Application of C1INH as senescence biomarker and intervention target in senescence degree evaluation and anti-senescence intervention

Abstract

The invention discloses application of C1INH as an aging biomarker and an intervention target in aging degree assessment and anti-aging intervention, relates to the field of aging biology, and particularly relates to application of C1INH as a biomarker for aging assessment and as an anti-aging intervention target in aging degree assessment and anti-aging intervention. The invention relates to application in products for evaluating the physiological aging degree of individuals and application in products for improving the aging-related physiological manifestations of the individuals. Clinical sample analysis finds that the content of C1INH in blood has a significant decreasing trend (plt; 0.05) for healthy young people, healthy middle-aged people and healthy old people. Animal experiments show that the C1INH protein is exogenously supplemented to old mice, and the aging of main organs can be remarkably improved. The invention provides a new scheme for aging evaluation and improvement.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of aging biology, and in particular to the application of C1INH as a marker of aging and intervention target in assessing the degree of aging and anti-aging intervention. BACKGROUND

[0002] Aging is a complex biological process that occurs over time, characterized by systemic decline in physiological function and increased susceptibility to age-related diseases. Its molecular mechanisms involve genomic instability, telomere loss, epigenetic changes, protein homeostasis imbalance, and abnormal intercellular communication. Currently, the core bottleneck in the field of aging research is the lack of a key biological indicator that can accurately quantify the degree of individual aging and directly translate into an effective intervention target. Existing methods for assessing aging rely on macroscopic characterization or complex molecular profiles, which have significant limitations in terms of operability, specificity, and coherence with treatment strategies.

[0003] The core breakthrough of the present application lies in the discovery and establishment of a key factor that serves both as a quantitative standard for assessing the degree of aging and as an intervention target for aging. This discovery provides a scientific basis and technical support for developing products and intervention strategies that target the degree of aging. SUMMARY

[0004] The first object of the present application is to provide a plasma marker for assessing the degree of physiological aging in individuals, namely plasma C1INH (C1 inhibitor).

[0005] The second object of the present application is to provide the application of plasma C1INH in the preparation of products for assessing the degree of aging in individuals. By detecting the content of C1INH protein or its encoding gene SERPING1 , the degree of aging in individuals can be assessed. Products for detecting the content of plasma C1INH protein include Elisa kits, Western blot detection reagents, or proteomics detection reagents. Products for detecting the content of encoding genes in plasma include nucleic acid detection products. SERPING1

[0006] The third object of the present application is to provide the application of C1INH protein or its encoding gene SERPING1 as an anti-aging target in the preparation of products for improving the physiological performance related to aging in individuals. This product can improve the physiological performance in any of the following ways: up-regulating the content of C1INH protein in the body of individuals; enhancing the activity of C1INH protein in the body of individuals; up-regulating the expression level of encoding genes in the body of individuals; and enhancing the activity of encoding genes in the body of individuals. SERPING1 SERPING1 ​​The transcription efficiency. The aging-related physiological manifestations include abnormal aging states of key organs, including the heart, liver, kidney, brain, and lungs. The improvement effect is manifested in: downregulating the expression levels of aging markers P21, P53, and P16 in organ tissues; and reducing the expression levels of aging-related secretory phenotype (SASP) markers TNF-α, IL-6, and MMP9 in organ tissues.

[0007] A fourth objective of this invention is to provide a composition for delaying the physiological aging process or improving age-related physiological characteristics, comprising an effective amount of C1INH protein or a functionally active fragment thereof, and a pharmaceutically acceptable carrier. The functionally active fragment of the C1INH protein satisfies the following condition: retaining the activity of the C1INH protein in regulating the expression of aging-related biomarkers, including P21, P53, P16, TNF-α, IL-6, and MMP9. The pharmaceutically acceptable carrier includes a diluent, binder, disintegrant, coating agent, adsorbent, wetting agent, solvent, suspending agent, or stabilizer.

[0008] The fifth objective of this invention is to provide a recombinant viral vector for delaying the physiological aging process or improving aging-related physiological characteristics, comprising encoding the C1INH protein. SERPING1 Genes and related SERPING1 The promoter sequence for operational gene linkage is used to increase C1INH protein levels in individuals, thereby delaying the physiological aging process or improving age-related physiological characteristics. The recombinant viral vector includes lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors. SERPING1 The gene has a nucleic acid sequence as shown in SEQ ID NO:2. The promoter sequence is a constitutive promoter or an inducible promoter, wherein the constitutive promoter includes the CMV promoter and the EF1α promoter, and the inducible promoter includes the tetracycline-inducible promoter.

