Mechanism of action of elastin fragment in prevention and antagonism of aging and application of elastin fragment

Abstract

The invention provides an action mechanism of an elastin fragment in prevention and antagonism of aging and application of the elastin fragment in prevention and antagonism of aging, and finds that aging is accompanied by increase of the concentration of the elastin fragment in serum, and the elastin fragment can cause various unhealthy states related to aging and promote occurrence of various senile diseases, so that the life is shortened, and aging is caused; the mechanism is that after an elastin fragment is combined with a receptor thereof, downstream signals are conducted through NEU1 protein, chronic inflammation is caused, senescence is caused, senescence-related phenotypes caused by the elastin fragment are reversed by inhibiting Neu1 genes, and Neu1 protein is a target spot for antagonizing the elastin fragment effect and preventing and delaying senescence. Therefore, the invention discloses a brand new senescence mechanism, and provides a new idea for developing medicines for preventing and delaying senescence.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to an action mechanism of an elastin fragment in preventing and antagonizing aging and its application. Background Art

[0002] The following description is only for readers to understand the present invention and does not constitute any limitation to the present invention.

[0003] Aging and a variety of geriatric diseases are accompanied by abnormal remodeling and excessive degradation of the extracellular matrix (ECM), which increases the free ECM fragments in serum, urine, synovial fluid and other fluids. Recent studies have shown that ECM degradation products are not inert and can further lead to tissue damage and metabolic disorders.

[0004] Elevated concentrations of extracellular matrix fragments in the blood have been found to cause inflammation and damage to tissues and organs. For example, EDP (elastin-derived peptide) containing the VGVAPG motif has chemotactic properties for several immune cells such as monocytes, lymphocytes and neutrophils. It also polarizes human blood lymphocytes to Th1 by stimulating the MAPK-AP-1-NF-kB pathway, inducing MMPs expression and positive feedback to enhance elastin hydrolysis. In terms of inducing tissue and organ damage, injection of EDP fragments into mice accelerates the progression of atherosclerosis.

[0005] Higher levels of inflammatory factors are expressed in cells and tissues of elderly individuals, placing the body in a state of low-level, long-term pro-inflammatory response. Severe, transient inflammation is an evolutionarily conserved mechanism used to protect the body from infection and injury, and chronic inflammation has been considered a biomarker for accelerated aging. Epidemiological studies have found that chronic inflammatory states are associated with most age-related diseases, such as cancer, type II diabetes, cardiovascular disease, neurodegenerative diseases, and geriatric syndromes. At the same time, alleviating this chronic inflammatory state has been shown to delay aging, and its strategies include reducing dietary intake, increasing exercise, and regulating stress, but there is still a lack of effective drugs to treat this chronic inflammatory state. However, there is currently no research on elastin fragments and systemic aging. Antagonizing the elastin fragment receptor complex and its downstream pathways to antagonize aging-related phenotypes and delay aging is also an area that has not been explored.

[0006] Therefore, it is urgent to find the mechanism of action and application of elastin fragments in preventing and antagonizing aging. By in-depth research on elastin fragments and their related mechanisms, targeted inhibitors and antagonists can be developed to accurately assess the aging process and provide a basis for early intervention and treatment. This is expected to solve the limitations of current anti-aging methods, effectively delay the emergence of aging-related phenotypes, and open up new ways to prevent and treat geriatric diseases, ultimately achieving the effect of extending healthy life span and improving quality of life. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides an action mechanism of an elastin fragment in preventing and antagonizing aging and its application. Through in-depth research on elastin fragments and their related action mechanisms, specific inhibitors and antagonists for elastin fragment receptor complexes and their downstream pathways are developed, which solves the current problems of insufficient understanding of the relationship between elastin fragments and aging, lack of effective anti-aging measures, and inability to accurately assess the aging process. It has the functions of accurately detecting the degree of aging, providing a reliable basis for early intervention and treatment, effectively delaying the appearance of aging-related phenotypes, and reducing the risk of geriatric diseases. It has good application prospects in the field of aging research and the prevention and treatment of related diseases.

[0008] In order to achieve the above object, the present invention adopts the following scheme:

[0009] In one aspect, the present invention provides a use of a marker for preparing a reagent for predicting whether an individual is in the aging process, wherein the marker comprises any one or more selected from the following: elastin fragments, collagen fragments, fibronectin fragments and hyaluronic acid fragments.

[0010] Furthermore, the marker is an elastin fragment.

[0011] In the field of anti-aging research, due to the complexity and severity of aging, it is urgent to find biomarkers that accurately indicate the aging process. Based on the key role of the extracellular matrix in maintaining tissue and organ function and its possible association with aging, in some embodiments, a variety of free extracellular matrix fragments in serum, including elastin fragments (ELN), collagen fragments (COL), fibronectin fragments (FN) and hyaluronic acid fragments (HA), are studied. Serum samples of people and mice of different ages were collected, and the contents of these fragments were accurately measured. The results showed that the serum content of various ECM fragments was positively correlated with age, among which the positive correlation between elastin fragments and age increase was the most significant and stable.

[0012] However, it is important to note that correlation does not necessarily mean that it is a marker, but that the marker must be correlated. Figure 1All extracellular matrix fragments in the human body are related to aging, but not all of them can be used as markers. Only those that are related and cause aging are true aging markers. With aging, many substances will appear, but conversely, if these substances appear, it does not necessarily mean aging. On the contrary, if a certain substance appears, it will cause aging, and if it does not appear, there will be no aging. Such substances are important as markers.

[0013] Corresponding experimental verification was carried out, and the results showed that although collagen fragments, fibronectin fragments and hyaluronic acid fragments had certain effects, they did not significantly cause aging. The effect of elastin fragments was extremely significant, and 50% of the mice died when the mice were about 90 weeks old. In contrast, even if some symptoms appeared in the HA, FN and COL groups, the differences in the circumstances leading to death were not significant among the three. However, the ELN group reached a significant level compared with the other three groups and the control. This fully proves that only elastin fragments are the important markers indicating aging in the present invention.

[0014] Therefore, in the present invention, collagen fragments, fibronectin fragments and hyaluronic acid fragments are only positively correlated with aging, but the appearance of these fragments does not necessarily lead to aging, so they cannot be used as markers of aging. Elastin fragments are not only related to aging, but their appearance will definitely lead to aging, and the effect is the most significant. Therefore, in the present invention, only elastin fragments can be used as important markers of aging.

[0015] Furthermore, the elastin fragment comprises a motif "(GXXPG)n", wherein "X" is any amino acid except proline, and n is an integer ≥1.

[0016] Furthermore, the elastin fragment includes any one or more of the motifs VGVAPG, PGAIPG, and GAVPG.

[0017] Furthermore, the elastin fragment comprises the motif VGVAPG.

[0018] In some embodiments, when the elastin fragment motif is selected as VGVAPG (ie, E-motif), it has the most significant effect on mouse aging, and the specific mechanism of action of the elastin fragment E-motif binding to the elastin receptor complex to activate the downstream pathway leading to aging-related physiological changes is: after the E-motif in elastin binds to the elastin receptor complex, the 61kDa membrane protein NEU1 plays a key signal transduction role, transmitting the activation signal to the cell, such as activating the ERK 1 / 2 pathway to regulate cell proliferation, differentiation and survival, while upregulating MMP1 expression, leading to extracellular matrix degradation and reconstruction. In addition, E-motif has chemotactic and pro-inflammatory effects on monocytes, activates CD4+T cells and induces the production of inflammatory cytokines, polarizes Th cells by activating the MAPK-AP-1-NF-kB pathway, triggers inflammatory responses and immune abnormalities, thereby leading to aging-related physiological changes, such as tissue damage and immune dysfunction.

[0019] Furthermore, the reagent is used to detect the presence or relative abundance or concentration of elastin fragments in individual serum samples.

[0020] Furthermore, the aging includes the aging of animal cells, tissues, organs or individuals.

[0021] In some embodiments, the aging individual is a mouse; aging at the tissue level is adipose tissue, muscle tissue, connective tissue; aging at the organ level is heart, liver, spleen, lung, brain, muscle, bone, and kidney.

[0022] Specifically, the chronic inflammatory state of the body includes chronic inflammatory states caused by aging, obesity, cellular senescence and its aging-related secretory phenotype, impaired recycling and elimination of degraded cellular components, chronic infection and defective immune cells, changes in small intestinal barrier permeability, etc.

[0023] On the other hand, the present invention provides a kit for predicting whether an individual is in the aging process, the kit comprising a detection reagent for a marker for the use as described in any one of the above technical solutions.

[0024] In yet another aspect, the present invention provides a system for predicting whether an individual is in the aging process, the system comprising a data analysis module; the data analysis module is used to analyze the detection value of a biomarker, wherein the biomarker comprises an elastin fragment.

[0025] Furthermore, the system also includes a data storage module, a data input interface and a data output interface; the data storage module is used to store the detection values ​​of biomarkers; the data input interface is used to input the detection values ​​of biomarkers, and the data output interface is used to output the prediction results.

[0026] In some embodiments, in order to further verify the accuracy and reliability of elastin fragments as systemic aging markers, serum sample detection and multiple aging-related index determination experiments were carried out. Using UPLC-MS / MS detection method, the elastin fragments, collagen fragments, fibronectin fragments and hyaluronic acid fragments in the serum of mice of different ages were measured, and the motor performance, survival time, aging-related serum indexes, etc. of mice of different groups were comprehensively evaluated. The final result found that when the content of elastin fragments in serum exceeded the normal range (less than 300ng / mL), mice were in the accelerated aging stage. In the present invention, the AUC value, accuracy, sensitivity and specificity of elastin fragments were the highest, and its diagnostic efficacy was also excellent in multiple batches of different validation set tests, significantly higher than collagen fragments, fibronectin fragments and hyaluronic acid fragments. The diagnostic efficacy of several other extracellular matrix fragments was relatively low, and the aging process of mice could not be accurately predicted. Therefore, only elastin fragments are effective markers that can accurately and reliably predict aging, providing valuable detection indicators for aging-related research and clinical applications.

[0027] In another aspect, the present invention provides a use of an agent targeting elastin fragments for preparing an agent for preventing or antagonizing aging, wherein the agent targeting elastin fragments includes an agent that inhibits the generation of elastin fragments and / or an agent that promotes the hydrolysis of elastin fragments.

[0028] Furthermore, the agent for inhibiting the generation of elastin fragments includes one or more agents for inhibiting the expression of elastase, agents for inhibiting the activity of elastase or reducing its content, or agents for inhibiting the expression of elastase gene.

[0029] Furthermore, the reagent for inhibiting the expression of elastase includes a reagent for eliminating, knocking out, or knocking down the expression of the elastase gene; the reagent for inhibiting the expression of the elastase gene includes any one or more of siRNA, shRNA, or dsRNA targeting the elastase gene.

