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Journal of Orthopaedic Surgery and Research logoLink to Journal of Orthopaedic Surgery and Research
. 2026 Jan 5;21:85. doi: 10.1186/s13018-025-06597-z

The role of electroacupuncture in modulating gut microbiota and alleviating lumbar disc herniation in rats

Yanbei Chen 1, Xiuting Cui 1, Zhihong Chen 1, Huilian Shi 1, Yanfei Qian 1, Meifang Yin 1, Xiaoju Zhu 1,✉
PMCID: PMC12870147  PMID: 41491531

Abstract

Background

Lumbar disc herniation (LDH) is a common disease, and the resulting low back pain seriously affects patients’ daily life and work. Previous studies have demonstrated the clinical efficacy of electroacupuncture (EA) treatment in alleviating LDH. Recent studies have shown a link between the gut microbiota and LDH. However, the relationship between the regulatory effect of electroacupuncture therapy on gut microbiota and the improvement of LDH is still not fully understood.

Methods

Eighteen SPF female Sprague-Dawley (SD) rats were randomly assigned to three groups: control group (CON), lumbar disc herniation group (LDH), and LDH + electroacupuncture group (EA). A rat model of lumbar disc herniation was established through autologous nucleus pulposus transplantation. Starting from the second day after LDH induction, the EA group received electroacupuncture treatment once a day for a total of 7 times (2 mA, 2/15 Hz, 30 min/day) at bilateral Weizhong (BL40). The pathological changes of intervertebral disc tissue of rats in each group were observed, and the expression of inflammatory factors and extracellular matrix (ECM)-related proteins in intervertebral disc tissue of rats in each group were detected. Changes in gut microbiota of rats in each group were detected by 16 S ribosomal DNA (rDNA) sequencing.

Results

EA treatment significantly improved the pathological damage and inflammatory response of intervertebral disc tissue in LDH rats, and maintained the balance of ECM in intervertebral disc tissue in LDH rats. Through 16 S rDNA sequencing analysis, it was found that the abundance of OTU/ASV in gut microbiota in LDH model rats was significantly reduced. However, EA treatment can partially reverse the changes in OTU/ASV abundance of EA treatment. At the genus level, the abundance of Flavonifractor, Christensenella, Lachnospiraceae_UCG-010, Fournierella, Prevotellace_Ga6A1_group, Eubacterium and Holdemania were increased in the LDH group, while the abundance of these bacterial groups was significantly down-regulated after EA treatment. The abundance of RF39_genus and Coriobacteria_genus were decreased in the LDH group, but the abundance of these bacterial groups increased significantly after EA treatment.

Conclusion

These findings suggest that EA at BL40 point has a significant therapeutic effect on LDH rats, which may be achieved by regulating the gut microbiota.

Keywords: Electroacupuncture, BL40 point, Lumbar disc herniation, Extracellular matrix, Gut microbiota

Introduction

Lumbar disc herniation (LDH) is a one of the most common disorders of the low back [1]. Prolonged mechanical stress on lumbar soft tissues leads to annular fibrosus rupture, resulting in nucleus pulposus herniation or extrusion. These compressions or irritations of the spinal nerve roots can cause radiating lumbosacral and lower extremity pain [2]. Patients typically exhibit varying degrees of neurological signs and symptoms such as pain, numbness, weakness and soreness [2]. Research demonstrates that inflammation significantly contributes to LDH pathogenesis. Inflammatory-mediated nerve root compression and associated radicular pain substantially impair patients’ daily function and occupational capacity [3]. While conservative therapies (e.g., firm bedding, analgesia, massage) may alleviate symptoms, persistent inflammation from nucleus pulposus-induced neural compression frequently limits their long-term efficacy [4].

Traditional Chinese medicine treatment methods for LDH are diverse, continuous and non-invasive. Over the past 10 years, there have been an increasing number of prospective studies on electroacupuncture (EA) in the treatment of soft tissue injuries and nerve damage, confirming that EA can activate the patient’s nervous system and improve pain sensation in situations of pain [5, 6]. Studies have shown that EA can cause rhythmic contraction of muscles in diseased areas through regular pulses of electrical stimulation, thereby improving the treatment effect [7]. EA in BL40 point for the treatment of low back pain has been widely used in clinical practice [8]. Studies have shown that EA at BL40 point can improve low back pain and/or leg pain, degree of disability, range of lumbar motion and quality of life in patients with LDH [9]. However, the mechanism of EA at BL40 point on LDH needs to be further explored.

