Therapeutic Effect Of Atorvastatin On Bleomycin-induced Pulmonary Fibrosis Of Rats And The Expression Of MMP-9 And TIMP-1

ObjectivePulmonary fibrosis, which is characterized with the proliferation of fibroblasts and deposition of extracelluar matrix(ECM) components, can be found in the late stage of many interstitial lung diseases and results in the dysfunction of organs. The fibrotic response, which is characterized by a striking increase in fibroblast population, a profound and complex change in ECM turnover, is generally considered an irreversible process leading to a progressive accumulation of connective tissue proteins or basement membrane disruption in early stage of the disease. In this context, an imbalance between the synthesis and degradation of ECM molecules in the local lung microenvironment appears to be of critical importance in the pathogenesis of the fibrosis process of IPF. Matrix metalloproteinases (MMPs), the mediators of matrix degradation, collectively capable of degrading essentially all ECM components, are a family of zinc endoproteinases that share structural domains. A pivotal extracellular control of MMP catalytic activity is accomplished by members of a specific family of inhibitors named tissue inhibitors of metalloproteinases (TIMPs). During fibrotic responses of the lung, there is an imbalance in remodeling mechanisms.Current therapies involving corticosteroid, cyclophosphamide and azathioprine are of limited value. It is urgent to investigate some effective drugs to control the fibrotic progress. In laboratory, HMG-CoA reductase inhibitor had been used in the treatment of atherosclerosis and glomerulosclerosis in animals. The investigations of statins show that the drugs may have an anti-fibrotic effect by the inhibition of MMPs, intervention of fibroblast proliferation and collagenic synthesis,the accommodation of ECM deposition. However, the anti-fibrorotic investigation of statins in vivo is rarely reported.In the experiment, the inhibitory effect of Atorvastatin on pulmonary fibrosis induced by bleomycin in rats was observed and the effect of the drugs on collagen secretion as well as MMP-9 and TIMP-1 expression, in vivo and vitro,were evaluated. This will provide experimental basis for Atorvastatin to treat pulmonary fibrosis.MethodsThirty-five Wistar rats were randomly enrolled into the control group(group C), model group(group M) and Atorvastatin-treatment group(group A). The group M and the group A were induced to produce pulmonary fibrosis with bleomycin endotracheally, while the group C was given normal saline instead in the same condition. From the second day the rats in the group A received orally Atorvastatin 10mg/ kg. d. Five rats in each group were sacrificed week 2, 4, 6 after intratracheal injection of bleomycin. Pathological changes in the lungs were evaluated by HE stain and Massons trichrome stain. Collagen content of the lung tissue was assessed by hydmxypmline concentration. Alveolar macrophages, polymorphonuclear leukocytes and lymphocytes in bronchoalveolar lavage fluid (BALF) were counted. The mRNA expression of MMP-9 and TIMP-1 were evaluated by RT-PCR semi-quantitative analysis, while the ELASA method was used to investigate the changes of MMP-9 and TIMP-1 in BALF and blood serum. Gelatin Zymography was used to analysis the activity of MMP-9 in BALF. Wistar rats were induced to produce pulmonary fibrosis with bleomycin endotracheally. On the week 1, the pulmonary fibroblast were isolated. The cells were exposed to Atorvastatin. Cell growth was evaluated by MTT. MMP-9, TIMP-1 expression in Fb was confirmed by reverse transcription-PCR (RT-PCR). Western Blot Analysis for MMP-9, TIMP-1, CollagenⅣ, and Fibronectin. Gelatin Zymography was used to assess the activity of MMP-9.[3H]-Proline Incorporation was used as an additional relative estimate of collagen production.ResultsThe degree of alveolitis and pulmonary fibrosis in group A was improved compared with that in group M. Hydroxypmline concentrations in group A were significantly lower than those in group M. There was a increase in the cellular secretion of MMP-9 after exposure to BLM, especially the group M2 (P＜0.01). There was a reduction in MMP-9 levels after exposure to Atorvastatin, especially the groups A2. Though there was no statistical significance between group A6 and group M6, a decreasing tendency was observed.Compared with those in the group Ms,MMP-9, TIMP-1 mRNA level in group As were lower. The ELISA analysis showed that the expression of MMP-9 in group Ms exposed to bleomycin was up-regulated, and reached the peak at week 2, After that the expression reduced markedly compared with those of group Ms. Compared with the group Ms, the levels of MMP-9 and TIMP-1 in BALF in group As were lower, while those in the blood serum there were no statistical significance. MTT indicate Atorvastatin had a pronounced antiproliferative effect, with the cell number falling to 75.5±8% (P＜0.01) at 1μM of control values, 49.8±12.6% (P＜0.001) at 5μM, 37.5±8.6% (P＜0.001) at 10μM. Gelatin Zymography show 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 5μM 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 h resulted in a reduction in secretion of both MMP-9 and TIMP-1. MMP-9, TIMP-1 secretion were significantly reduced by both 1μM Atorvastatin [(84.67±11.74)%,(48.89±1.92)%,P＜0.05,P＜0.001)] and 5μM Atorvastatin (29.15±6.03)%, (30.37±2.80) (P＜0.05, P＜0.001). Exposure of Fb to Atorvastatin 1 and 5μM for 72 h resulted in a decrease in the levels of secreted typeⅣcollagen levels to 65.9±9.32(P＜0.05),42.6±8.5%(P＜0.01), respectively. Similarly, exposure of Fb to 5μM Atorvastatin for 72 h 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 were 77.1±15.0% (P＜0.05 ) by 1μM Atorvastatin exposure and 54.7±6.6% (P＜0.05) by 5μM Atorvastatin exposure.Conclusion1,Atorvastatin could alleviate bleomycin-induced pulmonary fibrosis in rats.2,Atorvastatin may inhibit the expression of MMP-9 and TIMP-1 in the lung tissue of rat with pulmonary fibrosis.3,Atorvastatin may inhibit the proliferation of pulmonary fibroblasts and decrease the expression of MMP-9 and TEMP-1 in pulmonary fibroblasts...