Comparing the Analgesic Effects of Different Frequency EA

The cuff group exhibited significantly reduced PWT and TWL on Day 3 and Day 7 compared to the sham group, indicating successful sciatic nerve cuffing mouse modeling (Fig. 2A, p < 0.001; Fig. 2B, p < 0.05). EA at frequencies of 2 Hz, 100 Hz, and 18 kHz respectively was administered from Day 7 to Day 13 post-modeling. For each 30-minute stimulation session, the mice were maintained under general anesthesia using a 2% isoflurane gas mixture. One hour after stimulation, the PWT was reassessed to gauge the analgesic duration. The antinociceptive effect of 2 Hz EA on mechanically induced pain lasted for 1 h on Day 7 (Fig. 2C, p < 0.05), extended to 2 h on Day 9 (Fig. 2D, p < 0.05), 2 h on Day 11 (Fig. 2E, p < 0.05), and 2 h on Day 13 (Fig. 2F, p < 0.05). The anti-nociceptive duration induced by 100 Hz EA was 1 h on Day 7 (Fig. 2C, p < 0.05), 2 h on Day 9 (Fig. 2D, p < 0.05), 2 h on Day 11 (Fig. 2E, p < 0.05), and 3 h on Day 13 (Fig. 2F, p < 0.05). In contrast, the anti-nociceptive duration induced by 18 kHz EA was 2 h on Day 7 (Fig. 2C, p < 0.01), 3 h on Day 9 (Fig. 2D, p < 0.01), 5 h on Day 11 (Fig. 2E, p < 0.05), and 6 h on Day 13 (Fig. 2F, p < 0.05).

Fig. 2

Assessment of PWT and TWL before and after EA. The PWT(A) and TWL(B) of mice in the cuff group were notably lower than those in the sham group on Day 3 and Day 7 before EA. From Day 7 to Day 13, 2 Hz EA reduced mechanical pain for 1 h, followed by 2 h, another 2 h, and then 2 h. Similarly, 100 Hz EA reduced mechanical pain for 1 h, followed by 2 h, another 2 h, and then 3 h. Moreover, 18 kHz EA significantly alleviated mechanical pain for 2 h, 3 h, 5 h, and 6 h (C-F). All value represents the mean (n = 10; ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to the Sham group; ^p < 0.05, ^^p < 0.01, &p < 0.05, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 versus the cuff group

The results revealed that the duration of analgesia increased in correlation with the number of days of EA. Notably, the analgesic effect achieved with 2 Hz and 100 Hz EA peaks at approximately 3 h, whereas the intervention using 18 kHz EA extends this analgesic period to 6 h. The analgesic effect of 18 kHz EA exhibited a significant enhancement with the prolongation of EA days, surpassing the effects observed with 2 Hz and 100 Hz stimulations. There was no discernible difference in analgesic effect between 2 Hz and 100 Hz stimulations.

Impact of Varying EA Frequencies on Neuropeptide Release

To elucidate the effects of diverse EA frequencies on neuropeptide secretion within the spinal cord, we conducted an in-depth analysis of endorphin, enkephalin, and dynorphin levels across multiple experimental cohorts. qRT-PCR analysis demonstrated a marked reduction in the mRNA expression of POMC, PENK, and PDYN mRNA in the cuff group relative to the sham group (Fig. 3A, p < 0.001 for POMC; p < 0.01 for PENK; p < 0.001 for PDYN). However, treatment with 2hz EA significantly elevated POMC and PENK mRNA levels in cuff group (Fig. 3A, p < 0.05 for POMC; p < 0.05 for PENK), whereas 100hz EA effectively reversed the inhibitory state of PDYN mRNA (Fig. 3A, p < 0.05 for PDYN). Notably, there were no significant changes observed in POMC, PENK, and PDYN mRNA levels in the 18 kHz EA group when compared to the cuff group (Fig. 3A). Subsequent western blotting was employed to evaluate the protein expression of these neuropeptides in the spinal cord, revealing a pronounced downregulation of POMC, enkephalin and dynorphin A in the cuff group (Fig. 3B-G, p < 0.001 for POMC; p < 0.01 for enkephalin; p < 0.01 for dynorphin A). Treatment with 2 Hz EA increased the POMC and enkephalin protein levels in sciatic nerve cuffing mice (Fig. 3E-F, p < 0.01 for POMC; p < 0.05 for enkephalin), while 100 Hz EA effectively enhanced the expression of dynorphin A (Fig. 3G, p < 0.05 for dynorphin A). However, the 18 kHz EA did not alter the protein levels of POMC, enkephalin and dynorphin A in the spinal cord (Fig. 3B-G). To further validate these results, we utilized immunofluorescence analysis to assess the expression and activation of POMC and dynorphin A. We observed significant inhibition of POMC and dynorphin A in the cuff group (Fig. 4A-D, p < 0.01 for POMC; p < 0.01 for dynorphin A). Treatment with 2 Hz EA led to a notable increase in POMC expression compared to the cuff group (Fig. 4A-B, p < 0.01 for POMC), while 100HzEA resulted in increased expression of dynorphin A (Fig. 4C-D, p < 0.01 for dynorphin A). Notably, 18 kHz EA did not exert any influence on neuropeptide expression, thereby confirming the differential impact of varying EA frequencies on neuropeptide release within the spinal cord (Fig. 4A-D).

