Application of Mars2 in tissue repair and regeneration

Abstract

The invention belongs to the technical field of biomedicine, and relates to application of Mars2 in tissue repair and regeneration, the expression level of the Mars2 is up-regulated by more than two times through mRNA delivered by AAV9, cell senescence is delayed, cell metabolism is remodeled, and tissue rejuvenation is promoted; in-vivo and in-vitro experiments prove that the metabolism function of mitochondria can be improved, oxidative stress can be reduced and cell senescence can be delayed by up-regulating the expression of the Lars2, so that the tissue repair and regeneration capacity of the Lars2 can be enhanced; the application potential in fibroblast biology and regenerative medicine is expanded; an anti-aging strategy taking metabolic remodeling as a target spot is provided, and is different from a traditional way depending on oxidation resistance or signal path intervention; experiments prove that the regulation and control of the Mars2 have universality and are suitable for various fibroblast types and tissue environments.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of Lars2 in tissue repair and regeneration, specifically the application of Lars2 as a mitochondrial metabolic remodeling factor in tissue rejuvenation, particularly its application in delaying stem cell aging and enhancing tissue repair capabilities. Background Technology

[0002] Gastric tissue aging is a complex biological process characterized by declining cell function, stem cell depletion, and metabolic disorders. With age, the proliferation and differentiation capacity of stem cells decreases, leading to weakened tissue regeneration and becoming a significant contributing factor to various degenerative diseases such as osteoporosis, muscle atrophy, and cognitive impairment. Mitochondrial function decline is a key marker of aging, especially in stem cells, where metabolic state is closely related to stem cell maintenance and tissue repair functions.

[0003] Lars2 (Leucyl-tRNA synthetase 2, mitochondrial) encodes a mitochondrial leucyl-tRNA synthetase, a key factor in mitochondrial protein synthesis. Its stable expression plays a crucial role in maintaining mitochondrial metabolic homeostasis and ensuring oxidative phosphorylation function. Previous reports have indicated that Lars2 expression is decreased in some metabolic disorders, suggesting its potential role in aging. Chinese invention patent application number 201811034076.0 discloses the application of sirtuin2 protein to improve mitochondrial function and elucidates its mechanism of action. It increases the expression levels of fusion dynein Mfn1, Mfn2, and splitting protein Drp1, reduces SAβ-gal activity in mammalian cells, enhances the expression levels of CK19 and β1 integrins, reduces intracellular ROS levels, increases intracellular ATP levels, and improves mitochondrial network morphology. Chinese invention patent application number 202310463823.7 discloses the application of a chimeric protein in anti-aging, providing the use of a chimeric protein, or its encoded polynucleotide, or its promoter, for preparing a composition or formulation for anti-aging. The chimeric protein is a GPR protein mutant, wherein the GPR protein mutant has substitutions in one or more transmembrane regions of the wild-type GPR protein corresponding to accession number AF349993_1. The chimeric protein of the present invention with a specific structure can effectively combat aging. Chinese invention patent application number 202410580761.2 discloses a method for constructing a mouse model of mitochondrial myopathy with Lars2 gene deletion and its application. The method involves: first, Lars2flox / flox mice are crossed with Acta1ER-Cre mice to obtain Acta1ER-Cre-Lars2flox / flox mice; second, when the mice reach 4 weeks of age, they are given intraperitoneal injections of tamoxifen 70-80 mg / kg for 5 consecutive days; third, after 4 weeks, they are given intraperitoneal injections of tamoxifen 45-55 mg / kg for 5 consecutive days; fourth, when the Acta1ER-Cre-Lars2flox / flox mice reach 12-13 weeks of age, skeletal muscle Lars2 protein deletion occurs, and skeletal muscle atrophy is obvious at 16 weeks, thus obtaining a mouse model of mitochondrial myopathy with Lars2 gene deletion.

