3.1 positive sub-mucosal inflammatory cells. Scale bar = 50 μm.

Mentions: As depicted by immunofluorescence staining (Fig. 4), KCa3.1 positive cells were mainly epithelial cells among mucosal surface and surrounding basal lamina, and sub-mucosal inflammatory cells in the nasal mucosa. KCa3.1 was significantly overexpressed in AR group compared with control group; notably, LV-KCa3.1-shRNA treatment resulted in the reduction of KCa3.1 protein levels in LV-KCa3.1-shRNA treated group compared with untreated AR or negative control group, respectively (P < 0.01). These results were consistent with the changes in the nasal symptoms and cytokine levels following LV-KCa3.1-shRNA treatment.


Lentiviral shRNA against KCa3.1 inhibits allergic response in allergic rhinitis and suppresses mast cell activity via PI3K/AKT signaling pathway.

Lin H, Zheng C, Li J, Yang C, Hu L - Sci Rep (2015)

KCa3.1 immunolabelling assayed by immunofluorescence in UnT-AR group, NeC-AR group, Ksh-AR and normal control group.Arrows indicate KCa3.1 positive mucosal epithelial cells. Empty triangles indicate KCa3.1 positive epithelial cells surrounding basal lamina. Filled triangles indicate KCa3.1 positive sub-mucosal inflammatory cells. Scale bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4536635&req=5

f4: KCa3.1 immunolabelling assayed by immunofluorescence in UnT-AR group, NeC-AR group, Ksh-AR and normal control group.Arrows indicate KCa3.1 positive mucosal epithelial cells. Empty triangles indicate KCa3.1 positive epithelial cells surrounding basal lamina. Filled triangles indicate KCa3.1 positive sub-mucosal inflammatory cells. Scale bar = 50 μm.
Mentions: As depicted by immunofluorescence staining (Fig. 4), KCa3.1 positive cells were mainly epithelial cells among mucosal surface and surrounding basal lamina, and sub-mucosal inflammatory cells in the nasal mucosa. KCa3.1 was significantly overexpressed in AR group compared with control group; notably, LV-KCa3.1-shRNA treatment resulted in the reduction of KCa3.1 protein levels in LV-KCa3.1-shRNA treated group compared with untreated AR or negative control group, respectively (P < 0.01). These results were consistent with the changes in the nasal symptoms and cytokine levels following LV-KCa3.1-shRNA treatment.

Bottom Line: KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction.LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels.LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Otorhinolaryngology, Shanghai Jiao Tong University Affiliated Sixth People's hospital, Shanghai, China [2] Department of Otorhinolaryngology, Eye and ENT Hospital of Fudan University, Shanghai, China.

ABSTRACT
Calcium-activated potassium ion channel-3.1 (KCa3.1) plays a pivotal role in the potassium-calcium exchange involved in atopy. This study aimed to explore the impact of lentiviral-mediated shRNA silencing KCa3.1 on allergic response in a murine allergic rhinitis (AR) model. The BALB/c mice were divided into four groups: untreated AR group, negative control AR group, lentiviral KCa3.1-shRNA treated AR group and normal control group. Concentrations of ovalbumin (OVA)-specific IgE, histamine and leukotrienes C4 (LTC4) in serum, and IL-4, IL-9 and IL-17 in nasal lavage fluid (NLF) were analyzed. Goblet cells and mast cells were counted. KCa3.1 positive cells were counted after immunolabelling by immunofluorescence method. KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction. Furthermore, P815 cell line was used to explore the role and mechanism of lentiviral KCa3.1-shRNA on mast cells. The results showed that LV-KCa3.1-shRNA intervention effectively attenuated allergic responses in LV-KCa3.1-shRNA treated mice. LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels. LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway.

No MeSH data available.


Related in: MedlinePlus