| AIM:To investigate the effect of TGF-β1 on airway smooth muscle cells (ASMCs) proliferation and expression of ERK1/2 mRNA and P-ERK1/2 in different periods of asthmatic rats.METHODS:15 healthy male wistar rats were accustomed to feed for a week and divided into three groups, the experimental group rats were sensitized by intraperitoneal injection with ovalbumin (OVA) and exposed to aerosolized OVA two weeks later to be challenged, A group of excited 2 weeks (the early stages of asthma), another group of excited 6 weeks (chronic asthma model). Control group was injected with saline and exposed to aerosolized saline two weeks later. After the establishment of experimental asthma model is divided into two parts.1. The first part to investigate the effect of TGF-β1 on airway smooth muscle cells (ASMCs) proliferation in different periods of asthmatic rats.Primary cultures of ASMCs were established for experiments, ASMCs were treated with 1μg/L,10μg/L,100μg/L TGF-β1. ASMCs were divided into 5 groups randomly:(1) control group;(2) model group;(3),(4),(5) were different concentrations of TGF-β1 intervened groups. Proliferation of ASMCs were detected by flow cytometry and MTT colorimetric assay, observe the effect of different concentrations of TGF-β1 on the proliferation of ASMCs.2. The second part to investigate the effect of TGF-β1 on airway smooth muscle cells (ASMCs) Expression of ERK1/2 mRNA and P-ERK1/2 in different period asthmatic rats. Primary cultures of ASMCs were established for experiments, ASMCs were treated with 10μg/L TGF-β1 and extracellular signal-regulated kinase (ERK) inhibitor PD98059. ASMCs were divided into 5 groups randomly:(1) control group; (2) model group; (3) TGF-β1 group; (4) PD-98059 group; (5) TGF-β1+PD-98059 group. The expressions of ERK1/2 mRNA were detected by RT-PCR, the location and protein expressions of phosphorylated forms of ERK1/2(p-ERK1/2) were examined by immunocytochemical staining.RESULTS:1. (1) the percentage of cells at S phase,A value of 2 week and 6 week asthmatic ASMCs were significantly increased compared with control group (P<0.01); compared with model group, the percentage of cells of at S phase and A value of 2 week and 6 week asthmatic ASMCs, which were cultured in different concentration of TGF-β1, were significantly higher than the asthmatic group (P＜0.01). The percentage of cells of at S phase and A value of 10μg/L and 100μg/L TGF-β1 group were significantly higher than 1μg/L TGF-β1 group (P＜0.01); and there was no significant change in ASMCs proliferation compared 10μg/L TGF-β1 group with 100μg/L TGF-β1 group (P>0.05). There was no significant difference in the effect of TGF-β1 on ASMCs proliferation between 2 week and 6 week asthmatic model group (P>0.05). (2) the percentage of cells at S phase in different TGF-β1 intervened group were increased by TGF-β1 in a dose-dependent manner.2. The results showed that in2W,6W asthmatic groups, compared with that in the control group, The expressions of ERK mRNA, p-ERK1/2 protein and the activation ratio of ERK in ASMCs in asthmatic groups were significantly increased compared with those in the control group. After treatment with PD98059, the expressions of ERK mRNA, p-ERK1/2 protein and the activation ratio of ERK in ASMCs in asthmatic groups were significantly decreased compared with those in the control group. After treatment with group TGF-β1, the expressions of ERK mRNA, p-ERK1/2 protein and the activation ratio of ERK in ASMCs in 2W and 6W asthmatic group were significantly decreased compared with those in their asthmatic group. And this role can be partly inhibited by PD98059.There is no difference between the2W and 6W asthmatic model group with these results.3. ERK 1/2 mRNA and p-ERK1/2 expressions on ASMCs were positively correlated with the percentage of cells at S phase in asthmatic rats.CONCLUSION:1. The first part of the experiments show that, compared with normal rats, the proliferation of ASMCs in different periods of asthmatic rats were obvious, the percentage of cells at S phase was increasing. And ASMCs in different periods of asthmatic rats were intervened by TGF-β1, the percentage of cells at S phase was increased and the proliferation of ASMCs was enhanced. TGF-β1 may contribute to the proliferation of ASMCs in the early stage of asthmatic rats, and significantly promote the proliferation of ASMCs in different periods of asthmatic rats.2. The second part of the experiments show that, TGF-β1 up-regulated Expression of ERK1/2 mRNA and P-ERK1/2 in different periods asthmatic rats, At different stages of asthma, TGF-β1 is likely to continue through the promotion of ERK signal transduction pathway of the activity on the proliferation of airway smooth muscle cells regulate.
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