Application of isosteviol containing hydroxamic acid group and steviol derivative in nerve injury protection

Abstract

The invention discloses an application of isosteviol containing a hydroxamic acid group and a steviol derivative in nerve injury protection, relates to the field of nerve injury protection medicines, and aims to provide an HDAC6 inhibitor capable of improving nerve dysfunction. Isosteviol and steviol derivatives containing hydroxamic acid groups are applied in the field of nerve injury protection, especially in the treatment of neurodegenerative diseases. The isosteviol and the steviol derivative containing the hydroxamic acid group enhance the antioxidant defense capability of cells and reduce oxidative stress induced cell apoptosis by regulating and controlling an Nrf2 / HO-1 signal channel, a Bcl2 / Bax signal axis and an HDAC6 / Ac-Tubulin microtubule stabilizing signal axis, so that the neuroprotective effect is achieved. Important theoretical support is provided for development of tetracyclic diterpenoid natural compounds in the field of neuroprotection, and the application prospect of the tetracyclic diterpenoid natural compounds in treatment of neurodegenerative diseases is expanded.

Description

Technical Field

[0001] The present invention relates to the technical field of nerve damage protection drugs, and specifically to the application of isosteviol and steviol derivatives containing a hydroxamic acid group in nerve damage protection. Background Art

[0002] Histone deacetylase 6 (HDAC6) is a deacetylase primarily located in the cytoplasm that regulates the acetylation status of various non-histone substrates, including tubulin, and plays a crucial role in maintaining homeostasis and regulating nervous system function. In recent years, a growing number of studies have found that abnormal HDAC6 function is closely associated with various neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and ischemic stroke. Therefore, HDAC6 inhibitors, as potential neuroprotective agents, have gradually become a research hotspot in the field of neuroscience.

[0003] Research has shown that selective HDAC6 inhibitors can exert neuroprotective effects through multiple mechanisms. On the one hand, they can increase tubulin acetylation, thereby stabilizing microtubule structure, improving axonal transport, and promoting nerve regeneration and repair. On the other hand, HDAC6 inhibitors can effectively protect neurons by inhibiting oxidative stress damage, reducing the release of inflammatory factors, and lowering the rate of neuronal apoptosis. Furthermore, HDAC6 inhibitors are closely linked to the regulation of neurotransmitter homeostasis and have shown potential in improving neurological dysfunction.

[0004] Therefore, there is a need to provide an HDAC6 inhibitor that can improve neurological dysfunction. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the existing defects and provide an application of isosteviol and steviol derivatives containing hydroxamic acid groups in the protection of nerve damage, which can effectively solve the problems in the background technology.

[0006] In order to achieve the above-mentioned object, the present invention discloses an application of isosteviol and steviol derivatives containing a hydroxamic acid group in the protection of nerve damage. The technical solution adopted is that the structural formula of the hydroxamic acid compound is one of the following structural formulas:

[0007]

[0008] Wherein, n=5-8.

[0009] As a preferred technical solution of the present invention, the structural formula of the hydroxamic acid compound is:

[0010]

[0011] Wherein, n=5.

[0012] As a preferred technical solution of the present invention, the nerve damage protection includes the treatment of neurodegenerative diseases.

[0013] As a preferred technical solution of the present invention, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, ischemic stroke, and other neurodegenerative diseases.

[0014] Compared with the prior art, the present invention has the following beneficial effects: isosteviol and steviol derivatives containing hydroxamic acid groups enhance the antioxidant defense capacity of cells and reduce oxidative stress-induced cell apoptosis by regulating the Nrf2 / HO-1 signaling pathway, the Bcl2 / Bax signaling axis, and the HDAC6 / Ac-Tubulin microtubule stabilization signaling axis, thereby exerting a neuroprotective effect. Furthermore, isosteviol and steviol derivatives containing hydroxamic acid groups can enhance the selective binding of molecules to HDAC6, thereby promoting the stabilization of microtubules, and regulate the Nrf2 / HO-1 signaling pathway by inhibiting HDAC6 activity, thereby improving the antioxidant capacity of cells. The present invention provides important theoretical support for the development of tetracyclic diterpenoid natural compounds in the field of neuroprotection and expands their application prospects in the treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the relationship between cell survival rate and glutamate concentration;

[0016] Figure 2 This is a comparison chart of the effects of different concentrations of different compounds of the present invention on the survival rate of the HT22 cell injury model;

[0017] Figure 3 Effects of different concentrations of compound J01 and vitamin E on the survival rate of HT22 cell injury model;

[0018] Figure 4 This is a staining fluorescence comparison diagram of compound J01 of the present invention in the detection of ROS levels;

[0019] Figure 5 This is a quantitative analysis diagram of the compound J01 of the present invention in the detection of ROS levels;