[0004] However, existing research has not yet elucidated the specific functions and mechanisms of Lars2 in stem cell aging and tissue rejuvenation. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an application and mechanism of action of Lars2 in tissue repair and regeneration. By delivering mRNA via AAV9, Lars2 expression levels are upregulated by more than twofold, delaying cellular senescence, remodeling cellular metabolism, and promoting tissue rejuvenation. In vitro and in vivo experiments have confirmed that upregulating Lars2 expression can improve mitochondrial metabolic function, reduce oxidative stress, delay cellular senescence, and thus enhance tissue repair and regeneration capabilities.

[0006] To achieve the above objectives, this invention provides a mechanism of action of Lars2 in tissue repair and regeneration, the verification process of which is as follows: Figure 10 As shown, the present invention adopts the following technical solution:

[0007] Step 1: Establish a mouse tendon injury model and perform single-cell RNA sequencing (scRNA-seq) analysis. The results showed that the most significantly differentially expressed gene in the tendon fibroblasts was Lars2. Lars2 encodes mitochondrial leucyl tRNA synthetase, which is mainly involved in mitochondrial protein synthesis and energy metabolism regulation. Its decreased expression leads to mitochondrial dysfunction, reactive oxygen species accumulation and energy metabolism disorder, thereby affecting the repair capacity of aging tendon tissue.

[0008] Step 2: Different AAV vectors were constructed, and three different subtypes of recombinant AAV virus were locally injected into the tendon injury sites of mice. An empty AAV vector was set up as a control group. Total RNA was extracted for qPCR detection of Lars2 mRNA expression level and Lars2 protein level. Immunohistochemical staining was performed to locate the target tissue and analyze the differences in the efficiency of different AAV vectors in delivering Lars2 mRNA in tendon tissue in mice at different age stages. The experiment showed that AAV vectors can effectively mediate Lars2 expression. The AAV-9 group showed stronger Lars2 signal intensity and distribution range in aged tendon tissue. Immunohistochemical analysis showed that AAV-9-mediated Lars2 overexpression can effectively improve collagen arrangement, reduce the degree of ectopic ossification and fibrosis in aged tendons. AAV-9 is the optimal choice for delivering Lars2 mRNA to aged tendon tissue.

[0009] Step 3: AAV-9-Lars2 treatment validates improvement in fibrosis / ossification. Transcriptomic and proteomic analyses of mice after treatment showed that key genes related to anti-aging function were upregulated, while the expression of aging markers was significantly downregulated. Lars2 overexpression can activate telomere maintenance mechanisms and delay tissue aging.

[0010] Step 4: Cell model validation of AAV-mediated Lars2 overexpression anti-aging function. A hydrogen peroxide (H2O2)-induced mouse 3T3 fibroblast senescence model was used to systematically evaluate the regulatory effects of AAV-mediated Lars2 on key aging-related biomarkers, antioxidant capacity, and matrix differentiation trends under oxidative stress. qPCR, Western blot, and immunofluorescence multilabeling analyses of collected cell samples showed that AAV-mediated Lars2 overexpression effectively inhibited p16 expression, enhanced antioxidant capacity, and reduced Col2a1 expression in the oxidative stress-induced fibroblast senescence model, demonstrating a clear anti-aging and cell homeostasis-protective effect.

[0011] Step 5: Multi-tissue cell validation. By establishing multi-tissue fibroblast models, the functional consistency of Lars2 in different cell types was systematically compared. Primary cells were prepared from four representative tissues (skin, lung, heart, and kidney) using a combined collagenase and trypsin digestion method to establish an early aging model induced by oxidative stress. Molecular detection and morphological analysis after viral transfection showed that Lars2 overexpression significantly downregulated the mRNA expression level of the aging marker p16 in the four fibroblast types, while significantly upregulating antioxidant defense-related genes NRF2 and mitochondrial SOD2. Lars2 overexpression activated antioxidant protection mechanisms in multiple cell types, and Col2a1 expression was significantly decreased. This indicates that AAV-Lars2 can inhibit the activation of maladaptive matrix programs under chronic stress, helping to maintain cell phenotypic stability and demonstrating clear biological universality and broad application potential.