The gut microbiota is a complex ecosystem composed of various microorganisms. The gut microbiota plays a beneficial role in many physiological processes of the host [10]. It extracts energy and nutrients from food, prevents intestinal pathogens, and supports the development and maintenance of the host’s immune system. An imbalance in the gut microbiota triggers an inflammatory response that spreads from the gut to the intervertebral disc and is associated with LDH [11]. One study showed thatL.paracasei S16can improve symptoms in LDH rat by regulating inflammatory responses, reshaping intestinal flora structure, and influencing metabolic pathways [12]. Other studies have explored the mechanism of action of palmitic acid (PA) and trans-4-hydroxy-3-methoxycinnamic acid (THMC) in LDH through fecal microbiota transplantation (FMT). PA and THMC were found to relieve inflammation and pain by reshaping intestinal flora and restoring metabolic homeostasis after lumbar disc herniation, thereby activating autophagy and Wnt/β-catenin pathways, providing a new target for clinical LDH treatment [13]. In addition, several recent studies have shown that the therapeutic effects of EA are significantly correlated with regulation of the gut microbiota [14–16]. Therefore, in this study, we investigated whether the mechanism by which EA improves LDH is related to the regulation of gut microbiota in LDH rats.

Materials and methods

Animals

18 SPF female SD rats were purchased from Spfford (Beijing) Biotechnology Co., Ltd., license number: SCXK(Beijing)2024-0001. They were raised at room temperature of 20–26℃, relative humidity of 40%−70%, and 12 h of alternating light and dark environment. They were well ventilated, fed on pellet feed, and drank water ad libitum. After one week of adaptive feeding, rats were randomly divided into the following three groups (n = 6): control group (CON), lumbar disc herniation group (LDH), and EA group (LDH + EA treatment).

LDH rat model

The LDH rat model was established according to the methods described in the referenced literature [17]. One week after the rats were acclimated, the modeling process began. After inhalation of 3% isoflurane for anesthesia, the nucleus pulposus was extracted. The tail of the rat was tightly bound with an elastic band at the tail root, disinfected with iodophor, and then cut about 1 cm away from the root. The tail was sutured and pressed to stop the bleeding to prevent excessive bleeding after tail amputation. The cut rat tail was gently touched with the hand, and a small knife was used to quickly cut open the middle depression of the tail vertebra. After exposing the intervertebral disc, a transparent gel-like nucleus pulposus located in the middle was clamped with small curved forceps. Each rat was approximately 5 nuclei pulposus extracted. During the extraction, avoid excessive force to squeeze the veins on both sides of the rat tail. If blood flows out and contaminates the nucleus pulposus, re-extract the nucleus pulposus. Prepare the autologous nucleus pulposus suspension for the rat: take about 50 mg of the nucleus pulposus and mix it thoroughly with 50 µl of normal saline to form a suspension. Use a 1 ml syringe to aspirate it and store it in a−4℃ refrigerator for later use. Epidural injection: When the rat’s pain threshold recovers, inhalation of 3% isoflurane for anesthesia is followed by maintaining anesthesia with 1% isoflurane. The rat’s back is shaved using a hair removal machine. The rat is placed on the horizontal side of a cylindrical mineral water bottle, in a flexed lateral recumbent position, to fully expose the vertebrae, while widening the intervertebral foramina of the vertebrae. Locate the vertebrae by touching the transverse processes of the lumbar vertebrae, with the longest one being the fifth transverse process, the one before it being the fourth lumbar vertebra, and puncture through the intervertebral foramina of L4-5. Stop the puncture when there is a sense of emptiness after penetrating the ligamentum flavum, indicating that the epidural space has been reached. After successful puncture, inject 20 µl of the warmed autologous nucleus pulposus suspension at room temperature, followed by 30 µl of 2% lidocaine for mold verification. In the control group, sham operation (such as laminar puncture + injection of normal saline) was performed to eliminate the influence of the surgical trauma itself.

