The use of substances for regulating the pain microenvironment and remodeling the balance of the ascending perception and descending inhibition pathway of pain in the preparation of drugs for preventing and treating trigeminal neuralgia

Abstract

This invention discloses the application of substances that regulate the pain microenvironment and reshape the balance of the ascending pain perception-descending inhibition pathway in the preparation of drugs for the prevention and treatment of trigeminal neuralgia. It also verifies that after trigeminal nerve injury, the levels of reactive oxygen species (ROS) and TRPA1 channel protein expression increase in trigeminal neuralgia (TG). Improving the oxidative stress environment and silencing the NRF2 gene to assist in the regulation of TRPA1 channel protein can effectively downregulate the expression of pro-analgesic and inflammatory factors, significantly reducing orofacial pain sensitivity. This invention uses the pain microenvironment and the ascending pain perception-descending inhibition pathway as novel targets, providing specific drugs and diagnostic tools for the treatment of trigeminal neuralgia, and has significant clinical value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of substances that regulate the pain microenvironment and reshape the balance of ascending pain perception-descending inhibition pathways in the preparation of drugs for the prevention and treatment of trigeminal neuralgia. Background Technology

[0002] Trigeminal neuralgia (TN) is a common neurological disorder, ranking first in incidence among cranial nerve pain. Its etiology and pathogenesis remain unclear. Currently, treatment for trigeminal neuralgia mainly relies on medication and surgery. However, conventional drug therapy is often accompanied by decreased efficacy and adverse drug reactions, while surgical treatments such as trigeminal nerve decompression, percutaneous radiofrequency ablation, and percutaneous minimally invasive balloon compression of the trigeminal nerve carry high risks, high recurrence rates, and complications such as facial numbness and sensory loss.

[0003] The trigeminal ganglion, as a hub for pain signal transmission, includes not only sensory neurons but also satellite glial cells, Schwann cells, and immune cells such as macrophages. These cells work together through a complex network of intercellular signaling molecules to maintain the homeostasis of the neural microenvironment. Under pathological conditions, imbalances in the coordination among these cells disrupt the neural microenvironment, leading to a painful microenvironment and ultimately trigeminal neuralgia. The formation of this painful microenvironment involves multiple pathological factors, such as oxidative stress, inflammatory responses, abnormal activation of ion channels, and the production of pain mediators. Among these, abnormal activation of immune cells and oxidative stress activate the TRPA1 channel, promoting the synthesis and release of pain mediators such as SP, which in turn activates satellite glial cells to produce inflammatory factors IL-1β and TNF-α, creating a vicious cycle that exacerbates pain perception.

[0004] Furthermore, trigeminal neuralgia is closely related to the imbalance of the ascending pain perception-descending inhibition pathway. Patients with trigeminal neuralgia often exhibit pathological features of enhanced pain perception and weakened descending inhibition, with the activation and expression of the TRPA1 channel playing a crucial role in this process. TRPA1 channel expression is regulated by multiple factors. Besides directly activating the TRPA1 channel, ROS can also mediate the upregulation of Nrf2 nuclear transcription factor (a transcription factor that mediates the expression of antioxidant factors). Nrf2 can further act on the promoter region of the TRPA1 gene to directly upregulate TRPA1 channel expression, ultimately leading to hyperalgesia, i.e., an imbalance in the ascending pain perception-descending inhibition pathway. Therefore, it can be concluded that TRPA1 channel upregulation mediated by increased oxidative stress levels may play an important role in the sensitization of trigeminal neuralgia, and the TRPA1 channel is expected to become a key node in the regulation of the pain microenvironment and the reshaping of the balance of the ascending pain perception-descending inhibition pathway. Summary of the Invention

[0005] The purpose of this invention is to provide the application of substances that regulate the pain microenvironment and reshape the balance of ascending and descending pain pathways in the preparation of drugs for the prevention and treatment of trigeminal neuralgia, and the regulatory role of the pain microenvironment and the balance of ascending and descending pain pathways in trigeminal neuralgia.

[0006] The objective of this invention is achieved as follows:

[0007] Application of substances that regulate the pain microenvironment and reshape the balance of ascending and descending pain pathways in the preparation of drugs for the prevention and treatment of trigeminal neuralgia. Substances that regulate the pain microenvironment and reshape the balance of ascending and descending pain pathways include substances that improve the oxidative stress microenvironment and substances that reshape the balance of ascending and descending pain pathways.

