Preparation of siRNA (small interfering Ribonucleic Acid) targeting CCL2 and application of siRNA in treating pain

Abstract

The invention provides preparation of siRNA (small interfering Ribonucleic Acid) targeting CCL2 and application of the siRNA in treating pain, and relates to the technical field of biomedicine, and the technical key points are as follows: a cholesterol methoxy modified siRNA molecule for inhibiting CCL2 gene expression at least comprises one of the following three groups: Ccl2-mous-104, Ccl2-mous-246 and Ccl2-mous-329. The invention provides three groups of siRNA molecules, the three groups of siRNA molecules can obviously reduce CCL2 gene expression in vitro, the three groups of siRNA molecules can effectively reduce CCL2mRNA expression in vitro, and the mRNA level of the Ccl2-mous-246 group is the lowest; based on the data, the Ccl2-mous-246 has the most significant effect of inhibiting CCL2 gene and protein expression, effectively relieves neuropathic pain of mice, and lays a foundation for clinical treatment of pain.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the preparation of siRNA targeting CCL2 and the application thereof in treating pain. Background Art

[0002] Pain is an unpleasant sensation and emotional experience associated with actual or potential tissue damage, which is affected by many factors such as physiology, psychology, personal experience and social culture. From a neurophysiological perspective, pain can be divided into three types. The first type is an early warning issued by the physiological protection mechanism, prompting inspection and minimization of contact with noxious or harmful stimuli. The pain we feel when we touch something too hot, too cold or sharp is noxious pain, which is a high-threshold pain that is only activated when there is a strong stimulus. The second type of pain is also adaptive and protective, increasing sensitivity when the body is damaged and avoiding the body from engaging in certain movements to promote recovery, such as postoperative incision pain or joint inflammation, which is inflammatory pain or plastic pain caused by tissue damage or infection activating the immune system. The third type is caused by abnormal function of the nervous system, resulting in neuropathic pain and dysfunctional pain. The three are collectively referred to as pain, which drives pain in different ways, but often overlap and cause pain, making it difficult to separate them completely.

[0003] Pain can also be divided into acute pain and chronic pain. Acute pain is an unpleasant dynamic psychophysiological process, often caused by tissue damage and its related inflammatory process. It has survival value and is essential in the healing process, but once the acute danger period is over, pain is no longer a necessity, but a burden, a distressing disease. But if acute pain lasts for more than 12 weeks, it becomes chronic pain. The prevalence of chronic pain is between 11% and 40%, and research from the Centers for Disease Control and Prevention (CDC) in the United States shows that the prevalence is 20.4%. A review report from a British study showed that the pooled prevalence of chronic pain was 43.5%, and the prevalence of moderate to severe disabling pain ranged from 10.4% to 14.3%.

[0004] Pain treatment is guided by patient-reported severity of chronic disease, with treatment goals of “no more than mild pain” for acute pain and “severe,” “marked,” and “any” chronic pain. Epidemiological studies have shown increasing adverse relationships with employment status, interference with daily activities, and general health status.

[0005] Current pain treatment methods include psychotherapy, physical therapy, drug therapy, and gene therapy, etc. Drug therapy such as opioid drugs has far less effect on pain than side effects, and is prone to cause pain sensitization and sleep problems. Nerve block and psychotherapy have alleviated pain to a certain extent, but still lack satisfactory analgesic effects. Gene therapies targeting cannabinoid receptors, chemokine receptors, and angiotensin type 2 receptors have also emerged. With the rapid development of advanced gene sequencing technology, genome-wide association studies, human tissues, and stem cell differentiation and organoids, it is expected to design and screen reliable new drugs based on molecular targets.

