Application of Thrb or Thra inhibitor in preparation of medicine for treating nerve injury

Abstract

The invention belongs to the field of biological medicine, and discloses application of a thyroid hormone receptor gene Thrb or Thra inhibitor in preparation of a medicine for treating nerve injury. Researches show that thyroid hormone is not beneficial to recovery of spinal cord after zebra fish is completely transected, and siRNA fragments of Thrb or Thra have a remarkable effect on promoting growth of cortical neurons and can also promote expression of p-S6. Therefore, Thrb and Thra are taken as molecular intervention targets, and an inhibitor is used for inhibiting expression of the molecular intervention targets, so that growth and regeneration of cortical neurons can be promoted, expression of p-S6 is promoted, activation of a PI3K-AKT pathway is promoted, and a new possibility is provided for preparing the medicine for treating nerve injury.

Description

Technical Field

[0001] The present invention relates to the application of an inhibitor in preparing a drug for treating nerve damage, and in particular to the application of an inhibitor of Thrb or Thra in preparing a drug for treating nerve damage, and belongs to the field of biomedicine. Background Art

[0002] Nerve injuries include central nervous system injury and peripheral nerve injury. Because the central nervous system lacks intrinsic regenerative capacity and has more regeneration inhibitory molecules than peripheral nerve injury, regeneration of the central nervous system is more difficult than that of the peripheral nervous system. After central nervous system injury in adult mammals, the vast majority of neurons cannot regenerate axons, often leading to persistent neurological dysfunction. Spinal cord injury (SCI) severely damages the central nervous system and is the most serious complication of spinal cord injury, often leading to severe functional impairment of the limbs below the injured segment. Currently, the treatment methods for SCI are very limited.

[0003] Thra (Thyroid Hormone Receptor Alpha) and Thrb (Thyroid Hormone Receptor Beta) are genes encoding thyroid hormone receptors. Both proteins encode triiodothyronine nuclear hormone receptors, regulating the biological activity of thyroid hormones. Gene knockout studies in mice have shown that different receptors, while somewhat redundant, may mediate distinct functions of thyroid hormones. Alternatively spliced transcript variants encoding different isoforms are known. Mutations in the Thrb gene are responsible for systemic thyroid hormone resistance (GTHR), a syndrome characterized by goiter and high levels of circulating thyroid hormones (T3-T4), with normal or mildly elevated thyroid-stimulating hormone (TSH). Previous studies have shown that thyroid hormones promote recovery after brain injury and accelerate sciatic nerve regeneration. However, there is also evidence that thyroid hormones inhibit cardiac regeneration and participate in central nervous system development.

[0004] Small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA, is a double-stranded RNA of 20-25 nucleotides in length. siRNA is known to primarily participate in the phenomenon of RNA interference (RNAi), regulating gene expression in a specific manner. It participates in several RNAi-related pathways, such as antiviral mechanisms and alterations in chromatin structure. The siRNA mechanism proceeds as follows: The endoribonuclease Dicer cleaves long dsRNA to form short interfering RNA, which then forms the RNA-induced silencing complex (RISC). Once inside the cell, the siRNA is incorporated into other proteins to form the RISC, where it then unfolds into a single strand. The single-stranded siRNA finds its complementary messenger RNA (mRNA). Binding to the mRNA triggers mRNA cleavage, which is then recognized by the cell as abnormal, leading to mRNA degradation and affecting protein expression, thereby silencing the gene encoding the mRNA. siRNA is also similar to miRNA; however, miRNAs are derived from shorter stem-loop RNA products and typically silence genes by inhibiting translation and have a broader specificity of action, whereas siRNAs typically act by cleaving mRNA before translation and have 100% complementarity, resulting in very strict target specificity.

[0005] S6 is a component of the 40S ribosomal subunit. Its phosphorylation (e.g., at Ser235 / 236 or Ser240 / 244) is mediated by mTORC1 via S6 kinase (S6K). Phosphorylated S6 protein (p-S6) is a marker of mTORC1 activity, reflecting enhanced protein translation. Activation of p-S6 indirectly verifies the functional activity of AKT and the activation of the PI3K-AKT pathway.

