Neuropathic pain(NP) has similar phenotypes but different sequential neuroinflammatory mechanisms in the pathological process. It is of great significance to inhibit the initiation of neuroinflammation, which has become a new direction of NP treatment and drug development in recent years. Mongolian drug Naru-3 is clinically effective in the treatment of trigeminal neuralgia, sciatica, and other NPs in a short time, but its pharmacodynamic characteristics and mechanism of analgesia are still unclear. In this study, a spinal nerve ligation(SNL) model simulating clinical peripheral nerve injury was established and the efficacy and mechanism of Naru-3 in the treatment of NPs was discussed by means of behavioral detection, side effect evaluation, network analysis, and experimental verification. Pharmacodynamic results showed that Naru-3 increased the basic pain sensitivity threshold(mechanical hyperalgesia and thermal radiation hyperalgesia) in the initiation of SNL in animals and relieved spontaneous pain, however, there was no significant effect on the basic pain sensitivity threshold and motor coordination function of normal animals under physiological and pathological conditions. Meanwhile, the results of primary screening of target tissues showed that Naru-3 inhibited the second phase of injury-induced nociceptive response of formalin test in mice and reduced the expression of inflammatory factors in the spinal cord. Network analysis discovered that Naru-3 had synergy in the treatment of NP, and its mechanism was associated with core targets such as matrix metalloproteinase-9(MMP9) and interleukin-1β(IL-1β). The experiment further took the dorsal root ganglion(DRG) and the stage of patho-logical spinal cord as the research objects, focusing on the core targets of inducing microglial neuroinflammation. By means of Western blot, immunofluorescence, agonists, antagonists, behavior, etc., the mechanism of Naru-3 in exerting NP analgesia may be related to the negative regulation of the MMP9/IL-1β signaling pathway-mediated microglia p38/IL-1β inflammatory loop in the activation phase. The relevant research enriches the biological connotation of Naru-3 in the treatment of NP and provides references for clinical rational drug use.
Rats ; Mice ; Animals ; Matrix Metalloproteinase 9/metabolism* ; Rats, Sprague-Dawley ; Neuroinflammatory Diseases ; Interleukin-1beta/metabolism* ; Spinal Cord/metabolism* ; Signal Transduction ; Hyperalgesia/metabolism* ; Neuralgia/metabolism*

Nerve regeneration in adult mammalian spinal cord is poor because of the lack of intrinsic regeneration of neurons and extrinsic factors - the glial scar is triggered by injury and inhibits or promotes regeneration. Recent technological advances in spatial transcriptomics (ST) provide a unique opportunity to decipher most genes systematically throughout scar formation, which remains poorly understood. Here, we first constructed the tissue-wide gene expression patterns of mouse spinal cords over the course of scar formation using ST after spinal cord injury from 32 samples. Locally, we profiled gene expression gradients from the leading edge to the core of the scar areas to further understand the scar microenvironment, such as neurotransmitter disorders, activation of the pro-inflammatory response, neurotoxic saturated lipids, angiogenesis, obstructed axon extension, and extracellular structure re-organization. In addition, we described 21 cell transcriptional states during scar formation and delineated the origins, functional diversity, and possible trajectories of subpopulations of fibroblasts, glia, and immune cells. Specifically, we found some regulators in special cell types, such as Thbs1 and Col1a2 in macrophages, CD36 and Postn in fibroblasts, Plxnb2 and Nxpe3 in microglia, Clu in astrocytes, and CD74 in oligodendrocytes. Furthermore, salvianolic acid B, a blood-brain barrier permeation and CD36 inhibitor, was administered after surgery and found to remedy fibrosis. Subsequently, we described the extent of the scar boundary and profiled the bidirectional ligand-receptor interactions at the neighboring cluster boundary, contributing to maintain scar architecture during gliosis and fibrosis, and found that GPR37L1_PSAP, and GPR37_PSAP were the most significant gene-pairs among microglia, fibroblasts, and astrocytes. Last, we quantified the fraction of scar-resident cells and proposed four possible phases of scar formation: macrophage infiltration, proliferation and differentiation of scar-resident cells, scar emergence, and scar stationary. Together, these profiles delineated the spatial heterogeneity of the scar, confirmed the previous concepts about scar architecture, provided some new clues for scar formation, and served as a valuable resource for the treatment of central nervous system injury.
Mice ; Animals ; Gliosis/pathology* ; Cicatrix/pathology* ; Spinal Cord Injuries ; Astrocytes/metabolism* ; Spinal Cord/pathology* ; Fibrosis ; Mammals ; Receptors, G-Protein-Coupled