[0006] Small interfering RNA (siRNA) is a short RNA molecule, usually a double-stranded RNA composed of 20 to 25 nucleotides. siRNA binds to the mRNA of the target gene in a complementary manner, resulting in mRNA degradation or translational inhibition, thereby weakening or inactivating the function of the target gene. siRNA has been clinically trialed for various diseases such as cancer, poliomyelitis, obesity, and pain. After entering the human body, siRNA interferes with the specific gene mRNA of a specific disease, making its gene unable to normally express proteins, thus achieving the effect of treating diseases. Compared with commonly used therapeutic drugs, siRNA drugs have the advantages of low toxicity, few side effects, high efficiency, and strong specificity, and can specifically target pain-related genes such as ion channels, neurotransmitter receptors, and inflammatory mediators, and are one of the most promising therapeutic methods. Summary of the Invention

[0007] The object of the present invention is to provide a siRNA double-stranded sequence that inhibits the expression of the CCL2 gene and can effectively down-regulate or inhibit the expression of the CCL2 gene.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A cholesterol-methoxy modified siRNA molecule that inhibits the expression of the CCL2 gene, and the siRNA molecule contains at least one of the following three groups of siRNAs:

[0010] Ccl2-mouse-104

[0011] Sense sequence 5′-GCUACUCAUUCACCAGCAATT-3′

[0012] Antisense sequence 5′-UUGCUGGUGAAUGAGUAGCTT-3′

[0013] Ccl2-mouse-246

[0014] Sense sequence 5′-GGUCCAGACAUACAUUAAATT-3′

[0015] Antisense sequence 5′-UUUAAUGUAUGUCUGGACCTT-3′

[0016] Ccl2-mouse-329

[0017] Sense sequence 5′-GGUCUUCAGCACCUUUGAATT-3′

[0018] Antisense sequence 5′-UUCAAAGGUGCUGAAGACCTT-3′.

[0019] Preferably, the three groups of the siRNA molecules are used to inhibit CCL2 gene expression, and the target sequences are located in the coding region of the Ccl2 gene.

[0020] The present application also provides the use of the cholesterol methoxy-modified siRNA molecules for inhibiting CCL2 gene expression in the preparation of a medicament for treating pain.

[0021] Preferably, the pain includes neuropathic pain.

[0022] Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

[0023] The present application also provides the use of the SNI model in verifying the efficacy of the medicament prepared using the above-mentioned siRNA molecules in treating pain.

[0024] Preferably, during the verification process, mechanical pain behavior detection is used to verify the efficacy of the medicament.

[0025] Beneficial effects:

[0026] The present application provides three groups of cholesterol methoxy-modified siRNA molecules targeting the CCL2 gene. All three groups can significantly reduce CCL2 gene expression in vitro, and all three can effectively reduce Ccl2 mRNA expression in vitro, and the Ccl2-mouse-246 group has the lowest mRNA level; based on the above data, Ccl2-mouse-246 inhibits CCL2 gene and protein expression most significantly and can effectively relieve neuropathic pain in mice, laying a foundation for clinical treatment of pain and having great application and promotion value. Description of the drawings

[0027] Figure 1 : The target sequence of Ccl2-mouse-104 is located in the conserved region of the human and mouse CCL2 gene sequences;

[0028] Figure 2 : The target sequence of Ccl2-mouse-246 is located in the conserved region of the human and mouse CCL2 gene sequences;

[0029] Figure 3 : The Ccl2-mouse-329 target sequence is located in the conserved region of the human and mouse CCL2 gene sequences;

[0030] Figure 4 : Comparison chart of the changes in CCL2 mRNA expression in the DRG of neuropathic pain-induced mice by the SNI model (ligation injury of the tibial nerve and common peroneal nerve); 8 mice in each group, and the Real-time PCR results were analyzed by t-test (Student’s T-test). Compared with the solvent group at the corresponding time point, *P<0.05, **P<0.01, ***P<0.001.

[0031] Figure 5 : Comparison chart of the increased CCL2 protein expression in the DRG of neuropathic pain model-induced mice. The left immunofluorescence single-labeling results show that the CCL2 fluorescence signal in the DRG of mice after modeling is significantly enhanced compared with the Vehicle group, scale bar = 50μm, and the right is the statistical chart of the CCL2 fluorescence signal in the DRG of mice.

[0032] Figure 6 : Comparison chart of the silencing efficiency of CCL2 siRNA on CCL2 mRNA in BV2 cells verified by in vitro experiments in this application. In this application, the BV2 cell line was transfected with three groups of CCL2 siRNA for 24 hours, and the expression level of CCL2 mRNA was detected by Real-time PCR. The statistical results were analyzed by (One-way ANOVA) and compared with the NC group, *P<0.05, **P<0.01, ***P<0.001.