[0009] The C1INH protein described in this invention has the amino acid sequence shown in SEQ ID NO:1. SERPING1 The gene has a nucleic acid sequence as shown in SEQ ID NO:2.

[0010] To achieve the above objectives, this invention selected young, middle-aged, and elderly volunteers with clearly defined health status as research subjects, and conducted systematic analysis, screening, and verification of their plasma samples. This study reveals for the first time a close correlation between the blood factor C1INH and the individual aging process, specifically: the C1INH content in plasma exhibits a significant age-dependent trend, i.e., the C1INH content in healthy young volunteers > healthy middle-aged volunteers > healthy elderly volunteers, and the differences in content among the three age groups are statistically significant. p<0.05). This finding provides crucial experimental evidence for further in-depth research into the role of C1INH in aging regulation and its related applications.

[0011] This invention further validated the biological function of C1INH protein through animal experiments: after intravenous injection of C1INH protein into naturally aging C57BL / 6 mice, aging-related indicators of key organs such as the heart, liver, kidneys, brain, and lungs were detected. The results showed that the protein can effectively improve the aging state of the major organs in mice. Its regulatory effect is specifically manifested in that it can significantly downregulate the expression levels of aging markers (P21, P53, P16) in organ tissues, and also significantly reduce the expression levels of aging-related secretory phenotype (SASP) markers (TNF-α, IL-6, MMP9), providing in vivo experimental support for the application of C1INH protein in aging intervention.

[0012] Compared with the prior art, the technical solution provided by the present invention can produce the following significant beneficial effects: (1) This invention establishes for the first time blood C1INH content as an objective indicator for quantifying the degree of individual aging. Based on clear, age-specific critical reference values ​​(e.g., ≥315 ng / mL for young adults, ≥290 ng / mL for middle-aged adults, and ≥215 ng / mL for the elderly), a single test can achieve dual discrimination: firstly, it can distinguish the physiological age stage of an individual (young adults, middle-aged adults, and the elderly); secondly, it can identify individuals at risk of abnormally accelerated aging (i.e., aging status deviating from the normal range of the same age group) within the same age group, providing a precise basis for early intervention. Figure 1 ).

[0013] (2) This invention breaks through the bottleneck of the disconnect between assessment biomarkers and intervention targets in traditional research, and uses C1INH as both an aging biomarker and an anti-aging intervention target. This constructs a precise intervention paradigm that integrates "assessment-intervention", that is, when an individual's C1INH level is found to be low, an intervention plan with "supplementing or upregulating C1INH" as the core strategy can be directly adopted, realizing the coherence and targeting from the identification of aging status to the formulation of intervention plan.

[0014] (3) The intervention strategy of this invention demonstrates remarkable systemic improvement capabilities. Animal experiments have confirmed that peripheral intravenous injection of C1INH protein can effectively improve the aging phenotype of major peripheral organs (heart, liver, kidney, lung) and the brain simultaneously. Specifically, it significantly reduces the expression levels of cellular aging markers such as P21 and P53, as well as inflammation-related factors such as TNF-α and IL-6, providing strong in vivo experimental support for the effectiveness of the intervention strategy. Figures 2-6 ).

[0015] (4) This invention provides a diversified technical approach for anti-aging intervention targeting C1INH, and its scope of protection covers: a) protein replacement therapy (direct supplementation of C1INH protein or its functionally active fragments); b) gene regulation intervention (delivery via viral vectors such as AAV). SERPING1 (a) Gene-based long-term in vivo expression of C1INH; (b) Target-guided intervention screening (searching for intervention substances that can upregulate C1INH expression or activity). This provides flexible and feasible options for the development of anti-aging products in different application scenarios.

[0016] (5) The technical solution of the present invention provides a new direction for C1INH to be used for the status assessment of aging (including systemic aging and organ / tissue-specific aging) and as an intervention target in anti-aging intervention. Attached Figure Description

[0017] Figure 1 The graph shows the results of plasma C1INH content measurement in healthy young adults, healthy middle-aged adults, and healthy elderly subjects in this invention.

[0018] Figure 2 This diagram illustrates the effect of C1INH intervention on the expression of lung aging markers and SASP markers in aging mice, as described in this invention.

[0019] Figure 3 This diagram illustrates the effect of C1INH intervention on the expression of liver aging markers and SASP markers in aging mice, as described in this invention.

[0020] Figure 4 This diagram illustrates the effects of C1INH intervention on the expression of renal aging markers and SASP markers in aging mice, as described in this invention.

[0021] Figure 5 This diagram illustrates the effects of C1INH intervention on the expression of aging markers and SASP markers in the brains of aging mice, as described in this invention.

