Dietary supplementation of gingerols- and shogaols-enriched ginger root extract attenuate pain-associated behaviors while modulating gut microbiota and metabolites in rats with spinal nerve ligation

Abstract

Neuroinflammation is a central factor in neuropathic pain (NP). Ginger is a promising bioactive compound in NP management due to its anti-inflammatory property. Emerging evidence suggests that gut microbiome and gut-derived metabolites play a key role in NP. We evaluated the effects of two ginger root extracts rich in gingerols (GEG) and shogaols (SEG) on pain sensitivity, anxiety-like behaviors, circulating cell-free mitochondrial DNA (ccf-mtDNA), gut microbiome composition, and fecal metabolites in rats with NP. Sixteen male rats were divided into four groups: sham, spinal nerve ligation (SNL), SNL+0.75%GEG in diet, and SNL+0.75%SEG in diet groups for 30 days. Compared to SNL group, both SNL+GEG and SNL+SEG groups showed a significant reduction in pain- and anxiety-like behaviors, and ccf-mtDNA level. Relative to the SNL group, both SNL+GEG and SNL+SEG groups increased the relative abundance of Lactococcus, Sellimonas, Blautia, Erysipelatoclostridiaceae, and Anaerovoracaceae, but decreased that of Prevotellaceae UCG-001, Rikenellaceae RC9 gut group, Mucispirillum and Desulfovibrio, Desulfovibrio, Anaerofilum, Eubacterium siraeum group, RF39, UCG-005, Lachnospiraceae NK4A136 group, Acetatifactor, Eubacterium ruminantium group, Clostridia UCG-014, and an uncultured Anaerovoracaceae. GEG and SEG had differential effects on gut-derived metabolites. Compared to SNL group, SNL+GEG group had higher level of 1'-acetoxychavicol acetate, (4E)-1,7-Bis(4-hydroxyphenyl)-4-hepten-3-one, NP-000629, 7,8-Dimethoxy-3-(2-methyl-3-buten-2-yl)-2H-chromen-2-one, 3-{[4-(2-Pyrimidinyl)piperazino]carbonyl}-2-pyrazinecarboxylic acid, 920863, and (1R,3R,7R,13S)-13-Methyl-6-methylene-4,14,16-trioxatetracyclo[11.2.1.0~1,10~.0~3,7~]hexadec-9-en-5-one, while SNL+SEG group had higher level for (+/−)-5-[(tert-Butylamino)-2'-hydroxypropoxy]-1_2_3_4-tetrahydro-1-naphthol and dehydroepiandrosteronesulfate. In conclusion, ginger is a promising functional food in the management of NP, and further investigations are necessary to assess the role of ginger on gut-brain axis in pain management.

Introduction

Neuropathic pain (NP) arises from damage to the peripheral or central nervous system [1]. The burden of chronic NP is related to the complexity of NP symptoms (e.g., anxiety and depression), poor outcomes, and limited available treatment options. Opioid analgesics are the most common forms of treatment for NP; unfortunately, they have severe side effects and can result in opioid use disorder [2]. Nerve injury in NP leads to neuroinflammation and neuroplastic changes in peripheral and central neurons associated with sensitization and hyperexcitability [3]. Previous studies have shown that NP could be a consequence of an imbalance between reactive oxygen species (ROS) and endogenous antioxidants after nerve injury, leading to neuroinflammation [4]. Therefore, the development of new effective and safe analgesic and anti-inflammatory alternatives is urgently needed.

Newly developed evidence suggests that the gut microbiome may serve as an important element between the neuroimmune-endocrine and gut-brain axes, forming a sophisticated network that directly and indirectly affects critical factors in the manifestations of NP [5]. For instance, spinal cord injury (SCI)-induced NP increases intestinal permeability and bacterial translocation from the gut, resulting in gut dysbiosis [6, 7]. Gut dysbiosis is associated with profound changes in gut-associated lymphoid tissues immune cell activation, and dysbiosis also exacerbates spinal inflammation/lesion, leading to impaired recovery of neurological function [6, 8]. Moreover, gut dysbiosis has been implicated in the onset or progression of pain-associated behavior, such as pain sensitivity and depression [9-12].

Convincing evidence suggests that gut microbiome-derived metabolites (mediators) may play a key role in the direct or indirect regulation of peripheral and central sensitization mechanisms in the development of NP [9, 13, 14]. In the peripheral nervous system, gut microbiome-derived mediators (e.g., pathogen-associated molecular patterns) can also indirectly increase the excitability of DRG neurons by inducing pro-inflammatory factors (e.g., tumor necrosis factor-α, interluekin-1 β, and interluekin-6) and chemokine (e.g., monocyte chemoattractant protein-1) release from immune cells to enhance pain, whereas other mediators (e.g., short-chain fatty acids (SCFA) and bile acids) can indirectly decrease the excitability of dorsal root ganglion (DRG) neurons by releasing anti-inflammatory cytokines (i.e., interluekin-4, IL-4) or neuropeptides (e.g., opioids) from immune cells to inhibit pain [14]. Several cell types in the central nervous system (CNS), including microglia, astrocytes, and immune cells, can receive information from the periphery (e.g., gastrointestinal tract) [15-17]. In the CNS, gut microbiome-derived mediators may regulate energy metabolism and neuroinflammation, which involves the activation of cells in the blood-brain barrier, microglia, and infiltrating immune cells via their receptors, transporters, and histone deacetylase [18] to modulate induction and maintenance of central sensitization [9, 13, 14] and depression [18]. The activation of glial cells (e.g., microglia, astrocytes) can produce pro-inflammatory cytokines and chemokines, which have been shown to alter glutamatergic synaptic neurotransmission and GABAergic neurotransmission [19-22], contributing to central sensitization and leading to pain hypersensitivity [14]. Since the gut microbiome and metabolites may be linked to neuroinflammation, neuronal sensitization, and hyperexcitability in the development of NP, targeting gut microbiome and metabolites using bioactive compounds (in food) represents a new therapeutic strategy to manage NP.

