Application of mannosidase alpha2B enzyme gene MAN2B1 inhibitor in inhibiting physiological behaviors of neurons

Abstract

The invention discloses an application of a mannosidase alpha2B enzyme gene MAN2B1 inhibitor in inhibiting physiological behaviors of neurons. The MAN2B1 inhibitor inhibits substance metabolism, adhesion, spreading and protrusion branch growth of the neurons by inhibiting expression of mannosidase alpha2B enzymes. The invention proposes and verifies that the mannosidase alpha2B enzyme gene MAN2B1 inhibitor can effectively inhibit the physiological behaviors of neurons for the first time, and provides a new direction for research and development of drugs for treating diseases caused by excessive growth of neuronal axons of a peripheral or central nervous system.

Description

Technical Field

[0001] This invention relates to gene therapy, and more particularly to a mannosidase α2B enzyme gene. MAN2B1 Application of inhibitors in suppressing neuronal physiological behavior. Background Technology

[0002] Mannosidase α-member (Man2b1) is a lysosome-based glycosidic hydrolase belonging to the GH38 protease family. Its main function is to specifically hydrolyze the α-1,6-mannosidic bond in N-linked glycoproteins, participating in the pruning of oligosaccharide chains synthesized in the endoplasmic reticulum and Golgi apparatus. This enzymatic reaction is crucial for maintaining normal protein glycosylation, thereby affecting protein folding, stability, and function. At the physiological level, Man2b1 participates in various life activities such as programmed cell death, immune response regulation, and substance transport by regulating glycosylation, making it one of the key molecules for maintaining cellular homeostasis.

[0003] Although the functions of Man2b1 in various cell types have been preliminarily elucidated, its specific mechanism of action in neurons remains significantly unknown. Whether Man2b1 possesses substrate specificity or regulatory mechanisms distinct from those in other cell types in neurons remains unclear and requires further investigation. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide the application of a mannosidase α2B enzyme inhibitor in the preparation of a medicament for treating diseases caused by excessive neuronal growth in the peripheral or central nervous system.

[0005] Technical solution: The mannosidase α2B enzyme gene described in this invention MAN2B1 Application of inhibitors in suppressing neuronal physiological behavior.

[0006] Preferably, the MAN2B1 The inhibitor is any one of siRNA, shRNA, sgRNA, miRNA, or antisense oligonucleotide.

[0007] Preferably, the neuronal physiological behaviors include neuronal generation, differentiation, polarization, migration, adhesion, substance synthesis and metabolism.

[0008] Preferably, the application is in the inhibition of neuronal axonogenesis.

[0009] Preferably, the application is in the preparation of a drug for treating diseases caused by excessive axonal growth in the peripheral nervous system; more preferably, the diseases caused by excessive axonal growth in the peripheral nervous system include neuropathic pain, traumatic neuroma, and neurofibroma.

[0010] Preferably, the application is in the preparation of a therapeutic drug for treating diseases caused by excessive growth of axons in the central nervous system neurons; more preferably, the disease caused by excessive growth of axons in the central nervous system neurons is epilepsy.

[0011] Preferably, the drug contains a mannosidase α2B gene. MAN2B1 The inhibitor is the active ingredient and a pharmaceutically acceptable excipient.

[0012] Preferably, the dosage form of the drug is granules, tablets, capsules, oral liquid, pills, emulsions, suspensions, injections, or infusions.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention proposes and verifies for the first time the mannosidase α2B enzyme gene. MAN2B1 Inhibitors can effectively suppress neuronal physiological behavior; 2. They provide a new direction for the development of drugs to treat diseases caused by excessive growth of axons in peripheral or central nervous system neurons. Attached Figure Description

[0014] Figure 1 for Man2b1 Figure 1. Statistical results of knockdown efficiency of siRNA transfection into PC12 cells; Figure 2 Figure 10 shows the statistical results of PC12 cell viability on hydrogels with different hardnesses after Man2b1 expression interference. Figure 3 Figure 1 shows the statistical results of ATP production in PC12 cells on hydrogels of different hardnesses after interference with Man2b1 expression. Figure 4 The images show the morphology of PC12 cells on hydrogels of different hardness after interference with Man2b1 expression. In the images, A is the immunofluorescence result, B is the cell area statistics, C is the cell roundness statistics, and D is the cell length-to-width ratio statistics. Figure 5 Figure (A) shows the adhesion of PC12 cells on hydrogels of different hardness after interference with Man2b1 expression, and the statistical results (B). Figure 6 The figure shows the statistical results of PC12 cell adhesion-related gene expression on hydrogels of different hardnesses after interfering with Man2b1 expression. In this figure, A represents... Itga3 Statistical results graph, B is Lamb2 Statistical results chart; Figure 7 Figure (A) shows the staining results of PC12 cells on hydrogels of different hardness after interference with Man2b1 expression, and the statistical results (B). Figure 8 Figure 1 shows the statistical results of lysosome-related gene expression in PC12 cells on hydrogels of different hardness after Man2b1 expression was interfered with. Figure 9 This image shows the axonal regeneration of DRG neurons after sciatic nerve injury in rats following interference with Man2b1 expression. Detailed Implementation

