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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 NF-κB inhibition on IL-8 secretion in HepG2 cells. (a) The effect of NF-κB inhibitor on IL-8 secretion with or without palmitate treatment. The IL-8 secretion of Sc and Gi HepG2 cells treated with 10 μM NF-κB inhibitor (BAY11-7085) with or without 0.6 mM palmitate for 24 hr was measured 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 inhibitor (BAY)-treated HepG2 cells without palmitate. α indicates a significant difference (P < 0.05) between palmitate (PA)-treated cells without inhibitor (BAY) and palmitate (PA)-inhibitor (BAY) co-treated HepG2 cells. (b) The effect of p65 knockdown in Sc and Gi HepG2 cells on p65 protein level determined by Western blotting. The blot (cell lysate: 25 μg protein content) was incubated with anti-p65 antibody and subsequently stripped for incubation with anti-actin antibody as loading control. (c) The effect of p65 knockdown on IL-8 gene expression by qRT-PCR and (d) IL-8 secretion by ELISA. These results are representative of at least three separate experiments. *indicates a significant difference (P < 0.05) between SiRNA- control and SiRNA- p65.
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Fig4: The effect of NF-κB inhibition on IL-8 secretion in HepG2 cells. (a) The effect of NF-κB inhibitor on IL-8 secretion with or without palmitate treatment. The IL-8 secretion of Sc and Gi HepG2 cells treated with 10 μM NF-κB inhibitor (BAY11-7085) with or without 0.6 mM palmitate for 24 hr was measured 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 inhibitor (BAY)-treated HepG2 cells without palmitate. α indicates a significant difference (P < 0.05) between palmitate (PA)-treated cells without inhibitor (BAY) and palmitate (PA)-inhibitor (BAY) co-treated HepG2 cells. (b) The effect of p65 knockdown in Sc and Gi HepG2 cells on p65 protein level determined by Western blotting. The blot (cell lysate: 25 μg protein content) was incubated with anti-p65 antibody and subsequently stripped for incubation with anti-actin antibody as loading control. (c) The effect of p65 knockdown on IL-8 gene expression by qRT-PCR and (d) IL-8 secretion by ELISA. These results are representative of at least three separate experiments. *indicates a significant difference (P < 0.05) between SiRNA- control and SiRNA- p65.

Mentions: It has been reported that G6PD activates NF-κB in β-cells and adipocytes [33,34]. To investigate whether NF-κB pathway was involved in the enhancement of IL-8 secretion in G6PD-knockdown cells, Sc and Gi HepG2 cells with or without 0.6 mM palmitate treatment were exposed to 10 μM NF-κB inhibitor (BAY11-7085) followed by quantification of IL-8 secretion. As shown in Figure 4a, IL-8 secretion was significantly reduced by the NF-κB inhibitor in Sc HepG2 cells without palmitate treatment (75% decrease, P < 0.05) compared to that in untreated control cells. Similarly, the inhibitor reduced the IL-8 secretion in Gi HepG2 cells compared to that in untreated Gi HepG2 cells without palmitate treatment (64% decrease, P < 0.05). In the palmitate-treated cells, the NF-κB inhibitor significantly reduced IL-8 secretion (80% decrease, P < 0.05) in Sc HepG2 cells. Likewise, the NF-κB inhibitor significantly reduced the palmitate-induced IL-8 secretion in Gi HepG2 cells (72% decrease, P < 0.05) compared with palmitate-treated only Gi HepG2 cells. To further investigate the association of G6PD and NF-κB signaling on IL-8 secretion, a G6PD/ NF-κB(p65) double-knockdown cell line was established (Figure 4b). Because the immunoblotting result showed that the protein level of p65 was diminished markedly by SiRNA against p65 at 150 nM, such condition was used for the measurement of IL-8 level. The knockdown of NF-κB(p65) in Sc and Gi HepG2 cells resulted in a significant reduction of IL-8 mRNA (60% decrease, P < 0.05) compared with Sc and Gi HepG2 cells treated with control SiRNA (Figure 4c). In addition, Sc and Gi HepG2 cells treated with p65 SiRNA exhibited diminished IL-8 secretion (40% decrease, P < 0.05) compared to Sc and Gi HepG2 cells treated with control SiRNA (Figure 4d). The blockade of NF-κB due to selective inhibitor or specific SiRNA against NF-κB confirms that the up-regulation of IL-8 caused by G6PD deficiency is linked to NF-κB signaling. Nevertheless, the finding that the inhibition of NF-κB can not fully suppress the elevated IL-8 in Gi HepG2 cells suggests that besides NF-κB pathway there maybe alternative regulatory mechanism modulated by G6PD.Figure 4


