Application of ganciclovir in preparation of medicine for treating demyelination disease

Abstract

The invention relates to the field of biological medicine, in particular to application of a reagent for up-regulating PEX5 expression level or improving PEX5 protein activity in preparation of a medicine for treating demyelination diseases. A lentivirus infection experiment and lysolecithin are utilized to induce a mouse demyelination model, it is found that PEX5 can promote differentiation and maturation of oligodendroglia cells and remyelination and repair of demyelination mice, and ganciclovir can promote differentiation of the oligodendroglia cells and effectively relieve demyelination diseases by promoting expression of PEX5. PEX5 can be used as a drug intervention target, and a drug for promoting expression or function of PEX5 can be used as a potential therapeutic drug for demyelination diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to a reagent for upregulating PEX5 expression level or enhancing PEX5 protein activity and the use of ganciclovir in preparing a drug for treating demyelinating diseases. Background Art

[0002] Myelin plays a critical role in maintaining normal neural function in higher vertebrates. It not only provides insulation but also accelerates the conduction of nerve impulses and protects axons. Demyelination in the central nervous system is a common feature of many neuropsychiatric disorders, including multiple sclerosis (MS), hypoxic-ischemic encephalopathy, Alzheimer's disease, depression, and schizophrenia. Unfortunately, there are currently no definitive and effective treatments for demyelinating damage in the central nervous system, making the search for appropriate therapeutic strategies particularly urgent.

[0003] When demyelinating injury occurs, the body mobilizes its own oligodendrocyte progenitor cells (OPCs). Once activated, these cells proliferate and migrate to the damaged area, eventually differentiating into mature oligodendrocytes (OLs). These mature cells wrap around nerve axons, forming new myelin sheaths and achieving remyelination to repair the damaged lesion. However, in many demyelinating diseases, such as late-stage MS, this naturally occurring remyelination process often fails, leading to irreversible neurological dysfunction. Studies have shown that impaired OPC differentiation and maturation is a key factor in the failure of myelin regeneration in many demyelinating diseases. Therefore, these cells may serve as important targets for drug intervention.

[0004] Ganciclovir, a classic antiviral drug, is clinically used primarily to prevent and treat cytomegalovirus infection in immunocompromised patients. It is also used to treat herpes zoster and human herpesvirus-6 encephalitis. Ganciclovir also has pharmacological effects by inhibiting microglial inflammatory responses and suppressing immune cell infiltration.

[0005] Although specific signaling pathways have been shown to mediate the pharmacological effects of ganciclovir, research on its effects on oligodendrocytes has not yet been reported, and there are currently no literature reports on the relationship between ganciclovir and myelin regeneration and repair in demyelinating diseases. Summary of the Invention

[0006] The purpose of the present invention is to provide a reagent for upregulating the expression level of PEX5 or improving the activity of PEX5 protein and the use of ganciclovir in the preparation of a drug for treating demyelinating diseases.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The applicant's previous research found that the lack of PEX5 protein in OLs in the demyelinating lesions may be an important rate-limiting factor for the failure of myelin repair in chronic demyelinating lesions in MS, which also provides a potential target for regenerative treatment of demyelinating diseases. + When PEX5 was specifically knocked out in cells (mature OLs), mice were found to have extensive axonal degeneration and progressive subcortical demyelinating lesions. This result indicates that PEX5 in OLs plays an important regulatory role in the maintenance of myelin structure and the occurrence of demyelinating diseases. However, the effect of PEX5 on the pre-maturity stage of OLs remains unknown. In order to further study the effect of PEX5 on the in vitro differentiation of OPCs, we constructed PEX5 overexpression and interference lentiviruses, respectively. After infecting primary cultured OPCs with lentiviruses, they were cultured in differentiation medium for 72 hours, and then immunofluorescence staining was used to detect the proportion of myelin basic protein (MBP)-positive cells, and western blotting was used to detect the protein level of MBP. The results showed that overexpression of PEX5 can significantly promote MBP + The increase in the proportion of cells and the upregulation of MBP protein levels, while interference with PEX5 can lead to MBP + The above results indicate that PEX5 can promote the differentiation and maturation of OPCs in vitro and the expression of myelin-related proteins.

[0009] Since PEX5 primarily promotes OPC differentiation in vitro, does it affect their differentiation and myelination capacity in vivo? To this end, we established a mouse spinal cord lysolecithin (LPC) injury model and injected PEX5 overexpression or control viruses into the rostral and caudal regions of the LPC lesions. Twelve days later, spinal cords were harvested for fluorescein staining and electron microscopy. We found that compared with the control group, the PEX5 overexpression group showed a significant decrease in the area of ​​demyelination and a significant increase in the thickness of newly formed myelin sheaths. Statistically, the g-ratio values ​​in the PEX5 overexpression group were also significantly decreased compared with the control group, indicating a significant increase in myelin thickness. These results suggest that PEX5 overexpression significantly promotes the myelination capacity of OPCs, resulting in a reduction in the extent of LPC demyelination and an increase in remyelination. We further injected PEX5 or control virus into the rostral and caudal sides of LPC lesions. We found that after 14 days, the PEX5-treated group showed a significant increase in the area of ​​demyelination, a significant decrease in newly formed myelin, and an increase in the g-ratio compared to the control group. This indicates that PEX5-treated cells significantly inhibit the myelination capacity of OPCs, increasing the area of ​​demyelination and inhibiting remyelination in LPC lesions. These results suggest that PEX5 can significantly promote the differentiation of OPCs both in vitro and in vivo.