[0020] Figure 6 This is a fluorescence staining comparison diagram showing the effect of compound J01 of the present invention on mitochondrial membrane potential of HT22 cells;

[0021] Figure 7 This is a quantitative analysis diagram of the effect of compound J01 of the present invention on mitochondrial membrane potential of HT22 cells;

[0022] Figure 8This is a fluorescence staining comparison diagram of the effect of compound J01 of the present invention on HT22 cell apoptosis;

[0023] Figure 9 This is a flow cytometry test result showing the effect of compound J01 of the present invention on HT22 cell apoptosis;

[0024] Figure 10 This is a comparison chart of the effects of compound J01 of the present invention on α-tubulin levels;

[0025] Figure 11 This is a quantitative analysis diagram of the effect of compound J01 of the present invention on the level of α-tubulin;

[0026] Figure 12 This is a comparison chart of the effects of compound J01 of the present invention on HO-1 levels;

[0027] Figure 13 This is a quantitative analysis diagram of the effect of compound J01 of the present invention on HO-1 levels;

[0028] Figure 14 This is a comparison chart of the effects of compound J01 of the present invention on the cytoplasmic and nuclear expression levels of Nrf2 cells;

[0029] Figure 15 This is a quantitative analysis of the expression levels of Nrf2 in the cytoplasm and nucleus of the compound J01 of the present invention;

[0030] Figure 16 This is a comparison diagram of the effects of compound J01 of the present invention on the expression level of anti-apoptosis related protein Bcl2 / Bax;

[0031] Figure 17 This is a quantitative analysis diagram of the effect of compound J01 of the present invention on the expression level of anti-apoptosis related protein Bcl2 / Bax. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] In order to facilitate subsequent description, the hydroxamic acid compounds involved in the present invention are numbered:

[0034]

[0035]

[0036] Compounds J01-J16 are all existing compounds. Their preparation methods refer to Chinese patent CN118546075A, "An isosteviol and steviol derivatives containing a hydroxamic acid group, and their preparation method and application."

[0037] Example 1

[0038] This example discloses the first embodiment of the present invention, and the technical solution adopted is the use of compound J01 in the treatment of Alzheimer's disease.

[0039] Example 2

[0040] This example discloses a second embodiment of the present invention, and the technical solution adopted is the use of compound J02 in the treatment of Parkinson's disease.

[0041] Example 3

[0042] This example discloses a third embodiment of the present invention, and the technical solution adopted is the use of compound J03 in the treatment of ischemic stroke.

[0043] Example 4

[0044] This example discloses the fourth embodiment of the present invention, and the technical solution adopted is the use of compound J04 in the treatment of Alzheimer's disease.

[0045] Example 5

[0046] This example discloses the fifth embodiment of the present invention, and the technical solution adopted is the use of compound J05 in the treatment of Alzheimer's disease.

[0047] Example 6

[0048] This example discloses the sixth embodiment of the present invention, and the technical solution adopted is the use of compound J06 in the treatment of Alzheimer's disease.

[0049] Example 7

[0050] This example discloses the seventh embodiment of the present invention, and the technical solution adopted is the use of compound J07 in the treatment of Alzheimer's disease.

[0051] Example 8

[0052] This example discloses the eighth embodiment of the present invention, and the technical solution adopted is the use of compound J08 in the treatment of Alzheimer's disease.

[0053] Example 9

[0054] This example discloses the ninth embodiment of the present invention, and the technical solution adopted is the use of compound J09 in the treatment of Alzheimer's disease.

[0055] Example 10

[0056] This example discloses the tenth embodiment of the present invention, and the technical solution adopted is the use of compound J10 in the treatment of Alzheimer's disease.

[0057] Example 11

[0058] This example discloses the eleventh embodiment of the present invention, and the technical solution adopted is the use of compound J11 in the treatment of Alzheimer's disease.

[0059] Example 12

[0060] This example discloses the twelfth embodiment of the present invention, and the technical solution adopted is the use of compound J12 in the treatment of Alzheimer's disease.

[0061] Example 13

[0062] This example discloses the thirteenth embodiment of the present invention, and the technical solution adopted is the use of compound J13 in the treatment of Alzheimer's disease.

[0063] Example 14

[0064] This example discloses the fourteenth embodiment of the present invention, and the technical solution adopted is the use of compound J14 in the treatment of Alzheimer's disease.

[0065] Example 15

[0066] This example discloses the fifteenth embodiment of the present invention, and the technical solution adopted is the use of compound J15 in the treatment of Alzheimer's disease.

[0067] Example 16

[0068] This example discloses the sixteenth embodiment of the present invention, and the technical solution adopted is the use of compound J16 in the treatment of Alzheimer's disease.

[0069] A glutamate (GLU)-induced oxidative damage model in HT22 cells was used to simulate neuronal damage caused by oxidative stress in the brains of patients with Alzheimer's disease (AD).

