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INTRODUCTION
Neuropathic pain (NP) is a chronic pain disorder that results directly from injury or dysfunction of the somatosensory nervous system [1]. This particular type of pain can be triggered by a variety of pathological factors, such as metabolic abnormalities (e.g., diabetes), pathogenic infections, surgical procedures, primary neurological injury, trauma, stroke, and malignancy [2]. Epidemiological data suggest that the prevalence of this condition is 7%–10% worldwide [3], and it is recognized as one of the most problematic clinical issues in neurological disorders, as it is much more difficult to treat than other types of chronic pain [4]. It is therefore of great clinical importance and scientific value to further elucidate the molecular mechanisms of NP and find effective intervention targets.
It has been reported that non-coding RNAs (ncRNAs), as key regulatory molecules, play an important role in neurological disorders [5]. Recent studies confirm that multiple lncRNAs are involved in the pathological process of NP. For example, MEG3 lncRNA has been shown to exacerbate NP symptoms and induce astrocyte activation via the miR-130a-5p/CXCL12/CXCR4 signaling axis [6]. Distal transcription of HOXA (HOTTIP), an important regulatory molecule of the HOXA gene cluster, exhibits a wide range of biological functions in various diseases [7]. HOTTIP regulates the biological behavior of proliferation and differentiation of synovial fibroblasts in ankylosing spondylitis by modulating the microRNA (miRNA)-30b-3p/PGK1 signaling pathway [8]. Nevertheless, systematic studies on the exact function and mechanism of action of HOTTIP in NP are still lacking.
MiRNAs, another important class of post-transcriptional regulators, play an important role in NP. Among the many miRNAs, miR-216a-5p has attracted much attention for its significant neuroprotective effects. For example, this miRNA can effectively inhibit neuroinflammation caused by microglia activation by targeting the HMGB1-TLR4 (toll-like receptor 4)-NF-κB signaling cascade, thereby attenuating inflammation-related hyperalgesia [9]. These results justify the use of miR-216a-5p as a therapeutic target for inflammatory pain [10]. Bioinformatic methods predicted that there could be complementary binding regions between the long-stranded ncRNA HOTTIP and miR-216a-5p, suggesting that they could form a complex regulatory network via competing endogenous RNA (ceRNA) mechanisms.
Based on this research, this study hypothesized that the HOTTIP might be involved in the pathological process of chronic constriction injury (CCI) of the sciatic nerve. To test this hypothesis, we constructed an in vivo NP model using CCI and analyzed its effect on pain thresholds by regulating HOTTIP expression through intrathecal injection. In addition, we induced BV2 cells with lipopolysaccharide (LPS) and analyzed its effects on cellular inflammation by regulating HOTTIP. The aim was to provide important clues for elucidating a new mechanism of NP.
MATERIALS AND METHODS
1. Ethical review and preparation of experimental animals
Throughout this study, specific-pathogen-free male Sprague-Dawley rats were selected as experimental subjects. The rats used were 6–8 weeks old, with a body weight ranging from 180 to 200 grams. During the experiment, the animals were housed in a temperature-controlled environment maintained at 22°C ± 2°C under a diurnal cycle consisting of equal light and dark periods (12:12), with food and water provided without restriction, and with all husbandry conditions complying with international laboratory animal welfare standards. All animal procedures in this study were approved by the Animal Welfare Committee of the College of Mongolian Medicine, Inner Mongolia Medical University (no. 2023026). This study was carried out in compliance with the ARRIVE (Animal Research Reporting In Vivo Experiment) guidelines.
2. Establishment of a rat chronic constriction injury model
Before formal experiments, all laboratory rats underwent a 1-week acclimation period to the environment. For anesthesia, the animals were deeply anesthetized via intraperitoneal delivery of a 1% sodium pentobarbital solution, with the dose adjusted to 50 mg per kg of body weight. Following an incision in the rat’s thigh, the connective tissue separating the gluteus maximus and the biceps femoris muscles was carefully dissected to fully expose the right sciatic nerve trunk. Once the nerve was completely exposed, four intermittent ligatures were applied using a 5.0 suture thread, with a spacing of 1 mm to 1.5 mm between adjacent ligature points. To verify the effectiveness of the ligatures during the operation, the affected limb was observed for involuntary muscle spasms. After manipulation of the nerve, the muscle tissue and skin incision were closed by layered suturing. Control animals received the same surgical exposure of the nerve but did not undergo ligation. At the end of the experiment, euthanasia was performed via intraperitoneal injection of an overdose of sodium pentobarbital (dose: 120 mg/kg). Finally, dorsal root ganglion (DRG) tissue samples were collected for subsequent experimental studies.
