Use of mas-related g protein coupled receptor d in the peripheral nervous system in alleviating opioid tolerance

Abstract

The application discloses application of a peripheral nervous system Mas-related G protein-coupled receptor D as a target point in preparation of a medicine for screening for relieving opioid tolerance. Jun The application further provides a medicine composition for regulating opioid tolerance, containing a Mas-related G protein-coupled receptor D siRNA sequence shown in SEQ ID NO. 1 or a siRNA sequence shown in SEQ ID NO. 2. The application further provides an in-situ hybridization kit for detecting expression of a Mas-related G protein-coupled receptor D gene in a dorsal root ganglion tissue of a peripheral nervous system. The application expands a new path for opioid companion diagnosis and intervention.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a product for relieving opioid drug tolerance by Mrgprd in the peripheral nervous system. BACKGROUND

[0002] In clinical practice, opioids such as morphine and fentanyl are widely used for perioperative analgesia, cancer pain and chronic pain management due to their significant analgesic effect. However, the use of opioids can cause serious adverse reactions, and long-term use can lead to tolerance and addiction, limiting its application. The mechanism of opioid tolerance is complex, and there is still a lack of effective solutions in clinical practice.

[0003] In clinical practice, multi-modal analgesia is often used to reduce the dosage of opioids by providing analgesic effect through different mechanisms of action, such as opioid rotation, NMDA receptor antagonist combination, neural-glial cell regulation intervention, use of kappa-opioid receptor agonists / antagonists, multi-modal analgesia and dose reduction strategies. However, existing tolerance intervention strategies focus on the central nervous system, which has the following shortcomings: ① It is difficult to precisely target the root path of tolerance formation; ② Central action is easy to cause cognitive and mental side effects; ③ There is a lack of feasible intervention targets for peripheral analgesic pathways; ④ There is no effective prevention and control method for the early stage of opioid tolerance in clinical practice. Therefore, there is an urgent need for a new technology based on the peripheral nervous system that is precise and safe, to intervene in the occurrence of analgesic tolerance, thereby prolonging the efficacy period of opioid drugs, reducing the total dosage and side effect risk, which is still a difficult problem that needs to be solved in clinical practice.

[0004] The dorsal root ganglion (DRG) is a key node in the peripheral nervous system, which collects nociceptive information from the periphery to the central nervous system. Opioids directly act on the peripheral nervous system through the mu opioid receptor (MOR) in the DRG, reducing the transmission of pain signals. Therefore, selectively regulating MOR expression and function in peripheral afferent neurons will be a promising strategy to alleviate opioid tolerance such as morphine and fentanyl, while limiting central side effects. However, there is a lack of research on the regulation mechanism of opioid tolerance in DRG in existing technology, especially a lack of systematic study on peripheral gene changes during the early stage of opioid application.

[0005] The applicant found that Mas-related G protein-coupled receptor D (Mrgprd) specifically expressed in the peripheral nervous system plays an important role in the regulation of opioid tolerance. The present application adopts DRG transcriptome sequencing analysis, morphological experiment, cell molecular biology experiment, animal behavior pharmacology experiment and other methods, and for the first time finds that Mrgprd appears a dynamic expression pattern of rapid down-regulation in the early stage of opioid use (6 hours to 24 hours) and recovery in the later stage, and in animal experiments, it is confirmed that it has a negative regulation on the formation of tolerance: inhibiting Mrgprd expression (siRNA, gene knockout) significantly alleviates tolerance; activating Mrgprd accelerates the formation of tolerance; the expression regulation of Mrgprd is closely related to the response of specific neuron subgroups; Mrgprd and its downstream signals can regulate MOR expression. The present application finds that inhibiting the effect of Mrgprd in DRG regulates MOR expression to alleviate morphine tolerance, establishes a new opioid tolerance regulation path with DRG in the peripheral nervous system as the core, especially with Mas-related G protein-coupled receptor D (Mrgprd) as the key node, adjusts the expression and efficacy sensitivity of mu opioid receptor (MOR), finds a new target for clinical drug treatment of opioid tolerance, and the effect is accurate, completely acts on the peripheral DRG neurons, reduces the central side effects such as causes, consciousness disorders, cognitive side effects, is safer and does not cause cognitive disorders and mental side effects compared with the previous central target intervention, improves the safety and specificity of intervention, can realize intervention in the early stage of opioid use, is suitable for patients in perioperative period, chronic pain and cancer, and has a wide application prospect. SUMMARY