[0006] Currently, there are no reports on using thyroid hormone receptors as targets to repair spinal cord injury, nor are there any reports on the use of Thrb or Thra inhibitors in the preparation of drugs for treating nerve injury. Summary of the Invention

[0007] Purpose of the invention: The purpose of the present invention is to provide the use of Thrb or Thra inhibitors in the preparation of drugs for treating nerve damage.

[0008] Technical solution: The present invention provides the use of Thrb or Thra inhibitors in the preparation of drugs for treating nerve damage.

[0009] Based on the fact that interfering with the expression of thyroid hormone receptor encoding genes Thrb and Thra can significantly promote the growth of cortical neurons, promote the expression of p-S6, and promote the activation of the PI3K-AKT pathway, the present invention uses Thrb and Thra as molecular intervention targets and provides the use of their inhibitors in the preparation of drugs for treating nerve damage.

[0010] Preferably, the Thrb or Thra inhibitor can interfere with or inhibit the expression of Thrb or Thra.

[0011] Preferably, the Thrb or Thra inhibitor is siRNA of the Thrb or Thra gene.

[0012] Preferably, the sequence of the siRNA is as follows: si-Thrb-1: Justice chain: GCGAGACTCTAACCTTGAA Antisense strand: CGCTCTGAGATTGGAACTT si-Thrb-2: Justice chain: GCCAGGAATGTCGCTTTAA Antisense strand: CGGTCCTTACAGCGAATT si-Thrb-3: Justice Chain: CCCTGTGAAGACCAGATCA Antisense strand: GGGACACTTCTGGTCTAGT si-Thra-1: Justice chain: GCGTTTGAGCACTACGTCA Antisense strand: CGCAAACTCGTGATGCAGT si-Thra-2: Justice chain: GCAAGCTGATTGAGCAGAA Antisense strand: CGTTCGACTAACTCGTCTT si-Thra-3: Justice Chain: GCCGTACAATCCAGAAGAA Antisense strand: CGGCATGTTAGGTCTTCTT.

[0013] Preferably, the drug can promote the growth and regeneration of neurons.

[0014] Preferably, the neurons are cortical neurons.

[0015] Preferably, the neuronal growth and regeneration is manifested as axonal growth or activation of the PI3K-AKT pathway.

[0016] Preferably, the activation of the PI3K-AKT pathway is manifested as increased expression of phosphorylated S6 protein.

[0017] Preferably, the nerve damage is central nervous system damage.

[0018] Preferably, the central nervous system injury is spinal cord injury.

