Application of aging / aging related signal channel and channel molecule as aging inhibition or aging detection target

Abstract

The invention provides an aging / senescence related signal channel and application of a channel molecule as an aging inhibition or senescence detection target. The invention discloses a novel signal channel DKK1-LRP5 / 6-PreLamin A-Lamin A which is closely related to the evaluation or prognosis of senescence for the first time. The invention also discloses a new mechanism participated by the signal channel or the channel protein of the signal channel and used for regulating and controlling senescence and senescence-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and medicine; more specifically, the present invention relates to the application of the DKK1-LRP5 / 6-PreLaminA-Lamin A signaling pathway and its pathway molecules as assessment or prognostic markers of aging, or the application of regulators of the signaling pathway in inhibiting aging or aging-related diseases. Background Art

[0002] Health and longevity are eternal topics for human beings. Despite the rapid development of modern molecular biology and drug research and development, how to delay aging is still a major problem. Aging has never been regarded as a disease, but it is an independent risk factor for many diseases including cancer, cardiovascular and cerebrovascular diseases, osteoarthritis and systemic inflammation.

[0003] Aging is often accompanied by low immunity and reduced disease resistance, making people more susceptible to viral infections, tumors, cardiovascular diseases, atherosclerosis, arthritis, etc., and it is difficult to cure the disease. It can also cause a variety of complications, reduce the quality of life of patients, and seriously endanger people's lives and health. Aging is also closely related to sub-health of the body.

[0004] Unfortunately, there are currently no blood markers in the art that can concisely and effectively monitor individual aging; at the same time, there is an urgent need in the art for effective substances that inhibit aging. Summary of the invention

[0005] The purpose of the present invention is to provide an application of aging / senescence-related signaling pathways and pathway molecules as targets for inhibiting aging or detecting aging.

[0006] In the first aspect of the present invention, there is provided the use of a regulator of the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway (including signaling pathway molecules DKK1, LRP5, LRP6, PreLamin A and / or Lamin A) for preparing a composition for inhibiting aging; wherein the regulator comprises a member selected from the following group: an inhibitor of the expression or activity of DKK1, an inhibitor of the membrane endocytosis of LRP5 / 6, a stabilizer of LRP5 / 6 (stable at the corresponding position in the cell), and an intranuclear PreLamin A or Lamin upregulator.

[0007] In one or more embodiments, the expression or activity inhibitor of DKK1 includes (but is not limited to): an agent that inhibits the activity of DKK1 protein, an agent that knocks out or silences the DKK1 gene; preferably, it includes: an antibody (neutralizing antibody) that specifically inhibits the activity of DKK1 protein, a small molecule DKK1 protein inhibitor, an interfering molecule that specifically interferes with the expression of the DKK1 gene, a CRISPR gene editing agent, a homologous recombination agent or a site-directed mutagenesis agent for the DKK1 gene (the agent causes a loss-of-function mutation in DKK1).

[0008] In one or more embodiments, the LRP5 / 6 membrane endocytosis inhibitor includes: a TransGlutaminase-2 (transglutaminase 2, TG2) inhibitor; preferably, the TransGlutaminase-2 inhibitor includes a small molecule compound MDC (MonoDansyl-Cadaverine).

[0009] In one or more embodiments, the nuclear PreLamin A or Lamin A upregulator includes (but is not limited to): a vector expressing PreLamin A or Lamin A, a PreLamin A nuclear transport (nuclear import) promoter or a PreLamin A extracellular transport inhibitor.

[0010] In another aspect of the present invention, a method for preparing a composition for inhibiting aging by using a regulator of the DKK1-LRP5 / 6-PreLamin A-LAMIN A signaling pathway is provided; wherein the regulator comprises a member selected from the group consisting of an inhibitor of the expression or activity of DKK1, an inhibitor of the membrane endocytosis of LRP5 / 6, an LRP5 / 6 stabilizer (stabilized at the corresponding position in the cell), an intranuclear PreLamin A or Lamin A. A up-regulator; preferably, the expression or activity inhibitor of DKK1 includes: an agent that inhibits the activity of DKK1 protein, an agent that knocks out or silences the DKK1 gene; preferably, it includes: an antibody (neutralizing antibody) that specifically inhibits the activity of DKK1 protein, a small molecule DKK1 protein inhibitor, an interfering molecule that specifically interferes with the expression of the DKK1 gene, a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent for the DKK1 gene (the reagent causes DKK1 to undergo a loss-of-function mutation); preferably, the small molecule DKK1 protein inhibitor includes: a TransGlutaminase-2 (TG2) inhibitor (such as the small molecule compound MDC).

[0011] In one or more embodiments, the LRP5 / 6 membrane endocytosis inhibitor includes (but is not limited to): an agent that inhibits the LRP5 / 6 membrane endocytosis activity (such as an inhibitor of DKK1 expression or activity is also an agent that inhibits the LRP5 / 6 membrane endocytosis activity).

[0012] In one or more embodiments, the nuclear PreLamin A or Lamin A upregulator includes (but is not limited to): a vector expressing PreLamin A or Lamin A, a PreLamin A nuclear transport (nuclear import) promoter or a PreLamin A extracellular transport inhibitor.

[0013] In one or more embodiments, the senescence comprises aging.

[0014] In one or more embodiments, the aging includes: physiological aging (eg, aging in a natural state) and pathological aging (eg, aging caused by a disease state).

[0015] In one or more embodiments, the aging includes / encompasses aging-related diseases.

[0016] In one or more embodiments, the inhibition of aging includes: delaying disease development / progression.

[0017] In one or more embodiments, the delaying of disease development / progression includes: delaying tumor progression, delaying diabetes progression.

[0018] In one or more embodiments, the inhibition of aging includes: prolonging life span, alleviating metabolic syndrome, enhancing cardiac function (including reducing the risk of heart disease), preventing or alleviating (including treating) sub-health (maintaining a healthy state), enhancing athletic activity or endurance (reducing / alleviating weakness), promoting wound healing, enhancing hair gloss (reducing roughness), and reducing spinal curvature.

[0019] In one or more embodiments, the extended survival includes extended healthy life span.

[0020] In one or more embodiments, the alleviation of metabolic syndrome includes: lowering blood sugar, lowering blood lipids, lowering total cholesterol, lowering triglycerides, lowering LDL, increasing HDL, reducing the risk of coronary atherosclerosis, increasing body weight, and reducing inflammation levels.

[0021] In one or more embodiments, the inhibiting of aging includes preventing, alleviating or treating developmental delay.

[0022] In one or more embodiments, the aging includes: the aging of people / populations aged 30 years or older, such as 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 years or older; for example, the aging includes: the aging of people / populations aged 60-89 years or 60-90 years.

[0023] In one or more embodiments, when used to prevent, alleviate or treat developmental delay, or when used to enhance athletic activity or endurance, reduce / alleviate weakness, or when used to promote wound healing, or when used to reduce spinal curvature, or when used to alleviate sub-health, the aging includes aging / senescence of all age groups, for example, including aging / senescence in childhood, adolescence, youth, middle age or old age. A senescent state / aging environment / senescent cells will be produced in any age group.

[0024] In one or more embodiments, the up-regulation, improvement, enhancement, increase, strengthening or promotion is statistically (or significantly) up-regulation, improvement, enhancement, increase, strengthening or promotion, such as up-regulation, improvement, enhancement, increase, strengthening or promotion by 10% or more than 20% compared with the control or substrate, preferably by 40% or more than 50%, and more preferably by 80% or more than 100%.

[0025] In one or more embodiments, the down-regulation, inhibition, reduction, decrease or weakening is statistically (or significantly) down-regulation, inhibition, reduction, decrease or weakening, such as down-regulation, inhibition, reduction, decrease or weakening by 10% or more than 20% compared with the control or substrate, preferably 40% or more than 50%, and more preferably 80% or more than 100%.

[0026] In one or more embodiments, the small molecule compound also includes stereoisomers, geometric isomers, tautomers, racemates, hydrates, solvates, and pharmaceutically acceptable salts or prodrugs thereof.

[0027] In another aspect of the present invention, there is provided the use of DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway molecules for use as diagnostic markers and in the preparation of detection reagents for aging assessment or prognosis; wherein the signaling pathway molecules include: DKK1, LRP5 / 6, PreLamin A or Lamin A; preferably, the signaling pathway molecules include: DKK1, PreLamin A; more preferably, the DKK1 or PreLamin A is blood DKK1 or PreLamin A.

[0028] In another aspect of the present invention, there is provided a use of a reagent for specifically detecting DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway molecules, for preparing a detection reagent or a detection kit for aging assessment or prognosis; wherein the signaling pathway molecules include: DKK1, LRP5 / 6, PreLamin A or Lamin A; preferably, the signaling pathway molecules include: DKK1, PreLamin A; more preferably, the DKK1 or PreLamin A is blood DKK1 or blood PreLamin A.

[0029] In one or more embodiments, the assessment or prognosis is selected from the following: aging assessment or prognosis is performed based on the expression or activity of DKK1 protein in blood (including serum, plasma or whole blood), high expression or high activity of DKK1 protein indicates an aging state (or a subhealthy state); low expression or low activity of DKK1 protein (including normal expression or normal activity, and those with no significant difference from the control population are "normal") indicates a healthy state; preferably, the expression or activity of DKK1 protein is the expression or activity of DKK1 protein in blood (including serum, plasma or whole blood).

