Ginger alleviates mechanical hypersensitivity and anxio-depressive behavior in rats with diabetic neuropathy through beneficial actions on gut microbiome composition, mitochondria, and neuroimmune cells of colon and spinal cord

Abstract

The relationship among gut microbiota, mitochondrial dysfunction/neuroinflammation, and diabetic neuropathic pain (DNP) has received increased attention. Ginger has antidiabetic and analgesic effects because of its anti-inflammatory property. We examined the effects of gingerols-enriched ginger (GEG) supplementation on pain-associated behaviors, gut microbiome composition, and mitochondrial function and neuroinflammation of colon and spinal cord in DNP rats. Thirty-three male rats were randomly divided into 3 groups: control group, DNP group (high-fat diet plus single dose of streptozotocin at 35 mg/kg body weight, and GEG group (DNP+GEG at 0.75% in the diet for 8 weeks). Von Frey and open field tests were used to assess pain sensitivity and anxio-depressive behaviors, respectively. Colon and spinal cord were collected for gene expression analysis. 16S rRNA gene sequencing was done from cecal samples and microbiome data analysis was performed using QIIME 2. GEG supplementation mitigated mechanical hypersensitivity and anxio-depressive behavior in DNP animals. GEG supplementation suppressed the dynamin-related protein 1 protein expression (colon) and gene expression (spinal cord), astrocytic marker GFAP gene expression (colon and spinal cord), and tumor necrosis factor-α gene expression (colon, P < .05; spinal cord, P = .0974) in DNP rats. GEG supplementation increased microglia/macrophage marker CD11b gene expression in colon and spinal cord of DNP rats. GEG treatment increased abundance of Acinetobacter, Azospirillum, Colidextribacter, and Fournierella but decreased abundance of Muribaculum intestinale in cecal feces of rats. This study demonstrates that GEG supplementation decreased pain, anxio-depression, and neuroimmune cells, and improved the composition of gut microbiomes and mitochondrial function in rats with diabetic neuropathy.

Keywords: Diabetic neuropathy; Ginger root extract; Gut microbiome; Mitochondria; Neuroinflammation.

Fig. 1 –

Gingerol-enriched ginger (GEG) supplementation reduced pain sensitivity (A) and anxio-depressive behaviors, as assessed in the open field test as time spent (duration) in the central area (B) and frequency of entering the central area (C). Bar histograms shown mean ± SEM. n =6–8 per group. Data were analyzed by a-way ANOVA followed by post hoc Fisher LSD tests, *P < .05. ANOVA, analysis of variance; LSD, least significant difference; SEM, standard error of the mean.

Fig. 2 –

Gingerol-enriched ginger (GEG) supplementation decreased gene expression of DRP1 in colon (A) and spinal cord (B) of rats. Quantification of DRP1 fluorescence intensity in colon tissue sections, pictures acquired at 60× (C). Data are expressed as mean ± SEM. n = 6–8 per group. Data were analyzed by 1-way ANOVA followed by post hoc Fisher LSD tests, *P < .05, ^#.05 < P < .1. ANOVA, analysis of variance; DRP1, dynamin related protein 1; LSD, least significant difference; SEM, standard error of the mean.

Fig. 3 –

Gingerol-enriched ginger (GEG) supplementation had no effect on MFN2 gene expression in colon (A) but tended to suppress MFN2 in spinal cord (B) of rats. Data are expressed as mean ± SEM. n = 6–8 per group. were. Data were analyzed by 1-way ANOVA followed by post hoc Fisher LSD tests, *P < .05, ^#.05 < P < .1. ANOVA, analysis of variance; LSD, least significant difference; MFN2, mitofusin 2; SEM, standard error of the mean.

Fig. 4–

Gingerol-enriched ginger (GEG) supplementation lowered GFAP gene expression in colon (A) and spinal cord (B) of rats. Quantification of GFAP fluorescence intensity in colon tissue sections, pictures acquired at 60× (C). Data are expressed as mean ± SEM. n = 6–8 per group. Data were analyzed by 1-way ANOVA followed by post hoc Fisher LSD tests, *P < .05. ANOVA, analysis of variance; GFAP, glial fibrillary acidic protein; LSD, least significant difference; SEM, standard error of the mean.

Fig. 5 –

Gingerol-enriched ginger (GEG) supplementation lowered TNF-α gene expression in colon (A) and spinal cord (B) of rats. Data are expressed as mean ± SEM. n = 6–8 per group. Data were analyzed by 1-way ANOVA followed by post hoc Fisher LSD tests, *P < .05, ^#_.05 < P < .1. ANOVA, analysis of variance; LSD, least significant difference; SEM, standard error of the mean; TNF-α, tumor necrosis factor-alpha.

Fig. 6 –

Gingerol-enriched ginger (GEG) supplementation increased CD11b gene expression in colon (A) and spinal cord (B) of rats. Data are expressed as mean ±SEM. n = 6–8 per group. Data were analyzed by 1-way ANOVA followed by post hoc Fisher LSD tests, *P < .05. ANOVA, analysis of variance; CD11b, cluster of differentiation molecule 11b; LSD, least significant difference; SEM, standard error of the mean.

Fig. 7 –

Gingerol-enriched ginger (GEG) supplementation increased microbiome evenness but not richness. Data were analyzed by Kruskal-Wallis test followed by Dunn multiple comparison post hoc tests, * P < .05. n = 7 for control group, n = 9 for DNP group, and n = 7 for GEG group. DNP, diabetic neuropathic pain.

Fig. 8 –

Gingerol-enriched ginger (GEG) supplementation modified the composition of cecal microbiome. Data were analyzed by Kruskal-Wallis test followed by Dunn multiple comparison post hoc tests, *P < .05, **P < .01, ***P < .001. n = 7 for control group, n = 9 for DNP group, and n = 7 for GEG group. DNP, diabetic neuropathic pain.