[0010] Based on the PEX5 target, we screened a library of FDA-approved small molecule drugs with a history of safe clinical use. We ultimately identified three drugs that significantly upregulated PEX5 mRNA and protein levels in OPCs: ganciclovir, latanoprost, and olopatadine. We then added these three drugs to oligodendrocyte differentiation cultures and measured MBP protein levels 72 hours later. Ganciclovir significantly increased MBP expression. Therefore, we selected ganciclovir for subsequent studies.

[0011] The present invention further analyzed the effects of varying ganciclovir concentrations on the in vitro differentiation of OPCs. Western blotting revealed that ganciclovir significantly upregulated MBP protein levels at a concentration of 5 µM. Immunofluorescence staining was then used to verify the effects of ganciclovir on OPC differentiation at this concentration, demonstrating that ganciclovir significantly promoted OPC differentiation and maturation without affecting apoptosis or proliferation.

[0012] Based on the promoting effect of ganciclovir on the differentiation and maturation of OPCs in vitro, the present invention first used the LPC-induced myelin injury model in mice to evaluate the effect of ganciclovir on the differentiation and remyelination of OPCs in vivo.+ (OLs marker) cells and PEX5 + CC1 + The number of cells increased significantly. This is consistent with the reduction in the extent of demyelination revealed by MBP staining in the lesion area of ​​the treated mice on day 14. Electron microscopy analysis also revealed a significant increase in the degree of myelin regeneration in the treated mice: the proportion of myelinated axons in the lesions of the treated mice increased, and the g-ratio value was lower than that of the control group. In summary, the present invention discovered that ganciclovir can induce significant repair of LPC-induced demyelination lesions.

[0013] In order to further explore the therapeutic effect of ganciclovir in the MS animal model, the present invention also selected MOG, whose disease pathological changes highly simulate chronic progressive MS. 35–55 This study used a mouse model of experimental autoimmune encephalomyelitis (EAE) induced by ganciclovir. This study employed a therapeutic dosing regimen, with daily intraperitoneal administration beginning on day 15, the peak of the disease, to evaluate whether the drug could promote repair of demyelinating disease rather than prevent it. Results showed that animals treated with ganciclovir generally exhibited significant functional recovery, while most mice in the control group continued to exhibit severe hindlimb mobility impairment throughout the treatment period. Benztropine, a classic pro-myelinating drug, was also used as a positive control and found to have a therapeutic effect on EAE similar to that of ganciclovir. The significant functional recovery in ganciclovir-treated mice was associated with histological improvements in the spinal cord: ganciclovir-treated mice demonstrated restored MBP expression in the white matter of the spinal cord, indicating a reduction in the extent of demyelination, while control mice displayed persistent white matter damage. Electron microscopic examination of the spinal cords of the two groups of mice revealed a significant increase in the number of newly formed myelin sheaths and a significant decrease in the g-ratio in the ganciclovir-treated group compared with the control group. In summary, we found that ganciclovir significantly promoted the recovery of EAE in mice. Furthermore, we found that intraperitoneal administration of ganciclovir did not cause significant hepatotoxicity, renal toxicity, or weight changes in EAE mice, providing strong toxicological evidence for its systemic administration.

[0014] In summary, we found that ganciclovir has great potential clinical application prospects for the treatment of demyelinating diseases, especially the currently incurable chronic progressive MS.

[0015] Based on the above technical solution, the first aspect of the present invention provides the use of an agent for upregulating the expression level of PEX5 or increasing the activity of PEX5 protein in the preparation of a drug for treating demyelinating diseases.

[0016] Furthermore, the agent for increasing the expression level of PEX5 or enhancing the activity of PEX5 protein is selected from:

[0017] A) Drugs that upregulate Pex5 mRNA and protein levels in OPCs;

[0018] B) Recombinant vector containing the PEX5 encoding gene;

[0019] C) Recombinant virus containing the PEX5 encoding gene.

[0020] Furthermore, the drug for increasing the Pex5 mRNA content and PEX5 protein level in OPCs is ganciclovir.

[0021] Furthermore, the dosage of ganciclovir in the demyelinating disease treatment drug is 10 mg / kg.

[0022] Furthermore, ganciclovir is used in the preparation of drugs for promoting the differentiation and maturation of oligodendrocytes or the expression of myelin-related proteins.

[0023] Furthermore, the recombinant virus containing the PEX5 encoding gene is a recombinant lentivirus containing the PEX5 encoding gene (NM_000319.4).

[0024] Furthermore, the demyelinating disease includes but is not limited to multiple sclerosis.

[0025] Furthermore, the multiple sclerosis includes but is not limited to chronic progressive multiple sclerosis.

[0026] Furthermore, the drug for treating demyelinating diseases is a pharmaceutical composition comprising an agent that upregulates the expression level of PEX5 or increases the activity of PEX5 protein as an active ingredient and a conventional pharmaceutical carrier.

[0027] Furthermore, the pharmaceutical composition is in the form of a microcapsule, intravenous emulsion, liposome preparation, aerosol, prodrug preparation, injection, mixture, oral ampoule, tablet, capsule, pill, emulsion, ointment, rubber plaster, film, sponge, iontophoresis agent, or transdermal absorption agent.

[0028] In a second aspect, the present invention provides a pharmaceutical preparation for treating demyelinating diseases, wherein the active ingredient is an agent that upregulates the expression level of PEX5 or increases the activity of PEX5 protein.

[0029] Furthermore, the pharmaceutical preparation for treating demyelinating diseases also includes a pharmaceutically acceptable carrier or excipient.

[0030] The advantages of the present invention are:

[0031] 1. The present invention discovered a new medical application of the target PEX5 in central demyelinating diseases. PEX5 overexpression can significantly reduce the demyelinated area and significantly increase the thickness of thin new myelin sheaths. PEX5 overexpression can significantly promote the myelination ability of OPCs, thereby reducing the scope of LPC demyelination lesions and improving the level of remyelination.