Electroacupuncture (EA) treatment at BL40 point

The rats were fixed on a specially made fixture, exposing the hind limbs. A Huatuo brand disposable acupuncture needle of 0.30 mm× 13 mm was used. After acupuncture, it was connected to a Hanshi electroacupuncture instrument HANS-100 A. The compression-density wave of 2/15Hz and the current intensity was 2 mA for 30 min, once a day, and the intervention lasted for 7 consecutive days. The BL40 point is located in the depression directly behind the knee joint according to the commonly used animal acupuncture points and atlas in Experimental Acupuncture and Moxibustion. The model group was also fixed in a fixator, but no acupuncture intervention was performed.

HE staining

The rats were sacrificed after dislocation, and the L4-L5 intervertebral disc tissues of the rats were isolated and fixed in 4% paraformaldehyde for 1 day, embedded in paraffin, and cut into 3 μm thick slices with a microtome. The slices were dewaxed in xylene and dehydrated in gradient ethanol. Wash sections with PBS for 5 min. Sections were stained in hematoxylin and eosin solutions for 3 min respectively. After fully rinsing with PBS, dry and seal the film. The stained images were observed under a light microscope and disc tissue damage was evaluated.

Safranin O-fast green (SO-FG) staining

L4-L5 intervertebral disc tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 micron thick sections. Bone histopathological observation was performed using safranin O-fast green staining to reflect the structure of articular cartilage, subchondral bone and bone tissue. Next, the sections were stained with 0.02% malachite green and then with 0.1% safranin O solution. The stained images were observed under a light microscope.

Enzyme linked immunosorbent assay (ELISA)

The ELISA kits utilized for evaluation of supernatant of homogenized intervertebral disc tissue included rat IFN-γ (JYM0654Ra, Jiyinmei, Wuhan, China), rat IL-1β (JYM0419Ra, Jiyinmei) and rat TNF-α (JYM0635Ra, Jiyinmei).

Western blot (WB)

Intervertebral disc tissues were homogenized in liquid nitrogen and lysed in RIPA buffer on ice for 30 min. The supernatant was collected by centrifugation. Protein concentration was determined using the BCA method. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk at room temperature for 1 h and subsequently incubated with the following primary antibodies: Aggrecan (1:1000, DF7561, Affinity), SOX9 (1:1000, AF6330, Affinity), Collagen II (1:500, AF0135, Affinity), MMP13 (1:1000, AF5355, Affinity), and β-actin (1:1000, AF7018, Affinity). The membranes were then incubated with the appropriate secondary antibody, HRP-conjugated goat anti-rabbit IgG (1:500, Affinity) for 1 h. β-actin served as the internal control protein. Protein bands were visualized using a gel imaging analyzer, and quantitative analysis was performed using ImagePro Plus 6.0 software.

Immunohistochemistry

The intervertebral disc tissues were fixed with 4% paraformaldehyde and decalcified in EDTA solution for one week. After embedding and sectioning (4 μm), the samples were treated with 3% hydrogen peroxide for 20 min, followed by blocking with 5% BSA for 20 min. Primary antibodies against Collagen II (1:200, AF0135, Affinity) and MMP13 (1:200, AF5355, Affinity) were incubated at 4 °C, followed by incubation with secondary antibody (1:500) at 37 °C for 1 h. Nuclei were stained with DAB solution, and counterstaining was performed with hematoxylin. The sections were cleared with xylene, mounted with neutral balsam, and examined under a microscope. Positive expression was analyzed using ImageJ software.

Gut Microbiome analysis

Fecal samples from the CON, LDH, and EA groups were collected, and genomic DNA was extracted using the Fecal Genomic DNA Extraction Kit (D2700, Solarbio). DNA quantification was performed using Nanodrop, and the quality of DNA extraction was assessed via 1.2% agarose gel electrophoresis. The 16 S V3-V4 region was amplified using primers (Nobar_341F-CCTACGGGNGGCWGCAG; Nobar_805R-GACTACHVGGGTATCTAATCC). The PCR products were recovered through 2% agarose gel electrophoresis, and their concentrations were measured using the Qubit 3.0 Fluorometer. Sequencing of the library was conducted on the NovaSeq 6000 platform using the SP-Xp (PE250) paired-end sequencing strategy, followed by bioinformatics analysis. Alpha diversity was measured based on species richness derived from the rarefied OTU table. Beta diversity was estimated by calculating the Bray-Curtis dissimilarity and visualized through principal coordinate analysis.