[0015] The technical solution of the present invention will be further described below.

[0016] Example 1: Mannosidase α2B enzyme (Man2b1) gene Man2b1 Preparation of inhibitors Based on publicly available mannosidase α2B enzyme genes MAN2B1 Nucleotide sequence (NCBI Reference Sequence: NM_000528.4), designed MAN2B1 Inhibitors Man2b1- siRNA.

[0017] in, Man2b1- The target sequence of siRNA-001 is: 5'-GCCAGAAGTTTGTCTACAA-3' (SEQ ID NO: 1); Man2b1- The target sequence of siRNA-002 is: 5'-GTACATACGGGCCACATTT-3' (SEQ ID NO: 2); Man2b1- The target sequence of siRNA-003 is 5'-GCGCATTGACTACCAAGAT-3' (SEQ ID NO: 3), which was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.

[0018] The disordered sequence control siRNA (si-NC) was designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.

[0019] Example 2: Culture, transfection and validation of rat adrenal pheochromocytoma cell line PC12 PC12 cells were seeded at a density of 200,000 cells / dish in 90 mm diameter culture dishes, and 7 mL of DMEM high-glucose medium containing 10% fetal bovine serum was added. The culture was carried out in 5% CO2 at 37°C. The medium was replaced with fresh DMEM high-glucose medium containing 10% fetal bovine serum every 48 h. Using Invitrogen Lipofectamine TM Transfection was performed using the RNAiMAX kit, following the instructions. 0.1 nmol siRNA was transfected per 100,000 cells, as obtained in Example 1. MAN2B1 -siRNA or control siRNA, after incubation for 12 h, discard the culture medium containing transfection reagent and add pre-warmed complete culture medium; After culturing for another 36 hours, total RNA was extracted using the Yishan Bio RNA Rapid Extraction Kit (RN001-50Rxns), and reverse transcription was performed using the Novizan HiScript III RT SuperMix for qPCR (+gDNA wiper) kit. qRT-PCR detection was then performed using the Novizan ChamQ Universal SYBR qPCR Master Mix kit to identify the target genes. Man2b1 and internal reference genes Gapdh Primer sequences are shown in Table 1 below, and reaction conditions are shown in Table 2 below: Table 1 Target Genes Man2b1 and internal reference genes Gapdh Primer sequence

[0020] Table 2 qRT-PCR reaction conditions

[0021] Fluorescence values ​​were collected during the extension phase of each cycle; after the amplification reaction was completed, melting curve analysis of the products was performed to ensure the quality of the PCR products. Using... Gapdh As an internal reference, it detects target genes. Man2b1 and internal reference Gapdh The Ct value is calculated using the ΔΔCt method. Man2b1 The relative expression level.

[0022] The results are as follows Figure 1 As shown, compared to the si-NC group transfected with disordered sequences, among them Man2b1- The interference efficiency of siRNA-001 (si-Man2b1-1) was the most significant. MAN2B1 The expression efficiency decreased to 30%; Man2b1- The expression efficiency of siRNA-002 (si-Man2b1-2) decreased to 60%; Man2b1- The expression efficiency of siRNA-003 (si-Man2b1-3) decreased to 56%, which was significantly different from that of the si-NC group. Therefore, subsequent cell experiments all used transfection. Man2b1- PC12 cells were used to develop siRNA-001 (si-Man2b1-1).

[0023] Example 3: Interference with Man2b1 expression affects PC12 cell viability 1. Interference with Man2b1 expression leads to decreased viability of PC12 cells. The transfected PC12 cells obtained in Example 2 were seeded at a density of 125,000 cells / well in 24-well plates on polyacrylamide / chitosan hydrogel material with a hardness of 1.5 kPa or 13.75 kPa. Cell viability was detected using a CCK-8 assay kit. After incubation, the absorbance value at 450 nm was measured using a multi-functional microplate reader, and cell viability was calculated.