[0030] In one or more embodiments, aging assessment or prognosis is performed based on the expression or activity of PreLamin A protein in the blood. High expression or high activity of PreLamin A protein indicates an aging state (or a subhealthy state); low expression or low activity of PreLamin A protein (including normal expression or normal activity, with no significant difference from the control population being "normal") indicates a healthy state.

[0031] In one or more embodiments, based on the amount of PreLamin A or Lamin A in the cell nucleus, a low amount indicates an aging state (or a subhealthy state); a high amount (including normal) indicates a healthy state.

[0032] In one or more embodiments, aging assessment or prognosis is performed based on the LRP5 / 6 membrane endocytosis in the cell. A high level of LRP5 / 6 membrane endocytosis indicates an aging state (or a subhealthy state); a normal or low level of LRP5 / 6 membrane endocytosis indicates a healthy state.

[0033] In one or more embodiments, the detection reagent includes: an antibody specific against DKK1, LRP5 / 6, PreLamin A or Lamin A protein, a primer that specifically amplifies the coding gene of DKK1, LRP5 / 6, PreLamin A or Lamin A; and a probe that specifically recognizes the coding gene of DKK1, LRP5 / 6, PreLamin A or Lamin A.

[0034] In one or more embodiments, the aging state includes: poor health prognosis / sub-health. For example, it includes: increased inflammation level, high total cholesterol, triglyceride, LDL level, low HDL level, increased risk of heart disease, increased risk of coronary atherosclerosis, high blood sugar level, high blood lipid level, poor cardiac function prognosis (increased risk of heart disease).

[0035] In one or more embodiments, the health state includes: good health prognosis, for example, including: low inflammation level, low total cholesterol, triglyceride, LDL level, high HDL level, low risk of heart disease, low risk of coronary atherosclerosis, low blood sugar level, low blood lipid level, good cardiac function prognosis (low risk of heart disease).

[0036] In one or more embodiments, the high expression, high activity, high level, high presence, etc. is a statistically (or significantly) increase, such as an increase of 10% or 20% or more, preferably 40% or 50% or more, and more preferably 80% or 100% or more, compared to a control (e.g., a healthy person / healthy standard as a control) or a substrate.

[0037] In one or more embodiments, the low expression, low activity, low level, low presence, etc. is a statistically (or significantly) reduction, such as a reduction of 10% or more than 20% compared to a control (e.g., a healthy person / healthy standard as a control) or a substrate, preferably 40% or more than 50%, and more preferably 80% or more than 100%.

[0038] In another aspect of the present invention, a kit for evaluating or prognosing aging is provided, wherein the kit contains: a reagent for specifically detecting the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway or its signaling pathway molecules (including DKK1, LRP5, LRP6, PreLamin A and / or Lamin A).

[0039] In one or more embodiments, the kit further includes: enzyme chain immunoassay reagents, polymerase chain reaction reagents, protein immunoblotting reagents, and / or nucleic acid extraction reagents.

[0040] In another aspect of the present invention, the use of the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway is provided for screening substances that inhibit aging.

[0041] In another aspect of the present invention, a method for screening substances (including potential substances) that inhibit aging is provided, the method comprising: (1) contacting a candidate substance with a system containing a DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway; (2) screening out substances that regulate the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway, wherein the substances are substances (including potential substances) that are useful for inhibiting aging; wherein the regulation comprises: inhibiting the expression or activity of DKK1, inhibiting the endocytosis of LRP5 / 6 membrane, promoting the stability of LRP5 / 6 (stabilizing at the corresponding position in the cell), and upregulating PreLamin A or LaminA in the nucleus.

[0042] In one or more embodiments, step (1) comprises: adding a candidate substance to a system containing the DKK1-LRP5 / 6-PreLamin A-LAMINA signaling pathway.

[0043] In one or more embodiments, step (2) comprises: detecting changes in each protein or its encoding gene in the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway, and comparing with a control group, wherein the control group is a system containing the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway without adding the candidate substance; if the candidate substance inhibits the expression or activity of DKK1, inhibits LRP5 / 6 membrane endocytosis, promotes LRP5 / 6 stabilizer (stabilized at the corresponding position in the cell), and upregulates PreLamin A or Lamin in the nucleus, then the candidate substance is a substance useful for inhibiting aging.

[0044] In one or more embodiments, the system containing the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway is selected from: a cell (culture) system, a subcellular (culture) system, a tissue (culture) system or an animal system.

[0045] In one or more embodiments, the candidate substances include (but are not limited to): small molecule compounds designed for the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway, or its pathway proteins, or its upstream or downstream proteins or genes, regulatory molecules (such as but not limited to upregulators, interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands)), CRISPR constructs, compounds from a compound library, etc.

[0046] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The relationship between DKK1 upregulation and aging and aging-related diseases is shown.

[0048] Figure 1 a shows the effect of serum DKK1 expression level on the number of immune cells; mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

[0049] Figure 1 b shows the effect of serum DKK1 expression level on blood lipid expression level; mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

[0050] Figure 1 c shows the correlation between serum DKK1 expression level and cardiovascular disease; mean ± SD, ***P < 0.001.

[0051] Figure 1 d shows the effect of brisk walking on the expression level of serum DKK1 in the elderly; mean ± SD, **P < 0.01, ****P < 0.0001.

[0052] Figure 1 e shows the effect of statins on serum DKK1 expression level; mean ± SD, *P < 0.05.

[0053] Figure 1 f shows the trend of serum DKK1 increase and blood glucose or blood lipid abnormalities in different age groups; mean ± SD, serum DKK1 (red line, *P < 0.05, **P < 0.01, ***P < 0.001, *P < 0.05); blood glucose (yellow line, @P < 0.05, @@P < 0.01; HbA1c (black line, #P < 0.05, ##P < 0.001); total cholesterol (TC, pink line, $P < 0.05, $$P < 0.01); triglycerides (TG, purple line, P<0.05, P<0.01, P<0.001); HDL (green line, $P<0.05, $P<0.01) or LDL (blue line, P<0.05, P<0.01, P<0.001).

[0054] Figure 2 The results showed that the increase of serum DKK1 level had a damaging effect on the body of aged mice.

[0055] Figure 2 a shows the serum DKK1 expression levels in C57BL / 6 mice and BALB / c mice from 3 days to 14 months after birth. Mean ± SD, P<0.05, P<0.01 for comparison of DKK1 levels in C57BL / 6J and BALB / c at each time point. *P<0.05, **P<0.01, ****P<0.001 for comparison of DKK1 elevated levels at each time point

[0056] Figure 2 b, 2c show the effects of DKK1 neutralizing antibody on the spine of 12-month-old BALB / c mice. Scale bar = 1.0 cm

[0057] Figure 2 dj shows the effects of DKK1 neutralizing antibodies on body weight, grip strength, range of activity in a novel environment, walking speed and endurance, and cardiac function in 12-month-old mice. Figure 2 d Effect of DKK1 neutralizing antibody on body weight of 12-month-old mice; mean ± SD, *P < 0.05. Figure 2 e: Effect of DKK1 neutralizing antibody on the grasping force of 12-month-old mice; mean ± standard deviation, *P<0.05, ***P<0.001, ****P<0.0001. Figure 2 f shows the effect of DKK1 neutralizing antibody on open field locomotion in 12-month-old mice; mean ± SD, *P < 0.05. Figure 2 g: Effect of DKK1 neutralizing antibody on fatigue test in 12-month-old mice; mean ± SD, ***P < 0.001. Figure 2 h: Effect of DKK1 neutralizing antibody on running in 12-month-old mice; mean ± SD, **P < 0.01. Figure 2 i shows the effect of DKK1 neutralizing antibody on gait analysis of 12-month-old mice; mean ± standard deviation, *P<0.05, ****P<0.0001. Figure 2 j: Effect of DKK1 neutralizing antibody on cardiac function of 12-month-old mice; mean ± SD, ***P < 0.001.

[0058] Figure 2 k shows the effect of DKK1 and DKK1 neutralizing antibody on survival.

[0059] Figure 2 l shows the effects of DKK1 and DKK1 neutralizing antibody on wound healing in mouse tails. Mean ± SD, **P < 0.01, ***P < 0.001.

[0060] Figure 3 DKK1 was shown to exacerbate disease progression.

[0061] Figure 3 ac show the effects of one month of continuous injection of DKK1 on the body size, hair color, gripping strength, and range of activity in novel environments of CT26 colorectal cancer cell-bearing mice (BLAB / c mice) and Lewis lung cancer cell-bearing mice (C57BL / 6). Figure 3 a shows the effect of continuous injection of DKK1 on body size and hair color of CT26 colorectal cancer cell-bearing mice (BLAB / c mice) and Lewis lung cancer cell-bearing mice (C57BL / 6). Figure 3 b shows the effect of continuous injection of DKK1 on the grasping force of mice bearing CT26 colorectal cancer cells (BLAB / c mice) and Lewis lung cancer cells (C57BL / 6) mice. Figure 3c shows the effect of continuous injection of DKK1 on open field locomotion in CT26 colorectal cancer cell-bearing mice (BLAB / c mice) and Lewis lung cancer cell-bearing mice (C57BL / 6).