Ginger (Zingiber officinale Roscoe) consists of a complex combination of biologically active constituents, among which the compounds gingerols (6-, 8-, and 10-gingerol) and shogaols (6-, 8-, and 10-shogaol) reportedly account for the majority of ginger’s anti-inflammatory properties [23]. Various ginger compounds and extracts have been tested as anti-inflammatory agents, where the length of side chains determines the level of effectiveness [24]. Among all the gingerols, 6-gingerol is the most abundant and potent anti-oxidant that yields the greatest anti-inflammatory effects [24]. A combination of gingerols and shogaols is more effective in decreasing inflammatory mediators than the individual compounds [25]. Furthermore, ginger and its bioactive components have been shown to penetrate the blood-brain barrier via passive diffusion, suggesting the positive effects of ginger in CNS [26].

Due to ginger’s anti-inflammatory and antioxidant properties, ginger has been used for inflammation-associated knee osteoarthritis in vitro [27], ex vivo [28], and in humans [29]. Ginger elicits antinociceptive properties and intensifies morphine-induced analgesia during the rat radiant heat tail-flick test [30]. Recently Borgonetti et al. reported oral administration of CO₂-extracted ginger attenuated spared nerve injury (SNI)-induced NP symptoms by reducing spinal neuroinflammation [31]. Administration of ginger root extract or its individual bioactive compound (e.g., 6-gingerol, 6-shogaol, 8-shogaol) has also shown to mitigate pain in animals with SNI-induced NP [31-38]. Mata-Bermudez et al. reported that in a spinal nerve ligation (SNL)-induced rat model, the antiallodynic effect induced by 6-gingerol is mediated by the serotoninergic system involving the activation of 5-HT_1A/1B/1D/5A receptors and the NO-cyclic guanosine monophosphate-ATP-sensitive K⁺ channel pathway, but not by the opioidergic system [39]. On the other hand, ginger supplementation has shown to enhance the abundance of SCFA-producing favorable microbiota species in obese mice [40]. However, no study has evaluated the effects of two ginger root extracts, namely, gingerols-enriched ginger (GEG) and shogaols-enriched ginger (SEG), respectively, on pain sensitivity and anxiety-like behaviors and their potential impacts on gut microbiome composition and gut microbiome-derived metabolites in animals with spinal nerve ligation (SNL)-induced NP.

We designed this study to assess the effects of GEG and SEG on (1) pain- and anxiety-like behaviors, (2) the plasma circulating cell-free mitochondrial DNA (ccf-mtDNA) damage, a biomarker of excessive mitochondria-derived ROS linked to inflammation, (3) the composition of gut microbiota, and (4) gut microbiota-derived metabolites in the feces of SNL-treated rats. We hypothesized that dietary supplementation with GEG and SEG would reduce SNL-induced pain-associated sensory and affective behaviors compared to SNL control animals. Such changes in pain-associated behaviors may be mediated by reducing mitochondrial DNA damage and modulating gut microbiome composition and fecal metabolites. In this study, we combined microbiome and metabolome approaches to explore the effects of ginger bioactive compounds on metabolic pathways relevant to NP in the development of personalized nutrition therapy for NP management.

Materials and Methods

Animals

16 male Sprague-Dawley rats (4-5 week-old, 150-180 g, Harlan Laboratories, Indianapolis, IN) were housed under a 12-hour light-dark cycle with food and water ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Texas Tech University Health Sciences Center. Body weights, food intake, and water consumption were recorded weekly.

Introduction of neuropathic pain

The SNL model is widely used for the preclinical study of NP mechanisms and the development of new analgesic drugs/compounds. The nerve injury (SNL) results in acute hypersensitivity within 1 week that persists for weeks [41]. In this well-established SNL-induced NP model, prolonged changes in inflammatory and pronociceptive mediators, neurotransmitters, and receptor expression were observed, producing peripheral and central sensitization [42]. Spontaneous pain behaviors, increased emotional responses, anxiety-like and depression-like behaviors are also observed and have been studied by our group [3, 43, 44].

After 5-day acclimatization, we randomly assigned 4 animals as sham-controls receiving sham surgery, while the remaining 12 animals underwent SNL surgery. The SNL model of NP was used to induce peripheral neuropathy in the left hind paw, as described in our previous work [3, 43]. In brief, isoflurane was used for induction (3%) and maintenance (2%) of anesthesia throughout the procedure. After removing the L5/L6 level paraspinal muscles and underlying L6 transverse process, the L5 spinal nerve was removed from adjacent structures and tightly ligated with 6-0 silk thread. The paraspinal muscles were sutured closed, and the skin clipped together. Sham-operated animals served as controls for the NP model, receiving the same surgical procedure without the L5 spinal nerve ligation. After surgery, all animals were given antibiotic treatment (1 dose of gentamycin, 8 mg/kg, subcutaneously, s.c.; VetOne, Boise, ID) and were monitored for any signs of infection or distress. Throughout the study period, the animals were monitored to reduce unnecessary stress or pain following ethical guidelines of the International Association for the Study of Pain [45].

Dietary treatments

We randomly divided the 16 animals into four dietary treatment groups: sham group, SNL group, SNL+GEG group, and SNL+SEG group. All animals were given AIN-93G diet (catalog number # D10012G, Research Diet, Inc., New Brunswick, NJ, USA). For the SNL+GEG group and SNL+SEG group, after the SNL procedure, the animals were given GEG at 0.75% (wt/wt diet) and SEG at 0.75% (wt/wt diet) into AIN-93G diet for 30 days, respectively. GEG administration to rats (range between 100 mg and 400 mg/kg of body weight) has been shown to reduce inflammation in various inflammation models [46, 47]. In this study, the dose of 0.75% (wt/wt diet) corresponds 400 mg/kg body weight/day via oral gavage for rat, showing a reduction in mechanical hyperalgesia in streptozotocin-induced NP by ginger root extract [35].

Based on the results of gas chromatography-mass spectrometry, GEG consists of 18.7% 6-gingerol, 1.81% 8-gingerol, 2.86% 10-gingerol, 3.09% 6-shogoal, 0.39% 8-shogaol, and 0.41% 10-shogaol and SEG consists of 3.29% 6-gingerol, 0.06% 8-gingerol, 0.43% 10-gingerol, 17.4% 6-shogaol, 1.94% 8-shogaol, and 1.49% 10-shogaol [48]. Both GEG and SEG were obtained from Sabinsa, Inc., Piscataway, NJ.