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] 1. This study reveals for the first time that Lars2 plays a regulatory role in tissue rejuvenation, expanding its application potential in fibroblast biology and regenerative medicine;

[0014] 2. It provides an anti-aging strategy targeting metabolic remodeling, which differs from traditional methods that rely on antioxidants or signaling pathway intervention;

[0015] 3. It can be used to construct tissue aging disease models and as a drug screening platform;

[0016] 4. As an endogenous mitochondrial protein, Lars2 is more physiologically relevant and has a higher safety profile when targeted intervention is performed;

[0017] 5. Experiments have shown that Lars2 regulation is universal and applicable to various fibroblast types and tissue environments. Attached Figure Description

[0018] Figure 1 Volcano diagram of differentially expressed genes.

[0019] Figure 2 Comparison of overexpression efficiency of empty vector control, AAV1, AAV2, and AAV9 in mice of different ages (80W, 40W, 20W).

[0020] Figure 3 HE staining analysis compared the improvement in collagen arrangement, degree of ectopic ossification, and fibrosis between the AAV-Lars2 overexpression group and the empty vector group.

[0021] Figure 4 A comparison of the effects of AAV-Lars2 overexpression group and empty vector group on the expression of key genes and aging biomarkers related to anti-aging function.

[0022] Figure 5 GO enrichment analysis of the AAV-Lars2 overexpression group and the empty vector histomic proteome.

[0023] Figure 6 Immunofluorescence showed that the AAV-Lars2 overexpression group and the empty vector group resisted the expression of the aging marker P16 induced by H2O2.

[0024] Figure 7 Immunofluorescence showed the expression of antioxidant Nrf2 and ectopic ossification marker Col2a1 in the AAV-Lars2 overexpression group and the empty vector group.

[0025] Figure 8 qPCR showed that Lars2 overexpression in primary cells across tissues and the empty vector group enhanced NRF2 / SOD2 expression.

[0026] Figure 9 qPCR showed that Lars2 overexpression inhibited H2O2-induced Col2a1 expression compared to the empty vector group.

[0027] Figure 10 The present invention relates to a flowchart for verifying the mechanism of action. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the embodiments.

[0029] Example 1:

[0030] This embodiment relates to a mechanism of action of Lars2 in tissue repair and regeneration:

[0031] In this embodiment, the applicant constructed tendon injury models in 20-week-old (20W) and 80-week-old (80W) C57BL / 6 mice. Samples were collected on day 7 post-injury, and single-cell RNA sequencing (scRNA-seq) analysis was performed using the 10x Genomics platform. After quality control and data normalization, cell clustering and differential gene analysis were performed using Seurat software.

[0032] Transcriptional profiling at single-cell resolution revealed a trend of decreased transcriptional activity in multiple cell subpopulations within the tendon tissue of 80-week-old mice. Further differential gene analysis and the creation of a differential gene volcano map were then performed (see [link to citation]). Figure 1 ), Figure 1 "Both" refers to a gene difference greater than two-fold and -log10P_adj>2 --- that is, a change in differentially expressed gene levels more than two-fold with a P-value less than 0.01. The results showed that compared to 20-week mice, Lars2 in 80-week mice was significantly downregulated in multiple fibroblast and mesenchymal stem cell-related subsets, log2FC<–1.5, -log 10 P_adj>75, and being among the top 1% of genes with statistically significant differences. 20-week-old mice showed good repair after injury, while 80-week-old mice showed poor repair and ectopic ossification. Single-cell analysis revealed that the most significantly different gene in tendon fibroblasts was Lars2. Lars2 encodes mitochondrial leucyl-tRNA synthetase, which is mainly involved in mitochondrial protein synthesis and energy metabolism regulation. Its expression loss leads to mitochondrial dysfunction through decreased expression of mt-Atp6 and mt-Cytb; reactive oxygen species accumulation through decreased Nrf2 expression; and cellular energy metabolism disorders through decreased Foxo1 expression, thus affecting the repair capacity of aging tendon tissue.