[0062] Figure 3 dh showed the effects of intraperitoneal injection of DKK1 on body size, hair color, gripping strength, range of motion in a novel environment, and running activity in C57BL / 6 mice fed a high-fat, high-sugar diet for 8 months. Figure 3 d shows the effect of intraperitoneal injection of DKK1 on body size and hair color in C57BL / 6 mice fed with a high-fat, high-sugar diet for 8 months. Figure 3 e Effect of intraperitoneal injection of DKK1 on body weight of C57BL / 6 mice fed with a high-fat and high-sugar diet for 8 months; mean ± SD, **P < 0.01. Figure 3 f: Effect of intraperitoneal injection of DKK1 on the grasping force of C57BL / 6 mice fed with a high-fat and high-sugar diet for 8 months; mean ± SD, **P < 0.01. Figure 3 g Effects of DKK1 intraperitoneally injected into C57BL / 6 mice fed with a high-fat and high-sugar diet for 8 months on open field locomotion; mean ± SD, **P < 0.01. Figure 3 h: Effects of intraperitoneal injection of DKK1 on running cardiopulmonary function of C57BL / 6 mice fed with a high-fat and high-sugar diet for 8 months; mean ± SD, **P < 0.05.

[0063] Figure 3 i shows the effect of intraperitoneal injection of DKK1 on cardiac function in C57BL / 6 mice fed with a high-fat and high-sugar diet for 8 months. Mean ± SD, ***P < 0.001.

[0064] Figure 3 j shows the effect of intraperitoneal injection of DKK1 on the survival of C57BL / 6 mice fed with a high-fat and high-sugar diet for 8 months. Compared with high-fat and high-sugar diet + DKK1, *P<0.05, ***P<0.001.

[0065] Figure 3 k, l show the effects of DKK1 injection for 10 months on the appearance and spine of 12-month-old C57BL / 6 mice.

[0066] Figure 3 m shows the effect of DKK1 injection for 10 months on the cardiac function of 12-month-old C57BL / 6 mice. Mean ± SD, *P < 0.05.

[0067] Figure 3n shows the survival curve of C57BL / 6 mice after DKK1 injection for 12 months. *P<0.05 compared with the PBS control group.

[0068] Figure 4 The role of LRP5 / 6 in senescence is shown.

[0069] Figure 4 a shows signs of aging and progressive physical defects in conditional LRP5 / 6 whole-body knockout mice.

[0070] Figure 4 bi shows the changes in spinal curvature, body weight, grip strength, range of motion in a novel environment, walking speed, treadmill endurance training, walking speed, swing speed, and survival in conditional LRP5 / 6 whole-body knockout mice. Figure 4 b shows the effect of conditional LRP5 / 6 whole-body knockout mice on spinal curvature. Figure 4 c Effects of conditional LRP5 / 6 knockout mice on body weight; mean ± SD, ****P < 0.0001. Figure 4 d Effect of conditional LRP5 / 6 knockout mice on grip strength; mean ± SD, ****P < 0.0001. Figure 4 e Effect of activity range of conditional LRP5 / 6 knockout mice in a novel environment; mean ± SD, ***P < 0.001. Figure 4 f Effects of conditional LRP5 / 6 whole-body knockout mice on cardiopulmonary function in a fatigue device; mean ± SD, ****P < 0.0001. Figure 4 g Effects of conditional LRP5 / 6 whole-body knockout mice on treadmill endurance training; mean ± SD, *P < 0.05. Figure 4 h Effects of conditional LRP5 / 6 knockout mice on walking speed and swing speed in gait analysis; mean ± SD, **P < 0.01. Figure 4 i Effect of conditional LRP5 / 6 whole-body knockout mice on survival; compared with non-LRP5 / 6 knockout mice, ****P<0.0001.

[0071] Figure 4 j shows the changes in body size and hair color of LRP5 / 6 knockout mice bearing Lewis lung cancer cells.

[0072] Figure 4 km showed the effects of a high-fat, high-sugar diet on the body size, hair color, body weight, and survival of LRP5 / 6 whole-body knockout mice. Figure 4 k shows the effects of a high-fat, high-sugar diet on body size and hair color in LRP5 / 6 whole-body knockout mice. Figure 4l shows the effect of high-fat and high-sugar diet on body weight of LRP5 / 6 whole-body knockout mice; compared with whole-body knockout LRP5 / 6 + high-fat and high-sugar diet, ***P<0.001. Figure 4 m represents the effect of high-fat and high-sugar diet on the survival of LRP5 / 6 whole-body knockout mice; compared with non-knockout LRP5 / 6 + normal diet, *P<0.05.

[0073] Figure 5 LRP5 / 6 was shown to play a role in aging by regulating the transport of serum senescence-associated protein PreLamin A.

[0074] Figure 5 a shows the changes in the expression level of serum aging-related protein PreLamin A in LRP5 / 6 knockout mice and under the action of DKK1, DKK1 neutralizing antibody, and MDC; mean ± SD, *P < 0.05.

[0075] Figure 5 b shows the expression distribution of PreLamin A in HepG2 cells analyzed by immunostaining.

[0076] Figure 5 c shows the effect of FTase inhibitor (FTI) on the expression distribution of PreLamin A in HepG2 cells analyzed by immunostaining.

[0077] Figure 5 d shows the effect of LRP5 / 6 interference on the expression distribution of PreLamin A by immunostaining analysis.

[0078] Figure 5 e shows the effect of LRP5 / 6 interference and FTase inhibitor on PreLamin A secretion by Western blot analysis.

[0079] Figure 5 f shows the interaction between endogenous LRP5 / 6 and PreLamin A by co-immunoprecipitation analysis.

[0080] Figure 5 g shows the interaction between endogenous LRP5 / 6 and Lamin A by co-immunoprecipitation analysis.

[0081] Figure 5 h shows the distribution of the interaction between endogenous LRP5 / 6 and PreLamin A analyzed by proximity ligation technique.

[0082] Figure 6 showed the deleterious effects of elevated blood DKK1 in the elderly.

[0083] Figure 6a shows the correlation analysis of DKK1 levels with age and each clinically diagnosed abnormal blood glucose or lipid index in all populations.

[0084] Figure 6 b shows the changes in the expression levels of alpha-fetoprotein, acute phase protein, hemoglobin, blood glucose, and blood lipid-related indicators in the elderly population over 90 years old compared with the average levels of the population under 90 years old; mean ± SD, *P < 0.05, **P < 0.01.

[0085] Figure 6 c shows the changes in the expression levels of alpha-fetoprotein, acute phase protein, hemoglobin, blood glucose, and blood lipid-related indicators in the elderly population aged 94 years and above compared with the average levels of the population under 94 years old; mean ± SD, **P < 0.01.

[0086] Figure 6 d shows the change in DKK1 expression level in the elderly population aged 94 years and above compared with the average level of all populations.

[0087] Figure 7 The expression level of DKK1 in aged mice and its effects are shown.

[0088] Figure 7 a shows the difference in survival curves between BLAB / c mice and C57BL / 6 mice.

[0089] Figure 7 b shows the difference in apparent hair color between 18-month-old BLAB / c mice and C57BL / 6 mice.

[0090] Figure 7 ci showed differences in body weight, grip strength, range of motion in a novel environment, walking speed, treadmill endurance training, walking speed, swing speed, and cardiac function between 18-month-old BLAB / c mice and C57BL / 6 mice. Figure 7 c Difference in body weight between 18-month-old BLAB / c mice and C57BL / 6 mice; mean ± SD, ***P < 0.001. Figure 7 d Difference in grasping strength between 18-month-old BLAB / c mice and C57BL / 6 mice; mean ± SD, ****P < 0.0001. Figure 7 e The difference in activity range between 18-month-old BLAB / c mice and C57BL / 6 mice in a novel environment; mean ± SD, **P < 0.01. Figure 7 f shows the difference in cardiopulmonary function between 18-month-old BLAB / c mice and C57BL / 6 mice in the fatigue apparatus; mean ± SD, ***P < 0.001. Figure 7h is the difference in gait speed and swing speed between 18-month-old BLAB / c mice and C57BL / 6 mice; mean ± SD, **P < 0.01, ***P < 0.001. Figure 7 i shows the effect on cardiac function of 18-month-old BLAB / c mice and C57BL / 6 mice; mean ± SD, *P < 0.05.

[0091] Figure 7 jm shows the effects of continuous injection of DKK1 neutralizing antibody on grip strength, walking speed, treadmill endurance training, and cardiac function in C57BL / 6 mice. Figure 7 j Effect of continuous injection of DKK1 neutralizing antibody on the grasping force of C57BL / 6 mice; mean ± SD, ns, no statistical significance. Figure 7 k shows the effect of continuous injection of DKK1 neutralizing antibody on cardiopulmonary function in C57BL / 6 mice induced by fatigue device; mean ± SD, *P < 0.05. Figure 7 l shows the effect of continuous injection of DKK1 neutralizing antibody on the running function of C57BL / 6 mice; mean ± SD, ns, no statistical significance. Figure 7 m is the effect of continuous injection of DKK1 neutralizing antibody on cardiac function in C57BL / 6 mice; mean ± SD, ns, no statistical significance.

[0092] Figure 8 Negative effects of injection of DKK1 recombinant protein are shown.

[0093] Figure 8 ae show the effects of continuous DKK1 injection on body weight, grip strength, range of motion in a novel environment, maximum walking speed, and treadmill endurance training in 10-week-old C57BL / 6 mice. Figure 8 a Effect of continuous DKK1 injection on body weight of 10-week-old C57BL / 6 mice; mean ± SD, ns, not statistically significant. Figure 8 b Effects of continuous DKK1 injection on the grasping force of 10-week-old C57BL / 6 mice; mean ± SD, ns, not statistically significant. Figure 8 c Effects of continuous DKK1 injection on the activity range of 10-week-old C57BL / 6 mice in a novel environment; mean ± SD, ns, not statistically significant. Figure 8 d Effects of continuous DKK1 injection on cardiopulmonary function in fatigue apparatus of 10-week-old C57BL / 6 mice; mean ± SD, ns, not statistically significant. Figure 8 e Effects of continuous DKK1 injection on running motor function of 10-week-old C57BL / 6 mice; mean ± SD, ns, no statistical significance.