[0024] The hydrogel was prepared as follows: 1 mL of 5% chitosan solution was mixed with 4 mL of 0.1% (corresponding to 1.5 kPa hardness) or 0.5% (corresponding to 13.75 kPa hardness) bisacrylamide solution, and the mixture was allowed to stand to remove air bubbles. Then, 200 μL of 10% ammonium persulfate solution was added and mixed well to form a gel. The solidified hydrogel was placed in a beaker and soaked in pure water at 37℃ for 3 days, with the water changed once a day. Subsequently, the hydrogel was soaked in 75% ethanol for 30 min, sterilized by ultraviolet irradiation for 1 h, soaked in pure water for 1 h, soaked in sterile PBS buffer for 1 h, and finally soaked in complete culture medium for later use.

[0025] The results are as follows Figure 2 As shown, the cell viability decreased in both types of hydrogels after Man2b1 expression was interfered with, and the difference was significant compared with the si-NC group, indicating that interfering with Man2b1 expression affects cell viability on different materials.

[0026] 2. Interference with Man2b1 expression leads to a decrease in ATP production in PC12 cells. The transfected PC12 cells obtained in Example 2 were seeded at a density of 500,000 cells / well on 12-well plates with a hardness of 1.5 kPa or 13.75 kPa polyacrylamide / chitosan hydrogel material and cultured. After 12 h of culture, the culture medium was discarded and the cells were washed with PBS. The ATP content was measured using the Beyotime ATP assay kit (catalog number S0026).

[0027] The results are as follows Figure 3 As shown, after interfering with Man2b1 gene expression, the ATP content in both groups of materials decreased, showing a significant difference compared with the si-NC group. This result is consistent with the trend of Man2b1 affecting cell viability, indicating that knocking down Man2b1 does indeed affect cell viability and ATP content.

[0028] Example 4: Interference with Man2b1 expression affects PC12 cell spreading and adhesion 1. Interference with Man2b1 expression affects the spreading morphology and branching growth of PC12 cells. The transfected PC12 cells obtained in Example 2 were seeded at a density of 100,000 cells / well in 24-well plates on polyacrylamide / chitosan hydrogel material with a hardness of 1.5 kPa or 13.75 kPa. After culturing for 48 h, the culture medium was discarded, and the cells were gently rinsed with PBS and fixed with 4% paraformaldehyde at room temperature. Then, the cells were gently rinsed with PBS three times, and permeabilized by soaking in 0.5% Triton X-100 at room temperature for 20 min. After rinsing with PBS three times, the cells were blocked with cell immunostaining blocking solution at room temperature for 1 h.

[0029] On ice, use Yuyou Biotechnology primary / secondary antibody dilution buffer (catalog number U10008) to prepare primary antibody: Solarbio SF488-labeled phalloidin (catalog number CA1640) at a dilution ratio of 1:100; add 200 μL of primary antibody to each well and incubate overnight at 4°C; after incubation, remove the well, allow it to equilibrate at room temperature for 30 min, and gently wash three times with PBS.

[0030] On ice, primary / secondary antibody dilution buffer was used to prepare DAPI solution at a ratio of 1:500. 200 μL was added to each well and incubated at room temperature in the dark for 2 h. After incubation, the cells were gently rinsed 3 times with PBS. Images were acquired using a Zeiss LSM 900 laser scanning confocal microscope.

[0031] The results are as follows Figure 4 As shown, PC12 cells cultured in a hydrogel with a hardness of 13.75 kPa exhibited a more extended morphology with more branches, and the morphological changes before and after interference were more significant. In contrast, cells in the softer 1.5 kPa hydrogel group were more rounded and lacked branching. Further statistical analysis and quantification using ImageJ software revealed that the area of ​​the 13.75 kPa hydrogel group decreased and the roundness increased after interference, while the aspect ratio did not change significantly. This indicates that interfering with Man2b1 expression leads to a significant alteration in the morphology of the spread-out cells.

[0032] 2. Interference with Man2b1 expression affects PC12 cell adhesion. The transfected PC12 cells obtained in Example 2 were seeded at a density of 250,000 cells / well in 24-well plates on polyacrylamide / chitosan hydrogel material with a hardness of 1.5 kPa or 13.75 kPa. After 48 hours of culture, the cells were observed under a microscope.