Sample collection

At 1 day before- and 10, 20, and 30 days post-sham or SNL operation, (i) paw withdrawal mechanical thresholds were measured by von Frey test (VFT) using von Frey filaments for pain sensory assessment and (ii) center frequency and center duration in the open field test (OFT) were measured to assess anxiety-like behavior. After behavioral tests, the fecal samples of the animals were collected via individual metabolic cage and stored at −80°C for later microbiomes and metabolites analyses. On collection day, the animals were anesthetized, euthanized, and blood was drawn for plasma collection. Plasma samples were stored at −80°C until ccf-mtDNA concentration measurement.

Assessment of pain-related behaviors

The von Frey test was used to measure mechanosensitivity as described in our previous studies [3]. Mechanical withdrawal thresholds of spinal reflexes were measured using Electronic von Frey Aesthesiometer (IITC Life Science, Woodland Hills, CA). The tip made contact with the base of the third or fourth toe perpendicularly, with increasing force until a flexion reflex was provoked. The physical reaction was automatically recorded as the paw withdrawal threshold (in grams). The average of triplicate measurements at least 30s apart was utilized.

Open field test (OFT) was used to measure exploratory behavior of the animal in an arena (70 cm × 70 cm) with acrylic walls (height, 45 cm) for 15 min, using a computerized video tracking and analysis system (EthoVisionXT 11 software, Noldus Information Technology). Duration and entries in the center area (35 cm × 35 cm) were calculated for the first 5 min [3, 43]. Avoidance of the center area, in terms of duration and frequency, in the OFT suggests anxiety-like behavior.

Assessment of circulating cell-free mitochondrial DNA (ccf-mtDNA)

Quantitative PCR (qPCR) of COX2 and GAPDH genes was used to measure the copy numbers of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), respectively. Plasma DNA (mtDNA and nDNA) were isolated via the QIAamp DNA Blood Mini Kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s protocol. Standard curves were generated by dilution series of 20, 10, 5, 2, 1, and 0.1 ng of total rat liver DNA per reaction. We obtained the average threshold cycle (Ct) values for mtDNA and nDNA to determine the quantities of mtDNA and nucDNA present in the experimental samples. The cycle number (Ct) at which the fluorescent signal of a given reaction crosses the threshold value was used to quantify mtDNA and nucDNA copy numbers. We used the following primers to amplify the COX2 gene from mtDNA: forward – 5’CAC ACA AGC ACA ATA GAC GC-3’; reverse – 5’TAG GGA GGG AAG GGC AAT TA −3’. For amplification of the GAPDH gene from nDNA, the following primers were used: forward – 5’TGG CCT CCA AGG AGT AAG AAA C-3’; reverse – 5’-GGC CTC TCT CTT GCT CTC AGT ATC-3’. Primers were chosen using BLAST database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) so that the amplicon from mtDNA had no significant homology with the nuclear genome. Primers for qPCR were synthesized by SYBRGreenER Mix (Invitrogen, Paisley, Scotland), and qPCRs were performed in a 20-μl volume containing 10 μl of TaqMan Universal PCR Master Mix 2× Buffer (Applied Biosystems, Foster City, CA), 2 μl of the DNA solution, and 250 nM of each primer. The PCR cycles were as follows: 5 min at 95 °C followed by 40 amplification cycles (95 °C for 30 s, annealing, and elongation at 60 °C for 1 min). PCRs were performed in a 20-μl volume containing 10 μl of TaqMan Universal PCR Master Mix 2× Buffer, 2 μl of the DNA solution, and 250 nM of each primer. The gene relative expression level was analyzed by 2^−ΔΔCt method.

Gut microbiota profiling via 16S rRNA gene amplification and sequencing

Microbiota DNA was isolated from mouse feces using PowerFecal DNA isolation kit (Qiagen Inc., Germantown, MD, USA) following the manufacturer’s instructions. Amplicon sequencing of V4 region of the16S rRNA gene was performed by MR DNA (Molecular Research LP, Shallowater, TX, USA.). Briefly, V4 variable region was amplified using PCR primers 515F/806R. Samples were multiplexed and pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads, then used in Illumina DNA library preparation. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturer’s guidelines. Raw sequencing data have been deposited under BioProject accession numbers PRJNA715490 in the National Center for Biotechnology Information (NCBI) BioProject database.

Fecal metabolites profiling

Untargeted metabolomics profile in feces was determined using LC-MS/MS analysis [49]. In brief, 50 mg of fecal samples were homogenized using bead beater in PBS buffer followed by centrifugation. 100 μL of homogenized sample (supernatant) was transferred to a glass tube, and metabolites were extracted with dichloromethane/methanol/water (1:2:1 v/v) mixture and vortexed. After centrifugation, the aqueous phase was collected for LC-MS/MS analysis. The metabolite analysis was done by Q-Exactive HF mass spectrometer in both positive and negative ion mode using a Vanquish LC-system at a flow rate of 450 μL/min. An Acquity UPLC column (HSS T3, 1.7μm, 2.1 x 100mm) was used in a 15 min gradient for each run, and the temperature was kept at 50°C during the run.

Statistical analysis

Data analysis for body weight, food intake, water consumption, and pain parameters

The data of final body weight, food intake, water consumption, and pain-associated parameters were analyzed by one-way analysis of variance (ANOVA) test, followed by post hoc Fisher’s Least Significant Difference (LSD) test using GraphPad Prism software version 6.0. A significance level of P-value < 0.05 applies to all statistical tests.

Data Analysis for gut microbiome

16S rRNA gene sequencing data was analyzed using QIIME 2 [50]. In brief, reads were filtered, denoised, and merged. DADA2 was used to identify exact amplicon sequence variants (ASVs). For taxonomy assignment, Silva database version 132 database was used. To compare the relative abundance of taxa between groups, we used the non-parametric Kruskal–Wallis test followed by Dunn's test for multiple comparisons. Results were regarded as significant when P-value < 0.05.

