Yonsei Med J.  2016 Jan;57(1):260-264. 10.3349/ymj.2016.57.1.260.

alpha-Lipoic Acid Inhibits Expression of IL-8 by Suppressing Activation of MAPK, Jak/Stat, and NF-kappaB in H. pylori-Infected Gastric Epithelial AGS Cells

• Choi JH
• Cho SO
• Kim H

• KMID: 2466379
• DOI: http://doi.org/10.3349/ymj.2016.57.1.260

Abstract

The epithelial cytokine response, associated with reactive oxygen species (ROS), is important in Helicobacter pylori (H. pylori)-induced inflammation. H. pylori induces the production of ROS, which may be involved in the activation of mitogen-activated protein kinases (MAPK), janus kinase/signal transducers and activators of transcription (Jak/Stat), and oxidant-sensitive transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), and thus, expression of interleukin-8 (IL-8) in gastric epithelial cells. alpha-lipoic acid, a naturally occurring thiol compound, is a potential antioxidant. It shows beneficial effects in treatment of oxidant-associated diseases including diabetes. The present study is purposed to investigate whether alpha-lipoic acid inhibits expression of inflammatory cytokine IL-8 by suppressing activation of MAPK, Jak/Stat, and NF-kappaB in H. pylori-infected gastric epithelial cells. Gastric epithelial AGS cells were pretreated with or without alpha-lipoic acid for 2 h and infected with H. pylori in a Korean isolate (HP99) at a ratio of 300:1. IL-8 mRNA expression was analyzed by RT-PCR analysis. IL-8 levels in the medium were determined by enzyme-linked immunosorbent assay. NF-kappaB-DNA binding activity was determined by electrophoretic mobility shift assay. Phospho-specific and total forms of MAPK and Jak/Stat were assessed by Western blot analysis. ROS levels were determined using dichlorofluorescein fluorescence. As a result, H. pylori induced increases in ROS levels, mRNA, and protein levels of IL-8, as well as the activation of MAPK [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase 1/2 (JNK1/2), p38], Jak/Stat (Jak1/2, Stat3), and NF-kappaB in AGS cells, which was inhibited by alpha-lipoic acid. In conclusion, alpha-lipoic acid may be beneficial for prevention and/or treatment of H. pylori infection-associated gastric inflammation.

Keyword

alpha-lipoic acid; Helicobacter pylori; IL-8; NF-kappaB; MAPK; Jak/Stat

MeSH Terms

Enzyme-Linked Immunosorbent Assay
Epithelial Cells/metabolism
Gastric Mucosa/*drug effects/metabolism/microbiology
Gene Expression Regulation, Bacterial
Helicobacter Infections/immunology/*metabolism
Helicobacter pylori/drug effects/*pathogenicity
Humans
Interleukin-8/genetics/*metabolism
JNK Mitogen-Activated Protein Kinases
Janus Kinase 1
Mitogen-Activated Protein Kinases/*biosynthesis
NF-kappa B/*metabolism
RNA, Messenger/isolation & purification/metabolism
Reactive Oxygen Species/metabolism
STAT3 Transcription Factor
Stomach/metabolism/*microbiology
Thioctic Acid/*pharmacology
JNK Mitogen-Activated Protein Kinases
Janus Kinase 1
Interleukin-8
Mitogen-Activated Protein Kinases
NF-kappa B
RNA, Messenger
Reactive Oxygen Species
STAT3 Transcription Factor
Thioctic Acid

Figure

• 

  Fig. 1 mRNA and protein levels of IL-8 in H. pylori-infected AGS cells treated with or without α-LA. (A and B) The cells were cultured in the presence of H. pylori for indicated time points. mRNA levels of IL-8 were determined by real-time PCR. IL-8 mRNA levels were normalized to β-actin (A). IL-8 levels in the medium were assessed by ELISA (B). (C and D) The cells were pre-treated with α-LA for 2 h, and cultured in the presence of H. pylori for 3 h (IL-8 mRNA level, C) or 8 h (IL-8 level in the medium, D). All values are expressed as mean±SEM of four different experiments. *p<0.05 vs. 0 h (A and B) or control (C and D). Non (none), the cells cultured in the absence of H. pylori without treatment of α-LA; Con (control), the cells cultured in the presence of H. pylori without treatment of α-LA. H. pylori, Helicobacter pylori; α-LA, α-lipoic acid; ELISA, enzyme linked immunosorbent assay; IL, interleukin; SEM, standard error of means; AGS, gastric adenocarcinoma.

• 

  Fig. 2 ROS levels, activation of NF-κB, MAPK, and Jak/Stat in H. pylori-infected AGS cells treated with or without α-LA. The cells were pretreated with α-LA for 2 h and cultured in the presence of H. pylori for 30 min (ROS levels, activation of NF-κB, MAPK, and Jak/Stat) or 1 h (NF-κB). (A) ROS levels were determined using DCF fluorescence. All values are expressed as mean±SEM of four different experiments. (B) NF-κB activation was determined using EMSA, performing western blotting for phospho- and total forms of IκBα. (C and D) The levels of phospho-specific and total forms of MAPK (ERK1/2, JNK1/2, p38, C) and Jak1, Jak2, Stat3 (D) in whole cell lysates were determined by Western blot analysis. Non (none), the cells cultured in the absence of H. pylori without treatment of α-LA; Con (control), the cells cultured in the presence of H. pylori without treatment of α-LA. *p<0.05 vs. control. H. pylori, Helicobacter pylori; ROS, reactive oxygen species; MAPK, mitogen-activated protein kinases; α-LA, α-lipoic acid; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal protein kinase; Jak/Stat, janus kinase/signal transducers and activators of transcription; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; AGS, gastric adenocarcinoma; DCF, 2',7'-dichlorodihydrofluorescein.

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