[0033] The results are as follows Figure 5 As shown, interference with Man2b1 expression in PC12 cells inhibited the adhesion of PC12 cells on hydrogels of different hardnesses. Both groups showed significant differences compared to the si-NC group, and the difference in the number of adherent cells between the different hardness groups decreased, indicating that... MAN2B1 The expression level of [a specific substance] can affect the number of PC12 cells adhering to hydrogels of different hardness.

[0034] After 48 hours of culture, total RNA was extracted using the ESA RNA Rapid Extraction Kit, and reverse transcription was performed using the Novizuma HiScriptIII RT SuperMix for qPCR (+gDNA wiper) Kit. qRT-PCR detection was then performed using the Novizuma ChamQ Universal SYBR qPCR Master Mix Kit to detect integrin α-3 gene. Itga3 and laminin β-2 gene Lamb2 Primer sequences are shown in Table 3 below, and the internal reference gene is also included. GAPDH Primer sequences and reaction conditions are the same as in Example 2: Table 3 Integrin α-3 Genes Itga3 and laminin β2 gene Lamb2 Primer sequence

[0035] Fluorescence values ​​were collected during the extension phase of each cycle; after the amplification reaction was completed, melting curve analysis of the products was performed to ensure the quality of the PCR products. Using... GAPDH As an internal reference, it detects target genes. Itga3 , Lamb2 and internal reference Gapdh The Ct value is calculated using the ΔΔCt method. Itga3 , Lamb2 The relative expression level.

[0036] The results are as follows Figure 6 As shown, after interfering with the expression of Man2b1, the expression of PC12 cell adhesion-related genes in both groups of materials was reduced. Itga3 , Lamb2 Expression levels were all decreased, showing a significant difference compared to the si-NC group. Figure 5 The trend of PC12 cell adhesion is consistent.

[0037] Example 5: Interference with Man2b1 expression does not affect PC12 cell lysosomes 1. Interference with Man2b1 expression does not affect the lysosomal environment of PC12 cells. The transfected PC12 cells obtained in Example 2 were seeded at a density of 125,000 per well in 24-well plates on polyacrylamide / chitosan hydrogel material with a hardness of 1.5 kPa or 13.75 kPa and cultured. Lyso-Tracker Red (lysosomal red fluorescent probe) was mixed with pre-warmed complete culture medium at a ratio of 1:5000 to prepare lysosomal dye working solution. After removing the original culture medium of PC12, 200 μL of lysosomal dye working solution was added to each well and incubated in a 37℃, 5% CO2 incubator in the dark for 2 h. Images were taken using a Zeiss LSM 900 laser scanning confocal microscope.

[0038] The results are as follows Figure 7 As shown, live-cell lysosome staining was performed on PC12 cells on two groups of hydrogel materials with different hardness. It was found that there was no significant difference in fluorescence intensity of lysosomes in the two groups of PC12 cells after interfering with Man2b1 expression, indicating that Lyso-Tracker Red dye can still be protonated, that is, interfering with Man2b1 expression does not have a significant effect on the intralysosome environment.

[0039] 2. Interference with Man2b1 expression does not affect the lysosomal integrity of PC12 cells. After 48 h of culture, total RNA was extracted using the ESA RNA Rapid Extraction Kit and reverse transcribed using the Novizuma HiScript III RT SuperMix for qPCR (+gDNA wiper) Kit. qRT-PCR was then performed using the Novizuma ChamQ Universal SYBR qPCR Master Mix Kit to detect the lysosome-associated membrane protein 1 gene. Lamp1 Primer sequences are shown in Table 4 below, and the internal reference gene is also included. Gapdh Primer sequences and reaction conditions are the same as in Example 2: Table 4. Lysosome-associated membrane protein 1 gene Lamp1 Primer sequence

[0040] Fluorescence values ​​were collected during the extension phase of each cycle; after the amplification reaction was completed, melting curve analysis of the products was performed to ensure the quality of the PCR products. Using... Gapdh As an internal reference, it detects target genes. Lamp1 and internal reference Gapdh The Ct value is calculated using the ΔΔCt method. Lamp1 The relative expression level.

[0041] The results are as follows Figure 8 As shown, Lamp1 The expression levels of Man2b1 also showed no significant difference, a result consistent with the lysosomal fluorescence map, indicating that interfering with Man2b1 expression also had no significant effect on lysosomal integrity.