[0008] The substances that improve the microenvironment of oxidative stress include reactive oxygen species scavengers and enzyme-like nanomaterials.

[0009] The substances that reshape the balance of the ascending pain perception-descending inhibition pathway include substances that inhibit TRPA1 channel protein activity and TRPA1 channel protein gene expression. Substances that inhibit TRPA1 channel protein activity include RPA1 channel antagonists, and substances that inhibit TRPA1 channel protein gene expression include NRF2 gene silencing agents. TRPA1 channels regulate the ascending pain perception-descending inhibition pathway.

[0010] The NRF2 gene silencing reagents include nucleotide siRNA reagents, nucleotide shRNA reagents, nucleotide miRNA reagents, and nucleotide ASO reagents.

[0011] The substances that regulate the pain microenvironment and reshape the balance of ascending and descending pain perception pathways can alleviate orofacial pain sensitivity by improving the oxidative stress microenvironment and downregulating TRPA1 channel protein expression levels, thereby achieving the therapeutic effect of trigeminal neuralgia.

[0012] A kit for detecting trigeminal neuralgia, comprising reagents for detecting reactive oxygen species and TRPA1 channel protein expression levels.

[0013] The beneficial effects of this invention are:

[0014] 1. This invention reveals the key role of the TRPA1 channel in trigeminal neuralgia and is the first to use it as a therapeutic target.

[0015] 2. This invention regulates the pain microenvironment by improving the oxidative stress microenvironment, and reshapes the balance of the ascending-descending pain perception pathway by inhibiting TRPA1 channel protein activity or TRPA1 channel protein gene expression, thereby achieving the effect of treating trigeminal neuralgia.

[0016] 3. This invention has demonstrated through both in vivo and in vitro experiments that intervening in the pain microenvironment and reshaping the balance of the ascending pain perception-descending inhibition pathway can effectively alleviate pain behavior, thereby effectively relieving trigeminal neuralgia. Attached Figure Description

[0017] Figure 1 The figure shows the test results of an embodiment of the present invention; Figure 1 -A represents the change in the 50% mechanical pain threshold of the maxillofacial region in SD rats in the trigeminal neuralgia model group (TN group) and the sham-operated group (Sham group) within 21 days after surgery. Figure 1 -B represents the change in the 50% mechanical pain threshold of the maxillofacial region in SD rats in the model group (TN group), sham operation group (Sham group), reactive oxygen species scavenging group (TN + CS / GP@Mn-POM group), and TRPA1 channel antagonism group (TN + CS / GP-HC030031 group) within 3 weeks after treatment. Figure 1 -C and D represent the results of immunofluorescence staining and semi-quantitative fluorescence analysis of trigeminal ganglion tissue from SD rats in the model group (TN group), sham operation group (Sham group), reactive oxygen species scavenging group (TN + CS / GP@Mn-POM group), and TRPA1 channel antagonism group (TN + CS / GP-HC030031 group).

[0018] Figure 2 The figure shows the test results of an embodiment of the present invention; Figure 2 -A shows the Nrf2 gene silencing effect constructed using different siRNAs; Figure 2 -B and C show the Western blot (WB) results of the following trigeminal neuron cell groups: reactive oxygen species stimulation group (H2O2 group), control group (NS group), reactive oxygen species scavenging group (H2O2 + Mn group), TRPA1 channel antagonist group (H2O2 + HC030031 group), and NRF2 gene silenced trigeminal neuron cell group (H2O2 + Si-Nrf group).

[0019] Figure 3 This is a schematic diagram illustrating the mechanism of the application of the present invention in the preparation of drugs for the prevention or treatment of trigeminal neuralgia. Figure 3 -A, B, C, D, E, F, G, H, and I represent the sample correlation analysis diagram, inter-group difference Venn diagram, inter-group gene difference diagram, and GO enrichment analysis diagram after gene detection analysis of the model group (TN group), sham operation group (Sham group), and treatment group (POM group), respectively. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] The application of substances that regulate the pain microenvironment and reshape the balance of ascending and descending pain receptors in the preparation of drugs for the prevention and treatment of trigeminal neuralgia. These substances include those that improve the oxidative stress microenvironment and those that reshape the balance of ascending and descending pain receptors. In other words, the pain microenvironment and the balance of ascending and descending pain receptors serve as targets for the treatment of trigeminal neuralgia. The regulatory role of the pain microenvironment and the balance of ascending and descending pain receptors in trigeminal neuralgia is as follows: Immune cell oxidative stress: increased ROS levels → activation of TRPA1 channels in nerve cells → production of pain mediators → satellite glial cells: increased inflammatory factors → oxidative stress – pathological cycle. By scavenging reactive oxygen species (ROS) and inhibiting TRPA1 channel expression, the mechanical hyperalgesia of the oromaxillofacial region in model animals can be effectively alleviated.