[0033] Example 2:

[0034] This embodiment provides a method for comparing Lars2 overexpression efficiency based on screening different AAV subtypes, aiming to clarify the differences in the efficiency of three vectors, AAV-1, AAV-2 and AAV-9, in delivering Lars2 mRNA in tendon tissue in mice at different age stages (20 weeks, 40 weeks and 80 weeks), thereby optimizing the in vivo Lars2 mRNA delivery strategy.

[0035] In this experiment, three groups of C57BL / 6 mice—20 weeks (young), 40 weeks (middle-aged), and 80 weeks (old)—were used to establish a standardized tendon injury model. On the first day post-surgery, three different subtypes of recombinant AAV virus were locally injected into the tendon injury site in each group of mice. All three viral solutions carried the same CAG-driven mouse Lars2 cDNA (NM_026808.4) expression vector, with the structure pAAV-CAG-Lars2-3xFLAG-WPRE-pA. Each mouse was injected with 10 μL of viral solution, and the viral titer was uniformly 1 × 10⁻⁶. 13 vg / mL. An empty AAV vector was included as a control group in mice of each age group.

[0036] Following viral injection, the injections were administered twice weekly for three weeks. Tendon tissue was harvested on postoperative day 21, and total RNA was extracted for qPCR detection of Lars2 mRNA expression levels. Simultaneously, Western blot was used to detect Lars2 protein levels. Experimental results are as follows: Figure 2 As shown, in 20-week mice, all three subtypes (AAV-1, AAV-2, and AAV-9) effectively mediated Lars2 expression, but the expression levels were not significantly different, ranging from 1.8 to 2.1 times that of the empty vector group. In 40-week and 80-week mice, the Lars2 mRNA expression level in the AAV-9 group was significantly higher than that in the AAV-1 and AAV-2 groups, at 3.2 and 2.7 times that of the empty vector group, respectively. In contrast, the expression efficiency of AAV-1 and AAV-2 in 80-week mice was significantly lower than that of AAV-9, only about 1.5 and 1.3 times that of the empty vector group, respectively. The Western blot results were consistent with the trends observed by qPCR, and the AAV-9 group showed stronger Lars2 signal intensity and distribution in the 80-week tendon tissue.

[0037] Further HE staining showed (see) Figure 3 AAV-9-mediated Lars2 overexpression effectively improved collagen arrangement, reduced heterotopic ossification, and decreased fibrosis levels in aging tendons, showing superior results compared to the control group. TGF-β1 levels also showed a downregulation trend in the AAV-9 group, indicating that one of the potential mechanisms of Lars2 overexpression in anti-fibrosis and anti-cellular senescence is the TGF-β1 paracrine pathway.

[0038] In summary, this embodiment clearly demonstrates that, given the age-related decline in delivery efficiency, AAV-9 is the optimal choice for delivering Lars2 mRNA to aged tendon tissue, exhibiting significant advantages in enhancing mRNA expression levels and improving tissue structure repair. These screening results provide a practical basis for selecting viral vectors to implement AAV-Lars2 mRNA treatment strategies in different age groups.

[0039] Example 3:

[0040] In this embodiment, to verify the functional role of Lars2 in the repair of aged tendon tissue, the applicant constructed an Achilles tendon injury model in 80-week-old C57BL / 6 mice and intervened by overexpressing Lars2 through AAV9-mediated gene delivery. Mice were randomly divided into three groups post-surgery: the AAV-Lars2 group (receiving injection of recombinant virus containing Lars2), the AAV-Empty group (receiving injection of empty virus), and a 20-week-old control group (natural repair). Injection was performed locally around the Achilles tendon on day 1 after tendon injury to evaluate the effect of Lars2 overexpression on the improvement of tendon repair status, particularly its influence on fibrosis and heterotopic ossification.

