SiRNA targeting BACH1 and application of siRNA in preparation of chronic pain treatment medicine

Abstract

The invention discloses siRNA (small interfering Ribonucleic Acid) targeting BACH1 and application of the siRNA in preparation of chronic pain treatment medicines, belongs to the field of biological medicines, and discloses a compound, a composition and a method for regulating BACH1 gene expression, regulating response of activity and regulating characters, diseases or conditions of a BACH1 gene expression pathway. A behavioral result shows that the group of siRNAs can significantly inhibit mechanical hyperalgesia generated by peripheral nerve injury induction; the siRNA is injected into the trigeminal ganglion of a neuropathic pain model mouse, molecular biology experiments show that the siRNA can remarkably reduce expression of BACH1 in the trigeminal ganglion of the mouse in vivo, a new target and a new method are provided for research and development of chronic pain treatment drugs, non-chemical modification and chemical modification nucleic acid molecules of the targeted BACH1 gene are provided, and the application prospect is wide. The traditional Chinese medicine composition has a good medicine effect on chronic pain, lays a foundation for medicine preparation for treating chronic pain, and has great application and popularization value.

Description

A BACH1-targeting siRNA and its application in the preparation of drugs for treating chronic pain Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a BACH1-targeting siRNA 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 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 mRNA and protein expression of BACH1 in the trigeminal ganglion were significantly upregulated. This invention successfully designed and demonstrated a set of siRNAs that can effectively inhibit BACH1 gene expression, which can be used for drug development. Intraganglionic injection of Bach1 siRNA can alleviate mechanical hyperalgesia caused by peripheral nerve injury and downregulate Bach1 expression. Therefore, downregulating or inhibiting BACH1 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] Purpose of the invention: This application provides a BACH1-targeting siRNA and its application in the preparation of drugs for treating chronic pain. It provides a double-stranded siRNA sequence that inhibits BACH1 gene expression, which can effectively downregulate or inhibit the expression of BACH1 protein and reduce the secretion of BACH1 protein. Furthermore, it provides the application of siRNA that can inhibit BACH1 expression in pharmaceutical manufacturing.

[0008] To achieve the above objectives, this application provides the following technical solution: a siRNA targeting BACH1, wherein the sense strand sequence targeting BACH1 is shown in SEQ ID NO.1: 5′-GCAGAUGAAUUCUUGGAAATT-3′, and the antisense strand sequence is shown in SEQ ID NO.2: 5′-UUUCCAAGAAUUCAUCUGCTT-3′.

[0009] Furthermore, the siRNA targets are all designed in conserved regions of the Bach1 gene.

[0010] Furthermore, the siRNA targets are all designed in the coding region of the Bach1 gene.

[0011] Furthermore, the siRNA can effectively reduce the expression of the Bach1 gene.

[0012] This application also discloses the application of a BACH1-targeting siRNA 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 BACH1-targeting siRNA and its application in the preparation of drugs for treating chronic pain. Compared with the prior art, it has the following beneficial effects: 1. This application provides a chemically modified nucleic acid siRNA molecule that targets the Bach1 gene, which can effectively reduce the expression of the Bach1 gene; 2. The siRNA provided in this application can effectively alleviate pIONT-induced mechanical hyperalgesia by inhibiting the expression of the Bach1 gene; 3. This application lays the foundation for the preparation of clinical therapeutic pain drugs and has significant application and promotion value. Figure 1 shows the increased expression of Bach1 mRNA in the trigeminal ganglion of the pIONT neuropathic pain model of this application; Figure 2 shows the increased expression of BACH1 protein in the trigeminal ganglion of the pIONT neuropathic pain model of this application, where A is the immunofluorescence staining image of BACH1 in TG of control mice (Naive), B is the immunofluorescence staining image of BACH1 in TG of sham mice 10 days after surgery, C is the immunofluorescence staining image of BACH1 in TG of mice with infraorbital foramen ligation (pIONT) 10 days after surgery, D is the Western blot image of BACH1 in TG of Naive mice, sham group and pIONT group mice 10 days after surgery, and E is the grayscale statistical image of Western blot bands; Figure 3 shows the results of siRNA inhibition of mechanical hyperalgesia in mice, where the left image shows the effect of siRNA injection into the infraorbital foramen 7 days after pIONT surgery followed by the use of 0.02 g Von Frey The right image shows the evaluation of facial pain response in mice after facial stimulation with 0.02 g Von Frey filament 21 days after pIONT surgery. Figure 4 shows that the siRNA of this application can reduce Bach1 expression in vivo. A shows the knockdown of Bach1 mRNA expression in TG 24 hours after siRNA injection in the infraorbital foramen 7 days after pIONT surgery, and B shows the knockdown of Bach1 mRNA expression in TG 24 hours after siRNA injection in the infraorbital foramen 21 days after pIONT surgery. 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 BACH1. The design and synthesis of the siRNA are as follows: Regarding the siRNA sequence design, software such as RNAi Designer is used, with the following parameters set: GC content of 35%-55%; target region is gene coding region and evolutionarily conserved region.