[0019] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: the present invention uses Thrb and Thra as molecular intervention targets, and after inhibiting their expression using inhibitors such as siRNA, it can promote the growth and regeneration of cortical neurons, promote the expression of p-S6, and promote the activation of the PI3K-AKT pathway, providing new possibilities for the preparation of drugs for treating nerve damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The expression levels of thyroid hormone-related factors at different time points after SCI (A: KEGG top 20 differential pathway analysis. B: Schematic diagram of the process of thyroid hormone action. C: sNuc-seq, expression of Thra and Thrb in different subpopulations of pyramidal neurons after SCI. D: Expression of Thra and Thrb in four subpopulations of pyramidal neurons in the Lay5b layer of the cortex at different time points after SCI. E: Spatial mapping of Thrb and Thra in pyramidal neurons in the Lay5b layer of the cortex after SCI. F: Spatial transcriptome sequencing, expression of genes Thra, Thrb, and Dio2 in corticospinal neurons (CSN) at different time points after SCI; Figure 2 The interference efficiency after siRNA inhibition of Thrb and Thra in vitro (A: siRNA-Thra knockdown efficiency statistical graph verified by qPT-PCR, n=3, *p<0.05, **p<0.01, vs. si-NC. B: siRNA-Thrb knockdown efficiency statistical graph verified by qPT-PCR, n=3, **p<0.01, ****p<0.0001, vs. si-NC); Figure 3The effect of inhibiting Thrb and Thra expression using siRNA in vitro on the regeneration of cortical neurons (A: Immunofluorescence image of E18d fetal rat cortical neurons transfected with Thra and Thrb siRNA and dispersed cultured. Tuj1 labels cortical neurons, Scale bar = 75μm. B: Statistical image of dispersed cultured E18d fetal rat cortical neurons. n = 300, Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs si-NC); Figure 4 To show the expression of p-S6 in cortical neurons after inhibition of Thra and Thrb (A: Immunofluorescence histochemistry of dispersed cultured E18 fetal rat cortical neurons, green is Tuj1 signal, blue is DAPI, and black is p-S6. Scale bar = 70 μm, 50 μm. B: Under a 20x microscope, the number of co-labeled Tuj1 and p-S6 fluorescence was compared with the number of Tuj1 fluorescence in the si-NC, si-Thra-3, and si-Thrb-3 groups. **p<0.01 vs si-NC); Figure 5 This is the recovery of zebrafish spinal cord transection after drug activation of the thyroid hormone pathway (A: Schematic diagram of immunofluorescence staining 21 days after zebrafish spinal cord transection; red is NF200, green is GFAP, blue is DAPI, Scale bar = 100 μm. B: GFAP width and length of the bridging site, *p<0.05, **p<0.01, vs Negative Control. C: NF200 width and length of the bridging site, *p<0.05, vs Negative Control). DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0022] Example 1 Detection of Thrb and Thra expression by combining spatial transcriptome sequencing and single-cell sequencing Combined with single-cell data, the top 20 KEGG enrichment pathways of differentially expressed genes in the Lay5b layer corticospinal tract neurons and the 2 subpopulations of spinal cord anterior horn motor neurons in the cerebral cortex of SD rats (from the Animal Experiment Center of Nantong University) were analyzed, among which the thyroid signaling pathway was more significant ( Figure 1 A). Focus on the three factors Thra, Thrb and Dio2 in the thyroid hormone metabolism pathway ( Figure 1B). By observing the expression of Thra and Thrb in the cortex, it was found that Thra and Thrb were highly expressed in the Lay5b layer 4 subpopulation of pyramidal neurons (corticospinal tract neurons). At different time points after hemisection of the right spinal cord T10, the expression of Thrb was more significant than that of Thra ( Figure 1 C). We also examined the expression of Thra, Thrb, and Dio2 genes in the CSN after SCI in different samples. Thrb and Dio2 were expressed at extremely high levels 3 days and 7 days after SCI, respectively ( Figure 1 DF).

[0023] Example 2 Verification of Interference Efficiency by Inhibiting Thrb and Thra Using siRNA in Vitro Experimental method: RNA was extracted, reverse transcribed into cDNA, and then subjected to real-time quantitative polymerase chain reaction (qRT-PCR) as follows: 1) Anesthesia: 18-day pregnant SD rats were anesthetized with reanesthesia, and the abdomen of the pregnant rats was thoroughly disinfected with 75% alcohol.

[0024] 2) Sample collection: Under a stereomicroscope, the cerebral cortex of an 18-day gestational SD rat fetus was collected and placed in a medium dish containing dissection fluid.

[0025] 3) Digestion: After washing the cortex several times with PBS, add 2 ml of trypsin in a medium dish and digest for 15 minutes, pipetting thoroughly every 5 minutes.

[0026] 4) Terminate digestion: Complete digestion with 6 ml of DMEM (DMEM basal medium + 10% FBS + 1% PS) and pipette thoroughly.

[0027] 5) Filtration: Filter through a 70 μm cell strainer.

[0028] 6) Centrifugation: Centrifuge at 1200 rpm for 5 minutes at room temperature. Discard the supernatant, resuspend the pellet, count the cells using a cell counter, and then plate.

