The Effect Of Puerarin On The Expression Of Neutrophilic Nuclear Factor-κB And Pro-inflammatory Cytokines In Human Neutrophilic Granulocyte Activated With LPS In Vitro

Objective To investigate the effect of puerarin on expression of intracellular nuclear factor-κB(NF-κB), pro-inflammatory cytokines in human neutrophilic grannulocyte induced and activated with lipopolysaccharide(LPS) in vitro, and to explore the potential mechanism of its anti-inflammatory effect。Methods Human whole blood 15ml was withdrawn intravenously from an healthy adult volunteer each time, eight time for this study. The blood sample was divided into five test groups treated respectively with nothing (blank control group), immunoglobulin (isotype control group), endotoxin (1ug/ml) (activated control group), puerarin(10ug/ml) + endotoxin(1ug/ml ) (test group 1), and puerarin(50ug/ml) + endotoxin (1ug/ml)(test group 2). The intracellular expression of nuclear factor-кB and pro-inflammatory cytokines (interleukin-6 and interleukin-8) were detected by flow cytometry。Results (1) There is almost no NF-кB expression in the blank control group. Compared with the blank group, NF-кB expression increased significantly (p=0.000,p<0.01) in the endotoxin group ,NF-кB expression increased in both the test group 1 and 2(p=0.018,p<0.05).Compared with the endotoxin group, NF-кB expression decreased markedly (p=0.000,p<0.01) in both the test group 1 and 2. No statistical difference was found between the test group 1 and 2 (p>0.05). (2) Almost no IL-6 expression was detected in the blank control group. Compared with the blank group, IL-6 expression increased significantly (p=0.000,p<0.01) in the endotoxin group, IL-6 expression increased in both the test group 1and 2(p=0.000,p<0.01).Compared with the endotoxin group, IL-6 expression in both the test group 1 and 2 decreased markedly (p=0.000,p<0.01). No statistical difference was found between the test group 1 and 2 (p>0.05). (3)There is no IL-8 expression in the blank control group. Compared with the blank group, IL-8 expression increased significantly (p=0.000,p<0.01) in the endotoxin group ,IL-8 expression increased significantly in the test group 1 and 2. Compared with the endotoxin group, IL-8 expression decreased significantly (p=0.000,p<0.01) in both the test group 1 and 2. No statistical difference was found between the test group 1 and 2(p>0.05). (4)The immunoglobulin isotype control group is the immunoglobulin subtype, which is unrelated to the stain monoclonal antibody specificity. It was used to detect the non-specific staining that is induced by the non-specific binding of cells with the antibody。Conclusion This result revealed that Puerarin preconditioning may attenuate systemic inflammatory response syndrome by prohibiting activation of neutrophil characterized by over-expression of nuclear transcription factor-кB and over-production of pro-inflammatory cytokines.