[0006] In view of the deficiencies of the prior art and actual needs, the present application establishes a new opioid tolerance regulation path with DRG in the peripheral nervous system as the core, especially with Mas-related G protein-coupled receptor D (Mrgprd) as the key node, regulates the expression and efficacy sensitivity of mu opioid receptor (MOR) around the regulation system of the signal pathway "c-Jun-Mrgprd-DS-lncRNA-Ehmt2 / G9a-MOR", and realizes the purpose of alleviating opioid analgesic tolerance by interfering with the path. The present application for the first time finds that Mrgprd appears a dynamic expression pattern of rapid down-regulation in the early stage of opioid use (6 hours to 24 hours) and recovery in the later stage, and in animal experiments, it is confirmed that it has a negative regulation on the formation of tolerance: the experimental results show that inhibiting Mrgprd expression (siRNA, gene knockout) significantly alleviates tolerance; and activating Mrgprd accelerates the formation of tolerance; it is confirmed that the expression regulation of Mrgprd is closely related to the response of specific neuron subgroups; Mrgprd and its downstream signals can regulate MOR expression, and a new target for clinical drug treatment of morphine tolerance is found.

[0007] The application adopts the following technical solutions:

[0008] The application provides application of a peripheral nervous system Mas-related G protein-coupled receptor D, namely Mrgprd, as a target point in preparation of a medicine for relieving opioid drug tolerance.

[0009] Further, the medicine takes Mrgprd as a target point for regulation and control, reduces expression of the peripheral nervous system Mrgprd in a subject or inhibits activity of Mrgprd, thereby inhibits a c-Jun-Mrgprd-DS-lncRNA-Ehmt2 / G9a-MOR signal pathway, and further relieves opioid drug tolerance.

[0010] Further, the medicine reduces expression or activity of the Mas-related G protein-coupled receptor D by one of the following modes: a) antibody neutralization; b) small molecule inhibitor blocking an active site; c) epigenetic modifier regulating promoter activity; d) viral vector expression or gene editing means targeting an upstream regulatory factor of Mrgprd.

[0011] Further, the reduction of expression or inhibition of activity of Mrgprd is achieved by giving a siRNA sequence targeting the Mrgprd gene or using gene editing technology to knock out the Mrgprd gene or by inhibiting expression of a transcription factor c-Jun.

[0012] Further, the siRNA sequence targeting the Mrgprd gene is shown as SEQ ID NO. 1.

[0013] Further, the inhibition of expression of the transcription factor c-Jun is achieved by giving a Jun siRNA sequence shown as SEQ ID NO. 2.

[0014] The application further provides a siRNA targeting the Mas-related G protein-coupled receptor D gene, and a nucleotide sequence of the siRNA is shown as SEQ ID NO. 1.

[0015] The application further provides a Jun siRNA targeting the Mas-related G protein-coupled receptor D gene, and a nucleotide sequence of the Jun siRNA is shown as SEQ ID NO. 2.

[0016] The application further provides a medicine composition for relieving opioid drug tolerance, the medicine composition comprising an active ingredient and a pharmaceutically acceptable carrier, and the medicine composition delays analgesic tolerance caused by long-term use of opioids by reducing expression of Mrgprd.

[0017] Further, the active ingredient is the Mrgprd siRNA sequence shown in SEQ ID NO. 1 or the Jun siRNA sequence shown in SEQ ID NO. 2.

[0018] Further, the active ingredient is the Mrgprd siRNA sequence shown in SEQ ID NO. 1.

[0019] Further, the pharmaceutically acceptable carrier is a cationic liposome encapsulation system.

[0020] Further, the mass ratio of the active ingredient to the encapsulation system is 1:10.

[0021] Further, the above pharmaceutical composition is used for preclinical, perioperative, chronic pain and cancer patients, and can reduce the effective dosage of opioid drugs, prolong the duration of analgesia, and delay the tolerance of opioid drugs.

