Abstract
Highlights
Pregabalin shows efficacy in diabetic rats, improving mechanical and thermal pain thresholds, indicating its potential for diabetic neuropathic pain treatment.
The expression of circRNA_19601 is significantly altered in diabetic neuropathic pain models, being elevated in the model group and reduced by pregabalin treatment.
Knockdown of circRNA_19601 and its interaction with miR-324-5p influence pain -related proteins and physiological parameters, uncovering a potential regulatory mechanism for diabetic neuropathic pain.
Introduction
Diabetic neuropathic pain (DNP), a prevalent complication of diabetes, is characterized by persistent aching, tingling, or burning sensations, severely affecting patients’ daily activities and mental health.1–3 The pathogenesis of DNP is complex, involving a variety of biochemical and neurobiological changes.4,5 Pathologically, hyperglycemic states initially cause vascular dysfunction in peripheral nerves, leading to nerve ischemia and hypoxia, and ultimately to nerve fiber damage. 6 Additionally, high blood sugar levels can directly damage nerve cells, triggering inflammatory responses and the release of cytokines and chemokines, which can further exacerbate nerve damage and pain. 7
Current treatments strategies for diabetic neuropathic pain primarily include pharmacological and physical therapies. Commonly prescribed medications include antidepressants, anticonvulsants, and opioids, which aim to alleviate pain symptoms.8,9 However, these interventions typically offer only temporary relief and fail to halt the progression of nerve damage. Furthermore, prolonged use of these drugs is associated with various adverse effects, including dependence, tolerance, and systemic complications.10,11
Pregabalin, an anticonvulsant, binds to the α2δ subunit of calcium channels, inhibiting neurotransmitter release and reducing pain signals.12,13 Clinical trials demonstrate its efficacy in alleviating DNP symptoms and improving functional status. 14 Clinical trials demonstrate its efficacy in alleviating DNP symptoms and improving functional status. In addition to diabetic neuropathic pain, pregabalin has gained widespread clinical use for the treatment of postherpetic neuralgia (PHN), a chronic pain condition following herpes zoster. Its mechanism of action involves binding to the α2δ subunit of voltage-gated calcium channels in the central nervous system, thereby reducing the release of neurotransmitters such as glutamate, norepinephrine, and substance P, and ultimately diminishing pain transmission.15,16 Pregabalin’s ability to modulate pain pathways and potentially promote nerve repair makes it a promising treatment option. 17
Circular RNAs (circRNAs) represent a distinct class of non-coding RNAs that play critical roles in the regulation of gene expression. Recent studies have shown that circRNAs are particularly important in neurological diseases, potentially affecting neural function by influencing the activity of miRNAs and participating in protein coding.18–20 In the context of diabetic neuropathic pain, specific circRNAs, such as circ19601 (Chr4: 95149591-95205604, NCBI Reference Sequence: XM_039108695.1), are believed to affect the onset of neuropathic pain by regulating miRNAs like miR-324-5p.21–23 Moreover, transcriptional and post-transcriptional regulation of pain mediators and receptors has been shown to involve circRNAs. For instance, by modulating miR-324-5p expression, circ19601 may influence the activity of voltage-gated ion channels and inflammatory cytokines, both of which are critical drivers of neuronal sensitization in DNP. 24 Our study not only reveals how circ19601 regulates pain-related protein expression by modulating miR-324-5p but also demonstrates its role in regulating mechanical and thermal pain thresholds. Additionally, the research examines the efficacy of pregabalin in alleviating diabetic neuropathic pain, further uncovering the potential role of circ19601 in the modulation of pain mechanisms.
The dorsal root ganglion (DRG) is essential in neuropathic pain, acting as a key center for sensory neurons that relay pain signals from the peripheral nervous system to the central nervous system. Within the DRG, neuronal responses to injury or disease, such as diabetic neuropathy, can become hyperactive, leading to exaggerated pain sensations. 25 miRNAs are key regulators of these processes, as they modulate the expression of genes involved in neuronal excitability and inflammatory responses, thereby influencing pain sensitivity.26–28 Targeting dysregulated miRNAs in the DRG under disease conditions may thus provide new therapeutic opportunities for neuropathic pain.
