Stem Cell

Therapeutic effects and potential mechanisms of endoscopic submucosal injection of mesenchymal stem cells on chronic atrophic gastritis – Scientific Reports

Time Stamp: November 24, 2023 7:00 PM
Source Node: 359823

Cells

Human umbilical cord blood mesenchymal stem cells (hUC-MSCs) were purchased from Desheng Biological Engineering Co., Ltd. (Shandong, China), cultured in an α-MEM medium containing 10% (v/v) fetal bovine and 100 U/ml penicillin/streptomycin. The normal gastric mucosal epithelial cell line GES-1 were purchased from Procell Life Science & Technology Co., Ltd. (Hubei, China), cultured in an DMEM medium containing 10% (v/v) fetal bovine and 100 U/ml penicillin/streptomycin. Both cell lines were incubated at 37 °C in a humidified 5% CO₂ incubator.

Establishment of CAG rabbit model

Healthy adult male rabbits were obtained from Xietong Medicine Bioengineering Co., Ltd. (Jiangsu, China) and were housed under standard conditions with free access to food and water in a clean environment on a 12-h day-night cycle. To simulate the low gastric acidity state induced by H. pylori infection and bile reflux, a rabbit model of CAG was established by chemical stimulation. Rabbits were divided into the model group (n = 20) and the control group (n = 5). The model group was provided with free access to drinking water containing 150 μg/ml MNNG, 0.02% ammonia water, and 20 mmol/L deoxycholic acid, and was fasted twice a week. The control group was given normal water and food. The model was developed over a 12-week period, after which endoscopy and biopsy pathology assessment were conducted. Venous blood samples were also drawn from the ear margins to analyze serum biochemical indices, as outlined in the following procedure. Upon successful model completion, we proceeded with submucosal hUC-MSCs injections. The experimental procedures were approved by the Institutional Animal Care and Committee at Shandong Provincial Hospital, Shandong University, and conducted in compliance with the regulations.

Endoscopic examination

After a 24-h fasting period, the rabbits were anesthetized with propofol (i.v. maintenance) and provided with mechanical ventilation during the procedure. The gastroscopic procedure used in rabbits is illustrated in Fig. 1a. Briefly, the rabbit was positioned laterally on the examination table. An electronic transnasal gastroscope (Olympus, GIF-XP260N, Tokyo, Japan) was introduced through the rabbit’s oral cavity. To safeguard the endoscope from potential damage by the rabbit’s teeth, a five-milliliter syringe cartridge was used as a protective dental cushion. Real-time images from the endoscope’s camera were displayed on a monitor (Olympus, CV-170, Tokyo, Japan), aiding in the precise navigation through the esophagus to access the stomach. Gastroscopic images were captured during the examination, and biopsy forceps were utilized through an accessory channel to obtain samples of the gastric mucosa for subsequent histological analysis (Fig. 1b). Upon completion of the examination, the endoscope was carefully withdrawn from the rabbits’ digestive tracts. Post-surgery, all animals received 1 ml of preheated saline for postoperative fluid resuscitation. Gastroscopy was performed, and biopsies were taken at the end of the modeling period and at each injection session.

Submucosal hUC-MSCs injection

To perform submucosal injection, hUC-MSCs were first resuspended in saline at a concentration of 10 × 10⁶/10 ml. The rabbits in the CAG model were divided into the MSCs group (n = 10) and the saline-control group (n = 10). A standardized 10 ml of a different solution was then injected into the submucosal layer at multiple sites of the stomach using a disposable injector (Olympus, NM-201L-0423, Tokyo, Japan) through the accessory channel of the electronic transnasal gastroscope (Fig. 1c). Specifically, we followed the New Sydney System’s guidelines for biopsy locations and performed submucosal injections at five specified sites (the antrum at the lesser and greater curvature, the body at the lesser and greater curvature, and the incisura, corresponding to the anatomy of the human stomach). At each site, at least two distinct points were injected. Each injection site received 1 ml, approximately 1 × 10⁶ cells. The injection depth was restricted to the submucosa, and a noticeable elevation in the submucosal layer could be observed after the injection. Each group received two submucosal injections, with one month between treatments. One month after the final injection, gastroscopy and biopsy were once again performed on the rabbits, and peripheral blood samples were collected. At the conclusion of the experiment, all rabbits were humanely euthanized through the process of gas embolization.

