Application of epalrestat in preparation of medicine for treating spinal cord injury

Abstract

The invention relates to application of epalrestat in preparation of a medicine for treating spinal cord injury, and belongs to the technical field of biological medicine. The invention provides a novel pharmaceutical application of epalrestat, namely an application of epalrestat in preparation of a medicine for treating spinal cord injury. It is found for the first time that epalrestat can reduce the pyroptosis level of neurons after spinal cord injury by inhibiting an aldose reductase mediated NOX4 / ROCK / Poldip2 oxidative stress pathway, and it is proposed that epalrestat is used for preparing the medicine for treating spinal cord injury. Animal model experiments prove that after epalrestat gavage treatment, AR expression of a mouse spinal cord injury area and pyroptosis of neurons are remarkably reduced, and the motor function and nerve injury of a mouse are remarkably improved. The application of the epalrestat-containing medicine in clinical treatment of spinal cord injury is expected to provide benefits and help for improvement of prognosis of patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the use of epalrestat in preparing a medicine for treating spinal cord injury. Background Art

[0002] Epalrestat is an aldose reductase (AR) inhibitor primarily used to prevent, improve, and treat peripheral neuropathy associated with diabetes, manifested by symptoms such as numbness and pain. Multiple studies have shown that sorbitol accumulation within nerve cells caused by hyperglycemia is a key cause of diabetic neuropathy. Epalrestat inhibits AR, reducing the conversion of glucose to sorbitol. By reducing sorbitol accumulation within nerve cells, it reduces nerve cell damage and improves patients' neurological symptoms and dysfunction.

[0003] Spinal cord injury (SCI) refers to damage to the spinal cord structure or function due to external forces, resulting in severe functional impairment of the limbs below the injured segment. This injury not only causes severe physical and psychological harm to patients, but also imposes a huge economic burden on society.

[0004] Spinal cord injury (SCI) is an acute process caused by trauma, primarily physical damage, accompanied by inflammation and ischemia and hypoxia. Treatment focuses on acute treatment and rehabilitation training in the later stages. Acute treatment includes emergency care, surgery, and medication. Medications such as mannitol and dexamethasone are commonly used to reduce spinal cord edema and secondary damage, while gangliosides are used to promote neurological recovery.

[0005] Currently, there is no evidence that endogenous sorbitol accumulates in nerve cells of spinal cord injury; therefore, the use of epalrestat for the treatment of spinal cord injury has not been reported. Summary of the Invention

[0006] The present invention provides a new medicinal use of epalrestat, namely, use of epalrestat in preparing a medicine for treating spinal cord injury.

[0007] The technical solution of the present invention:

[0008] Use of epalrestat in preparing medicine for treating spinal cord injury.

[0009] Furthermore, the drug for treating spinal cord injury contains epalrestat as the sole active ingredient or one of the active ingredients.

[0010] Furthermore, the content of epalrestat in the drug for treating spinal cord injury is 0.1 wt% to 99 wt%.

[0011] Furthermore, the drug for treating spinal cord injury also includes pharmaceutically acceptable excipients and / or carriers.

[0012] Furthermore, the drug for treating spinal cord injury is administered orally.

[0013] Furthermore, when the drug for treating spinal cord injury is administered orally, the dosage of epalrestat is 250 mg / kg / d.

[0014] Furthermore, the medicine for treating spinal cord injury is in the form of granules, tablets, capsules, pills or oral liquid preparations.

[0015] Furthermore, the drug for treating spinal cord injury can reduce the expression level of aldose reductase in the spinal cord injury area.

[0016] Furthermore, the drug for treating spinal cord injury can improve the level of pyroptosis in spinal cord injured tissue.

[0017] Furthermore, the drug for treating spinal cord injury can reduce the level of neuronal pyroptosis after spinal cord injury by inhibiting the NOX4 / ROCK / Poldip2 oxidative stress pathway mediated by aldose reductase.

[0018] Beneficial effects of the present invention:

[0019] The present invention discovers for the first time that epalrestat can reduce the level of neuronal pyroptosis after spinal cord injury by inhibiting the aldose reductase-mediated NOX4 / ROCK / Poldip2 oxidative stress pathway, and proposes the use of epalrestat in the preparation of a drug for the treatment of spinal cord injury. Animal model experiments have confirmed that after oral gavage with epalrestat, AR expression and neuronal pyroptosis in the spinal cord injury area of ​​mice were significantly reduced, and the mice's motor function and nerve damage were significantly improved. The use of drugs containing epalrestat in the clinical treatment of spinal cord injury is expected to provide benefits and help improve patient prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 HE staining and Nissl staining of the spinal cord tissues of each group of mice in Example 1;

[0021] Figure 2 Figure 2 shows the BMS score comparison chart and the motion trajectory chart of the open field test for the three groups of mice in Example 2. A shows the BMS score comparison chart, and B shows the motion trajectory chart of the open field test.

[0022] Figure 3 This is a comparison of the total movement distance, average speed, and relative immobility time of the three groups of mice in the open field test in Example 2. A is the total movement distance, B is the average speed, and C is the relative immobility time;

[0023] Figure 4 These are the LFB staining images and staining intensity comparison images of the spinal cord tissues of the three groups of mice in Example 3, A is the LFB staining image, and B is the staining intensity comparison image;

[0024] Figure 5 These are the images of MAP2 and SYN / Neun immunofluorescence staining of spinal cord tissues of the three groups of mice in Example 3, the fluorescence intensity of MAP2 immunofluorescence staining, and the comparison of the number of SYN-positive cells in SYN / Neun immunofluorescence staining. A is the immunofluorescence staining image, B is the comparison of the fluorescence intensity of MAP2, and C is the comparison of the number of SYN-positive cells.

