2.1 Animals
Seventy-four male Sprague-Dawley rats with weights of 190 ± 10 g and ages of 6–7 weeks were provided by Chengdu Dashuo Biotechnology Co., Ltd and were housed in a laboratory at Chengdu University of Traditional Chinese Medicine on a 12 h/12 h light/dark cycle. The room temperature was maintained at 20 ± 2 ℃, and the relative humidity was maintained at 50–70%. The animal experiments in this study were approved by the Experimental Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (audit filing number: 2019-17).
2.2 Establishment and treatment of rat model of GPL
A rat model of GPL was established by the MNNG–ammonia composite modelling method. MNNG was purchased from Tokyo Chemical Industry Company, Japan (item number: CFFC-M0527-5G). The modelling process is shown in Fig. 1. First, the 74 rats were divided into normal (n = 12) and model (n = 62) groups using the digital random table method. The rats in the normal group were housed routinely, and the rats in the model group were given an ammonia solution with a concentration of 0.1% and were allowed to drink freely. Ammonia was purchased from China Sichuan Kebei Biological Company Limited (item number: GB/T631-2007). The rats in the model group received a 200 µg mL−1 MNNG solution by gavage and were fasted twice a week for 24 h each time without drinking [26]. At the end of week 8, two animals were randomly selected from the model group, from which the gastric mucosa at the gastric body–sinus junction was removed and stained with haematoxylin and eosin (H&E) to confirm the presence of early GPL in the form of mucosal intestinal epithelial metaplasia under light microscopy. The rats in the model group were then randomly divided into a model group, a folic acid group and high-, medium- and low-dose GRg3 groups, each of which comprised 12 rats. All groups except the normal group continued to receive the 0.1% ammonia solution ad libitum and the 200 µg mL−1 MNNG solution by gavage. Gavage treatment was started in week 9. The folic acid group received 2.7 mg kg−1 day−1 folic acid. Folic acid tablets were obtained from Changzhou Pharmaceutical Factory Co., Ltd (lot number: H32023302). The high-, medium- and low-dose GRg3 groups received 7.2 mg kg−1, 3.6 mg kg−1 and 1.8 mg kg−1 GRg3, respectively. GRg3 was supplied by Hefei Bomei Biotechnology Co., Ltd (item number: BZP0239). The normal and model groups received 1 mL/100 g saline by gavage. The rats were treated by gavage for 12 weeks.
2.3 Pathological analysis
After being fasted for 12 h, the animals were euthanized with sodium pentobarbital (2 mL g−1). The stomach was immediately removed, cut along the greater curvature and fixed in 4% paraformaldehyde fixative. Each sample was then embedded in paraffin and serially sectioned to a thickness of 3 µm. The sections were stained with H&E to observe histopathological changes in the gastric mucosa. In addition, the type of IM was determined using Alcian blue–periodic acid Schiff (AB-PAS) staining.
2.4 Terminal deoxynucleotidyl transferase dUTP nick end labelling staining
Paraffin sections (n = 10 per group) were dewaxed according to the instructions of a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining kit (Servicebio, No. G1507). The sections were shaken gently until dry, and the tissue was then circled with a histochemical pen (Servicebio, No. WG1066-1). A working solution of proteinase K was added dropwise inside the circle to cover the tissue, which was incubated at 37°C for 20 min. The sections were placed in phosphate-buffered saline (PBS) (pH 7.4) and washed three times with shaking on a decolorizing shaker for 5 min each time. The sections were placed in a 3% hydrogen peroxide solution and incubated for 20 min at room temperature while protected from light. The slices were shaken gently until dry, and buffer was added dropwise to cover the tissue in the circle. The slices were then incubated at room temperature for 10 min. After that, the equilibration solution was removed, and recombinant terminal deoxynucleotidyl transferase, biotin–dUTP labelling mix and equilibration buffer were mixed in a ratio of 1 µL: 5 µL: 50 µL and added to the circle to cover the tissue. The sections were placed flat in a wet box and incubated at 37°C for 1 h. A small amount of water was added to the wet box to maintain the humidity. After that, the sections were placed in PBS (pH 7.4) on a decolorizing shaker and washed three times for 5 min each. Streptavidin–horseradish peroxidase (HRP) and Tris-buffered saline–Tween-20 (TBST) were incubated at 1:200. The sections were placed in PBS (pH 7.4) and washed three times for 5 min each time with shaking on a decolorizing shaker. The sections were shaken gently, and freshly prepared 3,3'-diaminobenzidine (DAB) colour development solution (Servicebio, No. G1212) was added. The colour development time was controlled under a microscope, and the colour development was terminated by rinsing the sections with pure water. The sections were dehydrated in four cylinders of 100% alcohol for 5 min each time and were then immersed in n-butanol for 5 min, placed in xylene for more than 5 min until transparent, taken out to dry and sealed with neutral gum. Afterwards, the sealed slices were placed under a 200× high-magnification microscope. Five fields of view were randomly selected to count positively stained cells, and the apoptosis rate was calculated according to the following formula: apoptosis rate = number of positively stained cells/(number of positively stained cells + number of unstained cells) × 100%. Each set of data was measured three times, and the average value was taken. Brownish-yellow or brown granules in the nucleus were regarded as positive TUNEL staining.
