Effects Of UrsolicAcid On The HMGB1 And NF-κB Of THP1 Monocytes

Objectives To investigate the effects of UA on the expression of HMGB1 and NF-κB transcriptional activity in THP1 cells.Methods THP1 cells were cultured in 50ml culture bottle with RPMI-1640 medium including 100U/ml penicillinum,100U/ml phytomycin and 10% calf serum at 37℃, 5%CO2, saturated humidity incubator. After being stimulated by different concentrations UA, the cells were detected correspondingly.(1)MTT:When they growed favorably, the cells(1×104 cells/well) were seeded in culture plates of 96 wells. The cells were divided into 6 groups and 3 wells of every group at random. After being stimulated by different concentrations UA for 24 hours, the cell proliferation were checked by MTT.(2)RT-PCR:When they growed favorably, the cells(1×105 cells/well) were seeded in culture plates of 24 wells. The cells were divided into 5 groups and 3 wells of every group at random. After being stimulated by different concentrations UA for 24 hours, total RNA in THP1 cells was extracted with Trizol, reversey transcripted into cDNA. The HMGB1 and p65 genes were amplificated with PCR, and production was observed by agarose gel electrophoresis under viltalight lamp. (3)Western blot:When they growed favorably, the cells(2×10 cells/well) were seeded in culture plates of 6 wells. The cells were divided into 5 groups and 3 wells of every group at random. After being stimulated by different concentrations UA for 48 hours, total protein in THP1 cells was extracted and then HMGB1 and p65 protein was detected by western blot.(4)NF-κB-luciferase report gene: When they growed favorably, the cells(2×105 cells/well) were seeded in culture plates of 24 wells. The cells were divided into 5 groups and 3 wells of every group at random. Plasmid pG13-NF-KB-luciferase report gene was transfected into THP1 cells according to TfxTM-50 reagent. After transfected for 40 hours, different concentrations UA were added. Luciferase activity detection was performed when transfecting for 48 hours. At last, the NF-κB activation were analyzed in each group THP1 cells.Results (1)Effects of UA on cell proliferation:Compared with control group, the cell proliferation of 20 μmol/L UA group was inhibited significantly(P<0.05)and there was no changes in other groups.(2)Effects of UA on HMGB1 and p65 mRNA:(DHMGB1:Compar-ed with control group, the HMGB1 mRNA of 1 μmol/L UA group was more(P<0.05)and the other groups were not changed. (2)p65:The p65 mRNA of 1 μmol/L UA group was more(P<0.05)than control group and combination group of 1 μmol/L UA and BAY 11-7082 respectively. (3)Effects of UA on HMGB1 and p65 protein: ①HMGB1:Compared with control group, the HMGB1 protein of 1μmol/L UA group was more and there was no changes in other groups.② p65:The p65 protein of 1 μmol/L UA group was more(P<0.05) than control group and combination group of 1 umol/L UA and BAY 11-7082 respectively.(4)Effects of UA on NF-κB transcription activity:The NF-κB activity of 1 μmol/L UA group was more(P<0.05)than control group and combination group of 1 μmol/L UA and BAY 11-7082 respectively.Conclusions UA could modulate the HMGB1 expression and location in THP1 cell and NF-κB transcription activity and the influence exhibits a certain bidirectional.