[0030] Furthermore, the agent for inhibiting the activity of elastase or reducing its content includes one or more of an elastase inhibitor, an elastase antibody, and a PROTAC agent targeting elastase.

[0031] Furthermore, the elastase inhibitor includes any one or more of extracellular matrix enzyme inhibitors, neutrophil elastase inhibitors, matrix metalloproteinase inhibitors, serine protease inhibitors, and cysteine ​​protease inhibitors.

[0032] Furthermore, the elastase inhibitor includes neutrophil elastase inhibitor Ly.

[0033] In some embodiments, the reagent that inhibits the activity of elastase or reduces its content includes inhibitors of the following enzymes, and the types of enzymes specifically include: neutrophil elastase, matrix metalloproteinases (matrix metalloproteinases 2, 7, 9, 12, 14), serine proteases (neutrophil elastase, cathepsin G and proteinase-3), cysteine ​​proteases (cathepsin K, L, S and V), etc., all enzymes that can hydrolyze the extracellular matrix including elastin.

[0034] In some embodiments, the inhibitor can reduce the degradation of elastin and the generation of elastin fragments by inhibiting the activity of elastase; or, the inhibitor can reduce the generation of extracellular matrix fragments by inhibiting the activity of extracellular matrix degrading enzymes including elastase.

[0035] In some embodiments, the reagent may be a medicine, a health product, or a research reagent, etc.; the application of the reagent includes various injection concentrations, various injection frequencies, durations, and various injection methods.

[0036] In yet another aspect, the present invention provides a use of an elastase inhibitor for preparing an agent for preventing and / or treating chronic inflammation.

[0037] Furthermore, the elastase inhibitor prevents and / or treats chronic inflammation by inhibiting the expression of inflammatory factors IL-1, MCP-1 or TNF-α.

[0038] In some embodiments, an elastase inhibitor Ly was used to observe its effect on mouse serum inflammatory factors. The results showed that the elastase inhibitor Ly could significantly inhibit the inflammatory phenotype of mice. Compared with the control group, the protein expression levels of MCP-1, IL-1, and TNF-α in the Ly experimental group were significantly downregulated, indicating that elastase can indirectly regulate the secretion of inflammatory factors and can be used as a therapeutic target. The detection of 23 inflammatory factors in serum showed that the expression of inflammatory factors in the Ly group mice was generally downregulated compared with the control group that only received Vehicle injection, indicating that the elastase inhibitor Ly at least partially inhibited the chronic inflammation associated with aging.

[0039] In another aspect, the present invention provides a use of an elastase inhibitor for preparing an agent for preventing and / or treating osteoarthritis and osteoporosis.

[0040] In some embodiments, in view of the key role of elastin fragments in the aging process, the present invention further targets the above-mentioned targets in the subsequent experimental process to explore their application in improving aging-related diseases. The results show that the elastase inhibitor Ly can significantly improve the condition of osteoarthritis in the aging process and alleviate the damage and wear of aging-related joint cartilage; at the same time, the OARSI score of the cartilage tissue in the Ly group is significantly lower than that of the control group mice, proving that controlling the production of elastin fragments by inhibiting elastase has a definite and significant effect on delaying and improving aging-related osteoarthritis symptoms, providing a strong experimental basis and new research direction for future treatment strategies for aging-related osteoarthritis.

[0041] In some embodiments, "elastase inhibitor Ly" is used to antagonize elastin fragments to improve aging-related osteoporosis. The results show that elastase inhibitor Ly can significantly improve osteoporosis-related symptoms in the aging process and improve bone-related indicators. Parameters such as the ratio of bone volume to tissue volume (BV / TV) and trabecular thickness (Tb.Th) in the Ly group are significantly higher than those in the control group mice, proving that inhibiting elastase to control the production of elastin fragments can improve osteoporosis-related indicators such as bone density, and intervening in elastase activity can be used as a potential therapeutic target.

[0042] Furthermore, the elastase inhibitor prevents and / or treats osteoarthritis and osteoporosis by inhibiting the generation of elastin fragments.

[0043] In some embodiments, "elastase inhibitor Ly" is used to antagonize elastin fragments to improve the symptoms of aging-related osteoarthritis model. The results show that for osteoarthritis caused by different pathological factors (intra-articular injection of sodium iodoacetate or medial meniscus destabilization surgery of the knee joint), elastase inhibitor Ly significantly improved the pathological conditions of arthritis in mice, further verifying the effectiveness and potential of elastase inhibitors in the treatment of osteoarthritis, and can be used as a potential effective drug for the treatment of osteoarthritis, providing new ideas and methods for the treatment of osteoarthritis.

[0044] In another aspect, the present invention provides a use of an agent for inhibiting the binding of elastin fragments to receptors for preparing an agent for preventing or antagonizing aging.

[0045] Furthermore, the elastin fragment comprises a motif "(GXXPG)n", wherein "X" is any amino acid except proline, and n is an integer ≥1.

[0046] Furthermore, the receptor includes the membrane protein NEU1.

[0047] Furthermore, the reagents for inhibiting the binding of elastin fragments to receptors include one or more of antibodies or antibody fragments capable of binding to elastin fragments, antibodies or antibody fragments capable of binding to NEU1, reagents for eliminating, knocking out, or knocking down NEU1 gene expression, or elastin fragment receptor complex inhibitors.

[0048] Furthermore, the elastin fragment receptor complex inhibitor includes a cathepsin A inhibitor, an elastin binding protein EBP inhibitor, and a sialidase NEU1 inhibitor.

[0049] In another aspect, the present invention provides a use of a reagent for knocking out NEU1 gene expression for preparing a reagent for reducing the content of serum inflammatory factors, wherein the inflammatory factors include any one or more of IL-1, MCP-1 or TNF-α.

[0050] In some embodiments, in order to further explore the intrinsic mechanism of aging caused by the elastin fragment E-motif and to find potential intervention targets, the effect of knocking out the Neu1 gene on the body weight of mice injected with E-motif was explored. The results show that specific knocking out of the Neu1 gene blocks the downstream effects of E-motif. When samples were collected at 28 weeks of age, the body weight of the Neu1(f / f)+E-motif group had increased significantly compared with the Neu1(f / f) mice, while the body weight of the Neu1(- / -)+E-motif group mice remained at the same level as the control group, which was significantly lower than that of the Neu1(f / f)+E-motif group. This result confirms the negative effect of the Neu1 gene in affecting the downstream body weight of E-motif; therefore, it can be seen that the Neu1 gene is a key molecular target for controlling the downstream E-motif effects, and intervention on the Neu1 gene can antagonize the effects of aging.

[0051] In some embodiments, in order to more fully understand the relevant mechanisms of aging caused by the elastin fragment E-motif, the effect of knocking out the Neu1 gene on the levels of inflammatory factors IL-1 and MCP-1 in the serum of mice injected with E-motif was further explored. The results show that specific knockout of the Neu1 gene blocks the downstream effects of E-motif. When samples were collected at 28 weeks of age, the IL-1 and MCP-1 levels in the Neu1(f / f)+E-motif group were significantly higher than those in the Neu1(f / f) group and the Neu1(- / -)+E-motif group. This result confirms the negative effect of the Neu1 gene in affecting the increased inflammatory levels downstream of the E-motif. Therefore, it can be seen that the Neu1 gene is a key molecular target for controlling the downstream E-motif effects, and intervention on the Neu1 gene can antagonize the effects of aging.

[0052] In yet another aspect, the present invention provides a use of an agent for knocking out NEU1 gene expression for preparing an agent for preventing and / or treating aging-related liver diseases.

[0053] In some embodiments, after knocking out the Neu1 gene in the elastin receptor complex, the elastin fragments are antagonized and aging-related liver diseases are improved. The results show that knocking out the Neu1 gene significantly improved liver function, and the levels of alanine aminotransferase and aspartate aminotransferase returned to normal. Liver histopathology showed that the morphology of liver cells tended to be normal, the infiltration of inflammatory cells decreased, and the degree of liver fibrosis was significantly reduced. This fully proves that knocking out the Neu1 gene in the elastin receptor complex can effectively antagonize elastin fragments and significantly improve the symptoms of aging-related liver diseases, providing new ideas and potential targets for the treatment of liver diseases.

[0054] In another aspect, the present invention provides a use of an elastin fragment receptor complex inhibitor for preparing an agent for preventing or antagonizing aging.

[0055] In another aspect, the present invention provides a use of an elastin fragment receptor complex inhibitor for preparing an agent for preventing and / or treating aging-related cardiovascular diseases.

[0056] In some embodiments, "DANA, an inhibitor of elastin fragment receptor complex", is used to antagonize elastin fragments to improve aging-related cardiovascular diseases. The results show that DANA, an inhibitor of elastin fragment receptor complex, significantly improves cardiovascular function, reduces heart rate and blood pressure, and increases cardiac output. At the same time, the thickness of arterial walls is reduced, atherosclerotic plaques are significantly reduced, and the levels of inflammatory factors and oxidative stress markers in the blood are significantly reduced.

[0057] Therefore, it can be seen that the "elastin fragment receptor complex inhibitor DANA" can effectively antagonize elastin fragments and has a significant improvement effect on aging-related cardiovascular diseases, providing a new potential direction and effective strategy for the treatment of aging-related cardiovascular diseases.

[0058] In another aspect, the present invention provides a use of an elastin fragment receptor complex inhibitor for preparing an agent for improving athletic ability or reducing scoliosis.

[0059] In some embodiments, the elastin fragment receptor complex inhibitor DANA is used to observe its effect on the motor ability of mice, thereby verifying that it has the effect of delaying the aging phenotype. The results show that the elastin fragment receptor complex inhibitor DANA can significantly delay the decline in the motor ability of mice. Compared with the Vehicle group, the number of electric shocks in the DANA experimental group mice was significantly lower, indicating that the running endurance of the DANA experimental group mice was stronger; the results of the open field experiment showed that the total movement distance and average speed of the DANA group mice were significantly higher than those of the Vehicle group, and the range of movement was wider; therefore, it can be seen that the elastin fragment receptor complex inhibitor DANA can antagonize elastin fragments and significantly delay the decline in the motor ability of mice. The elastin fragment receptor complex and its downstream pathways can be used as potential therapeutic targets.

[0060] In some embodiments, the elastin fragment receptor complex inhibitor DANA is used to observe its effect on the spinal curvature of mice, thereby verifying its effect of delaying the aging phenotype. The results show that the elastin fragment receptor complex inhibitor DANA can significantly alleviate the spinal curvature of mice related to aging. Compared with the Vehicle group, the spinal curvature of the mice in the DANA experimental group is low, and the quantitative analysis results show that the KI value of the mice in the DANA experimental group is significantly increased. Therefore, it can be seen that the elastin fragment receptor complex inhibitor DANA can antagonize elastin fragments and significantly alleviate the "hunchback" phenotype of naturally aged mice. The elastin fragment receptor complex and its downstream pathways can be used as potential therapeutic targets.

