Effects And Mechanisms Of Andrographolide On Expression And Secretion Of Procoagulant And Fibrinolytic Inhibitory Factors In Alveolar Type Ⅱ Epithelial Cells Stimulated With LPS

Objective:To investigate the effect of Andrographolide on procoagulant and fibrinolytic inhibitory factors in a rat alveolar type II epithelial cell line(RLE-6TN)stimulated by lipopolysaccharide(LPS)and its mechanism.method:1.The safe dose range of Andrographolide for RLE-6TN was determined by CCK-8 method;2.RLE-6TN cells were pretreated with different doses of Andrographolide for 1 h,followed by LPS stimulation for 24 h;3.RLE-6TN cells were infected by lentivirus to construct a stable p65-silenced cell line(p65-/-)and a blank control cell line(NC),and were administered with andrographolide and LPS stimulation treatments via the same approach;4.The m RNA level of tissue factor(TF),plasminogen activator inhibitor-1(PAI-1)and tissue factor pathway inhibitor(TFPI)in cells was detected by real-time quantitative polymerase chain reaction(RT-q PCR);the protein expression of TF,PAI-1 and TFPI in cells was detected by immunoblotting(Western blotting);the expression of thrombin-antithrombin complex(TAT),pro-collagen type III peptide(PIIIP),antithrombin III(ATIII)and activated protein C(APC)in cell supernatant was detected by enzyme-linked immunosorbent assay(ELISA);5.The activation status of NF-κB signaling pathway in RLE-6TN cells was detected: intracellular p65 m RNA expression level was detected by RT-q PCR;cytoplasmic p-Iκκβ and p-p65 expression was detected by Western blotting;DNA binding ability of p65 was detected by ELISA,and endonuclear p65 expression level was detected by immunofluorescence.Results:1.RLE-6TN pretreated with Andrographolide showed decreased m RNA and protein expression levels of TF and PAI-1 and elevated m RNA and protein expression of TFPI upon LPS stimulation,while exhibited decreased level of TAT and PIIIP and increased level of ATIII and APC in the cell supernatant,in which the effect of 25mg/L andrographolide was greater than that of 12.5 mg/L and 6.25 mg/L.However,the alterations of different factors upon drug treatment of cells in each group did not reach the level of the control group(all p < 0.05);2.Andrographolide pretreatment significantly inhibited the activation of NF-κB signaling pathway,as indicated by the reduced DNA binding of p-Iκκβ,p-p65 and p65 and the decreased expression of p65 in the nucleus;3.Compared with NC group,p65-/-cells showed reduced expression of TF,PAI-1 and less secretion of PIIIP and TAT,whereas expression of TFPI and secreted ATIII and APC increased;compared with the p65-/-group,these factors in p65-/-cells under LPS stimulation showed completely opposite changes;The drug pretreatment group NC+ Andro +LPS showed similar results to the p65-/-+LPS group in terms of changes in these factors;compared with the p65-/-+LPS and NC+ Andro+LPS groups,the p65-/-+ Andro +LPS group showed significantly lower level of procoagulant factors TF,TAT and fibrinolytic inhibitory factors PAI-1 and PIIIP,and significantly higher level of anticoagulant factors TFPI,ATIII and APC,with more significant changes.Conclusion:Andrographolide pretreatment significantly inhibited the expression and secretion of procoagulant and fibrinolytic inhibitory factors in LPS-stimulated rat alveolar type II epithelial cells and promoted the expression and secretion of anticoagulant factors in a dose-dependent manner.The mechanism of action of andrographolide was at least partially related to the inhibition of NF-κB signaling pathway.