[0022] The sense strand sequence targeting BACH1 is shown in SEQ ID NO.1: 5′-GCAGAUGAAUUCUUGGAAATT-3′, and the antisense strand sequence is shown in SEQ ID NO.2: 5′-UUUCCAAGAAUUCAUCUGCTT-3′.

[0023] This invention designs a set of siRNAs targeting BACH1 that can target the BACH1 gene in mice, with the target sequence located in the coding region of the BACH1 gene.

[0024] Example 2, detection of changes in Bach1 mRNA expression in the trigeminal ganglion of a pain model mouse: Real-time quantitative PCR results showed that after pIONT, the expression of Bach1 mRNA in the trigeminal ganglion increased from day 1 and continued until day 21, as shown in Figure 1.

[0025] As shown in Figure 1, Bach1 mRNA expression is increased in the trigeminal ganglion of the pIONT neuropathic pain model.

[0026] Real-time PCR results showed that pIONT induced an increase in Bach1 mRNA expression in the trigeminal ganglion, with the increase starting on day 1 after surgery, peaking on day 3, and 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<0.001.

[0027] Preparation of a trigeminal neuralgia model mouse: 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 disposable sterile 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). A portion (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 was 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 employed throughout the procedure to ensure rapid and minimally invasive surgery.

[0028] The process of RNA extraction, reverse transcription and Real-time PCR of animal tissue samples, including RNA extraction: (1) Trigeminal ganglia obtained by perfusion with physiological saline were 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 mixture 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) 100 μl chloroform was added to each tube, and the mixture was shaken vigorously for 15 s and placed on ice for 5 min. (3) The mixture was centrifuged at 4℃ and 12000 rpm for 15 min. (4) The supernatant was aspirated into a 1.5 ml RNAase-Free EP tube, and an equal volume of isopropanol was added. The mixture was gently mixed until no visible filaments were found and placed on ice for 10 min. (5) The mixture was centrifuged at 4℃ and 12000 rpm for 10 min. (6) Discard the supernatant, add 1 ml of anhydrous ethanol and mix lightly, centrifuge at 4℃ and 12000 rpm for 10 min; (7) Discard the supernatant, invert it on 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 60℃ water bath for 10 min to promote dissolution, and place on ice after dissolution; (9) Detect the RNA concentration using an OD instrument.

[0029] Total RNA was reverse transcribed into cDNA, as shown in Tables 1 and 2: Table 1: Removal of genomic DNA (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 described 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 BACH1 protein expression in the trigeminal ganglion of model mice: Immunofluorescence results showed that, compared with the Sham 10 d group, the expression of BACH1 protein in the trigeminal ganglion of pIONT 10 d was significantly higher than that of the Sham 10 d group, as shown in Figures 2A-C.

[0036] As shown in Figures 2D-E, BACH1 protein expression is increased in the trigeminal ganglion of the pIONT neuropathic pain model.

[0037] Immunofluorescence single-label results showed that the BACH1 fluorescence signal was significantly enhanced after pIONT compared with the Sham group, indicating that the expression of BACH1 protein increased after pIONT. Scale bar = 100 m.

