Study On The Role Of APELIN/APJ In Inflammatory Reaction Of Human Bronchial Epithelial Cells And The Effect Of Elephantopus Scaber Linn On It

Objective:To explore the expression and localization of Apelin/APJ system in human bronchial epithelial cells(16HBE)and Inflammatory reaction,and to observe the effect of Apelin/APJ system on bronchial epithelial cell inflammation.To study the effects of Elephantopus scaber Linn on inflammation of bronchial epithelial cells and Apelin/APJ system,in order to provide possible intervention targets and new ideas for treatment of airway inflammatory diseases.Methods:Experimental grouping and establishment of acute bronchial inflammatory injury model: in this study,16 HBE cells were take into 6 groups according to different conditions(n=8).They were normanl group,LPS group,LPS+apelin-13 group,LPS+10,20,40μg/m LElephantopus scaber Linn.16 HBE cells were pretreated with APELIN-13 and Elephantopus scaber Linn of low,middle and high concentrations,and then stimulated with LPS(10μg/m L)for 16 h.Inflammatory response model of human bronchial epithelial cells: with the increase of LPS concentration and stimulation time,the secretion of IL-1β increased significantly,the increase of IL-1β reached the maximum at 16 h of 10μg/ml LPS,and decreased after16 h,and the results of TNF-α and IL-6 release were consistent.On this basis,it is best to establish inflammation model by stimulating LPS for 16 hours.There was no difference about cell survival rate of 10～80μg/ml Elephantopus scaber Linn.There was no difference about cell survival rate between the 0.1-1.0μmol/L group(apelin-13)and the normanl group(P>0.05).therefore,LPS(10μg/ml),and the Elephantopus scaber Linn was20,40,80μg / ml,1.0μmol/L(apelin-13)was selected to conduct the drug intervention trial.Observation index: first,the expression and localization of APELIN/APJ in 16 HBE cells and inflammatory models were detected by laser confocal scanning microscopy,then the effect of Elephantopus scaber Linn on the cell injury induced by LPS was detected by CCK8 method,and TNF-α,IL-1β and IL-6 of cells were checked by ELISA.The transcription and expression of APELIN/APJ were checked by RT-PCR and WB.Data analysis: all data are represented by mean ± standard deviation.We used SPSS22.0 software or Graph Pad Prism8 or image J software for statistical analysis,the comparison between groups using analysis of variance,P<0.05.There was significant difference.Results:1.To observe the localization expression of Apelin/APJ protein use master microscope: Apelin/APJ protein was localized on the cell membrane,and 10μg/m L LPS stimulation,Apelin-13 and Elephantopus scaber Linn increased the expression of Apelin/APJ protein on the cell membrane.2.CCK8 method was used to detect the infiuence of Elephantopus scaber Linn 、Apelin-13、LPS of cells: contrast the normanl group,different concentrations of LPS could affect the viability of 16 HBE cells,and 10μg/ml LPS reduced the cell viability by nearly half,so 10μg/m L LPS was selected to stimulate cells.Contrast LPS group,the vitality of cells was highly increased after Apelin-13 intervention(p<0.05).The infiuence of airway epithelial cells increased highly after the intervention of Elephantopus scaber Linn,and the higher the concentration was,the more obvious the cell viability was(p<0.05).3.The secretion of inflammatory was detected by ELISA : contrast the normanl group,IL-1β,IL-6 and TNF-α of the LPS group was highly increased.contrast the LPS group,IL-1β,IL-6 and TNF-α were decreased highly after the intervention of the Elephantopus scaber Linn(p<0.05),and the secretion decreased with the increase of the concentration.The inhibitory effect of 80ug/ml on the release of inflammatory cytokines was better than that of Apelin-13 group(p< 0.05).4.Apelin/APJ protein were checked by WB method: contrast the control group,LPS could increase the expression of Apelin/APJ about 16 HBE,and compared with LPS group,the expression level of Apelin/APJ in 16 HBE was further increased after intervention with Apelin-13 and Elephantopus scaber Linn(P<0.05).The results showed that LPS could up-regulate the expression of Apelin/APJ,and Apelin-13 and Elephantopus scaber Linn could further stimulate the expression of Apelin/APJ in bronchial epithelial cells.5.Detection of Apelin/APJ m RNA transcription by RT-PCR: compared with the control group,LPS(10μg/ml could increase the Apelin/APJm RNA transcription,and compared with the LPS group,the Apelin/APJ m RNA transcription level was further increased after the intervention of Apelin-13 and Elephantopus scaber Linn(P< 0.05).Conclusion:1.Apelin/APJ protein is located and expressed on the cell membrane.2.Apelin/APJ system is an endogenous protective mechanism against lung injury,which is activated in bronchial epithelial cell inflammation to combat lung injury.Apelin/APJ is a possible target for intervention of airway inflammatory diseases.3.Elephantopus scaber Linn plays an anti-inflammatory role by up-regulating the intracellular Apelin/APJ system of 16 HBE and reducing the secretion of inflammatory mediators.Elephantopus scaber Linn has the anti-inflammatory effect as apelin-13,which provides new ideas for the treatment of airway inflammatory diseases.