Transcriptome profiling analysis of the response to walnut polyphenol extract in Helicobacter pylori-infected cells

Abstract

Dietary intervention to prevent Helicobacter pylori (H. pylori)-associated gastric diseases seems to be ideal with no risk of bacterial resistance, safe long-term intervention, and correcting pathogenic mechanisms including rejuvenation of precancerous atrophic gastritis and anti-mutagenesis. A transcriptome as set of all RNAs transcribed by certain tissues or cells demonstrates gene functions and reveals the molecular mechanism of specific biological processes against diseases. Here, we have performed RNAseq and bioinformatic analysis to explain proof of concept that walnut intake can rescue from H. pylori infection and explore unidentified mode of actions of walnut polyphenol extract (WPE). As results, BIRC3, SLC25A4, f3 transcription, VEGFA, AZU1, HMOX1, RAB3A, RELBTNIP1, ETFB, INPP5J, PPME1, RHOB, TPI1, FOSL1, JUND.RELB, KLF2, MUC1, NDRG1, ALDOA, ENO1, PFKP, GPI, GDF15, and NRTN genes were newly discovered to be enriched with WPE, whereas CCR4, BLNK, CCR7, CXCR4, CDO1, KLSG1, SELE, RASGRP2, PIK3R3, TSPAN32, HOXC-AS3, HCG8, BTNL8, and CXCL3 genes as inhibitory targets by WPE in H. pylori infection. We identified additional genes what WPE afforded actions of avoiding H. pylori-driven onco-inflammation and rejuvenating precancerous atrophic gastritis. Conclusively, after applying RNAseq analysis in order to document walnut intake for precision medicine against H. pylori infection, significant transcriptomic profiling applicable for validation were drawn.

Keywords: H. pylori; RNAseq; pharmanutrient; transcriptome; walnut polyphenol extract.

Fig. 1

RNAseq analysis in non-treated gastric cells, gastric cells infected with H. pylori infection, and cells infected with H. pylori under WPE (walnut polyphenol extracts) treatment (A) in vitro AGS cells infected with H. pylori, and infected with H. pylori in the presence of WPE, triplicate repeated, analyzed with NextSeq 500 (Illumina) after Agilent 6000 Nanochip for RNA qualification and quantification. (B) Results of hierarchical clustering analysis (heatmap) for differential expressed gene (DEG) analyzed according to significantly changed after H. pylori infection and infected with H. pylori in the presence of WPE, 943 genes included within p<0.05. Red and green in the color bar represent high and low expression levels, respectively. Red and green in the y-axis represent upregulated and downregulated DEGs, respectively. (C) GO (gene ontology) function analysis of the upregulated/downregulated DEGs including biological process, cellular component and molecular function, gene analyzed according to significantly changed after H. pylori in the presence of WPE, 943 genes included showing p<0.05.

Fig. 2

(A) Expression heatmap for differential expressed genes showing significant trend like increased with H. pylori infection, but significantly decreased with co-treatment of WPE, description of gene category. GO enrichment analysis in box. (B,C) Kyoto Encyclopedia of Genes and Genomes enrichment analysis (KEGG) analysis. Annotation of KEGG HIF-1α and PI3K signaling pathways with transcriptomic data. (D) STRING-generated interaction network of the differentially expressed genes Amplification of the network for PPI associated with CCR4. PPI network of DEGs shows red nodes represent DEGs up-regulated in the model group, while green nodes represent DEGs down-regulated in the model group. The color shade of a red node is positively correlated with the degree of this node, while the darker the color is, the higher the expression of up-regulated DEGs is.

Fig. 3

Whole genes analyzed as significantly different, significantly increased after H. pylori infection, but significantly decreased after H. pylori infection in the presence of WPE (p<0.05). HP, H. pylori; HP-W, H. pylori + WPE.

Fig. 4

(A) Expression heatmap for differential expressed genes showing significant trend like decreased with H. pylori infection, but significantly increased with co-treatment of WPE, description of gene category, GO enrichment analysis in box. (B) KEGG analysis Annotation of KEGG lipid-related signaling pathways with transcriptomic data. (C) STRING-generated interaction network of the differentially expressed gene showing amplification of the network for PPI associated with HMOX1 (heme oxygenase 1).

Fig. 5

Whole genes analyzed as significantly different, significantly decreased after H. pylori infection, but significantly increased after H. pylori infection in the presence of WPE (p<0.05). HP, H. pylori; HP-W, H. pylori + WPE.

Fig. 6

(A) Expression heatmap for differential expressed genes showing significant trend like increased with H. pylori infection, but further significantly increased with co-treatment of WPE, description of gene category, GO enrichment analysis in box. (B–D) STRING-generated interaction network of the differentially expressed genes.

Fig. 7

Whole genes analyzed as significantly different, significantly increased after H. pylori infection, but further significantly increased after H. pylori infection in the presence of WPE (p<0.05). HP, H. pylori; HP-W, H. pylori + WPE.

Fig. 8

(A) Expression heatmap for differential expressed genes showing significant trend like decreased with H. pylori infection, but further significantly decreased with co-treatment of WPE, description of gene category, GO enrichment analysis in box. (B) STRING-generated interaction network of the differentially expressed genes.

Fig. 9

Whole genes analyzed as significantly different, significantly decreased after H. pylori infection, but further significantly decreased after H. pylori infection in the presence of WPE (p<0.05). HP, H. pylori; HP-W, H. pylori + WPE.

Fig. 10

Schematic presentation showing the transcriptomic profiling showing genes implicated in rescuing action of WPE against H. pylori infection.