The Effects Of Pirfenidone And N-acetylcysteine On The Expression Of NOX4in The Pulmonary Fibrosis In Mice Induced By Bleomycin

Objectives To observe the expression and role of NOX4in the bleomycin(BLM)-induced pulmonary fibrosis in mice, and to investigate the effects of pirfenidone and N-acetylcysteine on the expression of NOX4.Methods Seventy-two female adult (C57BL/6) mice were randomly divided into four groups as the following(each group includes eighteen mice):the control group (Group C), the bleomycin group(Group M), the pirfenidone group(Group P) and the N-acetylcysteine group(Group N). The model of pulmonary fibrosis was established through intratracheally instillation with bleomycin (3.5mg/kg) except group C. By instillation the same dose of normal saline (NS), Group C was selected for reference purpose.24hs after the establishment of the model, the mice in Group C and Group M were received NS lavage, yet Group P was force fed by pirfenidone every day, while mice of Group N began to force feeding N-acetylcysteine a week before establishing the model. Stochastically six mice in each group were killed separately on the7th,14th and28th day after instillation. Their pathological section of lung tissues were harvested for hemotoxylin and eosin stain and Masson’s trichrome stain, so as to observe the degree of pulmonary fibrosis and to determine hydroxyproline content. Therefore, it aims to examine the distribution and the expression of NOX4in pulmonary tissues in the above four groups through immunohistochemical method.Results1. With intratracheal instillation of bleomycin, we succeeded in making the mouse model of pulmonary fibrosis.2. The weight of mice and lung coefficients. There is no difference of the weights of mice among four groups before model establishment (P>0.05). However, the weights of mice had been decreasing after they had instillated with bleomycin. After five to seven days, their weight was increasing slowly. The weights of mice in four groups and their lung coefficients on the7th,14th and the28th day have statistical significant (P<0.05). The loss of weights in the mice of Group M was remarkably, and lung coefficient in Group M is much higher than that in Group C, which enjoys statistical significant (P<0.05).3. Masson staining showed that the degree of pulmonary fibrosis in Group M was severer than that in Group C at the7th,14th and28th day separately (P<0.05). The degrees of pulmonary fibrosis in Group P and N were lower than that in Group M (P<0.05).4. The content of hydroxyproline (Hyp) in the lung tissue of Group M was increased than that in Group C (P<0.05). The contents of Hyp in Group P and N were decreased obviously compared with that in Group M respectively (P<0.05).5. The immunohistochemical studies showed that NOX4was mainly observed in airway smooth muscle cells, alveolar epithelial cells, lung fibroblast, vascular smooth muscle cells and endothelial cells in lung tissues with a high level expression in Group M on the7th,14th or28th day respectively (p<0.05). Compared with that in Group M, the expression of NOX4of the lung tissues in Group C was weaker (p<0.05). The expressions of NOX4of lung tissues in Group P and Group N were a little higher than that in Group C (P<0.05), but still lower than that in Group M (P<0.05).Conclusion1. By intratracheal instillation of bleomycin, we succussfully made the mice pulmanory fibrosis of animal modle.2. NOX4was widely expressed in pulmonary fibrosis. The mechanism of up-reglation of the expression of NOX4may participate in pulmonary fibrosis induced by bleomycin.3. Both pirfenidone and N-acetylcysteine can alleviate the degree of pulmonary fibrosis induced by bleomycin, lesson sediment of lung tissue collagen. N-acetylcysteine plays an active role in down-regulating the expression of NOX4, while pirfenidone diminishes the expression of NOX4in the lung of mice. It suggests that NOX4may become the new target in the treatment of pulmonary fibrosis.