CN110564842B - Application of cytochrome enzyme CYP26A1 in preparation of medicine for treating neuropathic pain - Google Patents
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Abstract
The invention discloses application of cytochrome enzyme CYP26A1 as a target point in preparation of a medicine for treating neuropathic pain. The invention utilizes a mouse lumbar 5 Spinal Nerve Ligation (SNL) method to induce neuropathic pain, extracts mRNA of mouse spinal cord lumbar 5 segments of a model group and a sham operation group 10 days after an operation to carry out gene chip detection, screens related genes of the neuropathic pain, and searches a potential target spot for developing drugs for treating the neuropathic pain. The result shows that CYP26A1 has increased expression in the maintenance stage of neuropathic pain, promotes the maintenance and development of neuropathic pain, and inhibits the expression or function of neuropathic pain, thereby effectively relieving neuropathic pain. Based on this, the cytochrome enzyme CYP26A1 is taken as a target spot, and the inhibitor for inhibiting the expression and the function of the cytochrome enzyme can be used for screening the medicines for treating the neuropathic pain, preparing the medicines for treating the neuropathic pain or preparing the medicines for diagnosing the neuropathic pain.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of cytochrome enzyme CYP26A1 as a target spot in preparation of a medicine for treating neuropathic pain.
Background
Pain, as one of the somatic sensations, can alert the body when it is injured, causing a series of defensive protective responses in the body to prevent further injury. However, when the pain loses the normal protective function and persists for months or years, the pain becomes pathological pain (chronic pain) which is harmful to physical and mental health. According to the world health organization reports, chronic pain is the most common and most heavily burdened condition. Statistics show that about 30% of adults suffer from chronic pain. Neuropathic pain (neuropathic pain) is one type of chronic pain, and is caused by inflammation, operation, nerve trauma, viral infection (such as herpes zoster), cancer, metabolic disease (such as diabetes), immunological disease (such as multiple sclerosis and AIDS), etc. Patients develop hyperalgesia with increased sensitivity to noxious stimuli and decreased response thresholds, as well as touch-induced pain manifestations from non-painful stimuli (e.g., touch), which severely affect the physical and mental well-being and quality of life of the patient.
Neuropathic pain is a difficulty in clinical analgesia, and most of the existing analgesic drugs are opioid analgesics and non-opioid auxiliary analgesics (non-steroidal anti-inflammatory drugs, antidepressants and anticonvulsants). Opioid analgesics such as morphine, methadone, fentanyl, oxycodone and the like are often accompanied by respiratory depression and circulatory depression side effects, and symptoms such as constipation, dizziness, nausea, vomiting, pruritus, dry mouth and the like are common. The non-steroidal anti-inflammatory drug commonly uses the cyclooxygenase-2 inhibitor celecoxib, which is associated with gastrointestinal side effects including symptoms of abdominal pain, dyspepsia and nausea. Auxiliary analgesics mainly comprising anticonvulsants and antipsychotics, such as carbamazepine, clonidine, ketamine and the like, have different types of adverse reactions, such as symptoms of fatigue, headache, thrombocytopenia, skin pruritus, congestive rash, insomnia, constipation and the like. Therefore, the method further researches the occurrence and development mechanism of the neuropathic pain and has important academic value and practical significance for searching effective drug action targets from a new angle.
CYP26a1 belongs to the cytochrome P450 (CYP 450) superfamily, the CYP26 family, the CYP26A subfamily member, and is an oxidase that specifically metabolizes retinoic acid. Among the members of the CYP450 superfamily, CYP1, CYP2 and CYP3 families are studied more in the pain field, and participate in the metabolism of various narcotic analgesic drugs, which can cause the change of pharmacokinetics and the change of drug efficacy, thus having important significance for clinical medication guidance. However, it has been reported that CYP450 superfamily members are directly involved in the regulation of the development and progression of neuropathic pain. Researches report that cytochrome CYP26A1 is also an important target point for tumor treatment, and the tumor itself or the tumor treated by chemotherapeutic drugs can cause hyperalgesia and generate neuropathic pain, and the effect of one-arrow double-carving can be achieved by taking CYP26A1 as a drug target point in the treatment of the neuropathic pain caused by the cancer.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide the application of cytochrome enzyme CYP26A1 as a target spot in screening drugs for treating neuropathic pain, preparing drugs for treating neuropathic pain or preparing drugs for diagnosing neuropathic pain.
