The Effection Of The Three Tiao-bu Fei-shen Granules On Inflammatory Response In A549Alveolar Epithelial Cells Via Regulation NF-κB Signaling Pathway

Chronic Obstructive Pulmonary Disease(COPD) is a major disease endangeringhuman health. The pathogenesis of COPD is inflammatory response. Suppression ofinflammatory reaction is an important way to improve the symptoms of COPD andslow disease progression. The inflammatory response of COPD involved in a varietyof cells and multiple signaling pathways，and the alveolar epithelial cells play animportant role in it. The activation of nuclear factor-κB（NF-κB）signaling pathwaysis one of the reasons that leads to the excessive expression of inflammatory mediatorsand participation in inflammatory reaction. Therefore, suppress NF-κB signalingpathway is an important way to inhibit lung inflammation and treat COPD. Thepurpose of this paper is to research the adjust and control of NF-κB signaling pathwayof alveolar epithelial cells A549by three fang Tiaobu Feishen (Bufei Jianpi, BufeiYishen, Yiqi Zishen) of traditional Chinese medicine (TCM),and explore biologymechanism of suppression of COPD inflammatory reaction by Traditional ChineseMedicine.ObjectiveResearch the adjust and control of NF-κB signaling pathway of cells A549bythree fang Tiaobu Feishen of TCM Prove basis for the revealing of suppression ofCOPD inflammatory reaction mechanism.MethodSPF,60SD rats, divided into four groups: control, Bufei Jianpi, Bufei Yishen,Yiqi Zishen. Separately take normal saline、Bufei Jianpi fang, Bufei Yishen fang, Yiqi Zishen fang by gavage in rats from the four groups.Blood samples are obtained fromabdominal aorta in the7th gavage after1hour,centrifugal separation serum, and Savethe drug-containing serum in-70℃. Prepare cigarette smoke extract（CSE），and seekthe best reserve condition. Take different concentrations（40%，20%，10%，5%，2.5%）of CSE to stimulate alveolar epithelial cells A549through different time periods（6h，12h，24h，48h），Detect the cell proliferation and inhibition by MTT method；Test the secretion of cytokines of interleukin-6（IL-6）、interleukin-8（IL-8）、interleukin-10（IL-10）and tumor necrosis factor-α（TNF-α）in the supernatant byELISA method；Test protein expression of nuclear factor-κB(NF-κB）、Inhibitoryfactor–κBα(IκBα) and Myeloid differentiation factor88(Myd88）by western blotmethod. Choose the best condition of inflammation in vitro pattern according to theresult. Divided intervention cells into six groups: normal、model、blank serum、BufeiJianpi serum, Bufei Yishen serum, Yiqi Zishen serum. Each serum groupconcentration into high（20%）、medium（10%）and low（5%）, test according to theabove specification. Then add in the PDTC group，divided into threeconcentration(25μmol/L,50μmol/L,100μmol/L).Test NF-κB activity in cells by themethod of electrophoretic mobility shift assay（EMSA）.Result1CSE store conditionCSE light absorbance value under light avoiding condition has varied fromdifferent time points（0h、6h、12h、24h、48h）、different store conditions（roomtemperature、4℃、-20℃）and different wavelength（s236nm、259nm、282nm、320nm）.Compare to0h，CSE OD value have varied in room temperature in236nm at6h、12hand24h（p＜0.05），OD value varied in259nm at12h（p＜0.05）；OD value varied in4℃236nm at6h and24h（p＜0.01），OD value varied in259nm at6h（p＜0.05）；OD value varied in-20℃236nm at12h、24h、48h（p＜0.05）. Compare to6h，ODvalue varied in4℃259nm at12h、24h（p＜0.01，p＜0.05）. Compare to12h，ODvalue varied in4℃259nm at48h（p＜0.05）. Remaining OD value varies betweeneach time point have no obvious statistical significance（p＞0.05）.2Cells morphology and quantityHigh concentration（40%-10%）CSE will change cellular morphology：smallercells、cytoplasmic rough；Low concentration（5%-2.5%）CSE have a smaller influenceon cellular morphology. CSE in each concentration all have an inhibitory effect oncell growth；There is a positive correlation between inhibition ratio and concentration （r=0.957、0.941、0.965、0.953）；Negative correlation occurred between inhibition ratioand action time under the concentration