Effect Of Yi Fei Huo Xue Granule On The Inflammatory Response And Oxidative Stress Induced By Cigarette Smoke Extract In Macrophages And Its Possible Mechanism

Background and objectiveChronic obstructive pulmonary disease(COPD)is a common respiratory condition characterized by progressive airflow obstruction.At present,the pathogenesis of COPD is still unclear.Among these,the oxidative stress and chronic airway inflammation are the key factors in the pathogenesis of COPD.Macrophages function as the effector cells in COPD,play a pivotal role in the pathophysiology of COPD.The cigarette smoke is one of the main pathogenic factors of COPD and can induce the macrophage-related inflammation in COPD.NF-κB is one of the critical transcription factors required for maximal expression of many cytokines involved in the pathogenesis of COPD.Yi Fei Huo Xue Granule(YFHXG)was concocted by academician DONG Jian-hua.It has been used clinically to treat PAH and COPD in Chinese medicine for many years.However,the potential mechanisms of YFHXG are still unkown.With this in mind,the present study was performed to explore the effect of YFHXG on the inflammatory response and oxidative stress induced by cigarette smoke extract in macrophages and its possible mechanism.MethodsThe U937 monocytic cells were differentiated into macrophages using phorbol 12-myristate 13-acetate(PMA).The PMA-induced macrophages were identified by immunofluorescence and then were treated with various concentrations(1.2-9.6g/L)of YFHXG or 0.5-10%of cigarette smoke extract(CSE).At the 12,24,and 48 h time point,the cells viability was assessed by using the cell counting kit-8(CCK-8).Macrophages were divided into 5 groups:the normal group,the CSE group,the CSE+ YFHXG(1.2 g/L)group,the CSE+ YFHXG(2.4 g/L)group,and the CSE+YFHXG(4.8 g/L)group.The ROS level and SOD activity in macrophages were determined.The levels of IL-8 and TNF-a in cell culture supernatant were detected by(enzyme-linked immunosorbent assay,ELISA),and the gene expression of IL-8 and TNF-a were also measured by(real-time polymerase chain reaction,RT-PCR).For the study of mechanism,macrophages were divided into 4 groups:the normal group,the CSE group,the CSE+YFHXG(2.4 g/L)and CSE+Parthenolide group.The protein expression of nuclear factor-κB(NF-κB)p50/p65 and p-IκB was examined by Western blot.Meanwhile,the immunofluorescent assay was used to detect the expression and trans-location of p65 and p-IκB in macrophages.Results1.PMA could induce U937 cell differention towards the macrophage.And the U937 cell derived macrophages could express the specific marker of macrophage-CD 14.2.Nine point six g/L of YFHXG significantly reduced cell viability of the macrophage at 24 and 48 h time point.Ten percent of CSE could significantly decreased the cell viability at each time point.The optimal concentration of CSE for experiment is 5%and that of YFHXG for experiment is 1.2-4.8 g/L.3.YFHXG could inhibit the CSE-induced oxidative stress in macrophages.4.YFHXG could suppress the CSE-induced release of IL-8 and TNF-a in macrophages.Moreover,YFHXG could inhibit the activation of NF-κB induced by CSE.ConclusionsThe cigarette smoke is one of the main pathogenic factors of COPD and can induce the macrophage-related inflammation in COPD.These findings showed that YFHXG could suppress the oxidative stress,inhibit the secretion of IL-8 and TNF-a induced by CSE in macrophages.One of the mechanisms may be the inhibitory effect of YFHXG on NF-κB.