[0033] Figure 7 : Result chart of the behavior of treating mouse pain after siRNA interference in this invention. The leftmost one is the comparison chart of mechanical pain behavior detection, the middle one is the comparison chart of thermal hyperalgesia behavior test, and the rightmost one is the comparison chart of cold hyperalgesia behavior test. In this application, a SNI model was established to induce neuropathic pain, and Ccl2-mouse-246 siRNA was injected intrathecally on the 7th day after SNI, and then it was detected whether this siRNA could relieve the pain behavior induced by SNI. *P<0.05, **P<0.01, ***P<0.001, compared with the NC siRNA group at the corresponding time point. Detailed implementation manners

[0034] The following further elaborates on the present invention in detail in conjunction with specific embodiments.

[0035] A cholesterol-methoxy modified siRNA molecule that inhibits the expression of the CCL2 gene, and the siRNA molecule contains at least one of the following three groups of siRNA:

[0036] Ccl2-mouse-104

[0037] Sense sequence 5′-GCUACUCAUUCACCAGCAATT-3′

[0038] Antisense sequence 5′-UUGCUGGUGAAUGAGUAGCTT-3′

[0039] Ccl2-mouse-246

[0040] Sense sequence 5′-GGUCCAGACAUACAUUAAATT-3′

[0041] Antisense sequence 5′-UUUAAUGUAUGUCUGGACCTT-3′

[0042] Ccl2-mouse-329

[0043] Sense sequence 5′-GGUCUUCAGCACCUUUGAATT-3′

[0044] Antisense sequence 5′-UUCAAAGGUGCUGAAGACCTT-3′.

[0045] The above three groups of siRNA molecules are used to inhibit the expression of the CCL2 gene, and the target sequence is located in the coding region of the Ccl2 gene.

[0046] Based on the cholesterol-methoxy modified siRNA molecules that inhibit the expression of the CCL2 gene, the present application provides the use of cholesterol-methoxy modified siRNA molecules that inhibit the expression of the CCL2 gene in the preparation of drugs for treating pain. The pain includes neuropathic pain.

[0047] The drug further comprises a pharmaceutically acceptable carrier.

[0048] In addition, the present application provides a model for verifying pain treatment drugs, and the model is an SNI model (a spared nerve injury-induced neuropathic pain model).

[0049] Based on the SNI model, the present application also provides the use of the SNI model in verifying the efficacy of drugs prepared using the above siRNA molecules in treating pain. In one embodiment, the efficacy of the drug is verified by mechanical pain behavior detection.

[0050] The above content is elaborated below in combination with specific verification experiments:

[0051] Experimental materials and sources:

[0052]

[0053]

[0054] Example 1: Design and Synthesis of siRNA

[0055] In one embodiment, software such as RNAi Designer is used with the following parameter settings: the GC content is 35%-55%; the target region is the gene coding region and the evolution region. According to the Ccl2 mRNA sequence of mice ( NM_011333.3 ), the following three groups of double-stranded siRNAs are designed and synthesized:

[0056]

[0057] The three groups of siRNAs designed in the present invention target the Ccl2 gene of mice, and their target sequences are located in the coding region of the Ccl2 gene. The specific targeting positions are shown in Figures 1-3 .

[0058] Example 2: Changes in Ccl2 mRNA Expression in DRG of Mice with Neuropathic Pain Model

[0059] 2.1 Preparation of Mice with Neuropathic Pain Model

[0060] Retrograde sciatic nerve injury-induced neuropathic pain model: After anesthetizing the mice with isoflurane, the leg hair is removed with an animal hair clipper for sufficient skin preparation, and the skin preparation area is disinfected with alcohol. A longitudinal incision is made proximal to the knee using a scalpel, and the muscle layer is bluntly dissected with forceps. After the sciatic nerve is exposed through the biceps femoris, the common peroneal nerve and the tibial nerve are ligated with 7-0 silk thread at the trifurcation, then cut distal to the silk knot, and then 2-4 mm of the distal nerve end is removed. During the operation, the sural nerve should be kept intact. After the operation, the surrounding muscles are closed and the skin incision is sutured. The wound and its surrounding area are disinfected with iodophor. For the sham operation group of mice, only the sciatic nerve is isolated and exposed in the same way without any lesions. The lesion results in obvious hypersensitivity in the lateral area of the paw, which is innervated by the retained sural nerve.