Data Analysis for fecal metabolites

Principle Component Analysis (PCA) was performed to assess the different profiling of the metabolites detected and quantified among 4 groups. For data analysis, the following parameters were applied: mass tolerance for the precursor ion= 5 ppm, Intensity tolerance = 30%, minimum peak intensity= 1×10⁶, mass tolerance for alignment= 5 ppm, maximum shift for peak alignment= 2min and mass tolerance of fragment ion= 10 ppm. Data (peak areas of metabolites) were analyzed using Compound Discoverer software (3.1) to identify and quantify metabolites. Compound Discoverer 3.1 detected compounds with “Predicted Formula”, followed by automatic online library search against mzCloud and ChemSpider database. The compounds identified from the mzCloud library can be checked using the mirror plot of MS/MS spectra of identified compounds with library standards. The database search results yield spectra fit and matching score based on mass accuracy and isotope pattern and fragment ions obtained by MS/MS. For quantitative analysis, the area under the peak of each identified compound was calculated with proper peak alignment parameters described below. The t-test was performed to determine the number of statistically different metabolites in the SNL group compared to the other groups. Metabolites were filtered based on their statistical significance (P < 0.01), and results with >10 fold-change were presented.

Results

Final body weight, food intake, and water consumption

Throughout the study, there was no statistical difference in body weights among all groups, and the average final body weights of animals were 316.0±11.1 g, 312.8±11.6 g, 309.2±15.0 g, and 307.8±10.4 g for the sham, SNL, SNL+GEG, and SNL+SEG groups, respectively (P > 0.05). In addition, there was no significant difference in the food intake and water consumption of animals among all 4 groups (P > 0.05, data not shown).

Pain-like behaviors

The effects of GEG and SEG supplementation on NP-associated behaviors were assessed using von Frey test (Figure 1). Compared to the sham group, the SNL group had significantly greater sensitivity to mechanical stimuli starting at 10 days post-operation and sustained throughout the observation period (until 30 days after SNL induction). Compared to the SNL group, (i) both SNL+GEG and SNL+SEG groups showed significantly reduced pain sensitivity as early as 10 days post-operation and sustained through 20 and 30 days, as shown by increased mechanical thresholds; and (ii) there was no difference in pain sensitivity between the SNL+GEG group and the SNL+SEG group (Figure 1A). At the end of the study (30 days after supplements started), the order of pain sensitivity was SNL group > SNL+GEG group = SNL+SEG group > sham group.

Figure 1.

Effects of GEG and SEG on NP-associated behaviors. Pain sensitivity was assessed in the von Frey test (Figure 1A). Anxiety-like behaviors were assessed in the open field test as duration in the central area (Figure 1B) and frequency of entering the central area (Figure 1C) in animals with NP.

In terms of anxiety-like behaviors, relative to the sham group the SNL group had anxiety-like behaviors as shown by decreased duration in the center of an open field area (Figure 1B) and frequency of entering the central area (Figure 1C). Compared to the animals in the SNL group without supplements, the addition of GEG and SEG to the diet significantly improved NP-associated anxiety-like behaviors, as indicated by prolonged center duration at 30 days (Figure 1B) and increased center frequency at 20 and 30 days (Figure 1C).

Plasma ccf-mtDNA

We used ccf-mtDNA as a circulating biomarker indicative of oxidative stress from SNL and assessed the effect of GEG and SEG on reducing ccf-mtDNA. The SNL group had significantly higher plasma ccf-mtDNA levels relative to the sham group (Figure 2). When SNL-operated animals were fed a diet supplemented with either GEG or SEG, both SNL+GEG and SNL+SEG groups showed significant reduction in mitochondrial oxidative stress, as reflected in the decreased plasma ccf-mtDNA levels, relative to those in the SNL-only group (Figure 2).

Figure 2.

Effects of GEG and SEG on ccf-mtDNA concentration in animals with NP.

Gut microbiome composition and function

After data processing using QIIME 2, the number of unique amplicon sequence variants (ASVs) across all samples was 7,100, and the median number of ASVs per sample was 53,729 (range: 27,594–64,395). Beta-diversity analysis using weighted UniFrac distance metrics showed a slight separation between the different groups along PC2 (i.e., the sham group and the SNL group on one side and the SNL+GEG group and the SNL+SEG group on the other side) (Figure 3). This group separation was statistically significant using PERMANOVA (P = 0.003, pseudo-F = 2.34, and number of permutations = 999). Further, pairwise PERMANOVA was used to examine the separation of each group based on the distance between each pair. Only the distance between either sham group or SNL groups vs. SNL+SEG group was statistically significant (P = 0.03). In contrast, the distance between all other groups was not significantly different (P > 0.05). This indicates that while SNL didn't significantly impact the microbiome profile, the microbiome profile was strongly altered after SEG treatment.

Figure 3.

Alpha-diversity analysis of the gut microbiome. Principal component analysis (PCA) based on weighted UniFrac distance metric. Samples are colored based on the respective groups, while the size corresponds to the Shannon diversity index.

The sham and the SNL groups showed slightly higher alpha-diversity than other groups (Figure 3), calculated using the Shannon diversity index. The SNL+SEG microbiome showed lower diversity than either the Sham or the SNL groups (P = 0.043). In contrast, the SNL+GEG microbiome showed lower diversity than the sham group (P = 0.021) but not the SNL group (P = 0.083). We did not find a significant difference between the alpha-diversity of Sham and SNL groups (P = 0.772).

Then we identified taxa with the relative abundance that was altered with GEG or SEG supplementation. To achieve this, we used the Kruskal–Wallis test, which was followed by the post-hoc Dunn's multiple comparison test. We considered P < 0.05 as statistically significant (Figure 4). Overall, the SNL group has a small effect on the relative abundance of taxa in comparison with the sham group. The SNL group increased the abundance of Prevotellaceae UCG-001, and two Clostridia members, Roseburia and Eubacterium ruminantium group (Figure 4). In contrast, the SNL group showed a decrease in the abundance of Morganellaceae (Figure 4).