[0022] The application also provides a kit for detecting Mrgprd expression, comprising a targeting probe, a color developing system, and a tissue treatment solution.

[0023] Further, the targeting probe is an Mrgprd-mRNA probe labeled with Cy5 designed by RNAscope®, the color developing system comprises DAPI nuclear staining and TSA signal enhancer substrate, and the tissue treatment solution comprises a 4% polyformaldehyde fixing solution and a 0.1% Triton X-100 penetrating solution.

[0024] Further, the detection sample comprises a subject dorsal root ganglion tissue, and the detection method is in situ hybridization or in situ hybridization combined with immunofluorescent antibody detection.

[0025] Further, the detection sample is a subject dorsal root ganglion DRG tissue section, and the section thickness is 8–12 μm, that is, the kit is suitable for Mrgprd expression detection of a DRG frozen section tissue with a thickness of 8–12 μm.

[0026] Further, the detection method is to set the thickness of the frozen DRG tissue section to 10 μm, perform hybridization after fixation and penetration, incubate at room temperature for 2 hours, perform DAPI staining after color developing treatment, and take a picture for analysis using a laser confocal microscope.

[0027] The application also provides application of the above kit in molecular typing auxiliary judgment of individual use of opioid analgesic drugs, which judges the tolerance of a subject to opioid drugs by detecting the expression level of Mrgprd, and provides an expression genotyping auxiliary basis for individual use of opioid analgesic drugs.

[0028] Specifically, the prediction of the sensitivity or tolerance risk of a patient to an opioid drug is achieved by detecting the expression amount of Mas-related G protein-coupled receptor D in the sample and the correlation with analgesia.

[0029] The opioid drug tolerance relief disclosed in the present application is achieved by not only giving a synthetic siRNA sequence targeting Mrgprd gene or using a gene editing technology to knock out the Mrgprd gene, but also by expressing c-Jun (which is an upstream negative regulator of Mrgprd) through a viral vector, or by inhibiting the expression of Mrgprd by giving an ASO oligonucleotide, or by inhibiting the expression of a newly identified long-chain non-coding RNA, i.e., DS-lncRNA, through a viral vector (such as AAV), or by developing a small molecule drug to regulate the activities of Mrgprd and c-Jun, so as to restore the expression of MOR, relieve the tolerance, and prolong the analgesic effect.

[0030] The present application takes Mrgprd as the core target point: Mrgprd appears rapid down-regulation in the early use of opioids (6-24 hours); inhibiting the expression of Mrgprd can significantly enhance the analgesic effect of morphine and delay the occurrence of tolerance; activating Mrgprd will accelerate the formation of tolerance, suggesting that it is a negative regulator.

[0031] The present application also identifies the Mrgprd downstream lncRNA signal axis: when the expression of Mrgprd is up-regulated, a newly identified long-chain non-coding RNA (DS-lncRNA) is inhibited; DS-lncRNA combines with Ehmt2 (G9a), reduces the histone methyltransferase activity thereof; thereby removing the methylation inhibition of the Oprm1 promoter, and promoting the transcription of mu opioid receptor (MOR).

[0032] The intervention path of the present application is reversible and controllable: Mrgprd is inhibited by siRNA, or c-Jun (which is an upstream negative transcription factor of Mrgprd) is expressed by a viral vector; the above-mentioned methods can restore the expression of MOR, relieve the tolerance, and prolong the analgesic effect.

[0033] Meanwhile, the present application verifies the efficacy in animal models: the above-mentioned intervention strategies are used in morphine tolerance mouse models; the effectiveness of the signal path is verified by multiple means such as behavior, molecular detection, and RNAscope; and it is clear that the intervention effect is significantly better than the traditional strategy (such as dose reduction or opioid rotation).