[0032] 2. The present invention discovered a drug, ganciclovir, that can significantly increase the content of Pex5 mRNA and the level of PEX5 protein in OPCs, providing a new medical use in central demyelinating diseases. It was clarified that ganciclovir has the effect of promoting the differentiation and maturation of oligodendrocytes and inducing accelerated repair process in demyelinating disease model mice.

[0033] 3. The current treatment for the classic central demyelinating disease, MS, mainly relies on immune regulation strategies, which can only reduce the frequency of disease attacks in patients with relapsing-remitting multiple sclerosis. As the disease enters the chronic progressive stage, few drugs can effectively intervene and regulate. The reason for its failure is the lack of effective therapeutic drugs to reverse the failure of myelin regeneration. Ganciclovir discovered by the present invention shows a strong effect in promoting myelin regeneration in demyelinating disease model mice. Considering that the drug also has a certain anti-inflammatory effect, the present invention believes that the synergistic effect of these two effects may bring hope to the development of therapeutic drugs for demyelinating diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Overexpression or interference of Pex5 affects the in vitro differentiation of oligodendrocyte precursor cells (OPCs);

[0035] (A) Western blot analysis of PEX5 protein expression in OPCs infected with Pex5 overexpression and control lentiviruses after 72 hours of in vitro differentiation. N = 3. (B) Western blot analysis of PEX5 protein expression in OPCs infected with Pex5 knockdown and control lentiviruses after 72 hours of in vitro differentiation. N = 4. (C) Purified OPCs were infected with Pex5 overexpression and control lentiviruses and cultured in differentiation medium for 72 hours. Immunofluorescence staining was then used to analyze MBP expression in these cells. Nuclei were labeled with Hoechst. Scale bar = 50 μm. (D) Statistical results showed that Pex5 overexpression lentivirus significantly increased MBP expression in OPCs. N = 3. (E) Purified OPCs were infected with Pex5 knockdown and control lentiviruses and cultured in differentiation medium for 72 hours. Immunofluorescence staining was then used to analyze the expression of eGFP in these cells. +(F) Statistical results show that Pex5 knockout lentivirus significantly inhibited the increase in MBP expression in OPCs. N = 3. (G) Western blot analysis of MBP protein expression in OPCs infected with Pex5-overexpressing and control lentiviruses after 72 hours of in vitro differentiation. N = 4. (H) Western blot analysis of MBP protein expression in OPCs infected with Pex5 knockout and control lentiviruses after 72 hours of in vitro differentiation. N = 4. Data are expressed as mean ± standard error of the mean (SEM). *P < 0.05, **P < 0.01.

[0036] Figure 2 Overexpression or interference of Pex5 regulates remyelination in LPC-induced demyelination lesions;

[0037] (A) Representative fluorescein staining and electron microscopy images of lesions infected with Pex5 overexpression, knockdown, or corresponding control lentiviruses at day 12 or 14 after LPC-induced injury. (B) Quantitative analysis of the percentage of myelinated axons at the lesion site in each group of mice. Data are presented as mean ± standard error of the mean (SEM). *P < 0.05, **P < 0.01. (C) Statistical analysis showed that the G-ratio was decreased in the Pex5 overexpression group compared with the control group at day 12 after LPC-induced injury, while it was increased in the Pex5 infection group compared with the control group at day 14 after LPC-induced injury.

[0038] Figure 3 Validate the ability of candidate drugs identified through initial Pex5 screening to promote the differentiation of oligodendrocyte precursor cells (OPCs);

[0039] (A) Western blot analysis of MBP protein expression in OPCs treated with 5 μM ganciclovir, latanoprost, and olopatadine after 72 hours of in vitro differentiation. (B) Statistical results showed that ganciclovir significantly increased MBP levels in OPCs at a 5 μM concentration, while olopatadine and latanoprost had no such effect. N = 5. Data are expressed as mean ± standard error (SEM). ***P < 0.001; ns, not significant difference.

[0040] Figure 4 Ganciclovir modulates the differentiation level of oligodendrocyte precursor cells (OPCs) in a dose-dependent manner;

[0041] (A) Western blot analysis of MBP protein expression in OPCs treated with different concentrations of ganciclovir after 72 hours of in vitro differentiation. (B) Statistical results showed that ganciclovir significantly increased MBP levels in OPCs at concentrations of 1, 2, 5, and 10 μM, with the most significant effect observed at 5 μM. (C) Purified OPCs were treated with different concentrations of ganciclovir and cultured in differentiation medium for 72 hours. Immunofluorescence staining was then performed to analyze the expression of MBP and NG2 (an OPC marker). Nuclei were labeled with Hoechst. Scale bar = 50 μm. (D) Statistical analysis of the effect of different concentrations of ganciclovir on the percentage of MBP- and NG2-positive cells. N = 3. Data are expressed as mean ± standard error (SEM). *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

[0042] Figure 5 Ganciclovir had no significant effect on apoptosis and proliferation of oligodendrocyte precursor cells (OPCs);

[0043] (A) Purified OPCs were treated with 5 μM ganciclovir and cultured in differentiation medium for 72 hours. Immunofluorescence staining was used to analyze the expression of cleaved-Caspase 3 (a marker of apoptosis). Nuclei were labeled with Hoechst. Scale bar = 50 μm. (B) Quantification of the percentage of cleaved-Caspase 3-positive cells under the indicated conditions. N = 3. Data are expressed as the mean ± standard error of the mean (SEM). ns, no significant difference. (C) Purified OPCs were treated with 5 μM ganciclovir and cultured in proliferation medium for 48 hours. Immunofluorescence staining was used to analyze the expression of BrdU (a marker of proliferation). Nuclei were labeled with Hoechst. Scale bar = 50 μm. (D) Quantification of the percentage of BrdU-positive cells under the indicated conditions. N = 3. Data are expressed as the mean ± standard error of the mean (SEM). ns, no significant difference.