Statistical analysis

The data analysis was carried out using GraphPad Prism 9.5 (GraphPad Software, Inc., La Jolla, CA, USA). Quantitative results are presented as mean ± standard deviation (M ± SD). For comparisons across multiple groups, one-way analysis of variance (ANOVA) was employed, followed by pairwise comparisons using Tukey’s post hoc test. A P-value less than 0.05 was considered to indicate statistical significance.

Results

EA improves pathological damage of intervertebral discs in LDH rats

HE staining results clearly demonstrated the shape and structural characteristics of the intervertebral discs in each group. In the CON group, the intervertebral disc structure was complete; while in the LDH group, the intervertebral disc tissue showed loose structure and disorganized arrangement. However, after receiving EA treatment, the tissue structure and morphology of the disc were significantly improved (Fig. 1C-D). In addition, the results of SO-FG staining were consistent with HE staining (Fig. 1C-D). Together, the above results demonstrate that EA can effectively alleviate histopathological degeneration of the intervertebral disc.

Fig. 1.

Fig. 1

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Electroacupuncture (EA) improves pathological damage of intervertebral discs in LDH rats. A Schematic diagram of LDH rat model construction. B The study protocol of EA treatment in LDH rats in experiment. C The results of HE and SO-FG staining. D The HE scores. E The SO-FG scores. *P < 0.05, n = 6

EA alleviates the inflammatory response in LDH rats

The levels of inflammatory factors IFN-γ, IL-1β and TNF-α in serum of rats in each group were detected by ELISA. Compared with CON group, the serum IFN-γ, IL-1β and TNF-α contents of LDH group rats were significantly increased, indicating that LDH rats had inflammation. Compared with the LDH group, the serum IFN-γ, IL-1β and TNF-α contents of rats in EA group were significantly reduced, indicating that EA reduced the inflammatory response in LDH rats (Fig. 2A-C).

Fig. 2.

Fig. 2

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EA alleviates the inflammatory response in LDH rats. A The level of IFN-γ in the serum of each group. B The level of IL-1β in the serum of each group. C The level of TNF-α in the serum of each group. *P < 0.05, n = 6

EA maintains the ECM homeostasis of intervertebral disc tissue in LDH rats

The essence of intervertebral disc degeneration is the destruction of ECM homeostasis, which is manifested by catabolic significantly exceeding anabolic metabolism. Aggrecan, SOX9, Collagen II and MMP13 are key molecules involved in the synthesis, maintenance and degradation of extracellular matrix. Among them, Aggrecan, SOX9, and Collagen II are involved in the synthesis of extracellular matrix, while MMP13 is involved in the degradation of extracellular matrix. The expressions of Aggrecan, SOX9, Collagen II and MMP13 proteins in intervertebral disc tissues of rats in each group were detected by WB. Compared with the CON group, the expression levels of Aggrecan, SOX9, and Collagen II proteins in the LDH group were significantly reduced, while the level of MMP13 protein was significantly increased, indicating that the ECM homeostasis in the LDH group was destroyed. Compared with LDH group, the expression levels of Aggrecan, SOX9, and Collagen II proteins were significantly increased, while the level of MMP13 protein was significantly decreased in the intervertebral disc tissue of rats in EA group (Figs. 3A-E). At the same time, the expression of Collagen II and MMP13 proteins in rat disc tissues in each group was detected by immunohistochemistry, and the results were consistent with the WB test results (Figs. 4A-C). These results suggest that EA can maintain ECM homeostasis in LDH rat intervertebral disc tissues.

Fig. 3.

Fig. 3

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EA maintains the ECM homeostasis of intervertebral disc tissue in LDH rats. A The protein expression of Aggrecan, SOX9, Collagen II and MMP13 in the intervertebral disc tissue of each group. β-actin was used as an internal control. B-E The relative protein level of Aggrecan, SOX9, Collagen II and MMP13 in each group. *P < 0.05, n = 6

Fig. 4.