[0025] Figure 6 These are the WB images and expression level comparison graphs of AR expression in the spinal cord tissues of the sham-operated group and the four groups of mice at different times of spinal cord injury in Example 4. A is the WB image of AR expression, and B is the comparison graph of AR expression levels;

[0026] Figure 7 These are the WB images and expression level comparison graphs of AR expression in the spinal cord tissues of mice in the sham operation group, SCI model group, and treatment group in Example 4. A is the WB image of AR expression, and B is the comparison graph of AR expression levels.

[0027] Figure 8 AR / Neun immunofluorescence staining of mice in the sham operation group, SCI model group, and treatment group in Example 4;

[0028] Figure 9 This is a comparison of the number of AR-positive cells and the mean fluorescence intensity of AR in the sham-operated group, SCI model group, and treatment group in Example 4. A is the number of AR-positive cells, and B is the mean fluorescence intensity of AR.

[0029] Figure 10 This is a Western blot image of the expression of pyroptosis-related proteins in the spinal cord tissues of the sham-operated group and the four groups of mice at different times after spinal cord injury in Example 5;

[0030] Figure 11 This is a comparison of the expression levels of NLRP3, GSDMD, GSDMD-N, and IL-1β in the spinal cord tissues of the sham-operated group and the four groups of mice at different times after spinal cord injury in Example 5. A is NLRP3, B is GSDMD, C is GSDMD-N, and D is IL-1β;

[0031] Figure 12 This is a Western blot image of pyroptosis-related proteins in the spinal cord tissue of mice in the sham operation group, SCI model group, and treatment group in Example 5;

[0032] Figure 13Comparison of the expression levels of NLRP3, GSDMD, and caspase-1 in the spinal cord tissues of mice in the sham operation group, SCI model group, and treatment group in Example 5. A represents NLRP3, B represents GSDMD, and C represents caspase-1.

[0033] Figure 14 These are the immunofluorescence staining images of GSDMD and NLRP3 / Neun in the sham-operated group, SCI model group, and treatment group of mice in Example 5, as well as the comparison of the mean fluorescence intensities of GSDMD and NLRP3. A is the immunofluorescence staining image, B is the comparison of the mean fluorescence intensity of GSDMD, and C is the comparison of the mean fluorescence intensity of NLRP3.

[0034] Figure 15 The immunohistochemical staining images of IL-1β and IL-18 in the spinal cord tissues of mice in the sham operation group, SCI model group, and treatment group in Example 5;

[0035] Figure 16 This is a comparison of the percentages of IL-1β and IL-18 positive cells in the spinal cord tissues of mice in the sham operation group, SCI model group, and treatment group in Example 5. A represents IL-1β and B represents IL-18.

[0036] Figure 17 These are the WB images and expression level comparison graphs of the control group and the OGD injury model group at each time point in Example 6. A is the WB image of AR expression, and B is the comparison graph of AR expression levels.

[0037] Figure 18 Comparison of cell viability of blank control N2a cells and OGD-damaged cell models at various time points in Example 6;

[0038] Figure 19 Comparison of cell viability of the N2a cell model treated with different OGD injury times after stimulation with different concentrations of epalrestat in Example 6. A represents the OGD treatment group for 2 hours, B represents the OGD treatment group for 3 hours, and C represents the OGD treatment group for 4 hours.

[0039] Figure 20 These are WB images of AR expression and AR expression level comparison graphs in N2a cells of the blank control group, OGD injury group, and epalrestat treatment group in Example 7. A is a WB image of AR expression, and B is a comparison graph of AR expression levels.

[0040] Figure 21 The immunofluorescence staining images of N2a cells in the blank control group, OGD injury group, and epalrestat treatment group in Example 7;

[0041] Figure 22This is a comparison of the AR-positive cell count and AR mean fluorescence intensity of N2a cells in the blank control group, OGD injury group, and epalrestat treatment group in Example 7, where A represents the AR-positive cell count and B represents the AR mean fluorescence intensity;

[0042] Figure 23 This is a WB image of the expression of pyroptosis-related proteins in the blank control group and four groups of N2a cells damaged by OGD at different times in Example 8;

[0043] Figure 24 Comparison of the expression levels of NLRP3, GSDMD, and caspase-1 in the blank control group and four groups of N2a cells at different times of OGD injury in Example 8. A represents NLRP3, B represents GSDMD, and C represents caspase-1.

[0044] Figure 25 This is a WB image of pyroptosis-related proteins in N2a cells in the blank control group, OGD injury group, and epalrestat treatment group in Example 8;

[0045] Figure 26 Graph comparing the expression levels of NLRP3, GSDMD, and caspase-1 in N2a cells in the blank control group, OGD injury group, and epalrestat treatment group in Example 8, A represents NLRP3, B represents GSDMD, and C represents caspase-1;

[0046] Figure 27 These are immunofluorescence staining images of NLRP3 and caspase-1 in N2a cells of the blank control group, OGD injury group, and epalrestat treatment group in Example 8;

[0047] Figure 28 This is a comparison of the percentages of NLRP3 and caspase-1 positive cells in N2a cells in the blank control group, OGD injury group, and epalrestat treatment group in Example 8, A represents NLRP3, and B represents caspase-1;

[0048] Figure 29 This is a comparison of the secretion levels of inflammatory factors IL-1β, IL-18, TNF-α, and IFN-γ in N2a cells in the blank control group, OGD injury group, and epalrestat treatment group in Example 8. A is IL-1β, B is IL-18, C is TNF-α, and D is IFN-γ;

[0049] Figure 30 This is a comparison of the expression levels of SOD, MDA, GSH, and LDH in N2a cells of different treatment groups in Example 9, A is SOD, B is MDA, C is GSH, and D is LDH;

[0050] Figure 31This is a comparison of the secretion levels of IL-1β and IL-18 in N2a cells in different treatment groups in Example 10, A is IL-1β, B is IL-18;