[0038] Western blot results showed that BACH1 expression increased after pIONT. The results were analyzed using a t-test (Student's T-test), and **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: Take the slides out of the refrigerator and dry them thoroughly in the fume hood. Wash the slides with 0.01 M PBS, shake on a shaker for 10 min / time, 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℃ (12-16 h); (4) Washing: Take the slides out of the 4℃ refrigerator the next day, 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 washing the slides, add the corresponding fluorescent secondary antibody to the slides and incubate at room temperature in the dark for 2 h; (6) Washing: Wash with 0.01 M PBS, in the dark, shake on a shaker for 15 min / time, 15 h. min / time, 3 times; (7) Covering: After washing the slide, avoid light and dry the slide. Then add 60 μl of covering agent, use tweezers to pick up the coverslip, gently cover it on the slide, and avoid generating air bubbles. Store in a dark place and let it air dry at room temperature; (8) Photographing: After it is fully dried, it can be placed under a fluorescence microscope for observation and photographing.

[0041] Western blot results showed that BACH1 expression increased after pIONT, as shown in Figures 2D-E.

[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: Effect of Bach1siRNA on mechanical pain behavior in mice with trigeminal nerve pathological pain: In this invention, Bach1siRNA was injected into the trigeminal ganglion after partial infraorbital nerve transection, and then the effect of siRNA on relieving mechanical touch-induced pain in the face caused by pIONT was detected. Behavioral results showed that Bach1siRNA could relieve mechanical touch-induced pain caused by pIONT, as shown in Figure 3.

[0044] Figure 3 shows the results of the siRNA of this invention inhibiting mechanical hyperalgesia in mice.

[0045] ICR mice were injected with Bach1 siRNA into the trigeminal ganglion at 7 and 21 days after pIONT surgery. The efficacy of siRNA in alleviating pIONT-induced mechanical touch-evoked pain in the face was then assessed. Behavioral results showed that Bach1 siRNA could alleviate pIONT-induced mechanical touch-evoked pain. Behavioral statistical analysis was performed using a two-way ANOVA (*P<0.05, **P<0.01, ***P<0.001), compared with the NC siRNA group at the corresponding time points.

[0046] Intraganglionic injection of BACH1 siRNA in mouse trigeminal ganglia: siRNA was injected intraganglionically at 7 and 21 days after establishing the pIONT model. The model preparation method was the same as described above, with intraganglionic injection of siRNA 7 and 21 days after model establishment. NC siRNA served as a control, 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, verifying the effect of Bach1siRNA on BACH1 expression in vivo: To further verify the knockout effect of the above siRNA in vivo, Real-time PCR results showed that Bach1siRNA significantly reduced Bach1 mRNA expression after nerve injury, as shown in Figure 4.

[0050] RNA extraction, reverse transcription, and Real-time PCR were performed according to the methods described above.

[0051] As shown in Figure 4, the siRNA of this invention can reduce the expression of Bach1 in vivo.

[0052] The knockout effect of Bach1 siRNA in vivo was investigated using real-time PCR. Results showed that Bach1 siRNA significantly reduced Bach1 mRNA expression after nerve injury. Statistical analysis of the two groups was performed using a t-test (Student's 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 BACH1, characterized in that, The sense strand sequence targeting BACH1 is shown in SEQ ID NO.1: 5′-GCAGAUGAAUUCUUGGAAATT-3′, and the antisense strand sequence is shown in SEQ ID NO.2: 5′-UUUCCAAGAAUUCAUCUGCTT-3′.

2. The BACH1-targeting siRNA according to claim 1, characterized in that, The siRNA targets are all designed in conserved regions of the Bach1 gene.

3. The siRNA targeting BACH1 according to claim 1, characterized in that, The siRNA targets are all designed in the coding region of the Bach1 gene.

4. The siRNA targeting BACH1 according to claim 1, characterized in that, The siRNA can effectively reduce the expression of the Bach1 gene.

5. The use of the BACH1-targeting siRNA of claim 1 in the preparation of a medicament 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.

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