| Chronic Obstructive Pulmonary Disease(COPD) is a kind of limited airflow characteristics common clinical Disease, and its limited has not completely reversible airflow, and presents the characteristics of the progressive development[1].Inflammation is an important pathological features, so the research on airway inflammation is particularly important for elucidating the pathogenesis Lipopolysaccharide(LPS) is the main component of bacterial endotoxin can stimulate tissue cells to induce inflammation by releasing a variety of inflammatory substances(including cytokines,etc.) [2].Inflammatory response involves a variety of cells and multiple signaling pathways.Including macrophages which plays a very important role, and the molecular network system for the regulation of signaling pathways plays a central role [3].Through suppression of these signaling pathways can inhibit inflammation of COPD and have the effect of treatment.. In this study, by studying the LPS inducing mononuclear cell line(THP 1 cells) to secrete inflammatory factor, interleukin 6(IL-6) and interleukin 8(IL-8) and interleukin 10(IL-10), tumor necrosis factor alpha(TNF-a), matrix metalloproteinase 9(MMP-9), matrix metalloproteinases tissue inhibiting factor 1(TIMP-1), and the signal pathways in Nuclear factor kappa B predominate-(Nuclear factor kappa B, NF-κB), Janus kinase/Signal transduction and transcriptional activation factor(JAK/STAT), silk crack original activated protein kinase(MAPK), peroxidase pod body growth activated receptor(Peroxisome proliferator- activated receptor, PPAR) activation, optimization of LPS THP 1 cells in vitro induced inflammation model, to study the three Tiao-Bu Fei-Shen extracts(Bufei Jianpi,Bufei Yishen,Yiqi Zishen) the secretion of IL-8,TNF-a,MMP-9 and the transcription factor NF-κB, Signal transduction, transcriptional activation factor 3(Signal transducers and activators of transcription 3, STAT3) activity.ObjectiveTo observe inflammation model impact on inflammatory factors, and the LPS tothe NF-κB,JAK/STAT3, MAPK, PPAR signaling pathways in the NF-κB, STAT3, AP-1,PPARγthe change of their activity, screening the target channel.To observe the three Tiao-Bu Fei-Shen extracts(Bufei Jianpi,Bufei Yishen,Yiqi Zishen)THP 1 cells NF-κB,STAT3 transcription factors, so as to reveal molecular mechanism of the three Tiao-Bu Fei-Shen extracts and characteristics of the inflammatory response in the inhibition of COPD.Method1.Optimization of LPS induced inflammation model for THP 1 cellsAlamarblue method to test the LPS on THP 1 cells growth.The study has 2 large groups.Different concentration of LPS(including 2.5mg/m L,5mg/m L,10mg/m L,20 mg /m L,40 mg/m L) at different times(6h,12 h,24h,48h) impacted on THP-1 cells;Different concentration of LPS(including 0.125mg/m L,0.25mg/m L,0.5mg/m L,1mg/m L,2 mg/ m L)48h for THP-1 cells growth.By ELISA method to detect(includeing 1mg/m L,2 mg/m L in the different time points after the secretion of IL-6,IL-10, IL-8,TNF-a,MM P-9and TIMP-1,to select best concentration and action time for LPS.Electrophoretic mobility of the experiment method(EMSA)detected(1mg/m L LPS for 24h)the activation of transcription factors(NF-κB,AP-1,PPARγ, STAT3).2.The preparation of medicated serum96 Japanese big ear rabbits were randomly divided into:for the normal control group, and three Tiao-Bu Fei-Shen extracts(Bufei Jianpi,Bufei Yishen,Yiqi Zishen),giving Bufei Jianpi fang,Bufei Yishen fang,Yiqi Zishen fang for 2 times in a day, in the most seven 1 h after the treatment, by abdominal aortic blood, serum, centrifugal separation filter repackaging, 56 ℃, 30 min inactivated-70℃ after saved for later use.3.Observe three Tiao-Bu Fei-Shen extracts of transcription factors NF-κB,STAT3According to the results of the above model optimization grouping intervention for the blank group, model group,Bufei Jianpi,Bufei Yishen,Yiqi Zishengroup.Each group divided into 20%, 40%, two concentration, serum ELISA method to detect cytokine IL-8, TNF-a, MMP- 9.For the NF-κB signaling pathway, adding inhibitor Pyrrolidine dithiocarbamate salts(Pyrrolidine dithiocarbamate, PDTC) group;For JAK/STAT signaling pathway, adding inhibitor stattic group, EMSA method to detect the NF-κB and the activity of STAT3 transcription factors.Result1. The LPS induced inflammation model THP-1 cells1.1 The effect of LPS on THP-1 cells growingAlamarblue method to test the influence of LPS on THP-1cells growth.