Serum biochemical indices examination

Serum biochemical indices, including gastrin 17 (G-17), pepsinogen I (PG I), and pepsinogen II (PG II), were measured by enzyme-linked immunosorbent assay (ELISA). Kits were purchased from OriGene Wuxi Biotechnology. Inc. (Jiangsu, China).

Histopathological examination

The resected tissues were fixed in 10% formalin for 2 days, embedded in paraffin, sliced, and stained with hematoxylin and eosin (H&E). Five fields of gastric antrum mucosa were sampled from each section, and the thickness of glands in each field was measured using a micrometer. The average value of the five fields within each section was then calculated (μm). At 100X magnification, the total number of intact glands within the gastric antrum was counted and expressed as “units/mm”.

Co-culture of hUC-MSCs and GES-1

To establish an in vitro model of therapy, a cell–cell co-culture system was implemented using a 24-well plate with a 0.4 μm and 3 μm pore size membranes. The upper surface of the membrane was seeded with 1.0 × 10⁵ degenerative GES-1 cells, while the base of the well was seeded with 3.0 × 10⁵ hUC-MSCs at passage 4–6. The cells were co-cultured in α-MEM for 24 h at 37 °C and 5% CO₂ in a humidified atmosphere. Prior to co-culture, GES-1 cells were exposed to 40 μmol/L MNNG for 24 h to induce a cell atrophy model.

Cell migration assay

Following 24-h co-culture in a 24-well plate with a 3 μm pore size membrane, the nutrient solution was removed and the upper layer of the Transwell was gently wiped with a cotton swab. The membrane was then fixed with methanol for 30 min, washed with PBS, and stained with hematoxylin for 10 min. Five fields were randomly selected, and the number of invasive cells was counted under a microscope (Olympus, IX71-F22PH) to obtain the mean value, which was used for statistical analysis.

Cell proliferation assay

The proliferative capacity of GES-1 cells was assessed using an EdU (5-ethynyl-2′-deoxyuridine) assay, facilitated by the Cell-Light EdU DNA Cell Proliferation Kit (RiboBio, Shanghai, China). Following 24-h co-culture through a 0.4 μm pore size membrane, cells were treated with 100 μl of EdU and incubated for an additional 2 h. Post-treatment, the cells were fixed with 4% paraformaldehyde and stained with Apollo Dye Solution for proliferating cells, while nucleic acid was stained with Hoechst 33342. The cell proliferation rate was determined following the manufacturer’s guidelines. Fluorescence images were captured using a fluorescence microscope (Invitrogen, EVOS M7000).

Western blot

Total protein was extracted from cells using RIPA buffer containing 1/100 phenylmethylsulfonyl fluoride (PMSF; Solarbio, Beijing, China). The lysate was centrifuged at 12,500 g for 10 min at 4 °C and the supernatant was collected for protein quantification using a BCA protein assay kit (Solarbio, Beijing, China). Equal amounts of soluble protein were denatured by heating at 100 °C for 10 min in loading buffer (Solarbio, Beijing, China). The denatured proteins were then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% skimmed milk powder (SMP) in TBST at room temperature for 2 h. Next, the membranes were incubated overnight at 4 °C with specific primary antibodies. On the following day, the blots were washed and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, followed by another wash. Chemiluminescent signals were visualized using ECL Substrate (Sparkjade, Shandong, China) and detected on a ChemiDocMP System (Biorad, United States) for analysis.

Experimental design and statistical analysis

Experimental design, analysis and reporting followed the ARRIVE guidelines (https://www.nc3rs.org.uk/arrive-guidelines) where possible. Statistical analyses were conducted using GraphPad Prism 8 and SPSS 25.0. Differences with statistical significance were determined by unpaired Student’s t-test. Chi-square or Fisher’s exact test were applied to compare categorical variables across the groups. The pathological score per sample was computed as the aggregate of all recorded findings. A P-value of less than 0.05 was considered statistically significant.