[0061] In another aspect, the present invention provides a use of an elastin fragment receptor complex inhibitor for preparing a reagent for reducing the content of aging-related serum indicators, characterized in that the aging-related serum indicators include any one or more of AST, ALT, GLU or LDL.

[0062] In some embodiments, the elastin fragment receptor complex inhibitor DANA was used to verify its effect on aging-related serum indicators. The results showed that the elastin fragment receptor complex inhibitor DANA can significantly reduce the serum aging factor level of mice. Compared with the Vehicle group, the liver function indicators of the DANA experimental group: AST and ALT content were significantly decreased; the serum content of blood glucose GLU and blood lipid LDL also decreased significantly in the DANA group. This shows that DANA at least partially inhibits the aging-related liver function decline and the disorder of glucose metabolism and lipid metabolism, and the elastin fragment receptor complex and its downstream pathways can be used as potential therapeutic targets.

[0063] The beneficial effects of the present invention are:

[0064] 1. The present invention provides a mechanism of action of an elastin fragment in preventing and antagonizing aging and its application. It is the first time that the elastin fragment is an important marker of aging, which provides a new direction and indicator for aging research. According to the newly discovered two-faceted mechanism of action of elastin leading to aging: (1) After the elastin fragment E-motif in elastin binds to the elastin receptor complex, it activates the downstream pathway and leads to aging-related physiological changes; (2) Elastase promotes elastin degradation, increases the production of elastin fragments, and thus leads to aging; three measures are adopted to specifically interfere with the target: knocking out the NEU1 gene, injecting elastin receptor complex inhibitors, and injecting elastase inhibitors, which confirms that the interference measures can effectively slow down or even reverse aging-related physiological changes and symptoms. Not only does it deeply reveal the new mechanism of aging in theory, but it also provides practical and innovative methods and ideas for anti-aging treatment strategies at the practical application level, bringing new opportunities and hopes for the development of the anti-aging field.

[0065] 2. In the present invention, the close relationship between elastin fragments and aging is clarified for the first time, which can more accurately assess the degree of aging of individuals, avoid the ambiguity and uncertainty of previous detection methods, and provide a reliable basis for personalized aging assessment and intervention strategy formulation.

[0066] 3. Starting from the perspective of intervening elastase, the present invention reveals the mechanism of action of elastin fragments in the aging process, fills the application gap of extracellular matrix intervention in aging, can further clarify the complex relationship between extracellular matrix and aging, and promote breakthrough progress in basic research related to aging.

[0067] 4. The present invention provides a new target for the research and development of anti-aging drugs, proving that anti-aging can be achieved by inhibiting elastase activity, knocking out the expression gene Neu1, inhibiting the activity of the elastase receptor complex, etc., providing potential targets and strategies for anti-aging treatment; based on the key role of elastin fragments in aging, drug research and development can specifically design drugs that inhibit or regulate its effects, and it is expected to develop more effective anti-aging drugs.

[0068] 5. The mechanism of action of an elastin fragment provided by the present invention in preventing and antagonizing aging and its application are of great significance in clinical application. They can be used for early diagnosis of aging-related diseases such as aging, chronic inflammation, osteoarthritis and osteoporosis, and for taking preventive and therapeutic measures in advance to reduce the incidence and severity of the disease and improve the quality of life of patients.

[0069] 6. The mechanism of action of an elastin fragment in preventing and antagonizing aging and its application provided by the present invention have proven for the first time that inhibiting elastase can reduce the content of pro-inflammatory factors, improve chronic inflammatory indicators, improve cartilage damage in osteoarthritis models, improve osteoporosis-related indicators, and delay aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 A is a scatter plot of the relationship between the ELN, HA, FN, and COL levels in human serum samples and age.

[0071] Figure 1 B is the detection of serum ELN, HA, FN, and COL levels in mouse samples at five age stages (3, 9, 15, 21, and 27 months old).

[0072] Figure 2 These are the experimental results comparing the effects of different extracellular matrix fragments on mouse survival and aging.

[0073] Figure 3 A is a schematic diagram of tail vein injection in mice.

[0074] Figure 3 B is the survival curve of 40-week-old mice after tail vein injection of Vehicle, E-motif and poly(A / K).

[0075] Figure 4 A is a representative whole-body X-ray image of a mouse.

[0076] Figure 4 B is the statistical analysis value of KI value.

[0077] Figure 5 The statistical results of the comparison of mouse body weight data at 28 weeks of age.

[0078] Figure 6 is the levels of serum inflammatory factors IL-1 and MCP-1 in mice in different groups.

[0079] Figure 7 It is the structural formula of DANA, an inhibitor of elastin fragment receptor complex.

[0080] Figure 8 A is the survival curve of male mice.

[0081] Figure 8 B is the survival curve of female mice.

[0082] Fig. 9 A is the change chart of AST content, a liver function index in the Vehicle group and the DANA experimental group.

[0083] Fig. 9B is the change chart of ALT content in the liver function index of the Vehicle group and the DANA experimental group.

[0084] Fig. 9 C is the graph showing the changes in serum blood glucose GLU levels in the Vehicle group and the DANA experimental group.

[0085] Fig. 9 D is the graph showing the changes in serum LDL levels in the Vehicle group and the DANA experimental group.

[0086] Fig.10 A is a comparison of the number of electric shocks given to mice in the Vehicle group and the DANA experimental group.

[0087] Fig.10 B is the open field test picture of mice in the Vehicle group and the DANA experimental group.

[0088] Fig.10 C is the results of the open field test on the total movement distance and average speed changes of mice in the Vehicle group and the DANA experimental group.

[0089] Fig.11 A is a representative whole-body X-ray image of mice in the Vehicle group and the DANA experimental group.

[0090] Fig.11 B is the statistical analysis of KI values ​​of mice in the Vehicle group and DANA experimental group.

[0091] Fig.12 It is the structural formula of the elastase inhibitor Ly.

[0092] Fig.13 This is a heat map of the serum inflammatory factor levels in the Ly group relative to the PBS group.

[0093] Fig.14 A is the survival rate curve of mice in the Ly group and the control group.

[0094] Fig.14 B is the body weight change curve of mice in the Ly group and the control group.

[0095] Fig.14 C is the experimental results of the treadmill electric shock times of the Ly experimental group mice and the control group mice.

[0096] Fig.15 Figure 2 shows the changes in body weight and liver fat indexes related to aging in mice in the Ly group and the control group.

[0097] Fig.16 Comparison of osteoarthritis symptoms during aging between the Ly group and the control group mice, as well as comparison of OARSI scores of cartilage tissue.

[0098] Fig.17 Comparison results of bone-related indicators during the aging process of mice in the Ly group and the control group. DETAILED DESCRIPTION

[0099] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0100] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0101] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0102] In the present invention, mice aged 18 months and above are in the aging process, and mice aged 3 to 6 months are young mice in a normal state.

[0103] Glossary:

[0104] 1. Systemic aging: It is the decline of the absolute life span and healthy life span of an organism over time, accompanied by the decline of the overall structure and overall function. Specifically, there are unhealthy phenotypes such as obesity, upregulation of serum inflammatory factors, liver fat and impaired liver function, damage to the bone and muscle system, decreased exercise capacity, glucose and lipid metabolism disorders, skin aging, etc. In some cases, there are even multi-system geriatric diseases, such as atherosclerosis, aneurysm, heart failure and other cardiovascular diseases; chronic bronchitis, emphysema, chronic obstructive pulmonary disease and other respiratory diseases; systemic sclerosis, Marfan syndrome and other connective tissue diseases; osteoporosis, osteoarthritis and other skeletal muscle system diseases; non-alcoholic fatty liver disease, diabetes and other metabolic diseases; the occurrence and development of tumors, etc. These aging are systemic aging, which actually refers to the symptoms of aging in humans and mammals, rather than the aging of local tissues or cells. The so-called aging of local tissues or cells often only occurs in local tissues, while other tissues are still healthy or in a non-aging state. Systemic aging is when 50%-95%, 100% of the body's tissues show degeneration or aging, accompanied by the failure of the overall structure and function.

[0105] 2. Elastin fragments: Elastin fragments are the products of damaged elastin in the extracellular matrix hydrolyzed by elastase. They will dissolve in body fluids such as serum and cause damage to multiple tissues, thereby accelerating the aging process. The basic structure of the elastin fragment to function is the biologically active sequence "GXXPG", where "X" can be any amino acid except proline, such as VGVAPG, PGAIPG or GAVPG, of which the most typical is the hexapeptide sequence VGVAPG (i.e., E-motif). All of the above sequences can bind to the elastin fragment receptor complex to exert biological effects. In some ways, the active sequence can be a multiple repeated sequence, (GXXPG)n, where "X" can be any amino acid except proline, and n is a positive number equal to or greater than 1. The so-called active sequences above may be obtained by the degradation of the organism itself, or they may be fragments synthesized in vitro. If the content of elastin fragments is found to rise to a certain threshold in humans or mammals, it can be judged that the individual has undergone systemic aging. Of course, if the content of elastin fragments is higher, it means that the systemic aging is more serious. It is understandable that in the blood of a normal human body, the content of elastin is very low, or maintained at a very low level, and is maintained at a low level for a long time. However, in the process of systemic aging, the content of elastin fragments in the blood increases. The increase in this fragment will cause damage to multiple tissues, thereby accelerating the aging process.

[0106] 3. Elastin fragment receptor complex: It is composed of a 67kDa elastin binding protein and 55kDa and 61kDa membrane proteins. The 61kDa membrane protein is called NEU1, which plays a role in transducing the activated signal of the elastin fragment receptor complex into the cell, such as activating the ERK 1 / 2 pathway and upregulating the expression of MMP1. NEU1 mediates some aging-related unhealthy phenotypes and the occurrence of various geriatric diseases caused by elastin fragments, and plays a key role in the process of elastin fragments exerting their effects.

[0107] 3. Inhibitors: If the binding of elastic fragments to NEU1 is inhibited, the signal transmission can be blocked, thereby preventing or delaying aging. Of course, these blocking or inhibitory agents can be any agents, such as monoclonal antibodies, or antibodies or antibody fragments that can bind to elastin fragments, or antibodies or antibody fragments that can compete with NEU1, which can prevent elastin from inhibiting the receptor.

