Primary Investigaion Of The Function Of Glucocorticoid Induced Leucine Zipper In Inflammatory Reaction

Objective: Research the expression and function of glucocorticoid inducedleucine zipper in the THP-1 cells and the THP-1 cells transfected with GILZ gene inlipopolysaccharide. Primary investigation about the mechanism of the glucocorticoidinduced leucine zipper in inflammatory reaction.Methods: THP-1 cells were cultured in serum-free RPMI-1640 medium, thetotal RNA was extracted, the GILZ gene was amplified and connected with pMD 19-Tsimple vector, then transferred into E.coli Competent Cells JM109 and the sequencewas checked. The DNA of GILZ was purified after cutted by EcoRI/HindⅢincisionenzyme. The DNA was connected with pcDNA3.1(+) vector, transferred into E.coliCompetent Cells DH5a, the plasmids were cutted by EcoRI/HindⅢincision enzymeafter amplified, the target plasmids were picked out after the genes were identified bythe electrophoresis. The target plasmids were transfected into the THP-1 bylipofection regeant. Human monocyte cell line THP-1 cells and THP-1 cellstransfected with the GILZ gene were divided into two groups respectively. Treatedone group of THP-1 cells and one group of THP-1 transfected with LPS, the suspend was collected into Eppendorf tube. Twelve hours later total RNA and total proteinwere abstracted in all four groups. The mRNA encoding for expression of GILZ wasdetected by the methods of the RT-PCR. The protein expression of NF-κB and thec-Fos subunit of AP-1 protein were surveyed by Western Blotting. The inflammatorycytokines of TNF-α, IL-1βand IL-10 were surveyed by ELISA.Results: The results showed that the A values of GILZ/β-actin in groups A, B,C and D were 0.5728±0.0019, 0.4856±0.0025, 0.6878±0.0023, 0.7158±0.0033, thedifferences were significant among groups A, B, C, D(P＜0.05). The total gray valuesof NF-κB p65 protein/GAPDH in groups A, B, C and D were 0.2607±0.0014,0.6991±0.0024, 0.3693±0.0046, 0.1580±0.0027, the differences were significantamong groups A, B, C, D(P＜0.05). The total gray values of c-Fos subunit/GAPDH ingroups A, B, C and D were 0.2295±0.0004, 0.5799±0.0005, 0.3625±0.0007,0.1088±0.0010, the differences were significant among groups A, B, C, D(P＜0.05).The concentration of TNF-αin groups A, B, C and D were 32.86±0.48, 98.49±0.84,68.76±0.35, 17.76±0.71, the differences were significant among groups A, B, C,D(P＜0.05). The concentration of IL-1 in groups A, B, C and D were 56.80±0.42,85.43±1.98, 43.26±1.46, 27.03±0.22, the differences were significant among groupsA, B, C, D(P＜0.05). The concentration of IL-10 in groups A, B, C and D were53.52±0.60, 32.82±0.11, 65.76±0.58, 88.07±0.23, the differences were significantamong groups A, B, C, D(P＜0.05). The expression of GILZ mRNA was increased inthe stimulation groups, this result both in THP-1 cells and HP-1 transfected with theGILZ gene(P＜0.01). Whereas, protein expressions of NF-κB p65 and the c-Fossubunit of AP-1 protein were increased in the stimulation groups(P＜0.01).Conclusion: The expression of GILZ gene was down-regulated by LPS.Overexpression of GILZ inhibited NF-κB and AP-1 activity, it suggested that GILZpossess anti-inflammatory function.