[0044] Figure 6 Ganciclovir promotes OPCs differentiation and remyelination in LPC-induced demyelination lesions.

[0045] (A) Representative CC1 in the dorsal spinal cord lesion area (outlined by white dotted lines) of mice in the ganciclovir-treated and control groups at 10 days after LPC modeling (dpl). + cells, PEX5 +Immunofluorescence images of cells and MBP staining, nuclei were labeled with Hoechst, scale bar = 50 μm. (B) Quantitative analysis of CC1 in each lesion area in the ganciclovir-treated and control groups. + cells, PEX5 + Cells and PEX5 + CC1 + Cell numbers are normalized to those of the control group (N = 6). (C) Representative MBP staining and electron microscopy images of the dorsal spinal cord lesion region in ganciclovir-treated and control mice 14 days after LPC modeling (dpl). Nuclei were labeled with Hoechst. Scale bar = 50 μm (immunofluorescence staining) or 1 μm (electron microscopy). (D) Statistical graph showing the percentage of myelinated axons in the spinal cord lesion region of ganciclovir-treated and control mice 14 days after LPC modeling (dpl). N = 3. (E) Statistical graph showing the mean G-ratio of myelin in the spinal cord lesion region of ganciclovir-treated and control mice 14 days after LPC modeling, measured using ImageJ software. N = 3. Data are expressed as mean ± standard error (SEM). *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

[0046] Figure 7 Ganciclovir alleviates experimental autoimmune encephalomyelitis (EAE) in mice.

[0047] (A) Starting at the peak of EAE modeling (day 15), mice in each group were injected daily with ganciclovir (10 mg / kg), benztropine (10 mg / kg), and saline. EAE scores were continuously assessed in each group. N = 5. (B) Representative fluorescence and electron microscopy images of demyelinated areas in ganciclovir-treated and control groups on day 25 of EAE modeling. Nuclei were labeled with Hoechst. Scale bar = 50 μm (immunofluorescence staining) or 1 μm (electron microscopy). (C) Statistical graph showing the percentage of myelinated axons within the demyelinated area in ganciclovir-treated and control groups on day 25 of EAE modeling. N = 3. (D) Statistical graph showing the mean G-ratio of myelin within the demyelinated area in ganciclovir-treated and control groups on day 25 of EAE modeling. N = 3. Data are presented as mean ± standard error (SEM). *P < 0.05, ***P < 0.001. DETAILED DESCRIPTION

[0048] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0049] Example 1: Construction and concentration of lentivirus for overexpression and interference of Pex5

[0050] 1. Experimental methods:

[0051] 1. Plasmid construction and virus packaging:

[0052] The specific steps for plasmid construction and viral packaging are as follows: For the construction of the Pex5 overexpression lentiviral vector, the pWPT-GFP_V5 plasmid vector was first double-digested with BamHI / SalI or MluI / SalI, and the large fragment was recovered as a ligation vector; then, the synthetic Human peroxisomal biogenesis factor 5 isoform b (NM_000319.4) gene was amplified by PCR, double-digested with BamHI / SalI or MluI / SalI, and ligated with the above-mentioned vector. When packaging the Pex5 overexpression lentivirus, we used the commercial transfection reagent Lipofectamine2000. The target plasmid and two packaging plasmids were mixed in a certain mass ratio (pWPT-GFP_V5:psPAX2:pMD2g=4:2:1) and transfected into HEK293T cells in serum-free culture medium; fresh culture medium was replaced 24 hours after transfection, and the culture supernatant was collected once 48 hours after transfection and added to fresh culture medium. The supernatant was collected a second time about 72 hours after transfection and mixed with the previous one; after centrifugation and filtration, polybrene was added to it, and after mixing, it can infect cells cultured in vitro.

[0053] To construct a Pex5 knockout lentiviral vector, a second-generation lentiviral system was used to package a lentiviral plasmid carrying a Pex5 shRNA sequence into viral particles. The specific steps are as follows: the target plasmid (pLKD-CMV-GFP-U6-shRNA) and two packaging plasmids were mixed at a specific mass ratio (target plasmid: VSVG: Δ8.9 = 4:1.5:1.5) and transfected into HEK293T cells in serum-free culture medium. Fresh culture medium was replaced 24 hours after transfection. After 48 hours, the culture supernatant was collected and added to fresh culture medium. Approximately 72 hours after transfection, the supernatant was collected and mixed again. After centrifugation and filtration, polybrene was added to the mixture. The mixture was then ready to infect cells cultured in vitro.

[0054] The designed interference sequence for Pex5 is an oligonucleotide fragment: TGTAGTTCAAGGGCTCTGC (SEQ ID NO. 1). The detailed primer information is as follows:

[0055] The forward primers for shRNA are:

[0056] 5'-CCGGTGCAGAGCCCTTGAACTACATTCAAGAGATGTAGTTCAAGGGCTCTGCTTTTTTG-3' (SEQ ID NO. 2);

[0057] The reverse primers for shRNA are:

[0058] 5'-AATTCAAAAAAGCAGAGCCCTTGAACTACATCTCTTGAATGTAGTTCAAGGGCTCTGCA-3' (SEQ ID NO. 3);

[0059] 2. Virus concentration and purification:

[0060] For interfering lentivirus injected into LPC mice, the collected viral fluid requires concentration and purification. The specific steps are: centrifuge 200 mL of the collected viral fluid (500 g for 3 minutes); add 0.1x 50% PEG8000 (i.e., 21 mL) and shake overnight at 4°C; divide the viral fluid into centrifuge tubes and centrifuge at 6000 rpm for 20 minutes at 4°C; carefully remove and discard the supernatant, and pipette the precipitate with 1X PBS until clear; filter through 0.45µm and 0.22µm pore size filters, respectively, collect the fluid, and centrifuge at 16000 g for 40 minutes. Collect the precipitate and pipette again, and centrifuge at the same speed for another 40 minutes; finally, pipette the collected precipitate with an appropriate amount of 1X PBS (e.g., 50µl) to obtain a highly concentrated viral fluid.