3. Intrathecal administration of lentiviral vectors
After setting up the model in accordance with standard experimental methods, intrathecal injections were delivered. Intrathecal administration involved first implanting a sterile PE-10 catheter into the lumbar spinal cord region of rats. After securing the catheter, the surgical incision was sutured, and the exposed portion of the catheter was protected using a metal closure device. Subsequently, the lentiviral vector was injected into the rats via the microinjection system connected to the catheter, and the rats were randomly assigned to the following groups: the sham group, CCI group (no transfection), CCI + Lv-sh-NC (negative control) group, CCI + Lv-sh-HOTTIP group, CCI + sh-HOTTIP + antagomiR-NC group, and CCI + sh-HOTTIP + antagomiR-216a-5p group.
4. Quantifying pain response thresholds
To quantitatively assess mechanical pain perception, the plantar withdrawal threshold (PWT) was used as a detection indicator. On the day of surgery and 3, 7, and 14 days after surgery, the researchers placed the laboratory rats in a transparent glass box and allowed them to adapt to the environment for 30 minutes under soundproof conditions. Then, a standard calibrated Von Frey filament pain measurement device was used to deliver vertical mechanical stimuli to the plantar surface of the rats’ hind paws, with gradually increasing pressure intensity.
The heat sensitivity test is performed using a paw withdrawal latency (PWL) test. Before the experiment, the rats were placed in a specially designed plastic box and given a 30-minute acclimatization period to their environment. Using a precise temperature-controlled thermal radiation device, the surface of both hind paws was stimulated. To avoid tissue damage to the paw, the experiment was designed with a protective mechanism that automatically stopped the thermal stimulation after 20 seconds. Each test point required repeated measurements every 5 minutes.
To assess the sensitivity of the rats to cold stimuli, we gently applied a 0.1% acetone solution to the underside of the laboratory rats’ paws and recorded the frequency of pain-related behaviors in the laboratory animals (e.g., rapid withdrawal of claws, frequent licking of the stimulated area, involuntary tremors of the limbs, or lifting of the paws).
5. Cell culture and LPS treatment
The BV2 microglial cell line was stored and cultured under standard culture conditions. The culture system for this cell line is composed as follows: Dulbecco’s Modified Eagle’s Medium medium with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics is used, and the cells are cultured under standard conditions inside an incubator set to a stable temperature (37°C, 5% CO₂, saturated humidity). To establish an inflammatory response model, the experimental group was stimulated for 24 hours with 1 μg/mL LPS, while the control group (serum-free medium only) served as the standard control.
6. Cell transfection process
The induced BV2 cells were cultured at a density of 70%–80% and then transfected using Invitrogen Lipofectamine® 2000 reagent (Invitrogen) according to the manufacturer's instructions. Lv-sh-NC, Lv-sh-HOTTIP, NC inhibitor, and miR-216a-5p inhibitor were transfected into BV2 cells. Forty-eight hours after transfection, the cells were collected and used for the following experiments.
7. Cell viability assay
BV2 cells were inoculated at a density of 1 × 10⁴ cells per well in a 96-well culture plate, where the cells were in the exponential growth phase. After 24 hours of adhesion culture, cell survival was assessed using the cell counting kit-8 (CCK-8) cell proliferation assay kit (Solarbio Life Sciences). First, the original culture medium was removed from the plate, and then 100 μL of freshly prepared complete culture medium containing 10% CCK-8 reagent was added to each well. The treated culture plate was transferred to a 37°C incubator and cultured in the dark for 1 hour. Finally, the absorbance at 450 nm was measured for each well using a microplate reader (PerkinElmer).
8. Subcellular fractionation
The nuclear/cytoplasmic fractionation kit (Biovision) was used to successfully extract RNA components from the cytoplasm and nucleus, following the manufacturer’s protocol. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was selected as a marker for the cytoplasmic fraction, while U6 acted as a normalization control for the nuclear component.