In order to achieve the above purpose, the invention provides the following technical scheme:
the cytochrome enzyme CYP26A1 can be used for screening drugs for treating neuropathic pain, preparing drugs for treating neuropathic pain or preparing drugs for diagnosing neuropathic pain.
Further, the drug inhibits the expression or function of CYP26a1, and may be a nucleic acid molecule, a carbohydrate, a lipid, a small chemical drug, an antibody drug, a polypeptide, a protein, an interfering lentivirus, or an interfering adeno-associated virus.
The mouse SNL model induces the neuropathic pain, mRNA of the spinal cord waist 5 segment is extracted for gene chip detection, and the applicant finds that the level of CYP26A1mRNA is increased by about 22 times in the maintenance stage of the neuropathic pain model, and the CYP26A1 can play a role in the maintenance stage of the neuropathic pain. Real-time fluorescent quantitative PCR and Western Blot experiments further prove that CYP26A1mRNA level and protein level are obviously increased in the maintenance stage of a neuropathic pain model, and inhibition of CYP26A1 expression or inhibition of the function can effectively relieve neuropathic pain, which indicates that CYP26A1 is involved in the maintenance and development of neuropathic pain. The results all indicate that the cytochrome enzyme CYP26A1 can be used as a potential target for developing the medicine for treating neuropathic pain.
Based on the technical idea, the invention also provides a plurality of inhibitors of CYP26A1mRNA, which comprise:
a nucleic acid molecule for inhibiting CYP26A1mRNA, wherein the nucleic acid molecule is siRNA or shRNA; the siRNA or shRNA comprises a sequence similar to the sequence shown in SEQ ID NO: 1, and the RNA sequence has the same nucleotide sequence.
Further, the siRNA comprises a sense strand and an antisense strand, and the nucleotide sequences of the sense strand and the antisense strand are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
Further, the siRNA may be modified with methoxy or cholesterol.
A recombinant vector for inhibiting CYP26A1mRNA comprises a sequence capable of transcribing the nucleic acid molecule and a vector, wherein the sequence is embedded in the vector.
Further, the vector is a lentiviral vector or an adeno-associated viral vector.
A lentivirus for inhibiting CYP26A1mRNA is prepared from the recombinant vector through virus packing.
An adeno-associated virus for inhibiting CYP26A1mRNA is prepared from the recombinant vector through virus packing.
The application of the nucleic acid molecule, the recombinant vector, the lentivirus or the adeno-associated virus in preparing the medicine for preventing or treating neuropathic pain.
Use of an inhibitor of CYP26a1 function in the manufacture of a medicament for the prevention or treatment of neuropathic pain.
Further, the inhibitor of the CYP26a1 function is talarozole.
Has the advantages that: the invention induces neuropathic pain by utilizing a mouse spinal cord lumbar 5(L5) Spinal Nerve Ligation (SNL) method, finds that the expression of a CYP450 family member CYP26A1 is increased in the maintenance stage of the neuropathic pain, promotes the maintenance and development of the neuropathic pain, inhibits the expression or the function of CYP26A1, can effectively relieve the neuropathic pain, can take cytochrome enzyme CYP26A1 as a target point, and takes an inhibitor for inhibiting the expression and the function of the cytochrome enzyme CYP26A1 as an effective therapeutic drug for the neuropathic pain.
Drawings
FIG. 1 is a graph showing that Spinal Nerve Ligation (SNL) of L5 induced mechanical allodynia in the mouse hindpaw of example 1.
