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Glucose 6-phosphate dehydrogenase knockdown enhances IL-8 expression in HepG2 cells via oxidative stress and NF-κB signaling pathway.

Yang HC, Cheng ML, Hua YS, Wu YH, Lin HR, Liu HY, Ho HY, Chiu DT - J Inflamm (Lond) (2015)

Bottom Line: This study was designed to investigate the effect of glucose 6-phosphate dehydrogenase (G6PD) deficiency on pro-inflammatory cytokine secretion using a palmitate-induced inflammation HepG2 in vitro model.Cytokine array analysis indicated that IL-8 is most significantly increased in G6PD-knockdown HepG2 cells.The palmitate-induced inflammation in G6PD-deficient HepG2 cells could serve as an in vitro model to study the role of altered redox homeostasis in chronic hepatic inflammation.

View Article: PubMed Central - PubMed

Affiliation: Healthy Aging Research Center, Chang Gung University, Kwei-Shan, Tao-Yuan 333 Taiwan ; Department of Medical Biotechnology and Laboratory Sciences, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan 333 Taiwan.

ABSTRACT

Background: This study was designed to investigate the effect of glucose 6-phosphate dehydrogenase (G6PD) deficiency on pro-inflammatory cytokine secretion using a palmitate-induced inflammation HepG2 in vitro model. The modulation of cellular pro-inflammatory cytokine expression under G6PD deficiency during chronic hepatic inflammation has never been investigated before.

Methods: The culture medium of untreated and palmitate-treated G6PD-scramble (Sc) and G6PD-knockdown (Gi) HepG2 cells were subjected to cytokine array analysis, followed by validation with ELISA and qRT-PCR of the target cytokine. The mechanism of altered cytokine secretion in palmitate-treated Sc and Gi HepG2 cells was examined in the presence of anti-oxidative enzyme (glutathione peroxidase, GPX), anti-inflammatory agent (curcumin), NF-κB inhibitor (BAY11-7085) and specific SiRNA against NF-κB subunit p65.

Results: Cytokine array analysis indicated that IL-8 is most significantly increased in G6PD-knockdown HepG2 cells. The up-regulation of IL-8 caused by G6PD deficiency in HepG2 cells was confirmed in other G6PD-deficient cells by qRT-PCR. The partial reduction of G6PD deficiency-derived IL-8 due to GPX and NF-κB blockers indicated that G6PD deficiency up-regulates pro-inflammatory cytokine IL-8 through oxidative stress and NF-κB pathway.

Conclusions: G6PD deficiency predisposes cells to enhanced production of pro-inflammatory cytokine IL-8. Mechanistically, G6PD deficiency up-regulates IL-8 through oxidative stress and NF-κB pathway. The palmitate-induced inflammation in G6PD-deficient HepG2 cells could serve as an in vitro model to study the role of altered redox homeostasis in chronic hepatic inflammation.

No MeSH data available.


Related in: MedlinePlus

The effect of G6PD deficiency on IL-8 secretion in palmitate-treated HepG2 cells. (a) The IL-8 secretion of Sc and Gi HepG2 cells with or without palmitate treatment (0.6 mM, 24 hr) was measured by ELISA. (b) The IL-8 secretion of Sc and Gi HepG2 cells treated with different concentrations of palmitate (0.2, 0.3, 0.4 mM) for 24 hr was analyzed by ELISA. These results are representative of at least three separate experiments. *indicates a significant difference (P < 0.05) between Sc and Gi HepG2 cells. #indicates a significant difference (P < 0.05) between control and palmitate-treated HepG2 cells.
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Fig1: The effect of G6PD deficiency on IL-8 secretion in palmitate-treated HepG2 cells. (a) The IL-8 secretion of Sc and Gi HepG2 cells with or without palmitate treatment (0.6 mM, 24 hr) was measured by ELISA. (b) The IL-8 secretion of Sc and Gi HepG2 cells treated with different concentrations of palmitate (0.2, 0.3, 0.4 mM) for 24 hr was analyzed by ELISA. These results are representative of at least three separate experiments. *indicates a significant difference (P < 0.05) between Sc and Gi HepG2 cells. #indicates a significant difference (P < 0.05) between control and palmitate-treated HepG2 cells.

Mentions: Since palmitate induces pro-inflammatory cytokine IL-8 secretion in hepatocytes [21], we investigated whether G6PD deficiency affected pro-inflammatory cytokine secretion in the palmitate-treated cell model by cytokine array analysis. Among 36 different cytokines tested, several cytokines (IL-8, IL-1ra, MIF, sICAM-1, and Serpin E1) were detected (Additional file 5: Figure S4 and see the array format in Additional file 6: Table S1). Interestingly, G6PD deficiency enhanced secretion of these cytokines at basal and palmitate treatment conditions (Table 2). In particular, the IL-8 secretion showed a 9-fold increase in palmitate-treated Gi HepG2 cells compared with untreated Sc HepG2 cells, whereas the secretion of other high level cytokines showed a 5-fold increase. Since IL-8 showed a most dramatic increase in cytokine array, we validated its up-regulation in Gi HepG2 cells by ELISA (Figure 1a) and qRT-PCR (Figure 2). The ELISA data confirmed that IL-8 secretion was significantly increased (P < 0.05) in palmitate-treated Gi HepG2 cells (Figure 1a) in a palmitate-dependent manner (Figure 1b). Likewise, the qRT-PCR result also validated that G6PD deficiency significantly up-regulated IL-8 gene expression upon palmitate treatment in a palmitate-dependent manner (Figure 2).Table 2


Glucose 6-phosphate dehydrogenase knockdown enhances IL-8 expression in HepG2 cells via oxidative stress and NF-κB signaling pathway.