9. Enzyme-linked immunosorbent assay
An enzyme-linked immunosorbent assay (ELISA) kit (Beyotime) was used to quantitatively measure the expression levels of inflammatory mediators in the dorsal spinal cord tissue of rats and BV2 cell lines. The inflammatory mediators measured included interleukin (IL)-4, IL-6, and tumor necrosis factor (TNF)-α, and the experiment was performed according to the manufacturer’s instructions.
10. RNA extraction and RT-qPCR
Total RNA was extracted from bone marrow tissue and BV2 cells using the TRIzol kit (Invitrogen), and RNA purity and concentration were measured using the NanoDrop 2000c spectrophotometer (Thermo Scientific). The extracted RNA was converted to cDNA using the Prime Script RT reagent Kit (Takara), followed by polymerase chain reaction (PCR) amplification. GAPDH served as the internal control to normalize the expression of HOTTIP, mRNA, and inflammatory markers, including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (INOS), and TLR4. Meanwhile, U6 was employed as the endogenous reference for miR-216a-5p quantification. The 2−ΔΔCt approach was employed to compute the results.
11. Bioinformatics analysis
Using the lncBook and lncRNAWiki platforms and the DIANA tool, we predicted miRNA molecules that could bind to HOTTIP and selected miR-216a-5p as a potential target. To predict the target genes of this miRNA, we combined three prediction tools, TargetScan, miRDB, and miRWalk, and used an online graphical tool to generate a Venn diagram showing the intersection between the predictions of the different databases. To clarify the biological functions of these target genes, we constructed a protein-protein interaction network from the STRING database and identified the 10 most highly connected hub genes as key regulators.
12. Determination of dual luciferase activity
Oligonucleotide fragments containing potential binding sites for the wild-type (WT) and mutant (MUT) 3'-UTR regions of the HOTTIP and Janus kinase 2 (JAK2) genes were integrated into the pmirGLO reporter vector using molecular cloning techniques. Two recombinant plasmids encoding the WT and MUT HOTTIP and JAK2 genes were successfully constructed. During the cellular transcription stage, the experimental group used Invitrogen’s transfection reagent to transfect the miR-216a-5p mimic and the aforementioned plasmids into cells, while the control group established a NC mimic. After 48 hours, cell samples were collected and lysed. Luciferase activity was measured using Promega’s dual luciferase reporter system.
13. RNA pull-down
As per the directions included in the RNA pull-down detection kit (Thermo Scientific), the experimental steps were implemented. First, biotin-labeled RNA fragments were specifically bound to streptavidin-coated magnetic beads (Thermo Scientific). Meanwhile, biotin-miR-NC was created as a NC. Next, solutions prepared from cells were added to the aforementioned magnetic particle and RNA mixing system and reacted under mild conditions for 1 hour. After the reaction, the magnetic particles were cleaned several times with a special washing solution. Next, the RNA complex bound to the magnetic particles was eluted and collected. Finally, the expression levels of HOTTIP and JAK2 were measured using reverse transcription quantitative PCR (RT-qPCR).
14. Statistical analysis
GraphPad Prism 9.0 software (GraphPad) was used to process and analyze experimental data. All measurement results were expressed as mean ± standard deviation. Comparisons between groups were performed using Student’s t-test and one-way analysis of variance (ANOVA), and multiple comparisons were performed using Tukey’s post-hoc test. Statistical significance was set at P < 0.05.
RESULTS
1. Silence HOTTIP effectively relieves NP in CCI rats
To study the function of HOTTIP in NP in vivo, we surgically constructed a CCI rat model. The results showed that, compared with the sham-operated control group, CCI rats exhibited a significant increase in HOTTIP levels in DRG tissue. This enhanced expression was effectively suppressed by lentiviral shRNA-mediated HOTTIP knockdown (P < 0.0001, Fig. 1A). The experimental results revealed that following CCI surgery, the PWT (P = 0.004, Fig. 1B) and PWL (P < 0.0001, Fig. 1C) of the model group rats were markedly reduced in comparison to the control group, but sensitivity to cold stimulation was markedly enhanced (P < 0.0001, Fig. 1D). Importantly, when HOTTIP expression was downregulated, the rats’ PWT and PWL significantly recovered, and the frequency of cold-induced pain behavior also decreased significantly. In addition, we examined changes in the expression profiles of inflammation-related factors in DRG tissue. CCI surgery upregulates the expression of inflammatory markers like INOS, COX-2, and TLR4, while transfection with Lv-sh-HOTTIP effectively downregulates their expression levels (P < 0.001, Fig. 1E). ELISA results showed that a significant rise in the levels of pro-inflammatory cytokines TNF-α and IL-6 was detected in the CCI model group (P < 0.0001, Fig. 1F); however, the production of the anti-inflammatory factor IL-4 was inhibited (P < 0.001, Fig. 1G). By specifically inhibiting HOTTIP expression, these abnormal changes in the factors were significantly corrected.
