The Effect Of Baicalein On LPS/ATP-induced PBMCs To Secrete Inflammatory Factors And Its Mechanism

Objective:The aim of this study was to investigate the effect of baicalein on PBMCs secretion of inflammatory factors in LPS/ATP induced RA patients,and to further explore the possible mechanism of baicalein inhibiting the secretion of inflammatory factors,and then to clarify the mechanism of P2X7/TLR4 inflammatory related pathway in the occurrence and development of patients,and to provide a basis for baicalein application in clinic.Methods:1.50 RA patients were recruited for our study.Collecting all patients’relevant clinical indicators including the levels of CRP,ESR and the DAS28 scores.Besides,30 healthy volunteers in a healthy control group were also recruited.The cytokine concentrations IL-1β,TNF-αand IL-1βin plasma were detected by Enzyme-linked immunosorbent assay（ELISA）.2.The peripheral blood mononuclear cells（PBMCs）were isolated from peripheral blood by centrifugation on Ficoll-Hypaque solution.Then,the PBMCs were cultured in RPMI-1640 medium in plates and maintained in a 5%CO₂humidified atmosphere at 37℃.The treatment of drugs is as follows:（1）vcontrol group:No drug interference;（2）drug interference group:monocytes were cultured with LPS（100 ng/ml）for 1 hour,then treated with baicalein（0,20,40,80μM）for 12hours,and finally treated with 1 mM of ATP for 1 hour.3.ELISA was used to detect the cytokine IL-1β,IL-6 and TNF-αin culture supernatant of monocytes.4.To detect the effects of baicalein on the P2X7/NLRP3 and TLR4/NF-κB of peripheral blood monocytes in RA patients:（1）The expression of monocyte P2X7、NLRP3、TLR4mRNA was detected by RT-PCR method;（2）Detection of P2X7、NLRP3、TLR4、NF-κB、IκB and other protein expression by Western blotting.Results:1.The IL-1β、IL-6 and TNF-αlevels of inflammatory cytokines in plasma of RA patients were significantly higher than those of healthy controls（^*P<0.05）.4.LPS/ATP treatment induces the release of proinflammatory cytokines（IL-1β、IL-6 and TNF-α）;baicalein attenuates the release of IL-1β、IL-6 and TNF-α.^#P<0.05 compared with control group;compared with LPS ATP treatment,^*P<0.05。4.Baicalein can reduce the activity of P2X7/NLRP3 and TLR4/NF-κB related signaling pathways,thus affecting the secretion level of inflammatory factors,but hardly affecting the expression of p38、JNK and ERK1/2.Conclusion:Baicalein attenuates LPS/ATP-induced inflammatory responses PBMC patients,which may function by mediating P2X7/TLR4 pathways,and suggests that baicalein has the potential to act as an adjuvant for conventional antirheumatic drugs by inhibiting inflammation-related pathways.