Application of 11,12-EET in the preparation of drugs for promoting tissue repair and / or regeneration

Abstract

The present invention belongs to the technical field of drug preparation and relates to the application of 11,12-EET in the preparation of drugs that promote tissue repair and / or regeneration. The present invention provides the application of 11,12-EET in the preparation of drugs that promote tissue repair and / or regeneration. By adding conventional commercially available 11,12-EET or providing 11,12-EET that depends on the expression and secretion of Gsdmd or using reagents that can promote the accumulation of 11,12-EET, tissue repair and / or tissue regeneration can be promoted. The present invention has discovered the application of GSDMD in providing a microenvironment that promotes regeneration for tissue repair. GSDMD-mediated metabolic communication provides a microenvironment that promotes regeneration for tissue repair. Specifically, GSDMD-mediated secretion of 11,12-EET can promote the repair and / or regeneration of damaged or aging tissues such as muscle, cornea and skin.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug preparation, and specifically relates to the application of 11,12-EET in the preparation of drugs for promoting tissue repair and / or regeneration. Background Art

[0002] In multicellular organisms, the regeneration process depends on the coordinated interaction of various cell types within the injury microenvironment, among which macrophages play a central role. During tissue regeneration, macrophages have highly dynamic functions, including intercellular communication with stromal cells and other immune cells to regulate damage repair. This complex intercellular communication includes not only traditional ligand-receptor interactions, but also small molecule metabolite communication, which acts as information molecules to diffuse in the injury microenvironment and play a regulatory role. Although there have been preliminary studies on macrophage-mediated metabolite communication, there are few studies on how these metabolic signals are actively released into the injury microenvironment and their potential functions on neighboring cells.

[0003] Inflammation is an evolutionarily conserved process that protects the body from invasion by maintaining homeostasis and preserving the functional and structural integrity of tissues and organs. GSDMD plays a crucial role in defending against pathogen invasion and inflammatory diseases. Upon activation, GSDMD perforates membrane pores and triggers pyroptosis, which in turn promotes the release of inflammatory factors, ultimately triggering a diverse cascade of downstream inflammatory responses. However, in addition to its classic role in cell death, GSDMD also possesses non-pyroptotic functions that regulate cell homeostasis in various contexts, but its impact on tissue regeneration remains unclear. Under specific circumstances, such as in the "hyperactivated" state, GSDMD activation in macrophages does not necessarily lead to cell death. Rather, normal cellular function is preserved while pores are formed in the cell membrane. Therefore, cells in this state can actively secrete small molecules to regulate their microenvironmental niche. Although some small proteins, such as lectins, have been reported to be released through GSDMD pores, the secretory metabolome profile of these "hyperactivated" cells and their physiological and pathological significance have not been studied. Summary of the Invention

[0004] The present invention aims to provide the use of 11,12-EET in the preparation of a drug for promoting tissue repair and / or regeneration. The addition or accumulation of 11,12-EET can promote tissue repair and / or tissue regeneration.

[0005] The present invention provides use of 11,12-EET or an agent promoting the accumulation of 11,12-EET in the preparation of a drug for promoting tissue repair and / or regeneration.

[0006] Preferably, the tissue comprises damaged and / or aged tissue.

[0007] The present invention also provides the use of 11,12-EET or an agent that promotes the accumulation of 11,12-EET in the preparation of a method for promoting muscle tissue repair and / or regeneration.

[0008] The present invention also provides the use of 11,12-EET or an agent that promotes the accumulation of 11,12-EET in the preparation of a drug having any one or more of the effects 1) to 10);

[0009] 1) Increase muscle fiber area;

[0010] 2) Increase muscle mass;

[0011] 3) Increase muscle strength;

[0012] 4) Accelerate the resolution of muscle inflammation;

[0013] 5) Promote the expression of myogenesis-related genes;

[0014] 6) Promote the activation and / or proliferation of muscle stem cells;

[0015] 7) Improve the myogenic differentiation ability of muscle stem cells;

[0016] 8) Regulates liquid-liquid phase separation of fibroblast growth factors;

[0017] 9) Amplify FGF-FGFR signaling;

[0018] 10) Accelerate signal transduction of MAPK and PI3K-AKT-mTOR signaling pathways.

[0019] The present invention also provides the use of 11,12-EET or an agent that promotes the accumulation of 11,12-EET in the preparation of a drug that promotes corneal regeneration and / or prevents corneal fibrosis.

[0020] The present invention also provides the use of 11,12-EET or an agent that promotes the accumulation of 11,12-EET in the preparation of a drug for promoting the repair of skin damage.

[0021] The present invention also provides the use of 11,12-EET or an agent that promotes the accumulation of 11,12-EET in the preparation of a drug for restoring the vitality of aged muscles and increasing body weight and / or strength.

[0022] Preferably, the agent that promotes the accumulation of 11,12-EET comprises an agent that knocks out soluble epoxide hydrolase.

[0023] The present invention also provides the use of GSDMD in preparing drugs for promoting tissue repair and / or regeneration.

[0024] Preferably, the tissue comprises muscle tissue, cornea and / or skin.

[0025] The present invention provides the use of 11,12-EET in the preparation of a drug for promoting tissue repair and / or regeneration. Tissue repair and / or tissue regeneration can be promoted by adding conventional commercially available 11,12-EET, providing 11,12-EET that is dependent on the expression and secretion of Gsdmd, or using an agent that promotes the accumulation of 11,12-EET. The present invention has discovered the use of GSDMD in providing a microenvironment that promotes regeneration during tissue repair. GSDMD-mediated metabolic communication provides a microenvironment that promotes regeneration for tissue repair. Specifically, GSDMD-mediated secretion of 11,12-EET can promote the repair and / or regeneration of damaged or aged tissues such as muscle, cornea, and skin. Experimental results have shown that 11,12-EET can reduce the area of ​​muscle necrosis; reduce the area of ​​regenerating muscle; increase myofiber area; increase muscle mass; enhance muscle strength; accelerate the resolution of muscle inflammation; promote the expression of myogenic genes (Myod and Myog); promote the activation and / or proliferation of muscle stem cells; enhance the myogenic differentiation capacity of muscle stem cells; regulate the liquid-liquid phase separation of fibroblast growth factor; amplify FGF-FGFR signaling; and accelerate signaling in the MAPK and PI3K-AKT-mTOR pathways, thereby promoting the repair and / or regeneration of muscle tissue. 11,12-EET has also been shown to promote corneal regeneration, preventing corneal fibrosis and other complications; promote the repair of skin damage; and restore vitality to aging muscles. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 The results of tissue repair damaged by GSDMD deficiency in myeloid cells provided by the present invention are shown in Figure ac. In which, CTX was injected intramuscularly to establish an acute muscle injury model in mice; 0, 1, 3, 5, 10 and 14 days after CTX-induced injury, Gsdmd f / f and Gsdmd CKO Representative images of hematoxylin and eosin (H&E)-stained cross-sections of mouse TA (a); Scale bar: 50 μm; Graphs of necrotic area (black dashed line) (b) and percentages of regenerative area (yellow dashed line) at the indicated time points (n=4-6) (c); d, e, at 14 dpi Gsdmd f / f and Gsdmd CKORepresentative cross-sectional images of TA muscles of mice (d), and the corresponding frequency distribution of myofiber cross-sectional area (CSA) (n=4) (e); f, g, expression of early embryonic MyHC (eMyHC) at 7 dpi + Representative images (f) and quantitative images (n = 10) (g) of myofibers; h, Gsdmd at the indicated dpi f / f and Gsdmd CKO In muscles Pax7 、 Myod qRT-PCR analysis of Myog mRNA expression (n=4, 0 dpi; N=8, 1 dpi and 3 dpi; N=3, 6 dpi); ik, 3 days after CTX injection, resting period (n=8, PAX7 + , Ki67 - ) and proliferation phase (n=8, PAX7 + , Ki67 + ) muscle stem cells, and early (n=12, MyoD + nuclear) and late (n=11-12, MyoG + Representative immunofluorescence staining images (i) and quantitative statistical graphs (j, k) of nuclear differentiation markers; white arrows indicate PAX7 + , Ki67 + cells; scale bar, 50 μm; l, immunoblot of GSDMD in muscle extracts; shown are representative figures (ae, h) or replicates of at least two independent experiments (f, g, ik); (b, c, g, h, j, k) using unpaired two-tailed t-test; (e) using multiple t-test, mean ± SEM; abbreviation: dpi, days postinjury;

[0028] Figure 2 The GSDMD expressed by myeloid cells provided by the present invention is crucial for the function of MuSC in the regeneration process. Gsdmd f / f and Gsdmd CKO Single-cell sequencing of total muscle cells in mice; UMAP map of overall cell clustering (left) and changes in cell proportions during regeneration (right); bd, using Pax7 、 Myod 、 Mki67 and Myog MuSC subpopulations defined by equal markers; UMAP plots and dot plots of relative marker expression for six consecutive MuSC states (b); Trajectory analysis of the MuSC lineage using Monocle3 (c, left); Gsdmd f / f and GsdmdCKO Relative density of mouse MuSCs (c, right ） ; Red arrow indicates Gsdmd CKO Disturbance of proliferation lineages and terminal differentiation in mice Myog high The reduction of MuSCs; AddModuleScore Dot plots of the enrichment scores of the indicated gene sets calculated by function (d); e, f, visualization-based enrichment analysis to evaluate the functional status of each type of immune cell; FC dot plots showing the dynamics of each type of immune cell during tissue repair (e); dot size indicates the size of the cell population in the total cells; dot plots indicate gene set enrichment scores in highly dynamic immune cells (e), including Spp1 high Macrophages, Mrc1 high Macrophages and Ccr2 high Monocytes (f); g, Trajectory analysis of the monocyte-macrophage lineage using Monocle3; Gsdmd f / f and Gsdmd CKO Relative density map of mouse monocyte-macrophage lineage; h, Gsdmd f / f and Gsdmd CKO Dot plots of top-ranked ligand expression in mouse immune cells; i, j, OLINK analysis of muscle TIF protein levels; Gsdmd f / f and Gsdmd CKO PCA plot of each sample during mouse regeneration (i); boxplot of intramuscular IL-1b protein levels (j); (j) unpaired two-tailed t-test; WT and KO represent Gsdmd f / f and Gsdmd CKO mice;

[0029] Figure 3 The results of the GSDMD-dependent metabolite release promoting tissue regeneration provided by the present invention are shown in FIG; wherein a, b, C2C12 cells are respectively GsdmdCo-culture of <3kD and >3kD fractions of supernatants from hyperactivated macrophages of wild-type (WT) and knockout (KO) mice; representative images (a) and quantitative plots (b) of the size and fusion index of C2C12 myofibers after 3 days of culture in differentiation medium; c, d, Schematic diagram of the unbiased non-targeted metabolite screening strategy (c); FC-FC plots of metabolite levels in supernatants of macrophages after corresponding treatments (d); LN, LPS + nigericin; LP, LPS + poly (dA:dT); e, f, Gsdmd WT and Gsdmd KO Targeted oxylipin metabolomics of supernatants and lysates of highly activated mouse macrophages; PCA plot of metabolites in cell lysates and supernatants (e); Gsdmd WT and Gsdmd KO Heat map of metabolite levels in macrophage supernatant (f); g, h, Heat map of non-targeted metabolomics of muscle TIF at different time points after CTX-induced injury (g); Three metabolite types were identified based on k-means clustering; KEGG enrichment analysis of early secretory metabolites (h); i, Venn diagram showing significantly enriched metabolites in each experiment; j, Primary ex vivo MuSCs were cultured in proliferation medium for 4 days and then in differentiation medium for 3 days, and then supplemented with 11,12-EET or vehicle; Scale bar, 200 μm; kp, Flow cytometry analysis of the proliferation capacity of MuSCs under the indicated treatments; 11,12-EET or vehicle (km) or Gsdmd WT and Gsdmd KO Representative flow cytometric contour plots (k, n) and quantification (l, m, o, p) of Ki67+ levels in isolated MuSCs and non-MuSCs cultured for 2 days (n = 8) using supernatants collected from macrophages (np); Figures (a, b, j) or pools (k, p) from at least two independent experiments are shown; unpaired two-tailed t-test applied to (b); paired two-tailed t-test applied to (l, m, o, p); mean ± SEM.