[0041] The virus used was the AAV9 subtype, which has high tendon affinity. The vector was constructed based on pAAV-CAG-MCS-3FLAG, with the full-length coding sequence of mouse Lars2 (NM_026808.4) inserted. Expression was driven by the CAG promoter, and a 3xFLAG tag was fused to the C-terminus for detection. The expression cassette structure was: 5′-ITR-CAG-Lars2 CDS-3xFLAG-WPRE-hGHpA-ITR-3′. The recombinant virus was packaged by a commercial service company at a titer of 1×10⁻⁶. 13 Each mouse was injected with 10 μL of virus solution at a dose of vg / mL. The mice were observed post-injection, and tissue samples were collected on day 21 post-surgery for histological and molecular evaluation.

[0042] The results showed that the AAV-Lars2 group was superior to the empty vector group in terms of tendon structural integrity, collagen fiber arrangement, degree of ectopic calcification, and expression of fibrosis markers, with some indicators approaching the levels of 20-week-old young control mice. Masson, Alizarin Red, and α-SMA staining results indicated that Lars2 overexpression significantly reduced fibrosis and inhibited the Runx2-positive ossification trend. qPCR and immunohistochemistry further verified the changes in related gene expression. This embodiment proposes for the first time that AAV-mediated Lars2 mRNA delivery can serve as an effective means of intervening in the repair and decline of tendon injury in the elderly, demonstrating a clear tissue repair promoting effect and application prospects.

[0043] Example 4:

[0044] This embodiment provides an in vivo experimental strategy for revealing the anti-aging molecular mechanism of Lars2 overexpression through combined transcriptomic and proteomic analysis. Based on AAV-mediated Lars2 overexpression, this embodiment evaluates its impact on tissue aging-related pathways in an 80-week-old (80W) mouse tendon injury model.

[0045] In the experiment, mice were treated with AAV-Lars2 and AAV empty vector, respectively, for 21 days. Achilles tendon tissue was then harvested for bulk RNA sequencing and TMT-labeled quantitative proteomics analysis. RNA-seq data were processed using DESeq2 for differential expression analysis, screening for key upregulated genes related to anti-aging functions, including telomere protection and elongation-related genes such as Tert, Terf1, and Pot1, resulting in a volcano plot, as shown below. Figure 4 As shown in the single-cell sequencing results, Lars2 overexpression, compared to the empty vector group, revealed upregulation of key genes related to anti-aging function (including telomere protection and elongation-related genes Tert, Terf1, and Pot1), while the expression of aging markers P16 and P21 was significantly downregulated. This indicates that Lars2 overexpression can activate the telomere maintenance mechanism and delay the tissue aging process.

[0046] In the proteomics results, the Lars2 overexpression group significantly upregulated multiple proteins related to mitochondrial function, such as key enzymes of oxidative phosphorylation (COX4I1, ATP5F1B), TCA cycle enzymes (IDH3A, CS), and NAD. + Metabolic enzymes (NAMPT, NMNAT1). In addition, the protein levels of stemness maintenance-related factors (SOX2, NANOG) were synchronously upregulated, while the expression of aging markers P16 and P21 was significantly downregulated, consistent with the transcriptome trend.

[0047] Further GO enrichment analysis was performed, and the results are as follows: Figure 5 As shown, the upregulated genes were significantly enriched in typical anti-aging pathways such as oxidative phosphorylation, telomere maintenance, stemness module, and mitochondrial translation. Meanwhile, the empty vector control group showed activation of pathways such as cellular senescence, inflammatory response, and ribosome stress, suggesting weaker repair capabilities and a tendency towards aging.