[0094] Figure 8 fj show the effects of continuous DKK1 injection on body weight, grip strength, range of motion in a novel environment, maximum walking speed, and treadmill endurance training in 12-month-old C57BL / 6 mice. Figure 8 f Effect of continuous DKK1 injection on body weight of 12-month-old C57BL / 6 mice; mean ± SD, ****P < 0.0001. Figure 8 g Effects of continuous DKK1 injection on the grip strength of 12-month-old C57BL / 6 mice; mean ± SD, ***P < 0.001. Figure 8 h Effects of continuous DKK1 injection on the activity range of 12-month-old C57BL / 6 mice in a novel environment; mean ± SD, **P < 0.01. Figure 8 i Effects of continuous DKK1 injection on cardiopulmonary function in fatigue apparatus of 12-month-old C57BL / 6 mice; mean ± SD, **P < 0.01. Figure 8 j Effect of continuous DKK1 injection on running motor function of 12-month-old C57BL / 6 mice; mean ± SD, ***P < 0.001.

[0095] Figure 8 kl shows the effects of continuous DKK1 injection for 20 days on the appearance and survival of 20-month-old C57BL / 6 mice. Figure 8 k Effects of continuous DKK1 injection for 20 days on the appearance of 20-month-old C57BL / 6 mice. Figure 8 l Effect of continuous DKK1 injection for 20 days on the survival of 20-month-old C57BL / 6 mice; compared with PBS, *P<0.05, **P<0.01.

[0096] Fig. 9 It shows the alleviation of aging by the small molecule compound MDC.

[0097] Fig. 9 a shows the body weight changes of BALB / c mice during MDC injection for two months; *P<0.05, **P<0.01.

[0098] Fig. 9 bg shows the effects of continuous MDC injection on the grip strength, range of motion in a novel environment, and cardiac function of BALB / c mice. Fig. 9 b Effect of MDC injection on the gripping strength of BALB / c mice for two months; mean ± SD, *P < 0.05. Fig. 9 c Effect of MDC injection on the activity range of BALB / c mice in a novel environment for two months; mean ± SD, *P < 0.05. Fig. 9d Effects of MDC injection on cardiac function of BALB / c mice for two months; mean ± SD, **P < 0.01, ***P < 0.001. Fig. 9 KEGG enrichment Venn diagram of differentially expressed genes showed the number of signaling pathways involved by MDC and DKK1 neutralizing antibodies in aged mice. Fig. 9 g shows the effects of DKK1 and its neutralizing antibody on newborn mice.

[0099] Fig.10 LRP5 / 6 is shown to play a role in senescence by regulating the trafficking of PreLaminA.

[0100] Fig.10 a shows the Venn diagram of differentially expressed genes in different aging models.

[0101] Fig.10 b shows the expression level of PreLamin A in serum at different DKK1 expression levels; mean ± SD, *P < 0.05.

[0102] Fig.10 cg shows the effect of Lamin A overexpression on the range of activity, walking speed, treadmill endurance training, and cardiac function changes in a novel environment induced by DKK1 injection. Fig.10 c Effect of Lamin A overexpression on the activity range in a novel environment induced by DKK1 injection; mean ± SD, **P < 0.01. Fig.10 d Effect of Lamin A overexpression on cardiopulmonary function in fatigue device induced by DKK1 injection; mean ± SD, *P < 0.05. Fig.10 e is the mean ± standard deviation of the effect of Lamin A overexpression on running motor function induced by DKK1 injection, **P<0.01. Fig.10 f shows the effect of Lamin A overexpression on cardiac function induced by DKK1 injection; mean ± SD, *P < 0.05. Fig.10 g is the construct of Lamin A (Lamin A / AAV2 / 9Lamin A) transfected by adeno-associated virus AAV2 / 9.

[0103] Fig.11 Specific quantitative results of lifespan-related assays are shown. DETAILED DESCRIPTION

[0104] After in-depth research, the inventors have revealed for the first time a new signaling pathway closely related to the assessment or prognosis of aging: the DKK1-LRP5 / 6-PreLamin A-LAMIN A signaling pathway. The present invention discloses a new mechanism for regulating aging and aging-related diseases involving the signaling pathway or its pathway proteins.

[0105] The present invention relates to the application of elevated blood proteins Dickkopf-1 (DKK1) and PreLamin A as clinical biological effect markers of aging / senescence. LRP5 / 6 is a Wnt receptor protein located on the cell membrane, and the inventors have found that they are also normally located on the cell nuclear membrane. Furthermore, the inventors have found that LRP5 / 6 directly binds to PreLamin A to help it enter the nucleus, thereby forming a mature nuclear structural protein Lamin A. The inventors have also found that elevated blood DKK1 will induce changes in the position of LRP5 / 6 in the cell, leading to a decrease in LRP5 / 6 on the nuclear membrane, and then PreLamin A cannot enter the nucleus, thereby leading to Lamin A deficiency and nuclear malformation, and ultimately leading to cell aging and even individual aging. Preventing the DKK1-LRP5 / 6-PreLamin A-Lamin A pathway will inhibit cell aging and even individual aging. Therefore, the present invention also relates to the application of this pathway inhibitor in the development process of aging / senescence and aging-related diseases. The present invention uses anti-DKK1 neutralizing antibodies to remove DKK1 in serum, and small molecule inhibitors of TransGlutaminase-2 (TG2) (such as small molecule compound MDC) to prevent LRP5 / 6 from moving in cells, thereby delaying the aging process and preventing and treating aging-related diseases. Specifically, the use of DKK1 neutralizing antibodies and TG2 inhibitors has a rejuvenating effect on aged mice, enhanced the vitality of tumor-bearing mice and metabolic syndrome mice, and extended the healthy lifespan of these three types of mice.

[0106] DKK1-LRP5 / 6-PreLamin A-LAMIN A signaling pathway and its regulation

[0107] As used in the present invention, the "(signal) pathway" and "(signal) path" can be used interchangeably.

[0108] As used in the present invention, the "(signal) pathway" refers to a signal system formed by the mutual restriction or interaction between a series of genes or proteins or their metabolites (synthetic products or processed products), and also includes the interaction between pathway proteins and other elements or organelles in the cell, and sometimes also includes the joint participation of upstream and downstream genes or proteins, which generally leads to the occurrence of some cellular events. The DKK1-LRP5 / 6-PreLamin A-LAMIN A signaling pathway mainly includes the following elements: DKK1 protein (and / or the gene encoding it), LRP5 / 6 protein (and / or the gene encoding it), PreLamin A protein (and / or the gene encoding it), LAMIN A protein (and / or the gene encoding it).

[0109] As used in the present invention, the amino acid sequence of DKK1 protein is, for example, as shown in SEQ ID NO: 1 (human source). The amino acid sequence of LRP5 protein is, for example, as shown in SEQ ID NO: 2 (human source). The amino acid sequence of LRP6 protein is, for example, as shown in SEQ ID NO: 3 (human source). The amino acid sequence of PreLamin A protein is, for example, as shown in SEQ ID NO: 4 (human source). The amino acid sequence of LAMIN A protein is, for example, as shown in SEQ ID NO: 5 (human source).

[0110] The above-mentioned protein (polypeptide) also includes its variant forms, including (but not limited to): deletion, insertion and / or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. Any protein with high homology to the protein (such as 70% or higher homology to the polypeptide sequence; preferably 80% or higher homology; more preferably 90% or higher homology, such as 95%, 98% or 99% homology) and having the same function as the protein is also included in the present invention. The present invention also includes a mutant form of the protein or protein truncation, as long as the mutant protein or truncation substantially retains the function of the full-length protein.

[0111] The gene sequences mentioned above also include degenerate sequences thereof. The protein encoding polynucleotides (genes) may be natural genes or their degenerate sequences.

[0112] After extensive research and screening, the inventors found that serum DKK1 levels are often elevated in aged animal models (such as mice) and elderly people, and these aged animals and elderly people are often accompanied by poor prognosis. The use of DKK1 neutralizing antibodies and TG2 inhibitors in aged animals has played a role in making the aging state younger, enhancing the vitality of tumor-bearing animals and metabolic syndrome animals, and extending the healthy life span of these three types of mice. The inventors first discovered that the increase of DKK1 and PreLamin A in the blood can evaluate the aging / senescence status, and that blocking the DKK1-LRP5 / 6-PreLamin A-Lamin A pathway can delay the occurrence and development of aging and aging-related diseases.

[0113] As used in the present invention, the "down-regulator" and "inhibitor" can be used interchangeably, which also includes: blocker, antagonist, etc.

[0114] The inventors have found that in the signaling pathway, down-regulating the expression or activity of DKK1, inhibiting the nuclear membrane endocytosis of LRP5 / 6, promoting the stability of LRP5 / 6, and up-regulating PreLamin A or Lamin in the nucleus can inhibit aging. Therefore, this mode of action can be used to screen or design drugs suitable for targeted regulation.