[0030] Figure 4 The accumulation of 11,12-EET in vivo provided by the present invention promotes tissue regeneration results; wherein, a, b, 14dpi Ephx2 f / f and Ephx2 CKO Representative H&E staining images of mouse TA muscle cross-sections (a); scale bar, 50 μm; and corresponding percentages of necrotic area (black dashed line) and regeneration area (yellow dashed line) (n=5) (b); c, d, at 14 dpi Ephx2f / f and Ephx2 CKO Representative cross-sectional images of TA muscles of mice (c) and frequency distribution analysis of myofiber CSA (n = 4) (d); e, at the indicated dpi Ephx2 f / f and Ephx2 CKO In muscles Pax7 mRNA expression level graph (n=4); f, Representative microscopic images and quantitative graphs of eMyHC+ myofibers at 6 dpi (n=6); g, h, 3 d after CTX injection, resting period (n=6, PAX7 + Ki67 - ), proliferation phase (n=6, PAX7 + Ki67 + ), early stage (n=10, MyoD + nuclear) and late (n=10, MyoG + Representative immunofluorescence staining (g) and quantitative images (h) of MuSCs differentiation markers; white arrows, PAX7 + Ki67 + MuSCs; scale bar, 50 μm; im, CTX-induced Gsdmd CKO Ephx2 CKO Schematic diagram of acute muscle injury in mice and littermate control mice (i); representative immunostaining of TA muscle cross-sections at 14 dpi (j); mass of dissected TA muscles (n = 8); (k) frequency (l) and mean (m) of myofiber CSA distribution (n = 7); n, o, images of CTX-injected DSF-treated mice (n) or control mice (n) at 14 dpi Ephx2 CKOf / f Schematic diagram of Ephx2 and Ephx2 mice and mean cross-sectional area of ​​corresponding treatments (DMSO n = 4, DSF n = 6) (o); representative graphs (af, jo) or aggregated graphs (g, h) of at least two independent experiments are shown; unpaired two-tailed t-test was applied to (b, c, e, f, h, k, m); multivariate t-test was applied to (d, l); (o) Two-way ANOVA test was used, mean ± SEM; abbreviation: DSF, disulfiram;

[0031] Figure 5The results of the present invention provide that 11,12-EET promotes MuSC proliferation by enhancing FGF-FGFR signaling; wherein, a, b, BulkRNAseq analysis of MuSCs treated with 11,12-EET or vehicle for 2 days; Volcano plot of differentially expressed genes after 11,12-EET treatment (a); GSEA enrichment plot of MuSCs treated with 11,12-EET or vehicle (b); c, at a specified time with or without 11,12-EET treatment, from TA muscle Immunoblots of the PI3K-AKT-mTOR and MAPK signaling pathways downstream of FGF-FGFR in MuSCs (c) and NIH-3T3 cells (d) isolated from meat; e, GO enrichment analysis of the molecular function database using the top 300 genes associated with MuSCFGF signaling; fi, representative images (f) and dimensions (g) of eGFP-bFGF phase separation, mixed with increasing PEG concentrations of vehicle or 11,12-EET, and subjected to 340 nm ( h) Absorbance measurement of solution turbidity; Scale bar, 5 μm; Representative FRAP curves of eGFP-bFGF droplets (i); jl, Representative images (j), density (k) and corresponding FRAP curves (l) of cell surface FGF condensates; Scale bar, 10 μm; m, Immunoblotting results of eGFP-bFGF oligomerization levels in 3T3 cells at different time points after FGF treatment with or without 11,12-EET; nr, Evaluation of the in vivo effects of 11,12-EET on CTX-injured mice; Schematic diagram of 11,12-EET treatment and scRNAseq design (n); UMAP images of 6 consecutive MuSC states and changes in cell proportions after 11,12-EET treatment (o); Trajectory analysis of the MuSC lineage using Monocle3; Relative density of MuSCs under 11,12-EET treatment and control treatment (p); Red arrows indicate the changes in the number of MuSCs after 11,12-EET treatment. Myog high MuSCs have more proliferation lineages and a higher degree of terminal differentiation; AddModuleScore Enrichment score plots of corresponding gene sets calculated by the function (q); Correlation plots of FGF binding capacity and P38MAPK cascade levels of MuSCs with corresponding treatments (r); Figures show one representative (c, d, f, m, j) or one set (g, h, i, k, l) of at least two independent experiments; Unpaired two-tailed t-test applied to (g, h, k); Two-way analysis of variance (ANOVA) test for (i, l); Pearson correlation analysis (r). Mean ± SEM;

[0032] Figure 6The 11,12-EET provided by the present invention has the ability to promote tissue regeneration in various repair models; among them, af, TA muscle weight of 11,12-EET-treated or control (contralateral muscle) at 14 dpi (n=10) (a); muscle strength (n=3-4) (b), muscle fiber CSA (n=4) (c) and necrotic area (n=8) (d) of TA muscle after CTX injection with or without 11,12-EET treatment; representative images (e) and quantitative images (n=4) (f) of eMyHC+ muscle fibers; gh, schematic diagram of BAC-induced corneal injury model (g, left); representative images of cornea and corneal fluorescein staining before and after treatment (g, right); statistical data of axial length (h, left) and tear film breakup time (h, right) in the 15-s Schirmer test on the third day of corresponding treatment; i, UV-induced skin damage model (lower left); with or without 11,12-EET treatment. Images of the back of the ear of T-treated mice and time-dependent ear thickness statistics (n = 10); j, k, Immunoblots of EPHX2 expression (j) and associated 11,12-EET levels (k) in young and old muscle; ln, Schematic diagram of EET or vehicle supplementation in muscle of aged mice (left 1); Representative cross-sections of TA muscles (l, right) and frequency distribution analysis of myofiber CSA (m) of EET or vehicle-treated aged mice (n = 8); Representative images and quantification of Pax7 immunofluorescence staining (n); White arrows indicate Pax7 MuSCs; Scale bar, 100 μm; Representative images (right panels, bf, gh, jo) or pooled images (left panel, i) of at least two independent experiments are shown; Unpaired two-tailed t-test applied to (a, right panel, d, f, k, n); Paired two-tailed t-test applied to (left panel, h); Multivariate t-test applied to (c, m); Two-way ANOVA applied to (i); Mean ± SEM;

[0033] Figure 7 The results of tissue repair in myeloid GSDMD-deficient patients provided by the present invention are shown in Figure 1; a is a schematic diagram of the coupling of tissue repair process and inflammatory response; b is the average CSA, and Figure 1 Related to d in (n = 4); c, Schematic diagram of the MuSC myogenic gene program during regeneration; dg, F4 / 80 + Representative images and quantification of (d, e) (n=3, 0 dpi; n=4, 1-10 dpi) and CD31+ (f, g) (n=3, 1 dpi; n=4, 3-7 dpi) staining of injured TA muscles; the figures are representative results of at least two independent experiments; (b, e) unpaired two-tailed t-test was used; mean ± SEM;

[0034] Figure 8 The present invention provides Gsdmdf / f and Gsdmd CKO Results of mouse muscle single-cell data analysis; a, b, heat map (a) and feature map (b) show the expression of characteristic markers of each cell type; c, d, UMAP maps of cell population dynamics during regeneration; e.g., CytoTRACE score maps of various states of MuSCs (e, f); AddModuleScore Dot plot of calculated enrichment scores for the indicated gene sets and Figure 2 d(g) correlation in ;

[0035] Figure 9 The present invention provides Gsdmd f / f and Gsdmd CKO Results of single-cell data analysis of mouse intramuscular immune components; ac, Gsdmd f / f and Gsdmd CKO Analysis of intramuscular immune components in mice; Gsdmd f / f and Gsdmd CKO Dynamic changes in the percentage of each cell type in muscle (a); stacked violin plots of characteristic marker genes for each type of immune cell (b) and Gsdmd f / f and Gsdmd CKO Dynamic changes in the percentage of immune cells in muscle (c); d, flow cytometric analysis of myeloid cell composition at 2 dpi (n=8); g, h, cell-cell interaction analysis using NicheNet; expression of the top intramuscular ligands and the top receptors of each type of immune cell at 2 dpi (g) and (h), respectively. Figure 2 h correlation in [ Time Frame: 1 ]; The figure shows a representative graph of at least two independent experiments; (d) Unpaired two-tailed t-test was used; mean ± SEM;

[0036] Figure 10 The present invention provides Gsdmd The lack of effect on the muscle microenvironment after injury results in a small result; among them, a, immunoblotting of GSDMD and NINJ1 oligomerization in injured TA muscle at 2dpi (a); b, c, levels of intramuscular inflammatory proteins (b) and regeneration proteins (c) measured by OLINK, and Figure 2 The i in Figure 2j in Figure 5; d, Immunoblot (d) and statistical (e) graphs of HMGB1 secretion in muscle TIFs at 2 dpi; f, dot plots of pyroptosis index and corresponding gene expression graphs of three highly dynamic immune cell types; the figures show representatives of at least two independent experiments; (b, c, e) unpaired two-tailed t-tests were applied; abbreviations: BS3, amine-thiol crosslinker;

[0037] Figure 11 Graphs showing the results of the present invention's GSDMD-dependent metabolite release promoting tissue regeneration; a, schematic diagram of supernatant collection and ultracentrifugation after GSDMD activation by overactivated macrophages; bd, ELISA assays for IL-1β (b), IL-6 (c), and TNFα (d); e, f, LDH release assay for supernatant LDH levels, flow cytometry for PI + Cell results; gj, volcano plots comparing metabolite levels, showing total lysis product release (g), glycine-induced secretion (h), active secretion (i), and GSDMD pore-dependent active release (j), with Figure 3 In the c, Figure 3 The d correlations in (b, f) are shown; the figures show the pool of at least three independent experiments; unpaired two-tailed t-tests were applied to (b, f);

[0038] Figure 12 Figure 1 shows the results of 11,12-EET promoting the activation and proliferation of MuSCs provided by the present invention; wherein, a, schematic diagram of the biosynthesis and hydrolysis of 11,12-EET; b, ELISA diagram of the level of 11,12-EET in the supernatant; c, d, representative images (c) and quantitative graphs (d) of the C2C12 cell fusion index and myofiber diameter before and after 11,12-EET treatment; scale bar, 200 μm; e, schematic diagram of the original MuSC isolation procedure; f, g, representative scanning electron microscopy images (f) and statistical data (g) of MuSCs treated with 11,12-EET (n=30) and control group (n=40); shown are representative images (bd) or pooled images (f, g) of at least two independent experiments; (d, g) unpaired two-tailed t-test was applied; (b) one-way analysis of variance was used; mean ± SEM;

[0039] Figure 13 The accumulation of 11,12-EET in vivo provided by the present invention promotes tissue regeneration results; wherein, a, immunoblotting diagram of EPHX2 expression; b, average CSA (n=5) diagram, and Figure 4 c in the figure; c, after DSF treatment or without treatment, at 14dpi Ephx2 f / f and Ephx2 CKO In muscles Myod andMyog mRNA level graph (n=4); d, e, Ephx2 f / f and Ephx2 CKO Representative images of mouse TA muscle cross-sections (d) and myofiber CSA frequency distribution (n = 5) (e); shown are representative images of at least two independent experiments; unpaired two-tailed t-test applied to (c); multiple t-test applied to (e); mean ± SEM; abbreviation: DSF, disulfiram;

[0040] Figure 14 The results of the present invention show that 11,12-EET promotes MuSC proliferation by enhancing FGF-FGFR signal transduction; a, the correlation between the gene fold changes of 11,12-EET and the control group and the gene fold changes of activated and quiescent MuSCs (from GSE113631); b, according to Figure 2 scRNAseq data in b, expression dot plots of growth factor receptor genes in different types of MuSC cells; c, d, correlation levels of each gene with FGF signaling scores (c) and analysis pipeline results of the top 300 GO-enriched related genes (d), and Figure 5 e in correlation; using the gene set with “RESPONSE_TO_FIBROBLAST_GROWTH_FACTOR” AddModuleScore to determine the FGF signal score result map; ei, heat map of characteristic marker genes of each cell type (e, top), and the dynamic changes of cell populations after injury (e, bottom); UMAP map and the percentage of each cell type after corresponding treatment (f); characteristic map of characteristic marker genes of each type of cell (g); characteristic marker gene dot map of each state of MuSCs (h) and trajectory analysis result of MuSCs using Monocle3 (i), and Figure 5 o- Figure 5 q-related in;

[0041] Figure 15 The results of 11,12-EET promoting muscle regeneration provided by the present invention; a, average CSA results (n=4), and Figure 6 Related to b in [ 001 ]. b, Representative H&E staining of TA muscle sections with or without 11,12-EET addition and the percentage of regenerated area at 14 dpi ( n = 4 ). c, d, F4 / 80 immunofluorescence ( c ) and statistics ( d ) at 10 dpi ( n = 7 ). The figures show the representative data of at least two independent experiments. Unpaired two-tailed t-test was applied ( a , b , d ); Mean ± SEM.

