| Objective: Pulmonary fibrosis is a chronic and progressive fibrotic lung disorder of unknown etiology and unclear pathogenesis. No effective treatment was available. This study was to investigate the effect of artesunate on pulmonary fibrosis and on the expression of collagen-IV, MMP-2, MMP-9, TIMP-1 and TIMP-2 show more evidence for the treatment effect of artesunate on pulmonary fibrosis and provides a new sight for anti-fibrosis in clinic probably. Methods : The experiment consisted of two steps. The first stage is about histological detection, in which the effect of artesunate on the expression of collagen-IV, MMP-2, MMP-9, TIMP-1 and TIMP-2 and its possible mechanism in the lung tissues. Forty rats were randomly divided into saline blank control group(NS group), bleomycin-administered experimental group(BLM group), antesunate treatment group(AT group) and the artesunate blank control group(AC group). There were each ten rats in four groups. Rats in NS group and AC group were intratrachealy administered with 0.3ml saline water while the others were with 5mg/kg bleomycin. Rats in BLM group and AT group were consecutively intraperitoneal injected with 1ml saline water per day. The rats in artesunate group and intervention group were intraperitoneal injected with 10mg/100 g artesunate per day. The body weights of the rats were recorded. Lung coefficientwere calculated. The sections of the lungs was used for HE stain. The pulmonary inflammation severity and Ashcroft score of each group were graded. The protein of MMP-2, MMP-9, TIMP-1, TIMP-2 and collagen-IV were analyzed by Western blot. The second stage is about cytological detection, which observes the effect of artesunate on the expression of collagen-IV, MMP-2, MMP-9, TIMP-1 and TIMP-2 and its possible mechanism in the lung fibroblast. During this stage, the, forty rats were randomly divided into saline blank control group(NS group), bleomycin-administered experimental group(BLM group), antesunate treatment group(AT group) and the artesunate blank control group(AC group). There were each ten rats in four groups. The rats were sacrificed at 28 days for lung fibroblast(FB) culturing. FB were identified by immunohistochemical, and the MMP-2, MMP-9, TIMP-1, TIMP-2 and collagen-IV mRNA in lung fibroblast were detected by realtime quantitative reverse transcriptase polymerase chain reaction(RT-PCR). Results: The first part:(1) Compared with rats in NS and AC group, the general state in BLM and AT group were worse, they ate little and stayed inactive. However, the state in AT group kept btter.(2) The lung tissues in the NS group as well as in the NC group, which were of ruddy color and the uniform were soft and smooth. However, the BLM group’s tissues contained many gray nodules, that leading to the surface of the tissue in pale color, and of which’s uniform expressed roughness, sometimes we can see the bullae of lung. But in the AT group, we can see the lung tissues hyperemia dropsy and volume augmented, all of the tissues were in crimson. The lung coefficient of BLM were higher than other three groups.(3) In NS and AC groups, the inflammatory reaction were only mild, expect focal mild lymphocytic infiltration around small vessels. Fibrosis were scarce. In the BLM group, inflammatory cells infiltration into the lung tissue was investigated with HE staining, and was more seriously than other three groups.(4) The grading of alveolitis and pulmonary fibrosis in BLM group was significantly higher than other three groups.(5) The protein expression of TIMP-1, TIMP-2, collagen-IV are increased in BLM group than other three groups. But although the expression of MMP-2 and MMP-9 in BLM is higher than NS group and AC group, decresd in AT group. The part two:(1) We can see the some round like cells lined growth with adherence for 2 days, and extended to become fusiform or long striped for another 2 days. Around 7-8 days later, the cells formed like paliform, arranged radially, and proliferated to form a full layer.(2) Vimentin is expressed in the LF’s cytoplasm specifically, then we saw that the Vimentin have positive expression by immunohistochemical. The ration of the positive expression of Vimentin was 99.4%.(3) The mRNA expression of TIMP-1, TIMP-2, collagen-IV are increased in BLM group than other three groups. But although the expression of MMP-2 and MMP-9 in BLM is higher than NS group and AC group, decresd in AT group. Conclusions:(1)Bleomycin induced alveolitis and pulmonary fibrosis of rats was inhibited by artesunate.(2)Theses results highlight the significance of artesunate in alleviating alveolitis and pulmonary fibrosis, which is associated with decreased expression of collagen-IV, TIMP-1, TIMP-2, and also increased expression of MMP-2 and MMP-9 in lung tissues.(3)Theses results highlight the significance of artesunate in alleviating alveolitis and pulmonary fibrosis, which is associated with decreased expression of collagen-IV, TIMP-1, TIMP-2, and also increased expression of MMP-2 and MMP-9 in LF.(4)Artesunate may inhibit the development of pulmonary fibrosis via alleviating TIMP1/2 expressions, which can remove the effects of TIMP-1/2 to MMP-2/9 to a certain extent, reduce the formation of ECM and collagen type IV. |
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