Application of dimethyl fumarate targeted GSDME in preparation of anti-aging medicine

Abstract

The invention relates to the technical field of biological medicines, and provides an application of dimethyl fumarate targeted GSDME in preparation of an anti-aging medicine. Dimethyl fumarate relieves the aging process by inhibiting the activity of GSDME, and can be used for developing and preparing anti-aging drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of dimethyl fumarate targeting GSDME in the preparation of anti-aging drugs. Background Technology

[0002] Aging refers to the progressive decline in the body's physiological and psychological adaptability to the environment, gradually leading to death. It is a complex and multi-dimensional natural process. Aging-inducing factors include mitotic signaling disorders, DNA damage and gene mutations, telomere damage, protein toxicity stress (such as protein aggregation and unfolded proteins), increased metabolites (such as reactive oxygen species, ceramides, fatty acids, and high glucose), and increased nutrient signals (such as mTOR).

[0003] Aging is associated with several conserved mechanisms, such as nutrient pathways, mitochondrial dysfunction, metabolic pathways, energy homeostasis, DNA repair, and autophagy. Cells can, in turn, drive aging-related responses to damage: stem cell exhaustion and chronic inflammation. Inflammation is also one of the major exogenous influences on aging cells; in particular, chronic low-level inflammation is a serious and complex factor in many diseases, and its risk increases with age. The aging process is irreversible, but it can be slowed down. Recent studies have found that Gasdermin family proteins-mediated pyroptosis plays a crucial role in the release of inflammatory factors. However, the activation and regulatory mechanisms of gasdermin E (GSDME) in the aging lung environment remain unknown, and the specific role of GSDME in tissue aging has not been reported. Therefore, studying the regulatory matrix of GSDME in tissue aging, and the inhibitors targeting GSDME, holds promise for providing new strategies for the development of anti-aging drugs. Summary of the Invention

[0004] In view of this, the present invention proposes the application of dimethyl fumarate targeting GSDME in the preparation of drugs for treating aging.

[0005] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides the application of dimethyl fumarate targeting GSDME in the preparation of anti-aging drugs.

[0006] The structural formula of dimethyl fumarate is as follows:

[0007]

[0008] Based on the above technical solutions, preferably, the dimethyl fumarate alleviates the aging process by inhibiting the activity of GSDME.

[0009] Based on the above technical solutions, preferably, the aging refers to organ aging and aging-related inflammation.

[0010] On the other hand, the present invention also provides an anti-aging drug comprising dimethyl fumarate targeting GSDME.

[0011] Based on the above technical solutions, preferably, the drug also includes pharmaceutically acceptable excipients or carriers.

[0012] Based on the above technical solutions, preferably, the drug is an injectable preparation or an oral preparation.

[0013] The application of dimethyl fumarate targeting GSDME in the preparation of anti-aging drugs of the present invention has the following advantages over the prior art: The present invention proposes the application of dimethyl fumarate targeting GSDME in the preparation of anti-aging drugs. Dimethyl fumarate alleviates the aging process by inhibiting the activity of GSDME and can be used to prepare and develop anti-aging drugs. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 A schematic diagram of mouse modeling.

[0016] Figure 2 This is a volcano plot of RNA-seq results.

[0017] Figure 3 In the figure, Figure A shows the expression levels of GSDME, GSDMD, and Caspase-3 proteins in A549 cells and WI-38 cells; Figure B shows the Western blot (WB) detection levels of GSDME and GSDMD activation in young and aged A549 cells; Figure C shows the WB detection levels of GSDME and GSDMD activation in young and aged WI-38 cells; Figure D shows the detection levels of GSDME protein activation in lung tissue of young and 16-month-old aged mice; and Figure E shows the detection levels of GSDME protein activation in MLF cells of young and aged mice.

[0018] Figure 4 The diagram shows how DMF inhibits the activation of GSDME in senescent cells and tissues.

[0019] Figure 5 This is a graph showing the level of fibrosis in the tissues of naturally aging mice.

[0020] Figure 6 This is a graph showing the level of macrophage infiltration in naturally aging mice.

[0021] Figure 7 This is a map showing the level of tissue fibrosis in Dox-induced mice.

[0022] Figure 8 Map showing the level of Dox-induced macrophage infiltration in mice.

[0023] Figure 9 This is a graph showing the measurement of inflammatory factors in the mouse group. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] 1. Experimental Materials

[0026] 1.1 Animal Model

[0027] C57BL / 6 mice (8 weeks old).