[0042] Figure 16The results of the 11,12-EET provided by the present invention have the ability to promote regeneration of multiple organs; wherein, ae, representative H&E staining (a), epithelial and anterior stromal layer thickness statistics (b), and inflammatory infiltration (c) results of the cornea treated with 11,12-EET or vehicle (n=8); d, e, F4 / 80 staining (d) and quantitative (n=8) (e) results; f, representative H&E staining results of the ear 14 days after ultraviolet irradiation; g, micrograph and quantitative Mersenne trichrome staining method Figure 3. Collagen deposition in TA muscle of aged mice (n=8). h. Quantification of muscle weight (n=8) and strength (n=4) in vehicle-treated and 11,12-EET-treated aged mice. j, k. Representative cross-sections of TA muscle stained with H&E (j) and quantification of myofiber CSA (n=8) (k). Representative images of at least two independent experiments are shown. (b, c, e, g, h, k) Unpaired two-tailed t-test was used. Mean ± SEM.

[0043] Figure 17 Figure 3 is a graph showing the results of the 11,12-EET repair-promoting effect in the skin injury model of punch biopsy provided by the present invention, wherein a is a schematic diagram of the punch biopsy-induced mouse skin injury model, b is an image of mouse skin treated with 11,12-EET or a control solvent after a punch biopsy, c is a statistical analysis graph of the degree of wound healing in mice during the repair process, and d is the immunofluorescence staining results and statistical graph of the proliferation of mouse skin stem cells treated with 11,12-EET or a control solvent after a punch biopsy. DETAILED DESCRIPTION

[0044] The present invention provides the use of 11,12-EET or an agent that promotes the accumulation of 11,12-EET in the preparation of a drug that promotes tissue repair and / or regeneration. Gsdmd The expression and secretion of 11,12-EET, or agents that promote 11,12-EET accumulation, can promote tissue repair and / or tissue regeneration. In the present invention, the tissue preferably includes damaged and / or aged tissue. More preferably, the tissue includes damaged muscle, skin, or corneal tissue; or aged muscle tissue. 11,12-EET can amplify stem cell proliferation signals during self-limited injury repair (sterile inflammation).

[0045] The present invention also provides the use of 11,12-EET or an agent that promotes the accumulation of 11,12-EET in the preparation of a method for promoting muscle tissue repair and / or regeneration.

[0046] The present invention also provides the use of 11,12-EET or an agent that promotes the accumulation of 11,12-EET in the preparation of a drug having any one or more of the effects 1) to 10);

[0047] 1) Increase muscle fiber area;

[0048] 2) Increase muscle mass;

[0049] 3) Increase muscle strength;

[0050] 4) Accelerate the resolution of muscle inflammation;

[0051] 5) Promote the expression of myogenesis-related genes;

[0052] 6) Promote the activation and / or proliferation of muscle stem cells;

[0053] 7) Improve the myogenic differentiation ability of muscle stem cells;

[0054] 8) Regulates liquid-liquid phase separation of fibroblast growth factors;

[0055] 9) Amplify FGF-FGFR signaling;

[0056] 10) Accelerate signal transduction of MAPK and PI3K-AKT-mTOR signaling pathways.

[0057] In the present invention, the myogenesis-related genes preferably include Myod and Myog.

[0058] The present invention also provides the use of 11,12-EET or an agent that promotes the accumulation of 11,12-EET in the preparation of a drug that promotes corneal regeneration and / or prevents corneal fibrosis.

[0059] The present invention also provides the use of 11,12-EET or an agent that promotes the accumulation of 11,12-EET in the preparation of a drug for promoting the repair of skin damage.

[0060] The present invention also provides the use of 11,12-EET or an agent that promotes the accumulation of 11,12-EET in the preparation of a drug for restoring the vitality of aged muscles and increasing body weight and / or strength.

[0061] In the present invention, the reagent that promotes the accumulation of 11,12-EET includes a reagent that knocks out soluble epoxide hydrolase.

[0062] The present invention also provides the use of GSDMD in the preparation of a drug for promoting tissue repair and / or regeneration. In the present invention, the tissue preferably includes muscle tissue, cornea and / or skin.

[0063] The establishment of a pro-regenerative microenvironment in the early stage after tissue damage is crucial for tissue repair. It is known that the release of inflammatory factors depends on pyroptosis mediated by the GSDMD (Gasdermin D) protein, but the role of the GSDMD protein in tissue regeneration and maintenance of homeostasis is still poorly understood. The present invention found that knockout of macrophage GSDMD slowed down the process of tissue repair, but had little effect on the local inflammatory environment or pyroptosis process. By detecting the secretory metabolome of macrophages in a "superactivated" state, the present invention revealed a new function of GSDMD's non-classical metabolite secretion. Further studies found that 11,12-epoxyeicosatrienoic acid (11,12-EET, 11,12-epoxyeicosatrienoic acid) is an oxidized lipid with pro-repair activity secreted by "superactivated" macrophages in a GSDMD-dependent manner. By directly supplementing 11,12-EET or knocking out its hydrolase Ephx2 Elevated 11,12-EET levels accelerate muscle regeneration. Further research revealed that EPHX2 accumulation in aging muscle leads to a decrease in intramuscular 11,12-EET levels, and that 11,12-EET replenishment can restore the "juvenation" of aging muscle. Mechanistically, 11,12-EET regulates the liquid-liquid phase separation of fibroblast growth factor (FGF), thereby amplifying FGF-FGFR signaling and ultimately promoting the activation and proliferation of muscle stem cells (MuSCs). These findings elucidate a novel GSDMD-mediated metabolite communication between macrophages and MuSCs, providing new scientific basis for the treatment of damaged and aging tissue regeneration.

[0064] To further illustrate the present invention, the application of 11,12-EET provided by the present invention in the preparation of drugs for promoting tissue repair and / or regeneration is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0065] Example 1

[0066] Experimental materials and methods

[0067] Experimental Materials

[0068] experimental mice

[0069] Table 1 Experimental mouse information

[0070]

[0071] Lyz2-cre Mice and Gsdmd-flox Crossbreeding mice to obtain myeloid cell-specific knockoutGsdmd of Lyz2- cre-Gsdmd f / f Mouse, hereinafter referred to as Gsdmd cKO mice; Lyz2-cre Mice and Ephx2-flox Crossbreeding mice to obtain myeloid cell-specific knockout Ephx2 of Lyz2-cre- Ephx2 f / f Mouse, hereinafter referred to as Ephx2 cKO Mice. Knockout mice were used after genotyping by PCR. Gsdmd cKO Gsdmd mice and controls WT Mice were used for in vitro macrophage experiments. Mice were maintained in a specific pathogen-free facility at the Laboratory Animal Center of Zhejiang University School of Medicine in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Institutional Review Board of Zhejiang University School of Medicine (ZJU20230356), and the experimental procedures adhered to regulatory standards. During the modeling process, mice were monitored by a dedicated person, with temperature monitoring, respiratory status observation, and anesthesia status observed to prevent adverse reactions and harm. Mice of all ages and sexes were used throughout the study; sex- and age-matched controls were used in all experiments.

[0072] Experimental cells

[0073] Table 2 Experimental cell information

[0074]

[0075] Unless otherwise stated, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated FBS and 1% penicillin-streptomycin in a 37°C cell culture incubator.

[0076] Experimental antibodies

[0077] Table 3 Experimental antibody information

[0078]

[0079] Experimental methods

[0080] Cell culture and stimulation

[0081] In situ isolation of mouse muscle stem cells (MuSCs): After mice were sacrificed, hind limb muscles were dissected and minced using sterile surgical instruments in a cleanroom, and then placed on ice. Muscle fragments were then transferred to 10 mL of wash medium (WM) containing 800 U / mL type II collagenase (Gibco) in Han's F-10, 10% v / v horse serum, and 1% penicillin-streptomycin. Digestion was performed at 37°C at 100 rpm in a shaker for 1 hour. Digestion was stopped by placing the fragments at 4°C and adding 40 mL of cold WM. The cells were then centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was removed. The pellet was resuspended in 10 mL of fresh WM containing 100 U / mL type II collagenase and 1 U / mL type II dispase (Yeasen Biotechnology) and digested again at 37°C at 100 rpm for 30 minutes. Digestion was then stopped as described above. Isolated cells were sorted using a satellite cell isolation kit (Miltenyi Biotec, 130-104-268) according to the manufacturer's instructions. Non-satellite cell fractions were collected as controls when needed. Isolated satellite cells were plated on ECM gel (Sigma, E1270) containing 0.1 mg / mL of growth factor-containing medium (Hans F-10 with 20% FBS, 1% penicillin-streptomycin, and 2.5 ng / mL bFGF (Gibco, PHG0024)) or cultured in differentiation medium (DMEM with 5% horse serum and 1% penicillin-streptomycin) for a period of time before subsequent experimental analysis.

[0082] Mouse peritoneal macrophage collection: In the control Gsdmd WT Mice and Gsdmd cKOOn the fourth day after intraperitoneal injection of thioacetate medium (Merck, Fluid Thioglycollate medium), mice were sacrificed and collected by multiple peritoneal lavages using a sterile syringe in a clean bench with mouse peritoneal lavage fluid (prepared by taking a certain amount of PBS buffer prepared in (1) and adding 5% FBS (v / v), 2mM EDTA (pH 8.0), and 1% penicillin-streptomycin according to the corresponding content). The cells were cultured in DMEM containing 10% heat-inactivated FBS and 1% penicillin-streptomycin. To induce a "superactivated" state in vitro, macrophages were initially treated and stimulated with 500 ng / mL LPS (Sigma, L2630) in Opti-MEM (Gibco) for 4 hours. 10 mM glycine (Sigma) was added 30 minutes before the addition of the second signal. Secondary signals were then added to induce GSDMD activation by adding 2.5 mM ATP or 10 μM nigericin for 30 minutes, or by transfection with 1 μg / ml poly(dA:dT) for 1 hour using Lipofectamine 2000 (3 ml / mg DNA) according to the manufacturer's instructions (Invitrogen). Nigericin and poly(dA:dT) were obtained from Invitrogen. Supernatants and cells were collected for further analysis.

[0083] Mouse C2C12 cells were cultured in vitro with macrophage-conditioned medium. Macrophages pre-stimulated with LPS were stimulated with either ATP or nigericin in the presence of glycine for 30 minutes, or transfected with poly(dA:dT) for 1 hour. Cell supernatants were collected and separated by ultracentrifugation into fractions containing proteins less than 3 kD and greater than 3 kD. These fractions were mixed with the aforementioned growth medium and differentiation medium, respectively, and used to culture C2C12 cells, with the medium changed every two days. After three days of differentiation, cells were washed three times with PBS and fixed with 4% PFA and stained for myosin heavy chain (MYH). The fusion index (defined as the percentage of nuclei within myotubes, with ≥2 nuclei) and myotube size (defined as the average number of nuclei per tube) were calculated.

[0084] Construction of CTX muscle injury model in mice

[0085] To establish a CTX-induced injury model, mice were anesthetized with isoflurane, their legs wiped and disinfected with 75% alcohol, and 50 μL of saline containing 8 μM CTX (Latoxan, L8102) was injected into the tibialis anterior (TA) muscle using an insulin needle. Tibialis anterior muscles were harvested at various time points after injury for further analysis. 11,12-EET (Cayman, No. 50511, 500 ng / TA) was injected intramuscularly and disulfiram (MedChemExpress, HY-B0240, 50 mg / kg) was injected intraperitoneally at various time points after injury. The corresponding solvent was used as a control.

[0086] Mouse grip strength test

[0087] To assess the strength of the mouse TA muscles, a dynamometer (Chatillon, DF3 series digital dynamometer) was used. Briefly, the mouse's forelimbs were restrained with tape to prevent them from being affected by the force of their forelimbs. While the mouse was stable, the dynamometer was placed flat on a horizontal table and secured. The mouse was then placed on the dynamometer grid. Measurements were taken as the mouse's hindlimbs grasped the grid and moved backward in a continuous horizontal motion. Five measurements were repeated for each mouse to calculate the average hindlimb grip strength.

[0088] Mouse muscle interstitial fluid collection

[0089] Mouse muscle interstitial fluid (IMF) was collected using a two-step centrifugation method. Briefly, mice were sacrificed at various time points after injury for the desired experiment. The TA was excised using sterile surgical instruments in a cleanroom. The TA was then placed on a 40 μm sterile mesh, secured in a sterile 50 mL centrifuge tube and placed on ice to prevent evaporation of the ISF. Next, a two-step centrifugation method was performed. First, 100 μL of sterile saline was added to the TA surface. The entire 50 mL tube was centrifuged at 50 g for 5 minutes at 4°C to remove any residual fluid from the TA surface and wet the 40 μm mesh. The tube used in the first centrifugation step was discarded, and the mesh containing the TA was reattached to a new sterile 50 mL centrifuge tube. 50 μL of sterile saline was added to the TA surface. The entire 50 mL tube was centrifuged at 600 g for 10 minutes at 4°C. The resulting fluid, representing the ISF, was collected for further processing and analysis.