[0022] The substances that improve the microenvironment of oxidative stress include reactive oxygen species scavengers and enzyme-like active nanomaterials, with the enzyme-like active nanomaterial being Mn-POM (manganese-based polyoxometalate).

[0023] The substances that reshape the balance of the ascending pain perception-descending inhibition pathway include substances that inhibit TRPA1 channel protein activity and TRPA1 channel protein gene expression. The substances that inhibit TRPA1 channel protein activity include RPA1 channel antagonists, specifically the HC-030031 model. The substances that inhibit TRPA1 channel protein gene expression include NRF2 gene silencing reagents. TRPA1 channels regulate the ascending pain perception-descending inhibition pathway. The NRF2 gene silencing reagents include nucleotide siRNA reagents, nucleotide shRNA reagents, nucleotide miRNA reagents, and nucleotide ASO reagents. The siRNA, shRNA, and CRISPR-Cas9 gene editing system targeting the NRF2 gene uses NRF2 gene silencing reagents and induces NRF2 gene silencing in TG cells via liposome transfection, thereby resulting in a reduced TRPA1 channel expression level.

[0024] The substances that regulate the pain microenvironment and reshape the balance of ascending and descending pain perception pathways alleviate orofacial pain sensitivity by improving the oxidative stress microenvironment and downregulating TRPA1 channel expression levels, thereby achieving the therapeutic effect of trigeminal neuralgia.

[0025] A kit for detecting trigeminal neuralgia includes reagents for detecting reactive oxygen species (ROS) and TRPA1 channel protein expression levels. ROS and TRPA1 channel protein expression levels are diagnostic biomarkers for trigeminal neuralgia and are detected using methods such as RT-PCR, Western blot, and immunofluorescence.

[0026] The methods for screening drugs for the prevention and treatment of trigeminal neuralgia can be carried out from the following aspects: constructing trigeminal neuralgia model cells / animals, exposing candidate compounds, detecting changes in reactive oxygen species and TRPA1 channel expression and pain-related indicators (such as pain threshold and inflammatory factor levels), and screening for compounds that can improve the reactive oxygen species environment, downregulate TRPA1 channel expression levels, and alleviate pain.

[0027] Experimental example:

[0028] 1. Target: Establish a rat model of trigeminal neuralgia (CT-guided injection of talc suspension into the infraorbital foramen). Through related experiments, confirm the regulatory role of the pain microenvironment and the balance of ascending and descending pain perception pathways in trigeminal neuralgia.

[0029] In a rat model of trigeminal neuralgia, the downregulation of inflammatory mediators and related pain substances was detected by improving the pain microenvironment and regulating the ascending-descending pain perception-inhibition pathway using the TRPA1 channel.

[0030] Under CT guidance, 0.2 ml of 20% talc suspension and 0.2 ml of physiological saline were injected into the infraorbital foramen of SD rats from different groups to establish a trigeminal neuralgia model group (TN group) and a sham-operated group (Sham group). One week after surgery, reactive oxygen species scavengers (CS / GP@Mn-POM) and TRPA1 channel antagonists (CS / GP-HC030031) were injected around the trigeminal nerve into some model group rats, respectively, to establish a reactive oxygen species scavenging group (TN + CS / GP@Mn-POM group) and a TRPA1 channel antagonist group (TN + CS / GP-HC030031 group). Changes in the mechanical pain threshold of the rat maxillofacial region were detected by von Frey pain measurement fibers, and changes in the expression levels of related inflammatory factors and pain mediators in trigeminal neuralgia were detected by immunofluorescence (IF) assay.