[0028] 1.2 Reagents

[0029] Dimethyl fumarate, product number T0492, CAS number 624-49-7, purchased from Taoshu Biochemical.

[0030] Doxorubicin (Dox) is a DNA damaging agent, product number T1456, CAS number 23214-92-8, purchased from Taoshu Biochemical.

[0031] SA-β-Gal staining kit (Beyotime, C0602), ELISA kit (IL-6, IL-1β, IFN-β, purchased from biolegend), mitochondrial isolation kit (Beyotime, C3601 / C3606), and p16, p21, MAVS, IL-1β, and F4 / 80 antibodies were provided by the immunohistochemistry testing service provider.

[0032] 1.3 Experimental Apparatus

[0033]

[0034]

[0035] 2. Experimental Methods and Results

[0036] 2.1 Animal Model

[0037] Dox-induced aging in mice: C57BL / 6 mice received an intraperitoneal injection of Dox (5 mg / kg) on ​​day 0, followed by treatment with dimethyl fumarate (10 mg / kg) on ​​days 3, 9, and 12, and were analyzed on day 21 (see [link to analysis]). Figure 1 ).

[0038] Naturally aging mouse group: C57BL / 6 naturally aging mice were treated with dimethyl fumarate (10 mg / kg injection) at weeks 1, 3, and 5, and analyzed at week 7 (see [link to relevant documentation]). Figure 1 ).

[0039] Example 1: RNA Extraction and Sequencing (RNA-seq)

[0040] The specific steps are as follows:

[0041] (1) Take A545 cells that have been induced to age and extract total RNA using Trizol reagent according to the instructions to ensure that the sample is fully lysed and purified.

[0042] (2) The extracted RNA was evaluated using an Agilent 2100 Bioanalyzer to ensure RNA integrity and purity. Only RNA with a RIN value ≥ 7.0 could be used for subsequent experiments.

[0043] (3) RNA sequencing libraries were constructed using the NEBNext Ultra RNA Library Preparation Kit and library quality control was performed.

[0044] (4) The qualified libraries were subjected to high-throughput sequencing on the Illumina Novaseq 6000 platform. Sequencing data analysis was performed using tools such as Bowtie2, HISAT2, and DESeq2. The results are shown below. Figure 2 .

[0045] Analysis of RNA-seq sequencing results from induced senescent cells revealed that genes related to the pyroptosis pathway, such as IL-1β, IL-6, and CASP1, were generally upregulated in senescent A549 cells. Figure 2 This indicates that pyroptosis is crucial for the secretion of inflammatory factors. Studies have shown that it mainly promotes the secretion of inflammatory factors by activating two types of proteins, GSDME and GSDMD, which are then located on the cell membrane.

[0046] Example 2

[0047] To further demonstrate that GSDME is an anti-aging target, this embodiment performed protein analysis-Western Blot, the specific steps of which are as follows:

[0048] (1) After cell or tissue lysis, boil and perform SDS-PAGE electrophoresis.

[0049] (2) Transfer to PVDF membrane and seal with 5% skim milk.

[0050] (3) Incubate primary antibody (such as p16, p21, MAVS) at 4℃ overnight, and incubate secondary antibody at room temperature for 1 hour.

[0051] (4) Chemiluminescence development (Bio-Rad ChemiDoc system), results are shown in […]. Figure 3 A-3E.

[0052] The expression of GSDME and GSDMD proteins in A549 and WI-38 cells was detected by Western blot. Figure 3 A) The results showed that GSDME was expressed in both cell lines, while GSDMD was normally expressed in A549 cells but weakly expressed in WI-38 cells. Western blot results showed that GSDME activation to produce N-terminal protein was found in senescent A549 and senescent WI-38 cells, while GSDMD protein was not significantly activated. Figure 3 B, 3C). Activation of GSDME protein was also observed in the lung tissue of naturally aging mice and in MLF cells of aging mice. Figure 3 (D, 3E). This indicates that GSDME is a good target for anti-aging.

[0053] Example 3

[0054] GSDME plays a crucial role in the secretion of inflammatory factors. Further research is needed to identify drugs or gene-level therapies targeting this protein to inhibit GSDME activation and reduce inflammatory secretion in the aging environment. Recent studies have found that DMF can target key cysteine ​​residues in the GSDMD protein through succination, forming S-(2-succinyl)-cysteine. In GSDMD-deficient cells, DMF can also inhibit GSDME activation. This study aims to investigate whether DMF can inhibit the activation of GSDME in the aging lung environment, thereby suppressing the secretion of age-related inflammatory factors, alleviating degenerative diseases such as pulmonary fibrosis, and promoting healthy lung tissue aging.