[0042] Example 6: Interference with Man2b1 expression weakens sciatic nerve injury repair Adult male SD rats weighing 200g were anesthetized, and the hair near the hind limbs was shaved. After disinfection, the sciatic nerve was exposed and isolated at the iliac bone of the rat's hind limb. The sciatic nerve directly below the iliac bone was clamped vertically with hemostatic forceps at the third setting for 30 seconds, forming a 3 mm long clamping injury area. After the clamping injury was completed, the sciatic nerve at the clamping injury area appeared transparent and flat. The muscles and skin at the surgical site were sutured, and the suture site was disinfected. After suturing the surgical site of the rat, the hair on the spine of the hind limb was shaved off. The skin of the L4-L5 DRG area was cut along the midline of the back with surgical scissors. The muscles on both sides of the L4-L5 spine were removed with ophthalmic scissors to expose the lamina. The spinous process of the L5 segment was bitten off with bone forceps to expose the gap between the two vertebrae. The glass electrode needle was inserted horizontally. There was no resistance. The rat tail or hind leg reflexively bounced when the glass electrode needle was moved slightly, indicating that the needle insertion position was correct. Using Example 1 Man2b1 The siRNA-001 sequence and control sequence were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., and then subjected to in vivo adaptive modifications to obtain... Man2b1 -siRNA and control siRNA were used in the experiment. The results... Man2b1 The siRNA and control siRNA were diluted with sterile deionized water to 0.16 nmol / µL, and 10 μL was injected into each mouse, with 4 mice treated in each category. The injured areas were then sutured and disinfected. After the mice regained consciousness, they were returned to their cages for continued feeding.

[0043] Three days after the injection, an intrathecal injection was performed. Man2b1 Sciatic nerve tissue from the injured side of rats was inoculated with either siRNA or control siRNA. After fixation with 4% paraformaldehyde for 1 day, the tissue was dehydrated with sucrose, embedded in OCT, and frozen sectioned to a thickness of 12 μm. After sectioning, the sections were baked at 37°C for 30 min, then warmed to room temperature for 10 min, and rinsed three times with PBS. The sections were then blocked at 37°C for 1 h with blocking buffer. After blocking, the sections were incubated overnight at 4°C with 1:100 diluted Sigma NF200 (catalog number N2912) primary antibody, followed by gentle rinsing three times with PBS. The sections were then incubated for 2 h with a mixture of 1:200 diluted Abcam Donkey Anti-Mous lgG H&L (Alexa Fluor 488) (catalog number ab150109) secondary antibody and 1:200 diluted DAPI solution, followed by gentle rinsing three times with PBS. The sections were then mounted and incubated overnight at 4°C. Images were then acquired using a fluorescence microscope.

[0044] The results are as follows Figure 9 As shown, after interfering with the expression of Man2b1 in DRG neurons in rats, the number of regenerated axons after sciatic nerve injury was significantly reduced compared with the si-NC group, showing a significant difference.

Claims

1. A mannosidase α2B enzyme gene MAN2B1 Application of inhibitors in suppressing neuronal physiological behavior.

2. The application according to claim 1, characterized in that, The MAN2B1 The inhibitor is any one of siRNA, shRNA, sgRNA, miRNA, or antisense oligonucleotide.

3. The application according to claim 1, characterized in that, The physiological behaviors of neurons include neuronal generation, differentiation, polarization, migration, adhesion, substance synthesis and metabolism.

4. The application according to claim 1, characterized in that, The application described is in the inhibition of neuronal axonogenesis.

5. The application according to claim 4, characterized in that, The application is in the preparation of drugs for the treatment of diseases caused by excessive growth of axons in the peripheral nervous system neurons.

6. The application according to claim 5, characterized in that, Diseases caused by excessive axonal growth in the peripheral nervous system include neuropathic pain, traumatic neuroma, and neurofibroma.

7. The application according to claim 4, characterized in that, The application is in the preparation of drugs for treating diseases caused by excessive growth of axons in neurons of the central nervous system.

8. The application according to claim 7, characterized in that, Epilepsy is a disease caused by excessive growth of neuronal processes in the central nervous system.

9. The application according to claim 5 or 7, characterized in that, The drug contains mannosidase α2B gene. MAN2B1 The inhibitor is the active ingredient and a pharmaceutically acceptable excipient.

10. The application according to claim 1, characterized in that, The dosage form of the drug is granules, tablets, capsules, oral liquid, pills, emulsions, suspensions, injections, or infusions.

Images