[0031] The results are as follows Figure 1 As shown, in Figure 1 In the A-group, after CT-guided injection of talc suspension, the mechanical pain threshold of the face in the model group (TN group) was significantly lower than that in the sham-operated group (Sham group), and remained below 8g for 3 consecutive days, indicating that CT-guided injection of talc suspension into the infraorbital foramen successfully induced trigeminal neuralgia in rats; Figure 1 In the -B group, by improving the pain microenvironment and regulating the ascending-descending pain perception pathway, the mechanical pain threshold of the reactive oxygen species scavenging group (TN + CS / GP@Mn-POM group) and the TRPA1 channel antagonist group (TN + CS / GP-HC030031 group) was improved compared with that of the trigeminal neuralgia model group (TN group), and gradually recovered to the level of the sham surgery group (Sham group). Figure 1- Figures C and D show that the levels of corresponding neurotransmitters and pain mediators (SP, IL-1β, TNF-α) were significantly increased in the trigeminal neuralgia model group (TN group). After improving the pain microenvironment and regulating the ascending-descending pain perception pathway using TRPA1 channels, the expression of inflammatory factors and related pain mediators decreased in the reactive oxygen species scavenging group (TN + CS / GP@Mn-POM group) and the TRPA1 channel antagonist group (TN + CS / GP-HC030031 group).

[0032] 2. After constructing NRF2 gene-silenced trigeminal neurons, we verified that ROS upregulates TRPA1 channel protein expression mediated by the NRF2 gene, thereby clarifying its important role in sensory neuron overactivity and the imbalance of ascending pain perception-descending inhibition pathway.

[0033] Liposome transfection was used to introduce siRNA targeting the NRF2 gene into TG cells, inducing NRF2 gene silencing in TG cells. NRF2-silenced trigeminal neurons and normal trigeminal neurons were stimulated with hydrogen peroxide to construct a reactive oxygen species (ROS) stimulation group (H2O2 + Si-Nrf group) for NRF2-silenced trigeminal neurons and a ROS stimulation group (H2O2 group) for normal trigeminal neurons. A ROS scavenger (Mn) and a TRPA1 channel antagonist (HC030031) were added to some of the ROS-stimulated normal trigeminal neurons to construct an ROS scavenger group (H2O2 + Mn group) and a TRPA1 channel antagonist group (H2O2 + HC030031 group), respectively. The control group (NS group) was treated with saline only. Western blot analysis (WB) was used to detect changes in TRPA1 channel expression levels in TG cells.

[0034] The results are as follows Figure 2 As shown, Figure 2 The Nrf2 expression level in the siRNA-3 group (-A) was significantly lower than that in other groups, indicating that siRNA-3 can significantly reduce NRF2 gene transcription; after silencing the NRF2 gene in TG cells with siRNA-3, as... Figure 2As shown in B and C, the following groups were set up: normal trigeminal neuron cell reactive oxygen species stimulation group (H2O2 group), control group (NS group), reactive oxygen species scavenging group (H2O2 + Mn group), TRPA1 channel antagonist group (H2O2 + HC030031 group), and NRF2 gene silenced trigeminal neuron cell group (H2O2 + Si-Nrf group). Western blot experiments showed that ROS can upregulate TRPA1 channel protein expression through NRF2 gene mediation. Improving the reactive oxygen species environment and silencing the NRF2 gene in TG cells can produce a similar effect to channel antagonists, regulating the ascending perception-descending inhibition pathway of pain, thereby reducing the pain sensitivity of trigeminal neuralgia.

[0035] 3. Molecular mechanism verification:

[0036] To investigate the role of the pain microenvironment and the balance of ascending and descending pain perception pathways in trigeminal neuralgia.

[0037] Trigeminal neuralgia model group (TN group) and sham operation group (Sham group) were constructed by injecting 0.2 ml of 20% talc suspension and 0.2 ml of physiological saline into the infraorbital foramen of SD rats in different groups under CT guidance. One week after the operation, reactive oxygen species (POM) scavengers were injected into the peritrichus of some model rats to construct the reactive oxygen species scavenging group (POM group). Trigeminal ganglion tissues from different groups of SD rats were taken for RNA transcriptome gene sequencing.

[0038] The results are as follows Figure 3 As shown, Figure 3 - The correlation coefficients between replicate samples under the same experimental conditions in A are all >0.95, proving the reliability of the samples, while the correlation coefficients between samples from different treatment groups are <0.9, indicating that there are significant differences in genes between treatment groups; Figure 3 -B~E show the number of differentially expressed genes between the sham surgery group and the pain model group (Sham vs TN group), the sham surgery group and the treatment group (Sham vs POM group), and the pain model group and the treatment group (TN vs POM group); Figure 3-F~I, through a combination of differential gene expression analysis and GO enrichment analysis, for the first time reflected the direct regulation of TG gene activation, TRP channel inflammatory mediator release, pain perception, neuroactive ligand-receptor interaction, and differential expression in glial cells from two dimensions: differential gene expression and functional pathway enrichment. Furthermore, it revealed the biological functions of TG through pathways such as the MAPK signaling pathway and the calcium ion signaling pathway, systematically elucidating the pain microenvironment within TG and the mechanism of the ascending-descending pain perception-inhibition pathway: immune cell oxidative stress (increased ROS levels) → nerve cell TRPA1 channel activation → pain mediator production → satellite glial cell activation (increased inflammatory factors) → oxidative stress pathological cycle. This provides direct and comprehensive experimental evidence for new applications of pain microenvironment regulation and reshaping the ascending-descending pain perception-inhibition pathway balance in trigeminal neuralgia.