[0055] First, this example was validated in A549 cells, using doxorubicin (Dox) to induce senescence in lung A549 and WI-38 cells. Dox is a cytotoxic anthracycline antibiotic that inhibits DNA topoisomerase I and topoisomerase II, thereby inhibiting DNA replication. Low concentrations are generally used to induce cell senescence. Following previous laboratory procedures, 0.5 μM was used to induce senescence. Dox was prepared using DMSO, and an equal amount of DMSO was added to the control group. The specific steps are as follows:

[0056] 12 μL of the laboratory-prepared Dox stock solution (500 μM) was added to 12 mL of culture medium and mixed thoroughly with a pipette to a final concentration of 0.5 μM. The culture medium was removed from the 12-well plates containing the cells, the cells were washed once with PBS, and freshly prepared culture medium containing Dox was added to each well (final Dox concentration 0.5 μM). After 24 h of Dox treatment, the cells were washed once with PBS and the culture medium was replaced with fresh medium. At this point, the cells gradually entered a cell cycle inhibition state. The cells were cultured continuously for 7 days, with the medium being changed as needed based on the color of the medium. After 7 days, the senescent cell model was established. Subsequently, Western blot was used to detect the GSDME activation of senescent A549 cells after treatment with DMF (10 μM, 24 h). The results are shown below. Figure 4 .

[0057] Western blot results showed ( Figure 4 Senescent A549 cells treated with DMF showed reduced GSDME activation compared to untreated senescent A549 cells.

[0058] Example 4

[0059] This embodiment further explores the role of DMF in naturally aged WT C57BL / 6J mice and Dox-induced aged WT C57BL / 6J mice.

[0060] Naturally aging WT C57BL / 6J mice were treated with intraperitoneal injection of DMF (50 mg / kg) every three days for a total of 3 weeks. Figure 1 In 8-10 week old mice (n=5), an aging model was established by inducing aging with Dox (5 mg / kg), while mice were simultaneously raised to 18 months of age to establish a natural aging model. Subsequently, all different aged mice were treated with dimethyl fumarate (50 mg / kg). Immunohistochemical staining was performed to detect lung tissue lesions, fibrosis levels, and macrophage infiltration levels (see...). Figure 5-8 ), ELISA analysis of IL-1β protein levels in bronchoalveolar lavage fluid (see Figure 9 ).

[0061] 1. H&E staining

[0062] Mouse lung tissue was collected and immediately fixed in 4% paraformaldehyde (PFA) for 24 hours, followed by gradient dehydration (ethanol → xylene) and paraffin embedding. 4μm thick continuous sections were prepared using a microtome, attached to detachable glass slides, stained with hematoxylin and eosin, and inflammation and damage were observed.

[0063] 2. Masson's trichrome staining (for fibrosis assessment)

[0064] Dewaxing and hydration: xylene I / II for 10 min each → gradient ethanol (100% → 95% → 80%) until hydration.

[0065] Staining steps: Hematoxylin staining for 5-10 min → acidic ethanol differentiation → rinsing with running water → blueing → rinsing with running water. Ponceau red and fuchsin staining solution for 5-10 min → phosphomolybdic acid treatment for 1-2 min → aniline blue staining for 1-2 min.

[0066] Dehydration and mounting: Gradient ethanol dehydration → xylene clearing → neutral resin mounting.

[0067] Results interpretation: The degree of pulmonary fibrosis in mice was assessed by microscopic observation. Blue represents collagen fibers, red represents muscle fibers / cytoplasm, and dark blue represents cell nuclei.

[0068] 3. Immunohistochemical (IHC) staining

[0069] Slicing: Slice to a thickness of 3-5 micrometers, spread in water at 42℃, and bake in an oven at 60℃ for 30 minutes.

[0070] Dewaxing to water: xylene I for 5 minutes, xylene II for 5 minutes, xylene III for 5 minutes, anhydrous ethanol for 1 minute, 95% ethanol for 1 minute, 75% ethanol for 1 minute, and distilled water for 5 minutes.

[0071] Antigen retrieval: Microwave retrieval with EDTA buffer (pH 9.0) for 5-8 minutes, then cool to room temperature.