[0051] Figure 32 Comparison of the mRNA levels of NLRP1, NLRP2, NLRP3, and NLRC4 in N2a cells in different treatment groups in Example 10, A for NLRP1, B for NLRP2, C for NLRP3, and D for NLRC4;

[0052] Figure 33 The Western blot images of NOX4, ROCK and Poldip2 expressions in N2a cells of different treatment groups in Example 11 are shown;

[0053] Figure 34 Comparison of NOX4, ROCK, and Poldip2 expression levels in N2a cells in different treatment groups in Example 11, A represents NOX4, B represents ROCK, and C represents Poldip2;

[0054] Figure 35 Comparison of mRNA levels of NOX4, ROCK, and Poldip2 in N2a cells of different treatment groups in Example 11, A is NOX4, B is ROCK, and C is Poldip2;

[0055] Figure 36 The Western blot images of NOX4, ROCK and Poldip2 expression in the spinal cord tissues of mice in the sham operation group, SCI model group and treatment group in Example 12 are shown;

[0056] Figure 37 This is a comparison of the expression levels of NOX4, ROCK, and Poldip2 in the spinal cord tissues of mice in the sham operation group, SCI model group, and treatment group in Example 12. A represents NOX4, B represents ROCK, and C represents Poldip2.

[0057] Figure 38 This is a comparison of the mRNA levels of NOX4, ROCK, and Poldip2 in the spinal cord tissues of mice in the sham operation group, SCI model group, and treatment group in Example 12. A is NOX4, B is ROCK, and C is Poldip2;

[0058] Figure 39 This is a WB image of NOX4, ROCK and Poldip2 expression in N2a cells of different treatment groups in the AR knockdown experiment in Example 13;

[0059] Figure 40 This is a comparison of the expression levels of NOX4, ROCK, and Poldip2 in N2a cells in different treatment groups in the AR knockdown experiment in Example 13. A is NOX4, B is ROCK, and C is Poldip2.

[0060] Figure 41 This is a comparison of the mRNA levels of NOX4, ROCK, and Poldip2 in N2a cells in different treatment groups in the AR knockdown experiment in Example 13, A is NOX4, B is ROCK, and C is Poldip2;

[0061] Figure 42 This is a comparison of the secretion levels of IL-1β and IL-18 in N2a cells in different treatment groups in the AR knockdown experiment in Example 13, A is IL-1β, and B is IL-18;

[0062] Figure 43 This is a comparison of the secretion levels of IL-1β and IL-18 in N2a cells in different treatment groups in the epalrestat treatment experiment in Example 13, A is IL-1β, and B is IL-18;

[0063] Figure 44 This is a comparison of the expression levels of SOD, MDA, and LDH in N2a cells of different treatment groups in the AR knockdown experiment in Example 13, A is SOD, B is MDA, and C is LDH;

[0064] Figure 45 This is a comparison of the expression levels of SOD, MDA, and LDH in N2a cells of different treatment groups in the epalrestat treatment experiment in Example 13, where A represents SOD, B represents MDA, and C represents LDH. DETAILED DESCRIPTION

[0065] The technical solution of the present invention is further described below with reference to the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

[0066] Example 1

[0067] This example provides an SCI (spinal cord injury) animal model and screens the dosage of epalrestat for treating spinal cord injury in vivo.

[0068] In this example, C57BL / 6 mice were used to construct an animal model. A portion of the spinal cord was surgically cut to create a hemisection injury, thereby obtaining SCI model mice. Sham-operated (sham) mice were also obtained.

[0069] SCI model mice were divided into different groups and given 125 mg / kg, 250 mg / kg, or 500 mg / kg of epalrestat by oral gavage once daily. Three days later, spinal cord tissue was collected and stained with HE and Nissl to observe the extent of spinal cord injury.

[0070] like Figure 1 HE and Nissl staining of the spinal cord tissues of each group of mice showed that the normal spinal cord tissue in the sham group was dense, with regular arrangement of cells and Nissl structures. In contrast, the SCI group showed loose tissue structure, disorganized cell arrangement, loss of Nissl structures, and the presence of vacuoles, among other obvious histopathological changes. Epalrestat at doses of 125 mg / kg or 500 mg / kg had little effect on spinal cord tissue repair, while a dose of 250 mg / kg effectively alleviated the pathological changes in spinal cord tissue. Therefore, 250 mg / kg was selected as the therapeutic dose for subsequent experiments.

[0071] Example 2

[0072] This example investigates the effect of epalrestat on the recovery of motor function in mice with spinal cord injury.

[0073] In this example, the BMS (Basso Mouse Scale) scoring method was used to score the hind limb function of the mice in the sham operation group, SCI model group, and epalrestat treatment group (dose: 250 mg / kg) in Example 1 to evaluate the recovery of the hind limb motor function of the SCI mice after treatment. Figure 2 The BMS scores of the three groups of mice and the movement trajectories of the open field test showed that compared with the SCI model group, epalrestat treatment significantly increased the BMS scores of mice, with statistical differences ( ### P < 0.001), indicating a significant recovery of motor function.

[0074] This example uses an open field test to analyze the autonomous movement ability of mice from three aspects: total movement distance, average speed, and relative static time. Figure 3 The total movement distance, average speed and relative immobility time of the three groups of mice in the open field test were shown to show that compared with the SCI model group, the movement trajectory and total movement distance of the mice in the epalrestat treatment group were significantly increased, the average speed was significantly improved, and the immobility time was reduced. The differences were statistically significant ( ## P<0.01, ### P<0.001).

[0075] The above results indicate that epalrestat treatment can effectively improve motor dysfunction in mice with spinal cord injury.

[0076] Example 3

[0077] This example investigates the effects of epalrestat on pathological changes in mice with spinal cord injury.