[0115] It should be understood that after knowing the function of the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway (preferably, including its upstream and downstream proteins or genes), various methods well known to those skilled in the art can be used to regulate the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway. For example, various methods well known to those skilled in the art can be used to regulate the expression of pathway proteins or to cause their lack of expression.

[0116] As a preferred embodiment of the present invention, a regulator is provided which down-regulates the expression or activity of DKK1, inhibits the endocytosis of LRP5 / 6, promotes the stability of LRP5 / 6, or up-regulates PreLamin A or Lamin A in the nucleus.

[0117] As used in the present invention, the "down-regulator" includes any substance that can reduce the activity, stability, expression, effective action time, inhibition of transcription and translation of the corresponding pathway protein of the signal pathway, and these substances can be used in the present invention as substances that are potentially useful for inhibiting aging. They can be compounds, small chemical molecules, and biological molecules. The biological molecules can be at the protein level or at the nucleic acid level (including DNA, RNA). For example, the down-regulator is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of a pathway gene or its upstream gene; or an editing agent that specifically edits a pathway gene or its upstream gene, and the like.

[0118] As used in the present invention, the "up-regulator" includes any substance that can increase the activity, stability, expression, effective action time, and inhibit transcription and translation of the corresponding pathway protein of the signal pathway.

[0119] As a preferred embodiment of the present invention, the present invention provides a method for down-regulating the DKK1-LRP5 / 6-PreLaminA-Lamin A signaling pathway, comprising specifically down-regulating DKK1 with a DKK1 neutralizing antibody.

[0120] As another preferred embodiment of the present invention, another method for downregulating the DKK1-LRP5 / 6-PreLaminA-LAMIN A signaling pathway is provided, comprising: inhibiting LRP5 / 6 membrane endocytosis with an inhibitor that prevents the movement of LRP5 / 6 in the cell, or promoting the stability of LRP5 / 6 (stabilization at the corresponding position in the cell).

[0121] As other embodiments of the present invention, interfering molecules that interfere with the gene expression of corresponding pathway molecules in the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway can be transferred into cells, or cells can be treated through appropriate pathways to allow them to be introduced into cells, for example, by designing a transmembrane functional domain to enable it to have the ability to penetrate the membrane.

[0122] As other embodiments of the present invention, the genes of the corresponding pathway molecules in the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway can also be targeted for mutation, gene editing or gene recombination to achieve downregulation. As a more specific embodiment, the CRISPR / Cas system is used for gene editing to knock out or downregulate the target gene. Suitable sgRNA target sites will bring higher gene editing efficiency, so before embarking on gene editing, suitable target sites can be designed and found. After designing the specific target site, in vitro cell activity screening is also required to obtain effective target sites for subsequent experiments.

[0123] When used as a target for artificial regulation or when a screening system is artificially established, the above proteins or coding genes can be naturally occurring, such as being purified and isolated from mammals; or they can be recombinantly prepared, such as recombinant proteins can be produced according to conventional gene recombination techniques. In addition, any variation that does not affect the biological activity of these proteins is available, such as derivatives or variants whose functions have not been changed.

[0124] Application in aging assessment or prognostic evaluation

[0125] The inventors have found that the pathway molecules of the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway can also be used as aging / senescence markers. In particular, the increase of DKK1 or PreLamin A in the blood is closely related to the occurrence of senescence and can be used as ideal aging / senescence markers.

[0126] Based on the above new discovery of the inventors, the pathway molecules of the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway, especially blood DKK1 or PreLamin A, can be used as markers for aging assessment or prognosis: (i) for aging identification assessment and / or health status analysis; (ii) for assessing the disease status and prognosis of the relevant population, and selecting appropriate treatment methods. For example, the population with abnormal (high) expression of blood DKK1 or PreLamin A can be separated, so that more targeted tracking can be carried out and intervention can be implemented when necessary.

[0127] Generally, the method for determining the expression or presence level (amount) of the target molecule can adopt conventional methods in the art. For example, a threshold value can be specified to determine whether the expression or presence level is higher than or lower than the specified threshold value. The threshold value is easy to determine for those skilled in the art. For example, the corresponding threshold value can be obtained by comparing and analyzing the general expression or presence level of the target molecule of an aging patient, or the expression or presence level of the target molecule of a normal healthy person. Clinically, threshold values ​​have been specified for a large number of biological molecules or compound molecules, and this can be done.

[0128] When performing the detection, various techniques known in the art can be used to detect the presence or absence and expression of the target molecule gene or protein, and these techniques are all included in the present invention, for example, existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, etc. can be used, and these methods can be used in combination. Preferably, when performing the detection at the gene level, primers that specifically amplify the coding gene of the target molecule can be used; or probes that specifically recognize the coding gene of the target molecule can be used to determine the presence or absence of the target molecule; when performing the detection at the protein level, antibodies or ligands that specifically bind to the target molecule protein can be used to determine the expression of the target molecule.

[0129] The kit may also include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution, color development solution, washing solution, etc. In addition, the kit may also include instructions for use and / or nucleic acid sequence analysis software, etc.

[0130] Drug screening based on DKK1-LRP5 / 6-PreLamin A-LAMIN A signaling pathway

[0131] Based on the new discovery of the inventors, the research on the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway has many uses, including: screening substances that regulate the signaling pathway in order to inhibit aging. Among them, the regulation includes: inhibiting the expression or activity of DKK1, inhibiting LRP5 / 6 membrane endocytosis, promoting LRP5 / 6 stabilizers, upregulating PreLamin A or Lamin A in the nucleus, etc.

[0132] As used herein, the terms "inhibit", "increase" and "promote" all refer to "inhibit", "increase" and "promote" with statistical significance. That is, "inhibit", "increase" and "promote" significantly. For example, compared with the protein activity, protein expression, protein binding or methylation level of the control group, the protein activity, protein expression, protein binding or methylation level is significantly "inhibited", "increased" or "promoted" by 10%, 20%, 30%, 40%, 50% or more; preferably 60%, 70%, 80% or more.

[0133] The system containing the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway is selected from: a cell system (or a cell culture system), a subcellular system (or a subcellular culture system), a solution system, an animal system or a tissue system (or a tissue culture system).

[0134] As a preferred embodiment of the present invention, the method further comprises: conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and determine substances useful for inhibiting aging from the candidate substances.

[0135] When screening is performed, various techniques well known in the art can be used to determine changes in proteins or their encoding genes and interactions.

[0136] A variety of conventional techniques can be used to identify the transcription or expression of genes in the system. These techniques include, but are not limited to, oligonucleotide hybridization techniques (such as probes), polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, etc. The interaction between proteins and the strength of the interaction can be detected by a variety of techniques well known to those skilled in the art, such as immunoprecipitation, GST sedimentation, phage display or yeast two-hybrid system. Nuclear localization of proteins is also a well-known technique in the art.

[0137] The substances initially screened out by the above method can constitute a screening library, so that people can eventually screen out substances that are truly useful for inhibiting aging.

[0138] The present invention also provides potential substances obtained by the screening method and used for inhibiting aging.

[0139] The present invention also provides a method for preparing a drug for inhibiting aging (especially inhibiting aging), the method comprising: synthesizing and / or purifying the substance useful for inhibiting aging obtained by the aforementioned screening as a drug for inhibiting aging.

[0140] The obtained substance useful for inhibiting senescence can be used for preparing a pharmaceutical composition as described hereinafter in the present invention.

[0141] Methods for screening substances acting on a protein or gene or a specific region thereof as a target are well known to those skilled in the art, and all of these methods can be used in the present invention. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, organic small molecules, inorganic small molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art will know how to select an appropriate screening method.

[0142] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0143] Materials and methods

[0144] 1. Cell culture and reagents

[0145] CT26 colon cancer cell line and LLC mouse lung cancer cell line were cultured in RPMI-1640 medium containing 1% penicillin / streptomycin (Corning, USA) and 10% fetal bovine serum (Biosera, USA) in a 37°C, 5% CO2 incubator.

[0146] Recombinant mouse DKK1 protein was purchased from Genscript.

[0147] MDC was purchased from Sigma.

[0148] Anti-DKK1 neutralizing antibody (DKK1 neutralizing antibody) was purchased from Hankun Biotechnology Co., Ltd.

[0149] 2. Tumor-bearing model

[0150] BALB / c and C57BL / 6 mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd. and raised in an SPF environment. All animal studies were approved by the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine and conducted in accordance with the ethics guidelines of the National Institutes of Health. Tamoxifen was intraperitoneally injected at a dose of 30 mg / kg / day for 5 consecutive days to obtain inducible systemic knockout LRP5 / 6 mice. Male mice at different stages were selected and randomly divided into groups. Tumor-bearing mice were subcutaneously injected with 1*10 6 CT26 or LLC tumor cells, and the control group were injected with an equal volume of PBS at the same site.

[0151] The weight, food intake and tumor volume of mice were recorded at fixed intervals, and the activity, hair color and external manifestations (weakness, spinal curvature or not) of mice were observed. 30 days after tumor cell implantation and before drug treatment, tumor-bearing mice were randomly divided into groups to ensure that the average weight and average tumor volume of different groups were roughly the same. Subsequently, PBS, DKK1 (0.5 mg / kg), DKK1 neutralizing antibody DKK1 neutralizing antibody (0.2 mg / kg), and MDC (10 mg / kg) were intraperitoneally injected every two days, and the weight, hair color, external manifestations (weakness, spinal curvature or not), grip, range of activity in a new environment, walking speed and endurance, and changes in cardiac function of mice were observed. At the end of the experiment, mouse blood samples were obtained, placed in a centrifuge tube containing EDTA, centrifuged at 2000 rpm and 4°C for 10 minutes to obtain plasma samples, and stored at -80°C for testing. The ELISA kit for detecting mouse DKK1 (R&D, USA) was used to determine the plasma DKK1 level according to the instructions.

