HSPB1 targeting siRNA and application thereof in preparation of chronic pain treatment medicine
By interfering with siRNA targeting HSPB1, the tolerability and side effects of existing chronic pain treatments have been addressed, providing a low-cost, low-risk treatment for chronic pain that effectively relieves mechanical hyperalgesia.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-10
- Publication Date
- 2026-04-14
AI Technical Summary
Existing medications for chronic pain treatment suffer from drug tolerance, addiction, and high levels of adverse reactions and side effects. Furthermore, invasive treatments are costly and risky, limiting their clinical application.
We designed siRNAs targeting HSPB1, and through RNA interference, specifically degraded the HSPB1 gene, inhibiting its expression, to prepare drugs that relieve chronic pain.
It effectively inhibits HSPB1 protein expression, relieves mechanical hyperalgesia, and provides a new, low-side-effect, and low-cost treatment approach for chronic pain.
Smart Images
Figure CN121852373A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a siRNA targeting HSPB1 and its application in the preparation of drugs for treating chronic pain. Background Technology
[0002] According to the latest definition from the International Association for the Study of Pain (IASP), chronic pain is defined as pain that lasts longer than the normal tissue healing time (usually ≥3 months), is unrelated to actual or potential tissue damage, or persists after the damage has healed. In recent years, epidemiological data on chronic pain have shown significant changes, highlighting its public health burden worldwide. Recent research indicates that the global prevalence of chronic pain is as high as 38%, meaning that 38 out of every 100 people suffer from chronic pain long-term.
[0003] The number of patients with chronic pain is increasing annually, a trend closely related to the accelerating aging of the population, the rising incidence of chronic diseases, and changes in lifestyle. Current clinical treatments for chronic pain mainly include narcotic analgesics, antipyretic and anti-inflammatory analgesics, antiepileptic drugs, and antidepressants. While opioid analgesics (such as morphine and oxycodone) can effectively relieve moderate to severe pain, long-term use can easily lead to drug tolerance, addiction, and serious adverse reactions such as respiratory depression. Nonsteroidal anti-inflammatory drugs (NSAIDs) work by inhibiting cyclooxygenase (COX), but may cause side effects such as gastrointestinal ulcers and liver and kidney damage. Antiepileptic drugs (pregabalin and gabapentin) and antidepressants (duloxetine and venlafaxine) have some efficacy for neuropathic pain, but some patients experience low response rates (only 40%-60%) and adverse reactions such as dizziness and drowsiness. Furthermore, invasive treatments such as nerve blocks and spinal cord stimulation are limited in clinical application due to operational risks and high costs. Therefore, it is necessary to discover new analgesic targets and develop new drugs to inhibit or relieve neuropathic pain.
[0004] Small interfering RNA (siRNA) is a double-stranded RNA that, through RNA interference, can specifically degrade target mRNA, achieving post-transcriptional gene silencing. In the field of disease treatment, siRNA can precisely target disease-related abnormal genes, interfering with their RNA function and blocking protein translation, thereby achieving gene therapy effects. Compared with traditional clinical treatment drugs, siRNA drugs have significant advantages such as fewer toxic side effects, high specificity, and high efficiency, making them a highly promising new treatment approach.
[0005] The inventors discovered that in a mouse model of trigeminal nerve pathological pain induced by partial infraorbital nerve transection (pIONT), the trigeminal ganglion... Hspb1 Both mRNA and protein expression were significantly upregulated; this invention successfully designed and demonstrated a set of methods that can effectively inhibit... Hspb1 Gene-expressed siRNA can be used for drug development; intraganglionic injection of trigeminal nerve ganglion. Hspb1 siRNA can alleviate mechanical hyperalgesia caused by peripheral nerve injury and downregulate... Hspb1 Therefore, downregulating or inhibiting HSPB1 protein expression using small interfering RNA is an effective approach for preparing drugs to treat chronic pain. Summary of the Invention
[0006] Technical problems to be solved: The present invention addresses the following technical problems: long-term use of existing drugs can easily lead to serious adverse reactions such as drug tolerance, addiction and respiratory depression; nonsteroidal anti-inflammatory drugs may cause side effects such as gastrointestinal ulcers and liver and kidney damage; antiepileptic drugs and antidepressants have low response rates and adverse reactions such as dizziness and drowsiness; operational risks and high costs limit their clinical application.