[0061] 2. Experimental results: Overexpression or interference of Pex5 affects the in vitro differentiation of oligodendrocyte precursor cells (OPCs).

[0062] like Figure 1 A and Figure 1 As shown in Figure B, compared with the control group, Pex5 overexpression lentivirus can indeed upregulate the expression of myelin structural protein (MBP) in OPCs cultured in vitro; while Pex5 interference lentivirus can downregulate the expression of myelin structural protein (MBP) in OPCs cultured in vitro. Furthermore, immunofluorescence staining results confirmed that Pex5 overexpression can significantly increase the expression of MBP in differentiated OPCs. + The ratio of cell numbers (e.g. Figure 2 C and Figure 2 Pex5 knockdown can significantly downregulate MBP in virus-infected OPCs in differentiated culture. + The ratio of cell numbers (e.g. Figure 2 E and Figure 2 F). And as Figure 1 G and Figure 1As shown in Figure H, compared with the control group, Pex5 overexpression lentivirus significantly upregulated the expression of myelin sheath structural protein (MBP) in OPCs cultured in vitro; while Pex5 knockdown significantly downregulated the expression of myelin sheath structural protein (MBP) in OPCs cultured in vitro. These results indicate that PEX5 plays a role in promoting the differentiation and maturation of OPCs in vitro.

[0063] Example 2: Induction, drug administration and lentiviral injection of LPC model mice, animal perfusion, fixation and sampling, and immunohistofluorescence staining

[0064] 1. Experimental methods:

[0065] 1. Induction, Drug Administration, and Lentivirus Injection of LPC Model Mice

[0066] Eight-week-old C57BL / 6J female mice were deeply anesthetized with 3% chloral hydrate and underwent laminectomy. The spinal column was immobilized, and 1 μL of 1% LPC (62962, Sigma-Aldrich) prepared in sterile PBS was injected into the dorsal spinal cord at T11-T12 using a microinjection needle. The needle was held in place for 5 minutes to prevent LPC reflux. The day of injection was designated as 0 days postlesion (dpl).

[0067] For drug administration experiments in mice with LPC-induced injury, C57BL / 6J mice treated with DMSO or ganciclovir (10 mg / kg) were euthanized 10 or 14 days after dorsal injury. Mice euthanized 10 days later received DMSO or ganciclovir via intraperitoneal injection daily from 3 to 9 days post-injury; mice euthanized 14 days later received DMSO or ganciclovir via intraperitoneal injection daily from 4 to 13 days post-injury.

[0068] For Pex5 overexpression or interference lentivirus injection in mice with LPC-induced lesions, we injected 1 μl of Pex5 overexpression or interference lentivirus (0.5-1 × 10^9 TU / mL) into two locations on the dorsal spinal cord (0.5 cm above and below the center of the LPC lesion) immediately after LPC injection. The lentivirus was slowly injected over 2 minutes, and the needle was maintained in place for 3 minutes before being slowly withdrawn over 1 minute.

[0069] 2. Animal Perfusion, Fixation, and Sampling

[0070] Mice were anesthetized with an intraperitoneal injection of chloral hydrate. Placed in the supine position, their limbs were immobilized, the heart was dissected, the right atrial appendage was opened, and PBS was injected through the cardiac apex. After the liver and limbs became pale, the solution was replaced with PFA. Once the limbs and trunk were stiff, the needle was removed, and the specimen was fixed in PFA and stored in a refrigerator at 4°C. Six hours later, the specimen was removed, bone fragments removed, and the vertebrae were slowly cut open with scissors, starting from the ventral side (at a 45-degree angle, being careful not to injure the spinal cord). The posterior tissue was carefully cut open, and the spinal cord was carefully removed. The spinal cord was placed in a 20% sucrose solution, clearly labeled, and refrigerated at 4°C. After 24 hours, the solution was replaced with a 30% sucrose solution. Three days later, the specimen was ready for sectioning and staining.

[0071] 3. Immunohistofluorescence Staining

[0072] Take frozen sections or paraffin sections (need to be dewaxed and hydrated in advance), soak them in 1× PBS for 5 minutes × 3 times, place the sections in antigen retrieval solution in a 95℃ water bath for 10 minutes, take them out, wait until the antigen retrieval solution reaches room temperature, soak them in 1× PBS for 5 minutes × 3 times, punch with 0.2% Triton X-100 for 10 minutes, soak them in 1× PBS for 5 minutes × 3 times, block with donkey serum or BSA for 20 minutes, add primary antibody, incubate at 4℃ overnight, soak them in 1× PBS for 5 minutes × 3 times, add corresponding secondary antibody (with Hoechst) at room temperature for 1 hour, soak them in 1× PBS for 5 minutes × 3 times, seal the sections, store in dark, and take pictures.

[0073] 2. Experimental results:

[0074] 1. Overexpression or interference of Pex5 regulates myelin regeneration in LPC-induced demyelination lesions

[0075] like Figure 2 A. Figure 2 B and Figure 2 As shown in Figure C, 12 days after LPC-induced myelin injury, the Pex5 overexpression group showed a significant decrease in demyelination area, a significant increase in thin new myelin sheaths, and a significant decrease in g-ratio (indicating a significant increase in myelin thickness) compared to the control group. 14 days after LPC-induced myelin injury, the Pex5 interference group showed a significant increase in demyelination area, a significant decrease in thin new myelin sheaths, and a significant increase in g-ratio (indicating a significant decrease in myelin thickness) compared to the control group. These results suggest that PEX5 can indeed significantly promote the myelination ability of oligodendrocytes.