[0152] 3. High-fat and high-sugar model

[0153] (1) C57BL / 6 mouse hyperlipidemia model

[0154] 8-week-old C57BL / 6 mice were fed a high-fat diet (60% calories from fat, 20% calories from fructose) for one month, and then intraperitoneally injected with PBS and DKK1 (0.5 mg / kg) every two days for 8 months. During this period, the weight and food intake of the mice were recorded at fixed intervals, and the activity, hair color, external manifestations (weakness, spinal curvature, etc.) of the mice were observed, and the survival of the mice was recorded.

[0155] (2) LRP5 / 6KO mouse hyperlipidemia model

[0156] One month after tamoxifen induction (4-month-old LRP5 / 6floxed mice began tamoxifen induction), they were fed a high-fat diet (60% of calories from fat, 20% of calories from fructose) for 7 months. During this period, the weight and food intake of the mice were recorded at regular intervals, and the activity, hair color, and external manifestations (weakness, spinal curvature, etc.) of the mice were observed, and the survival of the mice was recorded.

[0157] 4. Determination of plasma DKK1 (ELISA method)

[0158] (1) Sample preparation: Place freshly obtained blood in an EDTA anticoagulant tube, let stand at room temperature for 10 minutes, centrifuge at 1000g for 15 minutes, and collect the supernatant. If the test cannot be performed immediately, the blood should be aliquoted and stored at -80°C to avoid repeated freezing and thawing. If precipitation occurs during storage, it should be fully thawed at room temperature and centrifuged again before use.

[0159] (2) All reagents were returned to room temperature for 30 minutes and then mixed thoroughly. Streptavidin-HRP (1:200), biotinylated goat anti-mouse DKK1 detection antibody (1:180), recombinant mouse DKK1 standard (1:100), and plasma sample (1:4) were first diluted to the specified concentration using reagent diluent; goat anti-mouse DKK1 coating antibody (1:180) was first diluted to the specified concentration using coating buffer. The diluted reagents were used in the following steps.

[0160] (3) Standard: Use reagent diluent to dilute the standard into seven concentration gradients: 1350, 675, 337.5, 168.8, 84.4, 42.2, and 21.1 pg / ml.

[0161] (4) Plate coating: Immediately add 100 μl of DKK1 coating antibody to each well of a 96-well plate for antibody coating, seal the plate with a sealing film, and incubate overnight at room temperature.

[0162] (5) Washing the plate: Carefully peel off the sealing film, discard the liquid and shake dry, fill each well with diluted washing solution, let it stand for 30 seconds and discard it, pat it dry thoroughly with paper, and thoroughly wash the sample plate 3 times.

[0163] (6) Blocking: Add 300 μl of reagent diluent to each well and incubate at room temperature for 1 h.

[0164] (7) Repeat step “(5)”.

[0165] (8) Sample loading: Add 100 μl ddH2O (blank well), DKK1 standard or plasma sample to each well, cover the sample loading plate, and incubate at room temperature for 2 h.

[0166] (9) Repeat step “(5)”.

[0167] (10) Detection: Add 100 μl of DKK1 detection antibody to each well, cover the plate, and incubate at room temperature for 2 h.

[0168] (11) Repeat step “(5)”.

[0169] (12) Add 100 μl of streptavidin-HRP to each well, cover the plate, and incubate at room temperature in the dark for 20 min.

[0170] (13) Repeat step “(5)”.

[0171] (14) Add 100 μl of colorimetric solution (substrate A and substrate B mixed in a ratio of 1:1 and used within 15 min) to each well, cover the plate, and incubate at room temperature in the dark for 20 min.

[0172] (15) Add 50 μl of stop solution to each well and gently shake the plate to mix the liquid.

[0173] (16) Plate reading: Use the blank well as the zero setting and read the OD value of each well at a wavelength of 450 nm.

[0174] (17) Statistics: Based on the concentration of the standard (abscissa) and its average OD value (ordinate), a standard curve and a linear regression equation were generated. The DKK1 concentration (pg / ml) in the sample wells was calculated using the formula and then multiplied by the dilution factor to obtain the actual concentration of DKK1 in plasma (pg / ml).

[0175] 5. Magnetic resonance imaging spine analysis

[0176] Mice were anesthetized with 1.2–1.5% isoflurane and subjected to magnetic resonance imaging (MRI) using a German 7.0T Bruker small animal magnetic resonance imager. They were placed in the center of the surface coil for MRI measurements, and respiratory rate and body temperature were monitored in real time. T1-weighted images were scanned from the sagittal, axial, and coronal planes, and T2-weighted images were scanned using relaxation-enhanced rapid scanning. The parameters were set to repetition time TR 2000ms, echo time TE 17ms, average 8, number of layers 40, layer thickness 0.6mm, and scanning time 17min 4s.

[0177] 6. Mouse front paw grip test

[0178] The experimental animal grasps the forepaw pull rod device with its forepaws, and the animal pulls the rod in a straight line in the direction of the sensor, records its maximum pulling force, and takes the average of 10 results.

[0179] 7. Maximum walking speed of mice

[0180] The rotarod uniform acceleration test was used to detect the maximum walking speed of mice. Before the formal experiment, mice were trained on the rotarod for 3 consecutive days, training at speeds of 4, 6 and 8 rpm / min for 200 seconds respectively. On the subsequent test day, the initial speed was 4 rpm / min, and the speed was accelerated from 4 rpm / min to 40 rpm / min within 5 minutes. The speed at which the mouse fell from the RotaRod device was recorded. The average value of 3-4 trials was used and standardized to the baseline speed. Mice that had been trained within the first two months were not trained repeatedly.

[0181] 8. Open field experiment

[0182] The Shanghai Xinruan open field test system was used to evaluate the autonomous behavior, exploratory behavior, and tension of mice in unfamiliar environments. The mice were placed in a 50cm x 50cm x 25cm black test box for 5 minutes, and the frequency and duration of their behaviors in the test box were tracked by video.

[0183] 9. Treadmill

[0184] Before the experiment, the experimental mice were subjected to three days of adaptive training. The daily intensity standard of the treadmill training process was: 5m / min for 2 minutes, 7m / min for 2 minutes, and 9m / min for 1 minute. On the day of the experiment, the mice ran on the treadmill at an initial speed of 5m / min for 2 minutes, and then increased the speed by 2m / min every 2 minutes until the mice were completely exhausted. At this time, even if the mice were given slight electric shock and mechanical stimulation, they were still unable to return to the treadmill.

[0185] 10. Catwalk, animal gait analysis system

[0186] The Dutch Noldus CatWalk XT analysis system was used to record the time and intensity differences between footprints and step length, and semi-automatically mark and analyze static and dynamic gait kinematics to determine the average running speed and swing speed, and evaluate the differences in gait changes between different groups of mice. Visual scale calibration was performed before each use. On the day of the experiment, the mice were pre-adapted in a dark room for 10 minutes, and then placed on the platform to cross the specified walkway 20 times. Parameter setting: The width of the T-stage channel is 8 cm, and the channel size for data recording is 8cm x 32cm, which enables 4 complete route cycles in the center of the channel. Only channels with speed changes of less than 10% were used for further analysis.

[0187] 11. Rat tail wound healing experiment

[0188] After the mice were anesthetized with 1.2–1.5% isoflurane, a 10 mm long and 3 mm wide wound was cut on the dorsal side of the tail of the experimental mouse, 10 mm away from the base of the tail, with a sterile scalpel. The bleeding was stopped by gentle pressure. The wounds of the mouse tails were photographed weekly with a digital camera, and the wound area was measured to compare the degree of wound healing between different groups.

[0189] 12. Echocardiography to detect cardiac function

[0190] Vevo2100 high-resolution small animal ultrasound imaging system was used for two-dimensional echocardiography. M-mode was used to measure left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular internal diameter at diastole (LVIDd), and left ventricular internal diameter at systole (LVIDs). EF and FS were calculated according to the formula. EF% = (LVIDd 3 -LVIDs 3 ) / LVIDd 3 ×100(%)

[0191] FS%=(LVIDd-LVIDs) / LVIDd×100(%).

[0192] 13. Western Blot

[0193] Serum protein and cell lysate were subjected to SDS-PAGE gel electrophoresis (80V 30min; 120V 1h), transferred to PVDF membrane, blocked with skim milk powder, incubated with specific antibodies at 4°C overnight, washed with TBST; incubated with secondary antibodies, washed with TBST; and detected the expression level of the target protein by ECL exposure.

[0194] 14. Immunofluorescence

[0195] When the cells were harvested, they were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, and then blocked with 5% BSA for 40 minutes at room temperature. Then, they were incubated with the following antibodies overnight at 4°C: PreLamin A (1:200; Millipore, USA), Lamin A / C (1:500; Active Motif, USA), Myc conjugated to Alexa Fluor 488 (1:200; Proteintech, USA). Next, the cells were washed three times with PBS at room temperature, and then incubated with secondary antibodies labeled with Alexa Fluor 555 and Alexa Fluor 647 (1:400; Abcam, USA) for 1 hour at room temperature, and then washed three times with PBS at room temperature. Finally, DAPI was incubated for nuclear staining.