[0022] Figure 6 This diagram illustrates the effects of C1INH intervention on the expression of cardiac aging markers and SASP markers in aging mice, as described in this invention.

[0023] Figures 2-6 In the study, AC represents aging markers P21, P53, and P16; DF represents SASP markers IL-6, TNF-α, and MMP9.

[0024] Figure 7The following figures illustrate the effects of C1INH intervention on the cognitive function of aging mice in this invention: A: Behavioral trajectory of mice in each group; B: Latency statistics of mice in each group; C: Platform crossing frequency statistics of mice in each group; D: Platform quadrant time percentage statistics of mice in each group; E: Platform quadrant distance percentage statistics of mice in each group. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] This study protocol was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine (Ethics Approval No.: NO.2024047XZ(KT)-014-03). All participants fully understood the purpose and process of the study and voluntarily signed written informed consent forms to participate in this study.

[0027] Example 1

[0028] This embodiment aims to study the detection of plasma C1INH levels in healthy subjects of different age groups, the establishment of reference ranges, and the assessment of aging tendencies.

[0029] By comparing plasma C1INH levels in healthy young, middle-aged, and elderly subjects, reference ranges for C1INH levels at each age stage were established, and their application value in assessing aging tendency was explored. A total of 90 subjects were selected and divided into three groups according to age: a healthy young subject group (n=30, 18-44 years old), a healthy middle-aged subject group (n=30, 45-59 years old), and a healthy elderly subject group (n=30, 60 years and older). All subjects were clinically confirmed to have no major organ diseases, chronic inflammation, tumors, or endocrine diseases affecting metabolism, and had not taken any anti-aging related drugs or health products in the past 3 months. After grouping and numbering the subjects, 5 mL of peripheral venous blood was collected in the morning on an empty stomach. The blood was anticoagulated with EDTA, centrifuged at 4℃ and 3000 r / min for 15 min to prepare plasma, which was then frozen at -80℃ for later use. The C1INH ELISA kit (brand: Boyan Biotechnology, catalog number: BY-EH115190) was used, and the procedure was strictly followed according to the kit instructions. The C1INH content in each plasma sample was measured, with three replicates per sample. The average value was taken as the final result for analysis and comparison. The experimental results are as follows: Figure 1As shown, plasma C1INH levels exhibited a significant age-related decreasing trend: plasma C1INH levels in young subjects were significantly higher than those in healthy middle-aged subjects ( p <0.001), plasma C1INH levels in healthy middle-aged subjects were significantly higher than those in healthy elderly subjects. p <0.01). The normal range for healthy young adults aged 18-44 is 926 ng / mL-315 ng / mL; the range for healthy middle-aged adults aged 45-59 is 760 ng / mL-290 ng / mL; and the normal range for healthy elderly subjects aged 60 and above is 615 ng / mL-215 ng / mL.

[0030] Based on the test results of 90 healthy subjects in this study, the normal reference ranges for plasma C1INH levels for each age group were determined using 95% confidence intervals: the lower limit of the normal reference range for plasma C1INH levels in healthy young adults aged 18-44 was 315 ng / mL; the lower limit of the normal reference range for healthy middle-aged adults aged 45-59 was 290 ng / mL; and the lower limit of the normal reference range for healthy elderly people aged 60 and above was 215 ng / mL. When a subject's plasma C1INH level was lower than the reference lower limit for their age group, it suggested a tendency for an abnormally accelerated aging process.

[0031] Example 2

[0032] This embodiment aims to investigate the effects of intravenous injection of C1INH on the expression of aging markers in major organs such as the heart, liver, kidney, brain, and lungs of naturally aging C57BL / 6 mice.

[0033] Healthy C57BL / 6 mice were selected and divided into three groups according to age and intervention method: a young control group (3 months old, n=6-8), in normal physiological state, without any drug treatment during the experiment; an aging model group (13 months old, n=6-8), naturally aging mice serving as a model control, receiving an equal volume of physiological saline via intravenous injection; and an aging + C1INH intervention group (13 months old, n=6-8), naturally aging mice receiving C1INH intervention via intravenous injection. All experimental mice were housed in an SPF-grade animal facility under consistent environmental conditions (temperature 22-25℃, humidity 50%-60%, 12h light / 12h dark cycle), with free access to food and water. The experiment officially began one week after the mice adapted to the above-mentioned housing environment.