This study aims to investigate the neuroprotective effect of tetramethylpyrazine on mice after spinal cord injury and its mechanism. Seventy-five female C57BL/6 mice were randomly divided into 5 groups, namely, a sham operation group, a model group, a tetramethylpyrazine low-dose group(25 mg·kg~(-1)), a tetramethylpyrazine medium-dose group(50 mg·kg~(-1)), and a tetramethylpyrazine high-dose group(100 mg·kg~(-1)), with 15 mice in each group. Modified Rivlin method was used to establish the mouse model of acute spinal cord injury. After 14 d of tetramethylpyrazine intervention, the motor function of hind limbs of mice was evaluated by basso mouse scale(BMS) and inclined plate test. The levels of inflammatory cytokines tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β) in the spinal cord homogenate were determined by enzyme-linked immunosorbent assay(ELISA). Hematoxylin-eosin(HE) staining was used to observe the histology of the spinal cord, and Nissl's staining was used to observe the changes in the number of neurons. Western blot and immunofluorescence method were used to detect the expression of glial fibrillary acidic protein(GFAP) and C3 protein. Tetramethylpyrazine significantly improved the motor function of the hind limbs of mice after spinal cord injury, and the BMS score and inclined plate test score of the tetramethylpyrazine high-dose group were significantly higher than those of the model group(P<0.01). The levels of TNF-α, IL-6, and IL-1β in spinal cord homogenate of the tetramethylpyrazine high-dose group were significantly decreased(P<0.01). After tetramethylpyrazine treatment, the spinal cord morphology recovered, the number of Nissl bodies increased obviously with regular shape, and the loss of neurons decreased. As compared with the model group, the expression of GFAP and C3 protein was significantly decreased(P<0.05,P<0.01) in tetramethylpyrazine high-dose group. In conclusion, tetramethylpyrazine can promote the improvement of motor function and play a neuroprotective role in mice after spinal cord injury, and its mechanism may be related to inhibiting inflammatory response and improving the hyperplasia of glial scar.
Rats ; Mice ; Female ; Animals ; Rats, Sprague-Dawley ; Neuroprotective Agents/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6 ; Mice, Inbred C57BL ; Spinal Cord Injuries/genetics* ; Spinal Cord/metabolism*

OBJECTIVE: To observe the analgesic effect of Tuina by pressing and kneading the Huantiao (GB30) acupoint on rats with chronic constriction injury (CCI) and to explore the analgesic mechanism of Tuina on sciatica rats. METHODS: Thirty-two SPF male SD rats weighing 180 to 220 g were randomly divided into fore groups:blank group (without any treatment), sham group (only exposed without sciatic nerve ligating), model group (sciatic nerve ligating) and Tuina group (manual intervention after lsciatic nerve ligating). The CCI model was prepared by ligating the right sciatic nerve of the rats, on the third day of modeling, the rats in the Tuina group were given pressing and kneading the Huantiao (GB30) point for 14 days, and the changes of paw withdrawal threshold(PWT), paw withdrawal latency(PWL) were measured before and on the 1st, 3rd, 7th, 10th, 14th and 17th days after modeling. The changes of sciatic functional index(SFI) were measured before and on the 1st and 17th day after modeling. The morphological changes of the sciatic nerve were observed by hematoxylin-eosin(HE) staining;and the differences in NF-κB protein expression in the right dorsal horn of the spinal cord of rats were detected. RESULTS: Following modeling, there was no significant difference in PWT, PWL and SFI between the blank group and the sham group (P>0.05), but the PWT, PWL and SFI of the model group and the Tuina group decreased significantly (P<0.01). After manual intervention, the pain threshold of rats in Tuina group increased. On the 8th day of manual intervention (the 10th day after modeling), PWT in Tuina group increased significantly compared with that in model group (P<0.01). On the 5th day of manual intervention (the 7th day after modeling), the PWL of the massage group was significantly higher than that of the model group (P<0.01). The pain threshold of rats in Tuina group continued to rise with the continuous manipulation intervention. After 14 days of manipulative intervention, the sciatic nerve function index of rats in the Tuina group increased significantly(P<0.01). Compared with the blank group and sham group, the myelinated nerve fibers of sciatic nerve in the model group were disordered and the density of axons and myelin sheath was uneven. Compared with the model group, the nerve fibers of rats in the Tuina group were gradually continuous and the axons and myelin sheath were more uniform than those in the model group. Compared with the blank group and sham group, the expression of NF-κB protein in the right spinal dorsal horn of the model group was significantly increased(P<0.01). Compared with the model group, the expression of NF-κB protein in the right spinal dorsal horn of rats in Tuina group decreased significantly(P<0.01). CONCLUSION Pressing and kneading the Huantiao (GB30) point restores nerve fiber alignment;and improves the PWT、PWL and SFI in the CCI model by decreasing NF-κB p65 protein expression in the spinal dorsal horn. There fore, Tuina demmstrates an analgesic effect and improves the gait of rats with sciatica.
Rats ; Male ; Animals ; Rats, Sprague-Dawley ; Sciatica/therapy* ; NF-kappa B/metabolism* ; Acupuncture Points ; Spinal Cord Dorsal Horn/metabolism* ; Spinal Cord ; Massage