[0196] Example 1: Relationship between DKK1 upregulation and aging and disease

[0197] The inventors selected 3478 Shanghai local elderly residents over 60 years old as subjects, obtained blood samples, and performed ELISA analysis on blood DKK1. The results showed that in these groups, the serum DKK1 level showed a progressive increase with age. Along with the increase in DKK1 expression level, the levels of immune cells such as white blood cells, neutrophils and lymphocytes also increased significantly, which was consistent with the increase in white blood cells and neutrophils caused by the sensitive and specific inflammatory marker C-reactive protein. The analysis results suggest that DKK1 can be used as a candidate for inflammatory markers ( Figure 1 a, Figure 6 a).

[0198] At the same time, the inventors also observed that the levels of total cholesterol, triglycerides, and LDL related to lipid metabolism were positively correlated with the expression level of DKK1; while HDL was negatively correlated with the level of DKK1, and CRP and alpha-fetoprotein (markers of cirrhosis and liver cancer) had no obvious correlation with the changes in triglycerides and cholesterol ( Figure 1 b, Figure 6 b).

[0199] In addition, in patients with heart disease diagnosed based on cardiac ultrasound results, the inventors detected a significant increase in the expression level of DKK1 in the blood ( Figure 1 c).

[0200] Interestingly, brisk walking of 10,000 steps per day can effectively reduce the level of DKK1 in serum ( Figure 1 d).

[0201] Statins, the first-line clinical drug for the treatment of coronary atherosclerosis, can improve coronary atherosclerosis and reduce the level of DKK1 in the blood ( Figure 1 e).

[0202] Compared with the normal population, the upregulation trend of DKK1 is more obvious in people with higher blood sugar and blood lipid levels. The data also show that from the age of 90, the blood DKK1 level stops rising and begins to decline ( Figure 1 f). Further studies on these people showed that their serum AFP, CRP and blood glucose levels were lower than baseline levels, and their blood lipid profiles also improved ( Figure 6 c).

[0203] These results indicate that the expression level of blood DKK1 is closely related to health status and is a key risk factor for the outcome of disease and health status. DKK1 can be used as a candidate for routine detection indicators, which is superior to the application of CRP or AFP in specific clinical examinations, and can better assess the overall health status of the population. High serum levels of DKK1 are warning signs of aging or subhealth, and poor prognosis / subhealth for disease and health status (increased inflammation levels, high levels of total cholesterol, triglycerides, and LDL, low HDL levels, increased risk of heart disease, increased risk of coronary atherosclerosis, and high blood sugar and blood lipid levels); low serum levels of DKK1 indicate better disease and health status (low inflammation levels, low levels of total cholesterol, triglycerides, and LDL, high HDL levels, low risk of heart disease, low risk of coronary atherosclerosis, and low blood sugar and blood lipid levels). This judgment interval is preferably for subjects aged 60-89 years.

[0204] In residents with at least one clinical abnormality of blood sugar or blood lipid index, it was found that serum Dkk1 had a significant increasing trend with age, indicating that DKK1 concentration has specificity in the detection index as age increases. The specific quantitative results of lifespan-related tests are as follows Fig.11 .

[0205] Example 2: Expression level of DKK1 in aged mice and its effects

[0206] Data from Jackson Lab showed that the survival period of C57BL / 6 mice was two and a half years, while that of BALB / C mice was only one and a half years. The inventors studied whether DKK1 was involved in this change in survival period. By monitoring the expression level of serum DKK1 in two strains of mice of different ages, the inventors found that, like humans, the expression level of serum DKK1 in mice showed a progressive upward trend with age, and the increase in the BALB / C strain was greater than that in the C57BL / 6 strain mice ( Figure 2 a, Figure 7 a).

[0207] Compared with C57BL / 6 mice, BALB / c mice at 12 months of age showed signs of aging, such as emaciation, spinal curvature, rough and dull hair, and weakness ( Figure 7 bi). Injection of DKK1 neutralizing antibodies can effectively restore the vitality of mice, reverse the spinal curvature caused by aging, the decline in grip, range of motion in a new environment, walking speed and endurance, and improve the heart function of mice, so that mice maintain the health of their youth and significantly increase their lifespan ( Figure 2 bk, Figure 7 jm).

[0208] Physiological aging delays wound healing, while injection of DKK1 neutralizing antibodies helps wound healing. In vivo injection of DKK1 neutralizing antibodies makes the wound healing ability of 12-month-old mice reach the healing level of 10-week-old mice ( Figure 2 l).

[0209] Continuous injection of DKK1 protein can cause C57BL / 6 mice to have rough and dull hair, weakness, spinal curvature, and poor heart function.

[0210] These results indicate that high expression of DKK1 can accelerate aging and delay wound healing, which is particularly evident in aged mice; this process can be reversed by using a downregulator of DKK1.

[0211] Example 3: Negative effects of high DKK1 expression levels in disease states

[0212] The inventors focused on the effects of DKK1 on tumors and diabetes. The results showed that the serum DKK1 expression level of CT26 and LLC tumor-bearing mice increased, and the injection of DKK1 recombinant protein accelerated the appearance of aging signs in tumor-bearing mice: emaciation, spinal curvature, rough and dull hair, weakness ( Figure 3 This suggests that high levels of DKK1 in the blood can accelerate aging.

[0213] On the other hand, in the study of high-sugar and high-fat diet, it was found that although high-sugar and high-fat diet can induce obesity, it did not cause obesity in mice injected with DKK1 recombinant protein. The inventors observed that these mice showed some aging characteristics, including significant decreases in body weight, grip strength, range of activity in a novel environment, maximum walking speed, and treadmill endurance training; impaired cardiac function; spinal curvature; and significantly reduced survival (especially the survival of the high-fat diet group) ( Figure 3 dn).

[0214] At the same time, compared with 10-week-old C57BL / 6 mice, 12-month-old C57BL / 6 mice injected with DKK1 recombinant protein showed significant weight loss, decreased activity, and impaired cardiac function ( Figure 8 ). These results suggest that aging is more sensitive to DKK1.

[0215] In summary, the increase of serum DKK1 levels can induce chronic aging. Under external stress conditions such as tumors, metabolic syndrome, and subhealth, DKK1 can accelerate the aging process. This suggests that serum DKK1 expression level is an independent risk factor for subhealth, and the clinical detection of serum DKK1 becomes particularly important in the evaluation of human health and aging. Anti-DKK1 neutralizing antibodies or other DKK1 inhibitors can become effective drugs to improve human subhealth and prevent aging.

[0216] Example 4: DKK1 induces aging by downregulating LRP5 / LRP6

[0217] LRP5 / 6 is a Wnt receptor protein located on the cell membrane. Analysis of its cell localization (experimental cells: liver cancer cell line HepG2 cells) showed that they are normally located on the cell nuclear membrane.

[0218] Ten-week-old LRP5 / 6 conditional knockout mice were injected with tamoxifen. The results showed that 4 months after the injection, the mice showed premature aging phenotypes: weight loss, muscle relaxation, spinal curvature, hair loss and hair loss, grip strength, range of motion in a novel environment, walking speed, treadmill endurance training, walking speed, swing speed, and shortened survival ( Figure 4 ai).

[0219] Although 1-month-old LRP5 / 6 knockout mice did not show obvious aging phenotypes, LLC tumor-bearing and long-term high-fat diet accelerated the aging process of LRP5 / 6 knockout mice and shortened their survival ( Figure 4 These results indicate that LRP5 / 6 exerts a protective effect on the body under various stresses and also suggest a role for LRP5 / 6 in the prevention of aging.

[0220] DKK1 can induce LRP5 / 6 membrane endocytosis. The use of the small molecule compound MDC (inhibiting DKK1-induced LRP5 / 6 membrane endocytosis) can delay the aging process and improve the cardiac function damage caused by DKK1 ( Fig. 9 ae).

[0221] In addition, the aging phenotype observed in LRP5 / 6 systemic knockout mice is more severe than aging induced by DKK1 alone, suggesting that persistent LRP5 / 6 downregulation has a more severe damaging effect on the body.

[0222] Example 5: Effects of DKK1 on neonatal mice

[0223] The inventors injected DKK1 continuously for two months into young mice (3 weeks old) of the two strains of C57BL / 6 and BALB / C that had just been weaned, and then analyzed the results. The results showed that mice given DKK1 (0.5 mg / kg) showed varying degrees of weakness, developmental delay, reduced gripping power, reduced range of activity in a new environment, and decreased cardiac function. Injection of DKK1 neutralizing antibody (0.2 mg / kg) can improve the cardiac function of these mice ( Fig. 9 g).

[0224] These results suggest that elevated serum DKK1 concentrations in larvae can produce a lasting insult and that high levels of DKK1 can disrupt the organism's health.

[0225] Example 6: LRP5 / 6 plays a role in aging by regulating the transport of serum aging-related protein PreLamin A. The results of serum protein proteomic sequencing analysis showed that the expression level of the nuclear scaffold protein laminin A precursor (PreLamin A) was upregulated in LRP5 / 6-deficient mice, mice injected with DKK1 recombinant protein, and aged mice. DKK1 neutralizing antibodies and MDC could prevent this increase ( Figure 5 a, Fig.10 a). The current view is that un-farnesylated PreLamin A is transported to the nucleus and converted into adult Lamin A and C. FTase inhibitors (FTIs) can inhibit the farnesylation of PreLamin A and cause it to aggregate in the nucleus.

