J Cancer Prev 2021; 26(4): 289-297
Published online December 30, 2021
© Korean Society of Cancer Prevention
1Institute of Pharmaceutical Sciences, College of Pharmacy, CHA University, Seongnam, 2SD Biotechnologies Co., Ltd., Seoul, Korea
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Ginger (Zingiber officinale) has traditionally been used as a treatment for inflammatory diseases in the Asian region. Recently, anti-inflammatory effects of steamed ginger extract (GGE03) have been reported, but its association with Helicobacter pylori (H. pylori)-induced gastritis has not been investigated. The purpose of this study was to assess the anti-inflammatory activity of GGE03 in H. pylori-infected gastric epithelial cells. Our studies revealed that the GGE03 suppressed the growth of H. pylori. GGE03 markedly reduced the expression of the H. pylori-induced pro-inflammatory cytokines including interleukin (IL)-8, TNF-α, IL-6, inducible NOS (iNOS) and IFN-γ. We also demonstrated that GGE03 treatment inhibited the H. pylori-activated NF-κB signaling pathway. In addition, the treatment with GGE03 significantly attenuated nitric oxide production and myeloperoxidase activity in H. pylori-infected gastric epithelial cells. These anti-inflammatory effects of GGE03 were more effective than ginger extract. Finally, we investigated the minimum effective concentration of GGE03 to inhibit H. pylori-induced inflammation. Our findings suggest that GGE03 not only inhibits the growth of H. pylori, but also attenuates H. pylori-induced inflammation.
Keywords: Steamed ginger extract, Helicobacter pylori, Gastritis, Anti-inflammation, NF-kappa B
GGE03 was prepared as previously published . Briefly, ginger was washed three times with distilled water, dried at 50°C for 30 hours, and then steamed at: 2 to 2.5 kgf/cm2, 97°C, for 2 hours. GGE03 was obtained by extracting steamed ginger with fifteen-fold 70% ethanol (v/v) for 15 hours at 85°C, 1.5 kg/cm2, and then passed through a 60-mesh filter, concentrated at –650 mmHg, 55°C. GE was prepared in the same way without steam process. The extract (GE and GGE03) was spray-dried to obtain a powder and stored at –20°C until use. GE and GGE03 were dissolved in distilled water and diluted with medium into appropriate concentrations.
AGS gastric adenocarcinoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained according to the ATCC’s instructions. Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (GE Healthcare, Chicago, IL, USA) supplemented with 10% (v/v) fetal bovine serum (ATCC), 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.
For measuring the viability of AGS cells, the cells were treated with GE and GGE03 at the concentrations of 1, 10, 100, or 200 μg/mL. After incubation for 24 hours, cells were rinsed with PBS and then MTT (Sigma-Aldrich, St. Louis, MO, USA) solution was added to the cells followed by incubation for additional 3 hours. After the conversion of the substrate to a chromogenic product by metabolically active cells, the medium was removed and the purple MTT formazan crystals were solubilized with dimethyl sulfoxide (DMSO). The absorbance of each samples measured at 570 nm as a reference wavelength.
This assay was performed as previously described . Briefly, proteins were separated by SDS PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with optimal concentrations of a primary antibody (pp65, p65, pp50, p50, p-IκBα, IκBα or β-actin) at 4°C overnight and then incubated with an appropriate secondary antibody for 1 hour at room temperature. The immune-labeled proteins were visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Waltham, MA, USA). The primary antibodies to detect pp65, p65, pp50, p50, p-IκBα, and IκBα used in this study were purchased from Cell Signaling Technology (Danvers, MA, USA) and β-actin from Santa Cruz Biotechnology (Dallas, TX, USA).
AGS cells were washed twice with PBS, and total mRNA was isolated from the cells using TRIzolTM Reagent (Invitrogen, Waltham, MA, USA), and cDNA was prepared using a Labopass cDNA synthesis kit (Cosmogenetech, Seoul, Korea) according to the manufacturer’s instructions. The mRNA levels were assessed by reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR). qRT-PCR was performed as previously and assessed on the ViiATM 7 real-time PCR system (Applied Biosystems, Waltham, MA, USA) using Luna universal qPCR master mix (New England Biolabs, Beverly, MA, USA). The relative quantities of target genes were calculated from triplicate samples after normalization by an internal control, 18S rRNA. The PCR primer sequences used in this study are listed in Table 1.
Samples obtained from cellular or bacterial pellets were washed twice with PBS and then lysed with a buﬀer containing 20 mM HEPES. Then, measurement of MPO activity was performed with kits (Abcam, Cambridge, UK) according to the manufacturer’s instructions. The activity was measured as the absorbance at 450 nm read by a 96-well microplate reader.
Culture medium supernatants were collected from the culture of AGS cells. The NO production in the culture medium was quantified colorimetrically using a nitric oxide detection kit (iNtRON Biotechnology, Seongnam, Korea). The absorbance at 550 nm was read with a 96-well microplate reader.
Results are expressed as the mean ± SD. The statistical significance was analyzed by one-way ANOVA. Statistical significance was accepted at
We conducted the investigation of relative inhibitory potency of GGE03 and GE against
It has been reported that
The NF-κB signaling pathway is one of the most important mediators of pro-inflammatory cytokine production upon
Several lines of evidence indicate that NO is involved in the pathogenesis of
MPO activity is used as an indicator of neutrophil infiltration in inflamed site . Because of the abundance of this enzyme, it is used as an index for
To assess the minimum concentration of GGE03 exerting anti-inflammatory responses in
It has been reported that
Next, we investigated that the anti-inflammatory responses using cultured AGS cells infected with
Production of NO is a feature of gastric epithelial cells as well as genuine immune cells including macrophages upon inflammatory insult [31-33]. In gastric epithelial cells,
Finally, we investigated the minimum inhibitory concentration of GGE03 for anti-inflammatory effect on
To sum up, GGE03 suppresses the expression of pro-inflammatory cytokines, NO production and MPO activity in
This work was supported by the GRRC program of Gyeonggi province (GRRC-201900830002-CHA2019-B01, Production of physiologically active substances), Republic of Korea.
No potential conflicts of interest were disclosed.
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