[0078] Luxol Fast Blue (LFB) is a copper-phthalocyanine dye that binds to myelin in alcoholic solutions. LFB staining can reveal the myelin structure of neural tissue and is therefore commonly used to assess myelin damage. In this example, LFB staining was performed on injured spinal cord tissue from the sham-operated, SCI-model, and epalrestat-treated mice (dose: 250 mg / kg) described in Example 1.

[0079] like Figure 4 As shown by the LFB staining images and staining intensity of the spinal cord tissues of the three groups of mice, the spinal cord tissue structure of the sham-operated control group was dense, and the myelin sheaths were arranged regularly and appeared bright blue; the fiber distribution in the spinal cord tissue of the SCI model group was reduced, and vacuoles were formed, indicating the loss of myelin sheaths, which was statistically significant (***P<0.001); epalrestat treatment alleviated the damage phenomenon in the above SCI model groups, which was statistically significant (###P<0.001).

[0080] Microtubule-associated protein 2 (MAP2) is a protein distributed in the dendrites and cell bodies of neurons. It is involved in microtubule stability and cytoskeletal maintenance. MAP2 staining can reveal the distribution and morphology of neuronal dendrites and is important for studying neuronal development, differentiation, and pathological changes. Synuclein (SYN) is a soluble protein expressed presynaptically and perinuclearly in the central nervous system. In this example, immunofluorescence staining was used to examine the expression levels of MAP2 and SYN in the injured spinal cord tissue of mice in the sham-operated group, SCI model group, and epalrestat-treated group (dose: 250 mg / kg) described in Example 1 to assess the effect of epalrestat on the degree of neuronal damage.

[0081] like Figure 5 Images of MAP2 and SYN / Neun immunofluorescence staining of spinal cord tissues of the three groups of mice. The fluorescence intensity of MAP2 immunofluorescence staining and the number of SYN-positive cells showed that compared with the sham-operated control group, the expression levels of MAP2 and SYN in the SCI model group were significantly reduced, with statistical differences (***P<0.001). After treatment with epalrestat, the expression levels of the two were restored, and there were statistical differences (#P<0.05).

[0082] These experimental results show that epalrestat can promote the recovery of motor function in mice with spinal cord injury, and can improve the degree of neuronal damage in spinal cord tissue, and has a certain therapeutic effect on mice with spinal cord injury.

[0083] Example 4

[0084] This example investigates the effect of epalrestat on the expression of aldose reductase (AR) in the spinal cord tissue of mice.

[0085] In this example, the same method as in Example 1 was used to establish spinal cord injury mouse models at 1 day, 3 days, 5 days, and 7 days, and a sham operation mouse model. Protein was extracted from the injured spinal cord tissue of each group of mice, and AR expression was detected by WB.

[0086] like Figure 6 As shown in the WB images and expression levels of AR expression in the spinal cord tissues of the sham operation group and five groups of mice at different times of spinal cord injury, AR expression in the spinal cord tissue was upregulated after spinal cord injury, indicating the aggravation of the pathological process, reaching a peak 3 days after spinal cord injury, with statistical differences (***P<0.001).

[0087] In this example, a 3-day SCI animal disease model and a sham operation model were constructed. Mice were given 250 mg / kg of epalrestat by gavage for 3 consecutive days, and the expression levels of AR protein in the spinal cord tissue of the sham operation control group, SCI model group, and epalrestat treatment group were detected.

[0088] like Figure 7 As shown in the WB images and expression levels of AR expression in the spinal cord tissues of mice in the sham operation group, SCI model group and epalrestat treatment group, compared with the sham operation group, the expression level of AR in the spinal cord tissue of the SCI model group was increased, while the AR expression level was significantly decreased after epalrestat treatment, and the difference was statistically significant (###P<0.001).

[0089] In this example, immunofluorescence staining was used to locate the expression level of AR in neurons (Neun) of mice in the sham operation group, SCI model group, and epalrestat treatment group.

[0090] like Figure 8 and Figure 9 As shown in the AR / Neun immunofluorescence staining images, the number of AR-positive cells, and the mean fluorescence intensity of mice in the sham operation group, SCI model group, and epalrestat treatment group, compared with the sham operation group, the AR expression level in the SCI model group was significantly increased, and the number of AR-positive neuronal cells increased, with statistical differences (***P<0.001); after epalrestat treatment, the AR expression level and the number of AR-positive neuronal cells decreased, with statistical differences (#P<0.05, ###P<0.001).

[0091] The above results show that AR expression increases significantly after spinal cord injury, and treatment with epalrestat can significantly reduce the expression level of AR in the spinal cord injury area of ​​spinal cord injured mice.

[0092] Example 5

[0093] This example investigates the effects of epalrestat on pyroptosis-related proteins after spinal cord injury in mice.

[0094] In this example, the same method as in Example 1 was used to establish a mouse SCI model. Proteins were extracted from spinal cord injury tissues at 1, 3, 5, and 7 days after spinal cord injury. The expression changes of pyroptosis markers NLRP3, GSDMD, and caspase-1 after spinal cord injury were detected by Western blotting.

[0095] IL-18 and IL-1β are classic inflammatory factors associated with pyroptosis. When cells undergo pyroptosis, IL-18 and IL-1β are activated in a caspase-1-dependent manner and secreted into the cell. IL-18 can induce the production of IFN-γ, thereby activating immune cells such as macrophages and T cells. IL-1β is an endogenous pyrogen that stimulates leukocyte migration and the expression of various cytokines and chemokines.

[0096] like Figure 10 and Figure 11 Western blot images of pyroptosis-related proteins in the spinal cord tissues of the sham operation group and the four groups of mice at different times of spinal cord injury and the expression levels of pyroptosis-related proteins NLRP3, GSDMD, GSDMD-N and IL-1β showed that the expression levels of pyroptosis markers NLRP3, GSDMD, GSDMD-N and IL-1β reached a peak on the third day of spinal cord injury, with statistical differences (**P<0.01, ***P<0.001), and then showed a downward trend.