[0226] Immunofluorescence analysis (experimental cells: liver cancer cell line HepG2 cells) showed that knockout of LRP5 / 6 inhibited the nuclear translocation of PreLamin A and also showed expression in the cytoplasm ( Figure 5 bd). The interaction between them was also confirmed by Western blotting ( Figure 5 eg) and proximity connection technology ( Figure 5 h) confirmed. Therefore, LRP5 / 6 directly binds to PreLamin A and helps it enter the nucleus to form the mature nuclear structural protein Lamin A.

[0227] Increased DKK1 in the blood will induce changes in the position of LRP5 / 6 in cells (experimental cells: liver cancer cell line HepG2 cells) (nuclear membrane endocytosis), resulting in a decrease in nuclear membrane LRP5 / 6, and then PreLamin A cannot enter the nucleus, leading to Lamin A deficiency and nuclear deformities, and ultimately leading to cell aging and even individual aging.

[0228] The above results indicate that DKK1 promotes the endocytosis of LRP5 / 6, thereby causing the extracellular transport of PreLamin A. The activation of the DKK1-LRP5 / 6 system induced the extracellular transport of PreLamin A, which is a new discovery.

[0229] 18-month-old C57BL / 6 mice were transfected with Lamin A using adeno-associated virus AAV2 / 9 (Lamin A / AAV2 / 9Lamin A, Fig.10 g) It can effectively restore the vitality of mice, reverse the decrease in activity range, walking speed and endurance in a novel environment caused by DKK2 injection, and improve the cardiac function of mice ( Fig.10 cf).

[0230] Analysis of the expression levels of serum PreLamin A at different DKK1 expression levels showed that high DKK1 expression levels corresponded to high PreLamin A expression levels; low DKK1 expression levels corresponded to low PreLamin A expression levels ( Fig.10 b). Therefore, the expression level of PreLamin A in serum is closely related to DKK1, and the expression level of PreLamin A in serum can also be used as an aging / senescence marker.

[0231] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

[0232] Sequence information

[0233] Amino acid sequence of DKK1 protein (SEQ ID NO: 1):

[0234] MMALGAAGATRVFVAMVAAALGGHPLLGVSATLNSVLNSNAIKNLPPPLGGAAGHPGSAVSAAPGILYPGGNKYQTIDNYQPYPCAEDEECGTDEYCASPTRGGDAGVQICLACRKRRKRCMRHAMCCPGNYC KNGICVSSDQNHFRGEIEETITESFGNDHSTLDGYSRRTTLSSKMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQRH

[0235] Amino acid sequence of LRP5 protein (SEQ ID NO: 2):

[0236]

[0237] Amino acid sequence of LRP6 protein (SEQ ID NO: 3):

[0238]

[0239] Amino acid sequence of preLamin A protein (SEQ ID NO:4):

[0240] METPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETENAGLRLRITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARNTKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGEAKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQREFESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQSRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRLSPSPTSQRSRGRASSHSSQTQGGGSVTKKRKLESTESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKRQNGDDPLLTYRFPPKFTLKAGQVVTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRTALINSTGEEVAMRKLVRSVTVVEDDEDEDGDDLLHHHHGSHCSSSGDPAEYNLRSRTVLCGTCGQPADKASASGSGAQVGGPISSGSSASSVTVTRSYRSVGGSGGGSFGDNLVTRSYLLGNSSPRTQSPQNCSIM

[0241] Amino acid sequence of LAMIN A protein (SEQ ID NO:5):

[0242] METPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETENAGLRLRITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARNTKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGEAKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQREFESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQSRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRLSPSPTSQRSRGRASSHSSQTQGGGSVTKKRKLESTESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKRQNGDDPLLTYRFPPKFTLKAGQVVTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRSGHAQAGALSDCG

Claims

1. Use of a regulator of the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway for preparing a composition for inhibiting aging; wherein: The regulator is selected from the following group: DKK1 expression or activity inhibitor, LRP5 / 6 membrane endocytosis inhibitor, LRP5 / 6 stabilizer, nuclear PreLamin A or Lamin upregulator.

2. The use according to claim 1, characterized in that The DKK1 expression or activity inhibitor includes: an agent that inhibits the activity of the DKK1 protein, an agent that knocks out or silences the DKK1 gene; preferably, it includes: an antibody that specifically inhibits the activity of the DKK1 protein, a small molecule DKK1 protein inhibitor, an interfering molecule that specifically interferes with the expression of the DKK1 gene, a CRISPR gene editing agent, a homologous recombination agent or a site-directed mutagenesis agent for the DKK1 gene; The LRP5 / 6 membrane endocytosis inhibitor includes: a TransGlutaminase-2 inhibitor; preferably, the TransGlutaminase-2 inhibitor includes a small molecule compound MDC; The nuclear PreLamin A or Lamin A up-regulator includes: a vector expressing PreLamin A or Lamin A, a PreLamin A nuclear transport promoter or a PreLamin A extracellular transport inhibitor.

3. Use of a regulator of the DKK1-LRP5 / 6-PreLamin A-LAMIN A signaling pathway in the preparation of a composition for inhibiting aging; wherein: The regulator is selected from the following group: DKK1 expression or activity inhibitor, LRP5 / 6 membrane endocytosis inhibitor, LRP5 / 6 stabilizer, nuclear PreLamin A or Lamin A upregulator; Preferably, the expression or activity inhibitor of DKK1 includes: an agent that inhibits the activity of DKK1 protein, an agent that knocks out or silences the DKK1 gene; preferably, it includes: an antibody that specifically inhibits the activity of DKK1 protein, a small molecule DKK1 protein inhibitor, an interfering molecule that specifically interferes with the expression of the DKK1 gene, a CRISPR gene editing agent, a homologous recombination agent or a site-directed mutagenesis agent for the DKK1 gene; preferably, the small molecule DKK1 protein inhibitor includes: a TransGlutaminase-2 inhibitor; The LRP5 / 6 membrane endocytosis inhibitor includes: an agent that inhibits the LRP5 / 6 membrane endocytosis activity; or The nuclear PreLamin A or Lamin A up-regulator includes: a vector expressing PreLamin A or LaminA, a PreLamin A nuclear transport promoter or a PreLamin A extracellular transport inhibitor.

4. Use of DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway molecules as diagnostic markers and for preparing detection reagents for aging assessment or prognosis; wherein, The signaling pathway molecules include: DKK1, LRP5 / 6, PreLaminA or Lamin A; preferably, the signaling pathway molecules include: DKK1, PreLamin A; more preferably, the DKK1 or PreLamin A is blood DKK1 or PreLamin A.

5. Use of a reagent for specifically detecting molecules of the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway for preparing a detection reagent or a detection kit for aging assessment or prognosis; wherein, The signaling pathway molecules include: DKK1, LRP5 / 6, PreLamin A or Lamin A; preferably, the signaling pathway molecules include: DKK1, PreLamin A; more preferably, the DKK1 or PreLamin A is blood DKK1 or blood PreLamin A.

6. The use according to claim 4 or 5, characterized in that The assessment or prognosis is selected from the following: Aging assessment or prognosis is performed based on the expression or activity of DKK1 protein in the blood. High expression or high activity of DKK1 protein indicates an aging state; low expression or low activity of DKK1 protein indicates a healthy state; preferably, the expression or activity of DKK1 protein is the expression or activity of DKK1 protein in the blood; or Aging assessment or prognosis is performed based on the expression or activity of PreLamin A protein in the blood. High expression or high activity of PreLamin A protein indicates aging; low expression or low activity of PreLamin A protein indicates a healthy state. According to the amount of PreLamin A or Lamin A in the cell nucleus, if the amount is low, it is in the aging state; The presence level is high and is healthy. Aging assessment or prognosis is performed based on the LRP5 / 6 membrane endocytosis in cells. A high level of LRP5 / 6 membrane endocytosis indicates an aging state; a normal or low level of LRP5 / 6 membrane endocytosis indicates a healthy state.

7. The use according to claim 4 or 5, characterized in that The detection reagent includes: an antibody specific for DKK1, LRP5 / 6, PreLamin A or Lamin A protein, and a primer for specifically amplifying the coding gene of DKK1, LRP5 / 6, PreLamin A or LaminA; Probes that specifically recognize genes encoding DKK1, LRP5 / 6, PreLamin A or Lamin A.

8. The use of DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway is used to screen substances that inhibit aging.

9. A method for screening a substance that inhibits aging, the method comprising: (1) contacting the candidate substance with a system containing the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway; (2) Screening out substances that regulate the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway, wherein the substances are substances useful for inhibiting aging; wherein the regulation includes: inhibiting the expression or activity of DKK1, inhibiting the endocytosis of LRP5 / 6 membrane, promoting the stability of LRP5 / 6, and upregulating PreLamin A or Lamin A in the nucleus.

10. The method according to claim 9, characterized in that Step (1) comprises: adding a candidate substance to a system containing a DKK1-LRP5 / 6-PreLamin A-LAMIN A signaling pathway; Step (2) comprises: detecting changes in each protein or its encoding gene in the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway, and comparing with a control group, wherein the control group is a system containing the DKK1-LRP5 / 6-PreLamin A-Lamin A signaling pathway without adding the candidate substance; if the candidate substance inhibits the expression or activity of DKK1, inhibits LRP5 / 6 membrane endocytosis, promotes LRP5 / 6 stabilizers, and upregulates PreLamin A or Lamin in the nucleus, then the candidate substance is a substance useful for inhibiting aging.

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