[0029] 7) Transfection: After centrifugation, discard the supernatant, resuspend the pellet, count the cells using a cell counting plate, and then inoculate into a tube containing transfection reagent (500 μl = 450 μl NB - culture medium + 50 μl Opti-MEM™ Reduced Serum Medium + 5 μl siRNA + 1.5 μl Lipofectamine RNAiMAX) in a 24-well plate.

[0030] The sequence of the siRNA is as follows: si-Thrb-1: Justice chain: GCGAGACTCTAACCTTGAA Antisense strand: CGCTCTGAGATTGGAACTT si-Thrb-2: Justice chain: GCCAGGAATGTCGCTTTAA Antisense strand: CGGTCCTTACAGCGAATT si-Thrb-3: Justice Chain: CCCTGTGAAGACCAGATCA Antisense strand: GGGACACTTCTGGTCTAGT si-Thra-1: Justice chain: GCGTTTGAGCACTACGTCA Antisense strand: CGCAAACTCGTGATGCAGT si-Thra-2: Justice chain: GCAAGCTGATTGAGCAGAA Antisense strand: CGTTCGACTAACTCGTCTT si-Thra-3: Justice Chain: GCCGTACAATCCAGAAGAA Antisense strand: CGGCATGTTAGGTCTTCTT.

[0031] 8) Change medium: 12-16 hours after transfection, change to NB ＋ Culture medium (NB basal medium + 2% B27 + 1% Glu + 1% PS).

[0032] 9) Verification of interference efficiency: After 48 h of culture, RNA was extracted for reverse transcription and qRT-PCR.

[0033] Test results: Figure 2 As shown, the expression levels of Thra and Thrb in cortical neurons were significantly reduced compared with the control group.

[0034] Example 3 Verification of the effect of inhibiting Thrb and Thra on promoting the growth of cortical neurons Test method: 1. Statistical Analysis of Axon Length in Dispersed Culture 1) Anesthesia: 18-day pregnant SD rats were anesthetized with reanesthesia, and the abdomen of the pregnant rats was thoroughly disinfected with 75% alcohol.

[0035] 2) Sample collection: Under a stereomicroscope, the cerebral cortex of an 18-day gestational SD rat fetus was collected and placed in a medium dish containing dissection fluid.

[0036] 3) Digestion: After washing the cortex several times with PBS, add 2 ml of trypsin in a medium dish and digest for 15 minutes, pipetting thoroughly every 5 minutes.

[0037] 4) Terminate digestion: Complete digestion with 6 ml of DMEM (DMEM basal medium + 10% FBS + 1% PS) and pipette thoroughly.

[0038] 5) Filtration: Filter through a 70 μm cell strainer.

[0039] 6) Centrifugation: Centrifuge at 1200 rpm for 5 minutes at room temperature. Discard the supernatant, resuspend the pellet, count the cells using a cell counter, and then plate.

[0040] 7) Transfection: After centrifugation, discard the supernatant, resuspend the pellet, count the cells using a cell counter, and then seed the cells into a 24-well plate containing transfection reagent. The transfection reagent is the same as in Example 2.

[0041] 8) Change medium: 12-16 hours after transfection, change to NB ＋ Culture medium (NB basal medium + 2% B27 + 1% Glu + 1% PS).

[0042] 9) Statistics: After 48 h of culture, dispersed cultured cells were immunohistochemically stained and axon length was counted.

[0043] 2. Cell Immunohistochemical Staining Sections were rinsed three times in 0.01 M PBS (10 min each time), permeabilized with PBST (PBS containing 0.5% Triton X-100) at room temperature for 15 min, and the tissue was circled with a histochemical pen. Approximately 200 μl of immunostaining blocking solution (Biyuntian, P0102) was added for 1 h at room temperature. The sections were then incubated with the primary antibody, Anti-beta III Tubulin (mouse, Abcam), overnight at 4°C. Sections were rinsed three times in 0.01 M PBS (10 min each time), and incubated with the secondary antibody, Donkey anti-mouse IgG 488 (1:500, Jackson), for 2 h at room temperature. Sections were then rinsed three times in 0.01 M PBS (10 min each). The sections were mounted with an appropriate amount of anti-quenching mounting solution (containing DAPI) (Biyuntian, P0131-25ml) and observed and photographed under a ZEISS upright fluorescence microscope.