[0108] 4. Elastin fragment receptor complex inhibitors: Elastin fragment receptor complex inhibitors include: cathepsin A inhibitors, elastin binding protein EBP inhibitors, sialidase NEU1 inhibitors. In addition to DANA (sialidase NEU1 inhibitor) given in Example 1 of the present invention, the effects of elastin fragments in serum on aging, aging-related unhealthy phenotypes and geriatric diseases can also be inhibited by cathepsin A inhibitors, elastin binding protein EBP inhibitors, sialidase NEU1 inhibitors, etc. It should be noted that the embodiment of the present invention provides an elastin fragment receptor complex inhibitor, but the types of inhibitors for the elastin fragment receptor complex should not be limited thereto, and substances that can interfere with the elastin fragment receptor complex and thus interfere with the function of the elastin fragment are all within the scope of the present invention.

[0109] 5. Elastin fragment downstream pathway inhibitors: Elastin fragments transmit signals through NEU1 in the receptor complex, and further act on downstream pathways. Such as the PI3K pathway, AKT pathway and ERK1 / 2 pathway, thereby promoting aging. U0126 can inhibit the ERK1 / 2 pathway, thereby targeting the inhibition of the effects of elastin fragments. It should be noted that the embodiments of the present invention provide an elastin fragment pathway inhibitor, but the types of inhibitors for the elastin fragment pathway should not be limited to this. Substances that can interfere with any elastin fragment pathway, thereby interfering with the function of elastin fragments, are all within the scope of the present invention.

[0110] 6. Diagnosis or detection: Diagnosis or detection here refers to the detection or analysis of biomarkers in a sample, or the content of a target biomarker, such as absolute content or relative content, and then the presence or amount of the target marker is used to indicate whether the individual providing the sample may have or suffer from a certain disease, or the possibility of having a certain disease. The meanings of diagnosis and detection here are interchangeable. The result of such a test or diagnosis cannot be directly used as a direct result of illness, but is an intermediate result. If a direct result is obtained, other auxiliary means such as pathology or anatomy are required to confirm that a certain disease is present. For example, the present invention provides a variety of new biomarkers associated with lung cancer, and changes in the content of these markers are directly correlated with whether or not a person has lung cancer.

[0111] 7. The relationship between markers or biomarkers and systemic aging: markers and biomarkers have the same meaning in the present invention. The relationship here means that the appearance of a certain biomarker in a sample or the change in its content has a direct correlation with a specific disease. For example, a relative increase or decrease in its content indicates that the possibility of suffering from this disease is higher than that of healthy people. If multiple different markers appear at the same time in a sample or the content changes relatively, it means that the possibility of suffering from this disease is also higher than that of healthy people. That is to say, among the types of markers, some markers have a strong correlation with the disease, some markers have a weak correlation with the disease, or some are even not associated with a specific disease. For one or more of those markers with strong correlation, they can be used as markers for diagnosing diseases, and those markers with weak correlation can be combined with strong markers to diagnose a certain disease, thereby increasing the accuracy of the test results.

[0112] Example 1 The present invention found that elastin fragments are markers of aging

[0113] In the field of anti-aging research, the aging phenomenon is manifested as the decline of the absolute lifespan and healthy lifespan of organisms over time, accompanied by the decline of structure and function, obesity, upregulation of serum inflammatory factors, liver fat and impaired liver function, damage to the bone and muscle system, decreased motor ability, glucose and lipid metabolism disorders, skin aging and other unhealthy phenotypes, and even the induction of multi-system geriatric diseases. Faced with such a complex and serious aging condition, it is urgent to find biomarkers that can accurately indicate the aging process in order to gain a deeper understanding of the aging mechanism and explore effective intervention strategies.

[0114] Based on the fact that the extracellular matrix plays a key role in maintaining the structure and function of tissues and organs, changes in its composition and metabolism may be closely related to aging, so the inventors speculate that its changes in the aging process may reflect the aging state of the body. Therefore, in this embodiment, a variety of free extracellular matrix fragments in serum were studied, including elastin fragments (ELN), collagen fragments (COL), fibronectin fragments (FN) and hyaluronic acid fragments (HA).

[0115] The specific steps are as follows:

[0116] Serum samples from people of different age groups and serum samples from mice of different age groups (the mouse strain used in the experiment was C57BL / 6, and wild-type mice were purchased from Jiangsu Jicui Pharmaceutical Co., Ltd. or Shanghai Slake Co., Ltd.) were collected, and the content of the above-mentioned free extracellular matrix fragments was accurately determined, and the Elisa kit was used for detection. Through comprehensive comparison and processing of a large amount of sample data, the final results are as follows Figure 1 A and Figure 1 As shown in B, Figure 1A is a scatter plot of the relationship between the ELN, HA, FN, and COL levels in human serum samples and age, n = 100 for all groups; Figure 1 B is the detection of serum ELN, HA, FN, and COL levels in mouse samples at five age stages (3, 9, 15, 21, and 27 months old). For mice of each age, n=5–8; the red dotted line represents the linear regression line.

[0117] The final results showed that as the human body ages, the concentrations of elastin fragments, collagen fragments, fibronectin fragments and hyaluronic acid fragments in the blood increased. The elastin fragments grew faster than the other growth rates. After regression analysis, the elastin fragment group reached a significant level (P less than 0.05). Similar results were also found in mice. Therefore, it can be seen that the serum levels of various ECM fragments are positively correlated with age, and the content of elastin fragments shows the most significant and stable positive correlation with age. However, it should be noted that correlation does not necessarily mean that it is a marker, but that the marker must be correlated. Although Figure 1 All extracellular matrix fragments in the human body are related to aging, but not all of them can be used as markers. Only those that are related and cause aging are true aging markers. With aging, many substances will appear, but conversely, if these substances appear, it does not necessarily mean aging. On the contrary, if a certain substance appears, it will cause aging, and if it does not appear, there will be no aging. Such substances are important as markers.

[0118] Specific as Figure 2 As shown, Figure 2 The same starting age mice were used, and elastin fragments (ELN), collagen fragments (COL), fibronectin fragments (FN) and hyaluronic acid fragments (HA) were injected into the blood of 40-week-old mice. According to the corresponding serum concentration, the injection doses of different ECM fragments were as follows: ELN fragments were 500 μg / kg, FN fragments were 5 mg / kg, COL fragments were 10 μg / kg, and HA fragments were 100 mg / kg. Vehicle was used as a control. The results showed that although collagen fragments, fibronectin fragments and hyaluronic acid fragments had certain effects, they did not significantly cause aging. The effect of elastin fragments was extremely significant, and 50% of the mice died when the mice were about 90 weeks old. In contrast, even if some symptoms appeared in the HA, FN and COL groups, the difference in the death caused by them was not significant among the three. However, the ELN group reached a significant level compared with the other three groups and the control. This fully proves that only elastin fragments are important markers indicating aging in the present invention.

[0119] In the present invention, collagen fragments, fibronectin fragments and hyaluronic acid fragments are only positively correlated with aging, but the appearance of these fragments does not necessarily lead to aging, so they cannot be used as markers of aging. Elastin fragments are not only related to aging, but their appearance will definitely lead to aging, and the effect is the most significant. Therefore, in the present invention, only elastin fragments can be used as important markers of aging.

[0120] Example 2 Validation of elastin fragments as aging markers

[0121] In this example, in order to further verify the accuracy and reliability of elastin fragments as systemic aging markers, a total of 50 serum samples of mice of different ages were collected, of which 25 were in the aging process and 25 were healthy controls, divided into two groups, one for the experimental group: mice aged 18 months and above in the aging process; one for the control group: normal healthy mice aged 3-6 months; the contents of elastin fragments, collagen fragments, fibronectin fragments and hyaluronic acid fragments in the samples were tested by UPLC-MS / MS, and the test results were analyzed. At the same time, multiple aging-related indicators such as the motor performance, survival time, and aging-related serum indicators of the above-mentioned mice were tested.

[0122] In the above test set results, it was detected that the concentration of elastin fragments in serum had a normal range: less than 300ng / mL. When it exceeded this range, the motor performance of mice was significantly reduced, manifested as shortened movement distance and slower movement speed; survival time was significantly reduced; and the levels of aging-related serum indicators: AST (aspartate aminotransferase), ALT (alanine aminotransferase), GLU (blood sugar) and LDL (low-density lipoprotein) were significantly increased.

[0123] By drawing the ROC curve to calculate the AUC value (area under the curve), the AUC value of the elastin fragment was 0.86, the accuracy was 88%, the sensitivity was 85%, and the specificity was 90%; the AUC value of the collagen fragment was 0.21, the accuracy was 36%, the sensitivity was 28%, and the specificity was 32%; the AUC value of the fibronectin fragment was 0.32, the accuracy was 28%, the sensitivity was 30%, and the specificity was 35%; the AUC value of the hyaluronic acid fragment was 0.37, the accuracy was 33%, the sensitivity was 29%, and the specificity was 34%.

[0124] Therefore, it can be seen that elastin fragments have significant advantages as aging markers. Their AUC value, accuracy, sensitivity and specificity are much higher than those of several other extracellular matrix fragments. This shows that elastin fragments have higher accuracy and reliability in reflecting the aging state of mice, and can more effectively identify whether mice are in the aging process, providing an important basis for aging-related research and the formulation of intervention measures.

[0125] In subsequent experiments, the above results were further verified: First, an additional batch of independent validation set data was collected. This time, a total of 60 serum samples from mice of different ages were collected, of which 30 were in the aging process and 30 were healthy controls. They were also divided into two groups, one was the experimental group: mice aged 18 months and above in the aging process; the other was the control group: normal healthy mice aged 3-6 months. The UPLC-MS / MS method was also used to detect the content of elastin fragments, collagen fragments, fibronectin fragments and hyaluronic acid fragments in these samples.

[0126] Then, the motor performance, survival time, and aging-related serum indicators (such as AST, ALT, GLU, and LDL) of this group of mice were also tested, and the test results were compared and analyzed with the data of the discovery set. The AUC value, accuracy, sensitivity, and specificity were calculated by drawing the ROC curve again. The results showed that in the validation set, the AUC value of the elastin fragment was 0.90, the accuracy was 86%, the sensitivity was 83%, and the specificity was 88%; the AUC value of the collagen fragment was 0.70, the accuracy was 70%, the sensitivity was 68%, and the specificity was 72%; the AUC value of the fibronectin fragment was 0.75, the accuracy was 73%, the sensitivity was 70%, and the specificity was 75%; the AUC value of the hyaluronic acid fragment was 0.78, the accuracy was 76%, the sensitivity was 73%, and the specificity was 78%.

[0127] In summary, the prediction results in the validation set are highly consistent with those in the discovery set, which once again proves the excellent performance of elastin fragments as aging markers, and their AUC values, accuracy, sensitivity, and specificity are significantly better than those of several other extracellular matrix fragments. This further confirms that elastin fragments have the highest diagnostic efficacy in reflecting the aging state of mice, providing a more solid and reliable basis for aging-related research and the formulation of intervention strategies.

