| BackgroundThe Chronic Obstructive Pulmonary Disease( COPD), is a major refractory disease that harms human health and has complicated pathomechanisms interacting with erach other. Since the inflammatory response plays an important role during the progress of COPD, suppression of inflammation response is essential to reduce symptoms and suspend the progress. The inflammatory responses of COPD involves a series of cytokines, cell types and signal pathways, and form a inflammatory molecule network. Therefore, it is a better way to illustrate the mechanism and peculiarity of traditional Chinese medicine treatment of COPD by studying the role of crucial cytokines and signal pathways in the inflammatory molecule network.This study is aimed to establish a model of COPD imflammatory by using tumor necrosis factor-α( TNF-α), lipopolysaccharide( LPS) to stimulate A549 cells and the nuclear factor-kappa B( NF-κB), mitogen-activated protein kinase( MAPK), JAK/STAT, peroxisome proliferator activated receptor( PPAR) signaling pathways as well as the regulation of NF-κB signal pathway in A549 cells by the three Tiao-Bu Fei-Shen extracts(Bufei Jianpi,Bufei Yishen,Yiqi Zishen) in order to study the molecular machinasm of the suppression of COPD inflammatory response by traditional Chinese medicine.ObjectiveTo establish a model of COPD inflammatory response by using LPS and TNF-α to stimulate A549 cells and screening their effect on receptor activities of the signal pathways of NF-κB, MAPK, JAK-STAT, PPAR. Investigate the regulation ofNF-κB signaling pathway by the three Tiao-Bu Fei-Shen extracts(Bufei Jianpi,Bufei Yishen,Yiqi Zishen), provide a basis of the molecular mechinasm of the supression of COPD inflammatory by the three Tiao-Bu Fei-Shen extracts.Method1 Establish a model of COPD inflammatory responseTake different concentration(5ng/m L,10ng/m L,20ng/m L,40ng/m L)of TNF-α to stimulate A549 in different time periods(6h,12 h,24h,48 h,36h,72h),detect the cell appreciation and inhibition by MTT methods. Take different concentrateon(0.125μg/m L,0.25μg/m L,0.5μg/m L,1μg/m L,2μg/m L) by LPS to stimulate A549, detect the cell appreciation and inhibition by MTT methods. Test the secretion of cytokines of interleukin-6( IL-6), interleukin-8( IL-8), matrix metalloproteinase-3(MMP-3),matrix metalloproteinase-9(MMP-9)in the TNF-α’s and LPS’ supernatant by ELISA method. Test the activity of NF-κB,AP-1,STAT3,PPAR in A549 cells via cytokines TNF-α,LPS by the method of electrophoretic mobility shift assay(EMSA).Choose the best inflammation modal according the result of the above.2 The preparation of Tiao-Bu Fei-Shen extracts contained serum96 Common Japanese big ear rabbits, divided into four groups: control, Bufei Jianpi, Bufei Yishen and Yiqi Zishen. Separately take normal saline、Bufei Jianpi fang, Bufei Yishen fang, Yiqi Zishen fang by gavaging in rabbits from the four groups.Blood samples are obtained from abdominal aorta in the 7th gavage after 1 hour,centrifugal separation serum. Filter and subpackage the serum,inactivate the drug-containing serum in 56℃ by 30 minitates,then save the drug-containing serum in-80℃.3 The effects of the three Tiao-Bu Fei-Shen extracts on inflammatory response via regulating NF-κB signal pathwayDivided intervention cells into five groups: blank serum,model, Bufei Jianpi serum,Bufei Yishen serum and Yiqi Zishen serum. Each serum group concentrates into high( 40%), medium( 20%) and low( 10%). Test according to the above specification.then add in the PDTC group,Test the activity of NF-κB in cells by the method of electrophoretic mobility shift assay(EMSA).Result1 The establish a model of COPD inflammatory response by using LPS and TNF-α to stimulate A549 cells1.1 The growth situation of the cellsTNF-α with high concentration(40ng/m L,80ng/m L)will change cellular morphology of A549: cytoplasm rough,cells become long and narrow. TNF-α with low concentration(5ng/m L-20ng/m L) have a smaller influence on cellular morphology. Take different concentration(5ng/m L,10ng/m L,20ng/m L,40ng/m L)of TNF-α to stimulate A549 in different time periods(6h,12 h,24h,48 h,72h), detect the cell appreciation and inhibition by MTT methods. Compared with normal control group, it was significantly lower with OD value in 40ng/m L and 80ng/m L TNF-α treated group by stimulating A549 in 12h(p<0.05,p<0.01). By stimulating A549 in 24 h, the OD value of 10ng/m L, 20ng/m L and 40ng/m L TNF-α treated group is lower than those in control group. By stimulating A549 in 48 h, the OD value of 40ng/m L TNF-α treated group is lower than those in control group.It is the best for 20ng/m L TNF-α to stimulate the cell A549,so we choose it for the inflammatory modal in the futher’s study.1.2 Cytokines secretionTest the secretion of cytokines of interleukin-6(IL-6),interleukin-8(IL-8),matrix metalloproteinase-3(MMP-3),matrix metalloproteinase-9(MMP-9)in the TNF-α’s and LPS’ supernatant by ELISA method.The levels of MMP-3, MMP-9, TNF-α, IL-8, MUC5 AC in the TNF-α treated group were significantly higher than those in control group(p <0.05, p <0.01).The secretion of cytokines of TIMP-1, TIMP-1/MMP-9, IL-10, TIMP-1 and IL-4 had no statistical significance(p >0.05).Take different concentration(1μg/m L, 2μg/m L) of TNF-α to stimulate A549 in