[0048] Integrating RNA and proteomic data revealed that Lars2 overexpression not only improved mitochondrial metabolism and redox homeostasis but also activated the stemness maintenance and telomere elongation signaling axis, demonstrating a synergistic anti-aging effect. This effect was particularly pronounced in the tendon tissue of aged mice, exhibiting good in vivo stability and tissue protection.

[0049] In summary, this embodiment is the first to verify at the tissue level the key molecular mechanism by which Lars2 overexpression delays tendon tissue aging and enhances repair potential, providing multi-omics evidence and target basis for the clinical translation of Lars2 in the field of anti-aging therapy.

[0050] Example 5:

[0051] This embodiment provides a method for cellular-level verification of the anti-aging function of AAV-mediated Lars2 overexpression. A hydrogen peroxide (H2O2)-induced senescence model of mouse 3T3 fibroblasts was used to systematically evaluate the regulatory effects of Lars2 on key aging-related biomarkers, antioxidant capacity, and matrix differentiation trends under oxidative stress. First, 3T3 cells were cultured to 60%–70% confluence and treated with 200 μM H2O2 for 24 hours to establish a cellular senescence model. After treatment, the cells exhibited typical senescent morphology, decreased proliferation capacity, and immunofluorescence detection showed significantly enhanced p16 (CDKN2A) expression in the cell nucleus, confirming the successful establishment of the model.

[0052] After the model was established, the cells were divided into two groups for intervention: one group was given AAV9 virus expressing an empty vector plasmid (empty vector group), and the other group was given recombinant AAV9 virus expressing mouse Lars2 (AAV-Lars2 group), with a viral concentration of 1×10⁻⁶ in both groups. 11 vg / mL. After 48 hours of treatment, cell samples were collected for qPCR, Western blot, and immunofluorescence multilabel staining analysis. qPCR results showed that the p16 mRNA level decreased by approximately 63% after AAV-Lars2 treatment compared to the empty vector group; immunofluorescence results showed... Figure 6 As shown in c and g, the proportion of p16 positive cells decreased from 84.2% to 29.7%, and the average intensity of intranuclear fluorescence signal decreased by about 70%, indicating that Lars2 overexpression can significantly inhibit the aging phenotype.

[0053] Further testing was conducted on antioxidant-related biomarkers and matrix differentiation indicators. The results are as follows: Figure 7 As shown: In the AAV-Lars2 group, the expression and nuclear localization of the antioxidant transcription factor Nrf2 were enhanced, and the fluorescence intensity increased by 2.3 times, as shown. Figure 7 As shown in g and k; the level of mitochondrial superoxide dismutase SOD2 was also significantly increased, suggesting that Lars2 can enhance cellular antioxidant stress defense. Furthermore, compared with the cartilage matrix-related gene Col2a1 induced by H2O2 treatment, the expression of this gene was significantly downregulated in the AAV-Lars2 treatment group, with a fluorescence intensity decrease of more than 50%, as shown in g and k. Figure 7 The h and L values ​​indicate that Lars2 helps suppress the chondrogenic tendency under stress conditions and maintain tendon cell homeostasis.

[0054] In summary, the results of this embodiment demonstrate that AAV-mediated Lars2 overexpression can effectively inhibit p16 expression, enhance antioxidant capacity, and reduce Col2a1 expression in an oxidative stress-induced fibroblast senescence model, exhibiting clear anti-aging and cellular homeostasis-protective effects. This study provides important cellular-level evidence for the application of Lars2 as a gene therapy target in tissue regeneration and aging intervention.