[0090] Construction of mouse corneal and skin injury models

[0091] To establish a mouse corneal injury model, 5 μL of eye drops containing 0.2% (w / v) benzalkonium chloride (TCI, B0414) were instilled into the mouse eyes twice daily for 5 consecutive days. The mice were then evenly divided into two groups: one group received 5 μL of eye drops containing 11,12-EET (10 μg / mL), while the other group received the corresponding solvent (normal saline) eye drops twice daily as a control. After three treatment cycles, the efficacy of 11,12-EET on corneal repair was assessed by measuring the Schirmer test (to assess lacrimal gland function), tear breakup time (TBUT) (to assess tear film stability), and corneal sodium fluorescein staining (to assess corneal epithelial cell viability). In the Schirmer test, tear volume was measured over 15 seconds using phenol red-soaked cotton thread (Tianjin Jingming Tech Co., Ltd.) according to the manufacturer's instructions. In the tear film breakup time test, the time from blinking to the first black dot observed under cobalt blue light using a slit lamp (YZ5T, 66 Vision-Tech Co., Ltd.) was recorded in seconds. This represents the interval between blinking and the first tear film breakup. In the corneal sodium fluorescein staining test, the lateral conjunctival sac of each mouse eye was stained with 2 μL of 1% sodium fluorescein (w / v, Jing Ming Tech Co., Ltd.). After corneal fluorescein staining, the corneas were examined under cobalt blue light using a slit lamp. After completion of 11,12-EET or control solvent eye drop treatment, the mice were sacrificed and their corneas were collected for subsequent H&E and immunofluorescence staining.

[0092] To establish a UV-induced skin injury model in mice, mice were anesthetized with isoflurane, their ears were cleaned with a depilatory (Veetcream), and their exposed ears were exposed to a UV lamp (λ = 254 nm, voltage, 8 W) at a distance of 30 cm for 25 minutes. The mice were then divided evenly into two groups. One group received a topical application of 200 ng of 11,12-EET to their ears every three days after model establishment, while the other group received a topical application of the corresponding solvent as a control. The thickness of the mice's ears was measured every three days with a vernier caliper (while under isoflurane anesthesia) and compared to the baseline value. Mice were sacrificed at different time points as needed, and their ears were collected for subsequent H&E and immunofluorescence staining.

[0093] To establish a mouse punch skin injury model, mice were anesthetized with isoflurane, and the hair in the center of the mouse's back was cleaned using a shaver and depilatory agent. The dorsal skin was exposed and disinfected with medical alcohol. A sterile circular biopsy punch was used to perform full-thickness skin excision in the center of the shaved and disinfected back, creating two circular full-thickness skin wounds. Within 24 hours after injury, a dressing was applied to the wound surface to prevent infection. 11,12-EET or a control solvent was applied to the wound once daily for 7 days, and the wound was disinfected before application. The observation endpoint was 8 days after injury, and the wound size during the repair process was recorded. The mice were sacrificed at specific time points after injury, and the mouse skin was collected for subsequent immunofluorescence staining.

[0094] H&E staining and immunofluorescence staining

[0095] Regarding H&E staining: After the mouse TA muscle was dissected, it was divided into two parts of similar volume along the maximum cross-section of the TA. Either part was immersed in 4% PFA and fixed at room temperature for at least 48 hours. The sample was dehydrated using a dehydrator and then embedded in paraffin. The maximum cross-section was used as the section surface, and the paraffin block was sectioned at a thickness of 10 μm using a microtome. The sections were dewaxed in xylene and rehydrated in graded ethanol, then stained with H&E and mounted. The sections were scanned using an Olympus VS200 scanner, and the images were processed and analyzed using Image J software.

[0096] Immunofluorescence staining: After the mouse TA muscle was peeled, it was divided into two parts of similar volume along the largest cross-section of the TA. One of the two parts was immersed in cold 30% w / v sucrose solution and the sample was dehydrated at 4°C for 30 minutes. After the solution on the sample surface was wiped dry, Tissue-Tek ®Specimens were embedded in OCT (Sakura) molds. After OCT embedding, the entire mold containing the embedded specimen was quickly frozen in liquid nitrogen and then stored in a -80°C ultra-low temperature freezer. Before cryosectioning, specimens were transferred to -20°C for 20 minutes. OCT-embedded specimens were sectioned at a thickness of 10 μm using a cryostat at -20°C using the largest cross-section. The resulting sections were flattened and mounted on tissue adhesive slides. Frozen tissue sections were allowed to thaw at room temperature for 5 minutes. 4% PFA was added to the specimens for 10 minutes, followed by three 5-minute washes in PBS on a shaker. After wiping the slides dry, the specimens were circled with a hydrophobic pen to prevent solution diffusion and drying in subsequent steps. 10 μL of permeabilization solution containing 5% donkey serum and 0.2% Triton X-100 was added to the specimens and permeabilized in a humidified chamber for 20 minutes at room temperature. The sections were then washed three 5-minute washes in PBS on a shaker. Slides were then placed in antigen retrieval solution (pH 6.0, Solarbio) in a 95°C waterbath for 20 minutes. After removal from the waterbath, the solution was allowed to cool to room temperature (approximately 40 minutes), and then washed three times with PBS on a shaker for 5 minutes each. Slides were then blocked with blocking solution containing 10% donkey serum for 1 hour at room temperature and treated with mouse Fab anti-mouse IgG (Jackson) for 30 minutes at room temperature. The slides were then washed three times with PBS on a shaker for 5 minutes each. The slides were wiped dry and placed in a humidified chamber at 4°C overnight. The slides were incubated with the following primary antibodies at corresponding dilution ratios according to experimental requirements: PAX7 (DSHB, 1:200), MyoD (Santa Cruz, 1:200), myogenin (DSHB, 1:200), Ki67 (Biolegend, 1:200), embryonic myosin (DSHB, 1:50), F4 / 80 (CST, 1:200), CD31 (Abcam, 1:500), and Laminin (Sigma, 1:500). All primary antibodies were diluted in permeabilization buffer. The next day, the slides were washed three times with PBS solution on a shaker for 5 minutes each time. The slides were then wiped dry and incubated with the following secondary antibodies (Abcam) for 1 hour at room temperature in the dark: anti-rabbit IgG H&L-Alexa Fluor 488, anti-rabbit IgG H&L-Alexa Fluor 555, anti-mouse IgG H&L-Alexa Fluor 555, and anti-rabbit IgG H&L-Alexa Fluor 647. The secondary antibodies were diluted 1:500 in PBS solution containing 5% donkey serum.Slides were washed three times with PBS in the dark on a shaker for 5 minutes each. DAPI (Solarbio, 1:500) was added to the slides to stain cell nuclei for 10 minutes. The slides were then washed once with PBS, wiped dry, and mounted with anti-fluorescence decay mounting medium. Slides were scanned using an Olympus VS200 scanner, and images were processed and analyzed using Image J software.

[0097] bFGF phase separation assay

[0098] First, a His-tagged eGFP-bFGF expression vector (FGF2 (ID: 2247)) was constructed. This expression vector was transformed into an E. coli expression system using genetic engineering techniques. The target protein was then expressed using the inducer IPTG. The E. coli cells were then disrupted to obtain a target protein extract. A nickel-ion affinity chromatography column was used to purify the target protein, leveraging its high affinity for the His tag. Briefly, the protein extract was added to a pre-equilibrated column. After binding, washing, and elution, the His-tagged target protein was separated from other impurities. The purified target protein was collected and diluted to 1 mg / mL in a buffer (8 mM Na2HPO4, 2 mM KH2PO4, 136 mM NaCl, and 2.6 mM KCl, pH 7.2).

[0099] To test in vitro phase separation, a 5 μM eGFP-bFGF protein solution was mixed with varying concentrations of PEG-8000 (0-10% w / v) at room temperature. The mixture was divided into two groups: one containing 11,12-EET and the other containing the solvent (ethanol) as a control. The absorbance at 340 nm was measured using a Nanodrop 2000c (Thermo Fisher Scientific) to reflect the turbidity of the mixture.

[0100] To detect cell surface-bound eGFP-bFGF, healthy NIH-3T3 cells were evenly plated on slides and starved for 8 hours in serum-free medium. A 3 μM eGFP-bFGF protein solution was then added. The cells were then divided into two groups: one group with 11,12-EET and the other with a control solvent (ethanol) as a control. The cells were incubated at 37°C for varying lengths depending on the experimental setup. The cells were washed three times with PBS, fixed with 4% PFA for 10 minutes, stained with DAPI for 10 minutes in the dark, and then mounted. The slides were photographed using a Zeiss LSM 800 laser confocal microscope, and the images were processed and analyzed using Image J software.

[0101] Fluorescence recovery after photobleaching (FRAP)

[0102] For in vitro FRAP detection, the initial processing is the same as that for in vitro phase separation detection in 1.2.7. Images are acquired using a Zeiss LSM 800 microscope equipped with a 100× objective lens. A circular region of interest (ROI) is placed at the center of the droplet. High-energy laser irradiation is used to reduce the fluorescence intensity of the ROI to approximately 40% of the original intensity. The fluorescence signal of the eGFP-bFGF protein mixture is then continuously recorded for a total of 250 seconds.

[0103] For cell surface FRAP analysis, healthy NIH-3T3 cells were evenly plated on glass-bottomed live cell culture dishes and starved for 8 hours in serum-free medium. A 3 μM eGFP-bFGF protein solution was then added. The cells were then divided into two groups: one group with 11,12-EET and the other with the control solvent, ethanol. The cells were incubated at 37°C for a specified period of time. The cells were washed three times with PBS and images were acquired using a Zeiss LSM 800 microscope equipped with a 40× objective. eGFP-positive cells were selected for measurement of eGFP-bFGF fluorescence signal intensity before and after photobleaching. A region of interest (ROI) was placed on the cell membrane. High-intensity laser irradiation was used to reduce the fluorescence intensity of the ROI to approximately 20% of its original intensity. The eGFP-bFGF fluorescence signal in the ROI was then continuously recorded for 250 seconds. Data were processed and analyzed using Zeiss Image software.

[0104] ELISA and LDH assays

[0105] Macrophage supernatants were collected according to experimental requirements. ELISA assays were performed using the corresponding kits according to the manufacturer's instructions to measure IL-1β, IL-6, TNF-α, and 11,12-EET in the supernatants. LDH levels were measured using an LDH release kit (Promega) according to the manufacturer's instructions. Results were analyzed using a multi-function microplate reader.

[0106] Immunoblot analysis

[0107] Proteins from cells or tissues were extracted for immunoblot analysis. Briefly, muscle tissue was minced, ground, and lysed in RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, containing a complete protease inhibitor cocktail). The lysis was allowed to stand on ice for 20 minutes to allow for complete lysis. The supernatant was then centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected. An equal volume of 5× SDS loading buffer (156 mM Tris-HCl, 5% SDS, 25% glycerol, 12.5% ​​β-mercaptoethanol, and 0.0125% bromophenol blue) was added and mixed thoroughly. The sample was boiled in a 100°C metal bath for 10 minutes, then loaded onto a 10% or 12% SDS-PAGE gel for electrophoresis and transferred to a nitrocellulose membrane (Pall, 66485) in transfer buffer. The membrane was placed on a shaker at room temperature and blocked for 1 hour (using 1× TBS solution to prepare blocking solution containing 0.05% Tween-20 and 10% nonfat milk powder), and washed three times (using 1× TBS solution to prepare washing solution containing 0.05% Tween-20), each time for 10 minutes. The membranes were then incubated at 4°C overnight and incubated with the following primary antibodies at corresponding dilutions: GSDMD (Abcam, 1:1,000), EPHX2 (Proteintech, 1:2,000), p38 (CST, 1:1000), phospho-p38 (CST, 1:1000), ERK1 / 2 (CST, 1:1000), phospho-ERK1 / 2 (CST, 1:1000), AKT (CST, 1:1000), phospho-AKT (CST, 1:1000), FGFR1 (CST, 1:1000), phospho-FGFR1 (Abclonal, 1:1000), NINJ1 (Santa Cruz Biotechnology Co., Ltd. Cruz, 1:1,000), p70S6K (Abclonal, 1:1,000), phospho-4E-BP1 (Thr37 / 46) (CST, 1:1,000), eIF4EBP1 (Abclonal, 1:1,000), and Vinculin (Abcam, 1:1,000). The next day, the membranes were washed three times for 10 minutes each, followed by incubation with the appropriate HRP-conjugated secondary antibodies (Diagbio) for 1 hour at room temperature. Signals were visualized using ECL reagent (FDbio), and the membranes were detected using a chemiluminescence imaging system (Clinxsci).