[0128] In addition, in the present invention, multiple batches of different validation sets were used for validation testing, and the results showed that the predictive performance of elastin fragments for an individual in the aging process can reach about 90%.

[0129] Therefore, it can be seen that when the content of elastin fragments in serum exceeds the normal range (lower than 300ng / mL), mice are in the accelerated aging stage. In the present invention, the AUC value, accuracy, sensitivity and specificity of elastin fragments are all the highest, and its diagnostic efficiency is also outstanding in multiple batches of different validation set tests, significantly higher than collagen fragments, fibronectin fragments and hyaluronic acid fragments. And the diagnostic efficiency of several other extracellular matrix fragments is all relatively low, and the aging process of mice cannot be accurately predicted. Therefore, only elastin fragments are effective markers that can accurately and reliably predict aging, and provide valuable detection indicators for the research and clinical application related to aging.

[0130] Example 3 Further verification of elastin fragments causing aging and the search for potential intervention targets

[0131] 1. Elastin fragments cause aging through the elastin receptor complex

[0132] 1. Composition and mechanism of action of elastin fragment receptor complex

[0133] The elastin fragment receptor complex is composed of a 67kDa elastin binding protein and 55kDa and 61kDa membrane proteins. The 61kDa membrane protein is called NEU1, which plays a role in transducing the activated signal of the elastin fragment receptor complex into the cell, such as activating the ERK 1 / 2 pathway and upregulating the expression of MMP1. NEU1 mediates some aging-related unhealthy phenotypes and the occurrence of various geriatric diseases caused by elastin fragments, and plays a key role in the process of elastin fragments exerting their effects.

[0134] 2. Elastin fragment E-motif causes aging and reduces the life span of mice

[0135] Elastin fragments are soluble products of elastin degradation by elastase, which can cause human immune abnormalities and tissue damage. For example, elastin fragments containing the VGVAPG motif (E-motif) have chemotactic and pro-inflammatory effects on monocytes, can activate CD4+T cells, induce them to produce inflammatory cytokines, and polarize Th cells in human peripheral blood lymphocytes to inflammatory Th1 by activating the MAPK-AP-1-NF-kB pathway, reducing Th2 polarization.

[0136] Therefore, in this example, the effect of the E-motif of the elastin fragment on the life span of mice was first investigated. The specific experimental steps are as follows:

[0137] 1. Experimental groups: Vehicle group (mice injected with PBS via tail vein), E-motif group (mice injected with elastin fragment E-motif via tail vein), and poly(A / K) group were set up respectively;

[0138] 2. Take the E-motif peptide (Sen'er Biotechnology, sequence: VGVAPGVGVAPG) and the poly(A / K) peptide (Sen'er Biotechnology, sequence: AAAAAAKAAAKAAK), and dissolve the fragments in 50 μL of PBS. The doses of the E-motif peptide and the poly(A / K) peptide are both 150 μg / kg;

[0139] 3. Administration: 40-week-old mice were injected through the tail vein twice a week, on the first and fourth day of each week, until 50% of the mice in the group with the shortest lifespan died, and then all groups were stopped at the same time.

[0140] Specific results such as Figure 3 A and Figure 3 As shown in B, Figure 3 A is a schematic diagram of mouse tail vein injection. Figure 3 B is the survival curve of 40-week-old mice after tail vein injection of Vehicle, E-motif and poly(A / K). The results showed that p<0.0001 was observed between the E-motif group and the Vehicle group, and there was no significant difference between the poly(A / K) group and the Vehicle group; this indicates that the life span of mice was significantly reduced after injection of the E-motif group compared with the poly(A / K) group or the Vehicle group. This result proves that the elastin fragment E-motif is a key factor affecting life span and can significantly reduce the life span of mice.

[0141] 2. Elastin fragment E-motif causes aging - affects spinal curvature in mice

[0142] In order to more comprehensively verify the effect of the E-motif of the elastin fragment on aging, this example also tested its effect on the spinal curvature of mice. The specific experimental steps are as follows:

[0143] 1. Experimental groups: Vehicle group (mice injected with PBS via tail vein) and E-motif group (mice injected with elastin fragment E-motif via tail vein) were set up respectively;

[0144] 2. Take the E-motif peptide fragment and dissolve it in 50 μL of PBS at a dose of 150 μg / kg;

[0145] 3. Administration: 40-week-old mice were injected through the tail vein twice a week, on the first and fourth day of each week, until 50% of the mice in the group with the shortest lifespan died, and then all groups were stopped at the same time.

[0146] Specific results such as Figure 4 A and Figure 4 As shown in B, Figure 4 A is a representative whole-body X-ray image of a mouse, in which the black line indicates the calculation basis of the KI value; Figure 4 B is the statistical analysis value of KI value; "*" indicates significant difference, p<0.05. The specific process of determining kyphosis index (KI) is as follows: (1) The mouse spine X-ray is taken by a mobile X-ray camera at Zhejiang University Animal Hospital; (2) The KI value is determined by the distance between two line segments, point A is defined as the caudal edge of the last cervical vertebra, and point B is defined as the caudal edge of the sixth lumbar vertebra (the cranial end of the iliac wing). C is defined as a point on the AB line segment, and a perpendicular line AB is drawn from C to the edge of the back spine, and the intersection with the back spine is defined as D. This perpendicular line should make the arc formed by A, B, and D obtain the maximum curvature. In this case, KI = AB / CD, that is, the value of KI is the distance between AB divided by the distance between CD. The smaller the KI value, the more severe the "hunchback".

[0147] The quantitative results showed that the KI value of the E-motif group was significantly lower than that of the Vehicle group, which means that E-motif caused significant abnormal curvature of the spine and hunchback phenomenon in mice. This indicates that the E-motif in the elastin fragment can cause hunchback phenomenon in mice, confirming that E-motif has a negative effect on the spine, indicating that the E-motif in the elastin fragment can cause aging-related signs in mice, thereby promoting aging.

[0148] 2. Experiment on how elastase promotes the degradation of elastin fragments and thus promotes aging

[0149] Elastase (Sigma) plays a vital role in the aging process. In order to verify that it promotes the degradation of elastin fragments and thus promotes the aging process, relevant studies were conducted in this example.

[0150] The results showed that as the activity of elastase increased, elastin was over-degraded, and a large number of elastin fragments accumulated in the body. These accumulated fragments triggered a series of aging-related changes, such as a significant increase in oxidative stress levels, intensified inflammatory responses, and an increase in cell aging markers. At the same time, at the tissue level, organs showed obvious aging-related pathological changes, such as cell damage and fibrosis, while these changes were relatively mild or not obvious when the activity of elastase was normal. In summary, it is fully proved that elastase has a significant role in promoting the aging process by promoting the degradation of elastin fragments.

[0151] In summary, elastin fragments cause aging-related signs in mice through the following two mechanisms:

[0152] 1. After the elastin fragment E-motif in elastin binds to the elastin receptor complex, it activates the downstream pathways to cause aging-related physiological changes. The specific mechanism of action is as follows: After the E-motif in elastin binds to the elastin receptor complex, the 61kDa membrane protein NEU1 plays a key signal transduction role. It transmits the activation signal to the cell, such as activating the ERK 1 / 2 pathway. Activation of the ERK 1 / 2 pathway can regulate processes such as cell proliferation, differentiation and survival; at the same time, it upregulates the expression of MMP1, which is a matrix metalloproteinase. Its increased expression may lead to the degradation and reconstruction of the extracellular matrix. In addition, E-motif can also produce chemotactic and pro-inflammatory effects on monocytes, activating CD4 T cells and activating CD8 T cells. 4+ T cells and induce them to produce inflammatory cytokines, and by activating the MAPK-AP-1-NF-kB pathway, Th cells in human peripheral blood lymphocytes polarize toward the inflammatory Th1 type, reduce Th2 polarization, induce inflammatory response and immune abnormalities, and thus lead to aging-related physiological changes, such as tissue damage, immune dysfunction, etc.

[0153] 2. The specific mechanism of elastase promoting elastin degradation and increasing the production of elastin fragments, which leads to aging, is as follows: the activity of elastase is enhanced, resulting in excessive degradation of elastin and the production of a large number of elastin fragments. These fragments accumulate in the body, which directly play a negative role, such as inducing inflammatory responses and destroying the balance of the extracellular matrix; on the other hand, by binding to the receptor complex, they activate a series of harmful intracellular signaling pathways mentioned above, further exacerbating cell and tissue damage, affecting normal physiological functions, and ultimately manifesting as signs and diseases related to aging.

[0154] Therefore, it can be seen that elastase, Neu1 gene and elastin receptor complex are all key targets that promote elastin degradation and thus lead to aging.

[0155] In view of the above two mechanisms of action, in subsequent experiments, the present invention antagonizes aging or the manifestations of aging by taking specific interference measures on the above targets and detecting relevant indicators.

[0156] Specifically, the following three measures were adopted: (1) knocking out the NEU1 gene and observing the effects on aging signs and aging-related indicators; (2) injecting an elastin receptor complex inhibitor and observing the effects on aging signs and aging-related indicators; (3) injecting an elastase inhibitor and observing the effects on aging signs and aging-related indicators.

[0157] Therefore, in subsequent experiments, by taking specific interference measures on the above-mentioned targets and detecting relevant indicators, we can verify the specific mechanism and key links of the targets in the aging process induced by elastin fragments, and then deeply analyze their precise impact on the aging process, so as to provide a solid theoretical basis and experimental foundation for exploring more effective anti-aging strategies, open up new ways to delay or even reverse the physiological changes and disease development related to aging, and provide potential intervention strategies for delaying the occurrence and development of aging-related diseases, which is of great significance.

[0158] Example 4 Experimental verification of antagonizing aging after knocking out the Neu1 gene in the elastin receptor complex

[0159] 1. Effect of Neu1 knockout on the body weight of mice injected with E-motif

[0160] In order to further explore the intrinsic mechanism of aging caused by the E-motif of elastin fragment and find potential intervention targets, the effect of knocking out the Neu1 gene on the body weight of mice injected with E-motif was investigated. The specific experimental steps are as follows:

[0161] 1. Construction of mice with conditional and specific knockout of Neu1 gene on myeloid cells (referred to as Neu1- / - mice):

[0162] ①By crossing LysM-Cre mice and Neu1-flox mice, LysM-Cre heterozygous and Neu1-flox homozygous hybrid mice were finally obtained, which are called LysM-Cre / Neu1-flox mice;

[0163] ② Dissolution of tamoxifen: Dissolve it in corn oil to a concentration of 20 mg / mL and place it in a 37°C shaker overnight to dissolve.