[0044] Test results: Figure 3 As shown, the results of dispersed culture indicate that the inhibition of Thrb and Thra using small interfering RNA can promote the axon growth of cortical neurons.

[0045] Example 4 Expression of p-S6 in cortical neurons after inhibition of Thra and Thrb Test method: 1. Dispersed culture of cortical neurons 1) Anesthesia: 18-day pregnant SD rats were anesthetized with reanesthesia, and the abdomen of the pregnant rats was thoroughly disinfected with 75% alcohol.

[0046] 2) Sample collection: Under a stereomicroscope, the cerebral cortex of an 18-day gestational SD rat fetus was collected and placed in a medium dish containing dissection fluid.

[0047] 3) Digestion: After washing the cortex several times with PBS, add 2 ml of trypsin in a medium dish and digest for 15 minutes, pipetting thoroughly every 5 minutes.

[0048] 4) Terminate digestion: Complete digestion with 6 ml of DMEM (DMEM basal medium + 10% FBS + 1% PS) and pipette thoroughly.

[0049] 5) Filtration: Filter through a 70 μm cell strainer.

[0050] 6) Centrifugation: Centrifuge at 1200 rpm for 5 minutes at room temperature. Discard the supernatant, resuspend the pellet, count the cells using a cell counter, and then plate.

[0051] 7) Transfection: After centrifugation, discard the supernatant, resuspend the pellet, count the cells using a cell counter, and then seed the cells into a 24-well plate containing transfection reagent. The transfection reagent is the same as in Example 2.

[0052] 8) Change medium: 12-16 hours after transfection, change to NB ＋ Culture medium (NB basal medium + 2% B27 + 1% Glu + 1% PS).

[0053] 9) Statistics: After 48 h of culture, disperse the cultured cells for immunohistochemical staining and calculate the co-labeling ratio.

[0054] 2. Cell Immunohistochemical Staining Sections were rinsed three times in 0.01 M PBS (10 min each time), permeabilized with PBST (PBS containing 0.5% Triton X-100) at room temperature for 15 min, and the tissue was circled with a histochemical pen. Approximately 200 μl of immunostaining blocking solution (Biyuntian, P0102) was added for 1 h at room temperature. The sections were then incubated with primary antibodies: anti-beta III tubulin (mouse, Abcam) and p-S6 (Rabbit, Cell Signaling) overnight at 4°C. Sections were then rinsed three times in 0.01 M PBS (10 min each), and incubated with secondary antibodies: Donkey anti-mouse IgG 488 (1:500, Jackson) and Donkey anti-rabbit IgG 555 (1:500, Jackson) for 2 h at room temperature. Sections were then rinsed three times in 0.01 M PBS (10 min each). After adding an appropriate amount of anti-fluorescence quenching mounting solution (containing DAPI) (Biyuntian, P0131-25ml), the slides were mounted and observed and photographed under a ZEISS upright fluorescence microscope.

[0055] Test results: Figure 4 As shown, the results of dispersed culture indicate that inhibition of Thrb using small interfering RNA can promote the axonal growth of cortical neurons and the expression of p-S6, while inhibition of Thra has no obvious effect.