[0070] Prepare cell culture medium:

[0071] When HT22 cells (purchased from Zhejiang Bio-Technology Co., Ltd.) were grown in a culture flask to a density of about 80%, they were digested with trypsin, collected by centrifugation, and the supernatant was discarded. After the cells were fully resuspended in complete culture medium, 5 × 10 3 The cells were seeded into a 96-well cell culture plate at a density of 100 cells / well and incubated in a cell culture incubator at 37° C. and 5% CO 2 for 24 h to ensure that the cells adhered to the wall and reached a stable growth state, thereby obtaining a cell culture medium.

[0072] This cell culture medium was used in all subsequent verifications.

[0073] 1. Establish a glutamate injury model and compare the effectiveness of compounds J01-J16:

[0074] (1) Preparation of main reagents

[0075] ① Preparation of complete culture medium

[0076] Take a 50mL centrifuge tube, place it in the clean bench, and turn on the ultraviolet light to ensure thorough sterilization. After sterilization is completed, use a 1mL pipette to accurately draw 5mL of fetal bovine serum and slowly inject it into the centrifuge tube. Then, while maintaining aseptic operation, add 45mL of DMEM high glucose medium to ensure that the medium and fetal bovine serum are evenly mixed. Next, add 0.5mL of penicillin-streptomycin mixture to prepare a complete medium containing double antibodies (penicillin-streptomycin). After all reagents are added, gently invert the centrifuge tube several times to ensure that the components are fully mixed. Finally, seal the centrifuge tube with sealing glue and store it in a 4°C refrigerator for use to maintain the stability and sterility of the culture medium.

[0077] ② Preparation of glutamate solution: Accurately weigh 147 mg of glutamate powder using an analytical balance and dissolve it in 10 mL of complete culture medium. Once fully dissolved, a 100 mM glutamate stock solution will be obtained. Seal the container with sealing tape and store at 4°C for later use.

[0078] ③ Preparation of Vitamin E Solution: Because Vitamin E appears as a thick, oily substance, the required amount is accurately weighed using the peeling method. Subsequently, DMSO is used as a solvent to fully dissolve the weighed Vitamin E to prepare a stock solution. After the concentration is calculated, the stock solution is stored in a -20°C refrigerator for dilution as needed in subsequent experiments.

[0079] (2) Model construction

[0080] After the cells in the cell culture medium adhered to the wall, the cells were treated with DMEM complete medium prepared with different concentrations of glutamate (0, 1.5mM, 3mM, 6mM, 12mM, 24mM) to induce oxidative damage and evaluate the cytotoxic effect induced by glutamate. Subsequently, the cells continued to be incubated in a 37°C constant temperature incubator for 24 hours. After the incubation, the CCK-8 kit was used to quantitatively detect cell viability and cell viability. The experimental data were statistically analyzed using GraphPaD Prism 8.0 software to evaluate the toxic effect of glutamate on HT22 cells. The test results are shown in Figure 2. Figure 1 As shown in the results, glutamate exposure significantly reduced cell viability, with a clear concentration-dependent (Log) decrease. When the glutamate concentration reached 6.76 mM, cell viability dropped significantly to approximately 50%. Based on this, 7 mM glutamate was selected as the optimal modeling concentration for subsequent experiments.

[0081] Based on this model, the glutamate-induced HT22 cell injury model was used to screen the antioxidant stress effects of compounds J01-J16.

[0082] (3) Comparison of the effectiveness of compounds J01-J16

[0083] According to the experimental grouping, the cell culture medium was prepared into the corresponding drug-treated medium and incubated at 37°C for the designated time. Five replicate wells were set up for each group to enhance data reliability and statistical power. After incubation, 10 μL of CCK-8 reagent was added to each well. The 96-well plate was gently shaken to ensure even distribution of the reagent, and then incubated at 37°C for another 2 h. After incubation, the optical density (OD) of each well was measured at a wavelength of 450 nm using a multi-function microplate reader, and the experimental data were recorded. Cell viability was calculated using the following formula: Viability (%) = (OD experimental group - OD blank control group) / (OD negative control group - OD blank control group) * 100%; where OD experimental group refers to the absorbance value of the wells treated with drug or other treatment; OD blank control group refers to the absorbance value of the wells without cells or any treatment; OD negative control group refers to the absorbance value of the wells containing only cells but without drug or treatment.