[0076] 2. Ganciclovir promotes OPCs differentiation and myelin regeneration in LPC-induced demyelination lesions

[0077] like Figure 6 A and Figure 6As shown in B, immunohistofluorescence staining revealed that CC1 in the spinal cord lesion area of ​​mice in the ganciclovir treatment group was positive on the 10th day after modeling. + Cell number and PEX5 + CC1 + The number of cells increased significantly compared to the control group, and the range of lesions shown by MBP staining also decreased. Figure 6 C. Figure 6 D and Figure 6 As shown in Figure E, immunohistofluorescence staining and electron microscopy revealed that on day 14 after modeling, compared with the control group, the demyelinated area in the ganciclovir-treated group was significantly reduced, the proportion of myelinated axons in the total axons was significantly increased, and the g-ratio value was significantly decreased (indicating a significant increase in myelin thickness). These results indicate that ganciclovir can promote the differentiation of OPCs and remyelination in LPC-induced demyelination lesions.

[0078] Example 3: Primary OPCs culture, OPCs differentiation experiment, and cell protein level detection

[0079] 1. Experimental methods:

[0080] 1. Primary OPCs Culture

[0081] Prepare two small dishes, one containing 5 mL of dissection solution and the other containing 2 mL of dissection solution. Asphyxiate the rat with alcohol, decapitate it, cut the skin, cut the skull, remove the brain, and excise the brainstem and hippocampus. Place the brainstem and hippocampus in a small dish containing 5 mL of dissection solution. Peel off the vascular membranes and place the brain in a small dish containing 2 mL of dissection solution. Mince the brain into 1 mm pieces. 3 Next, add 2 mL of trypsin and incubate at 37°C for 30 minutes (shaking gently every 5 minutes). Terminate digestion by adding pre-prepared DMEM medium (supplemented with FBS and double-antibody). Transfer the tubes to two centrifuge tubes containing 4 mL of culture medium and centrifuge at 300 g for 5 minutes. Discard the supernatant, resuspend in 8 mL of culture medium, and let it rest for 5 minutes. Pre-fill a T75 flask with PLL-coated cells with 4 mL of culture medium. Evenly distribute the rested supernatant to four T75 flasks and incubate. Change the medium the next day and every three days thereafter.

[0082] 2. OPCs differentiation experiment

[0083] Seed the primary cultured OPCs in appropriate well plates. After 6 hours of cell attachment, replace the culture medium with Neurobasal+B27 differentiation medium containing the corresponding drugs. After 72 hours, collect proteins for Western Blot (WB) detection or perform immunofluorescence staining on cell samples.

[0084] 3. Protein Level Detection (WB)

[0085] Harvest the desired cells, add RIPA lysis buffer, pipette over crushed ice, and let stand for 30 minutes. Centrifuge at 12,000 g for 10 minutes at 4°C. Collect the supernatant, transfer it to a fresh Eppendorf tube, add SDS-PAGE buffer, vortex evenly, and incubate at 100°C for 5 minutes. Assemble the electrophoresis apparatus, add the electrophoresis buffer, and load the sample. Apply 80V power for 30 minutes. Once the dye front enters the separating gel, adjust the voltage to 120V. After 1.5 hours, observe for slight bleed-out of bromophenol blue and turn off the power. Transfer the gel to a transfer cassette for transfer. Block the nitrocellulose membrane in blocking buffer for 2 hours at room temperature. Wash the membrane with 1× TBST, incubate with the corresponding primary antibody overnight at 4°C, then wash the membrane with 1× TBST, incubate with the secondary antibody for 2 hours at room temperature, wash the membrane with 1× TBST, and develop the color with ECL developer. Analyze the image using a chemiluminescence imager.

[0086] 2. Experimental results:

[0087] 1. Ganciclovir, a candidate drug, can promote the increase of MBP expression in OPCs

[0088] like Figure 3 As shown in the figure, OPCs were treated with 5 micromolar concentrations of ganciclovir, latanoprost, and olopatadine, respectively, and cultured in vitro for 72 hours. MBP protein expression was then analyzed by immunoblotting. Statistical results showed that at a 5 micromolar concentration, ganciclovir significantly increased MBP levels in OPCs, while olopatadine and latanoprost had no such effect.

[0089] 2. Ganciclovir promotes the increase of MBP expression in OPCs in a dose-dependent manner

[0090] The results are as follows Figure 4 A and Figure 4 As shown in Figure B, OPCs were treated with ganciclovir at concentrations of 0.5, 1, 2, 5, and 10 micromolar, cultured in vitro for 72 hours, and then analyzed for MBP protein expression by immunoblotting. Statistical results showed that ganciclovir significantly increased MBP expression in OPCs at concentrations of 1, 2, 5, and 10 micromolar, with the most significant effect at 5 micromolar.

[0091] Example 4: Cell Immunofluorescence and BrdU Staining

[0092] 1. Experimental methods:

[0093] 1. Cell Immunofluorescence

[0094] Primary OPCs were fixed in PFA at room temperature for 15 minutes, washed in 1× PBS for 5 minutes × 3 times, perforated with 0.2% Triton X-100 for 2 minutes, blocked with 10% goat serum for 60 minutes, incubated with the corresponding antibody of the target molecule at 4°C overnight, incubated with secondary antibody at room temperature for 2 hours, stained with Hoechst for 10 minutes, washed in 1× PBS for 5 minutes × 3 times, and sealed.