In light of the complexity of DNP and the constraints of current treatments, there is a pressing need for novel therapeutic strategies. This study focuses on Pregabalin as a positive control drug and aims to screen for potential circRNA therapeutic targets. By investigating the molecular mechanisms underlying the role of circ19601/miR-324-5p in diabetic neuropathic pain, we hope to uncover new avenues for more effective treatment of DNP.
Methods and materials
Animals
Adult Sprague-Dawley rats weighing 220–230 g were used in this study. Animals were housed in a pathogen-free environment under a 12 h light/dark cycle with controlled temperature and humidity. They were allowed free access to food and water and acclimatized for at least 1 week before any experimental procedures. Baseline behavioral assessments were conducted using a double-blind method. The animal grouping Settings were as follows: control group (Veh), model group, and positive drug (pregabalin) group. Each group consisted of six rats from which DRG tissues were collected.
Induction of a diabetic neuropathy rat model and treatment
After a 12 h fasting period, male SD rats (220–230 g) were intraperitoneally injected with streptozotocin (STZ, 35 mg/kg) and simultaneously fed a high-fat diet to accelerate the development of diabetes and neuropathic pain. Following STZ injection, rats received penicillin sodium to prevent infection, bedding was replaced daily, and food and water were provided ad libitum. At week 7 post-STZ injection, pregabalin treatment (50 mg/kg) was initiated in the 9th week and maintained for 12 h. Eight days after treatment, all rats were euthanized. During the study, rat body weight, blood glucose levels, mechanical paw withdrawal threshold (PWT), and thermal paw withdrawal latency (TWL) were measured weekly. 29
Blood glucose levels were measured using a handheld glucose meter with compatible test strips. Select the rat tail vein blood collection, disinfect it with an alcohol cotton ball before blood collection, and after the alcohol evaporates, squeeze the tail to fill the tail vein, pierce it quickly with a disposable blood collection needle, drop a drop of blood in the designated position of the test strip, insert the test strip into the blood glucose meter, and wait for the reading according to the operation prompts. During the process, ensure that the test strip is in good contact with the blood glucose meter.
Knockdown of circ19601 in vivo
To selectively knock down circ19601 expression in the central nervous system of diabetic neuropathy rats, a recombinant adeno-associated virus vector (AAV9-U6-sh-circRNA19601) was constructed by Anhui General Biological Co., Ltd. The shRNA sequence targeting circ19601 was 5′-agatctGGGAGAAAGAGTTCAAATAtacctgacccataTATTTGAACTCTTTCTCCCTTTTTggtacc-3′.
Adult rats were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneally), and the viral suspension (1 × 1012 vg/ml) was intrathecally injected into the L5–L6 intervertebral space at a rate of approximately 1 μl/10 s, with a total injection volume of 10 μl per rat. To enhance transduction efficiency, 4 μg/ml polybrene was added before injection. Fourteen days post-injection, three rats from each group were sacrificed, and dorsal root ganglia were collected to validate knockdown efficiency by RT-qPCR.
Thermal paw withdrawal latency (TWL)
Rats were placed individually on a plexiglass platform within an observation chamber. Once exploratory activity ceased, a radiant heat source was aligned with the plantar surface of the hind paw. Adjust the heat source to achieve a withdrawal latency of 8–15 s for rats with normal heat pain thresholds, setting a maximum exposure time of 15 s to prevent burns. Maintain the platform and ambient temperature at 26 ± 0.5°C. Start the heat exposure when the rat is still, recording the time until the rat lifts its paw. Measure each foot three times with 10-min intervals, and calculate the average latency.
Mechanical paw withdrawal threshold (PWT)
Determine the mechanical withdrawal threshold using Von Frey filaments on the rat’s hind paws. Place the rat on a metal mesh covered with a plexiglass box. After a 30-min acclimation period, apply increasing intensities of filaments (2–26 g) vertically to the central area of the hind paw, pressing for 6–9 s. Record a positive response (paw lift) as “X” and proceed to the next filament if there is no response. Repeat this three times per hind paw with 10-min intervals and calculate the threshold.