[0084] Statistical analysis was performed using GraphPaD Prism 8.0 software. Figure 2As shown in the results, compound J01 showed the best cell protection in this model. Therefore, compound J01 was selected for subsequent verification. HT22 cells were treated with compound J01 at different concentrations (0.5-10 μM) and vitamin E was used as a positive control to detect its protective effect against glutamate (7 mM)-induced cell damage. The results are shown in Figure 3 As shown (vs Control, ####P<0.0001; vs GLU, ***P<0.001, ****P<0.0001, n=3), compound J01 can significantly increase cell survival rate in the concentration range of 0.5~10μM, and shows a concentration-dependent trend. In the 1μM treatment group, cell survival rate recovered to 85.82%, which was statistically significantly different from the control group. However, when the concentration was higher than 10μM, cell survival rate did not increase further, and even showed a downward trend, suggesting that excessive concentration may induce cytotoxicity. Therefore, 1μM was determined to be the optimal protective concentration of compound J01 for subsequent mechanism verification.

[0085] 2. Verification of compound J01's ability to inhibit glutamate-induced oxidative stress in HT22 cells

[0086] (1) Preparation of main reagents

[0087] Preparation of ROS (Reactive Oxygen Species) Detection Solution: To detect ROS, dilute DCFH-DA (a fluorescent probe for reactive oxygen species, 2',7'-dichlorodihydrofluorescein diacetate) appropriately. Specifically, dilute DCFH-DA with serum-free culture medium at a ratio of 1:1500. After thorough mixing, a final concentration of 10 μM DCFH-DA working solution is obtained.

[0088] (2) Verification of oxidative stress damage

[0089] DCFH (dichlorodihydrofluorescein) can be oxidized by reactive oxygen species (ROS) within cells to form DCF (2',7'-dichlorofluorescein), which exhibits green fluorescence under a fluorescence microscope. Therefore, measuring the intensity of green fluorescence can indirectly reflect the level of ROS within cells.

[0090] After the cells in the cell culture medium have stabilized on the wall, they are grouped and treated accordingly according to the experimental design. Before experimental treatment, the original culture medium is first discarded and washed once with PBS to remove residual culture medium and metabolites. Subsequently, 1 mL of appropriately diluted DCFH-DA working solution is added to each well under light-proof conditions. Gently shake the 6-well plate to ensure that the probe solution is evenly distributed, and continue to incubate in a 37°C cell culture incubator in the dark for 20 minutes. After the incubation is completed, carefully remove the staining solution and wash the cells three times with serum-free culture medium to completely remove free probes that have not entered the cells and reduce background fluorescence interference. After staining is completed, use a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm) to observe the cell fluorescence signal and collect images within 1 hour. The obtained fluorescence image data are quantitatively analyzed using ImageJ software to calculate the fluorescence intensity and further perform statistical analysis.

[0091] The experimental results are as follows Figure 4 As shown in the figure, compared with the control group, the green fluorescence of the glutamate-treated group was significantly enhanced, indicating a significant increase in intracellular ROS levels. There was no significant change in cell fluorescence in the group treated with compound J01 alone. However, in the glutamate-induced injury model, the green fluorescence intensity was significantly reduced after compound J01 treatment, suggesting that compound J01 can effectively inhibit the excessive accumulation of ROS induced by glutamate, thereby alleviating oxidative damage in HT22 cells. Figure 5 In the quantitative analysis results (vs Control, ####P<0.0001; vs GLU, ****P<0.0001, n=3), the comparison of the relative fluorescence intensity of ROS further supported the protective effect of compound J01 in resisting oxidative damage.

[0092] 3. Verification of compound J01's inhibition of glutamate-induced apoptosis in HT22 cells

[0093] (1) JC-1 mitochondrial membrane potential detection

[0094] One of the key early hallmarks of apoptosis is changes in mitochondrial membrane potential (MMP), which can be detected using the fluorescent probe JC-1. When MMP is reduced, JC-1 cannot aggregate within the mitochondrial matrix and exists as a monomer, producing green fluorescence. Conversely, when MMP is elevated, JC-1 aggregates within the mitochondrial matrix, forming polymers that emit red fluorescence. Therefore, the degree of mitochondrial depolarization can be quantified using the red / green fluorescence ratio. In this study, the JC-1 fluorescent probe was used to detect the effect of compound J01 on the mitochondrial membrane potential of HT22 cells.

[0095] ①Prepare main reagents

[0096] Preparation of JC-1 working solution: Dilute JC-1 (200×) staining buffer at a ratio of 1:200. Mix thoroughly to obtain JC-1 working solution.