[0061] 2.2 Sampling of Animal Tissues, RNA Extraction, Reverse Transcription cDNA and Real-time PCR Processes:

[0062] 2.2.1 Tissue Sampling

[0063] (1) Use the anesthesia mask of Ruiwode to keep the mice in an anesthetized state with stable breathing. Cut open the chest cavity, insert the perfusion needle into the left cardiac apex, and perform cardiac perfusion with physiological saline;

[0064] (2) Observe that the liver turns white and the mesentery is transparent, indicating that the perfusion is completed. Cut open the spine to expose the spinal cord, separate the dorsal root ganglia beside the spinal cord, and place the extracted dorsal root ganglia into an RNAase Free EP tube containing 200 μL of Trizol, and place it in an ice box.

[0065] 2.2.2 RNA Extraction

[0066] (1) Homogenize the taken samples with a homogenizer once every 30 s for a total of 4 times until no tissue fragments can be seen, which is regarded as the completion of homogenization. Add 800 μL of Trizol and let it stand for 5 min;

[0067] (2) Add 200 μL of chloroform to each EP tube, shake vigorously for 1 min quickly, and let it stand for 5 min;

[0068] (3) Centrifuge under the conditions: 4 °C, 12000 rpm, 15 min;

[0069] (4) Gently aspirate the upper clear liquid into an RNAase Free EP tube, add an equal volume of isopropanol, gently invert up and down until no visible filaments can be seen with the naked eye, and let it stand for 10 min;

[0070] (5) Centrifuge under the conditions: 4 °C, 12000 rpm, 15 min;

[0071] (6) Discard the supernatant, add 1 mL of absolute ethanol and mix gently, centrifuge under the conditions: 4 °C, 12000 rpm, 15 min;

[0072] (7) Discard the supernatant, invert the EP tube on the filter paper, and let it dry at room temperature until a translucent precipitate appears at the bottom of the tube;

[0073] (8) Add 20 μL of RNase-free H2O to each tube, heat the water bath at 60 °C for 10 min to promote dissolution, and insert it into an ice box after dissolution;

[0074] (9) Measure the RNA concentration with an OD tester.

[0075] 2.2.3 Reverse Transcription of Total RNA into cDNA

[0076] (1) Remove DNA impurities in the sample (10 μL system)

[0077]

[0078] (2) Reverse transcribe RNA into cDNA (20 μL system)

[0079]

[0080] After reverse transcription into cDNA, add triple-distilled water to dilute 8-fold and store at -20°C for later use.

[0081] (3) Primer sequences

[0082] For primer design, the mouse Ccl2 mRNA sequence in the NCBI database was used in this application. Primers were designed on the NCBI website, and their specificity was verified by BLAST. The size of the amplified product was predicted, and the primer specificity was specifically verified according to the melting curve and the results of agarose gel electrophoresis. The primer sequences were synthesized by OBiO (Shanghai Heyuan).

[0083] The primer sequences required in the experiment are as follows in the table

[0084]

[0085] 2.2.4 Real-time PCR experiment

[0086] (1) Prepare the Real-time PCR reaction system (10 μL) according to the following table:

[0087]

[0088] (2) React with a PCR instrument (Life Technology), and the conditions are as follows:

[0089]

[0090] For experimental results, please refer to Figure 4 , and the results of real-time quantitative PCR showed that after SNI modeling, the expression of Ccl2 mRNA in the DRG of mice increased significantly.

[0091] Example 3: Detection of CCL2 protein expression in the DRG of model mice

[0092] 3.1 Preparation, staining, and filming of immunofluorescence samples

[0093] Tissue section preparation: After the mice were anesthetized by inhaling isoflurane, they were perfused through the heart with normal saline and 4% paraformaldehyde and then placed on ice. The mice were post-fixed by slowly shaking on a shaker. Then, the DRGs of L4-L6 were placed in a 5 mL tube containing 4% paraformaldehyde and kept at 4°C. After fixation for 6 - 14 h, the PBS solution containing 20% sucrose was changed. After the DRGs sank to the bottom of the tube, the PBS solution containing 30% sucrose was changed. After the DRGs sank to the bottom again, the DRGs were taken out, and the trimmed DRGs were placed in the model groove in order, and then quickly frozen on the quick-freezing table of the microtome. After solidification and molding, the embedding block was adhered to the freezing table with OTC, and frozen sections were made. The thickness of the DRG tissue sections was 12 μm, which were adhered to the adhesive glass slides and air-dried. They could be directly put into 0.01 mol / L PBS for immunofluorescence staining, or stored at -80°C for later use.