[0108] RNA extraction and real-time quantitative PCR

[0109] Mouse muscle tissue was isolated and placed on ice. The tissue was minced and ground in a total RNA extraction reagent (Vazyme, containing a complete protease inhibitor cocktail). The tissue was allowed to stand on ice for 20 minutes to allow for complete lysis. The tissue was then centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected. Chloroform was added and mixed thoroughly to dissolve the RNA and separate it from the aqueous phase. After standing at room temperature for 5 minutes, the tissue was centrifuged at 13,000 rpm for 15 minutes at 4°C. The supernatant was carefully aspirated into a new RNase-free centrifuge tube, an equal volume of isopropanol was added, and the mixture was gently mixed to precipitate the RNA. The tube was then allowed to stand at room temperature for 20 minutes. The supernatant was discarded and the pellet was washed twice with 80% ethanol, centrifuged again for 5 minutes, and the supernatant discarded. The pellet was dried until translucent and dissolved in RNase-free water. DNA was synthesized using a reverse transcription kit (Vazyme) according to the manufacturer's instructions.

[0110] Real-time quantitative PCR was performed using SYBR qPCR Mix on a LightCycler® 480 II (Roche). All samples were normalized to GADPH levels. Primer sequences are shown in Table 6.

[0111] Table 6 qPCR primer sequence list

[0112]

[0113] Flow cytometry

[0114] Using the same digestion method as described in 1.2.1, mouse TA muscle was prepared into a single-cell suspension, washed twice with pre-chilled PBS buffer, and centrifuged at 500 g for 5 minutes at 4°C. The cell pellet was resuspended in an appropriate amount of PBS buffer, and 10 μL of the solution was counted under a microscope. The solution was then diluted to the appropriate cell concentration based on the count results. A staining protocol was set up according to the experimental requirements. The flow cytometric antibody and cell solution were mixed evenly according to the antibody manufacturer's instructions at the appropriate ratio. The cells were stained on ice for 1 hour in the dark. After staining, the cells were washed twice with cold PBS buffer and centrifuged at 500 g for 5 minutes at 4°C. The cell pellet was resuspended and analyzed on a flow cytometer. Cells were analyzed using a NovoCyte (ACEA) flow cytometer, and data were analyzed using FlowJo 10 software. For cell sorting experiments, all procedures were sterile, and all solutions were filtered through a 0.22 μm filter before use. Cells were sorted using an Aria II (BD) flow cytometer and collected in sterile 15 mL centrifuge tubes. Cells were plated onto cell culture plates or stored for further processing, depending on the experimental requirements.

[0115] RNA sequencing library preparation and analysis

[0116] Primary MuSCs were isolated as described in 1.2.1 and cultured for 2 days in medium supplemented with or without 11,12-EET. Total RNA was isolated and purified using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The quantity and purity of each RNA sample were quantified using a NanoDrop ND-1000 (NanoDrop), and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent) (RNA integrity index >7.0) and further confirmed by denaturing agarose gel electrophoresis. Poly(A) RNA was purified from 1 μg of total RNA using Dynabeads Oligo(dT) (25-61005, Thermo Fisher) after two rounds of purification and fragmentation using the Magnesium RNA fragmentation Module (cat. e6150, NEB) at 94°C for 5–7 minutes. The fragmented RNA fragments were reverse transcribed using SuperScript™ II Reverse Transcriptase (cat. 1896649, Invitrogen) to generate cDNA. Next, uracil-labeled second-strand DNA was synthesized using Escherichia coli DNA polymerase I (cat. m0297, NEB), RNase H (cat. m0209, NEB), and dUTP solution (cat. R0133, Thermo Fisher). After PCR amplification, the final cDNA library had an average insert length of 300 ± 50 bp. Finally, 2 × 150 bp paired-end sequencing (PE150) was performed on an Illumina Novaseq™ 6000 according to the manufacturer's instructions. RNA libraries were prepared with the assistance of Hangzhou Lianchuan Biotechnology Co., Ltd.

[0117] In downstream analysis, FastQC Quality control was performed using default parameters. Cutadapt After adjusting the adapters, sequence pairs with length > 30 bp were retained and aligned to the mouse reference genome GRCm38.102 using asterisks. DESeq2 Name the differentially expressed genes. Application ClusterProfiler Perform GSEA enrichment analysis. ggplot2 Create volcano plots and other visualizations.

[0118] Single-cell library preparation and sequencing

[0119] TA muscle tissue was collected from mice of all treatments or genotypes. After washing with ice-cold PBS, the tissue was minced into 2-3 mm fragments and washed twice again with ice-cold PBS. Digestion was performed with digestion buffer [0.5 mg / mL collagenase I (Sigma), 0.5 mg / mL collagenase V (Sigma), 0.5 mg / mL dispase (Worthington)] at 37°C on a shaker at 100 rpm for 10 minutes. Digestion was stopped with an equal volume of cold PBS containing 10% FBS. The cell suspension was passed through a 70-30 μm strainer twice, counted using a cell counter (Countstar), and centrifuged at 400 g for 6 minutes at 12°C. The cells were resuspended in RPMI 1640 (Gibco) on ice. The remaining muscle tissue was digested with 6 ml of 0.25% Trypsin-EDTA at 37°C and 100 rpm for 10 min. The digestion was stopped with an equal volume of cold PBS containing 10% FBS, followed by two washes with PBS and two passes through a 70-30 μm strainer. Two cell fractions were collected and erythrocytes were removed with 4 ml of erythrocyte lysis buffer. The pellet was resuspended in 1× PBS containing 0.04% BSA and centrifuged twice at 300 g for 3 minutes at 4°C. The cell pellet was resuspended in 50 μL of 1× PBS containing 0.04% BSA. The overall cell viability was determined by trypan blue exclusion, with a requirement of >85%. The single-cell suspension was counted using a cell counter, and the final concentration was adjusted to 700-1200 cells / μL. Single-cell suspensions were loaded into 10× Chromium using the 10× Genomics Chromium Single Cell 3′ Kit (V3) according to the manufacturer's instructions, capturing 8,000–10,000 single cells. Subsequent cDNA amplification and library construction steps were performed according to standard protocols. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system at Hangzhou Lianchuan Biotechnology Co., Ltd. with a minimum depth of 20,000 reads per cell.

[0120] Single-cell data analysis

[0121] Sequencing results were decoded and converted to FASTQ format using Illumina bcl2fastq software (v2.20). Sample decoding, barcoding, and single-cell 3′ gene counts were performed using Cell Ranger (version 3.1.0). Single-cell RNA sequencing data were aligned to the Ensembl genome GRCm38 reference genome. These processes were performed with the assistance of Hangzhou Lianchuan Biotechnology Co., Ltd.

[0122] Downstream analysis was performed using the Seurat software package (v4.0.1). Low-quality cells with <200 or >1000 genes detected and cells with >20% mitochondrial-encoded transcripts were excluded from the analysis. The original gene expression matrix after quality control was normalized by total expression, and 2000 variable features were selected to integrate the six samples (WT_CON, WT_Day2, WT_Day10, KO_CON, KO_Day2, and KO_Day10), and then Gsdmd f / f and Gsdmd cKO Comparative analysis between the two groups; 3000 variable features were selected to conduct comparative analysis on 5 samples (WT_CON, Mock_Day3, Mock_Day10, EET_Day3, EET_Day10) after EET treatment. The collection was used FindInintegrationAnchors Function and IntegrateData The present invention performs regression processing on the following confounding factors: cell cycle (by CellCycleScoring Use FindAllMarkers To determine the characteristic marker genes of each cell population, and to identify each cell type based on known markers. Gsdmd f / f and Gsdmd cKO For comparative analysis between the two groups, there are the following cell populations: C01_Mac ( Lyz2 , C1qb ), C02_Mono( Plac8 , Ly6c2 ), C03_DC( H2- Eb1 , H2-Aa ), C04_T( Ccl5 , Trbc2 ), C05_Neu( S100a8 , S100a9 ), C06_Endo( Cdh5 , Fabp4 ), C07_Smooth_Muscle( Myl9 ), C08_Mesenchymal_Pro( Acta2 , Myh11 ), C09_Teno / FAP( Dcn , Col1a1 ), C10_MuSC( Pax7 , Gpx3 ), C11_Mature_SM( Tnnt3 , Tnnc2For comparative analysis after EET treatment, the following cell populations were used: C01_Mono / Mac ( C1qc , C1qb ), C02_DC( H2-Eb1 , H2-Aa ), C03_T( Ccl5 , Trbc2 ), C04_Neu( S100a8 , S100a9 ), C05_Endo( Cdh5 , Fabp4 ), C06_Smooth_Muscle( Myl9 ), C07_Teno / FAP( Dcn , Col1a1 ), C8_MuSC ( Pax7 , Gpx3 ), C9_Mature_SM( Tnnt3 , Tnnc2 ), C10_Peripheral_nerves( Mpz , Kcna1 ).

[0123] For VISION analysis, GO sets were filtered from the mouse native gene set in MsigDB. Vision (v2.1.0) getSignatureScores function to calculate feature scores. Feature autocorrelation was assessed using Geary's C method, and enrichment results were added back to the Seurat data. When measuring highly dynamic immune cell types, the inflammatory index and regenerative index were determined by the VISION enrichment scores of "GOBP_ACUTE_INFLAMMATORY_RESPONSE" and "GOBP_MUSCLE_ORGAN_DEVELOPMENT", respectively. ANOVA analysis of the two indicators over time was performed with P < 1e-30 as the cutoff. Other enrichment analyses were based on AddModuleScore , using a gene set selected from MsigDB. The correlation with FGF signaling is shown by the score between each gene and "GOBP_RESPONSE_TO_FIBROBLAST_GROWTH_FACTOR", and the top 300 genes are ranked ClusterProfiler (v4) GO enrichment. Use ggstatplot (v0.12.0) to plot the correlation levels. For trajectory analysis, CytoTRACE scores were calculated using CytoTRACE (v0.3.3) with default settings. Monocle3 (v1.2.9) for pedigree analysis. Pax7 high Starting with cells, use order_cellsFunctional determination of proliferation and differentiation lineages. For cell-cell interaction analysis, a subset of single-cell RNA sequencing data from the second day (WT_Day2 and KO_Day2) was used. NicheNet All cell types were considered as recipients and deliverers, and the top 15 enriched ligands were calculated.

[0124] OLINK analysis

[0125] After collecting interstitial fluid from mouse muscles, Olink ® Protein content is measured using the Target 96 Mouse Exploratory Panel (Olink Proteomics AB, Uppsala, Sweden). Briefly, proximity-dependent DNA polymerization generates unique PCR target sequences. The resulting DNA sequences are then detected and quantified using a microfluidic real-time PCR instrument (BiomarkHD, Fluidigm). The data are then quality-controlled and normalized to adjust for internal and external run-to-run variables. The final assay readout is expressed as normalized protein expression (NPX) values, which are expressed in relative units on a log2 scale, where high values ​​correspond to high protein expression. All assay validation data are available on the manufacturer's website.

[0126] Broad-target metabolomics

[0127] Interstitial fluid from mouse TA muscle tissue and supernatant from in vitro macrophages were extracted. To extract hydrophilic compounds, samples were thawed on ice, 3 volumes of ice-cold methanol were added to 1 volume of supernatant or TIF, vortexed for 3 minutes, and centrifuged at 12,000 rpm for 10 minutes at 4°C. Centrifugation was repeated, and the supernatant was analyzed by LC-MS / MS. To extract hydrophobic compounds, samples were thawed on ice and vortexed for 10 seconds. Samples were centrifuged at 3,000 rpm for 5 minutes at 4°C. 50 μL of sample was mixed with 1 mL of a mixture (methanol, methyl tert-butyl ether, and internal standard). The mixture was vortexed for 2 minutes, 500 μL of water was added, and vortexed again for 1 minute. The mixture was centrifuged at 12,000 rpm for 10 minutes at 4°C, and 500 μL of the sample was lyophilized. The powder was dissolved in 100 μL of mobile phase B and added to a sample vial for LC-MS / MS analysis.

[0128] For hydrophilic compounds, sample extracts were analyzed by LC-ESI-MS / MS system (UPLC, Shim-pack UFLC, Shimadzu CBM A system; MS, QTRAP ®The analytical conditions were as follows: UPLC: Waters Acquity UPLC HSS T3 C18 column (1.8 μm, 2.1 mm × 100 mm); column temperature, 40°C; flow rate, 0.4 mL / min; injection volume, 2 μL; solvent system, water (0.1% formic acid): acetonitrile (0.1% formic acid); gradient program: 95:5 V / V at 0 min, 10:90 V / V at 11.0 min, 10:90 V / V at 12.0 min, 95:5 V / V at 12.1 min, and 95:5 V / V at 14.0 min. For hydrophobic compounds, the analytical conditions were as follows: UPLC column: Thermo C30 (2.6 μm, 2.1 mm × 100 mm); solvent system: A: acetonitrile / water (60 / 40 V, 0.04% acetic acid, 5 mmol / L ammonium formate), B: acetonitrile / isopropanol (10 / 90 V, 0.04% acetic acid, 5 mmol / L ammonium formate); gradient program: A / B (80:20 V / V) at 0 min, 50:50 V / V at 3.0 min, 35:65 V / V at 5 min, 25:75 V / V at 9 min, and 10:90 V / V at 15.5 min; flow rate: 0.35 mL / min; temperature: 45°C; injection volume: 2 μL. The wastewater was connected to the QTRAP-MS.