[0097] ② Mitochondrial membrane potential detection

[0098] After the cells in the cell culture medium have stabilized on the wall, they are treated accordingly according to the experimental groups. Before cell treatment, the original culture medium is first discarded and the cells are washed once with PBS. Subsequently, 1 mL of fresh culture medium is added to each well, and 1 mL of JC-1 staining working solution is added under light-proof conditions, and the 6-well plate is gently shaken to ensure sufficient mixing. After that, the 6-well plate is placed in a 37°C incubator and incubated in the dark for 20 minutes. After the incubation is completed, the staining solution is carefully aspirated and the cells are washed twice with JC-1 staining buffer to remove free dye that has not entered the cells. Finally, 1 mL of culture medium is added to each well, and the images are observed and collected under a fluorescence microscope within 1 hour. The results are as follows. Figure 6 HT22 cells in the control group emitted primarily red fluorescence, with the red-to-green fluorescence ratio remaining high. In the glutamate-treated group, however, green fluorescence intensity significantly increased, while the red-to-green fluorescence ratio decreased significantly, suggesting that glutamate-induced oxidative damage can lead to increased mitochondrial depolarization and a significant decrease in MMP. While J01 treatment alone showed no significant change in cell fluorescence, in the glutamate-induced injury model, J01 treatment significantly weakened green fluorescence intensity and increased the red-to-green fluorescence ratio, suggesting that compound J01 can effectively inhibit the glutamate-induced decrease in mitochondrial membrane potential, alleviate mitochondrial depolarization, and restore MMP.

[0099] The resulting fluorescence images were quantitatively analyzed using ImageJ software to assess changes in mitochondrial membrane potential and further analyze the experimental results. Data were processed using GraphPaD Prism 8.0 software, and differences between groups were compared using one-way ANOVA or t-test.

[0100] The results of fluorescence intensity quantitative analysis were as follows Figure 7 As shown in the results (vs Control, ##P<0.01, n=3), the red / green ratio of HT22 cells after glutamate treatment was less than 1, further confirming a significant decrease in mitochondrial membrane potential. However, treatment with J01 restored this ratio, suggesting that compound J01 may have a protective effect in maintaining mitochondrial functional stability. Taken together, these results suggest that compound J01 can effectively ameliorate glutamate-induced mitochondrial membrane depolarization and may play an important role in protecting against oxidative damage.

[0101] (2) Hoechst 33258 detection of cell apoptosis

[0102] Glutamate can induce neurons to produce a large amount of ROS, thereby leading to an increase in abnormal apoptosis of neurons.

[0103] Hoechst 33258 staining is a fluorescent staining technique commonly used to detect apoptosis. Its principle is based on the changes in nuclear morphology caused by chromatin condensation during apoptosis. After staining, normal cell nuclei exhibit a uniform blue fluorescence, while the nuclei of apoptotic cells appear dense and bright blue, or fragmented, with enhanced staining and possibly a slightly pale appearance.

[0104] The cleaned and sterilized coverslips were placed in a 6-well plate and inoculated with 3×10 5 Cells were plated and incubated overnight in a 37°C, 5% CO2 cell culture incubator until the cell density reached 50% to 80%. Apoptosis was induced with 7 mM glutamate. After induction, the culture medium was discarded and 0.5 mL of fixative (such as 4% paraformaldehyde) was added. Fixation was performed at room temperature for 10 minutes or overnight at 4°C. After fixation, the fixative was removed and the wells were washed twice with PBS or 0.9% NaCl solution for 3 minutes each to ensure complete removal of residual fixative. During the washing process, appropriate shaking can be used to improve the washing effect. After washing, 0.5 mL of Hoechst 33258 working solution (final concentration 1-5 μg / mL) was added to each well and incubated in the dark for 5 minutes with gentle shaking to promote uniform staining. After staining, the staining solution was discarded and the wells were washed twice with PBS or 0.9% NaCl solution for 3 minutes each to remove unbound fluorescent dye. Subsequently, anti-fluorescence quenching mounting solution was added to the slide and then covered with a coverslip, ensuring that the cells were fully exposed to the mounting solution and minimizing the formation of bubbles. Finally, the nuclear fluorescence signal was observed using a fluorescence microscope with an excitation wavelength of 350 nm and an emission wavelength of 460 nm, and images were acquired within 1 hour.

[0105] Fluorescence microscopy results Figure 8 As shown, in the blank control group and the group treated with compound J01 alone, the cell nuclei exhibited uniform blue fluorescence, with intact nuclear morphology, round or oval, and clear outlines, with no obvious signs of apoptosis. In contrast, in the glutamate-treated group, significant changes in nuclear morphology occurred, including nuclear condensation, enhanced staining, and a change from round to irregular or spindle-shaped nuclear shape, indicating that glutamate induced apoptosis in HT22 cells. Notably, in the glutamate-induced injury model, treatment with compound J01 significantly improved abnormal nuclear morphology and reduced the incidence of nuclear condensation and deformation. These results demonstrate that compound J01 effectively inhibits glutamate-induced apoptosis in HT22 cells, thereby exerting a neuroprotective effect.