Fig. 3

The expression of neuropeptides in the spinal cord after different frequency EA. qRT-PCR was employed to evaluate the mRNA expression of POMC, PENK, and PDTN in the spinal cord. (A) Western blot analysis was performed to assess the protein expression levels of POMC, enkephalin and dynorphin A in the L4-L6 spinal cord. (B-G) All results were expressed as mean ± SEM and were statistically using one-way ANOVA followed by Tukey’s post-hoc test (n = 6). **p < 0.01, ***p < 0.001 compared to the Sham group; ^p < 0.05, ^^p < 0.01, & p < 0.05 compared to the cuff group

Fig. 4

The impact of EA on the expression levels of POMC and dynorphin A in the spinal cord. Immunohistochemical analyses and statistical histogram of the immunofluorescence of POMC and dynorphin A immunoreactive positive neurons with a scale bar set at 20 μm. (A-D) Results were presented as mean ± SEM and were analyzed using one-way ANOVA and Tukey’s post-hoc test (n = 6). Statistical significance was observed as **p < 0.01 compared to the Sham group, ^^p < 0.01 and &&p < 0.01 compared to the cuff group

Influence of Varying EA Frequencies on Inflammatory Cytokines Expression

To clarify the impact of various EA frequencies on the expression of inflammatory cytokines in the spinal cord, we conducted a comprehensive analysis of IL-1β and TNF-α levels. qRT-PCR analysis revealed a significant elevation in mRNA expression of both IL-1β and TNF-α in the cuff group compared to the sham group (Fig. 5A, p < 0.05 for IL-1β; p < 0.001 for TNF-α). Treatment with 2 Hz EA, 100 Hz EA, and 18 kHz EA significantly decreased mRNA levels of IL-1β and TNF-α in the cuff group (Fig. 5A). Subsequently, western blotting was employed to assess protein expression levels of these cytokines within the spinal cord, revealing a notable upregulation of TNF-α and IL-1β in the cuff group (Fig. 5B-E, p < 0.01 for both IL-1β and TNF-α). Moreover, treatment with 2 Hz EA, 100 Hz EA, and 18 kHz EA effectively suppressed protein levels of IL-1β and TNF-α in sciatic nerve cuffing mice (Fig. 5B-E).

Fig. 5

The expression of inflammatory cytokines in the spinal cord after different frequency EA. qRT-PCR was employed to evaluate the mRNA expression of IL-1β and TNF-α in the spinal cord. (A) Western blot analysis was performed to assess the protein expression levels of IL-1β and TNF-α in the L4-L6 spinal cord. (B-E) All results were expressed as mean ± SEM and were statistically using one-way ANOVA followed by Tukey’s post-hoc test (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the Sham group; ^p < 0.05, ^^p < 0.01, & p < 0.05, && p < 0.01, #p < 0.05, ## p < 0.01 compared to the cuff group

Sequencing and Assembly of Spinal Cord Tissue Data

In order to explore the mechanism of 18 kHz EA in relieving neuropathic pain, we conducted RNA-Seq on the sham group, the cuff group, and the 18 kHz EA group. Statistical analysis of three samples from each group revealed that the spinal cord tissue of the sham group yielded 149,692,394 original reads, whereas the cuff group produced 154,682,018 raw reads and the 18 kHz group generated 155,852,958 raw reads. After acquiring the data, each sample’s reads were compared to a reference sequence database using Star software. Subsequently, the alignment status was statistically analyzed with RSEQC. In the sham group, 142,068,444 read segments matched the database, compared to 149,077,817 in the cuff group and 150,198,646 in the 18 kHz group. Table 2 summarized the mapping rates for each sample.