[0095] 2. BrdU Staining

[0096] Cell proliferation assay, also known as BrdU incorporation assay. To test whether ganciclovir affects the proliferation of OPCs, we first treated OPCs with 5 micromolar ganciclovir and cultured them in proliferation medium for 48 hours. 10 micromolar BrdU was added to the culture medium 6 hours before the end of the culture. The culture medium was then aspirated and the cells were fixed with 4% paraformaldehyde for 20 minutes. The cells were then rinsed with PBS for 5 minutes three times and treated with 2N hydrochloric acid for 30 minutes. The hydrochloric acid was neutralized with 0.1M sodium borohydride solution for 10 minutes, and then perforated with 0.1% Triton-X100 for 2 minutes. After blocking with 10% donkey serum for 1 hour, the cells were incubated with mouse anti-BrdU antibody (1:100) at 4°C overnight. The cells were then washed with PBS for 5 minutes three times the next day, incubated with fluorescent secondary antibody and nuclear dye Hoechst 33342 for 2 hours, washed with PBS for 5 minutes three times, mounted with 50% glycerol, and photographed with a fluorescence microscope for BrdU incorporation. + / Hoechst + The cell numbers were statistically analyzed.

[0097] 2. Experimental results:

[0098] 1. Ganciclovir promotes OPCs differentiation in a dose-dependent manner

[0099] like Figure 4 C and Figure 4 As shown in D, OPCs were treated with 0.5, 1, 2, 5, and 10 micromolar concentrations of ganciclovir, and cultured in vitro for 72 hours. The expression of MBP and NG2 was then analyzed by immunofluorescence. The statistical results showed that ganciclovir at 1, 2, 5, and 10 micromolar concentrations significantly promoted MBP. + Increased cell percentage and NG2 + The reduction in cell percentage was most significant at a concentration of 5 micromolar.

[0100] 2. Ganciclovir does not affect the apoptosis of OPCs

[0101] like Figure 5 A and Figure 5As shown in Figure B, OPCs were treated with 5 micromolar ganciclovir and cultured in vitro for 72 hours, and then the expression of cleaved-Caspase 3 was analyzed by immunofluorescence. Statistical results showed that ganciclovir did not affect the apoptosis of OPCs.

[0102] 3. Ganciclovir does not affect the proliferation of OPCs

[0103] like Figure 5 C and Figure 5 As shown in Figure D, OPCs were treated with 5 micromolar ganciclovir and cultured in proliferation medium for 48 hours. Six hours before termination of culture, 10 micromolar BrdU was added to the culture medium, and BrdU expression was analyzed by immunofluorescence. Statistical results showed that ganciclovir did not affect the proliferation of OPCs.

[0104] Example 5: Induction and scoring of EAE model in mice, animal perfusion, fixation and sampling, and immunohistofluorescence staining

[0105] 1. Experimental methods:

[0106] 1. Induction, Scoring, and Drug Administration of the Mouse EAE Model

[0107] 1) Reagent preparation (the following dosages are for one mouse):

[0108] Carefully weigh 200 μg MOG using an electronic scale. 35–55 (Gill Biochemical, MK-21, 051716) and 0.15 mL of sterile 1× PBS were added to prepare a mixed solution. Carefully transfer the solution into a 10 mL glass syringe (a). Then, steadily weigh 500 μg of inactivated tauxin (Mtb, Difco, 231141) and place it in a mortar. While grinding, add 0.15 mL of incomplete Freund's adjuvant (IFA, Sigma-Aldrich, F5506) dropwise. Mix thoroughly. Aspirate the mixed complete Freund's adjuvant (CFA) into a 10 mL syringe (b). Expel the air from syringes a and b. Seal the syringes with a three-way fitting and insert them into crushed ice prepared in advance. Mix the two mixed solutions in a 1:1 ratio. Hold syringes a and b with your left and right hands, respectively, and push firmly into each syringe (never simultaneously) for approximately 200 times until the resistance increases and the solution stops. The solution in the tube should now appear turbid. Drop a small amount of the solution onto the water surface; the droplet should not disperse. The mixture was stored in crushed ice to keep it cold.

[0109] Weigh 200 ng of pertussis toxin (PTX, Calbiochem, 516562) and dissolve it in 0.2 mL of sterile 1× PBS. Prepare immediately before use.

[0110] 2) Mouse injection:

[0111] Prepare the operating tools (skin preparation knife, alcohol cotton ball, 1 mL syringe and gloves, etc.), shave the hair of adult 2-month-old female C57BL / 6 mice (20 g, Shanghai Bikai), expose the buttocks skin and disinfect it, and subcutaneously inject the prepared MOG into the middle of the buttocks and three points near the groin on both sides. 35–55 On days 0 and 2 after model establishment, 200 μL of PTX solution (1 μg / mL) was injected intraperitoneally per animal.

[0112] 3) Scoring and dosing:

[0113] Symptoms generally begin around 10 days after model establishment, and are scored according to Kono's 5-point scale: no symptoms (0 points); tail tip weakness (0.5 points); complete tail weakness (1 point); complete tail weakness with unsteady hindlimb gait (1.5 points); complete tail weakness with bilateral hindlimb weakness (2 points); complete tail weakness with unilateral hindlimb paw valgus (2.5 points); bilateral hindlimb weakness with abdomen touching the ground (3 points); bilateral hindlimb weakness with abdomen touching the ground and unilateral forelimb weakness (3.5 points); complete quadriplegia (4 points); and moribund or death (5 points). Starting from the peak of EAE model establishment (day 15), mice in each group were injected daily with ganciclovir (10 mg / kg), benztropine (10 mg / kg), and saline, and EAE scores were continuously assessed in each group.