[0034] Before the experiment, the basal expression levels of aging markers P21, P53, TNF-α, and IL-6 in the heart, liver, kidney, brain, and lung tissues of 13-month-old mice were measured. Compared with the 3-month-old young control group, the expression of the above markers in the model group was significantly increased. p<0.05), confirming the successful establishment of the natural aging model. The aging + C1INH intervention group was intervened by intravenous injection of C1INH at a dose of 4 IU / kg, once a week for 4 consecutive weeks. The aging model group received an equal volume of sterile saline intravenously during the same period, while the young control group received no intervention. 72 h after the last intervention, mice were euthanized by cervical dislocation, and heart, liver, kidney, brain, and lung tissues were quickly separated. Connective tissue and vascular impurities were removed, and the tissues were rinsed with pre-cooled PBS buffer. After blotting the surface moisture with filter paper, some tissues were placed in RNase-free cryovials and rapidly frozen at -80°C for subsequent RNA extraction.

[0035] Total RNA was extracted from various organs and tissues using Trizol extraction. RNA purity (A260 / A280 ratio of 1.8-2.0) and concentration were detected using Nanodrop to ensure that the RNA quality met the standards.

[0036] Real-time quantitative PCR (qRT-PCR) detection: Using qualified total RNA as a template, cDNA was synthesized using a reverse transcription kit (brand: Yisheng Biotechnology, catalog number: Cat: 11139ES60); using cDNA as a template, the mRNA expression levels of aging biomarkers and aging-related secretory phenotypic biomarkers P21, P53, P16, TNF-α, IL-6, and MMP9 were detected using a qRT-PCR kit (brand: Yisheng Biotechnology, catalog number: Cat: 11202ES08) on a real-time fluorescence quantitative PCR instrument. GAPDH was used as an internal control gene in the experiment. -ΔΔCt The relative expression level of the target gene was calculated using a method with three replicates for each sample.

[0037] The primer sequences used are as follows: P21 upstream 5'-TGAGCGGCCTGAAGATTCC-3', downstream 5'-ATCTGCGCTTGGAGTGATAGA-3'; P53 upstream 5'-AACTTACCAGGGCAACTATGGCTTC-3', downstream 5'-AACTGCACAGGGCACGTCTTC-3'; P16 upstream 5'-GCTCTTCTGCTCAACTACGGT-3', downstream 5'-CGATGTCTTGATGTCCCCGC-3'; TNF-α upstream 5'-ACTCCAGGCGGTGCCTATGT-3', downstream 5'-GTGAGGGTCTGGGCCATAGAA-3'; IL-6 upstream 5'-CCACTTCACAAGTCGGAGGCTTA-3', downstream 5'-TGCAAGTGCATCATCGTTGTTC-3'; MMP9 upstream 5'-GCCCTGGAACTCACACGACA-3', downstream 5'-TTGGAAACTCACACGCCAGAAG-3'.

[0038] like Figures 2-6 As shown (corresponding to lung, liver, kidney, brain, and heart tissues respectively), compared with the aging model group, the mRNA expression levels of P21, P53, P16, TNF-α, IL-6, and MMP9 in the above organs of mice in the aging + C1INH intervention group were significantly reduced. p <0.05, p <0.01 or p <0.001), with some indicators approaching the levels of the young control group, suggesting that C1INH can improve the organ aging phenotype of naturally aging mice by downregulating the expression of aging markers in major organs.

[0039] Statistical analysis: All experimental data were statistically analyzed using SPSS 26.0 software. Quantitative data were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons among multiple groups, and LSD-t tests were used for pairwise comparisons between groups. p <0.05 indicates a statistically significant difference.

[0040] Example 3

[0041] This embodiment aims to conduct the Morris water maze experiment, with the same grouping and intervention methods as in Embodiment 2, to examine the effect of the C1INH intervention regimen on the cognitive abilities of aging mice.

[0042] Movement trajectory recording: In the orientation navigation experiment (days 11-15) and spatial exploration experiment (day 16), the software captured the swimming trajectories of mice in each group in real time, visually presenting the mice's memory and orientation ability regarding the platform location; Latency comparison: During the orientation navigation experiment, the average escape latency (upper limit 60 s) was recorded for 4 training sessions per day, and the changes in latency for each group from day 11-15 were statistically analyzed to reflect learning ability; Comparison of platform crossing frequency: In the spatial exploration experiment (day 16, platform removed), the number of times mice crossed the original platform location within 5 minutes was recorded, reflecting spatial memory retention ability; Comparison of platform quadrant swimming distance: In the spatial exploration experiment, the proportion of swimming distance in the target quadrant within 5 minutes was statistically analyzed to verify the preference for the target area; Comparison of platform quadrant dwell time: In the spatial exploration experiment, the dwell time of mice in the target quadrant within 5 minutes was recorded to quantify spatial memory level. Latency was analyzed using repeated measures ANOVA (analysis time × between-group interaction effect); the number of crossings, swimming distance percentage, and dwell time were analyzed using one-way ANOVA, and pairwise comparisons were performed using the LSD-t test. p <0.05 indicates a statistically significant difference.