[0034] The molecular regulation mechanism of the present application is as shown in Figure 1 . Figure 1Figure 1: Schematic diagram of Mrgprd-mediated μ opioid receptor regulatory pathway, which describes the molecular regulatory mechanism disclosed by the present application: in DRG neurons, the expression of transcription factor c-Jun is reduced, which reduces the expression of Mrgprd; Mrgprd can negatively regulate DS-lncRNA, thereby affecting the expression of epigenetic regulatory factor Ehmt2 / G9a, and ultimately regulating the expression level of μ opioid receptor (MOR) gene Oprm1, affecting the analgesic effect and tolerance formation of opioid drugs (such as morphine)

[0035] The four core nodes that can be used for intervention in the present application are as shown in Figure 7 Figure 7 Figure 2: Schematic diagram of molecular intervention nodes in the Mrgprd regulatory pathway, which shows the four core nodes that can be used for intervention in the present application: c-Jun, Mrgprd, DS-lncRNA, and Ehmt2, and indicates the intervention methods (such as siRNA, overexpression vector, small molecule inhibitor, etc.) that can be used for each node.

[0036] The present application differs from the intervention methods of traditional focus on central system late tolerance mechanism, and for the first time focuses on the dynamic expression changes of peripheral DRG neurons in the early stage (6-24 hours) of opioid use, recognizes and intervenes in the tolerance process in advance, and has strategic value of forward-looking prevention. The present application discloses for the first time an epigenetic regulatory axis in peripheral DRG dominated by Mrgprd and mediated by DS-lncRNA, which has a decisive regulatory effect on the expression of μ-opioid receptor (MOR), and fills a key gap in the existing opioid tolerance mechanism research.

[0037] Compared with the prior art, the technical effect of the present application is positive and obvious. The present application discloses a peripheral non-canonical receptor regulatory opioid tolerance pathway, and clearly discloses that Mrgprd in DRG mediates opioid drug tolerance by regulating Ds-lncRNA, and constructs a complete and intervenable signal pathway axis, which is used for relieving the analgesic tolerance caused by long-term use of opioid drugs (such as morphine, fentanyl, etc.), and has great application value and prospect. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Figure 1: Schematic diagram of Mrgprd-mediated μ opioid receptor regulatory pathway.

[0039] Figure 2 Figure 2: Schematic diagram of molecular intervention nodes in the Mrgprd regulatory pathway.

[0040] Figure 3 Figure 3: Gene expression differences in DRG in a morphine tolerance model.

[0041] Figure 4 Figure 4: Expression of Mrgprd in DRG after intraperitoneal injection of morphine.

[0042] ​Figure 5 : Pain behavior of mice after Mrgprd expression in DRG is decreased after siRNA administration.

[0043] Figure 6 : Opioid tolerance intervention method for down-regulating Mrgprd by inhibiting expression of transcription factor c-Jun.

[0044] Figure 7 : Diagram of molecular intervention nodes in Mrgprd regulatory pathway. DETAILED DESCRIPTION

[0045] The specific embodiments of the present application and the technical solutions are further described in detail below in combination with the drawings and specific examples. In order to realize the new strategy for alleviating opioid tolerance proposed by the present application, the applicant has verified the Mrgprd related regulatory pathway from three dimensions of molecules, tissues and behaviors through multiple experiments. The specific embodiments give the preferred embodiments, and the following three preferred embodiments specifically describe the technical features, implementation modes and biological effects of the present application to illustrate that the present application has good feasibility and repeatability under multiple experimental conditions. The reagents, equipment and methods used in the present application are all conventional reagents, equipment and methods in the art. The conventional commercially available products with the source and specific model number are marked, and the test methods and specific conditions are carried out according to the test methods and conditions in the instruction manual of the corresponding products. If there is no special instruction, the raw materials, methods and equipment used can be purchased from conventional commercially available products. The reagents, equipment and test methods are all conventional reagents, equipment and methods in the art.

