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. 2015 Sep 15;8(9):15146-54.
eCollection 2015.

The effect of siRNA-mediated lymphocyte-specific protein tyrosine kinase (Lck) inhibition on pulmonary inflammation in a mouse model of asthma

Shikui Zhang  1 , Rongjia Yang  1 , Yonghua Zheng  2
Affiliations

The effect of siRNA-mediated lymphocyte-specific protein tyrosine kinase (Lck) inhibition on pulmonary inflammation in a mouse model of asthma

Shikui Zhang et al. Int J Clin Exp Med. .

Abstract

Objective: To explore the effect of siRNA-mediated inhibition of lymphocyte-specific protein tyrosine kinase (Lck) on pulmonary inflammation in a mouse model of asthma.

Methods: A total of 32 female BABL/c mice were used in the study. The mouse asthma model was established with ovabumin (OVA), and Lck specific siRNA or nonspecific siRNA was transfected through the tail vein before the first OVA challenge. Two days after the last challenge, mice were sacrificed and bronchoalveolar lavage fluid (BALF), plasma and lung tissue were collected. Levels of Lck mRNA and protein in lung were detected by quantitative real-time PCR and western blot. The levels of IL-4 and IgE in BALF and plasma were detected with ELISA.

Results: Lck specific siRNA significantly inhibited expression of Lck mRNA and protein in T cells. In vivo transfection of Lck siRNA down regulated the expression of Lck mRNA and protein in lung parenchymal homogenates. Sensitized mice treated with Lck siRNA prior to OVA challenge had fewer eosinophils in BALF and in lung sections and lower levels of IL-4 and IgE in BALF and plasma compared to those treated with nonspecific siRNA.

Conclusions: Pretreatment of OVA sensitized mice with Lck siRNA results in attenuation of pulmonary inflammation following OVA challenge. Inhibition of Lck gene expression should be investigated further as a potential therapy for asthma.

Keywords: IL-4; IgE; T lymphocyte; in vivo transfection.

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Figures

Figure 1
Figure 1
Primary T cells were transfected with Lck FAM-siRNA and observed by common light microscopy (A) and fluorescent microscopy (B). The magnification is 200×.
Figure 2
Figure 2
In vitro transfection of Lck siRNA decreases Lck expression in cultured primary T cells. Primary T cells were transfected with FAM-siRNA, nonspecific siRNA or Lck siRNA. A. Expression levels of Lck in primary T cell were detected by western blot. β-actin served as the loading control. B. Quantitative analysis of the changes of Lck. Data represent mean ± SEM (n=3, *P < 0.05 vs control group). C. Total RNA was extracted 48 hours after transfection and quantitative real time PCR was performed using Lck primers and β-actin primers. The amount of Lck mRNA relative to β-actin is shown. Data represents mean ± SEM (n=3 *P < 0.05 vs control group).
Figure 3
Figure 3
In vivo transfection of Lck siRNA reduces Lck expression in mouse lung tissue. Mice were sensitized with OVA and transfected via tail vein injection with Lck siRNA or nonspecific siRNA and then challenged with OVA. Control mice were sensitized and challenged with PBS and not transfected. Mice were sacrificed and lung tissue was removed following the final OVA challenge. A. Expression levels of Lck in lung tissue were detected by western blot. β-actin served as the loading control. B. Quantitative analysis of the changes of Lck. Data represent mean ± SEM (n=3, *P < 0.05 vs control group). C. Total RNA was extracted from lung and expressions of Lck mRNA levels were detected by quantitative real-time PCR. The amount of Lck mRNA relative to β-actin is shown. Data represent mean ± SEM (n=3, *P < 0.05 vs control group).
Figure 4
Figure 4
Effect of Lck siRNA on markers of inflammation. Mice were sensitized with OVA and transfected via tail vein injection with nonspecific siRNA or Lck siRNA, and then challenged with OVA. Control mice were sensitized and challenged with PBS and untransfected. Following OVA challenge, mice were sacrificed and BALF and plasma were collected. The total number of leukocytes (A) and the percentage of eosinophils (B) in BALF were determined. BALF was analyzed by ELISA to determine the concentration of IL-4 (C) and IgE (D). Plasma was analyzed by ELISA to determine the concentration of IL-4 (E) and IgE (F). *P < 0.05, **P < 0.01, compared to nonspecific siRNA (n=9 per group).
Figure 5
Figure 5
Effect of Lck siRNA on lung histology. Lung tissue was taken from control mice (A) sensitized and challenged with PBS and untransfected, or from mice sensitized with OVA and transfected via tail vein injection with nonspecific siRNA (B) or Lck siRNA (C), then challenged with OVA. Tissues were stained with hematoxylin and eosin (H&E). Being compared with control mice (A), the number of the inflammatory cells such as eosinophils in the lung tissues increased in nonspecific siRNA mice (B), while they decreased in Lck siRNA mice (C). Magnification is 200×.
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