[0164] ③Tamoxifen injection: Administered by intraperitoneal injection, the dose is 100 mg / kg, once a day (every 24 hours) for 5 consecutive days before the injection of E-motif, and start E-motif injection after confirming that the gene has been knocked out.

[0165] 2. Experimental groups: Neu1(f / f) group (control group), Neu1(f / f)+E-motif group (mice injected with E-motif), Neu1(- / -)+E-motif group (mice injected with E-motif after conditional specific knockout of Neu1 gene on myeloid cells);

[0166] 3. Administration: Different groups of 12-week-old mice were injected with E-motif via the tail vein twice a week, on the first and fourth day of each week, and the changes in the weight of the mice were monitored for 16 weeks (12 to 28 weeks of age).

[0167] Specific results such as Figure 5 As shown, Figure 5 The statistical results of the comparison of the weight data of mice at 28 weeks of age. "**" indicates a significant difference, p < 0.01. Figure 5 (Left) As can be seen, the Neu1 gene was successfully knocked out in Neu1- / - mice. The results showed that specific knockout of the Neu1 gene blocked the downstream effects of E-motif. When samples were collected at 28 weeks of age, the body weight of the Neu1(f / f)+E-motif group had increased significantly compared with the Neu1(f / f) mice, while the body weight of the Neu1(- / -)+E-motif group remained at the same level as the control group, which was significantly lower than that of the Neu1(f / f)+E-motif group. This result confirms the negative effect of the Neu1 gene in affecting the body weight downstream of E-motif; therefore, it can be seen that the Neu1 gene is a key molecular target for controlling the downstream E-motif effects, and intervention on the Neu1 gene can antagonize aging effects.

[0168] 2. Effect of Neu1 knockout on the levels of serum inflammatory factors IL-1 and MCP-1 in mice injected with E-motif

[0169] In order to more comprehensively understand the mechanism of aging caused by the E-motif of elastin fragment, we further explored the effect of knocking out the Neu1 gene on the levels of serum inflammatory factors IL-1 and MCP-1 in mice injected with E-motif. The specific experimental steps are as follows:

[0170] 1. Construction of mice with conditional and specific knockout of Neu1 gene on myeloid cells (referred to as Neu1- / - mice): the same operation as in the above exploration 3;

[0171] 2. Experimental groups: Neu1(f / f) group (control group), Neu1(f / f)+E-motif group (mice injected with E-motif), Neu1(- / -)+E-motif group (mice injected with E-motif after conditional specific knockout of Neu1 gene on myeloid cells);

[0172] 3. Administration: Different groups of 12-week-old mice were injected with E-motif via the tail vein twice a week, on the first and fourth day of each week, and the changes in the levels of serum inflammatory factors IL-1 and MCP-1 in mice were monitored for 16 weeks (12 to 28 weeks of age).

[0173] 4. The determination of inflammatory factor content is as follows:

[0174] (1) After the mice were deeply anesthetized with 1% (mass-volume ratio) sodium pentobarbital, blood was collected by removing the eyeballs;

[0175] (2) The blood was first placed in an EP tube at 4°C for 2 hours. After initial stratification, the blood was centrifuged at 3000 × g for 10 minutes at 4°C. The supernatant was collected and the centrifugation operation was repeated once to obtain serum, which was then stored in a -80°C refrigerator.

[0176] (3) Blood biochemical testing was performed by the Safety Assessment Center of Zhejiang University using a fully automatic biochemical analyzer.

[0177] (4) The levels of inflammatory factors MCP-1 and IL-1 in peripheral blood of different groups were determined by enzyme-linked immunosorbent assay (ELISA). The specific operations were as follows: ① The ELISA kit (Jianglai Biological Company) was balanced at room temperature for 1 hour; ② Standard wells and test sample wells were set up, and 50 μL of standard samples of known concentrations were added to the standard wells. After the test samples were diluted according to the recommended ratio in the instructions, 50 μL was added to the test sample wells; ③ The detection antibody coupled with a luminescent substrate catalyst was added to each well, the plate was sealed with a sealing film, and incubated at 37°C for 1 hour; ④ The liquid in the plate was discarded and patted dry, 350 μL of washing solution was added to each well, and it was allowed to stand for 1 minute, then the liquid was discarded and patted dry, and the above washing operation was repeated 5 times; ⑤ 50 μL of substrate A was first added to all wells, followed by 50 μL of substrate B, and the mixture was shaken and mixed, and incubated at 37°C in the dark for 15 minutes. The liquid in the well will change color due to the catalytic reaction; ⑥ Add 50μL of stop solution to all wells and measure the absorbance of each well at a wavelength of 450nm within 15 minutes.

[0178] Specific results such as Figure 6 As shown, Figure 6 The levels of IL-1 and MCP-1 in the serum of mice in different groups are shown in Table 1. “*” indicates p<0.05, “**” indicates p<0.01, and “***” indicates p<0.001. The results showed that specific knockout of the Neu1 gene blocked the downstream effects of E-motif. When samples were collected at 28 weeks of age, the levels of IL-1 and MCP-1 in the Neu1(f / f)+E-motif group were significantly higher than those in the Neu1(f / f) group and the Neu1(- / -)+E-motif group. This result confirms the negative effect of the Neu1 gene in affecting the increased level of inflammation downstream of E-motif. Therefore, it can be seen that the Neu1 gene is a key molecular target for controlling the downstream E-motif effects, and intervention on the Neu1 gene can antagonize aging effects.

[0179] Example 5 Experimental verification of anti-aging effect after injection of "elastin fragment receptor complex inhibitor DANA"

[0180] 1. After injection of "DANA, an inhibitor of elastin fragment receptor complex", the survival time and lifespan of mice were extended

[0181] According to the exploration of the mechanism of elastin fragments leading to aging and the search for potential intervention targets in Example 3, in this example, the elastin fragment receptor complex inhibitor DANA (GlpBio) was used to verify its inhibitory effect on the aging process and its effect on the life span of mice; wherein DANA is N-acetyl-2,3-dehydro-2-deoxyneuraminic acid, and its specific structural formula is as follows Figure 7 As shown, the CAS number is 24967-27-9. The specific operation is as follows:

[0182] 1. 40 male and 40 female mice in good health and aged 65 weeks were randomly divided into two groups: experimental group (mice injected with DANA) and control group (Vehicle group, mice injected with an equal amount of PBS);

[0183] 2. Prepare inhibitor solution: weigh an appropriate amount of DANA and dissolve it in PBS. The injection volume is 2.5 mg / kg.

[0184] 3. Injection operation: For the mice in the experimental group, 2.5 mg / kg of DANA solution was intraperitoneally injected once a week until the mice died of natural causes; for the mice in the control group, an equal amount of PBS solution was intraperitoneally injected once a week until the mice died of natural causes;

[0185] 4. Continuous observation and recording: Continuously observe the survival status of mice until all mice die. During the observation process, record the death time of each mouse in detail, count the life span of the mice, and draw a survival curve.

[0186] Specific results such as Figure 8 A and Figure 8 As shown in B, Figure 8 A is the survival curve of male mice. Figure 8 B is the survival curve of female mice. The results showed that the elastin fragment receptor complex inhibitor DANA can significantly prolong the lifespan of mice. Compared with the Vehicle group, the survival time of mice of both sexes was significantly prolonged, with the median lifespan of male mice prolonged by 17.4% and that of female mice prolonged by 12.2%. This indicates that antagonizing elastin fragments prolongs the absolute lifespan of mice, and the elastin fragment receptor complex and its downstream pathways can be used as potential therapeutic targets.

[0187] 2. After injection of "DANA, an inhibitor of elastin fragment receptor complex", serum indicators related to aging are reduced

[0188] Based on the exploration of the mechanism of elastin fragments leading to aging and the search for potential intervention targets in Example 2, in this example, DANA, an inhibitor of the elastin fragment receptor complex, was used to verify its effect on aging-related serum indicators.

[0189] The specific operations are as follows:

[0190] 1. Selection of experimental animals: 14 mice aged 60 weeks and in good health were selected and randomly divided into 2 groups, an experimental group (mice injected with DANA) and a control group (Vehicle group, mice injected with an equal amount of PBS), with 7 mice in each group.

[0191] 2. Prepare inhibitor solution: weigh an appropriate amount of DANA and dissolve it in PBS. The injection volume is 2.5 mg / kg.

[0192] 3. Injection operation: The mice in the experimental group were intraperitoneally injected with 2.5 mg / kg DANA solution once a week for 24 weeks; the mice in the control group were intraperitoneally injected with an equal amount of PBS solution once a week for 24 weeks;

[0193] 4. Continuous observation and record keeping: After 24 weeks, serum and other samples of the animals were collected and tested for serum aging factors, including liver function indicators (such as AST and ALT levels), blood sugar (GLU) and blood lipid (LDL) levels by the Safety Assessment Center of Zhejiang University using a fully automatic biochemical analyzer.

[0194] Specific results such as Fig. 9 A- Fig. 9 As shown in D, the results showed that the elastin fragment receptor complex inhibitor DANA can significantly reduce the serum aging factor levels of mice. Compared with the Vehicle group, the liver function indicators of the DANA experimental group: AST and ALT levels were significantly reduced; the serum levels of blood glucose GLU and blood lipid LDL were also significantly reduced in the DANA group. This shows that DANA at least partially inhibits the aging-related liver function decline and the disorders of glucose metabolism and lipid metabolism, and the elastin fragment receptor complex and its downstream pathways can be used as potential therapeutic targets.

[0195] 3. After injection of "DANA, an inhibitor of elastin fragment receptor complex", the aging phenotype was delayed: the motor ability of mice was enhanced

[0196] According to the exploration of the mechanism of elastin fragments leading to aging and the search for potential intervention targets in Example 3, in this example, the elastin fragment receptor complex inhibitor DANA was used to observe its effect on the motor ability of mice, thereby verifying its effect of delaying the aging phenotype. The specific operation is as follows:

[0197] 1. Selection of experimental animals: 20 mice aged 60 weeks and in good health were selected and randomly divided into 2 groups, an experimental group (mice injected with DANA) and a control group (Vehicle group, mice injected with an equal amount of PBS), with 10 mice in each group.

[0198] 2. Prepare inhibitor solution: weigh an appropriate amount of DANA and dissolve it in PBS. The injection volume is 2.5 mg / kg.

[0199] 3. Injection operation: The mice in the experimental group were intraperitoneally injected with 2.5 mg / kg DANA solution once a week for 24 weeks; the mice in the control group were intraperitoneally injected with an equal amount of PBS solution once a week for 24 weeks;

[0200] 4. Continuous observation and recording: After 24 weeks, the mice's motor ability was tested.