[0046] Example 1: Search for peripheral regulatory target

[0047] Experimental steps: as follows Figure 2As shown, bilateral DRGs from mice (L4 / L5) were collected (based on experimental grouping time points: Ctrl, 6h, 4d, 10d). Strict prevention of RNase contamination was maintained during the procedure, and tissues were immediately cryopreserved in liquid nitrogen after collection. Total RNA was extracted using the Trizol method, and RNA concentration and integrity were quality controlled using NanoDrop, Qubit, and Agilent Bioanalyzer. RNA meeting the standards was used for library construction. The library was constructed using the Illumina platform, including mRNA enrichment, fragmentation, cDNA synthesis, end repair, A addition, adapter ligation, and PCR amplification. After Qubit and Agilent sequencing, the library was sequenced using PE150 (NovaSeq 6000 platform), with a data volume of 6G / sample. Adapters, low-quality sequences, and rRNA contamination were removed from the raw reads to obtain Clean Reads. The Clean Reads were aligned to the mouse genome using Hisat2 software (GRCm38.p4, mm10 version), and a BAM file was output. Gene expression levels were normalized to FPKM values. Differentially expressed genes between different groups were analyzed using edgeR (threshold: q-value ≤ 0.05 and Fold change ≥ 2), and GO and KEGG enrichment analyses were performed.

[0048] Experimental results are as follows Figure 3 As shown, RNA transcriptome sequencing was performed on DRG tissues from four different time points (Ctrl, 6h, 4d, 10d) in a morphine tolerance model. Principal component analysis (PCA) was then used to remove outliers, allowing for in-depth data analysis. Differential gene analysis revealed a total of 2250 differentially expressed genes (DEGs). Figure 3 A). Among these differentially expressed genes, we particularly noted the expression of the Mrgprd gene. Comparison revealed that in samples 6 hours after morphine administration, the expression level of Mrgprd was significantly lower than that of the control group, while in samples 4 and 10 days after administration, the expression level of Mrgprd was not significantly different from that of the control group. Figure 3 B).

[0049] Furthermore, through in-depth analysis of single-cell sequencing data, we also observed a significant decrease in the proportion of neurons expressing Mrgprd in samples 6 hours after morphine administration. Figure 3 C). This finding suggests that in the early stages of morphine action, specific neuronal subsets (Mrgprd) may respond to drug exposure by adjusting their gene expression patterns, thereby influencing neural pathways for pain transmission.

[0050] Example 2: Expression of Mrgprd in DRG after morphine i.p.

[0051] Experimental procedure: To accurately explore the trend of Mrgprd expression, we continuously monitored the expression of Mrgprd in wild-type mice at different time points from 0 to 96 hours after morphine i.p. using QPCR method. Total RNA was extracted from mouse L4 / L5 DRG using Trizol method, and the expression levels of Mrgprd, Oprm1 and other genes were detected by real-time fluorescent quantitative PCR method, with Gapdh as the internal reference for normalization. At the same time, the spatial localization and co-expression relationship of Mrgprd gene in DRG tissue were clarified by using RNAscope in situ hybridization technology combined with frozen section samples. In this experiment, 4% chloral hydrate anesthesia, cardiac perfusion fixation were used to ensure the integrity of the tissue structure, and OCT embedding was performed after tissue dehydration for downstream in situ hybridization or immunostaining operation. The RNAscope probe (Cat NO. 417921) and Multiplex Fluorescent Detection Reagent v2 kit (Cat NO. 323110) used were purchased from Advanced Cell Diagnostics company, and the experimental procedure followed the standard operation steps to ensure the specificity of hybridization and signal intensity.

[0052] The experimental results are shown in Figure 4 Mrgprd expression showed a downward trend after morphine action, especially within 3 to 6 hours after administration, its expression decreased by about 30%, and decreased to the lowest point at 24 hours Figure 4 B). Further, we compared the Mrgprd expression in the control group (Ctrl group) and the morphine first administration 24 hours later and continuous administration for 4 days and 10 days in the morphine tolerance model. The results showed that 24 hours after single administration of morphine, the expression of Mrgprd decreased by 60-70% compared with the control group, and after continuous administration for 4 to 10 days, there was no significant difference in its expression compared with the control group, indicating that Mrgprd expression gradually recovered after long-term morphine use (Figure 4C). In addition, the results of RNAscope in situ hybridization (ISH) technology showed that in the control group, Mrgprd + Neurons account for about 30-40% of DRG neurons, and after 24 hours of morphine i.p. administration, it decreased to about 25%, and after continuous administration for more than 4 days, the proportion of Mrgprd + neurons showed no significant difference compared with the control group Figure 4 A, 4D). The experimental results of QPCR and RNAscope ISH further verified the accuracy and reliability of the transcriptome sequencing data.