[0129] For hydrophilic compounds, in QTRAP ®Linear ion trap (LIT) and QQQ scans were acquired on an LC-MS / MS system equipped with an ESI turbo ion spray interface, operating in positive and negative ion modes and controlled by Analyst 1.6.3 software (Sciex). ESI source operating parameters were: source temperature 500°C; IS, 5500 V (positive), -4500 V (negative); GSI, GSII, and CUR settings of 55, 60, and 25.0 psi, respectively; and CAD set to high. The instrument was calibrated and mass-calibrated using 10 μmol / L and 100 μmol / L polypropylene glycol solutions in QQQ and LIT modes, respectively. Multiple reaction monitoring (MRM) transitions specific to each cycle were monitored based on the metabolites eluting within each cycle. For hydrophobic compounds, ESI source operating parameters were: ion source turbo spray; source temperature 550°C; IS, 5500 V; GSI, GSII, and CUR settings of 55, 60, and 25 psi, respectively; and CAD set to medium. The instrument was commissioned and mass calibrated in QQQ and LIT modes using 10 μmol / L and 100 μmol / L polypropylene glycol solutions, respectively. QQQ scans were acquired during MRM experiments with a collision gas (nitrogen) setting of 5 psi. The cone voltage (DP) and collision energy (CE) were further optimized for individual MRM transitions. Specific MRM transitions were monitored within each cycle, based on the metabolites eluting within that cycle. Broad-target metabolomics was performed with the assistance of Wuhan Metawell Biotechnology Co., Ltd.

[0130] Targeted oxidative lipid metabolomics

[0131] All eicosanoids and deuterated internal standards were purchased from Cayman Chemical. Standard stock solutions were prepared in MeOH at a concentration of 0.1 mg / mL. All stock solutions were stored at -20°C. Working solutions were diluted with MeOH prior to analysis.

[0132] When analyzing cell samples, 10 7 Each macrophage was treated with 200 μL of oxidized lipid extract, vortexed for 5 minutes, and protein precipitated at -20°C. A 100 μL aliquot of the supernatant was added with 200 μL of oxidized lipid extract, vortexed for 5 minutes, and protein precipitated at -20°C. Twenty μL of a 1 μM internal standard mixture was added to each sample, vortexed for 10 minutes, and centrifuged at 5000 rpm for 10 minutes at 4°C. The extraction steps were repeated, and the supernatants were combined. Eicosanoid compounds in the supernatant were extracted using a Poly-Sery MAX SPE cartridge (ANPEL). Prior to analysis, the eluate was vacuum-dried and redissolved in 100 μL of methanol / water (1:1, v / v) for UPLC / MS / MS analysis.

[0133] HPLC conditions: LC-ESI-MS / MS system (UPLC, ExionLC AD; MS, QTRAP ® The sample extracts were analyzed using a Waters 6500+ system. The analytical conditions were as follows: a Waters Acquity UPLCHSS T3 C18 column (100 mm × 2.1 mm ID, 1.8 μm); the solvent system consisted of water containing 0.04% acetic acid (A) and acetonitrile containing 0.04% acetic acid (B); the gradient from 0.1% to 30% B was 0–2.0 minutes; to 50% B over 2.0–4.0 minutes; to 99% B over 4.0–5.5 minutes, then held for 1.5 minutes; then reduced to 0.1% B over 6.0–7.0 minutes and held for 3.0 minutes; the flow rate was 0.4 mL / min; the temperature was 40°C; and the injection volume was 10 μL.

[0134] ESI-MS / MS conditions: ® LIT and QQQ scans were acquired on a 6500+ LC-MS / MS system equipped with an ESI Turbo ion spray interface, operating in negative ion mode and controlled by Analyst 1.6.3 software (Sciex). ESI source operating parameters were: turbo spray, source temperature 550°C; I2S at −4500 V; and CUR at 35 psi. Eicosanoids were analyzed using pre-programmed MRMs. Data were acquired using Analyst 1.6.3 software (Sciex). All metabolites were quantified using Multiquant software (Sciex). Mass spectrometric parameters were optimized for DP and CE, encompassing individual MRM transitions. Specific MRM transitions were monitored for each cycle, depending on the metabolite eluting within that cycle. Eicosanoid content in the samples was determined using MetWare software on the AB Sciex QTRAP 6500 LC-MS / MS platform.

[0135] Transmission and field emission scanning electron microscopy imaging

[0136] For transmission electron microscopy imaging, mouse TA muscle tissue was harvested, the largest cross-section remaining, and cut into blocks. Samples were fixed overnight in 2.5% glutaraldehyde and washed three times with freshly prepared 0.1M PBS, pH 7.4, for 10–15 minutes each. Following post-fixation, the samples were fixed with 1% osmium hydroxide for 1 hour and washed three times with double-distilled water for 10–15 minutes each. Samples were then transferred to 2% uranyl acetate for 30 minutes and dehydrated in ascending ethanol gradients (50%, 70%, 90%, and 100%) for 20 minutes each. The samples were then treated in 100% acetone for 20 minutes, immersed in a 1:1 resin:acetone mixture for 2 hours at room temperature, and then immersed in a 3:1 resin:acetone mixture in a desiccator overnight at room temperature. Samples were then embedded in 100% resin at 30°C for 12 hours and heated at 60°C for 24 hours. The sections were stained with uranyl acetate and lead citrate and imaged using a transmission electron microscope (Talos 120 kV). The images were processed and analyzed using Image J software.

[0137] For field emission scanning electron microscopy imaging, after collecting mouse TA muscle tissue, the largest cross-section was retained and cut into blocks. The samples were fixed in 2.5% glutaraldehyde overnight. Washed three times with freshly prepared 0.1M pH7.4 PBS buffer for 10-15 minutes each time. Then, fixed with 1% osmium acid for 1.5 hours and washed again with 0.1M pH7.4 PBS buffer three times for 10-15 minutes each time. Then dehydrated in ascending gradient ethanol solutions (50%, 70%, 90%), each gradient dehydration for 15 minutes. Remove an appropriate amount of 100% ethanol in advance and store in a desiccating container. After dehydration in 90% ethanol, replace it with 100% ethanol and dehydrate for 20 minutes. This step is repeated once. After critical point drying (Balzers Union), the sample was first adhered to the sample stage with conductive glue. Then, the sample was carefully adhered to the conductive glue with tweezers. After spray coating (E-5100, Polaron Equipment Ltd.), it was imaged using a field emission scanning electron microscope (Nova Nano 450). The obtained images were processed and analyzed in Image J software.

[0138] Quantitative and statistical analysis

[0139] Statistical analysis was performed with Prism software (GraphPad 9) or R. Two-group comparisons were performed with the two-tailed unpaired Student's t-test, and comparisons between paired groups were performed with the two-tailed paired Student's t-test. Comparisons between more than two groups were performed with one-way analysis of variance. CSA analysis was performed with the multiple t-test. Single-cell RNA sequencing data were analyzed with the Wilcoxon rank-sum test. Statistical significance was assessed as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significantly different.

[0140] result

[0141] 1. GSDMD knockout in myeloid cells slows tissue damage and repair

[0142] Acute muscle injury induced by cardiotoxin (CTX) is a typical self-limited, sterile injury model commonly used to study tissue regeneration. This process consists of a series of tightly regulated events, including the activation of muscle stem cells (MuSCs or satellite cells) during the early inflammatory response after injury, the subsequent resolution of inflammation, and the proliferation and differentiation of MuSCs. Figure 7 In order to determine the potential role of myeloid cell GSDMD in tissue regeneration, the present invention constructed Gsdmd f / f and Gsdmd f / f -Lyz2-Cre (GSDMD myeloid cell conditional knockout, hereinafter referred to as Gsdmd CKO ) mice, and then CTX was injected intramuscularly into the tibialis anterior (TA) muscle of the mice. Muscles were harvested 0-14 days post-injury (dpi) and stained with hematoxylin and eosin (H&E) to assess the regeneration process after injury. Gsdmd CKO Mice exhibit delayed repair, as evidenced by the persistence of both necrotic and regenerative areas ( Figure 1 a- Figure 1 c) and the reduction of cross-sectional area (CSA) of myofiber in the late regeneration stage ( Figure 1 d in, Figure 1 The e in ​ b). At the same time ​ CKO Mouse muscles contain more early regenerating myofibers (marked by embryonic myosin heavy chain eMyHC) at the late repair stage (7 dpi) ( ​ f in​ g in the figure). The above data indicate that myeloid cells ​ When it is missing, the process of repairing muscle damage is slowed down.

[0143] Muscle regeneration during injury depends on the activation of quiescent MuSCs. Subsequently, MuSCs express ​ (a typical transcription factor of quiescent MuSCs), ​ and ​ (MuSCs early and late differentiation markers respectively) ​ c) and then upregulate its myogenesis-related pathways. Consistent with the delayed regeneration phenotype, Gsdmd CKO Mice showed downregulation of transcriptional levels of myogenesis-related genes (Pax7, MyoD, and MyoG) ( Figure 1 h in the figure). It is known that MuSCs undergo a large amount of proliferation after activation, then exit the cell cycle to differentiate, and finally participate in the fusion and repair of damaged muscle fibers or return to the quiescent phase. Based on this, the immunofluorescence staining results showed Gsdmd CKO Mouse MuSCs proliferation (PAX7 + Ki67 + , Figure 1 The i in Figure 1 The white arrow in j) was significantly reduced and the differentiation level was reduced ( Figure 1 The i in Figure 1 To determine the correlation between GSDMD activation and MuSC activation, the present invention performed WB detection on the cleavage form of GSDMD during the injury repair process. The results showed that the N-terminus of GSDMD (p30NT) appeared at 1 dpi and reached a peak at 3 dpi ( Figure 1 l in the figure), which is consistent with the degree of macrophage infiltration ( Figure 7 d in, Figure 7 Interestingly, GSDMD deficiency did not affect the acute infiltration of macrophages during the early regeneration phase (0-3 dpi), but macrophages had not yet subsided during the late repair phase (10 dpi). Figure 7 d in, Figure 7 This suggests that conditional knockout G / sdmd It may have less impact on the initial inflammatory stage after injury. In addition, the reconstruction of blood vessels can restore the oxygen and nutrient supply of tissues and secrete a variety of growth factors, which are crucial for tissue regeneration. The present invention found that the loss of GSDMD did not change the vascular density ( Figure 7 f in Figure 7 Taken together, these data suggest that GSDMD has an indispensable function in promoting tissue repair but has a minor impact on the inflammatory response after injury.

[0144] 2. Myeloid GSDMD is essential for MuSC function during regeneration

[0145] To achieve effective tissue repair, various cell types within the muscle need to function in an orderly manner and coordinate with each other to initiate stem cell activation, remove damaged debris, resolve inflammation, and restore homeostasis. Therefore, in order to obtain a cellular map of the muscle regeneration process under GSDMD knockout, the present invention collected mouse muscle tissue at two time points, early (day 2) and late (day 10) of tissue repair, and then performed single-cell sequencing. After quality control and SNN (shared nearest neighbor) clustering, the present invention collected a total of 51,945 cells, covering 11 major cell types ( Figure 2 a in Figure ), including immune cells (macrophages, monocytes, dendritic cells, T cells, and neutrophils), endothelial cells, fibro / adipocyte progenitor cells, MuSCs, and mature myocytes ( Figure 8 a- Figure 8 d in the figure). It is known that after muscle injury, MuSCs exit the resting state and enter the proliferation state, thereby initiating the regeneration process. Therefore, the present invention divides the injured MuSCs into 6 subpopulations according to the temporal state of MuSCs, namely, resting state, early / late activation state, proliferation state, Myod1 high and terminal differentiation Myog high Subpopulation ( Figure 2 CytoTRACE analysis further confirmed that this pseudo-temporal clustering of MuSCs was negatively correlated with their regenerative potential ( Figure 8 e in, extended data Figure 2 f in the figure), demonstrating the reliability of this method for predicting stem cell developmental potential. To explore the impact of myeloid cell GSDMD on MuSC-mediated repair, we performed pseudo-time series analysis and predicted two different fate trajectories after MuSC activation ( Figure 2 c). GSDMD deficiency leads to a disordered MuSC proliferation trajectory, manifested by a decrease in early proliferating cells (pseudo-time initiation stage) ( Figure 2 c, upper right panel), and terminal stage Myog high Inhibition of differentiation ( Figure 2 Specifically, maintenance of the quiescent state and the transition to a proliferative state after injury are tightly regulated by a series of cascade signals ( Figure 8 g in). The present invention found that Gsdmd CKOMouse MuSCs show persistent Notch and Wnt signaling after injury ( Figure 2 d in the figure), both of which are essential for maintaining the quiescent state of MuSCs. In contrast, GSDMD deficiency leads to decreased responsiveness of the PI3K-AKT and FGFR-MAPK pathways in MuSCs after muscle injury, thereby inhibiting the transition of MuSCs from a quiescent state to an activated state, further leading to decreased proliferation and differentiation levels ( Figure 2 Together, these data suggest that myeloid GSDMD may be required for the efficient transition of MuSCs from a quiescent to a proliferative state, which is crucial for the early establishment of a regenerative niche after acute injury.

