Study On The Effects Of Celastrol To The Promoter Activity Of HLA-B27 Gene

Purpose:To provide new evidence on molecular mechanism of treating ankylosing spondylitis (AS) for celastrol by observing the effect of celastrol to the activity of peripheral blood mononuclear cell (PBMC) in AS patients and inspecting the changes of expression of HLA-B27 gene as well as the activated factors relavent to its promoter under the impact of different concentration of celastrol.Methods:For the proof of cytotoxicity of PBMC in AS patients affected by celastrol, PBMC extracted from 3 cases of peripheral blood samples come from patients diagnosed as AS was cultured over 24 hours and co-cultured with different concentrations of celastrol for another 48 hours to evaluate the effect of celastrol on the activity of PBMC inspected by Cell Counting Kit-8 (CCK-8).For the proof of possible inhibition function of HLA-B27 promoter in PBMC of AS patients affected by celastrol, PBMC suspension adapoted from 10 cases of ten mililiter peripheral blood samples come from patients diagnosed as AS was cultured over 24 hours and co-cultured with different concentrations of celastrol for another 48 hours to extract RNA. The changes of mRNA expression on HLA-B27, TNF-α, IFN-γ, IL-1β, and IL-6 in cell were studied by Real-time PCR, while the changes of expression of cytokines such as HLA-B27, TNF-α, IFN-γ, IL-1β, and IL-6 were inspected by ELISA.Results:No obvious inhibition to the activity of PBMC on AS patients was observed for the concentration of celastrol lower than 1.0μmol/L (p>0.05), while significant inhibition to the activity of PBMC was observed for the concentration of celastrol greater than 3.0μmol/L (p< 0.05)The level of mRNA of HLA-B27, TNF-α, IFN-γ, IL-1β, and IL-6 in PBMC of AS patients showed a trend of downward as the concentration of celastrol increaed while comparing the medication group to the control group, especially when the concentration of celastrol was 1.0μmol/L, which exhibited a strongest inhibition with significant differences (p<0.05) on the changes of expression of mRNA of HLA-B27, TNF-α, IL-1β, and IL-6. The expression of cytokins of HLA-B27, TNF-α, IFN-γ, IL-1β, and IL-6 showed a trend of downward while comparing the medication group to the control group, also with a strongest inhibition when the concentration of celastrol was 1.0μmol/L.Conclusions:The celastrol exhibits an inhibition to the activity of PBMC in AS patients while its concentration is high; however, no obvious effect to the activity of PBMC was observed within a certain concentration range of celastrol, indicating that celastrol within a certain concentration range has no obvious cytotoxicity.The expression of HLA-B27, TNF-α, IFN-γ, IL-1β, and IL-6 in PBMC of AS patients decreased under the action of celastrol, indicating that celastrol might affect the activity of HLA-B27 promoter to reduce the expression of HLA-B27 by inhibiting the cytokines such as TNF-α, IFN-γ, IL-1β, and IL-6.