Omics data analysis and bioinformatics analysis
The locally conducted searches for the sequences of the identified genes with differential expression were carried out using InterProScan to pinpoint analogous sequences. Following this, the Gene Ontology (GO) terms were plotted, and the sequences received annotation through the Blast2GO software application. Proteins under investigation were processed through BLAST searches in the Kyoto Encyclopedia of Genes and Genomes (KEGG) online repository (accessible at https://www.genome.jp/kegg/) to acquire their KEGG annotations, which were then aligned with the pathways found within the KEGG database. Protein-Protein Interaction (PPI) data for these proteins were sourced from the IntAct molecular interaction database by utilizing their gene symbols or via the STRING application. The acquired data in XGMML file format were transferred into Cytoscape for graphical representation and for an in-depth examination of the functional PPI networks.
ELISA
The concentrations of substance P (catalog number ab288318, sourced from Abcam, United Kingdom), calcitonin gene-related peptide (CGRP; identified by MBS267126, obtained from MyBioSource), norepinephrine (NE; listed as ab287789, Abcam, UK), and cortisol (referred to as Cor; with the product code ab108665, from Abcam, UK) within rat serum were assayed utilizing ELISA kits as per the provided instructions. The absorbance readings were obtained at 450 nm with a microplate reader (3550 model by Bio-Rad), and a calibration curve was generated from the standard wells to determine the levels of the respective markers present in the sample wells.
Cell culture and treatment
The rat dorsal root ganglion (DRG) neurons were purchased from Shanghai Yaji Biotechnology Co., Ltd. (Catalog No. H10995). The neurons were cultured in a humidified atmosphere at 37°C with 5% CO₂. The recommended culture medium was Neurobasal™ medium supplemented with B27 and Glutamax. Cells were passaged every 2–3 days and maintained at a density of approximately 1 × 10⁶ cells/ml to ensure optimal growth conditions. The cell grouping was set as follows: Control group, HG, HG+sh-NC, HG+sh-circ19601, HG+sh-circ19601+NC inhibitor, HG+sh-circ19601+miR inhibitor.
Dual luciferase assay
Neural cells from the dorsal root ganglion were placed in a 24-well culture plate with a seeding density of roughly half. Initially, these cells were simultaneously transfected with either a wild-type or a mutated vector in conjunction with either miR-324-5p or a negative control, paired with the pRL-TK reporter construct. Subsequently, the DRG cells underwent processing with a dual-luciferase assay system (manufactured by Promega, based in the United States) and the luciferase activity was quantified utilizing a multifunctional microplate reader 24 h post-transfection.
Western blot
The dorsal root ganglia were meticulously dissected on ice and subsequently homogenized in a lysis solution supplemented with a protease inhibitor, PMSF (P7626, Sigma-Aldrich, USA), utilizing a high-frequency ultrasonic disruptor (30% amplitude, 10-s pulses with 5-s intervals, repeated six times; Lichen, Beijing, China). Post-homogenization, the resulting lysates underwent centrifugation at a force of 13,500 ×
Reverse transcription–quantitative polymerase chain reaction (RT–qPCR)
The rat-derived DRG tissues from the control group were finely minced utilizing an advanced tissue homogenizer. Following homogenization, each tissue sample was supplemented with 1 ml of TRIzol reagent for the isolation of comprehensive RNA. Subsequent to RNA conversion, quantitative reverse transcription polymerase chain reaction was carried out using the TaKaRa reverse transcription kit. 31 Following this, cDNA synthesis was carried out with a cDNA synthesis kit (D7168S, Beyotime, Beijing, China). PCR reactions were conducted on the ABI 7900 fluorescence quantitative PCR instrument (ABI, USA). The primer sequences used were as follows: circ19601 (rat): forward 5′-CAGGTTTGTGTGCAAGGTCG-3′ and reverse 5′-TGCTTACATGTCCTGGTGTGA-3′, miR-324-5p (rat): forward 5′-AACAATCGCATCCCCTAGGG-3′ and reverse 5′-GTCGTATCCAGTGCAGGGT-3′, GAPDH (rat): forward 5′-TGTGTCCGTCGTGGATCTGA-3′ and reverse 5′-CCTGCTTCACCACCTTCTTG-3′. The procedure began with an initial denaturation step at 95°C for 10 min. This was followed by 40 cycles consisting of denaturation at 95°C for 10 s, annealing at either 52.8°C or 56.2°C for 15 s, and extension at 72°C for 20 s. A temperature gradient from 72°C to 95°C was employed, increasing in 1°C increments per step, and gain calibration was automatically performed before the first run. GAPDH expression served as an internal control for mRNA quantification using the 2-ΔΔCT method.