[0201] The process of mouse motor ability test is as follows:

[0202] (1) First, the mouse's athletic ability was evaluated using a mouse treadmill. There is a charged metal coil at the end of the treadmill track used in the experiment. If the mouse's running speed cannot keep up with the speed of the conveyor belt, it will be shocked by the metal coil. In the first three days of the formal experiment, the mice were given adaptive training once a day. During the adaptive training, the speed of the treadmill was set to a constant speed of 5m / min and the time was set to 5min. The mice were allowed to adapt to the treadmill environment and the mice that could not adapt were eliminated. After three adaptive training sessions, the formal experiment began. In the formal experiment, in order to compare the athletic ability of the mice, the acceleration mode was set with an initial speed of 1m / min and an acceleration set to 2m / min. 2 , when the time reaches 10 min or the maximum number of electric shocks reaches 400 times, the experiment is stopped and the number of electric shocks for all mice is recorded;

[0203] (2) This study used an open field test to evaluate the spontaneous movement ability of mice. Starting from the first seven days of the open field test, the mice and the experimenters were familiar with each other through stroking and other means every day. The open field test was conducted in a container made of 40cm×40cm×40.5cm white acrylic boards. Before the formal experiment, the mice were allowed to enter the open field test room 45 minutes in advance to familiarize themselves with the environment. Before each mouse was placed in, 75% alcohol was used to remove the remaining odor. After the alcohol and water dried, the mice were placed in, and then the experimenters left quickly. The mice were allowed to move spontaneously in a quiet environment for 6 minutes. The movement trajectory, movement distance, average speed and other parameters of the mice were recorded by a camera and analyzed using the anymaze system.

[0204] Specific results such as Fig.10 A- Fig.10 As shown in C, the results showed that the elastin fragment receptor complex inhibitor DANA can significantly delay the decline in the motor ability of mice. Compared with the Vehicle group, the number of electric shocks in the DANA experimental group was significantly lower, indicating that the running endurance of the mice in the DANA experimental group was stronger; the results of the open field experiment showed that the total movement distance and average speed of the mice in the DANA group were significantly higher than those in the Vehicle group, and the range of movement was wider; therefore, it can be seen that the elastin fragment receptor complex inhibitor DANA can antagonize elastin fragments and significantly delay the decline in the motor ability of mice. The elastin fragment receptor complex and its downstream pathways can be used as potential therapeutic targets.

[0205] IV. Delaying aging phenotype after injection of "elastin fragment receptor complex inhibitor DANA": spinal curvature in mice

[0206] According to the exploration of the mechanism of elastin fragments leading to aging and the search for potential intervention targets in Example 2, in this example, the elastin fragment receptor complex inhibitor DANA was used to observe its effect on spinal curvature in mice, thereby verifying its effect of delaying aging phenotypes. The specific operation is as follows:

[0207] 1. Selection of experimental animals: Twelve mice aged 60 weeks in good health were selected and randomly divided into two groups: an experimental group (mice injected with DANA) and a control group (Vehicle group, mice injected with an equal amount of PBS), with 6 mice in each group.

[0208] 2. Prepare inhibitor solution: weigh an appropriate amount of DANA and dissolve it in PBS. The injection volume is 2.5 mg / kg.

[0209] 3. Injection operation: The mice in the experimental group were intraperitoneally injected with 2.5 mg / kg DANA solution once a week for 24 weeks; the mice in the control group were intraperitoneally injected with an equal amount of PBS solution once a week for 24 weeks;

[0210] 4. Continuous observation and recording: After 24 weeks, the spinal curvature of the mice was tested.

[0211] Specific results such as Fig.11 A and Fig.11 As shown in B, the results showed that the elastin fragment receptor complex inhibitor DANA can significantly alleviate the spinal curvature of mice associated with aging. Compared with the Vehicle group, the spinal curvature of the mice in the DANA experimental group was lower, and the quantitative analysis results showed that the KI value of the mice in the DANA experimental group was significantly increased. Therefore, it can be seen that the elastin fragment receptor complex inhibitor DANA can antagonize elastin fragments and significantly alleviate the "hunchback" phenotype of naturally aged mice. The elastin fragment receptor complex and its downstream pathways can be used as potential therapeutic targets.

[0212] Example 6 Experimental verification of anti-aging effect after injection of "elastase inhibitor Ly"

[0213] 1. After injection of "elastase inhibitor Ly", the expression of inflammatory factors in mouse serum was reduced

[0214] According to the exploration of the mechanism of elastin fragments leading to aging and the search for potential intervention targets in Example 3, in this example, an elastase inhibitor Ly (Sigma) was used to observe its effect on mouse serum inflammatory factors; wherein Ly is sivelestat sodium salt hydrate, and the specific structural formula is as follows: Fig.12 As shown, the CAS number is 127373-66-4. The specific operations are as follows:

[0215] Mice with a natural aging model (Jiangsu Jicui Pharmaceutical Co., Ltd. or Shanghai Slake Co., Ltd.) were intraperitoneally injected with the elastase inhibitor Ly (5 mg / kg, dissolved in PBS). After 8 weeks, the serum and other samples of the animals were collected and the expression of inflammatory factors was detected by ELISA. The serum inflammatory factors were detected by the Luminex multi-factor detection platform, and the inflammatory factor content was specifically determined as described in Example 3.

[0216] Specific results such as Fig.13 As shown, Fig.13 The figure is a heat map of serum inflammatory factor content in the Ly group relative to the PBS group (heat map values ​​are multiples of content). The results show that the elastase inhibitor Ly can significantly inhibit the inflammatory phenotype of mice. Compared with the control group, the protein expression levels of MCP-1, IL-1, and TNF-α in the Ly experimental group were significantly downregulated, indicating that elastase can indirectly regulate the secretion of inflammatory factors and can be used as a therapeutic target. The detection of 23 inflammatory factors in serum showed that the expression of inflammatory factors in the Ly group mice was generally downregulated compared with the control group that only received Vehicle injection, indicating that the elastase inhibitor Ly at least partially inhibited the chronic inflammation associated with aging.

[0217] 2. Delaying aging phenotype after injection of "elastase inhibitor Ly": exercise capacity and obesity in mice

[0218] Based on the exploration of the mechanism of elastin fragments leading to aging and the search for potential intervention targets in Example 3, in this example, the elastase inhibitor Ly was used to observe its effect on the motor ability of mice, thereby verifying its effect of delaying the aging phenotype. The specific operation is as follows:

[0219] Mice with a natural aging model (Jiangsu Jicui Pharmaceutical Co., Ltd. or Shanghai Slake Co., Ltd.) were randomly divided into two groups. The experimental group was injected intraperitoneally with the elastase inhibitor Ly (5 mg / kg, dissolved in PBS), and the control group was injected with an equal amount of PBS. After 6 months, their exercise capacity was tested on a mouse treadmill, their body weight was detected, and their survival status was recorded.

[0220] The athletic ability of mice was evaluated using a mouse treadmill. There is a charged metal coil at the end of the treadmill track used in the experiment. If the mouse's running speed cannot keep up with the speed of the conveyor belt, it will be shocked by the metal coil. In the first three days of the formal experiment, the mice were given adaptive training once a day. During the adaptive training, the speed of the treadmill was set to a uniform speed of 5m / min and the time was set to 5min to allow the mice to adapt to the treadmill environment and eliminate mice that could not adapt. After three adaptive training sessions, the formal experiment began. In the formal experiment, in order to compare the athletic ability of mice, the acceleration mode was set with an initial speed of 1m / min and an acceleration set to 2m / min. 2 When the time reaches 10 minutes or the maximum number of electric shocks reaches 400 times, the experiment is stopped and the number of electric shocks received by all mice is recorded.

[0221] Specific results such as Fig.14 A- Fig.14 As shown in C, Fig.14 A is the survival rate curve of mice in the Ly group and the control group. Fig.14 B is the curve of body weight changes of mice in the Ly group and the control group. Fig.14 C is the result of the treadmill electric shock experiment of mice in the Ly experimental group and the control group. The results showed that the elastase inhibitor Ly can significantly delay the aging phenotype of mice. Compared with the control group, the number of electric shocks recorded in the treadmill experiment of the Ly experimental group mice was less, the survival rate was better, and the obesity symptoms were significantly improved. Therefore, it can be seen that the elastase inhibitor Ly can antagonize elastin fragments and significantly alleviate the aging phenotype of naturally aged mice; elastin fragments regulate the aging phenotype and can be used as a potential therapeutic target.

[0222] 3. Delaying aging phenotype after injection of "elastase inhibitor Ly": aging-related weight and liver fat indicators

[0223] Based on the exploration of the mechanism of elastin fragments leading to aging and the search for potential intervention targets in Example 3, in this example, the elastase inhibitor Ly was used to observe its effect on the aging-related weight and liver fat indexes of mice, thereby verifying its effect of delaying the aging phenotype. The specific operation is as follows:

[0224] Mice with a natural aging model were randomly divided into two groups. The experimental group was injected with elastase inhibitor Ly (5 mg / kg, dissolved in PBS) by intraperitoneal injection, and the control group was injected with the same amount of PBS. After 6 months, the fatty state was observed by liver histological sections, and the triglyceride content was detected according to the following process:

[0225] (1) taking out the liver tissue frozen in liquid nitrogen, immersing it in NP-40 lysis buffer and grinding it, heating the ground homogenate to 80°C for 5 min, then cooling it to room temperature, and repeating the heating step to completely dissolve triglycerides;

[0226] (2) Centrifuge at 13,000 × g for 10 min, collect the supernatant, and repeat the centrifugation step once to remove undissolved matter;

[0227] (3) According to the instructions of the triglyceride detection kit, the standard and sample solutions were added to the wells of the 96-well plate, and the assay buffer and lipase were added and incubated for 20 minutes; then the triglyceride reaction mix was added and incubated for 60 minutes, and the absorbance was detected by an ELISA instrument. The triglyceride content was standardized by tissue weight.

[0228] Specific results such as Fig.15 As shown, the results showed that the elastase inhibitor Ly can significantly improve the aging-related body weight and liver fat indicators of mice. Compared with the control group, the liver steatosis of the Ly group mice was significantly improved and the triglyceride level was significantly reduced, which indicates that the elastin fragment regulates the aging-related liver steatosis phenotype and can be used as a potential therapeutic target.

[0229] Example 7 Application of “Elastase Inhibitor Ly” to Antagonize Elastin Fragments—Improving Aging-Related Osteoarthritis Symptoms

[0230] Through the search and verification of potential aging targets in the above examples, a solid foundation has been laid for the subsequent application of these research results to improve aging-related diseases. In view of the key role of elastin fragments in the aging process, the present invention further targets the above targets in the subsequent experimental process to explore their application in improving aging-related diseases.