[0097] In this example, a 3-day spinal cord injury (SCI) animal disease model and a sham operation model were constructed. Mice were given 250 mg / kg of epalrestat by gavage for 3 consecutive days. Western blotting was used to detect the expression levels of pyroptosis-related proteins in the spinal cord tissue of the sham operation control group, SCI model group, and epalrestat treatment group.

[0098] like Figure 12 and Figure 13 The WB images of pyroptosis-related proteins in the spinal cord tissue of mice in the sham operation group, SCI model group and epalrestat treatment group and the expression levels of NLRP3, GSDMD and caspase-1 showed that compared with the sham operation group, the expression of pyroptosis markers NLRP3, GSDMD and caspase-1 in the model group was significantly upregulated, with statistical differences ( *** P<0.001); after treatment with epalrestat, the expression levels of the above three proteins were significantly reduced, with statistical differences ( ## P<0.01).

[0099] Immunofluorescence staining was used to further detect changes in the level of cell pyroptosis in spinal cord tissue.

[0100] like Figure 14 The immunofluorescence staining of GSDMD and NLRP3 / Neun in the sham operation group, SCI model group and epalrestat treatment group, as well as the average fluorescence intensity of GSDMD and NLRP3, showed that the expression levels of GSDMD and NLRP3 in the model group were significantly increased compared with the sham operation group, with statistical differences ( *** P<0.001), the expression levels of both were significantly decreased in the epalrestat treatment group, with statistical differences ( ## P<0.01, ### P<0.001).

[0101] In this example, a 3-day spinal cord injury (SCI) animal disease model and a sham surgery model were established. Mice were given 250 mg / kg of epalrestat by gavage for 3 consecutive days. The expression levels of IL-18 and IL-1β were detected by immunohistochemical staining to further illustrate the effect of epalrestat on pyroptosis at the spinal cord injury site in mice.

[0102] like Figure 15 and Figure 16 The immunohistochemical staining of IL-1β and IL-18 in the spinal cord tissue of mice in the sham operation group, SCI model group and epalrestat treatment group, as well as the percentage of IL-1β and IL-18 positive cells, showed that compared with the sham operation group, the infiltration of IL-18 and IL-1β in the model group was significantly increased, with statistical differences ( *** P<0.001), while after treatment with epalrestat, the number of IL-18 and IL-1β positive cells decreased significantly, with statistical difference ( # P<0.05, ## P<0.01).

[0103] The above experimental results show that epalrestat treatment can improve the level of pyroptosis in the spinal cord tissue of mice with spinal cord injury.

[0104] Example 6

[0105] In this example, the dosage of epalrestat for in vitro experiments was obtained by screening the OGD (Oxygen and Glucose Deprivation) injury model in a cell experiment.

[0106] In this example, mouse neuroma cells (Neuro-2a, N2a) were used to construct OGD injury models at different time points (2 hours, 4 hours, 6 hours, and 8 hours). N2a cells were placed in a hypoxic chamber and the culture medium was replaced with sugar-free culture medium to cause cellular damage under ischemic / hypoxic conditions, simulating an in vivo SCI model.

[0107] Western blot was used to detect the changes in AR expression levels in N2a blank control cells and OGD injury model cells at different OGD time points.

[0108] like Figure 17 As shown in the WB images of AR expression in blank control N2a cells and OGD damaged cell models at different time points, the expression level of AR was significantly increased after 2 hours of glucose and oxygen deprivation treatment, with statistical differences (***P<0.001).

[0109] In this example, CCK8 (Cell Counting Kit-8) was used to count the cell viability of blank control N2a cells and OGD-damaged cell models at each time point. Figure 18 As shown in the blank control N2a cells and the cell viability of the OGD-damaged cell model at various time points, cell viability was significantly decreased after OGD treatment, with statistical significance (***P < 0.001). Based on these experimental results, we selected a 2-hour OGD treatment time as the condition for subsequent experiments.

[0110] In this example, 25 μM, 50 μM, 100 μM and 200 μM epalrestat were used to stimulate N2a cells treated with OGD for 2 h, 3 h and 4 h, respectively. Cell viability was detected by CCK8 to determine the appropriate epalrestat stimulation concentration in vitro. Figure 19 The cell viability of the OGD-injured cell model after stimulation with different concentrations of epalrestat, as shown in the results of different treatments, decreased N2a cell viability with all concentrations of epalrestat. However, at 50 μM epalrestat, cell viability stabilized. Based on these results, 50 μM epalrestat was used to stimulate cells in subsequent experiments.

[0111] Example 7

[0112] This example investigates the effect of epalrestat on AR expression levels in cells of an in vitro spinal cord injury model.

[0113] In this example, N2a cells were randomly divided into three groups: a blank control group, an OGD injury group (OGD injury time was 2 h), and an epalrestat treatment group (epalrestat dosage was 50 μM). The expression level of AR was detected by western blot. Figure 20As shown in the WB images and AR expression levels of N2a cells in the blank control group, OGD injury group and epalrestat treatment group, AR expression in N2a cells was significantly increased after OGD treatment, with statistical differences (***P<0.001); after stimulation with epalrestat, the expression level of AR was significantly decreased, with statistical differences (##P<0.01).

[0114] like Figure 21 and Figure 22 As shown in the immunofluorescence staining images of N2a cells in the blank control group, OGD injury group and epalrestat treatment group, as well as the number of AR-positive cells and the average fluorescence intensity of AR, compared with the blank control group, the AR expression level in N2a cells in the OGD injury group was significantly increased, with statistical differences (**P<0.01, ***P<0.001). After stimulation with epalrestat, the AR expression level in N2a cells was reduced, with statistical differences (#P<0.05, ###P<0.001).