FIG. 2 is a graph of SNL-induced spinal CYP26A1mRNA elevation in example 1. A is mRNA chip detection SNL model group and false operation group spinal cord differential expression mRNA clustering analysis. B is mRNA chip analysis cytochrome P450 family difference expression mRNA relative elevation fold. And C is the real-time fluorescent quantitative PCR verification of the CYP26A1mRNA expression level in the SNL model.
FIG. 3 is a graph of SNL-induced increase in spinal CYP26A1 protein expression in example 1. A. B is the Western Blot detection result of CYP26A1 protein. C-K is an immunofluorescence double-label staining for locating spinal cord CYP26A1 protein-expressed cell types.
FIG. 4 is a graph showing that inhibition of CYP26A1 expression by siRNA in example 2 relieves SNL-induced mechanical allodynia. A is selected to screen siRNA (siRNA-003) with high-efficiency interference effect aiming at mouse CYP26A1 mRNA. B is the result of mechanical pain induction by intrathecal injection of different doses of siRNA (siRNA-003-2OMe +5Chol) modified by methoxy and cholesterol. C, detecting the gene interference effect of injecting siRNA-003-2OMe +5Chol into the spinal cord sheath by real-time fluorescence quantitative PCR.
Figure 5 is a graph of the ability of the CYP26a1 inhibitor Talarozole of example 3 to alleviate SNL-induced mechanical allodynia. A is the detection result of mechanical touch induced pain of different doses of Talarozole intrathecally injected in spinal cord on the 7 th day of SNL. And B is a spinal intrathecal injection high-dose Talarozole on the 3 rd day of SNL, and a mechanical touch induced pain detection result. C is the real-time fluorescent quantitative PCR detection of the expression change of CYP26B1mRNA in the SNL model.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples.
Because experimental nerve injuries such as spinal nerve ligation, chronic compression of sciatic nerve or nerve roots generate thermal hyperalgesia and touch induced pain, which are similar to some clinical neuropathic pains, the invention utilizes a Spinal Nerve Ligation (SNL) method of mouse spinal cord lumbar 5(L5) to induce neuropathic pains, extracts mRNA of 5 segments of spinal cord lumbar of mice in an operation group and a sham operation group 10 days after the operation to carry out gene chip detection, screens related genes of the neuropathic pains and searches potential targets for developing drugs for treating the neuropathic pains. Clustering analysis revealed that the mRNA of Cytochrome P450 family member 26A1(Cytochrome P45026A 1, CYP26A1) was increased by nearly 22-fold in the neuropathic pain model group. Real-time fluorescent quantitative PCR is further used for verifying that the expression of CYP26A1 is obviously increased 7-14 days after SNL, and the expression is unchanged 3 days after SNL. The results suggest that CYP450 family member CYP26a1 functions during the maintenance phase of neuropathic pain.
Based on the above research, the present invention designs siRNA against CYP26a1 gene transcript, and performs methoxy modification and cholesterol modification to demonstrate that inhibition of CYP26a1 expression can alleviate mechanical allodynia induced by SNL. In addition, inhibition of CYP26a1 function was demonstrated by known CYP26a1 inhibitors to alleviate SNL-induced mechanical allodynia. In the following examples, the CYP26a1 inhibitor talarozole was selected for the validation test, but the CYP26a1 inhibitor is not limited to talarozole and includes other inhibitors.
Example 1
Increased expression of CYP26A1 during the SNL-induced neuropathic pain maintenance phase
1. Experimental methods
(1) Establishment of mouse Spinal Nerve Ligation (SNL) neuropathic pain model
After injecting the compound anesthetic into the abdominal cavity of SPF grade 8-week male ICR mice, the hairs on the back and the waist are shaved by a shaver, after being sterilized by iodine alcohol, a longitudinal incision is made at the position of about 5mm on the left outer side of the posterior midline, the incision is about 1cm long, and 2/3 is above the iliac crest. Then, thoracolumbar fascia was incised, the erector spinal muscles were bluntly separated and pulled outward, the transverse process of the 6 th lumbar vertebra was exposed under a surgical microscope and snapped off, the L5 spinal nerves were gently separated with a glass needle, the L5 spinal nerves were tightly ligated with 6-0 gauge silk, and then the incision was closed layer by layer. The procedure was the same as above in the sham group, but the spinal nerves were not ligated.
