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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 ROS production in HepG2 cells with or without palmitate treatment. The ROS production of palmitate-treated (0.3 mM, 24 hr) Sc and Gi HepG2 cells was determined by DCF-DA staining and analyzed by flow cytometry. 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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Fig5: The effect of G6PD deficiency on ROS production in HepG2 cells with or without palmitate treatment. The ROS production of palmitate-treated (0.3 mM, 24 hr) Sc and Gi HepG2 cells was determined by DCF-DA staining and analyzed by flow cytometry. 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: G6PD-deficient HepG2 cells are extremely sensitive to cytotoxic effects induced by oxidants with the concomitant production of oxidative stress [6,7]. To determine whether the enhanced IL-8 expression in palmitate-treated Gi HepG2 cells was due to oxidative stress, the ROS level of Sc and Gi HepG2 cells in control and palmitate-treated conditions was determined. As shown in Figure 5, Gi HepG2 cells without palmitate treatment displayed a 1.4-fold increase of ROS level (P < 0.05) compared with that of control Sc HepG2 cells, while in palmitate-treated Sc and Gi HepG2 cells there was an increase in ROS over that in control with a similar ratio between the two cell types. To examine the effect of oxidative stress on IL-8 secretion, both Sc and Gi HepG2 cells were treated with 0.5 mM H2O2 added exogenously according to a previous protocol [6] followed by quantification of IL-8 secretion. As shown in Figure 6, H2O2 treatment significantly enhanced IL-8 secretion in both Sc and Gi HepG2 cells (5-fold increase, P < 0.05) compared with that in control cells. The dramatic increased in IL-8 secretion by H2O2, particularly in Gi HepG2 cells, is consistent with the notion that IL-8 secretion in HepG2 cells is highly sensitive to oxidative stress.Figure 5


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 ROS production in HepG2 cells with or without palmitate treatment. The ROS production of palmitate-treated (0.3 mM, 24 hr) Sc and Gi HepG2 cells was determined by DCF-DA staining and analyzed by flow cytometry. 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

Fig5: The effect of G6PD deficiency on ROS production in HepG2 cells with or without palmitate treatment. The ROS production of palmitate-treated (0.3 mM, 24 hr) Sc and Gi HepG2 cells was determined by DCF-DA staining and analyzed by flow cytometry. 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: G6PD-deficient HepG2 cells are extremely sensitive to cytotoxic effects induced by oxidants with the concomitant production of oxidative stress [6,7]. To determine whether the enhanced IL-8 expression in palmitate-treated Gi HepG2 cells was due to oxidative stress, the ROS level of Sc and Gi HepG2 cells in control and palmitate-treated conditions was determined. As shown in Figure 5, Gi HepG2 cells without palmitate treatment displayed a 1.4-fold increase of ROS level (P < 0.05) compared with that of control Sc HepG2 cells, while in palmitate-treated Sc and Gi HepG2 cells there was an increase in ROS over that in control with a similar ratio between the two cell types. To examine the effect of oxidative stress on IL-8 secretion, both Sc and Gi HepG2 cells were treated with 0.5 mM H2O2 added exogenously according to a previous protocol [6] followed by quantification of IL-8 secretion. As shown in Figure 6, H2O2 treatment significantly enhanced IL-8 secretion in both Sc and Gi HepG2 cells (5-fold increase, P < 0.05) compared with that in control cells. The dramatic increased in IL-8 secretion by H2O2, particularly in Gi HepG2 cells, is consistent with the notion that IL-8 secretion in HepG2 cells is highly sensitive to oxidative stress.Figure 5

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