[0053] Example 3: An opioid tolerance intervention method based on siRNA regulation of Mrgprd expression.

[0054] Indications: For the treatment or prolongation of analgesia tolerance caused by prolonged use of opioids (such as morphine);

[0055] Operation steps: 1) Synthesize siRNA targeting the Mrgprd gene, with the sequence shown in SED ID No. 1:

[0056] SED ID No. 1: 5'-CCUGUCAUCAGUGGUAUCUTT-3'.

[0057] 2) The siRNA was encapsulated in cationic liposomes to prepare an injectable formulation;

[0058] 3) In mice with an opioid analgesia model, intrathecal injections of 2 μg / 10 μl were administered on days 1 and 3 of drug administration; morphine 10 mg / kg was administered intraperitoneally daily for 7 consecutive days.

[0059] 4) Use a tail-flick device to assess the duration and latency of analgesia;

[0060] The results are attached. Figure 5 As shown, Figure 5 To inhibit Mrgprd expression using siRNA, the control group received intrathecal injection of ScramblesiRNA ( Figure 5 A), or a morphine tolerance model can be established using Mrgprd-KO mice with Wild Type mice as littermates to detect pain behavior. Figure 5 D). The results showed that Mrgprd expression in the siRNA group was reduced to 30% of that in the Scramble control group (D). Figure 5 B), mice with suppressed Mrgprd expression showed a significantly prolonged duration of analgesic effect after multiple administrations, a rightward shift in the pain response curve, and a marked slowing of tolerance development. Figure 5 CD). This demonstrates the actual efficacy of the intervention strategy proposed in this invention and supports Mrgprd as a key regulatory factor.

[0061] Example 4: An opioid tolerance intervention method that downregulates Mrgprd by inhibiting the expression of transcription factor c-Jun.

[0062] Experimental model: Eight-week-old male C57BL / 6J mice were used; an opioid tolerance model was established by continuous intraperitoneal injection of morphine hydrochloride (10 mg / kg / day) for 10 days.

[0063] Intervention strategy: Construct Jun siRNA to inhibit its expression (e.g., Figure 6), with Scramble siRNA as control;

[0064] Jun siRNA sequence is shown in SED ID No. 2: SED ID No. 2: 5'-CCAAGAACUCGGACCUUCUTT-3'.

[0065] Experimental results: Bioinformatics search analysis found that the Mrgprd promoter has a conserved c-Jun binding site in the +185 to +191 region. ChIP-qPCR experiments verified that c-Jun can directly bind to the Mrgprd promoter in DRG neurons. And through the dual luciferase experiment, it is confirmed that c-Jun overexpression can significantly enhance the activity of Mrgprd promoter ( Figure 6 a-b). In vivo, intrathecal injection of Jun siRNA can reduce c-Jun expression by about 70%, and significantly down-regulate Mrgprd mRNA levels, while delaying the formation of morphine tolerance, which is similar to direct inhibition of Mrgprd ( Figure 6 c-d); while in Mrgprd knockout mice, further inhibition of c-Jun cannot further delay tolerance, suggesting that the role of c-Jun is achieved by regulating Mrgprd ( Figure 6 e-f). And the decline of Mrgprd does not affect the expression of c-Jun, indicating that the two are a one-way regulatory relationship (c-Jun→Mrgprd) ( Figure 6 g-h). In summary, c-Jun is a direct upstream regulator of Mrgprd, which plays a key role in the early molecular response of morphine exposure, and regulates the occurrence of opioid tolerance by regulating Mrgprd expression.

[0066] The above experimental results clearly prove that the path of reducing Mrgprd expression by inhibiting c-Jun can achieve tolerance relief.

[0067] Example 5: Nucleic acid pharmaceutical composition containing Mrgprd-siRNA

[0068] Product composition: siRNA sequence: 5'-CCUGUCAUCAGUGGUAUCUTT-3'; wrapping system: cationic liposome (DOTAP / Chol); mass ratio: siRNA:liposome = 1:10; dilution buffer: pH 7.4 HEPES buffer;

[0069] Preparation steps: siRNA and liposome are incubated at room temperature for 30 minutes to form a complex; sterilized by 0.22 μm filter; aliquot into light-proof cryotubes, stored at −80℃;

[0070] The present composition can be used for peripheral intrathecal injection for assisting analgesic treatment; it can also be combined with analgesic pump system as a continuous infusion solution.

