The Effect And Mechanism Of Vam3on Cigarette Smoke-induced Autophagy

Chronic obstructive pulmonary disease (COPD) is a major disease to threat global human health. At present, COPD is the forth common cause of death second only to heart disease, and also the second important reason for the loss of labour. As the adverse impact of COPD and of which the pathogenesis is not yet clear, there is still a big gap in its prevention, diagnosis and treatment, there are even some problems in the existing clinical drug therapies. With the development of research on the mechanism of oxidative stress in recent years, some antioxidants have been brough forth. The antioxidants are able to improve the symptoms of patients with COPD, delay the disease progression and so on. So, it would be important and has good commercial prospects if we successfully develop a kind of efficient, low toxic and easily absorbed natural antioxidant. The aim of this study is to appraise the efficacy of Vam3(Amurensis H), a dimeric derivative of resveratrol, on cigarette smoke-induced autophagy with the background of COPD, and we hope to develop an antioxidant better than resveratrol.In this study, we proved Vam3play the prevention and treatment role of COPD through inhibiting autophagy induced cigratte smoke both in vitro and in vivo. First, the human bronchial epithelial cells (Beas-2B) were treated with cigarette smoke condensates (CSC) in various concentrations for different time, the protein level of the autophagic mancer microtubule-associated protein1A/1B-light chain3(LC3) was detected by western blot and immunoflurescence, and the results indicated that the CSC could induce Beas-2B cells autophagy. Furthermore, we also treated the Beas-2B cells with autophagy inhibitor, balfiomycin A1, to comfirm the autophagy can be induced by CSC. Second, we observed Vam3inhibited CSC-induced autophagy in Beas-2B cells and was more effective than resveratrol. In order to investigate the molecular mechanism of Vam3in autophagy, we use the immunoprecipitation and western blot assays to detect the expressions and activities of Sirtl and FoxO3a, the results showed that after treatment with CSC, the expression and activity of Sirtl were down-regulated, and could be reversed by Vam3. In addition, CSC also reduced the expression of total FoxO3a and phosphorylated FoxO3a, increased the protein level of acetylated FoxO3a and promoted FoxO3a translocation into the nucleus, while Vam3could reverse all these changes of FoxO3a. Third, we knocked down the expression of Sirtl in Beas-2B cells in order to discuss if Sirtl is required for the effect of Vam3. We observed that Vam3failed to attenuate the CSC-induced autophagy without the present of Sirtl. Last, we found Vam3could inhibit oxidative stress, induced by cigratte smoke, by detecting intracellular ROS and GSH-Px content.In vivo experiments, we established the cigratte smoke-exposed mouse model, and we found the results that Vam3could improve the pathological changes in the lungs of mice by using HE staining. Then, by using western blot and immunohistochemistry assays, we got to know that Vam3inhibited autophagy and up-regulated or restored the expression levels of Sirtl and FoxO3a.In summary, we first discovered that Vam3has both anti-oxidative and anti-autophagical effects, and are better than resveratrol; Vam3inhibits cigratte smoke-induced autophagy by diminishing oxidative stress and modulating Sirtl/FoxO3a pathway, and thus plays the role of prevention and treatment in chronic obstructive pulmonary disease. This study also provides a theoretical basis and reliable lead compound for the further development of the antioxidant drug which prevents and treats COPD.