| Aims: The present study was designed to investigate the effects and mechanisms of zinc finger protein 667(ZNF667)on LPS-induced inflammation in Raw264.7 macrophages.Methods:(1)A macrophage inflammation model in vitro was established by lipopolysaccharides(LPS)induction in mouse Raw264.7 cells line,and the protein expression level of ZNF667 was detected by Western Blot(WB).(2)The ZNF667 overexpressing macrophages were generated by liposome-mediated transient transfection,and the mRNA and protein expression changes of inducible nitric oxide synthase(iNOS)and interleukin(IL)-1β were detected by WB and reverse transcription polymerase chain reaction(RT-PCR)in LPS-treated naive or ZNF667 overexpressing macrophages.The enzyme-linked immunosorbent assay(ELISA)was utilized to detect the level of IL-1β,IL-6 and tumor necrosis factor-α(TNF-α)in the macrophage culture supernatants.(3)The effects of enhanced ZNF667 expression on PI3K/AKT/mTOR expression andphosphorylation in LPS-treated macrophages were assessed by WB.(4)The effects of enhanced ZNF667 expression on transcriptional activity of nuclear factor-κB(NF-κB)were detected by dual-luciferase reporter assay,and NF-κB subunit p65,p38 mitogen-activated protein kinases(p38 MAPK),extracellular signal regulated kinase 1/2(ERK1/2)expression and phosphorylation in LPS-treated enhanced ZNF667 expression macrophage were also determined by WB.(5)The effects of enhanced or weakened ZNF667 expression on hexokinase 2(HK2)and6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3(PFKFB3)mRNA and protein expression in LPS-treated macrophage were determined by RT-PCR and WB,respectively,and the rate of glucose consumption and lactate production were also measured by chemical colorimetric assay.(6)The effects of 2-Deoxy-D-Glucose(2-DG)treatment on mRNA expression changes of IL-1β and iNOS resulting from ZNF667 knockdown in LPS-treated macrophages were detected by RT-PCR.(7)The effects of mTOR inhibitor rapamycin(RPM)treatment on mRNA expression changes of HK2 and PFKFB3 resulting from ZNF667 overexpression or ZNF667 knockdown in LPS-treated macrophages were measured by RT-PCR,and the rate of glucose consumption and lactate production were also determined by chemical colorimetric assay.Results:(1)Exposure of Raw264.7 macrophages to different concentrations of LPS(50ng/ml,100ng/ml,200ng/ml,400 ng/ml)for 12 hdemonstrated that LPS stimulated ZNF667 proteinexpression up-regulation,and the peak response in ZNF667 protein expression was detected at a concentrations of LPS 100 ng/ml.(2)The enhanced ZNF667 expression significantly inhibited LPS-stimulated up-regulation of iNOS and IL-1β mRNA and protein expression together with the secretion of IL-1β,IL-6 and TNF-α in macrophages upon exposure to LPS.(3)ZNF667 overexpression can confer resistance to LPS stimulation and that result in inhibition of PI3 K,AKT,mTOR hyperphosphorylation.(4)ZNF667 overexpression exerted no effect on the phosphorylation of NF-κB p65,ERK1/2,MAPK p38 in LPS-treated macrophages,and the transcriptional activity of NF-κB also remained unchanged when ZNF667 expression was up-regulated.(5)LPS-augmented mRNA expression of HK2 and PFKFB3,glucose consumptions and lactate productions were inhibited in the macrophages with ZNF667 overexpression compared with that in the control cells.(6)LPS-induced augmentation in the glycolytic genes HK2 and PFKFB3 mRNA expression,and the increases in proinflammatory cytokine IL-1β and iNOS mRNA expression were abolished by hexokinase inhibitor 2-DG in the ZNF667 knockdown macrophages.(7)The augmentation result from ZNF667 knockdown or the diminution result from ZNF667 overexpression in the LPS-induced the expression of HK2 and PFKFB3,glucose consumptions and lactateproductions in the macrophages were abrogated when the cells were treated with specific mTOR inhibitor RPM.Conclusion: ZNF667 suppressed LPS-induced mouse RAW264.7macrophages inflammation through mTOR-dependent aerobic glycolysis regulation. |
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