[0106] (3) Detection of HT22 cell apoptosis rate by flow cytometry

[0107] When the cells proliferate to about 80% density in the culture flask, trypsinize and collect the cells by centrifugation and discard the supernatant. Add 4 mL of complete culture medium to the cell pellet and gently pipette to resuspend to prepare a uniform cell suspension. 4 Cells were evenly seeded into 6-well plates at a uniform density and supplemented with complete medium to a final volume of 2 mL. The 6-well plates were then incubated overnight at 37°C, 5% CO2 in a cell culture incubator to ensure full cell adhesion. The next day, the cells were observed under a microscope. If there were a large number of floating cells in the culture medium, these cells should be collected for subsequent analysis. If there were a small number of floating cells, the old culture medium should be discarded. Subsequently, the plates were gently washed once with PBS to remove any residual culture medium and non-adherent cells. During the cell digestion step, 0.5 mL of EDTA-free trypsin was added to each well and digested at room temperature for approximately 50 seconds. The digestion reaction was then terminated by adding 0.5 mL of complete medium. Adherent cells were gently pipetted to fully disperse and the cell suspension was transferred to a 1.5 mL centrifuge tube. The plates were centrifuged at 3000 rpm for 5 minutes, the supernatant discarded, and the cell pellet washed twice with PBS to remove any residual culture medium and enzyme solution. Cells in the blank control group were equally divided into four centrifuge tubes for later use. Cells in the experimental treatment groups were not aliquoted and were stained directly. Subsequently, add 0.5 mL of staining buffer to each centrifuge tube to resuspend the cells. Depending on the experimental design, add the appropriate amount of Annexin V-FITC and PI staining solution to each tube. Gently mix and incubate at room temperature in the dark for 10 minutes. After staining, analyze the cells using flow cytometry within 1 hour to ensure fluorescence signal stability and data accuracy.

[0108] Test results such as Figure 9As shown in the figure, compared with the control group, the apoptosis rate in the glutamate-treated group was significantly increased, indicating that glutamate can induce apoptosis in HT22 cells. However, when compound J01 was treated alone, the apoptosis rate did not change significantly, indicating that compound J01 itself had no significant effect on the survival of HT22 cells. In the glutamate-induced cell injury model, the apoptosis rate was significantly reduced after compound J01 treatment, indicating that compound J01 can effectively inhibit glutamate-induced apoptosis in HT22 cells, further verifying its potential neuroprotective effect.

[0109] 4. Verification of the mechanism of action of compound J01

[0110] Glutamate-induced neuronal damage can trigger oxidative stress and inflammatory responses, ultimately leading to neuronal apoptosis. Heme oxygenase-1 (HO-1) is an important stress protein whose expression is regulated by nuclear factor E2-related factor 2 (Nrf2), playing a key role in the defense mechanism against oxidative damage. HO-1 activation is a common feature of multiple neurodegenerative diseases and plays an important role in the regulation of cellular oxidative stress. Furthermore, the stability and function of neuronal cells are also regulated by the cytoskeleton, with microtubule stability playing a crucial role in neuroprotection. Acetylated α-tubulin (Ac-Tubulin) is a marker of HDAC6 inhibition, and its expression level directly reflects the inhibitory status of HDAC6.

[0111] The expression level of anti-apoptosis related protein Bcl2 / Bax can also reflect the apoptosis of cells.

[0112] This validation verifies the mechanism of action of compound J01 by detecting related proteins in cells. The detection process is as follows:

[0113] Step 1, protein extraction and preparation: When the cells in the cell culture flask grow to 80% density, they are inoculated into a culture dish and cultured overnight. After experimental treatment, the cells are washed with sterile cold PBS buffer and then collected into a centrifuge tube. Pre-cooled lysis buffer (RIPA: PMSF: PI = 100: 1: 1) is added to each tube of cell suspension, and the sample is placed in an ice bath for lysis for 20 minutes, and vortexed every 5 minutes to ensure that the cells are fully lysed. After lysis, the supernatant, which is the extracted total cell protein, is obtained by high-speed centrifugation and placed in an ice bath for use;

[0114] Step 2, protein concentration determination: Use the BCA method to determine the optical density (OD) values of a series of BSA standard solutions with gradient concentrations, and draw a standard curve for calculating the protein concentration of subsequent samples. Calculate the concentration of each experimental group protein sample based on the standard curve, and dilute the protein sample appropriately according to the experimental requirements. Subsequently, denaturation treatment is performed to fully unfold the protein for SDS-PAGE electrophoresis analysis;

[0115] Step 3, SDS-PAGE electrophoresis: Prepare an SDS-PAGE gel, including a lower separating gel and an upper stacking gel. Load the denatured protein sample and molecular weight marker into the gel lanes. Perform electrophoresis under constant voltage or constant current conditions. SDS is used to impart a negative charge to the proteins, and the molecular sieving effect of the polyacrylamide gel allows for efficient separation of proteins based on their molecular weight.

[0116] Step 4, Protein Transfer: After electrophoresis, the separated proteins in the gel are transferred to a pretreated PVDF membrane. Using wet or semi-dry transfer, an electric field is used to transfer the proteins from the gel to the membrane while maintaining their relative position within the gel for subsequent immunoassay detection.

