Transcriptome profiling of microRNAs reveals potential mechanisms of manual therapy alleviating neuropathic pain through microRNA-547-3p-mediated Map4k4/NF-κb signaling pathway

Abstract

Background: Local neuroinflammation secondary to spinal nerve compression in lumbar disk herniation (LDH) is a key driver contributing to neuropathic pain. Manual therapy (MT), a widely used nonsurgical therapy, can relieve LDH-mediated pain by reducing inflammation. MT has attracted extensive attention; however, its mechanism remains poorly understood. MicroRNAs (miRNAs) are important regulators of pain signaling transduction, but are rarely reported in the chronic compression of dorsal root ganglia (CCD) model, and further investigation is needed to decipher whether they mediate anti-inflammatory and analgesic effects of MT.

Methods: We used a combination of in vivo behavioral and molecular techniques to study MT intervention mechanisms. Neuropathic pain was induced in a CCD rat model and MT intervention was performed according to standard procedures. Enzyme-linked immunosorbent assay (ELISA) was used to detect inflammatory cytokine levels in dorsal root ganglia (DRG). Small RNA sequencing, immunofluorescence, Western blot, and qRT-PCR were performed to screen miRNAs and their target genes and determine core factors in the pathway possibly regulated by miRNA-mediated target gene in DRG of MT-treated CCD rats.

Results: Compared with naive rats, small RNA sequencing detected 22 differentially expressed miRNAs in DRG of CCD rats, and compared with CCD rats, MT-treated rats presented 19 differentially expressed miRNAs, which were functionally associated with nerve injury and inflammation. Among these, miR-547-3p was screened as a key miRNA mediating neuroinflammation and participating in neuropathic pain. We confirmed in vitro that its function is achieved by directly regulating its target gene Map4k4. Intrathecal injection of miR-547-3p agomir or MT intervention significantly reduced Map4k4 expression and the expression and phosphorylation of IκBα and p65 in the NF-κB pathway, thus reducing the inflammatory cytokine levels and exerting an analgesic effect, whereas intrathecal injection of miR-547-3p antagomir led to opposite effects.

Conclusions: In rats, CCD-induced neuropathic pain leads to variation in miRNA expression in DRG, and MT can intervene the transcription and translation of inflammation-related genes through miRNAs to improve neuroinflammation and alleviate neuropathic pain. MiR-547-3p may be a key target of MT for anti-inflammatory and analgesia effects, which is achieved by mediating the Map4k4/NF-κB pathway to regulate downstream inflammatory cytokines.

Keywords: Inflammation; Lumbar disk herniation; Manual therapy; Map4k4; MiRNA; MicroRNA-547-3p; Neuropathic pain.

Fig. 1

Effects of MT on behavior and inflammatory cytokines in rats DRG. A Diagram of MT intervention. B Timeline of the experiment. C, D Radiographs presenting coronal and sagittal views of a rat after CCD surgery. Arrows indicate L1 and L6 to locate the lumbar spine, and circles indicate the L4 and L5 intervertebral foramen where the stainless-steel rod has been implanted. E, F PWT and PWL of rats were measured to assess their mechanical and thermal hyperalgesia. The data are expressed as mean ± SD (N = 8). Repeated measurement ANOVA was used to analyze the data at various time points between the groups and one-way ANOVA followed by LSD post hoc analysis was used for pairwise comparison (^##P < 0.01 versus sham group, ^**P < 0.01 versus CCD group). G–I ELISA experiments showed that the expression of TNF-α, IL-1β, and IL-6 in DRG varies between the groups. The data are expressed as mean ± SD (N = 8) and analyzed for statistical significance using one-way ANOVA followed by LSD post hoc analysis for multiple comparisons (^##P < 0.01 versus sham group, ^**P < 0.01 versus CCD group). ANOVA analysis of variance, CCD chronic compression of dorsal root ganglion, DRG dorsal root ganglion, ELISA enzyme-linked immunosorbent assay, LSD least significant difference, MT manual therapy, PWL paw withdrawal latency, PWT paw withdrawal threshold, SD standard deviation

Fig. 2

Analysis of differentially expressed miRNAs in DRG between the CCD and sham groups. A Volcano plot of differentially expressed miRNAs in each group. Red plots indicate upregulated miRNAs and green plots indicate downregulated miRNAs in CCD group compared with those in the sham group. B Heat map of differentially expressed miRNAs. For each miRNA, red indicates upregulated and green indicates downregulated miRNAs. The darker the color, the higher would be the expression. C, D GO functional enrichment and KEGG pathway analysis were performed to identify potential pathways or biological processes associated with CCD-induced neuropathic pain. The abscissa indicates the number of enriched target genes and − log₁₀(P-value), and the ordinate indicates the top 30 GO enrichment or KEGG pathway terms. CCD chronic compression of dorsal root ganglion, DRG dorsal root ganglion, GO Gene Ontology, KEGG Kyoto Encyclopedia of Genes and Genomes

Fig. 3

Analysis of differentially expressed miRNAs in DRG between the MT and CCD groups. A Volcano plot of differentially expressed miRNAs. Red plots indicate upregulated miRNAs and green plots indicate downregulated miRNAs in the MT group compared with those in the CCD group. B Heat map of differentially expressed miRNAs. For each miRNA, red indicates upregulated and green indicates downregulated miRNAs. The darker the color, the higher would be the expression. C, D GO functional enrichment and KEGG pathway analysis were performed to identify potential pathways or biological processes associated with the mechanism of MT. The abscissa indicates the number of enriched target genes and − log₁₀(P-value), and the ordinate indicates the top 30 GO enrichment or KEGG pathway terms. CCD chronic compression of dorsal root ganglion, DRG dorsal root ganglion, GO Gene Ontology, KEGG Kyoto Encyclopedia of Genes and Genomes, MT manual therapy