(2) Mechanical touch induced pain detection method
Before behavior detection, mice were individually placed in an organic glass box on a mechanical pain testing jig and acclimated to a quiet environment at room temperature of 23 + -1 deg.C for 3 days. The mechanical touch-induced pain perception threshold of the left hind paw of mice was measured using von Frey filenamet according to the "up and down" method reported by Dixon et al. The left rear Paw bottom of the mouse is stimulated from 0.16g of von Frey mouse, the left rear Paw bottom is stimulated, the mouse is continuously stimulated for 2 seconds by taking the small bending of the mouse as the standard of complete stress, if the mouse has phenomena of Paw withdrawal, Paw flinching, Paw licking and the like, the mouse is stimulated by using the small first gram number of the mouse, if the mouse does not have pain, the mouse is stimulated by using the first gram number of the mouse, the stimulation interval is 3 minutes, 6 stimulation results are recorded, and finally, a Paw withdrawal threshold (Paw withdry threshold) is checked according to a threshold table, wherein the unit is 'g'. A lower paw withdrawal threshold indicates a more severe mechanical touch-induced pain.
(3) RNA extraction and real-time fluorescent quantitative PCR
Total RNA was extracted by Trizol method, and after concentration measurement, 1. mu.g of total RNA was subjected to reverse transcription reaction according to the instructions of HiScript II 1st Strand cDNA Synthesis Kit of Vazyme. The obtained cDNA product is diluted 8 times and used as a template for real-time fluorescent quantitative PCR, 2 mu L of the template is added into each PCR system, the method is carried out by referring to the specification of an AceQ qPCR SYBR Green Master Mix of Vazyme company, and the sequence of the used primers is as follows:
CYP26A1forward,5′-TGCAAGAGCAATCAAGACAACA-3′(SEQ ID NO：4)；
CYP26A1reverse,5′-CTTCAGAGCAACCCGAAACC-3′(SEQ ID NO：5)；
CYP26B1forward,5′-CTCTGCCCCTTTGCTCTTG-3′(SEQ ID NO：6)；
CYP26B1reverse,5′-TCTTTCCACCTTACCTCTCTGCTT-3′(SEQ ID NO：7)；
GAPDH forward,5′-AAA TGG TGA AGG TCG GTG TGA AC-3′(SEQ ID NO：8)；
GAPDH reverse,5′-CAA CAA TCT CCA CTT TGC CAC TG-3′(SEQ ID NO：9)。
results analysis adopted 2-ΔΔCTAnd (4) carrying out analysis by a method.
(4) Protein extraction and Western Blot
After the mice are perfused by the heart with physiological saline, spinal cord lumbar 5(L5) segments are taken and put into tissue lysate containing protease inhibitor, and are electrically homogenized on ice, and the supernatant is taken after centrifugation, thus obtaining tissue protein solution. And (3) determining the total protein concentration of the sample by using the BCA protein content detection kit, adding a protein loading buffer solution, and calibrating the uniform protein concentration. After boiling for 5 minutes, the protein is denatured, and after SDS-polyacrylamide gel electrophoresis and wet membrane transfer, the membrane is placed into 5% skimmed milk powder sealing liquid for sealing for 2 hours at room temperature. After blocking, the primary antibody diluted by 5% skimmed milk powder was incubated overnight at 4 ℃, and the secondary antibody was incubated after membrane washing and imaged using an LI-COR Odyssey CLx imaging system. Image protein band analysis ImageJ software was used.