[0115] In vitro experimental results confirmed that epalrestat stimulation could reduce the expression level of AR in N2a cells treated with OGD.

[0116] Example 8

[0117] This example uses an in vitro spinal cord injury model to investigate the effect of epalrestat on the expression levels of pyroptosis-related proteins in the spinal cord injury model.

[0118] In this example, N2a cells were treated with OGD at different time points (2 h, 4 h, 6 h, and 8 h) in vitro, and proteins were extracted. Western blotting was used to detect changes in the expression levels of pyroptosis-related proteins in N2a cells after OGD treatment.

[0119] like Figure 23 and Figure 24 The Western blot images of pyroptosis-related protein expression in the blank control group and the four groups of N2a cells at different times of OGD injury, as well as the expression levels of NLRP3, GSDMD, and caspase-1, showed that the pyroptosis markers NLRP3, GSDMD, and caspase-1 reached their peak at 2 h of OGD, with statistical differences (***P<0.001), and then gradually decreased.

[0120] In this example, N2a cells were subjected to OGD for 2 h in vitro. Cell proteins were extracted from the blank control group, the OGD-induced injury group (OGD injury time was 2 h), and the epalrestat-treated group (epalrestat dosage was 50 μM). Western blot was used to detect changes in the expression levels of the pyroptosis-related proteins caspase 1, NLRP3, and GSDMD.

[0121] like Figure 25 and Figure 26WB images of pyroptosis-related proteins in N2a cells in the blank control group, OGD injury group, and epalrestat treatment group, as well as the expression levels of NLRP3, GSDMD, and caspase-1, showed that compared with the blank control group, the expression levels of caspase1, NLRP3, and GSDMD in N2a cells in the OGD injury group were significantly increased, with statistical differences (*P<0.05, **P<0.01), indicating that OGD treatment induced cell pyroptosis; after stimulation with epalrestat, the expression levels of caspase1, NLRP3, and GSDMD were significantly decreased, with statistical differences (#P<0.05, ##P<0.01).

[0122] Immunofluorescence staining results also confirmed this. Figure 27 and Figure 28 Immunofluorescence staining of NLRP3 and caspase-1 in N2a cells in the blank control, OGD-injured, and epalrestat-treated groups, as well as the percentage of positive cells, show that OGD-treated N2a cells significantly increased their NLRP3 and caspase-1 levels (**P<0.01, ***P<0.001). Epalrestat stimulation decreased their expression levels, with the decrease in caspase-1 being more significant (###P<0.001). These results suggest that epalrestat can inhibit OGD-induced pyroptosis.

[0123] In this example, N2a cells were subjected to OGD for 2 h in vitro. Cell proteins were extracted from the blank control group, the OGD-injured group (OGD injury time was 2 h), and the epalrestat-treated group (epalrestat dosage was 50 μM). The secretion levels of pyroptosis-related inflammatory factors in N2a cells were detected by ELISA.

[0124] like Figure 29 The secretion levels of inflammatory factors IL-1β, IL-18, TNF-α and IFN-γ in N2a cells in the blank control group, OGD injury group and epalrestat treatment group were shown. Compared with the blank control group, the secretion levels of IL-1β, IL-18, TNF-α and IFN-γ in N2a cells after OGD treatment were significantly increased, with statistical differences (***P<0.001); after epalrestat stimulation, the secretion levels of IL-1β and IL-18 were significantly reduced, with statistical differences (###P<0.001); while the secretion levels of TNF-α and IFN-γ were not significantly different from those in the OGD group.

[0125] The above experimental results show that epalrestat stimulation can significantly improve the level of neuronal pyroptosis treated with OGD.

[0126] Example 9

[0127] In this example, LPS / ATP-induced oxidative stress was inhibited by knocking down AR expression in N2a cells.

[0128] In this example, siAR was transfected into N2a cells to knock down AR expression. LPS and ATP were then co-stimulated to establish an oxidative stress model. The antioxidant NAC (N-acetyl-L-cysteine) was used as a positive control for the siAR group. MicroELISA was used to assess the expression of oxidative stress markers (superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and lactate dehydrogenase (LDH)) in four cell groups: the control group, the LPS / ATP group, the LPS / ATP + siAR group, and the LPS / ATP + NAC group.

[0129] like Figure 30 The expression levels of SOD, MDA, GSH and LDH in N2a cells of different treatment groups were shown in Figure 2. Compared with the control group, the SOD level in N2a cells of the LPS / ATP group decreased, with statistical differences ( *** P<0.001), indicating an increase in oxidative stress levels. After knocking down AR expression or adding the antioxidant NAC for stimulation, the SOD levels in both groups showed an increasing trend, with statistically significant differences ( ## P<0.01, ### P<0.001), and there was no significant difference between the two groups. Compared with the control group, the MDA level in N2a cells of the LPS / ATP group was increased, with statistical difference ( *** P<0.001); after knocking down AR expression or adding NAC stimulation, the MDA levels of the two groups decreased, with statistical differences ( ### P<0.001, and there was no significant difference between the two groups. After LPS / ATP stimulation of N2a cells, GSH levels decreased, with statistical differences ( *** P<0.001); after knocking down AR expression or stimulating with NAC, GSH levels increased, with statistical significance ( # P<0.05, ## P<0.01), and there was no significant difference between the two groups. Compared with the control group, the LDH concentration in the cell supernatant of the LPS / ATP group was significantly increased, with statistical difference ( * P<0.05); after knocking down AR expression, LDH concentration decreased, with statistical difference ( # P < 0.05), and the trend was the same as that of the NAC stimulation group.

[0130] These results indicate that knockdown of AR expression can inhibit the oxidative stress level in OGD-treated N2a cells.