[0094] 3.2 Immunofluorescence staining

[0095] (1) Place the sections in a container containing 0.01 mol / L PBS and wash 3 times, 10 min each time;

[0096] (2) After washing the slides, add the immunofluorescence blocking solution and block at room temperature for 2 h;

[0097] (3) After the blocking is completed, add the primary antibody prepared with the blocking solution and incubate overnight at 4°C;

[0098] (4) Take it out the next day, after restoring to room temperature, wash 3 times with 0.01 mol / L PBS, 10 min each time;

[0099] (5) Dilute the secondary antibody with 0.01 mol / L PBS and incubate in the dark at room temperature for 2 h;

[0100] (6) Discard the secondary antibody, wash the slides 3 times with 0.01 mol / L PBS, 10 min each time;

[0101] (7) Place it in the dark at room temperature. After the slides are half-dried, drop the mounting medium on the slides, cover with a coverslip, and take pictures with a fluorescence microscope after air-drying.

[0102] Experimental results: Please refer to Figure 5 , The immunofluorescence results showed that compared with the control group, the CCL2 protein and the number of positive nerve cells in the DRGs of SNI model for 7 d were significantly increased.

[0103] Example 4: Verification of the knockout efficiency of Ccl2 siRNA

[0104] In this example, first, the knockout effect of siRNA was verified by transfecting CCl2 siRNA into the BV2 cell line. Please refer to Figure 6, The results of real-time PCR showed that the expression of CCL2 could be inhibited in all three groups of CCL2-targeting siRNAs. The specific procedure is as described in 2.2.

[0105] Example 5: Effect of Ccl2 siRNA on mechanical pain behavior in mice with neuropathic pain

[0106] 5.1 Intrathecal injection of Ccl2 siRNA in mice

[0107] Seven days after establishing the SNI model, Ccl2 siRNA was intrathecally injected. Among them, the SNI model is described in 2.1, and the injection method is as follows: Dissolve the siRNA to the working concentration, 5 μg / 10 μL. At 7 days after SNI modeling, use an insulin syringe to aspirate 10 μL of Ccl2 siRNA. The control group was NC siRNA, and injection was performed in the same way.

[0108] 5.2 Behavioral detection of mice

[0109] Mice were allowed to adapt three days in advance during behavioral detection, with the same experimenters and environment maintained, and double-blind method was used for detection.

[0110] For the detection of mechanical pain behavior, place the mice in a transparent nine-square grid on an iron stand and let them adapt for 60 min. Perform plantar acupuncture according to different von Frey filaments (0.02 g, 0.04 g, 0.07 g, 0.16 g, 0.4 g, 0.6 g, 1 g, and 2 g). The von Frey filament is vertically stimulated on the outer side of the left plantar of the mouse until it bends to 45°, and repeat 5 times. Observe whether the mouse shows paw lifting, paw flicking, and paw licking behaviors. If the above behaviors occur 3 times, it is recorded as X; if less, it is recorded as O. First, stimulate with 0.16 g. If it is X, change to a smaller-grade stimulus; if it is O, change to a larger-grade stimulus, and so on. Finally, obtain four characters "XXXX" or five characters "OOOOO" or six characters such as "OXXXOX". Convert the characters to the paw withdrawal threshold (g) according to the threshold table. The larger the paw withdrawal threshold, the weaker the pain sensation.

[0111] For the behavioral test of thermal hyperalgesia in mice: Let the mice adapt on the hot plate of the hot plate pain tester for 3 days in advance; during the test, place each mouse on a hot plate instrument at (55.0 ± 0.5) °C. When positive reactions such as paw lifting, licking, and jumping first appear, read the time on the hot plate instrument, repeat 3 times, with an interval of 5 - 10 min each time, and calculate the average value of the three times as the thermal pain threshold of the mouse.