Data analysis
Differentially expressed circRNAs (DE-circRNAs) were identified using the DESeq2 package in R. Raw read counts were first normalized to account for library size and sequencing depth. CircRNAs with extremely low expression (average counts <10 across all samples) were filtered out to reduce noise. Statistical significance was determined using the Benjamini–Hochberg method to control the false discovery rate (FDR). CircRNAs with an adjusted
For other experimental data (body weight, blood glucose, behavioral tests, ELISA, etc.), statistical analysis was conducted using Prism and SPSS software. Results are expressed as means ± SD (standard deviation). A
Results
Effects of streptozotocin (STZ) on body weight, blood glucose levels, and pain sensitivity in diabetic rats
Figure 1(a) is the construction flow chart of the animal model. Diabetic rats received a streptozotocin (STZ) injection in week 7. In week 9, the rats were treated with pregabalin for 12 h, and 8 days later, all the rats were euthanized. We then observed changes in body weight, blood glucose levels, paw withdrawal threshold, and thermal withdrawal latency in both control and diabetic rats.
Effects of streptozotocin (STZ) on body weight, blood glucose levels, and pain sensitivity in diabetic rats. (a) Study design: STZ was administered to diabetic rats in week 7, followed by pregabalin treatment in week 9, and sacrifice of rats 8 days later.
In the control group, body weight remained stable and continued to increase. After STZ treatment in week 7, the weight gain in diabetic rats began to slow down. By week 10, the weight of the diabetic rats was significantly lower than that of the control group (
Prior to week 6, there were no statistically significant differences in either paw withdrawal threshold or thermal withdrawal latency between the control and diabetic rats. After STZ treatment in week 7, both the paw withdrawal threshold and thermal withdrawal latency in diabetic rats decreased sharply by week 8. By week 9, these measures were significantly lower in the diabetic rats compared to the control group (
Pregabalin improves mechanical hyperalgesia in diabetic rats
Pregabalin, a neuroregulatory medication, effectively alleviates mechanical hypersensitivity in rats both in the short and long term. Control group rats and diabetic rats received either pregabalin or a solvent control treatment for 12 h. As shown in Figure 2(a), the paw withdrawal threshold in diabetic rats significantly decreased compared to the control group (
The positive drug pregabalin can alleviate mechanical hypersensitivity and thermal hypersensitivity in diabetic rats. (a) Paw withdrawal threshold for 12 h. (b) Analysis of area under the curve (AUC) of paw withdrawal threshold. (c) Thermal withdrawal latency for 12 h. (d) Analysis of AUC of thermal withdrawal latency. (e) Paw withdrawal threshold for 8 days. (f) Analysis of AUC of paw withdrawal threshold. (g) Thermal withdrawal latency for 8 days. (h) Analysis of AUC of thermal withdrawal latency.
We also examined the long-term effects of pregabalin. After 8 days of treatment, the paw withdrawal threshold and its area under the curve (AUC) in diabetic rats were significantly reduced (
Pregabalin effectively improves mechanical and thermal sensitivity in diabetic rats in the short term, but mainly improves mechanical sensitivity in the long term.
Transcriptome profile of DRG neurons and identification of circ19601 in diabetic neuropathic pain rats
CircRNAs are a type of RNA molecule with a closed-loop structure, mainly found in the cytoplasm of eukaryotic cells. To investigate circRNA alterations in DRG neurons of diabetic rats, transcriptome analysis was performed. The circRNA distribution across chromosomes revealed that Chromosome 1 contained the highest number of circRNAs (Figure 3(a)). CircRNA lengths varied from hundreds to thousands of base pairs, with the majority ranging from 201 to 300 bp (Figure 3(b)). CircRNA lengths varied from hundreds to thousands of base pairs, with the majority ranging from 201 to 300 bp (Figure 3(b)). Based on their origin and structure, circRNAs were classified into exonic, intronic, and exon-intron types, which are known to regulate gene expression and cell signaling (Figure 3(c)). While these results provided an overview of circRNA characteristics, we further focused on differentially expressed circRNAs associated with DNP.