[0231] In this embodiment, "elastase inhibitor Ly" is used to antagonize elastin fragments to improve aging-related osteoarthritis symptoms. The specific operation is as follows:

[0232] Mice with a natural aging model were randomly divided into two groups. The experimental group was injected intraperitoneally with the elastase inhibitor Ly (5 mg / kg, dissolved in PBS), and the control group was injected with an equal amount of PBS. After 6 months, the mouse joints were collected, tissue sections and SO staining were performed, and the observed cartilage tissue was scored by OARSI.

[0233] Specific results such as Fig.16 As shown, the results showed that the elastase inhibitor Ly can significantly improve the condition of osteoarthritis in the aging process and alleviate the damage and wear of aging-related articular cartilage; at the same time, the OARSI score of the cartilage tissue in the Ly group was significantly lower than that in the control group mice, proving that controlling the production of elastin fragments by inhibiting elastase has a definite and significant effect on delaying and improving aging-related osteoarthritis symptoms, providing a strong experimental basis and new research direction for future treatment strategies for aging-related osteoarthritis.

[0234] Example 8 Application of "Elastase Inhibitor Ly" to Antagonize Elastin Fragments - Improving Aging-Related Osteoporosis

[0235] In this embodiment, "elastase inhibitor Ly" is used to antagonize elastin fragments to improve aging-related osteoporosis. The specific operation is as follows:

[0236] Mice with a natural aging model were randomly divided into two groups. The experimental group was injected with the elastase inhibitor Ly (5 mg / kg, dissolved in PBS) intraperitoneally, and the control group was injected with the same amount of PBS. After 6 months, the complete femoral tissue of the experimental mice was taken and photographed by the high-resolution mode of the small animal in vivo tomography imaging (micro-Compute tomography, micro-CT) system. The photographed images were reconstructed in three dimensions using the MILabs-Rec software, and the three-dimensional reconstructed bone tissue was analyzed using the IMALYTICS software. Starting from the lowest end of the femur (the knee cartilage), a region with a length of 7 mm was selected, and parameters such as the ratio of bone volume to tissue volume (BV / TV) and trabecular thickness (Tb.Th) in this region were analyzed.

[0237] Specific results such as Fig.17As shown, the results showed that the elastase inhibitor Ly can significantly improve osteoporosis-related symptoms in the aging process and improve bone-related indicators. Parameters such as the ratio of bone volume to tissue volume (BV / TV) and trabecular thickness (Tb.Th) in the Ly group were significantly higher than those in the control group mice, proving that inhibiting elastase to control the production of elastin fragments can improve osteoporosis-related indicators such as bone density, and intervening in elastase activity can be used as a potential therapeutic target.

[0238] Example 9 Application of "Elastase Inhibitor Ly" to Antagonize Elastin Fragments - Improving Symptoms of Osteoarthritis Model

[0239] In this example, "elastase inhibitor Ly" was used to antagonize elastin fragments to improve the symptoms of aging-related osteoarthritis model, so as to evaluate the therapeutic effect of elastase inhibitor on osteoarthritis caused by different pathological factors. The specific operation is as follows:

[0240] 1. Select 32 mice of 60 weeks old and in good health and randomly divide them into 4 groups, including experimental group 1, control group 1, experimental group 2 and control group 2, with 8 mice in each group.

[0241] 2. MIA osteoarthritis modeling: For experimental group 1 and control group 1, intra-articular injection of sodium iodoacetate (MIA) was used to establish osteoarthritis model in mice. Experimental group 1 was injected with elastase inhibitor Ly (5 mg / kg, dissolved in PBS) by intraperitoneal injection, and control group 2 was injected with an equal amount of PBS. After 6 months, Micro-CT, SO staining and other methods were used to observe the improvement of arthritis pathology, bone volume and other indicators, and cartilage morphology in the Ly group (experimental group) compared with the control group, in order to evaluate the therapeutic effect of elastase inhibitors on osteoarthritis caused by "intra-articular injection of sodium iodoacetate".

[0242] 3. DMM osteoarthritis modeling: For experimental group 2 and control group 2, osteoarthritis modeling of mice was established by destabilization of medial meniscus (DMM). Experimental group 2 was injected with elastase inhibitor Ly (5 mg / kg, dissolved in PBS) by intraperitoneal injection, and control group 2 was injected with an equal amount of PBS. After 6 months, Micro-CT, SO staining and other methods were used to observe the improvement of arthritis pathology, bone volume and other indicators, and cartilage morphology in the Ly group (experimental group) compared with the control group, in order to evaluate the therapeutic effect of elastase inhibitor on osteoarthritis caused by "destabilization of medial meniscus of the knee".

[0243] The results showed that for osteoarthritis caused by different pathological factors, the elastase inhibitor Ly significantly improved the pathological conditions of arthritis in mice. (1) Comparison of the results of experimental group 1 and control group 1 showed that the elastase inhibitor Ly could significantly improve the condition of osteoarthritis in the aging process and alleviate the damage and wear of articular cartilage related to aging; at the same time, the OARSI score of cartilage tissue in the Ly group was significantly lower than that in the control group mice; Micro-CT results also showed that the parameters such as the ratio of bone volume to tissue volume (BV / TV) in the Ly group were significantly higher than those in the control group mice, and the bone-related indicators were significantly improved; (2) Comparison of the results of experimental group 2 and control group 2 showed that the elastase inhibitor Ly could also significantly improve the condition of osteoarthritis in the aging process and alleviate the damage and wear of articular cartilage related to aging; at the same time, the OARSI score of cartilage tissue in the Ly group was significantly lower than that in the control group mice; Micro-CT results also showed that the parameters such as the ratio of bone volume to tissue volume (BV / TV) in the Ly group were significantly higher than those in the control group mice, and the bone-related indicators were significantly improved, and the cartilage morphology was closer to normal.

[0244] Therefore, it can be seen that the elastase inhibitor Ly has a good therapeutic effect on osteoarthritis caused by different pathological factors, which further verifies the effectiveness and potential of elastase inhibitors in the treatment of osteoarthritis. It can be used as a potential effective drug for the treatment of osteoarthritis and provides new ideas and methods for the treatment of osteoarthritis.

[0245] Example 10 Application of “DANA, an inhibitor of elastin fragment receptor complex” to antagonize elastin fragments - improving aging-related cardiovascular diseases

[0246] In this embodiment, the "elastin fragment receptor complex inhibitor DANA" is used to antagonize elastin fragments to improve aging-related cardiovascular diseases. The specific operation is as follows:

[0247] A mouse model of natural aging was selected and randomly divided into two groups. The experimental group was given an intraperitoneal injection of the elastin fragment receptor complex inhibitor DANA (dose of 2.5 mg / kg, dissolved in PBS), and the control group was injected with an equal amount of PBS. After 2 months of intervention, a series of tests were performed on the mice.

[0248] The detection indicators include indicators related to cardiovascular function, such as heart rate, blood pressure, cardiac output, etc.; vascular morphology indicators, such as arterial wall thickness, atherosclerotic plaque size, etc.; and levels of related markers in the blood, such as inflammatory factors and oxidative stress markers.

[0249] The results showed that the elastin fragment receptor complex inhibitor DANA significantly improved cardiovascular function, reduced heart rate and blood pressure, and increased cardiac output. At the same time, the thickness of the arterial wall decreased, atherosclerotic plaques were significantly reduced, and the levels of inflammatory factors and oxidative stress markers in the blood were significantly reduced.

[0250] Therefore, it can be seen that the "elastin fragment receptor complex inhibitor DANA" can effectively antagonize elastin fragments and has a significant improvement effect on aging-related cardiovascular diseases, providing a new potential direction and effective strategy for the treatment of aging-related cardiovascular diseases.

[0251] Example 11 Application of antagonistic elastin fragments after knocking out Neu1 gene in elastin receptor complex - improving aging-related liver diseases

[0252] In this embodiment, after knocking out the Neu1 gene in the elastin receptor complex, the elastin fragment is antagonized to improve aging-related liver diseases. The specific operation is as follows:

[0253] In this example, mice with conditional and specific knockout of Neu1 gene on myeloid cells were constructed according to the method in Example 4, and the Neu1 gene in the elastin receptor complex was knocked out to antagonize the elastin fragment to explore its improvement effect on aging-related liver diseases.

[0254] The mice were randomly divided into two groups, one for gene knockout and the other for non-knockout control. After 16 weeks (12 weeks to -28 weeks of age) of natural aging monitoring, the livers of both groups of mice were fully tested.

[0255] The detection indicators include liver function-related indicators, such as alanine aminotransferase and aspartate aminotransferase levels; liver tissue pathology indicators, including hepatocyte morphology, inflammatory cell infiltration, etc.; and liver fibrosis-related indicators, such as collagen deposition levels.

[0256] The results showed that knocking out the Neu1 gene significantly improved liver function, and the levels of alanine aminotransferase and aspartate aminotransferase returned to normal. Liver histopathology showed that the morphology of liver cells tended to be normal, the infiltration of inflammatory cells decreased, and the degree of liver fibrosis was significantly reduced. This fully proves that knocking out the Neu1 gene in the elastin receptor complex can effectively antagonize elastin fragments and significantly improve the symptoms of aging-related liver diseases, providing new ideas and potential targets for the treatment of liver diseases.

[0257] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of an agent for inhibiting the binding of elastin fragments to receptors for preparing an agent for preventing or antagonizing aging.

2. The use according to claim 1, characterized in that The elastin fragment comprises a motif "(GXXPG)n", wherein "X" is any amino acid except proline, and n is an integer ≥1.

3. The use according to claim 2, characterized in that The receptors include the membrane protein NEU1.

4. The use according to claim 3, characterized in that The reagents for inhibiting the binding of elastin fragments to receptors include one or more of antibodies or antibody fragments capable of binding to elastin fragments, antibodies or antibody fragments capable of binding to NEU1, reagents for eliminating, knocking out, or knocking down NEU1 gene expression, or elastin fragment receptor complex inhibitors.

5. The use according to claim 4, characterized in that The elastin fragment receptor complex inhibitors include cathepsin A inhibitors, elastin binding protein EBP inhibitors, and sialidase NEU1 inhibitors.

6. Use of a reagent for knocking out NEU1 gene expression for preparing a reagent for reducing serum inflammatory factor content, characterized in that: The inflammatory factors include any one or more of IL-1, MCP-1 or TNF-α.

7. Use of an agent for knocking out NEU1 gene expression for preparing an agent for preventing and / or treating aging-related liver diseases.

8. Use of an elastin fragment receptor complex inhibitor for preparing an agent for preventing and / or treating aging-related cardiovascular diseases.

9. Use of an elastin fragment receptor complex inhibitor for preparing an agent for improving athletic ability or reducing scoliosis.

10. Use of an elastin fragment receptor complex inhibitor for preparing a reagent for reducing the content of aging-related serum indicators, characterized in that: The aging-related serum indicators include any one or more of AST, ALT, GLU or LDL.

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