PAI-1对气道平滑肌增殖和ERK表达的影响

Effect of PAI-1 on proliferation of airway smooth muscle cells and expression of extracellular regulated protein kinase

目的探讨纤溶酶原激活物抑制物-1(PAI-1)对气道平滑肌增殖的调控作用,以及对细胞外调节蛋白激酶(ERK)表达的影响。方法体外培养小鼠气道平滑肌细胞(ASMCs)并分为7组:空白对照组、PAI-15μg/L组、PAI-1 10μg/L组、PAI-1 20μg/L组、PAI-1 40μg/L组、PAI-1 80μg/L组、PAI-1 100μg/L组。放入37℃培养箱分别培养12、24和48 h,采用CCK-8检测ASMCs的A值,计算增殖率。以增殖率最高的实验组的浓度和作用时间作为PAI-1作用最适浓度和最适作用时间,进一步分6组:A组(空白对照);B组(PAI-1 20μg/L);C组(PAI-1 40μg/L);D组(PAI-1 80μg/L);E组(PAI-1 100μg/L);F组(PAI-1最适浓度+ERK通道抑制剂PD98059 10μmol/L),用CCK-8检测ASMCs的增殖,Western blot检测ERK蛋白的表达,实时荧光定量聚合酶链反应检测ERK m RNA的表达。结果 5~100μg/L PAI-1作用ASMCs 12、24和48 h后细胞增殖率结果显示,在相同浓度下,不同时间各组比较,差异有统计学意义(P<0.05),以48 h时ASMCs增殖率最高;在相同时间下,不同浓度各组比较,差异有统计学意义(P<0.05),80μg/L PAI-1的ASMCs增殖率最高;故选取48 h为最适作用时间,80μg/L为最适浓度进行后续实验。再次分组的实验结果表明,B、C、D、E组的ERK磷酸化水平和ERK m RNA的相对表达量与A组比较,差异有统计学意义(P<0.05),B、C、D、E组增高;B、C、D、E组两两比较结果显示,除B组与E组比较,差异无统计学意义(P>0.05)外,其余各组两两比较,差异有统计学意义(P<0.05),当PAI-1浓度波动于20~80μg/L时,ERK磷酸化水平和ERK m RNA相对表达量随浓度升高而增高,>80μg/L后则不再增高;加入PD98059的F组与D组比较,差异有统计学意义(P<0.05),ERK磷酸化水平和ERK m RNA表达降低。结论外源性PAI-1可以通过促进ERK通路的表达,进而促进ASMCs的增殖。

Objective To investigate the regulation of plasminogen activator inhibitor-1(PAI-1) on the proliferation of airway smooth muscle cells(ASMCs) and its effect on extracellular regulated protein kinase(ERK). Methods The ASMCs of mice cultured in vitro were divided into 7 groups: control group, PAI-15 μg/L group, PAI-1 10 μg/L group, PAI-1 20 μg/L group, PAI-1 40 μg/L group, PAI-1 80 μg/L group and PAI-1 100 μg/L group. All groups were cultured respectively for 12, 24 and 48 h in a 37℃ incubator. The absorbance values were tested by CCK-8, and the proliferation rates were calculated. The concentration and intervention time of the experimental group with the highest proliferation rate were taken as the optimal concentration and time of PAI-1 action. Then the subjects were further divided into 6 groups: group A(blank control), group B(PAI-1 20 μg/L), group C(PAI-1 40 μg/L), group D(PAI-1 80 μg/L), group E(PAI-1100 μg/L) and group F(the optimal concentration of PAI-1+the inhibitor of ERK PD98059 10 μmol/L). The proliferation of ASMCs was detected by CCK-8. The expression of ERK protein was detected by Western blot.And the expression of ERK m RNA was detected by q RT-PCR. Results After 5-100 μg/L PAI-1 acted upon ASMCs for 12, 24 and 48 h, the proliferation rates of each test group at different time points were statistically different(P < 0.05), and the proliferation rates reached the maximal levels at the 48 th h; at the same time point the proliferation rates of the test groups with different concentrations were statistically different(P < 0.05),and the proliferation rates of ASMCs reached the peak at the concentration of 80 μg/L. Therefore, 80 μg/L of PAI-1 was chosen to be the optimum concentration, and 48 hours was set to be the optimal intervention period for the subsequent experiments. In the subsequent experiments, the results showed that compared to the group A, ERK phosphorylation levels and the expressions of ERK m RNA in the groups B, C, D and E were significantly increased(P < 0.05); in the pairwise comparison of the groups B, C, D and E, the ERK phosphorylation levels and the expressions of ERK m RNA in the ASMCs were statistically different only between the groups B and E(P < 0.05). When PAI-1 concentration varied from 20 to 80 μg/L, ERK phosphorylation level and the expression of ERK m RNA increased with the concentration; when the concentration went up over 80 μg/L, the phosphorylation level of ERK no longer increased. Compared with the group D, the ERK phosphorylation level and the expression of ERK m RNA were decreased in the group F(added with PD98059)(P < 0.05). Conclusions Exogenous PAI-1 may promote the proliferation of ASMCs through ERK pathway.