(5) Immunofluorescence staining
The SNL model group mice are fixed by 4% paraformaldehyde perfusion, spinal cord waist 5(L5) segments are taken, dehydrated in 20% and 30% sucrose solution in sequence, and frozen and sliced after sinking, and the thickness of the slices is 30 mu m. Spinal cord sections were rinsed 3 times with 0.01M PBS solution, 10 min/time, blocked with 1% BSA (containing 0.5% Triton X-100) incubated at room temperature for 2 hours, primary antibody incubated: CYP26A1 (rabbitt, 1:200, Bioss), the neuronal marker NeuN (mouse,1:800, Millipore), the astrocytic marker GFAP (mouse,1:6000, Millipore), the microglial marker Iba-1(goat,1:500, Abcam), were incubated overnight at 4 ℃. Sections were rinsed 3 times 10 min/time in 0.01M PBS solution, and secondary antibodies were incubated: cy 3-goat anti-rabbit (1:1000, Jackson), FITC-goat anti-mouse (1:1000, Jackson), Cy 3-donkey anti-rabbit (1:1000, Jackson), Alexa Fluor 488-donkey anti-goat (1:1000, Jackson), incubated at room temperature for 2 hours. Sections were rinsed 3 times 10 min/time in 0.01M PBS. Mounting the piece, sealing the piece by using a fluorescent sealing agent, and observing and photographing under a fluorescent microscope.
2. The experimental results are as follows:
(1) SNL induces mechanical touch-induced pain in the unilateral hind paw of the mouse
As shown in fig. 1, for the mechanical trigger-induced pain threshold (PWT) induced by von Frey filenament stimulation, the SNL model group (SNL model) was significantly decreased from 1 day after surgery and continued to 21 days after surgery compared to the Sham group (Sham), and the two-way anova was statistically different (p < 0.001). The result shows that the SNL-induced mice generate mechanical touch-induced pain, and the SNL neuropathic pain model is successfully established.
(2) SNL-induced spinal CYP26A1mRNA and protein expression increase
A L5 Spinal Nerve Ligation (SNL) method is used for inducing neuropathic pain of mice, mRNA of spinal cord lumbar 5(L5) segments of the mice in an SNL group and a sham operation group is extracted 10 days after operation for gene chip detection, and the result shows that the expression of CYP26A1mRNA in a CYP450 superfamily is increased most. As shown in FIG. 2A, clustering analysis was performed on all gene expression signal values detected by the chip using the hierarchical algorithm. The column shows the expression of all genes in a certain sample, the row shows the expression of a certain gene in four samples, the red shows that the gene expression level is high, the green shows that the gene expression level is low, the gene expression levels in the operation groups (SNL-1 and SNL-2) and the pseudo-operation groups (Sham-1 and Sham-2) are approximately the same, but the gene expression level difference between the groups is obvious, and the CYP450 superfamily members which meet the characteristics comprise CYP26A1, CYP4B1 and CYP1B 1. As shown in FIG. 2B, the expression level of CYP26A1mRNA in the CYP450 superfamily was increased by nearly 22 times and most by the SNL group compared with the sham group. In order to verify the detection result of the chip, the time course change of the spinal CYP26A1mRNA level in the SNL model is further detected by using real-time fluorescent quantitative PCR. The results showed that CYP26A1mRNA was unchanged on SNL 1 day, SNL 3 days (early pain phase), significantly increased on SNL7 days (pain maintenance phase), and continued to rise to 14 days post-model, compared to the sham group, with no significant difference in CYP26A1mRNA levels (. about.pp.) on day 7 of the sham group compared to the normal group, as shown in fig. 2C<0.05，***P<0.001 and
group comparisons, n-4-6, one-way analysis of variance and Bonferroni correction test). This result suggests that spinal nerve ligation induced significant increases in spinal CYP26A1mRNA levels in mice during the maintenance phase of the neuropathic pain model, suggesting that CYP26A1 may play a role during the maintenance phase of neuropathic pain. CYP216A1mRNA is translated into protein to exert physiological function, so according to the result shown by mRNA level change, spinal cord CYP26A1 protein expression change is detected by Western Blot on the 7 th day of normal group and false operation group and on the 7 th day after SNL, the result is shown as A and B in figure 3, and normal group and false operation groupCompared with the group, the SNL induced spinal cord CYP26A1 protein was significantly increased on day 7 after the operation, and the normal group and the sham operation group were not different (P)<0.001 and
group comparison of<0.05 compared to sham group, n-3, one-way analysis of variance and Bonferroni correction test). At the protein level, it was further confirmed by immunofluorescence double-labeling experiments that CYP26a1 is expressed in neurons and astrocytes of the spinal cord. As shown by C-K in FIG. 3, the immunofluorescence ditag indicates that CYP26A1 coexists with the neuronal marker NeuN (C-E) and the astrocytic marker GFAP (F-H), but not with the microglia marker Iba1 (I-K). Spinal nerve ligation induced significant increases in spinal CYP26a1 protein levels in mice during the maintenance phase of neuropathic pain models, suggesting that CYP26a1 is involved in the maintenance and development of neuropathic pain.