2. HOTTIP exhibits direct interaction with miR-216a-5p
Through intracellular localization analysis in the LncLocator database, we observed that HOTTIP is mainly distributed in the cytoplasmic region (Fig. 2A). This prediction was verified by subsequent intracellular fractionation experiments (Fig. 2B). During the screening phase of potential targets, we used a combination of three bioinformatics prediction platforms. The analysis data revealed the presence of two common candidate target genes (Fig. 2C). Through sequence alignment analysis, we identified potential specific base sequence regions between HOTTIP and miR-216a-5p (Fig. 2D). To verify their interaction, we performed a dual luciferase reporter gene assay. The experimental data showed that only the miR-216a-5p mimetic-treated group exhibited significant inhibition of luciferase activity under experimental conditions where the HOTTIP-WT vector was transcribed (P < 0.01, Fig. 2E). Furthermore, RNA pull-down experiments confirmed that biotin-labeled miR-216a-5p specifically binds to the HOTTIP molecule (P < 0.01, Fig. 2F). To further validate whether miR-216a-5p is regulated by HOTTIP, we used gene interference technology to alter the expression level of HOTTIP. RT-qPCR analysis showed that when HOTTIP was interfered with, the expression level of miR-216a-5p exhibited a highly significant upward trend (P < 0.0001, Fig. 2G). These experimental results collectively provide strong evidence that HOTTIP directly targets miR-216a-5p.
3. Inhibiting miR-216a-5p counteracted the suppressive effect of HOTTIP downregulation on NP
To investigate whether HOTTIP knockdown exerts a functional compensatory effect on NP behavior by upregulating miR-216a-5p expression, we first analyzed miR-216a-5p expression in the CCI group using RT-qPCR. The results showed that the expression level of miR-216a-5p was significantly reduced in the CCI group compared to the sham-operated group. After HOTTIP suppression, these expression levels increased significantly, and after transcription of antagomiR-216a-5p, they decreased significantly. This indicates that the inhibitor in question effectively inhibited HOTTIP-induced miR-216a-5p expression (P = 0.003, Fig. 3A). In terms of pain behavior, inhibition of miR-216a-5p reversed the increase in PWL (P = 0.036, Fig. 3B), PWT (P = 0.028, Fig. 3C), and reduction in frequency (P < 0.001, Fig. 3D) observed in CCI rats after HOTTIP inhibition.
4. Suppressing miR-216a-5p substantially diminishes the neuroprotection induced by HOTTIP knockdown
Subsequently, we investigated whether downregulation of HOTTIP expression exerts functional compensation for neural injury in rats by regulating the expression of miR-216a-5p. According to the experimental data, the levels of COX-2, INOS, and TLR4 increased significantly when the expression level of miR-216a-5p was reduced compared to the CCI + Lv-sh-HOTTIP + antagomiR-NC group. This change offset the increase in anti-inflammatory proteins caused by HOTTIP gene silencing in the CCI rats’ model (P < 0.0001, Fig. 4A). Further studies showed that inhibition of miR-216a-5p expression not only suppressed the inhibitory effect of reduced expression of HOTTIP on pro-inflammatory cytokines (P < 0.0001, Fig. 4B) but also weakened the positive regulatory effect on anti-inflammatory factors (P = 0.018, Fig. 4C), thereby significantly aggravating the neuroinflammatory response.