[0117] Step 5, blocking: To reduce the background signal of nonspecific binding of antibodies, incubate the PVDF membrane with 5% skim milk powder or BSA blocking solution at room temperature to block the membrane surface sites not occupied by proteins to improve the specificity of detection;

[0118] Step 6, primary antibody incubation: Soak the PVDF membrane in a diluted primary antibody solution and incubate at an appropriate temperature and time to allow the specific primary antibody to bind to the target protein and form an antigen-antibody complex, preparing for subsequent secondary antibody incubation and signal detection;

[0119] Table 1 Protein primary antibodies and their dilution ratios

[0120]

[0121] Step 7, Antibody Incubation: Add an enzyme-labeled secondary antibody that matches the species of the primary antibody and allows it to specifically bind to the primary antibody, thereby forming an antigen-antibody-enzyme complex. The secondary antibody is usually conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) for subsequent signal amplification and detection. After incubation, wash thoroughly with TBST buffer to remove unbound secondary antibody and reduce background signal;

[0122] Step 8, ECL development and protein expression analysis: ECL luminescent reagent was added to the PVDF membrane, and HRP catalyzed the substrate to produce chemiluminescent signals. A chemiluminescence imaging system was used to record protein band signals, and β-Actin antibody was used as an internal reference antibody during the experiment. The protein bands were quantitatively analyzed using the grayscale analysis software ImageJ, and the protein expression levels between different experimental groups were compared to evaluate the relative expression (relative density) and molecular weight of the target protein. The experimental data were processed using GraphPaD Prism 8.0 software, and one-way ANOVA or t-test was used to compare differences between groups.

[0123] (1) Detection of Nrf2 / HO-1 signaling pathway and HDAC6 / Ac-Tubulin signaling axis

[0124] like Figures 10 to 15 The results shown (vs Control, ##P<0.01, ####P<0.0001; vs GLU, *P<0.05, ****P<0.0001, n=3) indicate that compound J01 treatment increased the Ac-Tubulin / Tubulin ratio in a dose-dependent manner, indicating that J01 effectively inhibits HDAC6 activity, thereby enhancing microtubule stability and maintaining cytoskeletal integrity. Notably, this ratio remained unchanged in the glutamate-treated group, but remained significantly elevated in the glutamate-induced injury model after J01 treatment. This suggests that J01 not only directly inhibits HDAC6 activity but also maintains microtubule stability in the context of injury, thereby enhancing cytoskeletal integrity. This mechanism may contribute to maintaining neuronal functional homeostasis and provide mechanistic support for the neuroprotective effects of J01. Previous studies have shown that acetylation of α-tubulin can reduce microtubule dynamic instability and inhibit excessive dynamic changes in the plus ends of microtubules in neurons, thereby regulating proper axonal branching and growth and maintaining normal function of the central nervous system. Furthermore, inhibition of HDAC6 can significantly increase α-tubulin acetylation, improve axonal transport, reduce neuronal damage, and exert neuroprotective effects. Therefore, by inhibiting HDAC6 activity and increasing α-tubulin acetylation, J01 may play an important role in maintaining neuronal microtubule homeostasis, improving axonal transport, and promoting neuronal survival.

[0125] The above results show that J01 can significantly improve glutamate-induced HT22 cell damage. However, whether its mechanism of action is related to the Nrf2 / HO-1 signaling pathway remains unclear. Therefore, the expression levels of related proteins were detected by immunoblotting to explore its mechanism of action. Figure 8As shown, HO-1 expression levels were significantly decreased in cells treated with glutamate, indicating that GLU-induced oxidative stress can inhibit HO-1 activity. Furthermore, glutamate treatment increased nuclear Nrf2 expression, suggesting that cells enhance antioxidant defenses under oxidative stress through Nrf2 nuclear translocation. However, J01 alone did not induce significant changes in the expression of these proteins. However, treatment with compound J01 in the glutamate-induced injury model significantly increased HO-1 expression levels, while nuclear Nrf2 expression decreased, indicating that J01 can effectively alleviate oxidative stress and reduce cellular dependence on Nrf2 activation. Furthermore, J01's inhibition of HDAC6 further stabilizes microtubule structure, enhancing neuronal tolerance and playing a dual role in neuroprotection. These results suggest that J01 may synergistically alleviate glutamate-induced oxidative stress in HT22 cells by activating the Nrf2 / HO-1 signaling pathway to enhance antioxidant capacity and, through HDAC6 inhibition, enhance microtubule stability.

[0126] Taken together, these results suggest that compound J01 may alleviate glutamate-induced oxidative stress in HT22 cells by activating the Nrf2 / HO-1 axis, enhancing cellular antioxidant capacity, reducing the elevated nuclear expression of Nrf2 under oxidative stress, and enhancing microtubule stability through HDAC6 inhibition. This finding provides more comprehensive experimental evidence for J01's neuroprotective effects and suggests its potential application in the treatment of neurodegenerative diseases.