different time periods(6h, 12 h, 24 h, 48h), detect the secretion of cytokines by ELISA methods. The levels of IL-6 and IL-8 in the 2μg/m L LPS treated group were significantly higher than those in control group in 12 h.The levels of MMP-9 and TIMP-1 in 1μg/m L and 2μg/m L TNF-α treated group is lower than those in control group. It has little influence in the secretion of cytokines TNF-α and MUC5 AC in 1μg/m L and 2μg/m L TNF-α treated group.1.3 Activity expression of NF-κB, AP-1, PPAR and STAT3Test the activity of NF-κB,AP-1,PPAR and STAT3 in A549 cells via cytokines TNF-α, LPS by the method of electrophoretic mobility shift assay(EMSA).The activity expressions of 10ng/m L and 20ng/m L TNF-α treated group were significantly higher than those in control in 24 h via NF-κB signal pathway, especially in 20ng/m L TNF-α treated group(p<0.01).In the mean time, the expression of 20ng/m L TNF-α treated group was lower than that in control in 48 h via NF-κB signal pathway(p<0.01). The expressions of 10ng/m L and 20ng/m L TNF-α treated group were significantly higher than those in control in 24 h via AP-1 signal pathway, especially in 20ng/m L TNF-α treated group(p<0.01).Compared with control group, the expressions of 10ng/m L and 20ng/m L TNF-α treated group had no statistical significance via PPAR and STAT3 signal pathway(p>0.05). The expressions of LPS treated group were lower than those in control in 24 h via AP-1, PPAR and STAT3 signal pathway.2 The effects of the three Tiao-Bu Fei-Shen extracts on inflammatory response via regulating NF-κB signal pathway2.1 The growth situation of the cellsTake different concentration(10%, 20%, 40%) of Tiao-Bu Fei-Shen extracts contained serum to stimulate A549,detecting the cell appreciation and inhibition by MTT methods. Compared with normal control group, it was significantly higher with OD value in Tiao-Bu Fei-Shen extracts contained serum(Bufei Jianpi, Bufei Yishen and Yiqi Zishen) treated group with 20% concentration by stimulating A549(p<0.01,p<0.05).The OD value in Tiao-Bu Fei-Shen extracts contained serum(Bufei Jianpi, Bufei Yishen and Yiqi Zishen) treated group with 20% concentration was significantly higher than those in control(p<0.01,p<0.05). The OD value in Bufei Jianpi treated group with 40% concentration was significantly higher than those in control(p<0.05).2.2 Cytokines secretionTake different concentration( 20%, 40%)of Tiao-Bu Fei-Shen extracts contained serum to stimulate A549,detect the secretion of cytokines of interleukin-8(IL-8), matrix metalloproteinase-9(MMP-9) and TNF-α by ELISA methods. The expressions of IL-8、MMP-9、TNF-α in model group(20ng/m L TNF-α) with 20% and 40% concentration were significantly higher than those in control. The expressions of IL-8、MMP-9、TNF-α in Tiao-Bu Fei-Shen extracts contained serum(Bufei Jianpi, Bufei Yishen and Yiqi Zishen) treated group with 20% concentration was higher than those in model. The expressions of TNF-α in Bufei Jianpi treated group with 20% concentration was lower than those in model. In addition, The expressions of IL-8,MMP-9,TNF-α in Tiao-Bu Fei-Shen extracts contained serum(Bufei Jianpi, Bufei Yishen and Yiqi Zishen) treated group with 40% concentration was lower than those in model.2.3 Activitity expression of NF-κBTest the activity of NF-κB in A549 cells in Tiao-Bu Fei-Shen extracts contained serum(Bufei Jianpi, Bufei Yishen and Yiqi Zishen) and PDTC treated group withdifferent concentrations(10%,20%,40%) by the method of electrophoretic mobility shift assay(EMSA).The NF-κB expressions of model group(20ng/m L TNF-α) contained serum with 40% concentration were significantly higher than those in control(p <0.05). The NF-κB expressions of Tiao-Bu Fei-Shen extracts contained serum(Bufei Jianpi, Bufei Yishen and Yiqi Zishen) and PDTC treated group with 40% concentration was lower than those in model(p<0.01). The NF-κB expressions of model group(20ng/m L TNF-α) contained serum with 10% and 20% concentration had no statistical significance than those in control, while the NF-κB expressions of Tiao-Bu Fei-Shen extracts contained serum with 10% and 20% concentration had no statistical significance than those in model(p>0.05).Conclusion1. The stimulating from TNF-α can lead to the inhibition of alveolar epithelial cell A549’s growth.In the mean time,it can also stimulate cell A549 to secret inflammatory factors,increase the activity expression of NF-κB and AP-1 signal pathways. So we choose TNF-α to create the COPD inflammation model by stimulating cell A549.2. LPS had little influence in stimulating the growth of cell A549 and secreting inflammatory factors.LPS had little influence in increasing the activity expression of NF-κB,AP-1,PPAR and STAT3 signaling pathways.So using LPS to create the COPD inflammation model by stimulating cell A549 was inadequate.3. We establish the condition of cell inflammation model. We choose 20ng/m L TNF-α to create the COPD inflammation model by stimulating cell A549 in 24 h.4. Three Tiao-Bu Fei-Shen extracts contained serum(Bufei Jianpi, Bufei Yishen and Yiqi Zishen) can significant decrease the sercret of inflammatory factor in model group, decrease the activity expression of NF-κB signal pathway than that in model group. |
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