Example 2
Inhibition of CYP26A1 reduces SNL-induced mechanical pain
1. The experimental method comprises the following steps:
(1) primary astrocyte culture
Placing the cerebral cortex of a newborn mouse for 1-2 days into a small culture dish filled with precooled D-Hanks liquid, removing the meninges under a dissecting microscope, collecting the cerebral cortex after membrane stripping, shearing the tissue with scissors, blowing the tissue by a pipette until no fragment exists, and filtering by a mesh screen with the aperture of 100 mu m. Centrifuge at 4 deg.C, 3000g, 5 min. The supernatant was discarded, the cells were resuspended in complete medium and filtered through a 10 μm pore size filter. At 2X 106(per well) the number of cells is inoculated in a 6-well plate, the liquid is changed every day, then the liquid is changed every 3-4 days, when the growth confluency of the cells reaches 95%, d-cAMP (final concentration of 150 mu M) is added to induce the differentiation and maturation of astrocytes, and the drug intervention experiment is carried out after 3 days of induction.
(2) Design of synthetic mouse CYP26A1siRNA
siRNA design and synthesis are completed by Hibo Biotechnology Co., Ltd, Guangzhou City (address: Innovation mansion No. 182 science City in Guangzhou development area, C3, 13-14 layers, zip code: 510663): the full length of the sequence of mouse CYP26A1mRNA (NM-007811.2) is obtained from the National Center for Biotechnology Information (NCBI) database, 3 candidate siRNAs against the mouse CYP26A1 gene transcript are designed according to the RNAi principle and by combining with design software, and are compared and checked by BLAST to ensure that the candidate siRNAs have no homology with other genes, and then are chemically synthesized.
The target sequence information of 3 candidate sirnas is as follows:
(3) CYP26A1siRNA intervention experiment
After 3 days of the primary astrocytes induced with d-cAMP, the OPTI-MEM medium was replaced, different siRNAs (1ug) were transfected in advance for 24 hours, and then LPS (0.1ug/ml) was used to stimulate the cells for 6 hours, collect the cells, extract RNAs, and use them for real-time fluorescent quantitative PCR detection. The cell experiments were divided into 5 groups: siRNA-NC (control siRNA) group, siRNA-NC + LPS group, siRNA-001+ LPS group, siRNA-002+ LPS group, and siRNA-003+ LPS group. In the experiment, after the siRNA which is highly effective to the mouse CYP26A1 gene is screened from 3 candidate siRNAs, methoxy modification is carried out on the siRNA by Sharp Biotech limited of Guangzhou city to improve the stability in vivo, and cholesterol modification is carried out at the same time, so that the siRNA can directly enter cells without a carrier compound to play a role in interfering gene expression and is used for ethological detection after in vivo intrathecal injection.