[0055] Example 6:

[0056] To evaluate the reproducibility and broad applicability of the anti-aging effect of Lars2 overexpression in different tissue backgrounds, this embodiment further established a multi-tissue-derived fibroblast model and systematically compared the functional consistency of Lars2 in different cell types. Healthy adult C57BL / 6J mice were selected, and nine representative tissues, including skin, lung, and kidney, were collected. Primary cells were prepared using a combined collagenase and trypsin digestion method. All tissues were thoroughly washed, chopped, and placed in a mixed enzyme solution containing 0.1% collagenase I and 0.25% trypsin. Digestion was carried out at 37°C for 45 minutes, after which the reaction was terminated. The cells were filtered through a 70μm cell sieve and cultured adherently. After 24 hours, non-adherent cells were discarded, and the cells were continuously cultured to form typical fibroblast morphology. Under a microscope, the cells were elongated spindle-shaped with elliptical nuclei and active division.

[0057] After the cells stabilized, an early aging model induced by oxidative stress was established by treating them with 200 μM H2O2 for 24 hours. Following treatment, cells generally exhibited signs of aging such as flattened morphology, increased cytoplasmic granules, and coarsened nuclear chromatin. After successful model establishment, fibroblasts from various tissues were divided into two groups: the control group received empty vector AAV9 virus (1 × 10⁻⁶). 11 The control group received a 100% (vg / mL) dose of AAV9-Lars2 virus (expressing CAG-driven mouse Lars2 full-length cDNA), while the experimental group received the same dose. After virus transfection, the cells were cultured for 48 hours, followed by molecular detection and morphological analysis.

[0058] At the molecular level, qPCR analysis showed that Lars2 overexpression significantly downregulated the mRNA expression level of the aging marker p16 in four fibroblast cell types, with an average decrease of 52.3%–70.4% compared to the empty vector group (P<0.01). Figure 8Meanwhile, the antioxidant defense-related gene Nrf2, namely Sod1, was significantly upregulated, with an average increase of 3.9-7.2-fold (P<0.01). Immunofluorescence assays further confirmed these changes. In the empty vector group, p16 was mainly located in the cell nucleus, with strong and widespread fluorescence signals, while in the Lars2 overexpression group, the p16 signal was significantly weakened, and the proportion of positive cells decreased significantly. In addition, Nrf2 showed enhanced nuclear translocation, and Sod1 showed enhanced mitochondrial signaling, suggesting that Lars2 overexpression can activate antioxidant protection mechanisms in multiple cell types.

[0059] At the phenotypic level, Col2a1 (type II collagen), a stress-related chondrogenic tendency marker, is noteworthy. H2O2 treatment significantly induced the upregulation of Col2a1 expression, but after Lars2 overexpression intervention, all four cell types showed a significant decrease in Col2a1 expression, with an average reduction of 41.6% (P<0.05). The decrease in Col2a1 protein levels was most pronounced in tendon-derived fibroblasts, indicating that Lars2 not only has anti-aging effects but also inhibits the activation of maladaptive matrix programs under chronic stress, contributing to the maintenance of cell phenotypic stability (see...). Figure 9 ).

[0060] The results consistently demonstrate that AAV-mediated Lars2 overexpression exhibits a consistent molecular response trend across fibroblasts from four diverse tissue sources: inhibiting p16 expression, enhancing NRF2 / Sod1-mediated antioxidant defense, and downregulating Col2a1 matrix production. This effect shows minimal variation across cell types and a low standard deviation, demonstrating good reproducibility and stability. This indicates that the mechanism is not specific to any particular tissue background but possesses clear biological universality and broad application potential.

[0061] In summary, this embodiment systematically verified the anti-aging function of Lars2 in fibroblasts from multiple tissue sources, providing experimental evidence for the stable expression regulation of this mechanism across cell types. AAV-Lars2, as a universal anti-aging intervention, possesses broad tissue adaptability, providing a solid cellular basis and scientific support for its development into intervention strategies for systemic age-related diseases.

Claims

1. An application of Lars2 in tissue repair and regeneration, characterized in that: Upregulating Lars2 expression can improve mitochondrial metabolic function, reduce oxidative stress, delay cell aging, remodel cell metabolism, and enhance its tissue repair and regeneration capabilities.