[0071] Example 6: In situ hybridization kit for Mrgprd expression detection

[0072] Product composition: Target probe: Mrgprd-mRNA probe designed by RNAscope® (labeled with Cy5); color developing system: DAPI nuclear staining + TSA signal enhancer substrate; tissue treatment solution: 4% polyformaldehyde (fixing solution) and 0.1% Triton X-100 (penetrating solution);

[0073] Detection process: The thickness of the frozen DRG tissue section is set to 10 μm; after fixation and penetration, hybridization is performed, and incubation is carried out at room temperature for 2 hours; after color development treatment, DAPI staining is performed, and a laser confocal microscope is used for photographing and analysis;

[0074] The results are shown in Figure 4 Fig. 6. The proportion of Mrgprd+ neurons in the control group (Ctrl) was 38.5 ± 3.2%, and that in the morphine treatment group (24h) was 24.1 ± 2.9%. It can be seen that short-term morphine treatment can significantly down-regulate the proportion of Mrgprd-expressing neurons in DRG, verifying that the kit has high tissue resolution, strong sensitivity, and good repeatability.

[0075] Compared with the traditional RNAscope product which is suitable for a wide range of tissues (such as liver, lung, and brain), the present kit is specifically suitable for mouse / human L4-L5 DRG tissue sections, and the section thickness, staining time, and antigen repair scheme have been optimized, reflecting the specificity and repeatability of the applicable scenarios. The kit is particularly suitable for Mrgprd expression detection of DRG frozen section tissues with a thickness of 8-12 μm.

[0076] Secondly, the design of the kit allows for subsequent immunofluorescence antibody (such as anti-Oprm1) co-detection without affecting signal stability. The kit is suitable for combined application with immunofluorescence co-staining method for Mrgprd and its downstream protein co-expression localization analysis.

[0077] The kit is not only for expression "existence" detection, but also for bridging the expression detection and analgesic response correlation data (such as the correspondence between Mrgprd decrease and tolerance delay), suitable for animal experiment and clinical DRG sample detection, providing expression genotyping assistance for individualized use of analgesic drugs, and can be used for early molecular genotyping assistance for judgment of opioid analgesic tolerance risk.

[0078] In addition, the intervention strategy (including siRNA inhibition of Mrgprd expression or upstream regulatory transcription factor c-Jun expression) adopted by the present application exhibits good safety and tolerance in a mouse model. The survival rate of mice during the intervention is 100%, the behavioral activity is good, the body weight change is stable, and there is no obvious discomfort or toxicity reaction. Histopathological examination also does not find structural damage to important organs, which preliminarily verifies the biological safety of the strategy and has the potential for further preclinical development.

[0079] The above detailed the preferred embodiments of the present application, which are only preferred embodiments of the present application. In the above embodiments, the method part focuses on the required drug dosage, administration method, evaluation index and time node to achieve the purpose of the application, and the product part refines the preparation ratio, sequence, packaging and use method. The method and the structural scheme are independent and complementary to each other, and jointly support the practicability and implementability of the technical concept of the application. It should be pointed out that those skilled in the art can make many modifications and changes according to the concept of the present application without creative labor. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiment on the basis of the existing technology by those skilled in the art according to the concept of the present application shall be within the protection scope determined by the claims.

Claims

1. The use of siRNA that inhibits the expression of Mas-associated G protein-coupled receptor D in the peripheral nervous system in the preparation of a drug for alleviating opioid tolerance, wherein the siRNA sequence is selected from siRNA targeting the Mas-associated G protein-coupled receptor D gene as shown in SEQ ID NO.1 or c-Jun siRNA sequence that inhibits the expression of transcription factor c-Jun as shown in SEQ ID NO.2, wherein the opioid is morphine.

2. The application according to claim 1, characterized in that, The drug targets Mas-associated G protein-coupled receptor D (MAS-GCPD) and alleviates morphine tolerance by reducing its expression or activity in the subject.

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