[0007] Objective of the Invention: This application provides a siRNA targeting HSPB1 and its application in the preparation of drugs for treating chronic pain, providing inhibition of HSPB1. Hspb1 The double-stranded siRNA sequence of gene expression can effectively downregulate or inhibit the expression of HSPB1 protein, reduce the secretion of HSPB1 protein, and further provide the ability to inhibit Hspb1 Application of expressed siRNA in pharmaceuticals.
[0008] To achieve the above objectives, this application provides the following technical solution: A siRNA targeting HSPB1, wherein the sense strand sequence targeting HSPB1 is shown in SEQ ID NO.1: 5′-AGCUCACAGUGAAGACCAATT-3′, and the antisense strand sequence is shown in SEQ ID NO.2: 5′-UUGGUCUUCACUGUGAGCUTT-3′.
[0009] Furthermore, the siRNA targets are all designed in... Hspb1 Conserved regions of genes.
[0010] Furthermore, the siRNA targets are all designed in... Hspb1 The coding region of a gene.
[0011] Furthermore, the siRNA can effectively reduce Hspb1 Gene expression.
[0012] This application also discloses the application of a siRNA targeting HSPB1 in the preparation of a drug for treating chronic pain.
[0013] Furthermore, the chronic pain treatment drug also contains pharmaceutically acceptable excipients.
[0014] Furthermore, the pharmaceutically acceptable excipient is at least one of the following: sustained-release agent, excipient, filler, binder, humectant, disintegrant, absorption enhancer, adsorbent carrier, surfactant, and lubricant.
[0015] Furthermore, the medication for treating chronic pain can be administered orally, subcutaneously, intramuscularly, intravenously, or by inhalation.
[0016] Furthermore, the chronic pain treatment drug is in oral or non-oral form.
[0017] Furthermore, the chronic pain treatment drug is in the form of tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalers, or sprays.
[0018] This application provides a siRNA targeting HSPB1 and its application in the preparation of drugs for treating chronic pain, which has the following advantages compared with the prior art: 1. This application provides a chemically modified nucleic acid siRNA molecule targeting the HSPB1 gene, which can effectively reduce... Hspb1 Gene expression; 2. The siRNA inhibition provided in this application Hspb1 Gene expression can effectively alleviate pIONT-induced mechanical hyperalgesia; 3. This application lays the foundation for the preparation of clinically therapeutic pain drugs and has significant application and promotion value.
[0019] Attached image description: Figure 1 For the pIONT neuropathic pain model of the trigeminal ganglion in this application Hspb1 Graph showing increased mRNA expression; Figure 2The image shows the increased expression of HSPB1 protein in the trigeminal ganglion of the pIONT neuropathic pain model of this application. A is the immunofluorescence staining image of HSPB1 in TG of control mice (Naive), B is the immunofluorescence staining image of HSPB1 in TG 10 days after surgery of sham mice, C is the immunofluorescence staining image of HSPB1 in TG 10 days after surgery of mice with infraorbital foramen ligation (pIONT), D is the Western blot image of HSPB1 in TG 10 days after surgery of Naive mice, sham group, and pIONT group mice, and E is the grayscale statistical image of the Western blot bands. Figure 3 The figures show the results of siRNA inhibition of mechanical hyperalgesia in mice. Figure A shows the evaluation of facial pain response in mice after siRNA injection in the infraorbital foramen 7 days after pIONT surgery, followed by stimulation of the mouse face with 0.02 g Von Frey filament. Figure B shows the evaluation of facial pain response in mice after siRNA injection in the infraorbital foramen 21 days after pIONT surgery, followed by stimulation of the mouse face with 0.02 g Von Frey filament. Figure 4 The siRNA in this application can reduce [the risk of death] in vivo. Hspb1 The expression diagram shows that A represents the concentration of TG in the infraorbital foramen 48 hours after pIONT surgery. Hspb1 mRNA expression knockdown diagram, B represents the TG level 48 hours after siRNA injection in the infraorbital foramen 21 days post-pIONT surgery. Hspb1 mRNA expression knockdown diagram. Detailed Implementation
[0020] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.
[0021] Example 1: This example provides a siRNA targeting HSPB1. The design and synthesis of the siRNA are as follows: Regarding siRNA sequence design, using software such as RNAi Designer, the parameters are set as follows: GC content of 35%-55%; target regions are gene coding regions and evolutionarily conserved regions.