(including 10mg/m L LPS induced 6h reductiion ratio decreased(p<0.05),2.5-40mg/m L concentration of LPS induced 12 h,24h,48 h reduction ratio decreased(p<0.05).0.125-2 mg/m L concentration of LPS induced 48 h reduction ratio has no change.10mg/m L LPS at 6 h and 2.5-40 mg/m L LPS in 12 h,24h,48 h can make the cytoplasm retraction, not full,cell volume small, growing more slowly.1.2 The effect of LPS on THP-1 cells secreting cytokines IL-6, IL-10, IL-8, TNF-a, MMP-9 and TIMP-1ELISA method to detect 1mg/m L,2mg/m L LPS on THP 1 cells in different time points(6 h,12 h,24h,48h) impacting on the level of cytokine.For the influence of IL-6: compared with normal control group,1 mg/m L,2mg/m L LPS at all time points increased secretion of IL-6(p< 0.01);Compared with 12 h LPS,1mg/m L,2mg/m L LPS in 24 h, 48 h increased secretion of IL-6(p<0.01);2mg/m L group than 1mg/m L at 24 h, 48 h secretion of IL-6 decreased(p<0.01).For the influence of IL-8:compared with normal control group,1mg/m L,2mg/m L LPS IL-8 secretion decline when 12h(p<0.05),in 24 h,48h IL-8 secretion increased(p<0.05,p<0.01);2 mg/m L group at 48 h(2mg/m L than 1mg/m L secretion of IL-8 increased(p<0.05).For the effect of TNF-a:compared with normal control group, including 1 mg/m L LPS at 6h, 48 h TNF-a secretion increased(p<0.01),2 mg/m L LPS at 6h,12 h,48h TNF-a secretion increased(p<0.01);2mg/m L group than 1mg/m L at 6h,12 h,48h secreted TNF-a increased(p< 0.01).For the influence of IL-10:compared with normal control group,including 1 mg/m L LPS at 6 h IL-10 secretion increased(p<0.05),in 24 h secretion of IL-10 reduce(p<0.05),including 2 mg/m L LPS at all time points to the influence of IL-10 no statistical difference;2 mg/m L group than 1 mg/m L at 6h,24 h secretion of IL-10 decrease(p<0.05).For the influence of MMP-9: compared with normal control group,2 mg/m L LPS at 6h,48 h MMP-9 secretion increased(p< 0.01);2 mg/m L group than 1mg/m L at 6h,48 h secretion MMP-9 increased(p<0.01).For the influence of the TIMP-1:compared with normal control group,including 1 mg/m L LPS TIMP-1 at 6h,48 h secreted decrease(p<0.01),at the time of 12 h TIMP-1 secretion increased(p<0.05), 2 mg/m L LPS in 6h,12 h TIMP-1 secretion increased(p<0.01,p<0.05),in 24 h,48h TIMP-1 secretion decline(p<0.01);2mg/m L group than 1 mg/m L at 6h TIMP-1 secretion increased(p< 0.01),in 24 h TIMP-1 secretion decline(p<0.01).1.3 The effect of NF-κB, STAT3,AP-1,PPARγby LPS on THP-1 cellsEMSA method(1mg/m L LPS induced THP-1 cells for 24 h of transcription factor the NF-κB, STAT3, AP-1,the influence of the activity of PPARγ.Compared with normal controlgroup,1mg/m L LPS group of NF-κB,STAT3 probe bonding degree increased(p< 0.01);Compared with normal control group, 1 mg/m L LPS group, PPARγ probe for AP-1 considering the change of degree has no statistical significance.2.THP 1 cells of NF-κB, STAT3 regulation of the three Tiao-Bu Fei-Shen extracts drug containing serum in the influence of inflammatory reaction mechanism2.1 The three Tiao-Bu Fei-Shen extracts three different concentrations of serum medi cated effects on THP-1cells growthBy alamarblue method to determine different concentrations of serum medicated THP-1 cells growth.Compared with 20% of blank serum group,the reduction rate for the 20% Buyi Fei shen group was