[0146] The function of MuSCs is tightly regulated by various cell subpopulations in muscle tissue. Interestingly, the present invention found that conditional knockout of GSDMD in myeloid cells had little effect on the overall cell composition. Gsdmd CKO In mice, the composition of immune cells in muscle tissue remains largely unchanged early after injury ( Figure 2 a in Figure 9 Considering that immune cells show significant dynamic changes during muscle regeneration, the present invention divides immune cells into 11 cell types ( Figure 9 b). In the control group and Gsdmd CKO Similar levels of early inflammatory cell infiltration were found in the muscle tissue of mice ( Figure 9 c in Figure 9 d in the above), further indicating that Gsdmd CKO It may not directly affect the early inflammatory microenvironment within the muscle during muscle injury.

[0147] From a functional perspective, the muscle regeneration process is closely related to the transition from early inflammatory initiation to late regenerative microenvironment ( Figure 9 e in the figure). Many intercellular signaling molecules exert specific regulatory effects on MuSCs at different stages after injury. To reveal the key immune cell types involved in the highly dynamic muscle regeneration process, the present invention functionally analyzed each immune cell type, including the inflammatory gene set function (Inflammatory.Index) and the pro-regenerative gene set function (Regenerative.Index). The present invention defined three highly dynamic myeloid cell subsets among all immune cell types. Spp1 high Macrophages, Mrc1 high Macrophages and Ccr2 high Monocytes ( Figure 2The e in Figure 9 f), they upregulate inflammatory pathways in the acute phase after injury (from open squares to solid circles) and switch to a pro-regenerative phenotype in the later phase (from solid circles to solid triangles) ( Figure 2 Visual enrichment analysis further demonstrated that GSDMD loss did not affect the phenotypic switching of these dynamic myeloid populations ( Figure 2 f in the figure). The pseudo-series analysis results also showed that GSDMD deficiency did not affect the differentiation of monocytes into macrophages ( Figure 2 g in the figure), which is consistent with the previous results that GSDMD knockout did not affect the degree of macrophage infiltration ( Figure 7 d in, Figure 7 To further illustrate Gsdmd The present invention uses the NicheNet algorithm to analyze cell-cell interactions, which infers the active ligands and their gene regulatory effects on the interacting cells. Gsdmd Gene knockout had little effect on the expression of ligands enriched in immune components ( Figure 2 h in Figure 9 g in Figure 3 h), suggesting that GSDMD may have little effect on the intercellular interactions of known ligand-receptor pairs. In summary, single-cell sequencing analysis showed that myeloid cells Gsdmd It may not affect the changes in the composition of immune cells in muscles during regeneration.

[0148] GSDMD protein was originally considered to be the executioner of cell pyroptosis, secreting various pro-inflammatory factors such as interleukin-1β (IL-1β) through the pores formed by oligomerization of its N-terminal fragments. Therefore, in order to explore how GSDMD affects muscle regeneration after acute injury, the present invention first evaluated Gsdmd Whether knockout affects the pyroptosis process and the secretion of pro-inflammatory factors. In the early stage after injury, significant oligomerization of GSDMDN terminal fragments was found in the control group, representing the formation of GSDMD pores, while Gsdmd CKO This phenomenon was not observed in mice ( Figure 10 NINJ1 oligomerization is a key event in plasma membrane rupture secondary to the oligomerization of the N-terminal fragment of GSDMD. However, the present invention found that GSDMD knockout did not affect NINJ1 oligomerization ( Figure 10These data suggest that GSDMD pore formation during muscle regeneration may not occur without pyroptosis. Furthermore, given that typical pyroptosis leads to the massive release of proinflammatory mediators, the present inventors systematically analyzed the secretome of intermuscular fluid using the highly sensitive OLINK assay, targeting key proteins involved in mouse biological processes such as inflammation, regeneration, and stress response. Principal component analysis revealed significant differences in the secretome of intermuscular fluid between early and late stages ( Figure 2 However, GSDMD knockout had little effect on the overall interstitial fluid composition (including a range of pro-inflammatory and pro-regenerative factors) ( Figure 2 The j in Figure 10 b in Figure 10 IL-1β is one of the most important pro-inflammatory factors released during cell pyroptosis. Gsdmd Knockout did not affect IL-1β levels in interstitial fluid ( Figure 2 At the same time, the formation of GSDMD pores will eventually lead to the rupture of the plasma membrane, resulting in the passive release of macromolecules such as high-mobility group box 1 (HMGB1). Gsdmd CKO The levels of HMGB1 in the interstitial fluid of mice were comparable ( Figure 10 d in, Figure 10 In addition, based on the calculation of genes highly related to the pyroptosis process (pyroptosis index), the present invention found Gsdmd Knockout has little effect on the pyroptosis process of myeloid cells ( Figure 10 This further suggests that the oligomerization of the N-terminal fragment of GSDMD in the early stage of regeneration may not directly lead to lytic pyroptosis, but rather represents a "super-activated" state of macrophages. Together, these data reveal a role for GSDMD in promoting regeneration early after injury.

[0149] 3. GSDMD-dependent metabolite release promotes tissue regeneration

[0150] Considering that the pro-regenerative ability of GSDMD may not be related to its classical function, the present invention then established an in vitro culture experiment to explore why GSDMD activation is crucial for muscle regeneration. In order to simulate the "super-activated" state of macrophages after GSDMD activation in vitro without pyroptosis, the present invention added glycine to the culture medium to maintain osmotic pressure, protect the integrity of the plasma membrane, and allow active substances to pass through the oligomeric GSDMD pores. Next, the present invention tested the results of the experiment on the control group ( Gsdmd WT wild group (non-knockout group) and GsdmdThe supernatant collected from knockout macrophages was subjected to ultracentrifugation, and a 3 kDa cellulose membrane was used to separate most protein ligands (>3 kDa) and small molecule metabolites (<3 kDa) ( Figure 11 The present invention found that in the <3 kDa fraction, inflammatory factors such as IL-1β (GSDMD-dependent secretion), IL-6, and TNFα (GSDMD-independent secretion) were almost undetectable ( Figure 11 b- Figure 11 d in the figure), indicating the feasibility of the in vitro system. To determine which components promote muscle regeneration, the present invention then added the collected supernatant to the mouse myoblast cell line C2C12. Interestingly, the addition of Gsdmd WT Macrophages (from Gsdmd The <3 kDa fraction, but not the >3 kDa fraction, of peritoneal macrophages extracted from WT wild-type mice (GSDMD wild-type macrophages) resulted in increased C2C12 myogenic capacity compared to supernatants from knockout macrophages ( Figure 3 a), which is reflected by a larger myotube diameter and fusion level ( Figure 3 b) in the above example.

[0151] Based on the above results, the present invention hypothesizes that certain small molecules actively secreted through GSDMD pores can promote the muscle repair process. Next, the present invention treated cells with lipopolysaccharide (LPS) and nigericin (LN) or poly (dA:dT), LP, to stimulate GSDMD cutting and membrane pore formation. Propidium iodide (PI) staining was used to mark cells that formed pores, while the level of lactate dehydrogenase (LDH) in the supernatant represented the level of cell lysis and death. In order to identify the true GSDMD-mediated active secretion products, the present invention treated cells with glycine and used Gsdmd CKO Macrophages served as a negative control. As expected, glycine blocked pyroptosis in macrophages but did not affect GSDMD pore formation ( Figure 11 The e in Figure 11 f), confirming that macrophages are in a "hyperactivated" state. Therefore, the present invention established a screening strategy using these two GSDMD activation models (1. lipopolysaccharide plus nigericin (LN) plus glycine; 2. lipopolysaccharide plus poly(deoxyadenosine-thymidine) sodium salt (LP) plus glycine) to systematically analyze the secretome of "hyperactivated" macrophages using untargeted metabolomics ( Figure 3c in the present invention). A large number of metabolites were detected in the supernatant of pyroptosis induced by different stimuli ( Figure 11 Considering that glycine supplementation can lead to the direct accumulation of glycine itself and may induce corresponding artificial signals in the screening system of the present invention, the present invention excludes metabolites that undergo significant changes due to glycine addition. The present invention compares the LN plus glycine group with the LN group (LP plus glycine with the LP group), and the metabolites that are commonly upregulated under these two GSDMD activation models are regarded as glycine-dependent metabolite changes ( Figure 11 It is noteworthy that the present invention detected a metabolite 11,12-epoxyeicosatrienoic acid (EET), which was enriched in the supernatant of "superactivated" macrophages ( Figure 3 In d, Figure 11 i), the present invention also shows that the secretion of 11,12-EET depends on Gsdmd The expression ( Figure 11 in the j).

[0152] 11,12-EET is one of the arachidonic acids originally derived from phospholipids ( Figure 12 In order to quantify this result, the present invention used ELISA detection and found that GSDMD activation can indeed cause macrophages to actively secrete 11,12-EET ( Figure 12 b). To further verify, the present invention used targeted metabolomics of the arachidonic acid family and obtained the Gsdmd A total of 88 oxidized lipids were detected in the supernatants and cell lysates of knockout macrophages. Principal component analysis (PCA) showed that GSDMD knockout did not change the composition of intracellular oxidized lipids, but resulted in differences in the secretion levels of oxidized lipids ( Figure 3 e in ). Consistent with untargeted metabolomics, we discovered a series of arachidonic acid-containing proteins, including 11,12-EET, that are actively secreted in a GSDMD-dependent manner ( Figure 3 f in the figure). Subsequently, the present invention attempted to verify these findings in vivo. The present invention collected muscles and corresponding muscle tissue interstitial fluid (TIF) at different time points after CTX-induced injury. The metabolites in the muscle interstitial fluid were normalized to the levels in muscle tissue. The present invention classified the metabolites secreted after acute injury into three different patterns, "early secretion" (secreted throughout the early period, 1-5dpi), "transient secretion" (secreted briefly after injury) and "late secretion" (secreted starting in the late period, after 5dpi) ( Figure 3In addition to 11,12-EET itself, the present invention found that the early interphase fluid secretome is rich in a series of arachidonic acid-related metabolites ( Figure 3 In summary, using three independent omics strategies, we identified 11,12-EET as a representative metabolite actively secreted from “hyperactivated” macrophages upon GSDMD activation ( Figure 3 (i in the text).

[0153] Finally, the present invention set out to test whether this GSDMD-dependent metabolite secretion could directly affect muscle regeneration. The results showed that the myogenic fusion of C2C12 cells in the 11,12-EET treatment group increased, as reflected by a significant increase in the number of nuclei in myotubes. This indicates that 11,12-EET supplementation accelerated the myogenic differentiation ability of C2C12 myoblasts ( Figure 12 c in Figure 12 d in the present invention). Next, the present invention uses 11,12-EET and the control and Gsdmd The supernatant of knockout macrophages was treated with isolated mouse primary MuSCs ( Figure 12 e in the figure). Consistent with the findings of the present invention in the C2C12 cell line, 11,12-EET significantly accelerated the morphological changes of MuSCs after activation ( Figure 12 f in Figure 12 g in), enhancing their myogenic potential ( Figure 3 In addition, 11,12-EET ( Figure 3 k- Figure 3 m) and supernatant of control macrophages ( Figure 3 n- Figure 3 p) both enhanced the proliferation of MuSCs (Ki67 + staining ratio increased), while the effect on non-MuSC components was smaller.