2.5 Dihydroethidium fluorescent probe method
Frozen sections were warmed to room temperature while the humidity was controlled and were then dried. Circles were drawn around the tissue with a histochemical pen, and an autofluorescence quencher (Servicebio, No. G1221) was added for 5 min to the circles, which were then rinsed with running water for 10 min. An ROS staining solution (Sigma, D7008) was added dropwise to the circles, which were then incubated at 37°C for 30 min in a light-proof thermostatted box. The sections were placed in PBS (pH 7.4), shaken and washed three times for 5 min each time on a decolorizing shaker.. The sections were shaken, dried and sealed with an anti-fluorescence quenching sealer (Servicebio, No. G1401). The sections were observed under a fluorescence microscope, and images were collected with red fluorescence regarded as positive staining. 4',6-Diamidino-2-phenylindole: UV excitation wavelength 330–380 nm, emission wavelength 420 nm, blue light; fluorescein isothiocyanate: excitation wavelength 465–495 nm, emission wavelength 515–555 nm, green light; Cy3: excitation wavelength 510–560 nm, emission wavelength 590 nm, red light. The average absorbance values were calculated using ImageJ software.
2.6 Protein blotting
Gastric mucosal tissues (n = 10 per group) were homogenized and lysed in sample buffer, and then the lysates were centrifuged at 12000 rpm for 10 min at 4°C. The protein solution was then added to protein loading buffer in a ratio of 4:1, denatured in a boiling water bath for 15 min and stored in a freezer at −20°C as a backup. Equal amounts of protein from each sample were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were decolorized at room temperature on a shaker with 5% skimmed milk in a 0.5% TBST preparation and were then incubated with a diluted primary antibody (anti-TIGAR antibody; Abcam, ab189164) at 4°C overnight. A secondary antibody (HRP-labelled goat anti-rabbit; Servicebio, No. GB23303) was then added at room temperature, and the mixture was held for 1 h. The protein expression was assessed using an ultra-enhanced chemiluminescence reagent (K003; Affinity Biosciences, Cincinnati, OH, USA). β-Actin was used as an internal control, and the optical density (OD) of each band was determined using ImageJ software.
2.7 Biological data mining methodology
Clinical data on GC and data on TIGAR expression were downloaded from the Cancer Genome Atlas (TCGA) database and included data for 32 normal samples and 375 GC samples. To investigate the biological processes involved in low and high expression of TIGAR in GC, gene set enrichment analysis (GSEA) was performed on the transcriptomics data from the TCGA on GC, which included data on 19645 genes and 375 samples. The median value for the expression of TIGAR was used to classify these 375 samples into high- and low-expression groups, and GSEA software was used for analysis.
2.8 Immunohistochemical staining
Gastric tissue sections were fixed in 4% paraformaldehyde at room temperature, embedded in paraffin, cut into sections with a thickness of 4 µm and placed on slides. The sections were dewaxed, dehydrated and treated with sodium citrate buffer for antigen repair. The sections were then immersed in a 3% H2O2/ethanol solution to inhibit endogenous peroxidases and were then blocked with 3% bovine serum albumin. The sections were incubated overnight at 4°C with the primary antibody (anti-TIGAR antibody; Abcam, ab189164). After washing with PBS (pH 7.4) for 10 min, secondary antibodies (HRP-labelled goat anti-rabbit; Servicebio, No. GB23303) were added, and the sections were incubated for 20 min and reacted with DAB for 1 min. The sections were then restained with haematoxylin before being dehydrated with ethanol and xylene. Each slide was examined microscopically, and three fields of view were randomly selected for observation under a 100× light microscope. Brownish-yellow particles were regarded as indicating positive expression. The cumulative mean OD of the positively stained areas of each section was statistically analysed using Image-Pro Plus 6.0 software to semi-quantitatively determine the level of TIGAR.
