| Objective: Pulmonary fibrosis is a kind of chronic interstitial disease in lung, characterised by inflammatory cells infiltration in alveolar interstitium, fibroblast proliferation, extracellular matrix deposition. The pathogenesis of pulmonary fibrosis has not been clear, and the mechanism of pulmonary fibrosis has not been well clarified. It is generally accepted that the model of pulmonary fibrosis is made by intratracheal instillation of a single dose of bleomycin (BLM).Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of pulmonary fibrosis, as a downdream factor of many stimulus to promote fibrosis. It is known that CTGF expression is increased in pulmonary fibrosis induced by BLM. Baicalin is a flavanoid with phenolic hydroxyl group. Our previous study found that baicalin (12.5mg/kg/d, i.p, for 28d) alleviated pulmonary fiborsis induced by BLM in rats. But the mechanism of baicalin on pulmonary fibrosis has not been well known.In order to illuminate the mechanism of baicalin on pulmonary fibrosis, the present study is to investigate the effects of different doses of baicalin on fibrosis and CTGF expression in lungs of rats after intratracheal instillation of a single dose of BLM.Methods: 45 SD rats(♂) weighting 170-220g were randomly divided into six groups:①NS+NS group(n=6): After intratracheal instillation of a single dose of normal saline (NS, 0.5 ml/kg), the rats were given NS(1 ml/kg, i.p) for 28 days;②NS+Ba12.5mg group(n=6): After intratracheal instillation of a single dose of NS (1 ml/kg), the rats were given baicalin (12.5 mg/kg/d, i.p) for 28 days;③BLM+NS group(n=8): After intratracheal instillation of a single dose of BLM (5 mg/kg ), the rats were given NS (1ml/kg, i.p) for 28 days;④BLM+Ba6mg group(n=9): After intratracheal instillation of a single dose of BLM(5 mg/kg), the rats were given baicalin (6 mg/kg/d, i.p)for 28 days;⑤BLM+Ba12.5mg group(n=9):After intratracheal instillation of a single dose of BLM (5 mg/kg), the rats were given baicalin (12.5 mg/kg/d, i.p) for 28 days;⑥BLM+Ba50mg group(n=7): After intratracheal instillation of a single dose of BLM (5 mg/kg), the rats were given baicalin ( 50 mg/kg/d, i.p ) for 28 days.On day 29 after intratracheal instillation, all rats were killed after anesthetized by 10% chloral hydrate(3 ml/kg, i.p). Then left lungs were collected for immunohistochemistry, and the rest for hydroxyproline assay.The severity of pulmonary fibrosis was estimated by the content of lung hydroxyproline (HYP). The CTGF expression in lung was observed by immunohistochemistry. All data were shown as mean±standard deviation. Statistical significance was assessed by one-way ANOVA and least significant difference (LSD) by using SPSS-13.0 statistical software. A P values less than 0.05 was viewed as statistically significant.Results: 1 General healthy state of rats: In NS+NS group and NS+Ba12.5mg group it was well: their behavior being vivid, their reaction being agile, and their hair being slick. In BLM+NS group, the rats'act was lessened, reacted slowly and the hair was tarnished with poor appetite and hypoplasia. While the general healthy state of rats was improved in BLM+Ba6mg group, BLM+Ba12.5mg group, and BLM+Ba50mg group.2 Content of pulmonary HYP of rats: The contents of pulmonary HYP in NS+NS group and BLM+NS group were (6.95±0.81)μg/mg lung weight and 13.40±1.50μg/mg lung weight), respectively. Compared with NS+NS group, content of pulmonary HYP in BLM+NS group was increased (P<0.01). Content of pulmonary HYP of rats in BLM+Ba6mg group, BLM+Ba12.5mg group and BLM+Ba50mg group were (9.54±1.81)μg/mg lung weight, (8.87±1.31)μg/mg lung weight and (11.05±1.08)μg/mg lung weight, respectively. Compare with BLM+NS group, content of HYP in the three treatment groups (BLM+Ba6mg group, BLM+Ba12.5mg group and BLM+Ba50mg group) were decreased significantly(All P<0.01). The contents of pulmonary HYP either in BLM+6mg group or in BLM+12.5mg group was more lower than those in BLM+Ba50mg group (P<0.01, P<0.05 ) . There was no difference of pulmonary HYP between NS+NS group [(6.02± 0.58)μg/mg lung weight] and NS+Ba12.5mg group(P>0.05).3. Immunohistochemical staining of CTGF: There was slight positive signal of CTGF in NS+NS group, which was only in tracheal epitheliums. In BLM+NS group, the positive signal was increased, which was mainly in tracheal epithelial cells, fibroblasts, alveolar epithelial cells, and macrophages and so on. Compared with NS+NS group(2.23±0.39), integral optical density(IOD) of CTGF in BLM+NS group(19.80±1.55) was increased significatantly(P<0.01). IOD of CTGF in BLM+Ba6mg group, BLM+Ba12.5mg group and BLM+Ba50mg group was 12.01±1.60, 7.91±0.97 and 18.54±1.64, respectively. Compared with BLM+NS group, IOD of CTGF in the three treatment groups (BLM+Ba6mg group, BLM+Ba12.5mg group and BLM+Ba50mg group) markedly decreased (P<0.01, P<0.01, P<0.05). IOD of CTGF either in BLM+6mg group or in BLM+12.5mg group was lower than that in BLM+Ba50mg group (All P<0.01). There was no difference of IOD of CTGF between NS+NS group (3.12±0.8) and NS+NS group(P>0.05).Conclusion: 1 Baicalin prevents pulmonary fibrosis induced by BLM.2 Baicalin ameliorates the up-regulation of CTGF expression in fibrotic lungs of rats induced by BLM. This might be one of the mechanisms underlying the preventive action of baicalin on pulmonary fibrosis.
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