Effect Of ERK On Asthmatic Rat Airway Smooth Muscle Cells CyclinD1 And Role Of CyclinD1 In ERK Regulating The Proliferation Of Airway Smooth Muscle Cells In Rat

Objective:To investigate the effects of ERK on asthma airway remodeling and the expressions of CyclinD1, To investigate the effects of CyclinDl on the proliferation of air way smooth muscle cells regulated by ERK in rat.Methods:This study consists of two Parts.1. The rat asthma model was made through ovalbumin(OVA) sensitization and excitation. Primary cultures of ASMCs were established and cells between passages 3 and 6 were used for experiments. ASMCs were treated with ERK activator epidermal growth factor (EGF) and inhibitor PD98059, and then subdivided into 5 groups based on the drug interventions: (1)Normal control group; (3)Asthmatic Control group;(3)Asthmatic EGF group;(4)P+E group,PD98059 lOμmol/L+EGF 20ng/ml; (5) PD98059 group. The proliferations of ASMCs were examined with cell cycle analysis and MTT colorimetric assay, The expressions of CyclinD1 mRNA were detected by RT-PCR, and the protein expression of CyclinD1 was measured by FCM.2. The rat asthma model was made through ovalbumin(OVA) sensitization and excitation. Primary cultures of ASMCs were established and cells between passages 3 and 6 were used for experiments. ASMCs were treated with ERK activator epidermal growth factor (EGF) and inhibitor PD98059, and then subdivided into 4 groups based on the drug interventions:(1) control group; (2) EGF group;(3)P+E group,PD98059 lOμmol/L+EGF 20ng/ml;(2) PD98059 group. The proliferations of ASMCs were examined with cell cycle analysis and MTT colorimetric assay, The expressions of ERK 1/2 mRNA and CyclinDl mRNA were detected by RT-PCR, The data were subject to correlation analysis.RESULTS:1. (1)Compared to asthmatic (nomal) control group, there were significant differences in the percentage of S+G2/M phase and absorbance A value of MTT in EGF group and PD98059 group (P＜0.05).(2)Compared with asthmatic (nomal) control group, the ratios of A values of CyclinDl mRNA and protein in group EGF and PD98059 were significantly changed (P＜0.05),There were no significant differences between control and P+E group(P>0.05).2. (1)Compared to Asthmatic control group, there were significant differences in the percentage of S+G2/M phase and absorbance A value of MTT in Asthmatic EGF group and Asthmatic PD98059 group (P<0.05); Compared with Normal control group, there were significant differences else in that values.(2)Compared with Asthmatic control group, the ratios of A values of ERK-mRNA in group EGF and PD98059 were significantly changed (P<0.05) as well as the ratios of A values of CyclinD1 mRNA. There were no significant differences between Asthmatic control and Asthmatic P+E group (P＞0.05). The same tendency in Normal group was observed according with Asthmatic group.(3)There were positive correlations between ERK and CyclinDl in mRNA (r=0.565, P<0.01).CONCLUSIONS:1. The activation of ERK promotes ASMCs proliferation and expressions of CyclinDl in asthmatic rats.Indicating that the expression of CyclinD1 transcription may be dependent on the ERK activation in asthmatic rats.The finding will help to understand the mechanisms of airway remodeling.2. The activation of ERK promotes ASMCs proliferation in asthmatic rats, and there positive correlations between the ERK and CyclinD1 in mRNA and protein, indicating that the ERK signal pathway may be involved in the process of air way smooth muscle remodeling by regulating the expression of CyclinD1 in asthmatic rats. The finding will help to understand the mechanisms of asthmatic ASMCs involved in abnormal proliferation.