Figure 4.

A heatmap showing significant alterations in the relative abundance of the gut microbiome ASVs. The color scale represents the log₁₀ relative abundance of each ASV as the mean per group. The taxon at the genus level is shown on the left side, while the right side shows the respective phylum. Statistical test used was Kruskal–Wallis test followed by post-hoc Dunn's multiple comparison test (* and ** denote P < 0.05 and P < 0.01, respectively). Asterisks in SNL column represents significance in comparison to Sham, while asterisks in the SNL+GEG or SNL+SEG columns represents significance in comparison to SNL.

A common signature was observed with the GEG or SEG supplementation in the microbiome compared to the SNL group (Figure 4). Compared to the SNL group, both the SNL+GEG and the SNL+SEG groups had increased abundance of Lactococcus, Sellimonas, Blautia, Erysipelatoclostridiaceae (a family belonging to Firmicutes phylum), and Anaerovoracaceae (a family in the Clostridia class), but a decrease of Prevotellaceae UCG-001, Rikenellaceae RC9 gut group, Mucispirillum and Desulfovibrio (a member of Deferribacterota phylum), Desulfovibrio (a member of Desulfobacterota phylum), Anaerofilum, Eubacterium siraeum group (a member of Ruminococcaceae family, Firmicutes phylum), RF39 (belongs to Bacilli class, Firmicutes phylum), UCG-005 (a member of Oscillospiraceae family, Firmicutes phylum), Lachnospiraceae NK4A136 group, Acetatifactor (a member of Lachnospiraceae family, Firmicutes phylum), Eubacterium ruminantium group, Clostridia UCG-014, and an uncultured Anaerovoracaceae (Figure 4). We found that the abundance of Rikenellaceae and Prevotellaceae in the SNL+GEG and SNL+SEG groups was below or similar to the those in the Sham group (Figure 4).

The SNL+GEG group and the SNL+SEG group had a distinct effect on the gut microbiome as well (Figure 4). Relative to the SNL group, the SNL+GEG group showed an increase in the relative abundance of Parvibacter, Faecalitalea, Dubosiella, and Erysipelotrichaceae, and a decrease in the relative abundance of Muribaculaceae, Bacteroidales, and Ruminococcaceae. Furthermore, relative to the SNL group, the SNL+SEG group showed an increase in the relative abundance of Butryicimonas, Staphylococcus, Jeotgalicoccus, Erysipelatoclostridiaceae, Aerococcus, and Bacillales, and a decrease in relative abundance of Ruminococcaceae UCG-010, Ruminococcus, and Oscillospiraceae NK4A214 group.

Fecal metabolites

Overall, GEG and SEG supplementation had an impact on the fecal metabolite profiles (Figure 5). PCA of the experimental groups demonstrates less variance and good reproducibility among the 4 groups and significant difference between treated groups (i.e., the SNL+GEG group and the SNL+SEG group) and the sham/SNL groups (Figure 5). The level of 637 and 379 metabolites was significantly altered with GEG and SEG supplementation, respectively; fold-change ≥ 2 and adjusted P < 0.05.

Figure 5.

Effects of GEG and SEG on fecal metabolites of animals with NP, as shown in PCA

Due to the high number of metabolites, we focused on the top metabolites using these two filtering methods. First, we considered the top metabolites with more stringent statistical significance, i.e., adjusted P < 0.001 and fold-change ≥ 10 (Table 1). As a result of this filtering, compared to the SNL group the SNL+GEG group had a higher level of ‘seven’ metabolites including 1'-acetoxychavicol acetate, (4E)-1,7-Bis(4-hydroxyphenyl)-4-hepten-3-one, NP-000629, 7,8-Dimethoxy-3-(2-methyl-3-buten-2-yl)-2H-chromen-2-one, 3-{[4-(2-Pyrimidinyl)piperazino]carbonyl}-2-pyrazinecarboxylic acid, 920863, and (1R,3R,7R,13S)-13-Methyl-6-methylene-4,14,16-trioxatetracyclo[11.2.1.0~1,10~.0~3,7~]hexadec-9-en-5-one (Table 1). Relative to the SNL group, the SNL+SEG group had higher levels for two metabolites, including (+/−)-5-[(tert-Butylamino)-2'-hydroxypropoxy]-1_2_3_4-tetrahydro-1-naphthol and dehydroepiandrosteronesulfate (DHEAS) (Table 1).

Table 1.

Top enriched metabolites in either SNL+GEG or SNL+SEG vs SNL group

SNL+GEG group vs SNL group*
Metabolite Name	Formula	Log2 Fold Change (SNL+GEG/SNL)	P-value	Adj. P-value
1'-Acetoxyeugenol acetate	C14 H16 O5	13.10	5.52E-05	0.0009558
(4E)-1,7-Bis(4-hydroxyphenyl)-4-hepten-3-one	C19 H20 O3	11.20	2.30E-07	5.86E-05
NP-000629	C14 H16 O4	10.86	1.34E-05	0.00045946
7,8-Dimethoxy-3-(2-methyl-3-buten-2-yl)-2H-chromen-2-one	C16 H18 O4	10.32	1.57E-06	0.00015679
3-{[4-(2-Pyrimidinyl)piperazino]carbonyl}-2-pyrazinecarboxylic acid	C14 H14 N6 O3	10.30	1.60E-07	5.46E-05
920863	C16 H20 O5	10.27	3.01E-07	6.50E-05
(1R,3R,7R,13S)-13-Methyl-6-methylene-4,14,16-trioxatetracyclo[11.2.1.0~1,10~.0~3,7~]hexad ec-9-en-5-one	C15 H18 O4	10.11	5.83E-06	0.00029074
SNL+SEG group vs SNL group*
Metabolite Name	Formula	Log2 Fold Change (SNL+SEG/SNL)	P-value	Adj. P-value
(+/−)-5-[(tert-Butylamino)-2'-hydroxypropoxy]-1_2_3_4-tetrahydro-1-naphthol	C17 H27 N O3	11.57	1.93E-08	7.44E-05
Dehydroepiandrosterone sulfate (DHEAS)	C19 H28 O5 S	10.16	8.99E-07	0.00043157

^*Top metabolites were included if fold change ≥ 10 and adjusted P-value ≤ 0.001

Second, since our physiological results show that either GEG or SEG supplementation have a similar beneficial effect on NP-associated behaviors, we reported common metabolites that occurred in both the SNL+GEG and SNL+SEG groups. We filtered the metabolites for those with similar trends in both SNL+GEG and SNL+SEG groups and selected top shared metabolites (Table 2). Twenty-eight fecal metabolites were enriched with either GEG or SEG supplementation of SNL-treated animals with NP (Table 2).