A decline in the activities of oxidative phosphorylation (OXPHOS) complexes has been consistently reported in amyotrophic lateral sclerosis (ALS) patients and animal models of ALS, although the underlying molecular mechanisms are still elusive. Here, we report that receptor expression enhancing protein 1 (REEP1) acts as an important regulator of complex IV assembly, which is pivotal to preserving motor neurons in SOD1^G93A mice. We found the expression of REEP1 was greatly reduced in transgenic SOD1^G93A mice with ALS. Moreover, forced expression of REEP1 in the spinal cord extended the lifespan, decelerated symptom progression, and improved the motor performance of SOD1^G93A mice. The neuromuscular synaptic loss, gliosis, and even motor neuron loss in SOD1^G93A mice were alleviated by increased REEP1 through augmentation of mitochondrial function. Mechanistically, REEP1 associates with NDUFA4, and plays an important role in preserving the integrity of mitochondrial complex IV. Our findings offer insights into the pathogenic mechanism of REEP1 deficiency in neurodegenerative diseases and suggest a new therapeutic target for ALS.
Mice ; Animals ; Amyotrophic Lateral Sclerosis/metabolism* ; Superoxide Dismutase-1/metabolism* ; Superoxide Dismutase/metabolism* ; Mice, Transgenic ; Spinal Cord/pathology* ; Mitochondria/physiology* ; Disease Models, Animal

Effective treatments for neuropathic pain are lacking due to our limited understanding of the mechanisms. The circRNAs are mainly enriched in the central nervous system. However, their function in various physiological and pathological conditions have yet to be determined. Here, we identified circFhit, an exon-intron circRNA expressed in GABAergic neurons, which reduced the inhibitory synaptic transmission in the spinal dorsal horn to mediate spared nerve injury-induced neuropathic pain. Moreover, we found that circFhit decreased the expression of GAD65 and induced hyperexcitation in NK1R⁺ neurons by promoting the expression of its parental gene Fhit in cis. Mechanistically, circFhit was directly bound to the intronic region of Fhit, and formed a circFhit/HNRNPK complex to promote Pol II phosphorylation and H2B monoubiquitination by recruiting CDK9 and RNF40 to the Fhit intron. In summary, we revealed that the exon-intron circFhit contributes to GABAergic neuron-mediated NK1R⁺ neuronal hyperexcitation and neuropathic pain via regulating Fhit in cis.
Rats ; Animals ; Posterior Horn Cells/pathology* ; Spinal Cord Dorsal Horn/metabolism* ; Neuralgia ; Synaptic Transmission

Mechanical allodynia (MA), including punctate and dynamic forms, is a common and debilitating symptom suffered by millions of chronic pain patients. Some peripheral injuries result in the development of bilateral MA, while most injuries usually led to unilateral MA. To date, the control of such laterality remains poorly understood. Here, to study the role of microglia in the control of MA laterality, we used genetic strategies to deplete microglia and tested both dynamic and punctate forms of MA in mice. Surprisingly, the depletion of central microglia did not prevent the induction of bilateral dynamic and punctate MA. Moreover, in dorsal root ganglion-dorsal root-sagittal spinal cord slice preparations we recorded the low-threshold Aβ-fiber stimulation-evoked inputs and outputs of superficial dorsal horn neurons. Consistent with behavioral results, microglial depletion did not prevent the opening of bilateral gates for Aβ pathways in the superficial dorsal horn. This study challenges the role of microglia in the control of MA laterality in mice. Future studies are needed to further understand whether the role of microglia in the control of MA laterality is etiology-or species-specific.
Mice ; Animals ; Hyperalgesia/metabolism* ; Microglia/metabolism* ; Disease Models, Animal ; Spinal Cord/metabolism* ; Spinal Cord Dorsal Horn/metabolism* ; Ganglia, Spinal/metabolism*