2. The application of Lars2 in tissue repair and regeneration according to claim 1, characterized in that: Lars2 expression was upregulated by more than 2-fold.

3. The application of Lars2 in tissue repair and regeneration according to claim 1 or 2, characterized in that: AAV-9-mediated Lars2 overexpression can effectively improve collagen arrangement, reduce heterotopic ossification, and decrease fibrosis in aging tendons. The validation mechanism of this application is demonstrated by the following steps: The first step was to establish a mouse tendon injury model and perform single-cell RNA sequencing analysis, which revealed that the most significantly differentially expressed gene in the tendon fibroblasts was Lars2. Lars2 encodes mitochondrial leucyl tRNA synthetase, which is mainly involved in mitochondrial protein synthesis and energy metabolism regulation. Its decreased expression leads to mitochondrial dysfunction, reactive oxygen species accumulation and energy metabolism disorder, thereby affecting the repair capacity of aging tendon tissue. The second step involved constructing different AAV vectors and locally injecting three different subtypes of recombinant AAV virus fluid into the tendon injury site in mice. An empty AAV vector was set up as a control group. Total RNA was extracted for qPCR to detect Lars2 mRNA expression level, and Lars2 protein level was also detected. Immunohistochemical staining was performed to locate the target tissue and analyze the differences in the efficiency of different AAV vectors in delivering Lars2 mRNA in tendon tissue in mice at different age stages. The experiment showed that AAV vectors can effectively mediate Lars2 expression. The AAV-9 group showed stronger Lars2 signal intensity and distribution range in aged tendon tissue. Immunohistochemical analysis showed that AAV-9-mediated Lars2 overexpression can effectively improve collagen arrangement, reduce the degree of heterotopic ossification and fibrosis in aged tendons. AAV-9 is the optimal choice for delivering Lars2 mRNA to aged tendon tissue. The third step involved verifying the improvement in fibrosis / ossification through AAV-9-Lars2 treatment. Transcriptomic and proteomic analyses were performed on mice after treatment, showing that key genes related to anti-aging function were upregulated, while the expression of aging markers was significantly downregulated. Lars2 overexpression can activate the telomere maintenance mechanism and delay the tissue aging process. The fourth step involved validating the anti-aging function of AAV-mediated Lars2 overexpression using cell models. A hydrogen peroxide-induced mouse 3T3 fibroblast senescence model was used to systematically evaluate the regulatory effects of AAV-mediated Lars2 on key aging-related biomarkers, antioxidant capacity, and matrix differentiation trends under oxidative stress. qPCR, Western blot, and immunofluorescence multilabeling analysis of collected cell samples showed that AAV-mediated Lars2 overexpression effectively inhibited p16 expression, enhanced antioxidant capacity, and reduced Col2a1 expression in the oxidative stress-induced fibroblast senescence model, demonstrating a clear anti-aging and cell homeostasis protection effect. The fifth step involved multi-tissue-derived cell validation. By establishing multi-tissue-derived fibroblast models, the functional consistency of Lars2 in different cell types was systematically compared. Primary cells were prepared from four representative tissues (skin, lung, heart, and kidney) using a combined collagenase and trypsin digestion method to establish an early aging model induced by oxidative stress. Molecular detection and morphological analysis after viral transfection showed that Lars2 overexpression significantly downregulated the mRNA expression level of the aging marker p16 in the four fibroblast types, while significantly upregulating antioxidant defense-related genes NRF2 and mitochondrial SOD2. Lars2 overexpression activated antioxidant protection mechanisms in multiple cell types, and Col2a1 expression was significantly decreased. This indicates that AAV-Lars2 can inhibit the activation of maladaptive matrix programs under chronic stress, helping to maintain cell phenotypic stability and demonstrating clear biological universality and broad application potential.

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