The Effect Of Astaxanthin On Cigarette Smoke Extract-induced MUC5AC Hypersecretion

Airway mucus hypersecretion is an important pathological feature of chronic pulmonary disease. The mucins maintain airway mucus viscoelasticity.In airway diseases,MUC5AC is the major secretory protein. Smoking is the major risk factors of chronic obstructive pulmonary disease.The reactive oxygen species in cigarettes can active EGFR, sequential activation of downstream MAPK signaling pathway, which induce MUC5AC mucin expression. Astaxanthin is a carotenoid, has strong antioxidant activity, can scavenge oxygen free radicals and inhibit the production of ROS. Give the significant role that ROS regulate mucus production and the antioxidant activity of Astaxanthin, we investigate the role of astaxanthin in CSE-induced MUC5AC hypersecretion.Objective:To explore the effect of astaxanthin on cigarette smoke extract-induced airway mucin MUC5AC hypersecretion and to investigate the possible mechanisms.Method:(1)A549cell were exposed to CSE to establish mucus hypersecretion model in vitro. We assess the cell activity by MTT method and determine the dose of astaxanthin. Different concentrations of cigarette smoke extract (2.5%,5%, and10%) stimulated A549cells for24hours.The level of MUC5ACmRNA was detected by RT-PCR, the level of MUC5AC protein was measured by ELISA method. The cells were divided into five groups:the negative control group, CSE treatment group, the astaxanthin treatment groups were8uM16uM32uM...The same as before, we measure the level of MUC5AC mRNA and MUC5AC protein.(2)Again,the cell were divided into five groups:the negative control group, the CSE treatment group, the astaxanthin(32uM)group,the DMTU(20uM)group,the astaxanthin+DMTU group.We detect the change of ROS by microplate reader.AND the level of EGFR and p-EGFR protein were measured by western blotResult:(1) we successfully constructed the mucus hypersecretion by CSE. Different concentrations of cigarette smoke extract stimulates A549cells for24hours, MUC5ACmRNA and protein expression was increased in a dose-dependent manner.10%CSE were selected to establish the our model.MTT result show that the concentration of astaxanthin less than64uM had no significant damage. While the concentration of astaxanthin were more than64uM, the proliferation of cell was decreased significantly. So we choose8uM,16uM,32uM as the test concentrations. The astaxanthin can significantly reduce MUC5AC mRNA and protein expression induced by cigarette smoke extract in dose-dependent way.(2) Compared with the CSE group, astaxanthin (32uM)group can significantly reduce intracellular ROS levels, and Western blot showed that the level of EGFR, p-EGFR is also significantly reduced.Conclusion:(1) Cigarette smoke extract can stimulate MUC5AC gene transcription and protein expression in airway epithelial cells. Astaxanthin reduces MUC5AC gene transcription and protein expression induced by cigarette smoke extract (2) astaxanthin reduces cigarette smoke extract induced the MCU5AC gene transcription and protein expression via ROS/EGFR signaling pathway.