Fig. 4

Validations of miRNAs with high fold change between different groups. A Venn diagram of miRNAs differentially expressed between the groups. B–D Relative expression of miR-375-3p, miR-449a-5p, and miR-547-3p was detected using qRT-PCR to validate the sequencing results. The data are expressed as mean ± SD (N = 8) and analyzed for statistical significance using one-way ANOVA followed by LSD post hoc analysis for multiple comparisons (^##P < 0.01 versus sham group, ^**P < 0.01 versus CCD group). CCD chronic compression of dorsal root ganglion, LSD least significant difference, MT manual therapy, qRT-PCR real-time quantitative reverse transcription PCR

Fig. 5

Validation of Map4k4 as a target gene of miR-547-3p. A Prediction of binding sites between miR-547-3p and Map4k4. B Luciferase reporter gene assay was performed to validate the interaction between miR-547-3p and Map4k4. The data are expressed as mean ± SD (N = 3) and analyzed for statistical significance using one-way ANOVA followed by LSD post hoc analysis for multiple comparisons (^**P < 0.01 versus miRNA-NC + Map4k4-WT group). C, D Western blot and qRT-PCR analysis show the effects of miR-547-3p mimics or inhibitor on the expression of Map4k4 in the DRG cells. The data are expressed as mean ± SD (N = 3) and analyzed for statistical significance using one-way ANOVA followed by LSD post hoc analysis for multiple comparisons (^**P < 0.01 versus control group). DRG dorsal root ganglion, LSD least significant difference, NC negative control, WT wild-type, MUT mutant, qRT-PCR real-time quantitative reverse transcription PCR, SD standard deviation

Fig. 6

MT reduces neuropathic pain by regulating miR-547-3p targeting Map4k4. A, B PWT and PWL of rats were measured to assess their mechanical and thermal hyperalgesia. The data are expressed as mean ± SD (N = 8). Repeated measurement ANOVA was used to analyze the data at various time points between the groups and one-way ANOVA followed by LSD post hoc analysis was used for pairwise comparison (at the same time point, ^##P < 0.01 versus sham + NC group, ^**P < 0.01 versus CCD + NC group, and ^ΔΔP < 0.01 versus MT + NC group). C, D Immunofluorescence staining reveals the expression and cellular distribution of Map4k4 in different groups; white arrows indicate the co-expression of Map4k4 (green fluorescent) and IB4-positive nociceptors (red fluorescent) in DRG neurons (scale bar = 50 μm). E Quantification analysis of Map4k4 fluorescence intensity. The data are expressed as mean ± SD (N = 8) and analyzed for statistical significance using one-way ANOVA followed by Dunnett’s T3 post hoc analysis for multiple comparisons (^##P < 0.01 versus sham + NC group, ^**P < 0.01 versus CCD + NC group, ^ΔP < 0.05 versus MT + NC group). F, G qRT-PCR and Western blot analysis show the relative mRNA and protein expression of Map4k4, respectively. The data are expressed as mean ± SD (N = 8). One-way ANOVA followed by LSD post hoc analysis was used for protein expression, and one-way ANOVA followed by Dunnett’s T3 post hoc analysis was used for relative mRNA expression (^##P < 0.01 versus sham + NC group, ^**P < 0.01 versus CCD + NC group, ^ΔP < 0.05, and ^ΔΔP < 0.01 versus MT + NC group). ANOVA analysis of variance, CCD chronic compression of dorsal root ganglion, LSD least significant difference, MT manual therapy, NC negative control, SD standard deviation

Fig. 7

Inhibition of Map4k4/NF-κB signaling pathway may be a potential mechanism of miR-547-3p-mediated anti-inflammatory effect of MT. A, B Western blot analysis showed the relative expression of p65, p-p65, IκBα, and p-IκBα. The data are expressed as mean ± SD (N = 8) and analyzed for statistical significance using one-way ANOVA followed by LSD post hoc analysis for multiple comparisons (^##P < 0.01 versus sham + NC group, ^**P < 0.01 versus CCD + NC group, ^ΔΔP < 0.01 versus MT + NC group). C, D qRT-PCR results showed the relative mRNA expression of p65 and IκBα. The data are expressed as mean ± SD (N = 8) and analyzed for statistical significance using one-way ANOVA followed by Dunnett’s T3 post hoc analysis for multiple comparisons (^##P < 0.01 versus sham + NC group, ^**P < 0.01 versus CCD + NC group, ^ΔP < 0.05 and ^ΔΔP < 0.01 versus MT + NC group). (E–G) ELISA revealed the expression of TNF-α, IL-1β, and IL-6 in DRG. The data are expressed as mean ± SD (N = 8). One-way ANOVA followed by LSD post hoc analysis was used for TNF-α and IL-1β, and one-way ANOVA followed by Dunnett’s T3 post hoc analysis was used for IL-6 (^##P < 0.01 versus sham + NC group, ^**P < 0.01 versus CCD + NC group, ^ΔP < 0.05, and ^ΔΔP < 0.01 versus MT + NC group). ANOVA analysis of variance, CCD chronic compression of dorsal root ganglion, DRG dorsal root ganglion, ELISA: enzyme-linked immunosorbent assay, LSD least significant difference, MT manual therapy, NC negative control