Objective To explore the effect of shionone(SHI)on motor function in the mouse model of spinal cord injury(SCI)and probe into the underlying molecular mechanism.Methods C57BL/6 mice were treated to induce the SCI model and then assigned into a model group(SCI group),a SCI+SHI group,and a sham surgery(control)group.The Basso mouse scale(BMS)score was determined to evaluate the recovery of motor function in SCI mice.Hematoxylin-eosin(HE)staining,Nissl staining,and immunofluorescence staining were employed to examine the fibrosis,morphological changes of neurons,and neuron apoptosis in the spinal cord tissue of SCI mice,respectively.The mouse hippocampal neuronal cell line HT22 was cultured in vitro and then classified into tumor necrosis factor α(TNF-α)induction and SHI groups.Western blotting was employed to determine the expression of apoptosis-associated proteins.Network pharmacology,gene ontology annotation,and Kyoto Encyclopedia of Genes and Genomes pathway enrichment were employed to predict the possible molecular targets and signaling pathways of SHI in promoting functional recovery from SCI.Furthermore,the prediction results were verified by in vitro and in vivo experiments.Results Compared with the SCI group,the SCI+SHI group showed increased BMS score on days 21,28,35,and 42(P=0.003,P=0.004,P=0.023,and P=0.007,respectively),reduced area of spinal cord fibrosis(P=0.021),increased neurons survived(P=0.001),and down-regulated expression of cleaved cysteine aspastic acid-specific protease 3(cleaved Caspase-3)(P=0.017).Compared with the TNF-α group,the SHI group presented down-regulated expression levels of cleaved Caspase-3 and Bax(P=0.010,P=0.001)and up-regulated expression level of Bcl-2(P=0.001).The results of bioinformatics analysis showed that SHI might improve the motor function of SCI mice via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway.The results of in vivo and in vitro experiments showed that SHI inhibited the phosphorylation of PI3K and Akt in SCI mice or HT22 cells exposed to TNF-α(all P<0.05).The number of apoptotic HT22 cells after treatment with insulin-like growth factor 1 was higher than that in the SHI group(P=0.003).Conclusion SHI may inhibit neuron apoptosis via the PI3K/Akt signaling pathway,thereby promoting the recovery of motor function in SCI mice.
Mice ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; Caspase 3/metabolism* ; Phosphatidylinositol 3-Kinases ; Tumor Necrosis Factor-alpha/metabolism* ; Mice, Inbred C57BL ; Spinal Cord Injuries ; Apoptosis ; Neurons/pathology* ; Fibrosis