Table 2 The reads mapping rate of each sample in those three groupsAnalysis of the Differentially Expressed Genes (Cuff vs. 18 kHz EA)

We employed the DESeq2 software to analyze differential gene expression between the Cuff and 18 kHz EA groups, resulting in the detection of a total of 476 genes exhibiting varied expression patterns. In constructing the volcano plot, each data point represented an individual gene, with log2FC values depicted on the x-axis. We defined |log2FC| ≥ 1 as the threshold for significant differences in gene expression and observed that compared to the cuff group, there were 216 significantly upregulated genes (≥ 1) in the 18 kHz EA group, along with 260 significantly downregulated genes (≤-1). The y-axis represented -log10(P-Value), where genes were considered significantly different when P-Value was ≤ 0.05 (Fig. 6A). Additionally, we performed bidirectional hierarchical clustering on differentially expressed genes across samples and visualized these results using a heatmap (Fig. 6B).

Fig. 6

The upregulated and downregulated genes. Setting p < 0.05 and |log2FC| ≥ 1.0 as cutoff values

Investigation of Differential Gene Ontology Functions and KEGG Pathway Enrichment

GO encompasses molecular function (MF), biological process (BP), and cellular component (CC). In comparison to the cuff group, the 18 kHz group exhibited differential expression of 347 genes in the BP group, 36 genes in the CC group, and 84 genes in the MF group. GO enrichment analysis was conducted on the differentially expressed genes from both groups. The results revealed that 18 kHz EA influenced various biological processes such as cellular responses to cytokine stimulation and calcium-mediated signal transduction utilizing intracellular calcium sources. Additionally, it impacted cellular components including cluster of actin-based cell projections and muscle myosin complex, as well as molecular functions like neurotransmitter receptor activity (Fig. 7A). Furthermore, pathway enrichment analysis using the KEGG database indicated that differential gene expression in the 18 kHz group was predominantly enriched in pathways related to serotonergic synapses, G protein-gated Potassium channels, Inflammatory mediator regulation of TRP channels, TNF signaling pathway, voltage-gated Ca2 + channels via G beta/gamma subunits, and cell adhesion molecules (CAMs) pathways (Fig. 7B). The evidence indicated the potential involvement of the serotonin system in mediating the analgesic effects of KHES, and our sequencing findings also spurred further investigation into the role of endogenous spinal 5-HT in KHES within animal models.

Fig. 7

Gene enrichment analysis of the spinal cord concerning Cellular Component (CC), Biological Process (BP) and Molecular Function (MF) in the 18 kHz group as compared with the cuff group. (A) Enriched pathways from KEGG analysis of differentially expressed genes between the cuff group and 18 kHz group. (B)

18 kHz EA Relieving Neuropathic Pain Via the 5-HT Pathway

The qRT-PCR findings indicated a notable decrease in the mRNA levels of 5-HT1A, 5-HT1B, 5-HT3A, 5-HT4A, and 5-HT5A in cuff group (Fig. 8A, p<0.01 for 5-HT1A and 5-HT1B; p<0.05 for 5-HT3A, 5-HT4A, and 5-HT5A), accompanied by markedly reduced mRNA levels of GIRK1, GIRK2 and GIRK4 (Fig. 8B, p<0.01 for GIRK1 and GIRK2; p<0.05 for GIRK4). Additionally, there was a significant rise in the mRNA levels of 5-HT2A and 5-HT2B in cuff group (Fig. 8A, p<0.05 for 5-HT2A and 5-HT2B), accompanied by markedly increased mRNA levels of GIRK3, Cav2.2, CaMKII, and NMDAR2B (Fig. 8B, p<0.05 for GIRK3; Fig. 8C, p<0.01 for Cav2.2; p<0.05 for CaMKII and NMDAR2B). After 18 kHz EA, the mRNA levels of 5-HT1A, GIRK2 and GIRK4 were significantly increased in cuff mice (Fig. 8A, p<0.05 for 5-HT1A; Fig. 8B, p<0.01 for GIRK2; p<0.05 for GIRK4), while the levels of 5-HT2A, 5-HT2B, Cav2.2, CaMKII, and NMDAR2B were significantly decreased (Fig. 8A, p<0.01 for 5-HT2A and 5-HT2B; Fig. 8C, p<0.05 for Cav2.2, CaMKII and NMDAR2B).

Fig. 8

The mRNA levels of some related genes in the spinal cord detected by qRT-PCR. All the results were expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s post hoc tests (n = 6). * p < 0.05, ** p < 0.01 versus the Sham group; # p < 0.05, ##p < 0.01 versus the cuff group