Yang HC, Cheng ML, Hua YS, Wu YH, Lin HR, Liu HY, Ho HY, Chiu DT - J Inflamm (Lond) (2015)

The effect of G6PD deficiency on IL-8 secretion in palmitate-treated HepG2 cells. (a) The IL-8 secretion of Sc and Gi HepG2 cells with or without palmitate treatment (0.6 mM, 24 hr) was measured by ELISA. (b) The IL-8 secretion of Sc and Gi HepG2 cells treated with different concentrations of palmitate (0.2, 0.3, 0.4 mM) for 24 hr was analyzed by ELISA. These results are representative of at least three separate experiments. *indicates a significant difference (P < 0.05) between Sc and Gi HepG2 cells. #indicates a significant difference (P < 0.05) between control and palmitate-treated HepG2 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419400&req=5

Fig1: The effect of G6PD deficiency on IL-8 secretion in palmitate-treated HepG2 cells. (a) The IL-8 secretion of Sc and Gi HepG2 cells with or without palmitate treatment (0.6 mM, 24 hr) was measured by ELISA. (b) The IL-8 secretion of Sc and Gi HepG2 cells treated with different concentrations of palmitate (0.2, 0.3, 0.4 mM) for 24 hr was analyzed by ELISA. These results are representative of at least three separate experiments. *indicates a significant difference (P < 0.05) between Sc and Gi HepG2 cells. #indicates a significant difference (P < 0.05) between control and palmitate-treated HepG2 cells.
Mentions: Since palmitate induces pro-inflammatory cytokine IL-8 secretion in hepatocytes [21], we investigated whether G6PD deficiency affected pro-inflammatory cytokine secretion in the palmitate-treated cell model by cytokine array analysis. Among 36 different cytokines tested, several cytokines (IL-8, IL-1ra, MIF, sICAM-1, and Serpin E1) were detected (Additional file 5: Figure S4 and see the array format in Additional file 6: Table S1). Interestingly, G6PD deficiency enhanced secretion of these cytokines at basal and palmitate treatment conditions (Table 2). In particular, the IL-8 secretion showed a 9-fold increase in palmitate-treated Gi HepG2 cells compared with untreated Sc HepG2 cells, whereas the secretion of other high level cytokines showed a 5-fold increase. Since IL-8 showed a most dramatic increase in cytokine array, we validated its up-regulation in Gi HepG2 cells by ELISA (Figure 1a) and qRT-PCR (Figure 2). The ELISA data confirmed that IL-8 secretion was significantly increased (P < 0.05) in palmitate-treated Gi HepG2 cells (Figure 1a) in a palmitate-dependent manner (Figure 1b). Likewise, the qRT-PCR result also validated that G6PD deficiency significantly up-regulated IL-8 gene expression upon palmitate treatment in a palmitate-dependent manner (Figure 2).Table 2

Bottom Line: This study was designed to investigate the effect of glucose 6-phosphate dehydrogenase (G6PD) deficiency on pro-inflammatory cytokine secretion using a palmitate-induced inflammation HepG2 in vitro model.Cytokine array analysis indicated that IL-8 is most significantly increased in G6PD-knockdown HepG2 cells.The palmitate-induced inflammation in G6PD-deficient HepG2 cells could serve as an in vitro model to study the role of altered redox homeostasis in chronic hepatic inflammation.

View Article: PubMed Central - PubMed

Affiliation: Healthy Aging Research Center, Chang Gung University, Kwei-Shan, Tao-Yuan 333 Taiwan ; Department of Medical Biotechnology and Laboratory Sciences, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan 333 Taiwan.

ABSTRACT

Background: This study was designed to investigate the effect of glucose 6-phosphate dehydrogenase (G6PD) deficiency on pro-inflammatory cytokine secretion using a palmitate-induced inflammation HepG2 in vitro model. The modulation of cellular pro-inflammatory cytokine expression under G6PD deficiency during chronic hepatic inflammation has never been investigated before.

Methods: The culture medium of untreated and palmitate-treated G6PD-scramble (Sc) and G6PD-knockdown (Gi) HepG2 cells were subjected to cytokine array analysis, followed by validation with ELISA and qRT-PCR of the target cytokine. The mechanism of altered cytokine secretion in palmitate-treated Sc and Gi HepG2 cells was examined in the presence of anti-oxidative enzyme (glutathione peroxidase, GPX), anti-inflammatory agent (curcumin), NF-κB inhibitor (BAY11-7085) and specific SiRNA against NF-κB subunit p65.

Results: Cytokine array analysis indicated that IL-8 is most significantly increased in G6PD-knockdown HepG2 cells. The up-regulation of IL-8 caused by G6PD deficiency in HepG2 cells was confirmed in other G6PD-deficient cells by qRT-PCR. The partial reduction of G6PD deficiency-derived IL-8 due to GPX and NF-κB blockers indicated that G6PD deficiency up-regulates pro-inflammatory cytokine IL-8 through oxidative stress and NF-κB pathway.

Conclusions: G6PD deficiency predisposes cells to enhanced production of pro-inflammatory cytokine IL-8. Mechanistically, G6PD deficiency up-regulates IL-8 through oxidative stress and NF-κB pathway. The palmitate-induced inflammation in G6PD-deficient HepG2 cells could serve as an in vitro model to study the role of altered redox homeostasis in chronic hepatic inflammation.

No MeSH data available.


Related in: MedlinePlus