Resveratrol Alleviates Dextran Sulfate Sodium-Induced Acute Ulcerative Colitis in Mice by Mediating PI3K/Akt/VEGFA Pathway

Abstract

Ulcerative colitis (UC) is a chronic inflammatory disease that affects the colon, and its incidence is on the rise worldwide. Resveratrol (RSV), a polyphenolic compound, was recently indicated to exert anti-inflammatory effects on UC. Consequently, the current study was conducted to investigate the mechanism of RSV on alleviating UC in mice by mediating intestinal microflora homeostasis. First, potential targets that RSV may regulate UC were screened using the TCMSP database. Next, mice were treated differently, specifically subjected to sham-operation and dextran sulfate sodium (DSS) induction, and then treated or untreated with RSV. Disease Activity Index (DAI) and Hematoxylin-Eosin (HE) staining were employed to analyze the pathological changes of mice colon. In addition, the expression patterns of inflammatory factors in spleen tissues were detected using ELISA, while the protein expression patterns of phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), and vascular endothelial growth factor A (VEGFA) in colon tissues were determined by means of immunohistochemistry (IHC) and Western blot analysis. Moreover, changes in intestinal flora and metabolite diversity in UC were analyzed by metabonomics. It was found that RSV played inhibitory roles in the PI3K/Akt pathway in mice. Meanwhile, the administration of RSV induced downregulated the expressions of TNF-α, IFN-γ, IL-1β, IL-6, and IL-4. The six floras of Haemophilus and Veillonella were significantly enriched in UC, while Clostridium, Roseburia, Akkermansia, and Parabacteroides were found to be enriched in control samples. Lastly, it was noted that Akkermansia could regulate the intestinal flora structure of UC mice through triacylglycerol biosynthesis, glycerol phosphate shuttle, cardiolipin biosynthesis, and other metabolic pathways to improve UC in mice. Altogether, our findings indicate that RSV suppressed the activation of the PI3K/Akt pathway and reduced the VEGFA gene expression to alleviate UC in mice.

Keywords: phosphoinositide 3-kinase; protein kinase B; resveratrol; ulcerative colitis; vascular endothelial growth factor A.

FIGURE 1

Resveratrol (RSV) functions as a potential target for the therapy of ulcerative colitis (UC). (A) The overlapping genes between compound targets and UC targets in 92 RSG examined by Jvenn. (B) Analysis of KEGG enrichment of overlapping genes between drug targets and disease targets. (C) The overlapping zone between genes involved in enrichment analysis and genes involved in PI3K/Akt pathway. (D) The correlation and interaction analysis network between genes involved in the PI3K/Akt pathway; the color scale of the circle from orange to blue indicated that the degree value was from small to large. (E) Disease-compound-target relationship network diagram; orange represented targets, yellow compound, blue disease, and green traditional Chinese medicine. (F) Compounds that regulated core targets. (G) Compounds that controlled genes in the PI3K/Akt pathway. (H) Target genes regulated by RSV. VEGFA, vascular endothelial growth factor A.

FIGURE 2

RSV can bring positive results to UC. (A) Schematic diagram of animal experiment. (B) The colon length in MG, NG, RG, and PG mice was compared. (C) The curve of weight change of mice in different treatment groups was analyzed. (D) DAI disease scores of mice in different treatment groups were evaluated. (E) The pathological sections of the colon of mice in different treatment groups were observed using HE staining (100 ×, scale bar = 100 µm). (F) PCNA expression in the colon of mice in different treatment groups was measured using IHC (100 ×, scale bar = 100 µm). (G–K) The expression of IL-6, IL-8, IL-1β, TNF-α, and IL-10 in the colon tissues of mice in different treatment groups was monitored using ELISA. n = 20. Measurement data were expressed by mean ± SD. One-way ANOVA or repeated measures ANOVA was conducted for multiple group comparison, followed by Tukey’s post hoc test. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001. NG, normal control group; MG, model control group; RG, resveratrol group; PG, positive control group.

FIGURE 3

RSV inhibits the PI3K/Akt pathway activation and reduces the VEGFA gene expression. (A) The expression of VEGFA and PI3K and genes in colon tissues was analyzed by RT-qPCR. (B) The expression of VEGFA, PI3K, p-Akt, and Akt protein in colon tissues was detected by Western blot analysis. (C) Expression of VEGFA and Akt protein in colon tissues was analyzed by IHC. (D) The expression of VEGFA and PI3K in colon tissues after the addition of PI3K/Akt activator was determined with RT-qPCR. (E) Western blot analysis of the expression of VEGFA, PI3K, and p-Akt/Akt ratio was in colon tissues after the addition of PI3K/Akt activator. (F) The expression of VEGFA and p-Akt protein in colon tissue after adding PI3K/Akt activator was determined with IHC. ^# p < 0.05 vs. VEGFA, PI3K, and Akt expression in MG. ^## p < 0.01. n = 20. Measurement data were expressed by mean ± SD. One-way ANOVA was conducted for multiple group comparison, followed by Tukey’s post hoc test. NG, normal control group; MG, model control group; RG, resveratrol group; PG, positive control group; VEGFA, vascular endothelial growth factor A; 740Y-P, PI3K/Akt activator.

FIGURE 4

Inhibition of PI3K/Akt pathway alleviates DSS-induced UC in mice. (A–E) The expression of IL-6, IL-8, IL-1β, TNF-α, and IL-10 in the colon tissues of mice in different treatment groups was determined using ELISA. (F) The expression of PI3K in colon tissues was examined using RT-qPCR. (G) The expression of PI3K, p-Akt, and Akt protein in colon tissues was examined using Western blot. (H) The expression of VEGFA and Akt protein in colon tissues was detected using IHC. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001. n = 20. Measurement data were expressed by mean ± SD. One-way ANOVA was conducted for multiple group comparison, followed by Tukey’s post hoc test. MG, model control group; RG, resveratrol group; 740Y-P, PI3K/Akt activator.

FIGURE 5

RSV improves the intestinal flora structure of UC mice by inhibiting the PI3K/Akt pathway. (A) The Venn diagram of the intersection of intestinal bacteria associated with UC predicted by the databases GMrepo, gutMEGA, and gutMDisorder. (B) Significantly different intestinal flora in the log2Ratio of UC samples and normal samples. (C) Differential metabolites and the expression heat map of the differential metabolites were drawn. (D) Bar graph of enrichment analysis results of differential metabolites. (E) Dot chart of enrichment analysis results of differential metabolites. (F) Correlation network diagram of metabolites. (G) The presence of differential metabolites of microbes and UC of the differential microorganisms intersects Venn map was predicted using VMH database. (H) Distribution map of relative abundance of Akkermansia in healthy samples and UC samples. (I) Box plot of the relative abundance of Akkermansia in healthy samples and UC samples.

FIGURE 6

RSV suppressed the activation of the PI3K/Akt pathway and reduced the VEGFA gene expression to alleviate UC.