| Objective:Pulmonary fibrosis (PF) is a kind of serious lung diseases characterized by diffuse progressive fibroplasias and alveolar structure which finally leading to disorder of lung function, and is the common final of many lung diseases. The etiological factors of PF are diversity, its exact pathogenesis is not thoroughly understood, and there is no satisfactory therapy for it up to now. Glucocorticoid and cytotoxic drug are the first choices in treating PF in clinic, but the therapeutic effect is indefinite accompanied with obvious side effect. So the urgent question which the scholars need to solve is to seek effective and safe medicine for treating PF. Nowadays the anti-fibrosis research focus on the medicine which can regulate key cytokines effectively and can modulate the disequilibrium between deposition and degradation of extracellular matrix (ECM). In this study, we interfere in the fibrosis process of PF rat model induced by bleomycin with sodium ferulate (SF), and observe the effects of SF on observe the changes in pathomorphology and content of HYP as well as observe the effect of SF on the content of SOD,ET-1 and the expression of TGF-β1,TIMP-1 protein in lung tissue at different time point. The purpose is to study the protection of SF on experimental pulmonary fibrosis and explore the possible underlining mechanisms, then provide original thinking to clinical treatment for PF.Method:42 SD male rats were randomly divided into 3 group as normal control group (A group, n=6), BLM model group (B group, n=18) and sodium ferulate group (C group, n=18). The BLM model group and sodium ferulate groups each were separated into three time spot groups (7th, 14th and 28th day groups). Pulmonary fibrosis of rat model was induced by intratracheal instillation of bleomycin A5, while the control group was given sodium chloride in the same condition. From 2th day after modeling, the rats in sodium ferulate group received daily sodium ferulate 200mg/kg by intragastric administration, the other rats were given sodium chloride daily.The 6 rats in control group were all sacrificed on the 14th day after intratracheal instillation. On the 7th, 14th, 28th day after BLM administration, 6 rats in another two groups were randomly sacrificed. After fixed in 4% paraform the right lung tissues were embedded in paraffin and sliced. Some of the sections were stained with HE and Masson staining to observe the extent of alveolitis and collagen deposition, the degrees of alveolitis and fibrosis was classified by the method of Szapiel. The immunohistochemical techniques were used to investigate the expression of TGF-β1,TIMP-1 protein. The left lung tissue homogenate were used to determine the content of ET-1,HYP and the activity of SOD of lung tissue.Result:1. The content of HYP in lung tissue (μg/mg): The HYP content of B7 group(1.264±0.13) and C7 group(1.291±0.15) was not different from that of A group(1.154±0.11) significantly (P>0.05); The HYP content of B14 group(1.925±0.22) and B28 group(2.66±0.47) increased significantly compared with A group(P<0.01); The HYP content of C14 group(1.736±0.21) and C28 group(1.889±0.16) was lower significantly than that of B group at the corresponding time point(P<0.05), but was still higher than A group significantly (P<0.01). 2. The activity of SOD in lung tissue (U/mg prot): The SOD activity of B7 group(44.54±5.77) was lower than that of A group(76.72±4.87) significantly(P<0.01); after that the activity increased a little, the SOD activity of B14 group(57.73±4.04) was still significantly lower than that of A group(P<0.01); the activity of B28 group(72.24±3.06) was not statistic different from A group significantly(P>0.05). The SOD activity of C7 group (58.10±4.91) was significantly higher than that of B7 group(P < 0.01), but was still lower than A group significantly(P<0.01); the activity of C14 group(63.09±4.95) was higher than that of B14 group,but was no significant difference from that of A group (P>0.05); the activity of C28 group(72.17±3.71) maintain higher level, there were no significant differences between C28 group, B28 group and A group(P>0.05). 3. The content of ET-1 in lung tissue(pg/mg prot): The ET-1 content of B7group(317.988±20.95) was higher than that of A group(123.673±16.67) significantly(P<0.01); the content increased gradually along with the course of disease, B14 group(358.992±24.98) reach the peak level, after that the ET-1 content decreased gradually, B28 group(274.414±27.28) was still significantly lower than A group(P<0.01). The ET-1 content of C7 group(172.663±18.29) was significantly lower than that of B7 group(P<0.01), and was significantly higher than that of A group (P<0.01); the content of C14 group(239.585±29.16, P < 0.01) and C28 group(237.973±24.91, P<0.05) was significantly lower than that of corresponding B group, but was still higher than that of A group(P<0.01). 5. The change of pathomorphology of lung: The lung tissues of each B group and C group display the pathological process of alveolitis and fibrosis, these pathological changes proved the PF rat models induced by bleomycin was successfully duplicated. The degree of alveolitis and fibrosis of C group were alleviated compared to that of B group. After semiquantitative analysis of pathology, the alveolitis of C7 and C14 group were improved compared to that of corresponding B group significantly(P<0.05); the lung fibrosis of C28 group was significantly alleviated compared to that of B28 group(P<0.05). 6. The expression of TGF-β1 protein in lung tissue: In immunohistochemistry study, the TGF-β1 protein was weakly positive expression in tunica mucosa bronchiorum and alveolar epithelial cells,vascular endothelial cells in A group. In B group, besides the positive in the part mentioned above, TGF-β1 was strongly positive in fibroblast-like cells,macrophage and in the area of thickened lung alveolar septum and consolidation, In C group, the positive area decreased obviously compared to that in B group, furthermore, the positive intensity was weaker than that of B group. Semiquantitative analysis showed the expression of B7 group (4.57±0.46) was higher than that of A group(1.75±0.59) significantly(P<0.01), the expression of B14 group (6.20±0.36) continued to increase and reached the peak level, after that the level decreased gradually, the expression of B28 group(3.68±0.44) was still higher than A group significantly(P<0.01). The expression of C7(3.82±0.26 ) and C14 (5.37±0.51) group were significantly lower than that of corresponding B group(P<0.05). The expression of C28 group(3.32±0.29) was not significantly different from B28 group(P>0.05). 7. The expression of TIMP-1 protein in lung tissue: In immunohistochemistry study, the TIMP-1 protein was weakly positive expression in tunica mucosa bronchiorum and alveolar epithelial cells,mesenchyma macrophage in A group. In B group, besides the positive in the area mentioned above, TIMP-1 was strongly positive in fibroblast focus,macrophage and consolidation area; In C group, the positive area and intensity decreased obviously compared to that in B group. Semiquantitative analysis showed the expression of B7 group (2.67±0.40) was higher than that of A group(2.18±0.48),but there was no significant difference(P>0.05). The expression of B14 (5.10±0.40) and B28 (6.18±0.34) group was was higher than that of A groupsignificantly(P<0.01). The expression of C28 group (5.38±0.43) significantly was significant lower that of B28 group (P<0.05), the expression of C7(2.65±0.35) and C14 group (4.58±0.39) was lower than that of corresponding B group, but there were no statistic differences (P>0.05).Conclusion: 1. In the course of BLM-induced rat pulmonary fibrosis, the activity of SOD and content of ET-1 in lung tissue increase, the expression of TGF-β1 and TIMP-1 in lung tissue upregulate. 2. Sodium ferulate, administrating in earlier period, can alleviate alveolitis and lung fibrosis, can downregulate the expression TGF-β1 and TIMP-1 in lung tissue and content of ET-1 in lung tissue, can also increase the activity of SOD in lung tissue. 3. Sodium ferulate can alleviate BLM-induced rat pulmonary fibrosis by antioxidant effect, antagonizing biology effect of ET-1 and downregulating the expression of TGF-β1 and TIMP-1 protein in lung tissue.
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