Table 2.

Top enriched and shared metabolites in both SNL+GEG and SNL+SEG groups vs SNL group

Metabolite Name*	Log2 Fold Change	Log2 Fold Change
	(SNL+GEG/SNL)	(SNL+SEG/SNL)
(+)-(1R_2R)-1_2-Diphenylethane-1_2-diol	7.19	6.19
[ST(3:0)]estra-1_3_5(10)-triene-2_4_17beta-triol	8.15	6.95
1-(4-Hydroxy-3-methoxyphenyl)-3-oxo-5-decanesulfonic acid	6.7	8.61
1-[2-(3-Hydroxy-1-propen-2-yl)-2,3-dihydro-1-benzofuran-5-yl]ethanone	9.79	10.6
16a-Hydroxyestrone	6.24	6.18
1'-Acetoxychavicol acetate	5.35	6.55
1-Hydroxy-4-isopropyl-7-methoxy-1,6-dimethyl-2(1H)-naphthalenone	7.6	7.12
3,7,15-Trihydroxy-12,13-epoxytrichothec-9-en-8-one	6.81	7.67
3-{[4-(2-Pyrimidinyl)piperazino]carbonyl}-2-pyrazinecarboxylic acid	10.3	8.15
4,4'-(3,4-Dimethyltetrahydrofuran-2,5-diyl)bis(2-methoxyphenol)	6.35	6.59
5-[(1E)-3-hydroxy-3-methylbut-1-en-1-yl]-2-methylcyclohex-5-ene-1,2,4-triol	8.26	5.99
6-{4-Oxo-5-[(2E)-2-penten-1-yl]-2-cyclopenten-1-yl}hexanoic acid	5.16	6.23
7,8-Dihydroxy-3,7-dimethyl-6-oxo-7,8-dihydro-6H-isochromene-5-carbaldehyde	7	6.64
8'-Hydroxyabscisate	6.45	8.38
alpha-Santonin	6.87	5.19
Benzylideneacetone	6.56	6.21
Dihydrokavain	6.03	7.52
Di-n-Amyl phthalate	5.78	6.7
Ethyl vanillin isobutyrate	5.91	11.34
Mazindol-d4	9.04	5.68
Methyl (2E,4E)-5-(3,8-dihydroxy-1,5-dimethyl-6-oxabicyclo[3.2.1]oct-8-yl)-3-methyl-2,4-pentadienoate	6.91	5.35
methyl 2-[4-ethenyl-2,6-dihydroxy-3-(3-hydroxyprop-1-en-2-yl)-4-methylcyclohexyl]prop-2-enoate	5.49	5.17
Methyl N-formylnorleucylleucylphenylalaninate	8.3	8.7
Resorcinol diglycidyl ether	6.35	8.32
Sakuranetin	8.1	5.48
Trepibutone	8.39	6.9
Ubiquinone-1(CoQ1)	5.31	8.36
zinniol	6.04	5.94

^*Shared metabolites were included if fold change for both SNL+GEG/SNL and SNL+SEG/SNL ≥ 5 and adjusted P-value ≤ 0.05

DISCUSSION

In the present investigation, the SNL model of NP was successfully employed to investigate the impact of two ginger extracts, GEG and SEG, supplemented into the diet for 30 days in NP-associated behaviors. Compared to Sham rats, SNL rats showed pain-like behaviors (mechanical hypersensitivity) and anxiety-like behavior consistent with previous studies [3, 43]. Ginger and its bioactive components have been shown to reduce pain in a variety of NP models [31, 32, 39, 51]. The present study demonstrated potent effects of dietary GEG and SEG supplementation in mitigating mechanical hypersensitivity in the SNL-operated male rats during chronic pain. The capacity of 30-day GEG and SEG supplementation post SNL to decrease indices of mechanical allodynia in SNL-treated male rats corroborates the findings of Mata-Bermudez et al [39] and Fajirin et al. [35], respectively.

The current findings that both GEG and SEG supplementation decrease anxiety-like behaviors in the SNL model of NP further corroborates ginger’s anxiolytic-like role in vivo through serotonin [5-HydroxyTryptamine (5-HT)] and its receptors (5-HT1, 5-HT1A, 5-HT2, 5-HT3, 5-HT7 receptors) [52-57]. Ginger and its constituents 6-shogaol, 1-dehydro-6-gingerdione, 8- and 10-gingerol have shown to attenuate serotonin-induced hypothermia in mice [25]. Nievergelt et al. demonstrated 10-shogaol and 1-dehydro-6- gingerdione partially activated the serotonin 5-HT1A receptor [53]. Gingerols or the diterpenoid galanolactone are potent antagonists at the 5-HT3 receptor [56, 57].

Although we did not measure pro-inflammatory cytokines in the present study, previous studies have shown the inhibitory impacts of GEG or SEG on mechanical hypersensitivity may be mediated, in part, through inhibition of neuroinflammation and oxidative stress in vitro studies [31, 58-60]. Significant anti-neuroinflammatory effects of GEG and SEG have been reported in animals with NP. For instance, Mata-Bermudez et al. demonstrated that the antiallodynic effect induced by 6-gingerol in SNL-treated rats is mediated by activating the serotoninergic system and NO-cyclic guanosine monophosphate-ATP-sensitive K⁺ channel pathway [39]. Fajrin et al. reported 6-shogaol oral feeding reduced mechanical hyperalgesia in streptozotocin-induced NP mice via reduction in axon myelination damage from the sciatic nerve [35] and mRNA expression of transient receptor potential vanilloid-1 (TRPV-1) and N-methyl-D-aspartate receptor subunit 2B (NMDAR2B) in the spinal cord [51].