[0056] Example 5 Recovery of zebrafish spinal cord transection after drug activation of the thyroid hormone pathway Test method: 1. Complete transection of the zebrafish spinal cord 1) Place zebrafish (provided by the Experimental Animal Center of Nantong University) in a homemade fixed container and anesthetize them with anesthetic; 2) Using microtweezers, the spinal cord was completely transected at the T10 region of the zebrafish dorsal fin; 3) Apply a layer of waterproof material to the wound of the zebrafish after complete spinal cord transection; 4) Place the zebrafish in water and let it sink to the bottom. Observe it after it wakes up. If it tilts to one side when swimming, the model is successful. 5) The zebrafish with successful modeling were divided into two groups. The experimental group was treated with 5 nM thyroid hormone T3 (Sigma, D6397) in water, while the control group was not treated with the drug. 6) After 21 days, the zebrafish were fixed with 4% PFA solution, embedded, and sectioned. Immunofluorescence staining was performed to observe the recovery of the injured area.

[0057] 2. Tissue Immunohistochemical Staining Sections were rinsed three times in 0.01 M PBS (10 min each time). The membrane was permeabilized with PBST (PBS containing 0.5% Triton X-100) at room temperature for 15 min. The tissue was circled with a histochemical pen and approximately 200 μl of immunostaining blocking solution (Biyuntian, P0102) was added for 1 h at room temperature. The sections were then incubated with primary antibodies: GFAP (mouse, Abcam) and NF200 (rabbit, Sigma) overnight at 4°C. Sections were rinsed three times in 0.01 M PBS (10 min each time). Secondary antibodies: Donkey anti-mouse IgG 488 (1:500, Jackson) and Donkey anti-rabbit IgG 555 (1:500, Jackson) were incubated for 2 h at room temperature. Sections were then rinsed three times in 0.01 M PBS (10 min each time). After adding an appropriate amount of anti-fluorescence quenching mounting solution (containing DAPI) (Biyuntian, P0131-25ml), the slides were mounted and observed and photographed under a ZEISS upright fluorescence microscope.

[0058] Test results: Figure 5 As shown in the figure, immunofluorescence results showed that after applying 5 nM thyroid hormone T3, the width and length of the cytoskeletal protein NF200 and glial fibrillary acidic protein GFAP in the nerve cells of the experimental zebrafish bridge site were both lower than those in the control group. This suggests that less thyroid hormone is more conducive to the recovery of the zebrafish spinal cord after complete transection.

Claims

1. Use of Thrb or Thra inhibitors in the preparation of drugs for treating nerve damage.

2. The use according to claim 1, characterized in that The Thrb or Thra inhibitor can interfere with or inhibit the expression of Thrb or Thra.

3. The use according to claim 1, characterized in that The Thrb or Thra inhibitor is siRNA of Thrb or Thra gene.

4. The use according to claim 3, characterized in that The sequence of the siRNA is as follows: si-Thrb-1: Justice chain: GCGAGACTCTAACCTTGAA Antisense strand: CGCTCTGAGATTGGAACTT si-Thrb-2: Justice chain: GCCAGGAATGTCGCTTTAA Antisense strand: CGGTCCTTACAGCGAATT si-Thrb-3: Justice Chain: CCCTGTGAAGACCAGATCA Antisense strand: GGGACACTTCTGGTCTAGT si-Thra-1: Justice chain: GCGTTTGAGCACTACGTCA Antisense strand: CGCAAACTCGTGATGCAGT si-Thra-2: Justice chain: GCAAGCTGATTGAGCAGAA Antisense strand: CGTTCGACTAACTCGTCTT si-Thra-3: Justice Chain: GCCGTACAATCCAGAAGAA Antisense strand: CGGCATGTTAGGTCTTCTT.

5. The use according to claim 1, characterized in that The drug can promote the growth and regeneration of neurons.

6. The use according to claim 5, characterized in that The neurons are cortical neurons.

7. The use according to claim 5, characterized in that The growth and regeneration of the neurons are manifested as axon growth or activation of the PI3K-AKT pathway.

8. The use according to claim 7, characterized in that The activation of the PI3K-AKT pathway is manifested by increased expression of phosphorylated S6 protein.

9. The use according to claim 1, characterized in that The nerve damage is central nervous system damage.

10. The use according to claim 9, characterized in that The central nervous system injury is spinal cord injury.

Images