5. The participation of the HOTTIP- miR-216a-5p mechanism in LPS-mediated BV2 cell damage
To further investigate whether the effects of HOTTIP knockdown could be rescued by miR-216a-5p overexpression, we treated LPS-induced BV2 cell lines using recombinant plasmid transcription technology. RT-qPCR results showed that LPS stimulation significantly increased HOTTIP expression in BV2 cells compared to the control group and that the introduction of Lv-sh-HOTTIP effectively inhibited the expression of this gene (P < 0.0001, Fig. 5A). Experimental data also showed that miR-216a-5p inhibitors significantly inhibited the expression of this miRNA (P = 0.008, Fig. 5B). In LPS-treated cell models, a reduction in HOTTIP expression significantly promoted cell proliferation, but this effect was inhibited by the miR-216a-5p inhibitor (P = 0.012, Fig. 5C). Lv-sh-HOTTIP significantly reduced the secretion of COX-2, INOS, and TLR4 in LPS-activated microglial cells, and the miR-216a-5p inhibitor partially reversed this inhibitory effect (P = 0.043, Fig. 5D). In particular, the reduction in inflammatory factors (TNF-α and IL-6) and the increase in the level of the anti-inflammatory factor IL-4, induced by HOTTIP gene silencing under conditions where miR-216a-5p expression was specifically inhibited, were significantly inhibited (P = 0.032, Fig. 5E, F).
6. A direct regulatory interplay exists between JAK2 and miR-216a-5p
Three bioinformatic techniques were utilized to systematically predict the target genes of miR-216a-5p, aiming to investigate its potential function in NP. As shown in Fig. 6A, a total of 101 common target genes were identified by cross-comparison. The results of the protein interaction network analysis for these genes showed that the network consisted of 101 protein molecules and 124 interaction edges (Fig. 6B), and the 10 central genes with the highest connectivity value were highlighted. Fig. 6C shows the specific binding site between miR-216a-5p and JAK2. To verify this prediction, we performed a dual luciferase reporter assay. The data showed that transcription of miR-216a-5p mimetic significantly reduced the fluorescence signal in the WT JAK2 reporter system (P = 0.004, Fig. 6D), confirming a direct regulatory relationship between the two. The results of RNA pull-down experiments also confirmed the binding relationship between miR-216a-5p and JAK2 (P = 0.003, Fig. 6E). In addition, LPS treatment significantly promoted JAK2 expression. Silencing the HOTTIP gene effectively blocked this promoting effect, but when administered simultaneously with the miR-216a-5p inhibitor, the JAK2-inhibiting effect of the HOTTIP defect was partially attenuated (P = 0.028, Fig. 6F).
DISCUSSION
NP is a chronic and difficult-to-treat pain syndrome resulting from damage to the nervous system. It is among the most clinically challenging neurological disorders to treat, with a steadily rising incidence [11]. Although substantial advances have been made in understanding the pathophysiology, existing treatments still show limited efficacy [12,13]. In this context, there is an urgent need to systematically clarify the molecular mechanism underlying this disease and to develop new, highly effective therapeutic strategies.
Recent studies have revealed that lncRNA participates in the NP through several pathways, including the regulation of neuroinflammatory responses, the regulation of oxidative stress levels, the regulation of glial cell activation, and the regulation of autophagy processes [14,15]. For example, five prime to XIST significantly improves NP symptoms and neuroinflammation through the miR-320a/RUNX2 signaling pathway [16]; lncRNA Miat participates in pain regulation through the miR-362-3p/BAMBI pathway [17]; Inhibition of PCAT19 expression inhibits the development and progression of NP via the miR-182-5p/JMJ D1A signaling axis, thereby blocking the progression of NP [18]. LncRNA 4933431K23Rik participates in the pathological process of NP by negatively regulating miR-10a-5p to influence the transformation of microglia after spinal cord injury [19]. These results clearly show that lncRNA plays a central role in the development of neuroinflammation and the maintenance of NP. Clarifying the mechanism of action of lncRNA could provide new targets for intervention in the prevention and treatment of NP. According to existing research, lncRNA HOTTIP is not only closely related to the development of myeloid malignancy but also determines the differentiation direction of neural stem cells [20,21]. In a study on amyotrophic lateral sclerosis using the SOD1-G93A transcription model, abnormal expression of HOTTIP is closely related to neurological developmental disorders and tumor formation [22]. Pang et al. [23] found that HOTTIP was differentially expressed in a bilateral CCI rat model. In this study, we combined the CCI rat model with an LPS-stimulated BV2 cell model to confirm that HOTTIP expression activity was significantly elevated in the DRG tissue of CCI rats and LPS-stimulated BV2 cells. Lun et al. [24] previously reported that downregulation of HOTTIP expression can alleviate neuronal damage. This study confirmed through gene knockout experiments that HOTTIP deficiency could effectively alleviate NP symptoms and reduce neuroinflammatory responses. However, the specific molecular mechanism by which HOTTIP regulates NP requires further investigation.