[0127] (2) Bcl2 / Bax detection

[0128] like Figure 16 、 Figure 17 As shown (vs Control, ##P<0.01; vs GLU, *P<0.05, n=3), GLU treatment significantly increased the expression of the pro-apoptotic protein Bax in HT22 cells, while decreasing the expression of the anti-apoptotic protein Bcl2, leading to a decrease in the Bcl2 / Bax ratio. This suggests that GLU-induced injury may accelerate cell death through the mitochondrial apoptotic pathway. However, treatment with compound J01 alone did not significantly alter Bcl2 / Bax expression levels. Notably, in a glutamate-induced injury model, J01 treatment significantly upregulated Bcl2 expression while decreasing Bax expression, restoring the Bcl2 / Bax ratio and inhibiting GLU-induced apoptosis. This suggests that compound J01 may inhibit glutamate-induced apoptosis in HT22 cells by regulating Bcl2 / Bax protein expression, thereby exerting its neuroprotective effects.

[0129] The aforementioned validations demonstrated that compound J01 significantly increased cell survival, reduced intracellular ROS levels, inhibited mitochondrial membrane potential loss, and reduced apoptosis in a glutamate-induced HT22 cell oxidative stress injury model. Western blot results further revealed that J01 may exert neuroprotective effects by regulating the Nrf2 / HO-1 signaling pathway, the Bcl2 / Bax signaling axis, and the HDAC6 / Ac-Tubulin microtubule stabilization axis, thereby enhancing cellular antioxidant defenses and reducing oxidative stress-induced apoptosis.

[0130] Structure-activity relationship (SAR) analysis revealed that shorter fatty chain substituents are more favorable for neuroprotective activity. This may be because these structural features enhance the selective binding of the molecule to HDAC6, thereby promoting tubulin stabilization and modulating the Nrf2 / HO-1 signaling pathway by inhibiting HDAC6 activity, thereby enhancing the antioxidant capacity of cells.

[0131] 5. Verification of Alzheimer's disease (AD) treatment

[0132] Anxiety Behavior Assessment: The novel tank diving test (NTT) was used to evaluate the effects of compound J01 on anxiety-like behaviors in the SCOP (scopolamine)-induced zebrafish AD model. Compared with the positive control drug IMP, compound J01 significantly increased the zebrafish's exploratory behavior in the upper water tank and reduced the number of freezing episodes and erratic movements, indicating its anxiolytic effect.

[0133] Memory and Cognitive Assessment: Compound J01's ability to improve memory and cognition in the SCOP-induced zebrafish AD model was evaluated using a T-maze and object recognition tasks. Compared to the positive control drug GAL, compound J01 significantly increased the time zebrafish spent in the enriched zone (EC) of the maze and increased the total swimming distance, indicating that it has the effect of improving memory and cognition.

[0134] Overall, isosteviol derivatives are significantly better than steviol derivatives in neuroprotection. Isosteviol is a structural rearrangement product of steviol. The opening of its D ring forms new hydroxyl and ketone groups at C13 and C16, making the molecule more flexible. This may be more conducive to binding to protein targets, improving the selectivity of HDAC6, and playing a greater role in microtubule stability regulation, oxidative stress resistance, and neuronal protection. In addition, the neuroprotective effect of isosteviol derivatives mainly depends on shorter fatty chain substituents and more polar substituents. These structural features can improve the selectivity of HDAC6, thereby stabilizing microtubule structure, reducing oxidative stress, and protecting neurons. Isosteviol derivatives can significantly improve anxiety-like behavior and memory deficits in zebrafish, indicating that they have the potential to treat AD.

[0135] The present invention provides important theoretical support for the development of tetracyclic diterpenoid natural compounds in the field of neuroprotection and expands their application prospects in the treatment of neurodegenerative diseases.

[0136] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An application of isosteviol and steviol derivatives containing hydroxamic acid groups in protecting against nerve damage, characterized in that: The structural formula of the hydroxamic acid compound is one of the following structural formulas: Wherein, n=5-8.

2. The use of isosteviol and steviol derivatives containing hydroxamic acid groups in the protection of nerve damage according to claim 1, characterized in that: The structural formula of the hydroxamic acid compound is: Wherein, n=5.

3. The use of isosteviol and steviol derivatives containing hydroxamic acid groups in the protection of nerve damage according to claim 1, characterized in that: The protection against nerve damage includes the treatment of neurodegenerative diseases.

4. The use of isosteviol and steviol derivatives containing hydroxamic acid groups in the protection of nerve damage according to claim 3, characterized in that: The neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, and ischemic stroke.

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