Ginsenoside Rg3 induces apoptosis and inhibits proliferation by down-regulating TIGAR in rats with gastric precancerous lesions

Abstract

Background: Ginsenoside Rg3 (GRg3) is one of the main active ingredients in Chinese ginseng extract and has various biological effects, such as immune-enhancing, antitumour, antiangiogenic, immunomodulatory and anti-inflammatory effects. This study aimed to investigate the therapeutic effect of GRg3 on gastric precancerous lesion (GPL) induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the potential mechanism of action.

Methods: The MNNG-ammonia composite modelling method was used to establish a rat model of GPL. Histopathological changes in the rat gastric mucosa were observed by pathological analysis using haematoxylin-eosin staining to assess the success rate of the composite modelling method. Alcian blue-periodic acid Schiff staining was used to observe intestinal metaplasia in the rat gastric mucosa. Apoptosis was detected in rat gastric mucosal cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling staining. The production level of reactive oxygen species (ROS) was determined by the dihydroethidium fluorescent probe method, and that of TP53-induced glycolysis and apoptosis regulator (TIGAR) protein was determined by immunohistochemical staining and western blotting. The production levels of nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate dehydrogenase (G6PDH) were determined by an enzyme-linked immunosorbent assay, and that of glutathione (GSH) was determined by microanalysis.

Results: GRg3 significantly alleviated the structural disorganization and cellular heteromorphism in the form of epithelial glands in the gastric mucosa of rats with GPL and retarded the progression of the disease. Overexpression of TIGAR and overproduction of NADP, GSH and G6PDH occurred in the gastric mucosal epithelium of rats with GPL, which in turn led to an increase in the ROS concentration. After treatment with GRg3, the expression of TIGAR and production of NADP, GSH G6PDH decreased, causing a further increase in the concentration of ROS in the gastric mucosal epithelium, which in turn induced apoptosis and played a role in inhibiting the abnormal proliferation and differentiation of gastric mucosal epithelial cells.

Conclusion: Grg3 can induce apoptosis and inhibit cell proliferation in MNNG-induced GPL rats. The mechanism may be related to down-regulating the expression levels of TIGAR and production levels of GSH, NADP and G6PD, and up-regulating the concentration of ROS.

Keywords: Apoptosis; Gastric precancerous lesions; Ginsenoside Rg3; Reactive oxygen species; TP53-induced glycolysis and apoptosis regulator.

Fig. 1

Scheme of the experimental design (n = 12 rats per group)

Fig. 2

Effect of GRg3 on the body weight and histopathology of the gastric mucosa in MNNG-induced GPL rats. A Observations of gastric mucosa from rats with gastric precancerous lesion. B Histopathological changes in gastric mucosa from rats in various groups (haematoxylin and eosin staining, 100× and 200× magnification). C Integrity of gastric mucosa from rats in various groups (Alcian blue–periodic acid Schiff staining, 100× and 200× magnification). D Folded graph of body weight of rats from 0 to 20 weeks，the body weight in the 20th week results are expressed as the mean ± SEM (n = 10 for each group). E haematoxylin and eosin staining score are expressed as the mean ± SEM (n = 10 for each group). F Alcian blue–periodic acid Schiff staining score are expressed as the mean ± SEM (n = 10 for each group). #P < 0.05, ##P < 0.01 vs normal group; *P < 0.05, **P < 0.01 vs model group; ▲P < 0.05, ▲▲P < 0.01 vs folic acid group

Fig. 3

Effects of GRg3 on the production levels of apoptosis, proliferation and ROS in in MNNG-induced GPL rats. A Apoptosis in gastric mucosa from rats in various groups shown by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining (400× magnification). B Determination of PCNA in rat gastric mucosa samples from various groups by an immunohistochemistry (IHC) assay (400× magnification) C Effect of GRg3 on ROS in gastric mucosa (40× magnification). D Apoptosis ratio shown by TUNEL staining. The results are expressed as the mean ± SEM (n = 10 for each group). E PCNA protein expression determined from cumulative mean optical density (F) Production of ROS in gastric mucosa. The results are expressed as the mean ± standard error of the mean (SEM) (n = 10 for each group). #P < 0.05, ##P < 0.01 vs normal group; *P < 0.05, **P < 0.01 vs model group; ▲P < 0.05, ▲▲P < 0.01 vs folic acid group

Fig. 4

Effects of GRg3 on the production levels of G6PDH, NADP, GSH in MNNG-induced GPL rats. A Effect of GRg3 on production of glucose-6-phosphate dehydrogenase in gastric mucosa. B Effect of GRg3 on production of nicotinamide adenine dinucleotide phosphate in gastric mucosa. C Effect of GRg3 on production of glutathione in gastric mucosa. The results are expressed as the mean ± SEM (n = 10 for each group). #P < 0.05, ##P < 0.01 vs normal group; *P < 0.05, **P < 0.01 vs model group; ▲P < 0.05, ▲▲P < 0.01 vs folic acid group

Fig. 5

TIGAR expression is up-regulated in human GPL samples than in normal mucosa samples, and GRg3 can effectively reduce the TIGAR expression level in MNNG-induced GPL rats. A Expression of TP53-induced glycolysis and apoptosis regulator (TIGAR) in gastric cancer from data retrieved from the Cancer Genome Atlas database. B Results of enrichment analysis of a TIGAR gene set. C Determination of TIGAR in human gastric mucosa samples from various groups by an immunohistochemistry (IHC) assay (200× and 400× magnification). D Determination of TIGAR in rat gastric mucosa samples from various groups by an IHC assay (400× magnification). E TIGAR protein expression determined from cumulative mean optical density. The results are expressed as the mean ± SEM (n = 10). F TIGAR protein expression in human gastric mucosa determined from the immunoreactivity score. The results are expressed as the mean ± SEM (n = 10). G Determination of TIGAR in rat gastric mucosa from various groups by WB. H TIGAR protein expression determined by western blotting (WB). The results are expressed as the mean ± SEM (n = 3). #P < 0.05, ##P < 0.01 vs normal group; *P < 0.05, **P < 0.01 vs model group; ▲P < 0.05, ▲▲P < 0.01 vs folic acid group