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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

Results of P815 cell culture and intervention experiments.Western blotting analysis of KCa3.1 levels (KCa3.1/β-actin) and phosphorylation of AKT (p-AKT/AKT) (a–c) tryptase mRNA levels assayed by real-time PCR (d). Concentrations of IL-6 and IL-18 in the culture supernatants (e–f). One-way ANOVA test was employed for intergroup comparison. *P < 0.01 vs blank control group. #P < 0.01 vs Ksh group. *P > 0.05 vs blank control group. The cropped blots images are shown in the figure and the full-length blots images are presented in Supplementary Information.
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f5: Results of P815 cell culture and intervention experiments.Western blotting analysis of KCa3.1 levels (KCa3.1/β-actin) and phosphorylation of AKT (p-AKT/AKT) (a–c) tryptase mRNA levels assayed by real-time PCR (d). Concentrations of IL-6 and IL-18 in the culture supernatants (e–f). One-way ANOVA test was employed for intergroup comparison. *P < 0.01 vs blank control group. #P < 0.01 vs Ksh group. *P > 0.05 vs blank control group. The cropped blots images are shown in the figure and the full-length blots images are presented in Supplementary Information.

Mentions: To further illustrate the roles and mechanism of LV-KCa3.1-shRNA on mast cell, P815 cell line was intervened with LV-KCa3.1-shRNA and LY294002 serving as a specific inhibitor of PI3K/AKT signaling pathway. As indicated in Fig 5a–c, strong bands of KCa3.1 and p-AKT protein were found in the blank control or negative control group, respectively; whereas weak bands were detected in LV-KCa3.1-shRNA group or LV-KCa3.1-shRNA +LY294002 group; notably, weaker bands were found in LV-KCa3.1-shRNA +LY294002 group compared with LV-KCa3.1-shRNA group. KCa3.1 levels (KCa3.1/β-actin) and phosphorylation of AKT (p-AKT/AKT) were suppressed by LV-KCa3.1-shRNA, and LY294002 enhanced these effects.


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)

Results of P815 cell culture and intervention experiments.Western blotting analysis of KCa3.1 levels (KCa3.1/β-actin) and phosphorylation of AKT (p-AKT/AKT) (a–c) tryptase mRNA levels assayed by real-time PCR (d). Concentrations of IL-6 and IL-18 in the culture supernatants (e–f). One-way ANOVA test was employed for intergroup comparison. *P < 0.01 vs blank control group. #P < 0.01 vs Ksh group. *P > 0.05 vs blank control group. The cropped blots images are shown in the figure and the full-length blots images are presented in Supplementary Information.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Results of P815 cell culture and intervention experiments.Western blotting analysis of KCa3.1 levels (KCa3.1/β-actin) and phosphorylation of AKT (p-AKT/AKT) (a–c) tryptase mRNA levels assayed by real-time PCR (d). Concentrations of IL-6 and IL-18 in the culture supernatants (e–f). One-way ANOVA test was employed for intergroup comparison. *P < 0.01 vs blank control group. #P < 0.01 vs Ksh group. *P > 0.05 vs blank control group. The cropped blots images are shown in the figure and the full-length blots images are presented in Supplementary Information.
Mentions: To further illustrate the roles and mechanism of LV-KCa3.1-shRNA on mast cell, P815 cell line was intervened with LV-KCa3.1-shRNA and LY294002 serving as a specific inhibitor of PI3K/AKT signaling pathway. As indicated in Fig 5a–c, strong bands of KCa3.1 and p-AKT protein were found in the blank control or negative control group, respectively; whereas weak bands were detected in LV-KCa3.1-shRNA group or LV-KCa3.1-shRNA +LY294002 group; notably, weaker bands were found in LV-KCa3.1-shRNA +LY294002 group compared with LV-KCa3.1-shRNA group. KCa3.1 levels (KCa3.1/β-actin) and phosphorylation of AKT (p-AKT/AKT) were suppressed by LV-KCa3.1-shRNA, and LY294002 enhanced these effects.

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