We also determined the expression level of TIGAR in human gastric mucosa using the immunoreactivity score (IRS) in the following way: we invited two pathologists to assess the intensity of immunohistochemical (IHC) staining of TIGAR in the human gastric mucosa in a randomized manner using magnifications of 100× and 200× without clinicopathological data. The percentage of positive cells was calculated by IPP software, and the IRS was calculated for the semi-quantitative determination of human TIGAR. The IRS was determined from the sum of the percentage of positive cells (0 points, 0–5%; 1 point, 6–25%; 2 points, 26–50%; 3 points, 51–75%; and 4 points, 76–100%) and the staining intensity score (0 points, no staining; 1 point, weak staining; 2 points, moderate staining; and 3 points, strong staining). A final IRS of >4 indicates strong positive expression, whereas a score of <4 indicates weak positive expression.
2.9 Enzyme-linked immunosorbent assay
According to the instructions of an enzyme-linked immunosorbent assay (ELISA) kit (mlbio, ml202850, ml059197), 20 mg tissue was taken and added to 20 µL PBS before grinding and homogenization at 4°C. After centrifugation at 2000 rpm for 20 min, the supernatant was taken. Blank wells, standard wells and wells for the samples to be tested were set up. The concentrations of the standard in the wells were 8, 4, 2, 1, 0.5 and 0 ng mL−1, respectively. The blank wells were set up without samples or enzyme standard reagent. The standard wells were set up with 50 µL aliquots of the standard with different concentrations. The sample wells were set up with 40 µL sample diluent, followed by 10 µL of the sample to be tested (the final dilution of the sample was thus 5×), which were mixed with gentle shaking. Except for the blank wells, 100 µL enzyme standard reagent was added to each well, mixed with gentle shaking, covered with a plate sticker and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the wells were shaken until dry. A 350 µL aliquot of washing solution was added to each well, allowed to stand for 30 s and then discarded; this process was repeated five times, and the wells were then patted dry. Aliquots of 50 µL chromogenic agent A and 50 µL chromogenic agent B were added to each well, shaken gently and mixed well, and the colour was allowed to develop at 37°C for 15 min. A 50 µL aliquot of terminating solution was added to each well to terminate the reaction. The measurements were zeroed against the blank wells, and the OD of the sample in each well was measured sequentially at 450 nm with an enzyme marker within 15 min of the termination of the reaction. The perigastric tissues were weighed, added to the corresponding amount of PBS and homogenized, and the supernatant was taken for analysis. According to the instructions of the ELISA kit, ELISA Calc software was used to generate the standard curve and calculate the regression equation according to the concentration and OD value of the standard, and the protein contents of the samples to be tested were calculated according to the regression equation.
2.10 Microdetermination
According to the instructions of a GSH assay kit (Hailian Biologicals, m1076450), tissues were first washed twice with PBS, and then a 0.1 g sample was weighed out. Liquid nitrogen was added, and then 1 mL reagent I was added. The mixture was ground rapidly and centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was kept at 4°C to be tested. In the blank tube assay, to a 96-well plate were added 20 µL distilled water, 140 µL reagent II and 40 µL reagent III, which were mixed well. A 1 mg sample of the standard was weighed out and dissolved in 1 mL distilled water to give a concentration of 1 mg mL−1. The resulting solution was taken to prepare the standard with concentrations of 200 µg mL−1, 100 µg mL−1, 50 µg mL−1, 25 µg mL−1 and 12.5 µg mL−1 (diluted tenfold with reagent I). A 1.5 mL Eppendorf tube was taken, to which 20 µL standard, 140 µL reagent II and 40 µL reagent III were added, mixed and allowed to stand for 2 min. The absorbance at 412 nm was measured, and the absorbance minus that of a blank well (A1) was used as the horizontal coordinate to generate the standard curve of absorbance (x) versus concentration (y, µg mL−1). In the sample tube measurements, to a 96-well plate 20 µL sample, 140 µL reagent II and 40 µL reagent III were added, mixed and left for 2 min. The absorbance at 412 nm (A2) was measured, and ΔA was defined as A2 − A1. The standard curve was generated from the standard concentrations and OD values using ELISA Calc software, and the regression equation was derived, namely, [GSH] = y × volume of supernatant added to the reaction system/(volume of supernatant added to the reaction system/total volume of supernatant × sample mass), from which the GSH concentration was calculated.
2.11 Statistical processing
SPSS 25.0 software was used for statistical analysis. One-way analysis of variance was used to analyse comparisons among groups. Tukey’s test was used to compare the mean square deviations for the two groups. When the variance was not uniform, Dunnett’s T3 test was used. The data were expressed as the mean ± standard error of the mean. A P-value of less than 0.05 was considered to be statistically significant.