Fig. 4

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Immunohistochemical detection of the protein levels of Collagen II and MMP13 in intervertebral disc tissues of rats in each group. A The protein expression of Collagen II and MMP13 in the intervertebral disc tissue of each group. B, C The relative protein level of Collagen II and MMP13 in each group. *P < 0.05, n = 6

Analysis of the characteristics of differential gut microbiota in feces of rats in each group

Changes in gut microbiota of rats in each group were detected by 16srRNA sequencing. Venn plot was used to screen the unique OTUs/ASVs in each group and the common OTUs/ASVs between groups. The results show (Fig. 5A) that there are 252 common species in the three groups of samples. Alpha diversity analysis results showed that the Chao1 index of rats in the LDH group decreased, but there were no significant changes in Shannon, Simpson and Goods_species indices between groups (Fig. 5B). β diversity indicators represented by principal component analysis (PCA) and principal coordinate analysis (PCoA) showed that there were differences in the gut microbiota among the three groups (Fig. 5C-D). LEfSe analysis was conducted to identify the dominant species in each sample group, and the top 10 species with the lowest P-values (all selected if fewer than 10) were selected for bar chart visualization. The results show (Fig. 5E) that the dominant species in the CON group are g_Clostridia_UCG_014_genus.s_uncultured_bacterium, g_Prevotellaceae_genus.s_uncultured_bacterium, s_human_gut, g_Eubacterium_ruminantium_group.s_uncultured_bacterium, s_Clostridium leptum, s_uncultured_Clostridiaceae, g_Lachnospiraceae_NC2004_group and s_uncultured_Bacteroidales; The dominant species in the LDH group are s_Clostridiales_bacterium, g_Lachnospiraceae_UCG_010, g_Lachnospiraceae_UCG_010.s_uncultured_bacterium, s_Ruminococcus_sp, s_Alistipes_finegoldii, s_Christensenella_massiliensis, g_CAG_196.s_uncultured_bacterium, g_Christensenella g_Flavonifractor and s_uncultured_marine༛The dominant species in the EA group are s_uncultured_Lachnospiraceae and g_Monoglobus.s uncultured bacterium. The KEGG pathways predicted from the metagenomic sequences were analyzed by PICRUSt2, and the results are shown in Fig. 5F.

Fig. 5.

Fig. 5

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The 16 s rRNA sequencing results of intestinal microorganisms in the feces of rats in each group. A Venn diagram of OTU/ASV distribution comparison. B α-Diversity measured by Chao1, Shannon, Simpson and Goods_species. C, D PCA, PCoA of β-diversity, based on weighted UniFrac distances. E Mean relative abundance of the genera that showed significant differences between CON, LDH and EA mice by LDA coupled with effect size measurements; F Heatmap of the KEGG analysis results. *P < 0.05, n = 3

Figure6A shows the changes in the abundance of intestinal microorganisms at the Genus level in each group of samples. The Kruskal-Wallis test is a non-parametric test method commonly used to analyze whether there are differences in the overall distribution between multiple groups of samples. It does not make special assumptions about the data distribution and is suitable for a variety of data distribution situations. The Kruskal-Wallis test was used to compare more than 3 groups of intergroup samples to find species with significant differences between groups at each classification level. The Kruskal-Wallis test was then used to analyze species differences between groups at the genus level. At the genus level, compared to the CON group, the abundance levels of Prevotellaceae_genus, RF39_genus, and Coriobacteriales_genus were decreased in the LDH group (Figs. 6C, H, and J), while the abundance levels of Flavonifractor, Lachnospiraceae_UCG-010, Christensenella, Fournierella, Prevotellaceae_Ga6A1_group, Eubacterium, and Holdemania were increased (Figs. 4B, D-G, I, and K). In contrast to the LDH group, the EA group exhibited significantly elevated abundance levels of RF39_genus and Coriobacteriales_genus (Figs. 6C and J), whereas the abundance levels of Flavonifractor, Lachnospiraceae_UCG-010, Christensenella, Fournierella, Prevotellaceae_Ga6A1_group, Eubacterium, and Holdemania were markedly reduced (Figs. 4B, D-G, I, and K).

