Dendrobium officinale Polysaccharides Inhibit 1-Methyl-2-Nitro-1-Nitrosoguanidine Induced Precancerous Lesions of Gastric Cancer in Rats through Regulating Wnt/β-Catenin Pathway and Altering Serum Endogenous Metabolites

Abstract

Dendrobium officinale is a herb in traditional Chinese medicine where D. officinale polysaccharides (DOP) are the main active ingredient. This study aimed at evaluating DOP efficiency at inhibiting 1-Methyl-2-nitro-1-nitrosoguanidine (MNNG) induced precancerous lesions of gastric cancer (PLGC) in rats through the Wnt/b-catenin pathway and analyzing the variations of serum endogenous metabolites. PLGC was established in male Sprague-Dawley (SD) rats by administering 150 μg/mL MNNG in drinking water for 7 months and giving 0.1 mL of 10% NaCl once weekly during the initial 20 weeks. Treatment with DOP inhibited the progress of PLGC through decreasing the expression of β-catenin by immunohistochemical analysis. The futher study indicated DOP downregulated gene expression of Wnt2β, Gsk3β, PCNA, CyclinD1, and β-catenin, as well as protein expression of Wnt2β, PCNA, and β-catenin. On the other hand, there were nine endogenous metabolites identified after the DOP treatment. Among these, the most significant one is betaine because of its strong antioxidant activity, leading to an anti-tumor effect. DOP can inhibit MNNG-induced PLGC models via regulating Wnt/β-catenin pathway and by changing endogenous metabolites.

Keywords: Dendrobium officinale polysaccharides (DOP); Wnt/β-catenin pathway; precancerous lesions of gastric cancer (PLGC); serum endogenous metabolites analysis.

Figure 1

The protein expression of β-catenin in stomach tissues by immunohistochemistry (400 × original magnification). (A): The CTRL group: the normal rat were administered with free water and diet; (B): The rat were given 150 μg/mL 1-Methyl-2-nitro-1-nitrosoguanidine (MNNG) in drinking water for 7 months and given 0.1 mL of 10% NaCl once weekly during the initial 20 weeks, which was regarded as the PLGC model; (C–E): The PLGC model were administered with 9.6, 4.8, 2.4 g/kg/day Dendrobium officinale polysaccharide (DOP), respectively, and they were administered DOP 2 weeks earlier before starting the PLGC model. (F): The intensity of β-catenin measured by Image J. Data was presented as mean ± SEM (n = 6). Statistical analyses were carried out by using one-way ANOVA and Tukey post-hoc test. ** p < 0.01 vs. CTRL; ^# p < 0.05 vs. PLGC model.

Figure 2

The effect of DOP on Wnt2β, Gsk3β, PCNA, and β-catenin gene expression. Results from administering DOP 7 months on PLGC rat model. The relative expression for a particular gene was calculated by using Ct method, a comparative 2^−ΔΔCT method. Data was presented as mean ± SEM (n = 6). Statistical analyses were carried out by using one-way ANOVA and Tukey post-hoc test. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. CTRL; ^# p < 0.05 and ^## p < 0.01 vs. PLGC model.

Figure 3

The effect of DOP on Wnt2β, PCNA and β-catenin protein expression ((A) the western blot bands; (B–E) the relative level of Wnt2β, PCNA, β-catenin and CyclinD1). Results from administering DOP 7 months on PLGC rat model. The relative expression for protein was analyzed and calculated by using Image lab software. Data was presented as mean ± SEM (n = 3). Statistical analyses were carried out by using one-way ANOVA and Tukey post-hoc test. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. CTRL; ^# p < 0.05 and ^## p < 0.01 vs. PLGC model.

Figure 4

PLS-DA analysis between different groups in positive and negative ion pattern. After the component identification was completed, the identified data was imported into the Ezinfo software for partial least squares-discriminant analysis (PLS-DA) analysis, the circle showed that they tend to be grouped together in the PLS-DA analysis, and could be separated between each groups of observations, then cited there have the change in PLGC group (A,B). After treatment with DOP (C,D), the gather was changed, which indicated that the endogenous metabolites could be different from the PLGC group.

Figure 4

PLS-DA analysis between different groups in positive and negative ion pattern. After the component identification was completed, the identified data was imported into the Ezinfo software for partial least squares-discriminant analysis (PLS-DA) analysis, the circle showed that they tend to be grouped together in the PLS-DA analysis, and could be separated between each groups of observations, then cited there have the change in PLGC group (A,B). After treatment with DOP (C,D), the gather was changed, which indicated that the endogenous metabolites could be different from the PLGC group.

Figure 5

The effect of DOP on height values of nine changing endogenous metabolites in each group under positive and negative ion mode. Results from administering DOP 7 months on PLGC rat model: the compound was analyzed in the serum by UPLC/Q-TOF-MS and then selected variables with a VIP > 1.0 and p < 0.05 and that compound was regarded as potential biomarkers for further statistical analysis. Statistical analyses were carried out by using one-way ANOVA and Tukey post-hoc test. * p < 0.05, *** p < 0.001 vs. the CTRL; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 vs. the PLGC model.

Figure 6

To confirm the compound by two stages mass spectrometry analysis. Taking the metabolite m/z 252.1095 as an example, the first stage of mass spectrometry (MS1) is 252.1095 and second stage of mass spectrometry (MS2) is 136.0612 (A), next to compare with the METLIN metabolomics database, the MS2 136.0612 was also appeared (B), and then confirmed the MS1 of this compound was 252.1095. Student’s t-test was used for statistical analysis to evaluate significant differences of endogenous metabolites. (A): Two stages mass spectrometry of the sample; (B): Two stages mass spectrometry from the database.

Figure 7

The effect of DOP on the metabolites pathway. By uploading the nine endogenous metabolites to the MetaboAnalyst software, metabolite enrichment analysis could be conducted.

Figure 8

The diagrammatic sketch of the experiment.