[0039] Treatment methods: Based on the above experiments, any substance that can improve the oxidative stress microenvironment, inhibit TRPA1 channel protein activity, or inhibit the gene can be used to prepare the drug of this invention. Including but not limited to:

[0040] Nucleic acid drugs: siRNA, shRNA, miRNA, and ASO (antisense oligonucleotides) targeting the TRPA1 channel gene.

[0041] Protein / antibody drugs: Specific antibodies, nanobodies, or peptides that can scavenge reactive oxygen species and bind to and antagonize the function of TRPA1 channel protein.

[0042] Small molecule compounds: Small molecule agonists obtained through high-throughput screening that can improve the oxidative stress microenvironment, antagonize TRPA1 channel protein binding, or inhibit its function.

[0043] In summary, this invention discloses the application of substances related to the pain microenvironment and the ascending-descending pain perception-inhibition pathway in the preparation of drugs for the prevention or treatment of trigeminal neuralgia. It also verifies that after trigeminal nerve injury, the levels of reactive oxygen species (ROS) and TRPA1 channel protein expression increase in trigeminal neuralgia (TG). Improving the oxidative stress environment and silencing the NRF2 gene to assist in the regulation of TRPA1 channel protein can effectively downregulate the expression of pro-analgesic and inflammatory factors, significantly reducing orofacial pain sensitivity. This invention uses the pain microenvironment and the ascending-descending pain perception-inhibition pathway as novel targets, providing specific drugs and diagnostic tools for the treatment of trigeminal neuralgia, and has significant clinical value.

Claims

1. Application of substances that regulate the pain microenvironment and reshape the balance of ascending and descending pain receptor pathways in the preparation of drugs for the prevention and treatment of trigeminal neuralgia. Substances that regulate the pain microenvironment and reshape the balance of ascending and descending pain receptor pathways include substances that improve the oxidative stress microenvironment and substances that reshape the balance of ascending and descending pain receptor pathways.

2. The use of the substance for regulating the pain microenvironment and remodeling the balance of the pain ascending perception and descending inhibition pathway according to claim 1 in the preparation of drugs for preventing and treating trigeminal neuralgia, characterized in that, The substances that improve the microenvironment of oxidative stress include reactive oxygen species scavengers and enzyme-like nanomaterials.

3. The application of the substance according to claim 1, which regulates the pain microenvironment and reshapes the balance of ascending and descending pain pathways, in the preparation of drugs for the prevention and treatment of trigeminal neuralgia, characterized in that, The substances that reshape the balance of the ascending pain perception-descending inhibition pathway include substances that inhibit TRPA1 channel protein activity and TRPA1 channel protein gene expression. Substances that inhibit TRPA1 channel protein activity include RPA1 channel antagonists, and substances that inhibit TRPA1 channel protein gene expression include NRF2 gene silencing agents. TRPA1 channels regulate the ascending pain perception-descending inhibition pathway.

4. The application of the substance according to claim 1, which regulates the pain microenvironment and reshapes the balance of ascending and descending pain pathways, in the preparation of drugs for the prevention and treatment of trigeminal neuralgia, characterized in that, The NRF2 gene silencing reagents include nucleotide siRNA reagents, nucleotide shRNA reagents, nucleotide miRNA reagents, and nucleotide ASO reagents.

5. The application of the substance according to claim 1, which regulates the pain microenvironment and reshapes the balance of ascending and descending pain pathways, in the preparation of drugs for the prevention and treatment of trigeminal neuralgia, characterized in that, The substances that regulate the pain microenvironment and reshape the balance of ascending and descending pain perception pathways can alleviate orofacial pain sensitivity by improving the oxidative stress microenvironment and downregulating TRPA1 channel protein expression levels, thereby achieving the therapeutic effect of trigeminal neuralgia.

6. A reagent kit for detecting trigeminal neuralgia, characterized in that, This includes reagents for detecting reactive oxygen species and TRPA1 channel protein expression levels.

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