OBJECTIVE: To observe the effect of moxibustion at "Guanyuan" (CV 4) and "Shenque" (CV 8) on acetylcholine (Ach), adenosine triphosphate (ATP) and muscarinic-type choline receptor (M2) and purine receptor P2X3 in bladder tissue in the rats with neurogenic bladder (NB) of detrusor areflexia after lumbar-sacral spinal cord injury and explore the underlying mechanism of moxibustion for promoting detrusor contraction. METHODS: Sixty SD rats were randomly divided into a model preparation group (n=45) and a sham-operation group (n=15). In the model preparation group, the modified Hassan Shaker spinal cord transection method was used to prepare the model of NB. In the sham-operation group, the spinal cord transection was not exerted except laminectomy and spinal cord exposure. Among the rats with successfully modeled, 30 rats were selected and divided randomly into a model group and a moxibustion group, with 15 rats in each one. On the 15th day after the operation, moxibustion was applied at "Guanyuan" (CV 4) and "Shenque" (CV 8) in the moxibustion group, 10 min at each acupoint, once a day. The consecutive 7-day treatment was as one course and the intervention for 2 courses was required. Urodynamic test was adopted to evaluate bladder function in rats. Using HE staining, the morphological changes in bladder tissue were observed. The content of Ach and ATP in bladder tissue was measured with biochemical method, and the protein and mRNA expression levels of M2 and P2X3 receptors in bladder tissue were detected with Western blot and real-time fluorescence quantification PCR method. RESULTS: Compared with the sham-operation group, the maximum bladder capacity, leakage point pressure and bladder compliance were increased in the rats of the model group (P<0.05). Compared with the model group, the maximum bladder capacity, the leakage point pressure and bladder compliance were decreased in the rats of the moxibustion group (P<0.05). In the model group, the detrusor fibres were arranged irregularly, bladder epithelial tissues were not tightly connected and cell arrangement was disordered, combined with a large number of vacuolar cells. In the moxibustion group, compared with the model group, the detrusor fibres were arranged regularly, bladder epithelial cells were well distributed and vacuolar cells were reduced. Compared with the sham-operation group, the content of Ach and ATP in bladder tissue was decreased (P<0.05), the protein and mRNA expression levels of M2 and P2X3 receptors were reduced (P<0.05) in the model group. In the moxibustion group, the content of Ach and ATP in bladder tissue was increased (P<0.05) and the protein and mRNA expression levels of M2 and P2X3 receptors were increased (P<0.05) as compared with the model group. CONCLUSION Moxibustion at "Guanyuan" (CV 4) and "Shenque" (CV 8) may effectively improve bladder function in the rats with NB of detrusor areflexia after lumbar-sacral spinal cord injury and its underlying mechanism is related to promoting the release of Ach and up-regulating the expression of M2 receptor, thereby enhancing the release of ATP and increasing the expression of P2X3 receptor. Eventually, detrusor contraction is improved.
Animals ; Moxibustion/methods* ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X3/metabolism* ; Spinal Cord Injuries/therapy* ; Urinary Bladder ; Urinary Bladder, Neurogenic/therapy*

Neuropathic pain is one of the common complications of diabetes. Tetrahydropalmatine(THP) is a main active component of Corydalis Rhizoma with excellent anti-inflammatory and pain-alleviating properties. This study aims to investigate the therapeutic effect of THP on diabetic neuropathic pain(DNP) and the underlying mechanism. High-fat and high-sugar diet(4 weeks) and streptozotocin(STZ, 35 mg·kg~(-1), single intraperitoneal injection) were employed to induce type-2 DNP in rats. Moreover, lipopolysaccharide(LPS) was used to induce the activation of BV2 microglia in vitro to establish an inflammatory cellular model. Fasting blood glucose(FBG) was measured by a blood glucose meter. Mechanical withdrawal threshold(MWT) was assessed with von Frey filaments, and thermal withdrawal latency(TWL) with hot plate apparatus. The protein expression levels of OX42, inducible nitric oxide synthase(iNOS), CD206, p38, and p-p38 were determined by Western blot, the fluorescence expression levels of OX42 and p-p38 in the dorsal horn of the rat spinal cord by immunofluorescence, the mRNA content of p38 and OX42 in rat spinal cord tissue by qRT-PCR, and levels of nitric oxide(NO), interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), interleukin-10(IL-10), and serum fasting insulin(FINS) by enzyme-linked immunosorbent assay(ELISA). RESULTS:: showed that the mo-del group demonstrated significant decrease in MWT and TWL, with pain symptoms. THP significantly improved the MWT and TWL of DNP rats, inhibited the activation of microglia and p38 MAPK signaling pathway in rat spinal cord, and ameliorated its inflammatory response. Meanwhile, THP promoted the change of LPS-induced BV2 microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, suppressed the activation of the p38 MAPK signaling pathway, decreased the expression levels of inflammatory factors NO, IL-1β, IL-6, and TNF-α, and increased the expression level of anti-inflammatory factor IL-10. The findings suggested that THP can significantly ameliorate the pain symptoms of DNP rats possibly by inhibiting the inflammatory response caused by M1 polarization of microglia via the p38 MAPK pathway.
Animals ; Berberine Alkaloids ; Blood Glucose/metabolism* ; Diabetes Mellitus ; Diabetic Neuropathies/genetics* ; Interleukin-10 ; Interleukin-6/metabolism* ; Lipopolysaccharides/pharmacology* ; Microglia ; Neuralgia/metabolism* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Spinal Cord/metabolism* ; Streptozocin/therapeutic use* ; Tumor Necrosis Factor-alpha/metabolism* ; p38 Mitogen-Activated Protein Kinases/metabolism*