Research findings have shown that a certain type of lncRNA acts as a ceRNA and can regulate various pathological processes by exploiting miRNA binding sites [25]. Recent research data have shown that miRNA expression disturbances are closely related to the development of various human diseases [26,27], particularly in the context of nerve function regulation and disease progression [28,29]. For example, miR-216a-5p exhibits abnormal expression profiles in various malignant tumors, such as renal cell carcinoma [30], hepatocellular carcinoma [31,32], and respiratory diseases such as bronchopneumonia [33]. Neuroscience studies have shown that this molecule not only relieves NP [34] but also promotes the restoration of nerve function after spinal cord injury to a large extent [35]. In an Alzheimer’s disease model, this miRNA effectively improved cognitive dysfunction and reduced neuroinflammation by regulating the HMGB1/NF-κB signaling pathway [36]. Based on the research data, miR-216a-5p is likely to play an important role in the onset and development of neurological diseases. Liu et al. [37] found that miR-216a-5p was significantly downregulated in NP. This study confirmed that HOTTIP downregulates miR-216a-5p expression by sponging it under NP conditions. Additionally, Xin et al. [38] found that reducing miR-216a-5p exacerbates pain responses in rats. This study demonstrates that blocking the abundance of miR-216a-5p significantly alleviates the reduced cell survival rate and exacerbated neuroinflammation caused by the decreased expression of HOTTIP. Bioinformatics predictions indicate that HOTTIP could contain complementary binding sequences for miR-216a-5p, and subcellular localization analysis shows that HOTTIP is primarily localized in the cytoplasm, suggesting that HOTTIP may function as a molecular sponge for miR-216a-5p. To further elucidate the molecular mechanisms of NP, we conducted functional rescue experiments in both the CCI rat model and the LPS-stimulated cell model. The results showed that knocking down HOTTIP increased miR-216a-5p expression, improved pain behavior, and regulated inflammatory factor expression, while inhibiting miR-216a-5p reversed these effects. These findings suggest that HOTTIP participates in NP progression by targeting miR-216a-5p.
This study still has some limitations. First, the activation status of glial cells has not been directly detected; instead, it has been indirectly inferred through inflammatory factor levels, which may not fully reveal the cell-specific mechanisms of HOTTIP in regulating neuroinflammation. Therefore, future studies plan to use techniques such as immunofluorescence staining to further observe the effects of HOTTIP inhibition on the activation status of glial cells in the CCI model, aiming to more deeply elucidate the molecular mechanisms by which HOTTIP regulates neuroinflammation. Second, although it has been preliminarily confirmed that HOTTIP may function through the miR-216a-5p/JAK2 pathway, the regulatory mechanisms of the downstream signaling pathways of JAK2 have not been thoroughly explored. Additionally, while the functional association between HOTTIP and miR-216a-5p has been validated through reverse experiments, there is a lack of direct evidence from forward complementary experiments (such as HOTTIP knockdown combined with miR-216a-5p overexpression). Additionally, while the functional association between HOTTIP and miR-216a-5p has been validated through reverse experiments, there is a lack of direct evidence from forward complementary experiments (such as HOTTIP knockdown combined with miR-216a-5p overexpression). Future studies will further clarify how JAK2 transmits signals through its downstream molecules and influences the pathological processes of microglia, as well as perform bidirectional rescue experiments with HOTTIP knockdown and miR-216a-5p overexpression. This will provide a more comprehensive understanding of the entire functional network of HOTTIP in NP progression, laying a solid theoretical foundation for subsequent mechanistic studies and potential clinical applications.
Overall, this study has clarified for the first time the underlying mechanism of the HOTTIP/miR-216a-5p signaling pathway in NP. Inhibiting HOTTIP expression can significantly alleviate NP symptoms and reduce neuroinflammatory responses by targeting the miR-216a-5p/JAK2 signaling pathway. This major discovery provides a critical theoretical basis for clinical treatment of NP and lays an important foundation for the development of novel targeted drugs.
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