(4) Intrathecal injection of medicine for spinal cord
After the hair on the back and the waist of the mouse is shaved by electric hair clippers, the mouse is inhaled and anesthetized by isoflurane, the skin is disinfected by alcohol, 10 mu l of medicine is injected between the spinous processes of the fourth lumbar vertebra and the fifth lumbar vertebra to the subarachnoid space of the mouse by using a 29gauge (BD company) injection needle, and the sign of successful puncture is that the tail of the mouse is shaken or suddenly swung sideways. After the inhalation anesthesia is stopped, the mouse can wake up quickly, the subsequent pain ethology detection and other researches are not influenced, and the reliability of the experimental result is ensured.
2. The experimental results are as follows:
(1) screening out high-efficiency siRNA aiming at mouse CYP26A1 gene
F-H experimental junction as in FIG. 3The results show that spinal cord astrocytes express CYP26A1, and after 3 candidate siRNAs synthesized by the company are obtained, experiments are carried out by using primary astrocytes cultured in vitro, so that the siRNA which most effectively interferes with the expression of CYP26A1 is screened out. In recent years, a new concept in chronic pain research is that neuroinflammatory reaction is an important cause for starting and maintaining chronic pain, so that Lipopolysaccharide (LPS) capable of causing inflammatory reaction is used for stimulating astrocytes in vitro experiments, and real-time fluorescence quantitative PCR is used for detecting CYP26A1mRNA level change. The LPS induced a nearly 26-fold increase in CYP26A1mRNA levels in astrocytes as shown by the a results in figure 4. siRNA-001 and siRNA-003 in 3 candidate siRNAs have significant interference effect, inhibit CYP26A1mRNA rise induced by LPS, and the knockdown rate reaches nearly 76.9% and 92.3% respectively, and siRNA-002 has no interference effect (P + P)<0.001 compared with the siRNA-NC group,###P<0.001 compared to the siRNA-NC + LPS group, n-4, one-way anova and Bonferroni correction test). Therefore, the effect of screening siRNA-003 interfering target genes is best, and the siRNA is selected to carry out methoxy modification and cholesterol modification, so that the siRNA is more stable in vivo and can directly play an interference role in vivo without the help of a transfection reagent, and the siRNA is used for behavioral experiments.
The sequence information of siRNA-003 is:
sense strand 5'-GCAAGAGCAAUCAAGACAA-3' (SEQ ID NO: 2)
Antisense strand 5'-UUGUCUUGAUUGCUCUUGC-3' (SEQ ID NO: 3)
(2) Inhibition of CYP26A1 expression by siRNA can relieve SNL-induced mechanical touch-induced pain
Injecting siRNA which is subjected to methoxy modification and cholesterol modification aiming at CYP26A1 gene intrathecally on day 7 of SNL, specifically inhibiting CYP26A1 expression, and detecting the change of mechanical touch-induced pain threshold value of mice by behavior. As shown in fig. 4, B results, SNL7d intrathecally injected siRNA-003-2OMe +5Chol at low dose (2ug) had a tendency to inhibit mechanical allodynia in mice without significant difference, while at high dose (5ug) there was a clear effect of alleviating mechanical allodynia in mice at 6 hours and 1 day (P <0.05 compared to SNL + siRNA-NC-2OMe +5Chol group, n ═ 6-8, two-way repeated measures anova, Bonferroni correction test for pairwise comparison). Real-time fluorescent quantitative PCR results showed that CYP26A1mRNA levels in spinal cord of SNL model mice were significantly decreased after intrathecal injection of siRNA-003-2OMe +5Chol (5ug) (P <0.01 compared to the SNL + siRNA-NC-2OMe +5Chol group, n-4-5, Student's t-test) as shown in fig. 4C. These results indicate that inhibition of CYP26a1 expression can alleviate SNL-induced mechanical allodynia.
Example 3
Inhibition of CYP26A1 relieves SNL-induced mechanical pain
1. The experimental method comprises the following steps:
intrathecal injection (same procedure as example 2) of various doses of the CYP26a1 inhibitor Talarozole (Talarozole) at the early (3 days) and maintenance (7 days) stages of Spinal Nerve Ligation (SNL) -induced pain: 0.1ug, 0.5ug, 1ug, behaviorally detect the change in mechanical touch-induced pain threshold in mice. Talarozole used in the examples was commercially available from MedChem Expree company (NJ08852, USA). Talarozole (Talarozole) is an oral all-trans retinoic acid metabolic blocker, effectively and selectively inhibits CYP26A1 and CYP26B1, increases the intracellular level of endogenous all-trans Retinoic Acid (RA), and is used for treating psoriasis and acne.