of2.5%、5%、2.5%（r=-0.512、-0.706、-0.620），inhibition ratio and action time have no correlation under the rest of theconcentrations.3Cytokine expression in each group3.1The influence of CSE to cytokinesNormal and the other CSE groups secreting IL-6、IL-8reach the secretion peakat48h；10%CSE group and2.5%CSE groupsecreting IL-10reach the secretion peak at48h，5%CSE groups secrete the least of IL-10at48h；Each cell group secreting TNF-αhave no obvious correlation with action time. Compare to normal group at48h，both2.5%CSE group and10%CSE group reduced the secretion of IL-6（p＜0.01），5%CSEgroup and20%CSE group increased secretion of IL-6（p＜0.01，p＜0.05）；2.5%CSEgroup reduced the secretion of IL-8，there was no significant statistical difference（p＞0.05）.5%CSE group increased secretion of IL-8（p＜0.01），10%CSE groupincreased secretion of IL-8，there was no significant statistical difference（p＞0.05），20%CSE group reduced the secretion of IL-8（p＜0.01）；Each CSE group all increasedsecretion of IL-10（p＜0.01），among them5%CSE group has the least increase，10%CSE group has the most increase；2.5%CSE group、5%CSE group、10%CSE grouphave a slight increase on the secretion of TNF-α，20%CSE group have a slightdecrease on the secretion of TNF-α，there was no significant statistical difference（p＞0.05）.5%CSE group has a higher standard of the secretion of IL-6、IL-8than otherCSE groups（p＜0.01，p＜0.05）；5%CSE group has a lower standard of the secretionof IL-10than other CSE groups（p＜0.01）.3.2The influence of Thress Fang Tiaobu Feishen to cytokinesThe secretion of IL-6and IL-8of Bufei Jianpi serum, Bufei Yishen serum andYiqi Zishen serum have a significant decrease compared to blank serum group（P＜0.01），among them nourish the Bufei Yishen serum group decreased the most（P＜0.05，P＜0.01）；Bufei Jianpi serum and Yiqi Zishen serum increased the secretion ofIL-10（P＜0.01）， Bufei Yishen serum decreased the secretion of IL-10，there was nosignificant statistical difference（P＞0.05）.4Protein expression of Myd88、IκBα and NF-κBp654.1The influence of CSE to protein expressionThe model group of Myd88、IκBα and NF-κBp65standard of protein expressionhave significantly increase compared with normal group（P＜0.01）. 4.2The influence of Three Fang Tiaobu Feishen to protein expressionIn the three groups-Bufei Jianpi serum（20%，10%）、Bufei Yishen serum （20%，10%）and Yiqi Zishen （20%）,protein expression of Myd88decreased compared toblank serum group（P＜0.01，P＜0.05）；in the Bufei Yishen serum（10%）theexpression of IκBα decreased compared to blank serum group（P＜0.05）； in nourishthe Bufei Yishen serum （20%）、Bufei Jianpi serum（20%，10%）,the expression ofNF-κBp65decreased compared to blank serum group（P＜0.01，P＜0.05）；each5%serum group to the expression of protein have no significant statistical difference（P＞0.05）；20%serum group is significantly lower than10%serum group and5%serumgroup（P＜0.01，P＜0.05）.5Activity expression of NF-κBThe combination of cells stimulated by CSE and NF-κB probe has increased；Thecombination of Three Fang Tiaobu Feishen, cells after the intervention of PDTC andNF-κB probe has decreased，NF-κB activity declined.Conclusion1.OD value varied between after-stored CSE and fresh preparation of CES,it isrecommended to use fresh preparation of CES.2. The stimulate from CSE lead to the inhibition of alveolar epithelial cells A549growth. Secretion of inflammatory factors by stimulating cells can be increased, andit can establish cell inflammatory reaction model by the stimulating of CSE.3. The key conditions of the preparation of model is determined，suggest to useconcentration of5%CSE as alveolar epithelial cells and48h to establish cellinflammation model.4.In Three Fang Tiaobu Feishen, COPD inflammatory response can be inhibited bylower related cytokines expression level and adjusting NF-κB Signaling Pathway.5. Among Three Fang Tiaobu Feishen, Bufei Yishen Fang has a more outstandingcurative effect on lower cytokines level and inhibit protein expression. Highconcentration（20%）has a better effect on the inhibition of IκBα、Myd88expression.