Crosstalk Among Gut Microbiota, Fecal Metabolites, and Amygdala Neuropathology Genes After Ginger Polyphenol Administration in Female Rats with Neuropathic Pain: Evidence for Microbiota-Gut-Brain Connection

Abstract

Objectives. The relationships among neuropathic pain, gut microbiota, microbiome-derived metabolites, and neuropathology have received increasing attention. This study examined the effects of two dosages of gingerol-enriched ginger (GEG) on mechanical hypersensitivity, anxiety-like behavior, gut microbiome composition and its metabolites, and neuropathology markers in female rats in the spinal nerve ligation (SNL) model of neuropathic pain. Methods. Forty female rats were assigned to 4 groups: sham-vehicle, SNL-vehicle, SNL+GEG at 200 mg/kg BW, and SNL+GEG at 600 mg/kg BW via oral gavage. All animals were given an AIN-93G diet for 5 weeks. Mechanical hypersensitivity was assessed by the von Frey test. Anxiety-like behavior was assessed by the open field test. Fecal microbiota composition and metabolites were determined using 16S rRNA gene sequencing and GC-MS, respectively. Neuropathology gene expression profiling of the amygdala was assessed by an nCounter^® Neuropathology pathway panel. Results. Both GEG-treated groups showed decreased mechanical hypersensitivity and anxiety-like behavior in the SNL model. Gut microbiome diversity in both GEG groups was decreased compared with untreated SNL rats. In the SNL model, phyla such as Bacteroidota, Proteobacteria and Verrucomicrobiota were decreased. Compared with the untreated SNL group, both GEG groups exhibited increased abundance of the phyla Bacteroidota (i.e., Rikenella, Alistipes, Muribaculaceae, Odoribacter), Firmicutes (i.e., UBA1819, Ruminococcaceae, Oscillospiraceae, Roseburia), and Verrucomicrobiota (i.e., Victivallis). GEG groups had higher levels of nine hydrophilic positive metabolites [val-glu, urocanic acid, oxazolidinone, L-threonine, L-norleucine, indole, imino-tryptophan, 2,3-octadiene-5,7-diyn-1-ol, and (2E)-3-(3-hydroxyphenyl) acrylaldehyde] and two hydrophilic negative metabolites [methylmalonic acid and metaphosphoric acid], as well as lower levels of five hydrophilic metabolites [xanthine, N-acetylmuramic acid, doxaprost, adenine, and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine]. Among the 770 neuropathology genes, 1 gene (PLA2G4A) was upregulated and 2 genes (CDK5R1 and SHH) were downregulated in SNL rats. GEG caused the upregulation of nine genes (APC, CCNH, EFNA5, GRN, HEXB, ITPR1, PCSK2, TAF9, and WFS1) and downregulation of three genes (AVP, C4A, and TSPO) in the amygdala. Conclusions. GEG supplementation mitigated pain-associated behaviors in female rats with neuropathic pain, in part by reversing the molecular neuropathology signature of the amygdala. This was associated with changes in the gut microbiome composition and fecal metabolites, which could play a role in mediating the effects of GEG on neuropathic pain.

Keywords: brain; ginger; gut microbiome; neuropathic pain; neuropathology; rats.

Figure 1

Effects of GEG on mechanosensitivity assessed by an electronic von Frey anesthesiometer. Data are expressed as mean ± SEM and were analyzed by two-way ANOVA with the Bonferroni multiple comparison test, n = 9 per group. *** p < 0.001 compared with Sham-V group. + p < 0.05, ++ p < 0.01, +++ p < 0.001 compared with SNL-V group.

Figure 2

Effects of GEG on anxiety-like behavior assessed in the open-field test (OFT). Data are expressed as the mean ± SEM and were analyzed by one-way ANOVA with the Bonferroni multiple comparison test, n = 5–8 per group. ** p < 0.01 compared with Sham-V group. + p < 0.05 compared with SNL-V group.

Figure 3

Effects of GEG on gut microbiota. (A) Alpha diversity analysis of the microbiome in different groups. Alpha diversity was described using two metrics, one for evenness and one for richness. The Wilcoxon test was used to determine statistical significance. The SNL group was used as a reference group for comparison. * p < 0.05, *** p < 0.001. (B) Compositional analysis of the microbiome ASVs across different groups using LOCOM. Female 0 indicates differences between SNL-V and Sham-V; Female 200 indicates differences between SNL+200GEG and SNL-V; and Female 600 indicates differences between SNL+600GEG and SNL-V. Only ASVs with p < 0.05 are reported, and labels indicate FDR-adjusted p-values.

Figure 4

Effects of GEG on fecal hydrophilic positive metabolites (A) and negative metabolites (B). Data were analyzed by ANOVA on each dataset, followed by the Benjamini–Hochberg procedure for selected compounds with p ≤ 0.001 for pairwise comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, comparing abundancy of fecal metabolites between groups. Abbreviation: val-glu: valyl-glutamate.

Figure 4

Effects of GEG on fecal hydrophilic positive metabolites (A) and negative metabolites (B). Data were analyzed by ANOVA on each dataset, followed by the Benjamini–Hochberg procedure for selected compounds with p ≤ 0.001 for pairwise comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, comparing abundancy of fecal metabolites between groups. Abbreviation: val-glu: valyl-glutamate.

Figure 5

NanoString neuropathology pathway panel analysis of amygdala tissue. (A) Principal component analysis of the gene expression profiles of neuropathology-related genes. (B) Differential expression analysis between SNL-V and Sham-V. (C) Differential expression analysis between SNL+200GEG and SNL+600GEG and SNL-V. * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001, t-test. (D) Gene expression levels were assessed by qRT-PCR. * p < 0.05 compared with Sham-V group. + p < 0.05, ++ p < 0.01, and +++ p < 0.001 compared with SNL-V group, one-way ANOVA with Bonferroni multiple comparisons tests. Abbreviations: APC: adenomatosis polyposis coli, AVP: arginine vasopressin, C4A: complement C4A, CAMK2G: calcium/calmodulin dependent protein kinase II gamma, CCNH: cyclin H, CDK5R1: cyclin dependent kinase 5 regulatory subunit 1, EPFN5: ephrin A5, GRN: granulin, HEXB: hexosaminidase subunit beta, ITPR1: inositol 1,4,5-trisphosphate receptor 1, NR4A2: nuclear receptor subfamily 4 group A member 2, PCSK2: proprotein convertase subtilisin/kexin type 2, PLA2G4A: phospholipase A2 group IVA, SHH: Sonic hedgehog signaling molecule, TAF9: TATA-box binding protein associated factor 9, TPSO: translocator protein, WFS1: Wolfram syndrome 1 homolog.

Figure 6

Pathway enrichment analysis using NanoString fundamental themes to map top genes identified as differentially regulated by GEG treatment.