[0072] Inactivation: Draw circles with an immunohistochemical pen to prevent reagent leakage, add endogenous peroxidase blocking solution (3% H2O2), incubate at room temperature for 10 minutes, wash 3 times with PBS buffer, 5 minutes each time.

[0073] Blocking: Add blocking serum and incubate at 37°C for 30 minutes. Remove excess serum and do not wash.

[0074] Primary antibody incubation: Add primary antibody (F4 / 80, IL-1β) (Abcam ab9722, 1:200 dilution), incubate overnight at 4°C, wash 3 times with PBS buffer for 5 minutes each time.

[0075] Secondary antibody incubation: Add HRP-labeled secondary antibody (Goat Anti-Rabbit IgG), incubate at 37°C for 30 minutes, wash three times with PBS buffer for 5 minutes each time.

[0076] Developing color: DAB developing solution. Prepare DAB: Mix 1 ml of B solution + 1 drop of A solution, add DAB developing solution, and observe under the microscope until the positive color is significantly enhanced and the background is clean, then stop developing color. If the color is faint, repeat the previous step to enhance the color.

[0077] Counterstaining: Add Mayer's hematoxylin for 30 seconds, wash with distilled water, soak in blue solution for 1 minute, and wash with water.

[0078] Dehydrated clear mounting: 75%-95%-100% alcohol gradient dehydration, 1 minute per tank, xylene clearing in three tanks, 2 minutes per tank, neutral resin mounting.

[0079] Image analysis: Microscopic (×200 / 400) images, ImageJ quantitative analysis of the percentage of positive areas.

[0080] 4. ELISA analysis of IL-1β protein levels in bronchoalveolar lavage fluid

[0081] Bronchoalveolar lavage fluid (BALF) collection: On day 7 after the mouse experiment, mice were anesthetized and intubated. Sterile PBS (500 μL each time, 2-3 times) was injected into the lungs, and the lavage fluid was collected. The collected BALF was slowly centrifuged (e.g., 300g, 10 minutes) to remove cell debris, and the supernatant was preserved.

[0082] Protein quantification: Determine protein concentration using the BCA or Bradford method, ensuring the total sample volume is within the range of ELISA detection. Dilute the sample to an appropriate concentration as needed.

[0083] ELISA assay: A commercially available mouse IL-1β-specific ELISA kit was used. The specific method was as follows: Add 100 μL of sample / standard to each well, cover with sealing film, and incubate at 37°C for 2 hours. Wash the plate (automatic plate washer or manual 5 times, washing buffer: 0.01M PBS + 0.05% Tween-20). Add biotin-labeled detection antibody (100 μL per well) and incubate at 37°C for 1 hour. After washing, add HRP-streptavidin (100 μL per well) and incubate in the dark for 20 minutes. For color development: Add TMB substrate (100 μL / well), incubate at room temperature in the dark for 15 minutes, then add stop solution (50 μL / well). Reading: Detect at 450 nm (reference wavelength 570 nm) using a microplate reader. Calculate the concentration using a standard curve. Results are shown below. Figure 9 .

[0084] The results showed that dimethyl fumarate treatment significantly inhibited the expression levels of Dox-induced pneumonia and fibrosis in both senescent and naturally senescent mice, and reduced the levels of inflammatory factors in bronchoalveolar lavage fluid. Histopathological analysis showed ( Figure 5-8 Dimethyl fumarate treatment significantly inhibited Dox-induced tissue damage, reduced fibrosis in various tissues, and suppressed inflammatory cell infiltration in these tissues.

[0085] These findings suggest that dimethyl fumarate is a potential strategy for treating systemic aging. In summary, our results demonstrate that dimethyl fumarate effectively targets GSDME in aging tissues, significantly inhibiting various age-related inflammatory responses and tissue degeneration, showcasing its promising therapeutic efficacy and application potential.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of dimethyl fumarate targeting GSDME in the preparation of anti-aging drugs.

2. The application as described in claim 1, characterized in that: The dimethyl fumarate described herein slows down the aging process by inhibiting the activity of GSDME.

3. The application as described in claim 1, characterized in that: The aging referred to here includes organ aging and age-related inflammation.

4. An anti-aging drug, characterized in that: The anti-aging drug includes dimethyl fumarate, which targets GSDME.

5. The anti-aging drug as described in claim 4, characterized in that: The drug also includes pharmaceutically acceptable excipients or carriers.

6. The anti-aging drug as described in claim 4, characterized in that: The drug is an injectable or oral formulation.

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