[0022] The sense strand sequence targeting HSPB1 is shown in SEQ ID NO.1: 5′-AGCUCACAGUGAAGACCAATT-3′, and the antisense strand sequence is shown in SEQ ID NO.2: 5′-UUGGUCUUCACUGUGAGCUTT-3′.
[0023] This invention designs a set of siRNAs targeting HSPB1 that can target the HSPB1 gene in mice, with the target sequence located in the coding region of the HSPB1 gene.
[0024] Example 2, Trigeminal ganglion in a pain model mouse Hspb1 mRNA expression change detection: Real-time quantitative PCR results showed that after pIONT, in the trigeminal ganglion Hspb1 mRNA expression begins to increase from day 1 and can continue until day 21, such as... Figure 1 As shown.
[0025] like Figure 1 As shown, in the pIONT neuropathic pain model, the trigeminal ganglion... Hspb1 Increased mRNA expression.
[0026] Real-time PCR results showed that pIONT induces... Hspb1 The expression of mRNA in the trigeminal ganglion increased, starting on day 1 after surgery and peaking, with the increased expression continuing until day 21. Real-time PCR results were analyzed using a t-test (Student's T-test). Compared with the Sham group at the corresponding time points, ** P <0.01, *** P <0.001.
[0027] Preparation of a mouse model of trigeminal neuralgia: Male ICR mice were fasted and deprived of water for 3-4 hours prior to surgery. Tribromoethanol was then injected intraperitoneally with 1 ml of the syringe to induce deep anesthesia. The mice were placed on a sterile disposable mat, the light source was turned on, and they were fixed in a supine position. Using curved forceps, the mouse's tongue was placed on the right side of the cheek to fully expose the oral cavity, revealing a white tendon on the left cheek. A 3 mm incision was made 2 mm lateral to the nose, at approximately 45 degrees to the midline. The fascia and muscle were bluntly dissected using curved forceps, while simultaneously exposing the infraorbital nerve (ION). Approximately half of the ION was gently dissected using a glass electrode and ligated with 8-0 suture. The distal end of the ligated nerve was then cut, gently reinserted, and the incision sutured. The procedure in the sham surgery group was identical to that in the model group, except that the infraorbital nerve was not ligated. Aseptic techniques were maintained throughout the entire procedure to ensure rapid and minimally invasive surgery.
[0028] The process of RNA extraction, reverse transcription, and real-time PCR from animal tissue samples, including RNA extraction: (1) The trigeminal ganglion obtained by perfusion with physiological saline was placed in a 1.5 ml RNAase-free EP tube containing 100 μl Trizol, placed on ice, and homogenized with an electric homogenizer for 3 min. After homogenization, 400 μl Trizol was added and the tube was placed on ice for 5 min. For cell samples, 1 ml Trizol was added directly and aspirated into a 1.5 ml RNAase-free EP tube and placed on ice. (2) Add 100 μl of chloroform to each tube, shake vigorously for 15 s, and let stand for 5 min; (3) Centrifuge at 4℃ and 12000 rpm for 15 min; (4) Aspirate the supernatant into a 1.5 ml RNAase-Free EP tube, add an equal volume of isopropanol, mix gently until no visible filaments remain, and let stand for 10 min. (5) Centrifuge at 4℃ and 12000 rpm for 10 min; (6) Discard the supernatant, add 1 ml of anhydrous ethanol and mix lightly, then centrifuge at 4°C and 12000 rpm for 10 min. (7) Discard the supernatant, invert it onto filter paper and air dry at room temperature until the precipitate is translucent; (8) Add 10 μl of RNase-free H2O to each tube, incubate in a water bath at 60℃ for 10 min to promote dissolution, and place on ice after dissolution; (9) Detect RNA concentration using an OD instrument.
[0029] Total RNA was reverse transcribed into cDNA, as shown in Tables 1 and 2: Table 1: Genomic DNA removal (10 μl) .
[0030] Table 2: RNA reverse transcription to cDNA (20 μl) .
[0031] After reverse transcription to cDNA, dilute 8-fold with ddH2O and store at -20℃ for later use.
[0032] Primer design: The mRNA sequences of specific mouse genes in the NCBI database were selected. Primers were designed on the NCBI website and their specificity was verified by BLAST. The size of the amplification product was predicted. The specificity of the primers was further verified by melting curves and agarose gel running results. The primer sequences were synthesized by Invitrogen (Shanghai).
[0033] Real-time PCR experiments are shown in Tables 3 and 4: Table 3: Prepare the Real-time PCR reaction system (10 μl) according to the following table: .