[0131] Example 10

[0132] This example downregulates oxidative stress-induced cell pyroptosis by knocking down AR expression in N2a cells.

[0133] In this example, siAR was transfected into N2a cells to knock down AR expression, and N2a cells were co-stimulated with LPS and ATP to establish an oxidative stress model. At the same time, NAC was used as a positive control for the siAR group. ELISA was used to detect the secretion levels of pyroptosis-related inflammatory factors IL-1β and IL-18 in the supernatant of four groups of cells: control group, LPS / ATP group, LPS / ATP+siAR group, and LPS / ATP+NAC group.

[0134] like Figure 31 The secretion levels of IL-1β and IL-18 in N2a cells in different treatment groups are shown in Figure 2. Compared with the control group, LPS / ATP combined stimulation of N2a cells promoted the secretion of IL-1β and IL-18, with statistical differences ( *** P<0.001), indicating that LPS / ATP combined stimulation can induce cell pyroptosis; after knocking down AR expression, the secretion levels of IL-1β and IL-18 were significantly reduced, with statistical differences ( ### P < 0.001), and this trend was consistent with that in the NAC group.

[0135] This example also detected the expression of other pyroptotic bodies by qPCR. Figure 32 As shown in the mRNA levels of NLRP1, NLRP2, NLRP3 and NLRC4 in N2a cells of different treatment groups, the combined stimulation of LPS / ATP group in N2a cells significantly increased the mRNA levels of NLRP1, NLRP2, NLRP3 and NLRC4, with statistical differences ( ** P<0.01, *** P<0.001); after AR expression was knocked down, the mRNA levels of NLRP1, NLRP3, and NLRC4 were significantly decreased, with statistical differences ( # P<0.05, ## P<0.01, ### P < 0.001), and this trend was similar to that in the NAC group. These results suggest that combined LPS / ATP stimulation increases pyroptosis, which is subsequently reduced after AR expression is reduced. Therefore, AR may regulate pyroptosis by inhibiting oxidative stress.

[0136] Example 11

[0137] This example demonstrates that AR regulates oxidative stress levels through the NOX4 / ROCK / Poldip2 pathway.

[0138] In this example, siAR was transfected into N2a cells to knock down AR expression, and N2a cells were co-stimulated with LPS and ATP to establish an oxidative stress model. NAC was used as a positive control for the siAR group. Protein and RNA were then extracted from four groups: a control group, an LPS / ATP group, an LPS / ATP + siAR group, and an LPS / ATP + NAC group. Western blot was used to detect changes in the expression of NOX4, ROCK, and Poldip2.

[0139] like Figure 33 and Figure 34 As shown in the WB images of NOX4, ROCK and Poldip2 expression in N2a cells of different treatment groups, compared with the control group, LPS / ATP stimulation significantly upregulated the expression levels of NOX4, ROCK and Poldip2, with statistical differences ( ** P<0.01, *** P<0.001); knocking down AR expression significantly downregulated the expression levels of NOX4, ROCK, and Poldip2, with statistical differences ( # P<0.05, ## P<0.01, ### P < 0.001), and there was no significant difference compared with the NAC group.

[0140] like Figure 35 As shown in the mRNA levels of NOX4, ROCK, and Poldip2 in N2a cells of different treatment groups, after LPS / ATP stimulation, the mRNA levels of NOX4, ROCK, and Poldip2 increased significantly, with statistical differences ( *** P<0.001); after knocking down AR expression, the mRNA levels of the three were significantly reduced, with statistical differences ( ## P<0.01, ### The NAC group showed the same trend as the siAR group (P < 0.001). These results indicate that knocking down AR expression in N2a cells can downregulate the expression levels of NOX4 / ROCK / Poldip2, a molecule involved in the oxidative stress pathway.

[0141] Example 12

[0142] This example demonstrates that epalrestat treatment can reduce the expression of NOX4 / ROCK / Poldip2 in mice with spinal cord injury.

[0143] In this example, a 3-day spinal cord injury (SCI) animal disease model and a sham operation model were constructed. Mice were given 250 mg / kg of epalrestat by gavage for 3 consecutive days. Protein and RNA were extracted from the injured spinal cord tissue, and the expression changes of NOX4, ROCK, and Poldip2 were detected by Western blot and qPCR.

[0144] like Figure 36 and Figure 37 As shown in the WB images and expression levels of NOX4, ROCK and Poldip2 in the spinal cord tissues of mice in the sham operation group, SCI model group and treatment group, compared with the sham operation group, the expression levels of NOX4, ROCK and Poldip2 in the SCI model group were increased, with statistical differences (***P<0.001); after treatment with epalrestat, the expression levels of the three were decreased, with statistical differences (#P<0.05, ###P<0.001).

[0145] like Figure 38 The mRNA levels of NOX4, ROCK, and Poldip2 in the spinal cord tissues of mice in the sham, SCI, and treatment groups are shown in Figure 2. Compared with the SCI model group, the mRNA levels of NOX4, ROCK, and Poldip2 in the epalrestat-treated group were significantly decreased (#P<0.05, ###P<0.001). This indicates that epalrestat treatment can reduce the expression levels of NOX4 / / ROCK / Poldip2 in SCI mice.

[0146] Example 13

[0147] This example demonstrates that in an in vitro SCI model, AR inhibition can improve cell pyroptosis by downregulating oxidative stress mediated by the NOX4 / / ROCK / Poldip2 pathway.

[0148] In this example, siAR was transfected into N2a cells to knock down AR expression. The NOX4 agonist dimethyl fumarate (DMF) was also added to activate NOX4 pathway expression. Protein and mRNA were extracted from four groups: a control group, an OGD injury group, an OGD injury + siAR group, and an OGD injury + DMF group. Western blot analysis was used to measure NOX4, ROCK, and Poldip2 expression levels.

