| Various pathological changes and functional changes of respiratory system caused by air pollution,smoking,aging of population and other factors affect people’s life.Respiratory disorders and diseases seriously threaten human health.Inflammation and oxidative stress of airway epithelial cells have been proved to be the most common causes of respiratory diseases.As an important oxidative stress regulation pathway,NRF2/ARE pathway has been reported to exert the great impact on the pathogenesis of respiratory diseases.NRF2/ARE pathway is involved in the complex oxidative stress process,and nuclear factor erythroid 2 related factor 2（NRF2）has been demonstrated to play an important role to reduce oxidative damage.Among NRF2 target genes,heme oxygenase-1（HO-1）is a crucial transcriptional regulation factor in the oxidative stress response.Atmospheric pressure cold plasma（ACP）has been found to have impactful biological effects.In the medical field,ACP has been widely used in sterilization,tissue removal and cauterization.The recent research has shown that ACP is a selective anti-cancer treatment with a promising prospect,because some cancer cells are highly susceptible to reactive oxygen species（ROS）produced in APC,but non-tumor cells are highly resistant to it,which suggests that one of APC’s roles is mainly mediated by ROS production.Objective: To investigate the regulation and mechanism of ACP on inflammation and oxidative stress in normal bronchial epithelial cells and human non-small cell lung cancer cells.Methods: HBEC and A549 cells were first stimulated with tunicamycin（TM）to simulate inflammatory response and oxidative stress,then irradiated with ACP.Butylated hydroxy anisole（BHA）and Trigonelline（TRI）were used as agonists and inhibitors of NRF2.Methyl thiazolyl tetrazolium（MTT）assay and Colony formation assay were used to detect the proliferation of cells.Enzyme-linked immune sorbent assay（ELISA）was used to detect the concentration of inflammatory factors in culture medium.m RNA levels of NRF2 and inflammatory factors were detected by Reverse transcription-polymerase chain reaction（RT-PCR）.Western blot was used to detect the expression levels of NRF2/ARE signal pathway transcription factors.Fenton reaction and Griess reaction were used to detect the concentration of ·OH and NO in culture medium respectively.Results:1、ACP inhibits TM-induced inflammation and oxidative stress in HBEC and A549 cells,alleviating the inhibition of cell proliferation caused by oxidative stress.MTT and Colony formation assay showed that TM could inhibit the survival of HBEC and A549 cells,and the survival cells were increased after ACP irradiation.The results of Fenton,Griess and ELISA showed that the levels of ·OH,NO and the concentrations of inflammatory factors TNF-α,IL-6 and IL-1β in HBEC and A549 cell culture medium were increased after TM stimulation,but decreased significantly after ACP treatment.q RT-PCR showed the same results of m RNA.2.NRF2 pathway is positively related to the regulation of ACP on inflammation and oxidative stress.Agonist BHA and inhibitor TRI of NRF2 pathway were used respectively,q RT-PCR and WB showed that,in comparison with A549 cells with TM and ACP treatment,the additional TRI pretreatment was able to greatly enhance transcriptions and expressions of TNF-α,IL-6 and IL-1β,whereas BHA further decreased the levels of TNF-α,IL-6 and IL-1β.Moreover,NRF2 and HO-1 levels were greatly suppressed by TRI,but moderately promoted by BHA.3.ACP facilitates A549 cells to resist TM-induced proliferation inhibition.MTT and Colony formation assay showed that TM inhibited A549 cells survival,the proliferation inhibition of A549 cells was greatly relieved by ACP irradiation.Conclusion: ACP inhibits inflammation and oxidative stress response by activating NRF2 pathway,but causes cytotoxicity and oxidative stress resistance induced by antitumor drugs in A549 cells,indicating that ACP has the potential to treat respiratory diseases,but cannot be used in combination with anti-tumor drugs. |
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