Circular RNAomics study of DRG neurons in diabetic rat neuropathic pain. (a) Distribution of circular RNA on chromosomes. (b) Length of circular RNA. (c) Types of circular RNA.
In the comparison between the Model group and the Veh group, 844 circRNAs were upregulated and 831 were downregulated. When comparing the Pregabalin group with the Model group, 871 circRNAs were upregulated and 829 were downregulated (Figure 4(a)). Among them, 753 differentially expressed circRNAs overlapped between the 2 comparisons (Figure 4(b)). The intersection of Model-vs-Veh_up and Pregabalin-vs-Model_down had 531 genes. Further data screening (removing the data with an expression value of 0 in duplicate samples) revealed two circRNAs (circRNA_19601, circRNA_03894). The intersection of Model-vs-Veh_down and Pregabalin-vs-Model_up had 202 genes, and after further screening, one circRNA (circRNA_17205) was found (Figure 4(c)). The volcano plots demonstrated the distribution of differentially expressed circRNAs (Figure 4(d)), and the clustering heatmap illustrated the expression patterns among groups (Figure 4(e)). Importantly, compared with the Veh group, circRNA_19601 was significantly upregulated in the Model group, whereas pregabalin treatment reversed this increase, leading to a significant reduction in circRNA_19601 expression (Figure 4(f)). These findings suggest that circ19601 may play a pivotal role in the pathogenesis of DNP and could represent a potential target of pregabalin’s therapeutic effect.
Circular RNA expression analysis of DRG neurons in diabetic rat neuropathic pain. (a) Number of differentially expressed circular RNAs. (b) Venn diagram analysis of differentially expressed genes between groups. (c) Venn diagram analysis was used to analyze the number of differentially expressed genes with high or low expression in the comparison between groups. (d) Volcano plot analysis of differentially expressed circRNAs. (e) Heatmap analysis of expression of differentially expressed genes. (f) Expression level of circ19601.
Functional analysis of differentially expressed circRNAs
To further explore the potential biological roles of differentially expressed circRNAs, we performed functional enrichment analyses for the two comparison groups (Veh vs Model and Model vs Pregabalin). GO functional analysis classified these circRNAs into biological processes, molecular functions, and cellular components, providing insight into their potential involvement in regulatory networks, signaling pathways, and DNP mechanisms (Figure 5(a)). Functional analysis of the top 30 go of the total differentially expressed circRNAs in Veh versus Model and Model versus Pregabalin (Figure 5(b)). Similarly, KEGG pathway enrichment analyses were carried out for both comparisons (Figure 5(c)), and the top 20 enriched KEGG pathways were identified (Figure 5(d)). For the Veh versus Model comparison, the most significantly enriched pathways were regulation of actin cytoskeleton, endocytosis, and Ras signaling pathway. For the Model versus Pregabalin comparison, the most significantly enriched pathways were non-homologous end-joining, nucleocytoplasmic transport, and chemical carcinogenesis.
Functional analysis of differentially expressed circRNAs. (a) GO functional analysis of total differentially expressed circRNAs in Veh versus Model and Model versus Pregabalin groups. (b) GO functional analysis of top 30 differentially expressed circRNAs (Veh vs Model and Model vs Pregabalin). (c) KEGG pathway analysis of differentially expressed circRNAs in both comparisons (Veh vs Model and Model vs Pregabalin). (d) Top 20 ranked KEGG pathway in the Veh versus Model and Model versus Pregabalin groups.
Circ19601 binds with rno-miR-324-5p
Constructing an interaction network of the top 300 miRNAs and circRNAs is a multi-step bioinformatics process designed to reveal the regulatory relationships between these molecules. This network analysis aids in understanding gene expression regulation and disease pathogenesis by selecting the top 300 miRNAs based on high expression levels or significant biological relevance from databases (Figure 6(a)). The binding site of circ19601 and rno-miR-324-5p were predicted using the miRanda tool (https://www.bioinformatics.com.cn/local_miranda_miRNA_target_prediction_120; Figure 6(b)). The dual-luciferase reporter assay is employed to investigate the interaction between circ19601 and rno-miR-324-5p by assessing the effect of their binding on luminescence signals. The findings reveal that the presence of rno-miR-324-5p significantly decreases the ratio compared to the control group, indicating that the miRNA effectively binds to circ19601 and may inhibit its expression (Figure 6(C)).