[0154] 4. Accumulation of 11,12-EET promotes tissue regeneration in vivo

[0155] Considering the potential of 11,12-EET to promote regeneration, the next step of this invention is to explore whether tissue regeneration can be promoted by increasing the level of 11,12-EET in the body. The biosynthesis of 11,12-EET involves many cooperating enzymes from the cytochrome P450 pathway, so it is difficult to increase the biosynthesis level of 11,12-EET by manipulating specific upstream genes. However, it is known that soluble epoxide hydrolase (EPHX2) hydrolyzes EETs into non-biologically active DHETs ( Figure 12 a), knocking out EETs will lead to the accumulation of EETs levels. Ephx2f / f and Ephx2 CKO (EPHX2 conditional knockout in myeloid cells, Ephx2 f / f -Lyz2-cre) mice to evaluate the effects of 11,12-EET accumulation on muscle regeneration. （ Figure 13 a) in the above example. Ephx2 CKO Mice showed accelerated muscle repair at 14 dpi, including muscle necrosis and a reduction in the regenerating area ( Figure 4 a in Figure 4 b) and an increase in muscle fiber area ( Figure 4 c in Figure 4 In d, Figure 13 b). In line with this, Ephx2 Knockout promoted the expression of myogenic-related genes during the proliferation and differentiation of MuSCs ( Figure 4 The e in Figure 13 c in ). In addition, Ephx2 CKO eMyHC in mice at 6 dpi + There are fewer regenerating muscle fibers in the early stage ( Figure 4 f), indicating that the regeneration process was accelerated compared with the control mice. Ephx2 Accelerated the proliferation of MuSCs, thereby enhancing the expression of MYOD and MYOG proteins ( Figure 4 h in the text).

[0156] To confirm whether the contribution of 11,12-EET accumulation to muscle regeneration is GSDMD-dependent, the present invention Ephx2 - f / f Lyz2- cre mice and Gsdmd f / f Hybridization, construction Gsdmd f / f Ephx2 f / f -Lyz2-cre mice and controls Gsdmd f / + Ephx2 f / f -Lyz2-cre mice were then subjected to CTX-induced TA muscle injury ( Figure 4 In Ephx2 Knockout of GSDMD in knockout mice impeded the pro-repair effect of 11,12-EET, resulting in reduced muscle weight and myofiber size at 14 dpi ( Figure 4 j- Figure 4 As an effective inhibitor of GSDMD pore formation, disulfiram (DSF) treatment also counteracted the effect of Ephx2 Knockout-mediated promotion of muscle regeneration （ Figure 4 n in, Figure 4 The o in Figure 13 d in, Figure 13 Taken together, these in vivo results suggest that the pro-regenerative potential of macrophage-released 11,12-EETs requires the pore-forming function of GSDMD.

[0157] 5. 11,12-EET promotes MuSC proliferation by enhancing FGF-FGFR signaling

[0158] To explore the mechanism by which 11,12-EET promotes muscle regeneration by upregulating the myogenic capacity of MuSCs, we isolated primary MuSCs and induced their activation and proliferation in vitro. The activation and proliferation of MuSCs are strictly regulated by time and are triggered by various cascade signals, such as the MAPK and PI3K-AKT pathways. These signaling pathways, in turn, initiate a series of transcriptional, post-transcriptional, and metabolic programs to provide sufficient cells to support regeneration. By analyzing the transcriptome data of control (DMSO (dimethyl sulfoxide)) and 11,12-EET-treated MuSCs, we found that 11,12-EET significantly promoted the activation and proliferation of MuSCs, as manifested by increased levels of cytoplasmic translation and mitochondrial respiratory activity, as well as the expression of several cell cycle-related genes (such as H3c7 and H2ac10) and differentiation myosin genes (such as Myh4 and Mylpf) ( Figure 5 a in Figure 5 In addition, compared with the resting state, the gene expression pattern of MuSCs after 11,12-EET treatment was positively correlated with that of activated MuSCs (GEO: GSE113631) ( Figure 14 Interestingly, although 11,12-EET-treated MuSCs had entered a proliferative state, control MuSCs were still enriched in the MAPK and PI3K signaling pathways at this time point ( Figure 5 b), indicating that 11,12-EET accelerates these signal transductions during MuSC activation, leading to enhanced proliferation levels. Next, the present invention directly isolated MuSCs from TA muscles 3 days after CTX injury and found that 11,12-EET treatment indeed induced an increase in mTOR (p-AKT, p-P70, and p-4EBP) and MAPK (p-P38) signaling ( Figure 5(c) Consistent with previous results, early activated MuSCs expressed higher levels of FGFRs compared to other growth factor receptors. (Fgfr1 and Fgfr4 )( Figure 14 (b) suggests that the FGF-FGFR axis is one of the major upstream signals of the muscle PI3K-AKT-mTOR and MAPK pathways. Therefore, the present inventors next set out to determine whether 11,12-EET affects the upstream FGF-FGFR axis. Strikingly, 11,12-EET directly amplifies FGF signaling, with elevated activation of the MAPK and PI3K-AKT-mTOR pathways in FGF-responsive NIH-3T3 cells ( Figure 5 d) in the above example.

[0159] During the repair process, due to the limited concentration of pro-regenerative ligands in the muscle injury microenvironment, the body requires specific strategies to amplify signal transduction to achieve effective repair. Therefore, using MuSC single-cell transcriptome data, the present invention ranked the correlation between each gene and the FGF signaling score to reveal the underlying biological processes required to amplify FGF signaling ( Figure 14 c in the figure). The present invention found that genes highly positively correlated with FGF signal transduction were significantly enriched in biological processes such as extracellular structure, membrane organization and growth factor binding ability ( Figure 5 The e in Figure 14 d in), which are all required for the effective initiation and transduction of FGF-FGFR signaling. It is worth noting that the functions of these enriched molecules are closely related to the biochemical properties of FGF. Specifically, the disordered N- and C-terminal sequences of FGF contribute to its oligomerization, providing sufficient multivalent weak interactions for liquid-liquid phase separation (LLPS) on the cell membrane surface. Therefore, the present invention hypothesizes that 11,12-EET, as a lipid, may enhance the FGF phase separation on the membrane surface, thereby effectively amplifying downstream signals. The present invention first used polyethylene glycol (PEG-8000) as an aggregating agent and found that the addition of 11,12-EET significantly increased the formation of PEG-induced green fluorescent protein fused FGF (eGFP-FGF) droplets ( Figure 5 f in Figure 5 g in), while increasing the turbidity of the solution ( Figure 5 In addition, fluorescence recovery after photobleaching (FRAP) data further demonstrated that 11,12-EET enhanced the fluorescence recovery of FGF droplets, suggesting a higher mobility ( Figure 5Given that 11,12-EET promotes FGF phase separation in the extracellular system, the present invention next determined whether this effect occurs on the cell surface. Interestingly, the supplementation of 11,12-EET enhanced the formation of punctate condensates of eGFP-FGF at the cell edge ( Figure 5 j in Figure 5 k in ), and 11,12-EET enhances the liquid properties of these condensates ( Figure 5 Consistent with the formation of cell surface condensates, biochemical data further demonstrated that 11,12-EET enhanced the oligomerization of FGF, thereby promoting its signal transduction ability ( Figure 5 Taken together, these data suggest that 11,12-EET amplifies FGF-FGFR signaling by enhancing FGF phase separation at the membrane surface.

[0160] Considering that the activation capacity and proliferation level of MuSCs in vitro culture systems cannot fully simulate the dynamic changes of MuSCs in vivo after injury, the present invention further explored whether 11,12-EET promotes FGF signaling in vivo. The present invention injected saline or 11,12-EET into the muscles of CTX-injured mice and then performed single-cell sequencing ( Figure 5 n in Figure 14 e- Figure 14 g in the control group and Gsdmd CKO Consistent with the data from mice, the present invention identified six pseudo-temporally classified MuSC cell types ( Figure 5 O in Figure 14 h in ). This is consistent with the in vitro experimental results ( Figure 3 j- Figure 3 p in the ), 11,12-EET injection induced injury Myod high and Myog high MuSCs differentiation level increased ( Figure 5 o in), accelerated the proliferation and differentiation lineage of MuSCs ( Figure 5 In the p, Figure 14 Enrichment analysis showed that 11,12-EET upregulated the FGF binding ability and downstream signaling cascades of MuSCs ( Figure 5 In addition, the present invention found that EET treatment enhanced the correlation between FGF binding and p38MAPK signaling pathway activation, while Gsdmd Knockout abolished this correlation ( Figure 5r in the figure), suggesting that 11,12-EET amplifies the FGF-FGFR signaling cascade by promoting its binding ability in vivo. In summary, using biochemical and bioinformatics data, the present invention demonstrates that 11,12-EET amplifies FGF-FGFR signaling by enhancing FGF LLPS.

[0161] 6. 11,12-EET promotes multi-organ regeneration

[0162] The present invention ultimately sets out to elucidate the therapeutic potential of 11,12-EET in tissue repair in vivo. Intramuscular injection of 11,12-EET significantly improves muscle regeneration after CTX-induced acute injury, as evidenced by increases in muscle weight and strength ( Figure 6 a) in the figure), the diameter of muscle fibers increases ( Figure 6 b in Figure 6 In,c, Figure 15 (a) and the reduction of necrotic and regenerating areas on day 14 after injury ( Figure 6 In d, Figure 15 b). The present invention also noted that 11,12-EET-treated muscles had eMyHC in the late regeneration stage. + Reduced muscle fiber count ( Figure 6 The e in Figure 6 f) and inflammation subsided faster ( Figure 15 c in Figure 15 (d) These data indicate that 11,12-EETs promote muscle regeneration.

[0163] Given that FGF has a broad regenerative capacity in various tissue regeneration processes (such as corneal and skin injuries), the present invention next examined whether 11,12-EET could promote tissue regeneration in other injury models. The present invention established a corneal injury model by administering an overdose of the ophthalmic agent benzalkonium chloride, which causes dry eye symptoms and corneal epithelial damage. The results showed that treatment with 11,12-EET eye drops significantly reduced corneal sodium fluorescein staining, indicating a reduction in epithelial damage ( Figure 6 g in the figure), while also improving clinical manifestations such as tear secretion and tear film stability ( Figure 6 Pathological analysis further demonstrated that 11,12-EET treatment improved corneal thickening and reduced inflammatory cell infiltration in the late stage ( Figure 16 a- Figure 16 e in the figure). These results indicate that 11,12-EET promotes corneal regeneration and prevents corneal fibrosis and other complications. The present invention also used a classic UV-induced skin damage model and found that 11,12-EET treatment significantly reduced auricular swelling ( Figure 6 i) and decreased leukocyte infiltration ( Figure 16 f in ).

[0164] In addition to the UV-induced skin injury model, the present invention also constructed another skin injury model using a punch biopsy method to further explore the role of 11,12-EET in it. After performing a skin punch biopsy on the back of mice, 3 μL of 11,12-EET or a control solvent (ethanol) was dripped into the skin wound once a day for 7 consecutive days ( Figure 17 Compared with the control group, 11,12-EET treatment significantly promoted wound healing ( Figure 17 b in Figure 17 c), and 11,12-EET treatment significantly increased the proliferation rate of epidermal stem cells (KRT14+) ( Figure 17 d in the figure). This result provides new evidence that 11,12-EET promotes repair in skin injury models. In summary, this invention demonstrates that 11,12-EET can be used in a variety of mouse injury models and has a significant effect on promoting tissue regeneration.

[0165] Although the age-related decline in tissue regeneration capacity is multifactorial, we wanted to know whether 11,12-EET levels are physiologically related to age-related repair defects. Surprisingly, we found that EPHX2 protein expression is upregulated in aged muscle ( Figure 6 j in the figure), leading to a decrease in 11,12-EET levels in muscle tissue ( Figure 6 k in). Therefore, the present invention supplemented old mice with 11,12-EET (500 ng single injection) or solvent control (ethanol) every 3 days for a total of 15 cycles. Although 11,12-EET supplementation slightly alleviated age-induced fibrosis ( Figure 16 g in), but it can significantly rejuvenate aging muscles, increase weight and strength ( Figure 16 h in the figure), while the muscle fiber area increases ( Figure 6 1 in, Figure 6 The m in Figure 16 j in Figure 16 These “rejuvenating” effects may be due to an increase in the satellite cell pool caused by a local elevation of 11,12-EET concentrations ( Figure 6 Thus, the present invention demonstrates that the metabolite 11,12-EET released from the GSDMD pore has a universal pro-repair potential and the ability to rejuvenate aged muscle.

[0166] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of 11,12-EET in the preparation of drugs for promoting the repair and / or regeneration of tibialis anterior muscle tissue.

2. The use according to claim 1, characterized in that The 11,12-EET promotes the repair and / or regeneration of tibialis anterior muscle tissue through any one or more of the effects 1) to 10); 1) Increase muscle fiber area; 2) Increase muscle mass; 3) Increase muscle strength; 4) Accelerate the resolution of muscle inflammation; 5) Promote the expression of myogenesis-related genes; 6) Promote the activation and / or proliferation of muscle stem cells; 7) Improve the myogenic differentiation ability of muscle stem cells; 8) Regulates liquid-liquid phase separation of fibroblast growth factors; 9) Amplify FGF-FGFR signaling; 10) Accelerate signal transduction of MAPK and PI3K-AKT-mTOR signaling pathways.

3. Use of 11,12-EET in the preparation of drugs for promoting corneal regeneration and / or preventing corneal fibrosis.

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