[0043] Experimental results: such as Figure 7 As shown, compared with the model group, C1INH significantly improved the spatial memory ability of aging mice.

[0044] Conclusion: Exogenous intravenous supplementation of C1INH protein (4 IU / kg, 4 times at 3-day intervals) can effectively restore plasma C1INH levels in 13-month-old C57BL / 6J aging mice and significantly improve their spatial learning and memory abilities (manifested as shortened latency, increased number of platform crossings, and prolonged time spent in the target quadrant). This demonstrates that C1INH protein is an effective intervention for anti-aging and provides a new strategy for improving individual aging performance.

Claims

1. The application of plasma C1INH as an aging biomarker in the preparation of products for assessing the degree of physiological aging in individuals, characterized in that, An assessment of age-related reference ranges based on plasma C1INH levels was conducted. These reference ranges were determined using data from 90 healthy subjects and 95% confidence intervals: the normal reference range for plasma C1INH levels in healthy young adults aged 18-44 years was 315 ng / mL-926 ng / mL, with a lower limit of 315 ng / mL; the normal reference range for healthy middle-aged adults aged 45-59 years was 290 ng / mL-760 ng / mL, with a lower limit of 290 ng / mL; and the normal reference range for healthy elderly individuals aged 60 years and older was 215 ng / mL-615 ng / mL, with a lower limit of 215 ng / mL. When a subject's plasma C1INH level was lower than the lower limit of the reference range for their age group, it suggested a tendency for abnormally accelerated aging.

2. The application according to claim 1, characterized in that, The plasma C1INH includes the C1INH protein and its encoding gene. SERPING1 The C1INH protein has the amino acid sequence shown in SEQ ID NO:1, and the encoding gene... SERPING1 It has the nucleic acid sequence shown in SEQ ID NO:2; by detecting the content of the C1INH protein in plasma or the encoding gene. SERPING1 The content of [something] enables the assessment of an individual's physiological aging level.

3. The application according to claim 1, characterized in that, The product is a detection product for detecting the C1INH protein content in plasma, including ELISA kits, Western blot assays, or proteomics assays; and / or the product is for detecting gene-encoding substances in plasma. SERPING1 Nucleic acid testing products with high content.

4. C1INH protein or its encoding gene SERPING1 Its application as an anti-aging target in the preparation of products that improve individual aging-related physiological manifestations is characterized by... The C1INH protein has the amino acid sequence shown in SEQ ID NO:1, and the encoding gene SERPING1 It has a nucleic acid sequence as shown in SEQ ID NO:

2.

5. The application according to claim 4, characterized in that, The aging-related physiological manifestations include abnormal aging states of key organs, including the heart, liver, kidneys, brain, and lungs.

6. The application according to claim 4, characterized in that, The product exerts its beneficial effects through at least one of the following mechanisms: upregulating the level of C1INH protein in an individual; enhancing the activity of C1INH protein in an individual; upregulating the coding gene in an individual. SERPING1 Expression levels; enhance the expression levels of gene-encoding genes in individuals. SERPING1 transcription efficiency.

7. The application according to claim 6, characterized in that, The improvement is manifested in the following ways: downregulating the expression levels of aging markers P21, P53, and P16 in organ tissues; and reducing the expression levels of aging-related secretory phenotypic markers TNF-α, IL-6, and MMP9 in organ tissues.

8. A composition for delaying the physiological aging process of an individual or improving aging-related physiological characteristics, characterized in that, The invention comprises an effective amount of an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is C1INH protein or a functionally active fragment thereof; wherein the C1INH protein has an amino acid sequence as shown in SEQ ID NO:1; and the functionally active fragment of the C1INH protein satisfies the following condition: retaining the activity of C1INH protein in regulating the expression of aging-related biomarkers, including P21, P53, P16, TNF-α, IL-6, and MMP9.

9. A recombinant viral vector for delaying the physiological aging process or improving aging-related physiological characteristics, characterized in that, Contains the protein encoding C1INH SERPING1 Genes and related to SERPING1 The promoter sequence for gene operational linkage; wherein, the SERPING1 The gene has a nucleic acid sequence as shown in SEQ ID NO:

2.

10. The recombinant viral vector according to claim 9, characterized in that, The recombinant viral vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector; the promoter sequence is a constitutive promoter or an inducible promoter, the constitutive promoter includes the CMV promoter and the EF1α promoter, and the inducible promoter includes the tetracycline inducible promoter.

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