Interaction between miRNA and circRNA. (a) Network of interactions between top 300 miRNAs and circRNAs. (b) Binding site of circ19601 with rno-miR-324-5p. (c) Dual-luciferase assay to detect binding between circ19601 and rno-miR-324-5p.
Knockdown of circ19601 alleviates changes in body weight, blood glucose levels, and mechanical hyperalgesia in diabetic rats
To assess the role of circ19601 in diabetic neuropathy, we measured its expression levels across different treatment groups in a rat DNP model using RT-qPCR. Relative expression levels of circ19601 were significantly increased in the Model group compared to the Veh group (
Knockdown of circ19601 improves diabetic neuropathic pain. (a) Detection of circ19601 expression levels in rat models using RT-qPCR. (b) RT-qPCR analysis of the effect of circ19601 knockdown on miR-324-5p expression. (c) Impact of circ19601 knockdown on rat body weight. (d) Effect of circ19601 knockdown on rat blood glucose levels. (e) Influence of circ19601 knockdown on mechanical withdrawal threshold in rats. (f) Effect of circ19601 knockdown on thermal withdrawal latency in rats. (g) ELISA measurement of levels of pain neurotransmitters SP and CGRP in model serum. (h) ELISA detection of levels of norepinephrine (NE) and cortisol (Cor) in model serum.
Knockdown of circ19601 alleviates changes in neurotransmitter levels, and hormone levels in diabetic rats
Serum levels of neurotransmitters substance P (SP), calcitonin gene-related peptide (CGRP), norepinephrine (NE), and cortisol were analyzed by ELISA. sh-circ19601 significantly decreased serum levels of SP and CGRP in model rats (
Knockdown of circ19601 decreased the levels of pain-related membrane proteins in cells
The expression of circ19601 was increased in the HG-induced cell model (
The knockdown of circ19601 affects pain-related membrane proteins in cells. (a) RT-qPCR detection of circ19601 expression. (b) RT-qPCR detection of miR-324-5p expression. (c) Western blot analysis of changes in expression of pain-related membrane proteins.
MiR-324-5p inhibitor reversed the downregulation of pain-related membrane proteins induced by sh-circ19601 in cells
Then, we further explore the role of miR-324-5p in the expression of pain-related membrane proteins. A significant decrease in miR-324-5p expression was observed in the miR inhibitor group, indicating successful suppression (
Rescue experiment of the interaction between circ19601 and rno-miR-324-5p in pain. (a) RT-qPCR detection of miR-324-5p inhibition efficiency. (b) Western blot analysis of changes in expression of pain-related membrane proteins. (c) Statistical analysis of pain-related membrane protein expression.
Discussion
In this study, we conducted an in-depth investigation of the function of circ19601 in the dorsal root ganglia and its role in DNP, specifically how it regulates pain transmission through miR-324-5p. 32 We noted a marked upregulation of circ19601 in diabetic rat models, which was strongly associated with increased pain sensitivity. Further functional assays confirmed miR-324-5p as a direct target of circ19601, revealing a regulatory network that may provide novel theoretical insights and potential therapeutic targets for the management of DNP.33–35
STZ-induced diabetes led to significant metabolic and sensory changes in rats, including reduced body weight, elevated blood glucose levels, and decreased mechanical and thermal pain thresholds. These findings are consistent with previous research demonstrating STZ’s ability to induce diabetic neuropathy characterized by hyperglycemia and altered pain sensitivity. 36 The observed decrease in body weight and increase in blood glucose following STZ administration corroborate the results of other studies showing similar effects of STZ in diabetic rodent models. 37 Pregabalin treatment for 12 h in diabetic rats resulted in significant improvements in both mechanical hyperalgesia and thermal withdrawal latency, demonstrating its short-term effectiveness in alleviating neuropathic pain. However, after 8 days of treatment, while pregabalin continued to improve mechanical sensitivity, it did not significantly affect thermal pain sensitivity in diabetic rats. This aligns with clinical and preclinical data indicating pregabalin’s efficacy in managing diabetic neuropathic pain by modulating calcium channels and reducing neuronal excitability. 17 The ability of pregabalin to restore both mechanical and thermal pain thresholds supports its role as a valuable therapeutic option for DNP.