[0034] Table 4: PCR reaction using a Life Technology instrument, under the following conditions: .
[0035] Example 3: Detection of HSPB1 protein expression in the trigeminal ganglion of model mice: Immunofluorescence results showed that, compared with the Sham 10-day group, the expression of HSPB1 protein in the trigeminal ganglion of the pIONT 10-day group was significantly higher than that of the Sham 10-day group. Figure 2 As shown.
[0036] like Figure 2 As shown, HSPB1 protein expression is increased in the trigeminal ganglion of the pIONT neuropathic pain model.
[0037] Immunofluorescence single-label results showed that the fluorescence signal of HSPB1 was significantly enhanced after pIONT compared with the Sham group, indicating that the expression of HSPB1 protein increased after pIONT. Scale bar = 100 m.
[0038] Western blot results showed that HSPB1 expression increased after pIONT. The results were analyzed using a t-test (Student's T-test). P <0.01.
[0039] Tissue section preparation: Trigeminal ganglia were harvested from mice after perfusion with saline and paraformaldehyde. They were then fixed in 4% paraformaldehyde at 4°C for 6 hours, followed by dehydration in 20% sucrose at 4°C. After the sample settled, the solution was replaced with 30% sucrose and dehydrated again at 4°C. Following gradient dehydration, the samples were removed, trimmed, embedded in embedding medium, and placed on the stage of a cryostat for cryosectioning. Sections were prepared to a thickness of 14 μm and then subjected to immunofluorescence staining.
[0040] Immunofluorescence staining: (1) Drying and washing the slides: Take the slides out of the refrigerator and let them dry thoroughly in the fume hood. Wash the slides with 0.01 M PBS and shake them on a shaker for 10 min each time, for a total of 3 times. (2) Blocking: Add 200 μl of 1% BSA blocking solution to each slide and incubate at room temperature for 2 h; (3) Incubation of target antibody (primary antibody): Add 200 μl of primary antibody to each slide and incubate overnight at 4°C (12-16 h); (4) Washing: The next day, take the slides out of the 4℃ refrigerator, warm them for 1 hour, then recover the target antibody, wash the slides with 0.01 M PBS, shake on a shaker for 15 min / time, 3 times; (5) Incubation of fluorescent secondary antibody: After cleaning the slide, add the corresponding fluorescent secondary antibody to the slide and incubate at room temperature in the dark for 2 hours; (6) Washing: Wash with 0.01 M PBS, protected from light, shake on a shaker for 15 min / time, 3 times; (7) Mounting: After the slides are washed, keep them away from light and dry them. Then add 60 μl of mounting medium, use tweezers to pick up the coverslip and gently place it on the slide, being careful to avoid air bubbles. Store in a dark place and let it air dry at room temperature. (8) Photographing: After drying thoroughly, the film can be placed under a fluorescence microscope for observation and photographing.
[0041] Western blot results showed that HSPB1 expression increased after pIONT treatment, such as... Figure 2 As shown.
[0042] Western blot: After perfusion of the heart with physiological saline in mice, the trigeminal ganglion was collected and placed in tissue lysis buffer containing protease inhibitors. Homogenization was performed on ice, and the supernatant was collected after centrifugation to obtain the tissue protein solution. The total protein concentration of the sample was determined using a BCA protein assay kit, and protein loading buffer was added to standardize the protein concentration. After boiling for 5 minutes to denature the proteins, gel electrophoresis and wet transfer were performed. The membrane was then blocked in 5% skim milk blocking buffer at room temperature for 2 hours. After blocking, the membrane was incubated with a 5% skim milk diluted primary antibody at 4°C overnight. After washing, the membrane was incubated with fluorescent secondary antibody, washed again, and then developed.
[0043] Example 4, Hspb1 Effects of siRNA on mechanical pain behavior in mice with trigeminal neuralgia: This invention employs intraganglionic injection after partial infraorbital nerve transection. Hspb1 siRNA was used, and then its effectiveness in alleviating pIONT-induced facial mechanical touch-induced pain was assessed. Behavioral results showed that... Hspb1 siRNA can alleviate mechanical touch-induced pain caused by pIONT, such as Figure 3 As shown.
[0044] like Figure 3 The figure shown illustrates the results of the siRNA of this invention inhibiting mechanical hyperalgesia in mice.