Excessive oxidative stress has been associated with the progression of chronic NP. NP is initiated by increases in ROS, which leads to increased mitochondrial damage and increased plasma-free mitochondrial DNA (mtDNA) [41, 61, 62]. The present study is the first study to show that both GEG and SEG significantly reduced ccf-mtDNA levels in the SNL-treated animals with NP, further corroborating the previous studies, showing ginger root extract improved mitochondrial function through enhancing antioxidant capacity and decreasing ROS production [18, 63].

Microbiome analysis conducted in the present study showed a shift in the composition of the gut microbiome in the SNL-operated rats with mechanical hypersensitivity and anxiety-like behaviors compared to sham-operated rats, suggesting a link between the gut microbiome and behavioral abnormalities in these SNL rats. From our collective knowledge, this is the first study establishing that mechanical hypersensitivity and anxiety-like behaviors in SNL-operated rats were mitigated by GEG and SEG supplementation, by means of an alteration of the gut microbiome, namely a decrease in the abundance of Eubacterium ruminantium, Clostridia UCG-014, and especially Prevotellaceae UCG-001 [11, 64]. Yang et al. reported that compared to the sham group, SNI-treated rats with depression-like/anhedonia-like phenotypes had increased levels of Prevotellaceae UCG-001, and fecal transplantation from SNI rats with or without anhedonia can exaggerate or mitigate pain and depression-like/anhedonia-like phenotypes in the pseudo-germ-free mice, respectively [64]. The current observation that GEG or SEG caused a decrease in Prevotellaceae UCG-001 in SNL rats with less anxiety-like phenotypes agrees with Yang’s study [64].

Ginger has been widely used for centuries to treat intestinal inflammation, both in vitro and in vivo. For instance, 6-gingerol has been shown to maintain barrier function in a cellular model of gut inflammation [65]. In animals, 6-gingerol abates colonic injury [66] and restores colonic permeability [67], in part mediated through downregulation of oxidative stress and inflammation in the inflamed colon [67-69]. Zingerone (chemical components of ginger) significantly reduced colonic transit and attenuated anxiety-like behavior in rats with irritable bowel disorder [70]. 6-shogoal prevents TNF-α-induced barrier loss, as shown by downregulation of claudin-2 via inhibition of PI3K/Akt and NF-κB signaling [71]. 6-gingerol prevents chronic ulcerative colitis via anti-inflammatory and anti-oxidative mechanisms and preservation of Wnt/β-catenin signaling pathway [72]. In the present study, the findings that the increased intestinal “potential pro-inflammatory” taxa (Prevotellaceae UCG-001 [73], Eubacterium ruminantium group, and Mucispirillum [74] in the SNL-operated rats were decreased when treated with GEG or SEG supplementation, further corroborating the anti-inflammatory role of GEG and SEG on colon health via modification of gut microbiome composition. Furthermore, Sun et al. recently reported that an increase in the abundance of Rikenellaceae RC9 gut group has been associated with increased intestinal permeability and oxidative stress, subsequently impairing the intestinal barrier and stimulating gut inflammation in coronary heart disease [75]. In the present study, we found that both GEG and SEG supplementation reduced the abundance of Rikenellaceae RC9 gut group which was even below the sham level along with a reduction of ccf-mtDNA in SNL-treated rats, supporting the protective role of GEG and SEG in gut health, presumably through decreased intestinal permeability and oxidative stress.

In the present study, GEG and SEG had differential effects on the gut microbiome composition. In general, the present study shows the gut micro-ecosystem of GEG and SEG-fed rats was shifted at the phylum level by favoring Firmicutes at expense of Bacteroidetes. Numerous studies in mice and humans indicated that the higher ratio of Firmicutes to Bacteroidetes might play an important role in energy uptake and nutrient utilization [76, 77]. The findings that an increase in abundance of SCFA-associated beneficial gut microbiome in the present study suggests that GEG and SEG supplementation can contribute to the improvement of gut function [78].

Wang et al. recently reported that ginger supplementation modulated the gut microbiota composition by increased amounts of species belonging to the Bifidobacterium genus as well as SCFA-producing bacteria (Alloprevotella and Allobaculum), along with increases in fecal SCFA (e.g., butyrate) concentrations in obese mice [40]. Feng et al. also showed that 6-gingerol increased the relative abundance of beneficial Bacteroidetes while decreasing Firmicutes on the phylum level in rats treated with cisplatin [79]. The present study supports the findings that GEG and SEG supplementation restores gut dysbiosis by elevating SCFA-producing beneficial bacteria, e.g., Lactococcus [80], Sellimonas [81], Blautia [82], and Erysipelatoclostridiaceae [83], while decreasing potential pro-inflammatory bacteria, such as Mucispirillum [84], Desulfovibrio [84], Anaerofilum [85], Eubacterium siraeum group, Eubacterium ruminantium group [85], Lachnospiraceae NK4A136 group [86], and Clostridia UCG-014 [87].

Ginger has been shown to benefit glucose homeostasis in diabetic animals [88, 89]. Gut microbiome is considered to be closely related to the occurrence and development of Type 1 diabetes mellitus in recent years [90, 91]. Specifically, Ma et al. reported an imbalance of the gut microbiota in individuals with Type 1 Diabetes presented by higher pathogenic bacteria (e.g., Ruminococcaceae, Shigella, Enterococcus, Streptococcus, Rothia, and Alistipes) and decreased beneficial bacteria (e.g., Lactobacillus, Faecalitalea, Butyricicoccus, Allobaculum, and Parvibacter) that are associated with infection and inflammation in diabetic subjects [90]. Although our SNL model is not a diabetic model, the findings that GEG supplementation increased the abundance of Faecalitalea and Parvibacter in animals further supports the beneficial role of GEG in gut health of SNL-treated animals. Moreover, recent studies have shown that the decreased abundance of Butryicimonas [92], Staphylococcus [91], Jeotgalicoccus [91], Aerococcus [93], and Bacillales [94] is associated with depression-like behaviors in animals. Intriguingly, in addition to an increased abundance of Prevotellaceae UCG-001, we have also found increased abundance of Butryicimonas, Staphylococcus, Jeotgalicoccus, Aerococcus, and Bacillales, while improving NP-associated negative anxiety-like behaviors. Together, these collective findings corroborate the SEG’s potential in ameliorating depression-like behaviors in SNL-treated rats.