[0114] 2. Animal Perfusion, Fixation, and Sampling

[0115] After scoring on day 25 after model establishment, mice were anesthetized with an intraperitoneal injection of chloral hydrate. Placed in a supine position, their limbs were immobilized, the heart was dissected, the right atrial appendage was opened, and PBS was injected through the cardiac apex. After the liver and limbs became pale, the solution was replaced with PFA. Once the limbs and trunk were stiff, the pillow was removed, and the specimen was fixed in PFA and stored in a refrigerator at 4°C. Six hours later, the specimen was removed, bone fragments removed, and starting from the ventral side, the vertebrae were slowly cut open with scissors (at a 45-degree angle, taking care not to injure the spinal cord). The posterior tissue was then carefully cut open, and the spinal cord was carefully removed. The spinal cord was placed in a 20% sucrose solution, clearly labeled, and refrigerated at 4°C. After 24 hours, the solution was replaced with a 30% sucrose solution. Three days later, the specimen was ready for sectioning and staining.

[0116] 3. Immunofluorescence Staining

[0117] Take frozen sections or paraffin sections (need to be dewaxed and hydrated in advance), immerse in PBS for 5 minutes three times, place the sections in sodium citrate antigen retrieval solution in a 95℃ water bath for 10 minutes, take out, wait until the antigen retrieval solution reaches room temperature, immerse in PBS for 5 minutes three times, punch with 0.2% Triton X-100 for 10 minutes, immerse in PBS for 5 minutes three times, block with 10% donkey serum or BSA for 20 minutes, add corresponding primary antibody, incubate at 4℃ overnight, immerse in PBS for 5 minutes three times, add corresponding secondary antibody (with Hoechst) at room temperature for 1 hour, immerse in PBS for 5 minutes three times, seal the sections, store in dark, and take pictures.

[0118] 2. Experimental results:

[0119] 1. Ganciclovir promotes behavioral improvement in EAE mice

[0120] like Figure 7 As shown in Figure A, EAE behavioral scores indicate that mice in the ganciclovir-treated group generally showed significant behavioral improvement, with behavioral scores dropping below approximately 2 points, and their hind limbs essentially returning to normal. In contrast, behavioral scores in the control group generally exceeded 2 points, still demonstrating significant neurobehavioral dysfunction.

[0121] 2. Ganciclovir promotes myelin regeneration in EAE mice

[0122] like Figure 7 B, Immunofluorescence staining results showed that mice in the ganciclovir treatment group showed recovery of MBP expression in the white matter of the spinal cord, indicating a reduction in the extent of demyelination, while mice in the control group showed continued destruction of the white matter. Figure 7 B. Figure 7 C and Figure 7 As shown in D, electron microscopy analysis showed that the number of newly formed myelin sheaths in the ganciclovir-treated mice was significantly increased compared with that in the control mice, the proportion of myelinated axons in the total axons was significantly increased, and the g-ratio value was significantly decreased, indicating an increase in the thickness of the myelin sheath.

[0123] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Use of reagents that upregulate PEX5 expression levels or enhance PEX5 protein activity in the preparation of therapeutic drugs for demyelinating diseases.

2. Use of the reagent for upregulating PEX5 expression level or increasing PEX5 protein activity according to claim 1 in the preparation of a drug for treating demyelinating diseases, characterized in that: The reagent for increasing the expression level of PEX5 or enhancing the activity of PEX5 protein is selected from: A) Drugs that upregulate Pex5 mRNA and protein levels in OPCs; B) Recombinant vector containing the PEX5 encoding gene; C) Recombinant virus containing the PEX5 encoding gene.

3. Use of the reagent for upregulating PEX5 expression level or increasing PEX5 protein activity according to claim 2 in the preparation of a drug for treating demyelinating diseases, characterized in that: The drug for increasing the content of Pex5 mRNA and the level of PEX5 protein in OPCs is ganciclovir.

4. Use of the reagent for upregulating PEX5 expression level or increasing PEX5 protein activity according to claim 3 in the preparation of a drug for treating demyelinating diseases, characterized in that: The dosage of ganciclovir in the demyelinating disease treatment drug is 10 mg / kg.

5. Use of the reagent for upregulating PEX5 expression level or increasing PEX5 protein activity according to claim 3 in the preparation of a drug for treating demyelinating diseases, characterized in that: Use of ganciclovir in preparing drugs for promoting differentiation and maturation of oligodendrocytes or promoting expression of myelin-related proteins.

6. Use of the reagent for upregulating PEX5 expression level or increasing PEX5 protein activity according to claim 2 in the preparation of a drug for treating demyelinating diseases, characterized in that: The recombinant virus containing the PEX5 encoding gene is a recombinant lentivirus containing the PEX5 encoding gene.

7. Use of the reagent for upregulating PEX5 expression level or increasing PEX5 protein activity according to claim 1 in the preparation of a drug for treating demyelinating diseases, characterized in that: The demyelinating disease includes but is not limited to multiple sclerosis.

8. Use of the reagent for upregulating PEX5 expression level or increasing PEX5 protein activity according to claim 7 in the preparation of a drug for treating demyelinating diseases, characterized in that: The multiple sclerosis includes but is not limited to chronic progressive multiple sclerosis.

9. Use of the reagent for upregulating PEX5 expression level or increasing PEX5 protein activity according to claim 1 in the preparation of a drug for treating demyelinating diseases, characterized in that: The demyelinating disease therapeutic drug is a pharmaceutical composition prepared from an agent that upregulates the expression level of PEX5 or increases the activity of PEX5 protein as an active ingredient and a conventional pharmaceutical carrier.

10. A pharmaceutical preparation for treating demyelinating diseases, characterized in that: The active ingredient is an agent that upregulates the expression level of PEX5 or increases the activity of PEX5 protein.

Images