*4.7. Mice Management and HCl/EtOH-Induced Gastric Ulcer Model*

Six-week-old ICR mice were handled in accordance with the published method [34] and in accordance with the guide for the care and use of laboratory animals (Eighth Edition, 2011, published by The National Academies Press, Washington, DC, USA) and Institutional Animal Care and Use Committee (IACUC; CBNU 2017-0126) of the Chonbuk National University Laboratory Animal Center in Korea. An experimental gastric ulcer model in mice was induced using HCl/EtOH [44]. Forty ICR mice were randomly divided into four groups. Mice fasted overnight before the experiment. Fasted mice were orally given RI (400 mg/kg) and ranitidine (40 mg/kg) in respected groups for 1 h. After 1 h, 60% EtOH in 150 mM HCl (10 μL/g) was given orally. Control mice were treated with normal saline. Mice were anesthetized with Zoletil 50 (10 mg/kg) 1 h after administration of HCl/EtOH and samples were collected for experimental analysis.

#### *4.8. Gross and Histopathology of Gastric Mucosal Tissue*

To evaluate the gross and histological changes in glandular stomach tissue, we followed previous study [34]. For quantifying the degree of gross and histological lesions index, we followed a prescribed method with slight modification [45]. Briefly, the gross lesions of gastric mucosa were measured using following formula; (number of lesions of type I) + (number of lesions of type II) × 2 + (number of lesions of type III) × 3. Here, the gross lesions were characterized by: the presence of single submucosal punctiform hemorrhage, edema, type I; the presence of submucosal hemorrhagic lesions with slight erosions, type II; and the presence of deep ulcer with erosions and invasive lesions, type III. Besides, the histological lesions of gastric mucosa were measured using following formula; (% type I lesion) × 1 + (% type II lesion) × 2 + (% type III lesion) × 3. Here, the histological lesions were characterized by: gastric mucosal cells appeared intact and had a normal shape, type 0 lesion; surface epithelial cells and the uppermost 2 or 3 cells lining the glands were damaged, type I damage; damage greater than I but involving <50% of the thickness of the gastric mucosa, type II damage; and damage involving >50% of the thickness of the gastric mucosa, type III damage.

#### *4.9. Analysis of Lipid Peroxidation and NO Production*

Malondialdehyde (MDA) and NO are important indicators of oxidative stress. The gastric tissue was homogenized and centrifuged at 10,000 rpm at 4 ◦C for 10 min. The supernatant was collected and kept at −80 ◦C for experimental analysis. MDA and NO concentration were measured in the gastric tissue samples according to the commercial kit instructions.

#### *4.10. RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qPCR)*

The RNA was extracted from a gastric tissue according to the manufacturer's instructions. The concentration of total RNA was quantified with the BioSpec-nano spectrophotometer (Shimadzu Biotech, Tokyo, Japan) at a 260/280 nm ratio. For complementary DNA (cDNA) synthesis, total RNA (3 μg) was used, and cDNA synthesis was maintained according to the manufacturer's instructions. qPCR was performed SYBR Green Real-Time PCR master mix according to Roche LightCycler™. Relative expression of target genes was normalized to the reference gene: glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences of the primers (Bioneer, Daejeon, Korea) used are shown in Table 2 [46].

#### *4.11. Immunohistochemical (IHC) Analysis*

COX-2 immunopositive cells expression in the gastric tissue was performed using according to Vectastain ABC kit recommendations. Briefly, paraffin section was deparaffinized in xylene and hydrated in ethanol. Citrate buffer was used for antigen retrieval and 3% hydrogen peroxide (H2O2) was used for inactivating the endogenous peroxidase activity. Tissue was blocked with normal serum for 1 h. Anti-rabbit monoclonal COX-2 antibody (dilution 1:200) was incubated overnight at 4 ◦C. Subsequently, the section was incubated with biotinylated secondary antibody for 1 h and Vectastain ABC reagent for 30 min at room temperature. The sections were incubated with diaminobenzidine (DAB) in the dark until brown color development. After counterstain, the section was dehydrated in ethanol, cleared in xylene and mounted on a glass slide. The section was imaged at a fixed 100× magnification using Leica DM2500 microscope (Leica Microsystems, Wetzlar, Germany).

### *4.12. Western Blot Analysis*

RAW 264.7 cells and gastric tissue were harvested and washed twice with ice-cold PBS. Cells and tissues were lysed by the lysis buffer; radioimmunoprecipitation assay buffer (RIPA), and/or tissue protein extraction reagent (T-PER), phenylmethanesulfonyl fluoride (PMSF), sodium orthovanadate (Na3VO4), and protease inhibitor cocktail. The total concentration of protein of lysate cells and tissues were measured with a bicinchoninic acid (BCA) protein assay protein kit. An equal amount of protein was separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was incubated with blocking serum; 5% bovine serum albumin (BSA) in Tris-buffered saline with tween twenty (TBST) for 2 h at room temperature and by primary antibodies for overnight at 4 ◦C. Then, the blot was washed and incubated with secondary antibodies for 2 h. Bands were detected using an enhanced chemiluminescence (ECL) detection kit, and bands images were taken by a LAS-400 image system, (GE Healthcare, Little Chalfont, UK); β-actin was used as the reference antibody.

#### *4.13. Statistical Analysis*

Data were analyzed with Graph Pad Prism 5.0 (Graph Pad Software, Inc., San Diego, CA, USA) and are expressed as mean ± standard error (SEM). Statistical analyses were assessed by analysis of variance (ANOVA) followed by Bonferroni post-hoc tests. The minimum statistical significance was considered to be *p <* 0.05 for all analyses.

#### **5. Conclusions**

RI mitigates inflammation and maintains normal gastric mucosal integrity. The present study validates that RI protects against inflammation and gastric ulcer by mitigating the inflammation response and oxidative stress via downregulation of the pro-inflammatory cytokines mediated by MAPK/NF-κB signaling pathways. Therefore, RI could be a promising phytomedicine and has advantages for prospective clinical applications in the future for an oxidative stress-mediated gastric ulcer.

**Acknowledgments:** This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2017R1D1A1B03035765) and Biomedical Research Institute, Chonbuk National University Hospital.

**Author Contributions:** Md Rashedunnabi Akanda and Byung-Yong Park conceived and designed the study. Md Rashedunnabi Akanda performed the experiment, analyzed the data and wrote the original manuscript. Md Rashedunnabi Akanda, In-Shik Kim, Dongchoon Ahn, Hyun-Jin Tae, Hyeon-Hwa Nam, Byung-Kil Choo, Kyunghwa Kim and Byung-Yong Park read and approved the final version of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
