The Migration Of THP-1Cells And The Expression Of HMGB1Induced By LPS And The Effects Of Polydatin Intervention

Objective:To investigate the effects of polydatin intervention on THP-1cells migration and HMGB1expression induced by LPS and to explor the molecular mechanism.Methods:(1)The well-growing THP-1cells were randomly assigned to the following6groups (n=3):①control group:only THP-1;②LPS stimulated group:THP-1+LPS(5μg/ml);③0.05mmol/L polydatin intervention group:THP-1+PD(0.05mmol/L)+LPS;④0.4mmol/L polydatin intervention group:THP-l+PD(0.4mmol/L)+LPS;⑤1.0mmol/L polydatin intervention group:THP-l+PD(1.0mmol/L)+LPS;⑥PDTC intervention group:THP-1+PDTC(0.01mmol/L)+LPS。 The three polydatin intervention groups of THP-1were preincubated with corresponding concentration of polydatin in RPMI1640culture medium for2hours, and the PDTC group of THP-1were preincubated with0.01mmol/L PDTC in RPMI1640culture medium for30minute,after washing,RPMI1640was supplied to THP-1in control group while RPMI1640containing5μg/ml LPS was supplied to THP-1in the rest5group and incubation continued for24hours.(2) To observe the migration of THP-1cells stimulated by the supernatants from the six group by using the transwell chamber, and tested the concentration of MCP-1and HMGB1in the supernatants from the six group.(3) Real-time PCR were applied to measure the HMGB1mRNA expression, the western blot was applied to measure the HMGB1and NF-kappaBp65protein expression, the electrophoretic mobility shift assay was applied to measure the activity of NF-kappaBp65in THP-1cells in the①,②and⑤group, and the immunofluorescence was applied to observe the relocation of HMGB1in the THP-1cells in the three groups.Results:(1) LPS induced the significant migration of THP-1cells(P<0.05vs. control group),and the migration stimulated by LPS was inhibited by PD in a dosage-dependent manner and the intervention of PDTC (P<0.01), LPS induced the MCP-1and HMGB1significantly elevation in the supernatants of THP-1cells(P<0.01vs. control group),the effects were inhibited by polydatin and PDTC(P<0.01).(2)The HMGB1mRNA expression in LPS+polydatin was lower than in LPS alone(P<0.01),and the polydatin also inhibited the HMGB1protein expression, NF-kappaBp65protein expression and the activation of NF-kappaBp65induced by LPS.(3)LPS stimulated the transfer of HMGB1from nucleu to cytoplasm, and polydatin can partially inhibite this process.Conclusion:(1)LPS induces the migration of THP-1cells significantly, this effect is related to the activation of NF-kappaB, the secretion of THP-1and HMGBl; (2) Polydatin inhibits the LPS-induced migration of THP-1cells, this effect is related to the inhibition of HMGB1-NF-kappaB signaling pathway,and the reduction of the inflammatory cytokines and chemokines;(3)The inhibition of the HMGB1-NF-kappaB signaling pathway, the reduction of inflammatory factor and chemokines expression,as well as the inhibition of the chemotaxis of the inflammation cells may help us to understand the mechanisms of the anti-inflammation and anti-atherosclerosis from polydatin.