The present study is the first study to demonstrate that compared to the SNL group, the SNL+GEG and SNL+SEG groups had relative higher intensity of fecal 1'-acetoxychavicol acetate that may be involved in inhibition of NF-κB [95, 96] and ERK/MAPK signaling [97, 98, 99]. Our findings of decreased neuroinflammation in SNL-treated animals due to GEG and SEG can be partially explained by the increased 1'-acetoxychavicol acetate levels in both SNL+GEG and SNL+SEG groups.

In the present study, we also demonstrated that the anti-neuroinflammatory effects of GEG and SEG are not only due to their anti-inflammatory property, as shown by increased levels in alpha-santonin [100], dihydrokavain [101], trepibutone [93], and sakuranetin [102], but also due to their anti-oxidant property, as shown by increased levels of sakuranetin [102, 103] and ubiquinone-1 (also called CoQ1) [104]. alpha-Santonin has exhibited diverse bioactivities including antioxidant [105, 106], anti-inflammation [107], and analgesic [108]. Dihydrokavain has exhibited a greater analgesic effect than aspirin in morphine-induced pain [109] due to its anti-inflammatory activity [110]. Dihydrokavain was reported to contribute significantly to the stress-induced anxiolytic effects in chicks under social separation stress [111]. In the present study, the observation that both GEG and SEG supplementation into the diet significantly reduces pain and anxiety-associated behaviors in SNL-treated animals may be, in part, due to elevation in the dihydrokavin levels. Sakuranetin, a group of methoxylated flavanones, was reported to have potent anti-inflammatory, antioxidant, antimicrobial, antiparasitic, antimutagenic, and antiallergic properties [102]. Ubiquinone-1 (also called CoQ1), a member of polyprenylbenzoquinones, acts as an antioxidant with the ability to transfer electrons in the electron transport chain and to stabilize mitochondrial complex in the status of inflammation [104]. Our observation that the elevation of fecal anti-inflammatory, anti-oxidant, and anxiolytic effects of GEG and SEG mitigated the SNL-induced NP parameters are corroborated with properties of metabolites as discussed above [102, 104, 109-111].

Based on our best knowledge, the present research is the first study to show that both GEG and SEG have antinociceptive effects in SNL-treated animals through a modulation of fecal metabolites, as shown in the increased levels of mazindol-d4 [112] and trepibutone [93, 113]. Mazindol is a non-selective catecholamine reuptake inhibitor, which blocks dopamine reuptake and thus could be used as an enhancer of dopamine release by neuropsychological tasks [114]. Rothman et al. reported that mazindol attenuates cocaine craving and euphoria by the inhibition of dopamine reuptake [115]. Robledo-González et al. showed that repeated mazindol administration significantly decreased spontaneous pain-like behaviors in arthritic joints animals through activation of dopaminergic and opioid receptors [112]. In clinic, trepibutone is widely used to mitigate cholestatic disease-associated pain [116]. In the mice with cholestatic disease, the G-protein–coupled BA receptor 1 (TGR5) is expressed by primary sensory neurons, and TGR5’s activation by bile acid induces neuronal hyperexcitability and scratching [113], resulting in the spinal cord releasing neuropeptides [117]. Trepibutone promotes secretion of bile and pancreatic juice, accelerating flaccidity of the smooth muscle in the gastrointestinal tract to decrease internal pressure of the gallbladder and bile duct, resulting in pain reduction in cholestatic disease [116]. The fact that elevated levels of mazindol and trepibutone in SNL-treated animals due to GEG and SEG further elucidate their roles in SNL-induced pain reduction.

In this study, we observed there were differential effects of GEG and SEG on fecal metabolites in SNL-treated rats. In general, these fecal metabolites possessed antioxidant and anti-inflammatory activities in the SNL+GEG group. In the SNL+GEG group, the elevated levels of 1’-acetoxyeugenol acetate [118, 119], (4E)-1,7-bis(4-hydroxyphenyl)-4-hepten-3-one [120], and 3-{[4-(2-pyrimidinyl)piperazino]carbonyl}-2-pyrazinecarboxylic acid [121] possessed antioxidant and anti-inflammatory activities. On the other hand, in the SNL+SEG group, the increased fecal level of (+/−)-5-[(tert- Butylamino)-2'-hydroxypropoxy]-1_2_3_4-tetrahydro-1-naphthol was involved in conversion of NADP⁺ to NADPH, suggesting SEG’s role in energy metabolism [122]. Dehydroepiandrosterone sulfate (DHEAS), an endogenous androstane steroid that is produced by the adrenal cortex, is considered a neuroactive neurosteroid [123]. DHEAS can modulate inhibitory gamma-aminobutyric acid (GABA) and excitatory N-methyl-D-aspartate (NMDA) receptors, producing complex neuronal effects. Naylor et al. reported that serum DHEAS levels were inversely correlated to the level of chronic low back pain in female Veterans, indicating a role for DHEAS in pain analgesia [124].

CONCLUSION

Both GEG and SEG supplementation into the diet decreased mechanical hypersensitivity and improved anxiety-like behavior mediated in part by suppressing oxidative stress (ccf-mtDNA). GEG and SEG exhibited differential effects on the microbiome composition and fecal metabolites, suggesting a prebiotic potential for dietary ginger root intake in the management of NP.

Highlights.

• Beneficial effects of two ginger root extracts rich in gingerols and shogaols on pain sensitivity and anxiety-like behaviors, and circulating cell-free mitochondria DNA in the development of neuropathic pain

• Dietary gingerols and shogaols favored microbiome composition in rats with neuropathic pain

• Differential impacts of gingerols and shogaols on fecal metabolites in rats with neuropathic pain