Regulatory Effect Of All-trans-retinoic Acid To The Expression Of MMP-12,TIMP-2 And TNF-α In PPE-induced Rat Model Of Emphysema

Introduction:Chronic obstructive pulmonary disease(COPD) is a serious hazard to human health respiratory diseases, but the pathogenesis is not clear. Emphysema is the main pathological types of COPD, which is defined as the abnormal permanent enlargement of airspaces distal to the terminal bronchioles, accompanied by destruction of the walls and without obvious fibrosis. To clarify the pathogenesis of emphysema and to seek treatment and prevention of a reliable measure is the aim for many scholars.All-trans retinoic acid (ATRA), the biologically active metabolite of vitamin A , plays an important role in lung development,mature and maintaining alveolar architectural stability,It induces alveolar formation and increase elastic recoil in adult rat lung with elastase-induced emphysema, so it is probably an effective drug for treatment of pulmonary emphysema , but the mechanism is unclear. This experiment using porcine pancreatic elastase induced rat model of emphysema found that a certain concentration of ATRA in rats with emphysema could promote alveolar repair. Through the observation of MMP-12, TIMP-2 and TNF-αin lung tissue expression and serum TNF-αconcentration, exploring the interventions mechanism of ATRA effecting COPD is the main purpose of the study.Objectives:1. To build the emphysema model in SD rat with porcine pancreatic elastase(PPE)2. At the basis of this model to the rat intra-abdominal injection of different doses of ATRA, observe the severity of emphysema and further to observe the expression of MMP-12, TIMP-2 and TNF-αin different groups.3. Determination of the serum concentration of TNF-αin different groups , to observe the regulatory effect of different doses of ATRA for emphysema rats.Methods:Male Sprague-Dawley (SD) rats (weight range,170 to 220g) were randomly divided into the following six groups: a control group(A), an emphysema group(B), a low-dose ATRA treatment group(C1), a middle-dose ATRA treatment group(C2), a high-dose ATRA treatment group(C3) and a cottonseed oil group(D). In addition to control group, we constructed the rat model of emphysema by a single intratracheal instillation of porcine pancreatic elastase (PPE) to rats. After 76 days, The ATRA treatment groups and cottonseed oil group were individually treated with ATRA(500μg/kg,1000μg/kg,2000μg/kg) and cottonseed oil(1 mL/kg), intraperitoneally daily for 2 weeks. All rats were killed at 90 days, The rat lung tissue were immersed in 10% neutral formalin for more than 24 hours, Conventional paraffin-embedded sections, to observe the pathological changes of the rat lung in each group by HE staining. The expression of protein of MMP-12, TIMP-2 and TNF-αwere detected by immunohistoc- hemistry. Using average optical density value reflect the intensity of protein expression. The serum concentration of TNF-αwas detected by enzyme-linked immunosorbent assay (ELISA).Rusults:1. Histopathological changes in lung tissue Under the light microscope, the alveolar duct, alveolar sacs and alveolar were enlarged in model group. As to the high-dose ATRA treatment group and a cottonseed oil group .the alveolar wall became thinning and disrupted, resembling the central lobular emphysema. But low-dose and middle-dose ATRA treatment group lesions lesser extent.2. The expression of MMP-12, TIMP-2 and TNF-αin lung tissue of rats of each group Compared with control group, the expression of MMP-12 and TIMP-2 protein in model group emphysema wall were significantly enhanced (P < 0.01), with MMP-12 mainly enhanced. the expression of MMP-12 in ATRA treatment were not distinct from the group B and D (P > 0.05). Compared with group B and D, the expression of TIMP-2 in ATRA treatment groups were increased (P < 0.05), the ratio of MMP-12/TIMP-2 increased in group B and D compared with group A. Compared with group B and D, the expression of TNF-αin ATRA treatment group were decreased(P < 0.05),the distinction between treatment groups were not significant. However, the serum concentration of TNF-αin normal group was lower than other groups (P < 0.01), there were no differences between ATRA treatment groups and emphysema group.3. The serum concentration of TNF-αin each group The mean serum concentration of TNF-αin model group, ATRA treatment groups and the cottonseed oil group were hingher than the control group (P <0.01), but between the treatment group and model group and there were no significant differences ( P>0.05). At the same time, there were no differernces between the three treatment groups( P>0.05).Conclusion:1. We constructed the rat model of emphysema by a single intratrachcal instillation of porcine pancreatic elastase (PPE) to rats.2. All-trans-retinoic acid may be the whole way through the adjustment of the ratio of MMP-12/TIMP-2 and inhibition of TNF-αformation in lung tissue , thus exert on the alveolar structure repair.3. As the solvent ,cottonseed soil had no effect on the experimental results.