Fig. 6.

Fig. 6

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The Kruskal-Wallis test was used to analyze the differences in species among groups at the genus level. A Bar charts showing the abundance changes of gut microbiota at the genus level in each group of samples. CON: A1-A3; LDH: B1-B3; EA: C1-C3. B-K The abundances of Prevotellaceae_genus, RF39_genus, Coriobacteriales_genus, Flavonifractor, Lachnospiraceae_UCG-010, Christensenella, Fournierella, Prevotellaceae_Ga6A1_group, Eubacterium and Holdemania at the genus level. *P < 0.05

To further elucidate the association between gut microbiota and the progression of LDH, we performed a Pearson correlation analysis to evaluate the relationships between the relative abundances of Christensenella and RF39_genus and the protein expression levels of Aggrecan, SOX9, Collagen II, and MMP13, as well as the concentrations of the inflammatory cytokines TNFα and IL-1β. The analysis revealed that Christensenella abundance was positively correlated with the protein levels of Aggrecan, SOX9, and Collagen II, as well as with TNFα and IL-1β levels, but negatively correlated with MMP13 protein expression (Fig. 7A and C, and E). In contrast, RF39_genus abundance was negatively associated with Aggrecan, SOX9, and Collagen II protein levels and exhibited inverse correlations with TNFα and IL-1β levels, while showing a positive correlation with MMP13 expression (Fig. 7B and D, and F).

Fig. 7.

Fig. 7

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The correlation between the abundances of the Christensenella and RF39_genus communities and the protein expressions of Aggrecan, SOX9, Collagen II and MMP13, as well as the contents of TNF-α and IL-1β. AChristensenella and Aggrecan, SOX9. BRF39_genus and Aggrecan, SOX9. CChristensenella and Collagen II, MMP13. DRF39_genus and Collagen II, MMP13. EChristensenella and TNF-α, IL-1β. F RF39_genus and TNF-α, IL-1β

Discussion

In this study, we employed 16 s rDNA sequencing of fecal microbiota to investigate alterations in the gut microbiome and the potential mechanisms underlying EA-mediated LDH. Our findings demonstrate that EA exerts an influence on LDH and induces changes in the gut microbiota of LDH-afflicted rat, suggesting that EA may contribute to the amelioration of LDH. These results indicate that the gut microbiota could serve as a potential therapeutic target.

Extensive research indicates that EA intervention can ameliorate symptoms of LDH. A systematic review demonstrates that EA therapy reduces serum levels of inflammatory factors and alleviates pain in patients with LDH [18]. A randomized controlled trial assessing the efficacy and safety of EA for LDH patients revealed that this intervention has been supported by preclinical studies and represents a promising alternative treatment for LDH [19]. In the present study, EA at the BL40 point was found to significantly improve histopathological damage in intervertebral disc tissues of LDH rats and reduce serum concentrations of the inflammatory cytokines IFN-γ, IL-1β, and TNF-α. These findings align with previous research, suggesting that EA at the BL40 point may serve as an effective therapeutic strategy for LDH.

The dynamic balance of extracellular matrix (ECM) synthesis and degradation is the core basis for maintaining the normal structure, hydration state and biomechanical function of the disc [20]. Aggrecan is the most important proteoglycan core protein in the nucleus pulposus of the intervertebral disc, carrying a large number of negatively charged chondroitin sulfate glycosaminoglycan (GAG) chains [21]. It attracts a large number of water molecules, giving the disc the ability to absorb shock and maintain the expansion pressure of the nucleus pulposus[22]. Collagen II is the main collagen type of intervertebral disc extracellular matrix, providing tensile strength and structural support [23]. SOX9 is a key transcription factor that is critical for chondrocyte differentiation and maintenance. In intervertebral discs, SOX9 directly regulates the expression of Aggrecan and Collagen II genes [23, 24]. MMP13 (collagenase-3) is one of the most effective enzymes for degrading Collagen II [25]. The increase in MMP13 activity is a key factor leading to the degradation and destruction of Aggrecan and Collagen II, directly driving the catabolic metabolism of disc matrix [26]. In this study, we found that the expression of Aggrecan, SOX9 and Collagen II was significantly reduced and the expression of MMP13 was significantly increased in LDH rat disc tissue, indicating that ECM homeostasis was disrupted in LDH rat disc tissue. However, EA intervention reversed the changes in these protein expression, indicating that EA intervention can reshape ECM homeostasis in LDH rat discs.