obviously increased(p< 0.05),20% Bufei Jianpi and Yiqi Zishen group reduction rate was significantly higher(p<0.01);Compared with 20% model group, 20% Bufei Jianpi and Yiqi Zishen group reduction rate was significantly higher(p<0.01);Compared with 40 % model group,40% reduction rate of Bufei Jianpi group significantly decreased(p<0.05), the residual concentration of medicated serum reduction rate has no obvious statistical significance.2.2 The influence of IL-8, TNF-?, MMP-9 by the three Tiao-Bu Fei-Shen extracts drug containing serum of THP-1 cellsELISA method to detect 20%,40% the three Tiao-Bu Fei-Shen extracts(Bufei Jianpi, Bufei Yishen,Yiqi Zishen) medicated serum of THP-1 cells secrete IL-8,the influence of TNF-?, MMP-9.Compared with 20% blank serum group, MMP-9, TNF-? of 20% model group increased(p<0.01,p<0.05),20% 100mmol/L stattic group, IL-8 secretion decreased(p<0.01),20% 100 mmol/L PDTC group secretion of IL-8 reduced(p<0.01);Compared with 20% mod el group, 20% 100mmol/L stattic group, IL-8, TNF-? secrete decrease(p<0.01), 20% 100 mm ol/L PDTC group for secretion of IL-8 reduced(p<0.01).20% the three Tiao-Bu Fei-Shen e xtracts drug-containing serum of MMP-9, the secretion of TNF-? has a tendency to reduce,bu t had no significant statistical difference.Compared with 40% blank serum group,40%model group,MMP-9,IL-8,TNF-? secretion increased(p<0.01),40% Yiqi Zishen group TNF-? secrete decreased(p<0.01),40% 100 mmol /L stattic group,40% 100 mmol/L PDTC group MMP-9,IL-8 secretion reduced(p<0.01),the secretion of TNF-? decreased(p<0.05);Compared with 40% model group,40% the three Tiao-Bu Fei-Shen extracts containing medicine serum levels of MMP-9 secretion declined(p<0.05),the secretion of IL-8, TNF-? decreased(p<0.01),the tendency for 100mmol/L stattic, 100mmol/L PDTC group MMP-9, IL-8 secretion reduced(p<0.01),the secretion of TNF-? de creased(p<0.05).2.3 The three Tiao-Bu Fei-Shen extracts containing serum drug and inhibitors of THP- 1 cells NF-κB, the influence of STAT3 transcription factorsEMSA method to detect the three Tiao-Bu Fei-Shen extracts and inhibitor, and the formulas were 20%, 40% concentration of medicated serum groups of NF-κB,STAT3 activity.For the activity of transcription factor NF-κB: compared with 40% of blank serum group,40% model group with NF-κB probe level increased(p<0.01);Compared with 40% model group,40% the three Tiao-Bu Fei-Shen extracts and inhibitor PDTC group and NF-κB probe bonding degree decreased(p<0.01);Compared with 20% blank serum group,20% the three Tiao-Bu Fei-Shen extracts containing medicine serum with NF-κB probe bonding degree has a tendency to increase,but no obvious statistical significance;Compared with model group 20%,there is no obvious statistical significance.For transcription factor of STAT3:activity:compared with 40% blank serum group, 40% model group with STAT3 probe combination degree increased(p<0.01);Compared with 40% model group, 40% the three Tiao-Bu Fei-Shen extracts containing serum and inhibitor stattic group combined with STAT3 probe decreased(p<0.01);compared with 20% blank serum group, 20% model group with STAT3 probe degree has a tendency to increase,but no obvious statistical significance;Compared with 20%model group,20% the three Tiao-Bu Fei-Shen extracts containing serum and inhibitor stattic group has no obvious statistical significance.Conclusion1. 1μg/m L LPS effect 24 h can activate the NF-κB, JAK/STAT pathway.The conditions of the optimization cell inflammation model, it is suggested that 1 μg/m L LPS induction THP 1 cells for 24 h to inflammation model.2. The three Tiao-Bu Fei-Shen extracts(Bufei Jianpi,Bufei Yishen,Yiqi Zishen) can cut in model group cells the level of inflammatory cytokines IL-8, TNF-?,MMP-9.It can weaken the expression of NF-κB,STAT3 transcription factors. |
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