[0112] For the behavioral test of cold hyperalgesia in mice: Place the mice in a transparent plexiglass box on a metal mesh rack; evaluate cold sensitivity by acetone evaporation cooling. Through the bottom of the metal mesh, gently spray acetone (50 μL) onto the plantar surface of the hind paws of the mice using a syringe, repeating 5 times with an interval of at least 5 minutes between each time. Score according to 0 - 2 points, where 0 = no response or brief and rapid or shaking of the hind paw and immediate subsidence; 1 = lifting, licking or shaking of the hind paw, persisting after the first use but subsiding within 5 seconds; 2 = long-term, repeated lifting, licking or shaking of the hind paw. The individual score is the average of the five times.

[0113] In this application, 7 days after ligating the sural nerve and tibial nerve, Ccl2 siRNA was injected intrathecally, and then it was detected whether the siRNA alleviated the scratching caused by SNI. Please refer to Figure 7 , and the behavioral results showed that Ccl2 siRNA could alleviate the mechanical pain behavior caused by SNI, starting to take effect 6 hours after injection, and then the curative effect gradually decreased.

[0114] To sum up, this experiment used a neuropathic pain model to verify the role of siRNA in pain. The neuropathic pain model was induced by ligating two branches of the sciatic nerve (tibial nerve and common peroneal nerve) while preserving the sural nerve. We took the dorsal root ganglion (DRG) of the mice after modeling, and transcriptome sequencing found that the expression of CCL2 was significantly increased. In vivo experiments verified that the expression of CCL2 mRNA and protein in the DRG of the model mice was significantly increased. The present invention successfully designed and proved three groups of siRNAs that could effectively inhibit the expression of the CCL2 gene, and selected the best-performing siRNA-2 for further research, providing a potential treatment strategy for drug development. Ccl2 siRNA in the dorsal root ganglion could alleviate the neuropathic pain of the model mice and simultaneously down-regulate the expression of CCL2 mRNA. Therefore, down-regulating or inhibiting the expression of the CCL2 gene and protein by applying small interfering RNA is an effective way to treat pain.

Claims

1. A cholesterol methoxy-modified siRNA molecule for inhibiting CCL2 gene expression, characterized in that: The siRNA molecule comprises at least one of the following three groups of siRNAs: Ccl2-mouse-104 Sense sequence 5′-GCUACUCAUUCACCAGCAATT-3′ Antisense sequence 5′-UUGCUGGUGAAUGAGUAGCTT-3′ Ccl2-mouse-246 Sense sequence 5′-GGUCCAGACAUACAUUAAATT-3′ Antisense sequence 5′-UUUAAUGUAUGUCUGGACCTT-3′ Ccl2-mouse-329 Sense sequence 5′-GGUCUUCAGCACCUUUGAATT-3′ Antisense sequence 5′-UUCAAAGGUGCUGAAGACCTT-3′.

2. The cholesterol methoxy-modified siRNA molecule for inhibiting CCL2 gene expression according to claim 1, characterized in that: The three groups of siRNA molecules are used to inhibit the expression of CCL2 gene, and their target sequences are located in the coding region and evolutionary region of Ccl2 gene.

3. Use of the cholesterol methoxy-modified siRNA molecule for inhibiting CCL2 gene expression according to claim 1 or 2 in the preparation of a drug for treating pain.

4. Use of the cholesterol methoxy-modified siRNA molecule for inhibiting CCL2 gene expression according to claim 3 in the preparation of a drug for treating pain, characterized in that: The pain includes neuropathic pain.

5. Use of the cholesterol methoxy-modified siRNA molecule for inhibiting CCL2 gene expression according to claim 3 in the preparation of a drug for treating pain, characterized in that: The medicine also contains a pharmaceutically acceptable carrier.

6. Application of the SNI model in verifying the efficacy of a drug prepared using the siRNA molecule of claim 1 or 2 in treating pain.

7. The use of the SNI model according to claim 6 in verifying the efficacy of a drug prepared using the siRNA molecule according to claim 1 or 2 in treating pain, characterized in that: During the validation process, mechanical pain behavior testing was used to verify the efficacy of the drug.

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