[0149] like Figure 39 and Figure 40 As shown in the WB images and expression levels of NOX4, ROCK and Poldip2 in N2a cells of different treatment groups in the AR knockdown experiment, the expression levels of oxidative stress-related pathway molecules NOX4, ROCK and Poldip2 in N2a cells treated with OGD increased, with statistical differences (** P<0.01); after knocking down AR expression, the expression levels of the three decreased, with statistical differences ( # P<0.05, ### P<0.001). The decrease in expression levels of pathway molecules caused by AR knockdown was reversed by DMF, with statistical significance ( $ P<0.05, $$ P<0.01).

[0150] like Figure 41 As shown in the mRNA levels of NOX4, ROCK, and Poldip2 in N2a cells of different treatment groups in the AR knockdown experiment, compared with the OGD group, the mRNA levels of NOX4, ROCK, and Poldip2 in the siAR group were decreased, with statistical differences ( ## P<0.01, ### P<0.001); the addition of DMF reversed this phenomenon.

[0151] Next, in this example, siAR was transfected or epalrestat was added to inhibit AR expression, and DMF was added to activate the NOX4 / ROCK / Poldip2 pathway and the effects of the above interventions on OGD-mediated cell pyroptosis were detected.

[0152] like Figure 42 The secretion levels of IL-1β and IL-18 in N2a cells of different treatment groups in the AR knockdown experiment showed that OGD treatment promoted the secretion of IL-1β and IL-18 in N2a cells, with statistical differences ( *** P<0.001); after siAR transfection and knockdown of AR expression, the secretion levels of IL-1β and IL-18 decreased, with statistically significant differences ( ### P<0.001); the addition of DMF reversed the decrease in the secretion level of pyroptotic inflammatory factors caused by AR inhibition, with statistical significance ( $$ P<0.01, $$$ P<0.001).

[0153] like Figure 43 The secretion levels of IL-1β and IL-18 in N2a cells of different treatment groups in the epalrestat treatment experiment showed that after using epalrestat (Eps) to inhibit AR expression, the change trend of IL-1β and IL-18 secretion levels was the same.

[0154] Subsequently, the expression levels of oxidative stress-related indicators SOD, MDA, and LDH in N2a cells were detected using AR knockdown experiments and epalrestat-treated groups to prove that AR depends on the NOX4 / ROCK / Poldip2 pathway to regulate cell pyroptosis caused by oxidative stress.

[0155] like Figure 44 The expression levels of SOD, MDA and LDH in N2a cells of different treatment groups in the AR knockdown experiment showed that compared with the control group, the SOD level in N2A cells after OGD treatment was significantly decreased, with statistical differences ( ** P<0.01), indicating that the oxidative stress level increased. After knocking down AR expression, SOD levels increased significantly, with statistical differences ( ## P<0.01); knocking down AR and adding DMF reversed this phenomenon. Compared with the normal group, the level of MDA in the OGD group was significantly increased, with statistical differences ( *** P<0.001), the MDA level in the siAR group was significantly decreased, with statistical difference ( ## P<0.01); after using DMF to activate the oxidative stress pathway, there was no significant difference in MDA levels, which was not statistically significant. Compared with the OGD group, knocking down AR expression reduced the secretion level of LDH, which was statistically different ( ## P<0.01); however, the addition of DMF to activate the oxidative stress pathway while inhibiting AR expression reversed this phenomenon.

[0156] Subsequently, we used epalrestat (Eps) instead of siAR to inhibit AR expression and also detected the expression levels of oxidative stress-related indicators to explore whether epalrestat affects the oxidative stress level. Figure 45 The expression levels of SOD, MDA, and LDH in N2a cells treated with epalrestat showed the same trend as the above siAR expression. Taken together, these results suggest that epalrestat may inhibit oxidative stress-induced pyroptosis through the AR / NOX4 / ROCK / Poldip2 pathway.

Claims

1. Use of epalrestat in the preparation of a drug for treating spinal cord injury.

2. The use of epalrestat according to claim 1 in preparing a medicament for treating spinal cord injury, characterized in that: The medicine for treating spinal cord injury uses epalrestat as the only active ingredient or one of the active ingredients.

3. Use of epalrestat according to claim 1 or 2 in the preparation of a medicament for treating spinal cord injury, characterized in that: The content of epalrestat in the medicine for treating spinal cord injury is 0.1 wt% to 99 wt%.

4. Use of epalrestat in the preparation of a medicament for treating spinal cord injury according to claim 3, characterized in that: The medicine for treating spinal cord injury also includes pharmaceutically acceptable excipients and / or carriers.

5. Use of epalrestat in the preparation of a medicament for treating spinal cord injury according to claim 4, characterized in that: The drug for treating spinal cord injury is administered orally.

6. Use of epalrestat in the preparation of a medicament for treating spinal cord injury according to claim 5, characterized in that: When the drug for treating spinal cord injury is administered orally, the dosage of epalrestat is 250 mg / kg / d.

7. Use of epalrestat in the preparation of a medicament for treating spinal cord injury according to claim 6, characterized in that: The medicine for treating spinal cord injury is in the form of granules, tablets, capsules, pills or oral liquid preparations.

8. Use of epalrestat in the preparation of a medicament for treating spinal cord injury according to claim 7, characterized in that: The medicine for treating spinal cord injury can reduce the expression level of aldose reductase in the spinal cord injury area.

9. Use of epalrestat in the preparation of a medicament for treating spinal cord injury according to claim 8, characterized in that: The drug for treating spinal cord injury can improve the pyroptosis level of spinal cord injury tissue.

10. Use of epalrestat in the preparation of a medicament for treating spinal cord injury according to claim 9, characterized in that: Reduce the level of neuronal pyroptosis after spinal cord injury by inhibiting the aldose reductase-mediated NOX4 / ROCK / Poldip2 oxidative stress pathway.

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