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 NF-κB inhibition on IL-8 secretion in HepG2 cells. (a) The effect of NF-κB inhibitor on IL-8 secretion with or without palmitate treatment. The IL-8 secretion of Sc and Gi HepG2 cells treated with 10 μM NF-κB inhibitor (BAY11-7085) with or without 0.6 mM palmitate for 24 hr was measured 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 inhibitor (BAY)-treated HepG2 cells without palmitate. α indicates a significant difference (P < 0.05) between palmitate (PA)-treated cells without inhibitor (BAY) and palmitate (PA)-inhibitor (BAY) co-treated HepG2 cells. (b) The effect of p65 knockdown in Sc and Gi HepG2 cells on p65 protein level determined by Western blotting. The blot (cell lysate: 25 μg protein content) was incubated with anti-p65 antibody and subsequently stripped for incubation with anti-actin antibody as loading control. (c) The effect of p65 knockdown on IL-8 gene expression by qRT-PCR and (d) IL-8 secretion by ELISA. These results are representative of at least three separate experiments. *indicates a significant difference (P < 0.05) between SiRNA- control and SiRNA- p65.
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Related In: Results  -  Collection

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Fig4: The effect of NF-κB inhibition on IL-8 secretion in HepG2 cells. (a) The effect of NF-κB inhibitor on IL-8 secretion with or without palmitate treatment. The IL-8 secretion of Sc and Gi HepG2 cells treated with 10 μM NF-κB inhibitor (BAY11-7085) with or without 0.6 mM palmitate for 24 hr was measured 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 inhibitor (BAY)-treated HepG2 cells without palmitate. α indicates a significant difference (P < 0.05) between palmitate (PA)-treated cells without inhibitor (BAY) and palmitate (PA)-inhibitor (BAY) co-treated HepG2 cells. (b) The effect of p65 knockdown in Sc and Gi HepG2 cells on p65 protein level determined by Western blotting. The blot (cell lysate: 25 μg protein content) was incubated with anti-p65 antibody and subsequently stripped for incubation with anti-actin antibody as loading control. (c) The effect of p65 knockdown on IL-8 gene expression by qRT-PCR and (d) IL-8 secretion by ELISA. These results are representative of at least three separate experiments. *indicates a significant difference (P < 0.05) between SiRNA- control and SiRNA- p65.
Mentions: It has been reported that G6PD activates NF-κB in β-cells and adipocytes [33,34]. To investigate whether NF-κB pathway was involved in the enhancement of IL-8 secretion in G6PD-knockdown cells, Sc and Gi HepG2 cells with or without 0.6 mM palmitate treatment were exposed to 10 μM NF-κB inhibitor (BAY11-7085) followed by quantification of IL-8 secretion. As shown in Figure 4a, IL-8 secretion was significantly reduced by the NF-κB inhibitor in Sc HepG2 cells without palmitate treatment (75% decrease, P < 0.05) compared to that in untreated control cells. Similarly, the inhibitor reduced the IL-8 secretion in Gi HepG2 cells compared to that in untreated Gi HepG2 cells without palmitate treatment (64% decrease, P < 0.05). In the palmitate-treated cells, the NF-κB inhibitor significantly reduced IL-8 secretion (80% decrease, P < 0.05) in Sc HepG2 cells. Likewise, the NF-κB inhibitor significantly reduced the palmitate-induced IL-8 secretion in Gi HepG2 cells (72% decrease, P < 0.05) compared with palmitate-treated only Gi HepG2 cells. To further investigate the association of G6PD and NF-κB signaling on IL-8 secretion, a G6PD/ NF-κB(p65) double-knockdown cell line was established (Figure 4b). Because the immunoblotting result showed that the protein level of p65 was diminished markedly by SiRNA against p65 at 150 nM, such condition was used for the measurement of IL-8 level. The knockdown of NF-κB(p65) in Sc and Gi HepG2 cells resulted in a significant reduction of IL-8 mRNA (60% decrease, P < 0.05) compared with Sc and Gi HepG2 cells treated with control SiRNA (Figure 4c). In addition, Sc and Gi HepG2 cells treated with p65 SiRNA exhibited diminished IL-8 secretion (40% decrease, P < 0.05) compared to Sc and Gi HepG2 cells treated with control SiRNA (Figure 4d). The blockade of NF-κB due to selective inhibitor or specific SiRNA against NF-κB confirms that the up-regulation of IL-8 caused by G6PD deficiency is linked to NF-κB signaling. Nevertheless, the finding that the inhibition of NF-κB can not fully suppress the elevated IL-8 in Gi HepG2 cells suggests that besides NF-κB pathway there maybe alternative regulatory mechanism modulated by G6PD.Figure 4

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