2. The experimental results are as follows: talarozole, an inhibitor of CYP26A1, relieves SNL-induced mechanical touch-induced pain
Talarozole was intrathecally injected on SNL day 3 and day 7 to inhibit CYP26a1 function and behavioural tests of mice for mechanical touch-induced pain threshold changes. As shown in the A results in FIG. 5, different doses of Talarozole injected intrathecally on day 7 of SNL showed varying degrees of relief from mechanical allodynia in mice, with Talarozole 0.5ug injected intrathecally for 3 hours showing significant relief from the control group, and Talarozole 1ug showed significant relief at 1 hour for up to 6 hours, which was dose-dependent in mice to relieve mechanical allodynia ([ P ])<0.001，###P<0.001 compared to the Vehicle group, n-8-10, two-factor repeated measures analysis of variance, Bonferroni correction test for pairwise comparison). While the intrathecal injection of Talarozole on day 3 of SNL showed no analgesic effect, as shown in fig. 5B, further demonstrates that CYP26a1 plays a role during the neuropathic pain maintenance phase,not early. Since the CYP26a1 inhibitor Talarozole also had inhibitory effects on CYP26B1, but real-time fluorescence quantitative PCR assay showed C in fig. 5 that CYP26B1mRNA was not expressed in the SNL-induced neuropathic pain model (n-5-6, one-way anova and Bonferroni correction test), the analgesic effects of intrathecally injected Talarozole were through its inhibition of CYP26a 1.
Sequence listing
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Application of cytochrome enzyme CYP26A1 in preparation of medicine for treating neuropathic pain
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Claims (10)
1. Application of cytochrome enzyme CYP26A1 in screening drugs for treating neuropathic pain.
2. Use according to claim 1, characterized in that: the drug inhibits the expression or function of CYP26A1, and is selected from nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, interfering lentiviruses or interfering adeno-associated viruses.
3. A nucleic acid molecule that inhibits CYP26a1mRNA, wherein: the nucleic acid molecule is siRNA or shRNA; the siRNA or shRNA is similar to the siRNA or shRNA shown in SEQ ID NO: 1, and the RNA sequence has the same nucleotide sequence.
4. The nucleic acid molecule of claim 3, wherein: the siRNA comprises a sense strand and an antisense strand, wherein the nucleotide sequences of the sense strand and the antisense strand are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
5. The nucleic acid molecule of claim 4, wherein: the siRNA is modified by methoxy or cholesterol.
6. A recombinant vector that inhibits CYP26a1mRNA, wherein: comprising a sequence capable of transcribing the nucleic acid molecule of any one of claims 3-5 and a vector, said sequence being embedded in the vector.
7. A lentivirus which inhibits CYP26a1mRNA, wherein: the recombinant vector of claim 6, which is packaged with a virus.
8. An adeno-associated virus that inhibits CYP26a1mRNA, wherein: the recombinant vector of claim 6, which is packaged with a virus.
9. Use of the nucleic acid molecule of any one of claims 3-5, the recombinant vector of claim 6, the lentivirus of claim 7 or the adeno-associated virus of claim 8 in the manufacture of a medicament for the prevention or treatment of neuropathic pain.
Use of an inhibitor of CYP26a1 function in the manufacture of a medicament for the prevention or treatment of neuropathic pain, wherein: the CYP26A1 function inhibitor is talarozole.
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| Title |
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| Molecular targeting of retinoic acid metabolism in neuroblastoma:the role of the CYP26 inhibitor R116010 in vitro and in vivo;JL Armstrong 等;《British Journal of Cancer》;20070508;第96卷(第11期);第1675-1683页 * |
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