阿托伐他汀对博莱霉素诱导大鼠肺成纤维细胞MMP-9及TIMP-1表达影响

Effect of atorvastatin on the expression of of MMP-9 and TIMP-1 in bleomycin-induced pulmonary fibroblast of rats

目的本研究观察阿托伐他汀对博莱霉素诱导大鼠肺成纤维细胞的干预作用及其分泌的MMP-9、TIMP-1的影响，为探讨阿托伐他汀抗肺纤维化作用的机制提供实验依据。方法博莱霉素制备大鼠肺纤维化模型，1周后处死，提取肺成纤维细胞，阿托伐他汀进行干预。MTT法测定细胞存活率；底物酶谱法分析IV型胶原酶的分泌及活性；[3H]-脯氨酸渗入评价脯氨酸含量的变化；RT—PCR检测细胞分泌的MMP-9、TIMP—1mRNA的表达；Western—blot检测细胞培养液中MMP-9、TIMP-1的蛋白表达。结果阿托伐他汀具有显著抑制成纤维细胞增殖的作用，与对照组比较，1μM的阿托伐他汀组的细胞数降至（78．5±8）％（P〈0．001），5μM组的细胞数降至（49．8±12．6）％（P〈0．0001），10μM组的细胞数降至（36．1±10．7）％（P〈0．0001），0．01μM和0．1μM的阿托伐他汀对细胞的抑制与对照组相比较无显著的差异，分别为（98．3±10．8）％（P=0．35），（92．2±8．4）％（P=0．13）。Gelatin Zymography明胶酶谱分析结果表明：暴露于1μM和5μM的阿托伐他汀72h的肺成纤维细胞分泌的MMP-9分别减少至（79．11±14．39）％（P=0．1）和（56．74±12．98）％（P〈0．01），而MMP-2分别为（103．90±5．46）％，（79．07±16．75）％；后者虽有下降趋势但并无统计学意义。Westernblot分析细胞培养液中MMP-9、TIMP-1酶活性示：暴露于1μM和5μM的阿托伐他汀72h的肺成纤细胞分泌的MMP-9分别减少至（84．67±11．74）％（P=0．31）和（29．15±6．03）％（P〈0．05）。TIMP-1分别减少至（48．89±1．92）％（P〈0．01）和（30．37±2．80）％（P〈0．001）。Westernblot显示暴露于1μM和5μM的阿托伐他汀72h的肺成纤细胞分泌的胶原IV分别减少至（65．9±9．32）（P〈0．05）、（42．6±8．5）％（P〈0．01）。与之类似，暴露于5μM的阿托伐他汀72h的肺成纤细胞分泌的纤维连接蛋白减少至对照组的（20．6±4．2）％fP〈0．01）。[3H]-脯氨酸渗入结果显示：1μM的阿托伐他汀减少脯氨酸的结合至（77．1±15．0）％（P〈0．05），5μM的阿托伐他汀减少至（54．7±6．6）％fP〈0．05）。RT—PCR的分析阿托伐他汀对成纤维细胞的MMP-9、TIMP-1的mRNA表达的影响结果显示，不同浓度的阿托伐他汀处理肺纤维化成纤维细胞72h后MMP-9、TIMP-1InRNA表达均减少（P〈0.05）。结论阿托伐他汀具有抑制博莱霉素诱导大鼠肺成纤维细胞增殖的作用，并能抑制成纤维细胞分泌的MMP-9、TIMP-1。

Objective To investigate the effect of atorvastatin on the expression of MMP-9 and TIMP-1 in bleomycin-induced pulmonary fibroblast of rats. Methods On the week 1, the pulmonary fibroblast were isolated, and the cells were exposed to Atorvastatin. Cell growth was evaluated by MTT. MMP-9, TIMP-1 expression in fibroblast was confirmed by reverse transcription-PCR （RT-PCR）. Western Blot Analysis was used for evaluating the expression of MMP-9, TIMP-1, Collagen IV, and Fibroneetin. Gelatin Zymography was used to assess the activity of MMP-9. [3H] -Profine Incorporation was used as an additional relative estimate of collagen production. Results MTT indicated atorvastatin had a pronounced antiproliferative effect, with the cell number falling to （77.9 ± 3 ） % （P 〈 0.001） at 1 μM of control values, （49.0 ± 7.9）% （P 〈 0.0001） at 5μM, （37.5 ± 8.6）% （P 〈 0.0001） at 10 μM. Gelatin Zymography demonstrated the change of MMP-9 secretion after exposure atorvastatin. There was a decrease in the cellular secretion of MMP-9 to （79.11 ± 14.39）%, （56.74± 12.98） % （P 〈 0.01） at 1μM and 5 μM of control values, respectively. There was a reduction in MMP-2 levels secreted after exposure to atorvastatin but the difference did not reach statistical significance. Exposure of Fb to atorvastatin for 72 hours resulted in a reduction in secretion of both MMP-9 and TIMP-1. MMP-9, TIMP-1 secretion were significantly reduced by both 1 μM atorvastatin [（84.67 ± 11.74）%, （48.89± 1.92）%, P〈0.05, P〈0.001）] and 5 μM Atorvastatin （29.15±6.03）%, （30.37 ± 2.80） （P〈0.05, P〈0.001）. Exposure of Fibroblat to Atorvastatin 1 and 5 IX M for 72 hours resulted in a decrease in the levels of secreted type IV collagen levels to （65.9±9.32）（P〈0.01）, （42.6±8.5）%（P 〈0.05）, respectively. Similarly, exposure of Fibroblat to 5μM Atorvastatin for 72 hours resulted in a reduction in the levels of secreted fibronectin levels to （20.6±4.2） （P〈0.01） of that observed in control cells. Furthermore, atorvastatin resulted in the progressive reductions in proline incorporation, which was （77.1 ± 15.0）% （P〈0.05） by 1μMAtorvastatin exposure and （54.7 ±6.6）% （P〈0.05） by 5 IX M atorvastatin exposure. Conclusion Atorvastatin may inhibit the proliferation of pulmonary fibroblasts and decrease the expression of MMP-9 and TIMP-1 in pulmonary fibroblasts.