Our analysis revealed that circ19601 was significantly upregulated in diabetic rats and that this upregulation was reversed by pregabalin treatment. This observation highlights circ19601 as a potential biomarker for diabetic neuropathy and a target for therapeutic intervention. Previous studies have demonstrated that circRNAs can modulate pain pathways and are involved in neuropathic pain conditions. 38 The interaction network analysis and dual-luciferase assays confirmed that circ19601 binds to miR-324-5p, suggesting a regulatory mechanism where circ19601 may sequester miR-324-5p, thereby affecting its availability to target mRNAs. This interaction underscores the importance of circRNA-miRNA networks in regulating pain pathways. Our findings are consistent with recent reports demonstrating that circRNAs can act as miRNA sponges, influencing the expression of genes involved in pain. 39
Knockdown of circ19601 led to significant improvements in body weight, blood glucose levels, and pain thresholds in diabetic rats. This suggests that circ19601 contributes to the pathology of DNP and that targeting it may ameliorate diabetes-induced sensory deficits. Moreover, the knockdown of circ19601 resulted in increased levels of miR-324-5p, further supporting its role as a negative regulator of miRNA expression. Our results also showed that knocking down circ19601 decreased the serum levels of neurotransmitters and hormones associated with pain, such as SP, CGRP, NE, and cortisol. This finding aligns with studies indicating that circRNA modulation can influence neurotransmitter levels and pain perception. 40 In vitro experiments revealed that circ19601 knockdown led to decreased expression of pain-related membrane proteins such as Nav1.7, TRPV1, and COX-2. These proteins are well-documented contributors to pain signaling and neuropathic pain. 41 The restoration of their levels following miR-324-5p inhibition further reinforces the regulatory role of circ19601 and miR-324-5p in neuropathic pain mechanisms.
Notably, the knockdown of circ19601 in this study was achieved by intrathecal injection of an AAV9-U6-sh-circ19601 vector into the L5–L6 intervertebral space, ensuring selective suppression of circ19601 expression in the dorsal root ganglia rather than systemic knockdown. This localized delivery provides stronger evidence that the observed analgesic effect is directly attributable to modulation of DRG neuronal activity. Moreover, in addition to pain relief, circ19601 knockdown mitigated body weight loss and hyperglycemia in diabetic rats. These systemic improvements may be secondary to reduced chronic pain–induced stress responses and lowered circulating levels of stress-associated hormones (e.g. cortisol, norepinephrine), which are known to exacerbate metabolic dysregulation in diabetes.42–44 Thus, circ19601 may influence not only nociceptive signaling but also neuroendocrine and metabolic pathways, providing a broader therapeutic potential in DNP.
Functional enrichment analysis revealed that the non-homologous end-joining (NHEJ) pathway was significantly enriched in the Model versus Pregabalin comparison. NHEJ is a key DNA double-strand break repair mechanism critical for maintaining genomic stability and preventing neuronal apoptosis.45,46 In diabetic conditions, hyperglycemia and oxidative stress can induce DNA damage in dorsal root ganglion neurons, and dysregulation of NHEJ may exacerbate neuronal injury, contributing to neuropathic pain.47,48
This study investigates how dorsal root ganglion circ19601 regulates DNP via miR-324-5p. In a DNP rat model and high-glucose DRG neuronal cells, circ19601 expression increased significantly, suggesting its role in DNP. Reducing circ19601 levels decreased pain sensitivity, indicating its involvement in pain pathways. Bioinformatics and dual-luciferase assays revealed circ19601 inhibits miR-324-5p, affecting pain-related genes like TRPV1 and Nav1.8. This insight offers a potential new therapeutic target for DNP. However, a limitation of the present study is that only male rats were used; since sex hormones, particularly estrogen, may influence pain mechanisms, future studies should include both sexes to comprehensively assess gender-specific effects.
Footnotes
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