[0045] ICR mice were injected into the trigeminal ganglion on days 7 and 21 after pIONT surgery. Hspb1 siRNA was used, and then its effectiveness in alleviating mechanical touch-induced facial pain caused by pIONT was assessed. Behavioral results showed that... Hspb1 The siRNA can alleviate mechanical touch-induced pain caused by pIONT. Behavioral statistical analysis was performed using two-way ANOVA. P <0.01, *** P <0.001, compared with the NC siRNA group at the corresponding time point.
[0046] Intraganglionic injection in mice Hspb1 siRNA: siRNA was injected into the trigeminal ganglion 7 and 21 days after the establishment of the pIONT model. The model preparation method was the same as described above, with siRNA injected into the ganglion 7 and 21 days after model establishment. NC siRNA was used as a control and injected using the same method.
[0047] Behavioral testing in mice: To ensure that behavioral changes caused by unfamiliar environments are excluded, mice should be placed in the experimental environment 2-3 days before the behavioral test to allow them to adapt. During the test, the room should be quiet with minimal noise, soft lighting, humidity controlled at 40%-60%, and room temperature at 22-24°C. Testing should be conducted within a reasonable timeframe, from 9:00 AM to 6:00 PM. A randomized, double-blind method should be used for the behavioral test.
[0048] Mice were placed in wire cages and allowed to acclimatize for 30 minutes. Two different strengths of Von Frey filament (0.02 g and 0.16 g) were used to stimulate the mice's faces, and their facial pain response was assessed using a 0-5 rating scale. Higher scores indicated greater mechanical pain sensitivity in the mouse's face. Initially, a low-intensity 0.02 g stimulus was used to observe the facial pain response and record the corresponding rating. Subsequently, a higher-intensity 0.16 g stimulus was used, and the rating was recorded again.
[0049] Example 5, verification in vivo Hspb1 Effects of siRNA on HSPB1 expression: To further verify the knockout effect of the above siRNA in vivo, Real-time PCR results showed that... Hspb1 siRNA levels were significantly reduced after nerve injury. Hspb1 mRNA expression, such as Figure 4 As shown.
[0050] RNA extraction, reverse transcription, and Real-time PCR were performed according to the methods described above.
[0051] like Figure 4 As shown, the siRNA of this invention can reduce [the risk of death] in vivo. Hspb1 The expression.
[0052] Hspb1 The knockout effect of siRNA in vivo was shown by Real-time PCR results. Hspb1 siRNA levels were significantly reduced after nerve injury. Hspb1 mRNA expression. The statistical results of the comparison between the two groups were analyzed using a t-test (Student's test). t -test),** P <0.01, *** P <0.001.
[0053] The embodiments selected in the above materials are for ease of understanding and not for limiting the process method. Those skilled in the art can easily modify the process flow or transfer it to other cases without inventive change. If these modifications also fall under the category of similar claims or similar technology of this invention, then the intent of this invention also includes these modifications.
Claims
1. A siRNA targeting HSPB1, characterized in that, The positive strand sequence targeting HSPB1 is shown in SEQ ID NO.1: 5′-AGCUCACAGUGAAGACCAATT-3′, and the negative strand sequence is shown in SEQ ID NO.2: 5′-UUGGUCUUCACUGUGAGCUTT-3′.
2. The siRNA targeting HSPB1 according to claim 1, characterized in that, The siRNA targets are all designed in Hspb1 Conserved regions of genes.
3. The siRNA targeting HSPB1 according to claim 1, characterized in that, The siRNA targets are all designed in Hspb1 The coding region of a gene.
4. The siRNA targeting HSPB1 according to claim 1, characterized in that, The siRNA can effectively reduce Hspb1 Gene expression.
5. The use of the siRNA targeting HSPB1 as described in claim 1 in the preparation of a drug for treating chronic pain.
6. The application according to claim 5, characterized in that, The medication for treating chronic pain also contains pharmaceutically acceptable excipients.
7. The application according to claim 6, characterized in that, The pharmaceutically acceptable excipients are at least one of the following: sustained-release agents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, adsorbents, surfactants, and lubricants.
8. The application according to claim 5, characterized in that, The medication for treating chronic pain can be administered orally, subcutaneously, intramuscularly, intravenously, or by inhalation.
9. The application according to claim 5, characterized in that, The medication for treating chronic pain is available in oral or non-oral form.
10. The application according to claim 5, characterized in that, The chronic pain treatment drugs are tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalers, or sprays.