Studies have shown that the gut microbiota not only has diverse and exquisite functions, but also plays an indispensable and important role in the occurrence and development of LDH [27–29]. For example, Rajasekaran et al. performed 16SrRNA sequencing on 24 lumbar disc samples and found that there were significant differences in the diversity and abundance of bacteria in healthy and diseased discs. There were more protective bacteria in healthy discs (includingSphingonas, Herbaspirillum, Devosia, Lentibacillus, Planomicrobium and Virgibacillus), while pathogenic bacteria were the dominant ones in degenerative and herniated discs (including Pseudomonas, etc.) [28]. Another study analyzed the microbial structure of disc tissue from 99 patients with degenerative disc disease using mNGS technology and found that hypoxic infections may be involved in disc degeneration, and that this occult infection is more diverse than previously thought [30]. Dysfunction of the gut microbiota may disrupt the homeostasis of disc cells and promote degenerative changes through immune regulation, bacterial transfer and colonization, and decomposition and absorption of intestinal metabolites [31]. For example, colonization ofL.paracasei S16in a mouse model of intervertebral disc degeneration (IDD) promoted the proliferation of intervertebral disc cells, inhibited apoptosis, and significantly improved the behavioral score of IDD mice, showing a significant therapeutic effect compared to the non-intervention group. In addition, this treatment effectively alleviated abnormal inflammatory responses in IDD mice [12]. In the IDD rat model, FMT treatment by gavage of a fecal suspension not only significantly reduced pathological damage to disc tissue, reduced the secretion of inflammatory factors such as TNF-α, IL-1β, IL-6, etc., but also maintained the stability of disc cells [32]. In addition, the Mendelian randomized study also found a causal relationship between specific gut microbiota and disc degeneration, which further supports the existence of a gut-disc axis [33, 34]. In this study, we found that OTU/ASV abundance in the gut microbiota was significantly reduced in the LDH group compared to the CON group. At the same time, EA treatment inhibited the decrease in OTU/ASV abundance in the gut microbiota of LDH rats. This suggests that EA treatment can restore the diversity of intestinal microorganisms in LDH rats.

Our study showed that changes occurred in the gut microbiota of LDH rats. Related studies have shown that the abundance of Christensenellais significantly increased in sciatica mice [35]. Our results also showed thatChristensenella abundance was also significantly increased in LDH rats, and EA treatment reduced Christensenella genus abundance. On the other hand, EA treatment can regulate the abundance of other flora in LDH rats. The abundance of Flavonifractor, Lachnospiraceae_UCG-010, Fournierella, Prevotellacea_Ga6A1_group, Eubacterium and Holdemania increased in the LDH rat group, but the abundance of these bacterial groups was significantly down-regulated after EA treatment. The abundance of RF39_genus and Coriobacteria_genus decreased in the LDH rat group, but the abundance of these bacterial groups increased significantly after EA treatment. Flavonifractor is closely related to inflammation. In rats with post-inflammatory irritable bowel syndrome, the abundance of Flavonifractor is significantly increased, while EA intervention can reduce the abundance of Flavonifractorin rats with post-inflammatory irritable bowel syndrome [36]. TheLachnospiraceae_UCG-010 genusproduces short chain fatty acids (SCFAs) and is associated with Alzheimer’s disease [37]. ThePrevotellace_Ga6A1_groupgenus is positively correlated with levels of interleukin-1 β, tumor necrosis factor-alpha and interleukin-10, which may mediate pro-inflammatory effects through these inflammatory factors [38]. Some species of the genusEubacterium can convert bile acids and affect immune regulation, and a Mendelian randomized study suggested that Eubacteriummay be associated with disc degeneration [34]. An increase in the abundance ofHoldemania was observed in Parkinson rats, while the abundance of Holdemaniawas significantly down-regulated in Parkinson rats after EA treatment [39].Coriobacteria_genus is involved in estrogen metabolism and maintains immune homeostasis. Reduced abundance may affect the anti-inflammatory response. In rats with nonalcoholic steatohepatitis, the abundance of Coriobacteria_genuswas significantly reduced, and significantly increased after metformin and berberine intervention [40]. This study found that after EA treatment, at the genus level, the abundance ofFlavonifractor, Lachnospiraceae_UCG-010, Fournierella, Prevotellaceae_Ga6A1_group, Eubacterium, and Holdemania in the intestines of LDH rats was significantly reduced, while the abundance of RF39_genus and Coriobacteriales_genus was significantly enriched.

