Roxithromycin treatment inhibits TGF-β1-induced activation of ERK and AKT and down-regulation of caveolin-1 in rat airway smooth muscle cells

Abstract

Background: Roxithromycin (RXM) has been widely used in asthma treatment; however, the mechanism has not been fully understood. The aim of our study was to investigate the underlying mechanism of RXM treatment in mediating the effect of transforming growth factor (TGF)-β1 on airway smooth muscle cells (ASMCs) proliferation and caveolinn-1 expression.

Methods: Firstly, the rat ovalbumin (OVA) model was built according to the previous papers. Rat ASMCs were prepared and cultured, and then TGF-β1 production in ASMCs was measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the proliferation of ASMCs was determined using cell counting kit (CCK-8) assay. Additionally, the expressions of caveolin-1, phosphorylated-ERK1/2 (p-ERK1/2) and phosphorylated-AKT (p-AKT) in ASMCs treated with or without PD98059 (an ERK1/2 inhibitor), wortannin (a PI3K inhibitor), β-cyclodextrin (β-CD) and RXM were measured by Western blot. Finally, data were evaluated using t-test or one-way ANOVA, and then a P value < 0.05 was set as a threshold.

Results: Compared with normal control, TGF-β1 secretion was significantly increased in asthmatic ASMCs; meanwhile, TGF-β1 promoted ASMCs proliferation (P < 0.05). However, ASMCs proliferation was remarkably inhibited by RXM, β-CD, PD98059 and wortmannin (P < 0.05). Moreover, the expressions of p-ERK1/2 and p-AKT were increased and peaked at 20 min after TGF-β1 stimulation, and then suppressed by RXM. Further, caveolin-1 level was down-regulated by TGF-β1 and up-regulated by inhibitors and RXM.

Conclusion: Our findings demonstrate that RXM treatment inhibits TGF-β1-induced activation of ERK and AKT and down-regulation of caveolin-1, which may be the potential mechanism of RXM protection from chronic inflammatory diseases, including bronchial asthma.

Figure 1

TGF-β1 production in ASMCs and the effect of TGF-β1 on proliferation of ASMCs. (A) Supernatants were collected after culture for 24 hours. Cytokine TGF-β1 was measured by ELISA. Data are expressed as mean ± SD for each group. (*P < 0.05 versus control). (B) Proliferation of ASMCs in each group was measure by CCK-8 assay. Data are expressed as mean ± SD for each group. TGF-β1 significantly promoted cell proliferation, which could be inhibited by PD98059, wortmannin and β-CD (*P < 0.05 versus control and asthma group, ^#P < 0.05 versus control group, **P < 0.05 versus TGF-β1 group). No significant change between inhibitor group and TGF-β1 + inhibitor group was observed.

Figure 2

The presence of caveolae and caveolin-1 in ASMCs. (A) Normal caveolae structures were observed in the control group by scanning electron microscope (×7000). All the scale bars are 2 μm. However, there were fewer caveolae structures observed in the asthma group compared with control group. The locations of caveolae were indicated with arrows. (B) Western blot was used for analysis of caveolin-1 in separate group. GAPDH served as a loading control. The expression of caveolin-1 protein was significantly decreased in the asthma group. Data are expressed as mean ± SD for each group. (*P < 0.05 versus control).

Figure 3

The time course of ERK and AKT activation in ASMCs stimulated with TGF-β1. (A, C) Asthmatic ASMCs were incubated with TGF-β1 (10 ng/ml) for 5, 10, 15, 20 and 60 min. The expression of p-ERK1/2 protein was assessed by western blot and analyzed by densitometry compared to ERK1/2 expression. The expression of p-ERK1/2 protein in ASMCs was increased and peaked at 20 min after TGF-β1 stimulation (*P < 0.05 versus control, 5, 10, 15, 60 min groups). (B, D) Asthmatic ASMCs were incubated with TGF-β1 (10 ng/ml) for 5, 10, 15, 20, and 60 min. The expression of p-AKT protein was assessed by western blot and analyzed by densitometry compared to AKT expression. The expression of p-AKT protein in ASMCs was increased and peaked at 20 min after TGF-β1 stimulation (*P < 0.05 versus control, 5, 10, 15, 60 min groups).

Figure 4

Caveolin-1 suppressed expression of phosphorylated PI3K and ERK1/2 in ASMCs. Cultured and serum-deprived ASMCs were treated with TGF-β1 for 20 min for p-ERK1/2 (B, D) and for p-AKT (F, H) measurement, with or without preincubation with PD98059, wortmannin, β-CD and RXM. The expressions of p-ERK1/2 and p-AKT were up-regulated by TGF-β1 and significantly down-regulated by PD98059, wortmannin, β-CD and RXM. (A, C, E, G) Asthmatic ASMCs were treated with TGF-β1 for 48 h for caveolin-1 measurement, with or without preincubation with PD98059, wortmannin, β-CD and RXM. The expression of caveolin-1 was down-regulated by TGF-β1and up-regulated by PD98059, wortmannin and RXM. Data are expressed as mean ± SD for each group (*P < 0.05 versus asthma and control group, & P < 0.05 versus control group, ^#P < 0.05 versus TGF-β1 group).