In recent years, accumulating evidence has highlighted a significant association between the gut microbiota and musculoskeletal degenerative disorders. For example, studies have demonstrated that gut microbial dysbiosis is closely linked to chronic low-grade systemic inflammation, a condition that may exacerbate the development of low back pain and intervertebral disc degeneration through its detrimental effects on metabolic and immune homeostasis [41, 42]. In addition, gut microbiota dysregulation may impair central nervous system function, contributing to neuroinflammation and altered pain perception [43]. These effects can influence the onset and progression of musculoskeletal disorders, supporting the existence of a “gut microbiota-joint axis” in skeletal degenerative diseases. However, a critical question remains: how does electroacupuncture stimulation applied to the lumbar and dorsal regions exert remote regulatory effects on gut microbial composition? We propose that this bidirectional gut microbiota-joint axis may be mediated through several interconnected mechanisms. First, electroacupuncture may modulate intestinal motility, secretion, and barrier permeability by restoring balance within the autonomic nervous system—particularly between sympathetic and parasympathetic activity—thereby reshaping the intestinal milieu and altering microbial survival conditions Second, electroacupuncture has been shown to produce robust systemic anti-inflammatory effects; as demonstrated in this study, it reduces serum levels of pro-inflammatory cytokines. This attenuation of circulating inflammatory mediators may directly or indirectly foster an intestinal microenvironment that suppresses the expansion of pro-inflammatory microbial taxa and promotes the restoration of microbial homeostasis[46]. Notably, the significant associations observed in this study between specific bacterial taxa—such as Christensenella and RF39_genus—and markers of intervertebral disc extracellular matrix (ECM) homeostasis and inflammation provide empirical support for these proposed mechanisms. Nevertheless, the precise neuro-immuno-endocrine pathways underlying electroacupuncture-induced modulation of the gut microbiota require further investigation in future studies.Although our research has identified certain microbial communities that appear to influence the efficacy of EA treatment for herniated discs, the specific mechanisms of action of these microbial communities remain unclear. Future research will focus on elucidating these mechanisms, which is crucial for gaining a deeper understanding of the interaction between the microbiome and this disease. Further exploration is needed to investigate the specific mechanisms of action of differential microbes in pain regulation and EA treatment for herniated discs.

Conclusion

To sum up, the results of this study show that EA at BL40 point can significantly improve the pathological damage and inflammatory response of intervertebral disc tissue in LDH rats, and maintain ECM balance. In addition, the effect of EA treatment may be closely related to the diversity and abundance of gut microbiota.

Ethical and ARRIVE statement

The experiments adhered to the Animal Protection and Use Committee of BST Biotechnology Co., Ltd. (Ethics No. BST-PZ-RAT-20241129-01). The study is reported in accordance with ARRIVE guidelines.

Author contributions

Yanbei Chen: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft; Xiuting Cui: Investigation, Data curation, Validation, Writing - review & editing; Zhihong Chen: Resources, Supervision, Project administration; Huilian Shi: Methodology, Validation; Yanfei Qian: Investigation, Visualization; Meifang Yin: Formal analysis, Data curation; Xiaoju Zhu: Conceptualization, Resources, Writing - review & editing, Supervision, Funding acquisition.

Funding

This research was supported by Yunnan Provincial Department of Science and Technology-Yunnan University of traditional Chinese Medicine applied basic research (No.202101AZ070001-228).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Data Availability Statement

No datasets were generated or analysed during the current study.

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