Application of mannan-binding lectin in inhibiting cell senescence

Abstract

The present invention belongs to the field of inhibitors of cell aging, and specifically relates to the application of mannan-binding lectin in inhibiting cell aging. Application of mannan-binding lectin in inhibiting cell aging for non-therapeutic purposes. The present invention confirms for the first time that MBL is closely related to cell aging induced by oxidative stress, and can inhibit the in vitro aging of NIH-3T3 mouse embryonic fibroblasts induced by hydrogen peroxide, including inhibiting the activity of β-galactosidase associated with aging, maintaining the normal morphology of the cell nucleus under conditions of DNA damage caused by oxidative stress, inhibiting the accumulation of aging marker proteins P53 and P21, enhancing the expression level of cell longevity gene SIRT1mRNA, inhibiting cell arrest in the G1 / G0 phase, thereby enhancing the ability of cell proliferation, and reducing the level of intracellular reactive oxygen species (ROS).

Description

Technical Field

[0001] The invention belongs to the field of inhibitors of cell aging, and specifically relates to the application of mannan-binding lectin in inhibiting cell aging. Background Art

[0002] Cell senescence refers to the shortening of telomeres caused by cell replication or external stress, which manifests as cell cycle arrest.

[0003] Cell senescence mainly presents in two forms. The first refers to the shortening of telomeres after several replications, the cells no longer proliferate, and the cell growth arrest occurs. The second is that cell senescence can be induced by DNA damage or the activation of abnormal oncogenes. Although senescent cells show growth arrest, they are still metabolically active and have obvious molecular and cellular change characteristics, including cell cycle arrest, increased levels of intracellular reactive oxygen species (ROS), morphological changes, activation of p53 and p21 senescence-related pathways, changes in chromatin structure, and significant changes in secreted factors such as cytokines and chemokines.

[0004] Studying the inhibitory mechanism of cell aging is of great significance for the screening of related drugs, and for slowing down and treating aging-related diseases. Summary of the invention

[0005] The purpose of the present invention is to provide an application of mannan binding lectin in inhibiting cell senescence, and to prove for the first time that mannan binding lectin (MBL) is closely related to cell senescence.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] Non-therapeutic applications of mannan-binding lectins in inhibiting cellular senescence.

[0008] Mannan-binding lectin (MBL) is a plasma protein mainly secreted by hepatocytes and is a member of the collectins family in the C-type lectin superfamily. MBL can recognize sugar structures distributed on the surface of various pathogens (bacteria, viruses, parasites, fungi, etc.), and exert lytic and indirect conditioning effects by activating the complement lectin pathway; or exert direct conditioning effects by binding to the gelling receptors on the surface of phagocytes, thereby protecting the body from the harm of pathogens.

[0009] The present invention confirms for the first time that MBL is closely related to oxidative stress-induced cell senescence, and can inhibit hydrogen peroxide-induced senescence of NIH-3T3 mouse embryonic fibroblasts in vitro, including inhibiting senescence-associated beta-galactosidase (SA-β-Gal) activity, maintaining normal nucleus morphology under conditions of oxidative stress-induced DNA damage, inhibiting the accumulation of senescence marker proteins P53 and P21, enhancing the expression level of cell longevity gene SIRT1 mRNA, inhibiting cell arrest in the G1 / G0 phase, thereby enhancing the ability of cell proliferation, and reducing intracellular reactive oxygen species (ROS) levels.

[0010] Preferably, the cell senescence is induced by DNA damage. Further preferably, the DNA damage is caused by H2O2 through oxidative stress. The induction concentration of H2O2 can be 100-700 μM, preferably 100-400 μM.

[0011] Preferably, the cells are embryonic cells. Further preferably, the embryonic cells are embryonic fibroblasts. More preferably, the embryonic fibroblasts are NIH-3T3.

[0012] Preferably, after cell senescence, by upregulating the content or expression level of mannan-binding lectin in the cell growth environment, the activity of β-galactosidase associated with aging is inhibited, the normal morphology of the cell nucleus is maintained, the accumulation of aging marker proteins P53 and P21 is inhibited, the expression level of the cell longevity gene SIRT1 mRNA is enhanced, and the cell arrest in the G1 / G0 phase is inhibited, thereby enhancing the cell proliferation ability.

[0013] Preferably, after cell senescence, the intracellular reactive oxygen level is reduced by upregulating the content or expression level of mannan-binding lectin in the cell growth environment.

[0014] Preferably, the application is to prepare a drug for preventing or treating cell senescence and its related diseases.

[0015] Preferably, the application is to screen candidate drugs for preventing or treating cellular senescence and its related diseases, and the screening of candidate drugs comprises the following steps:

[0016] (1) treating a system expressing or containing MBL with a test substance;

[0017] (2) detecting the expression level of MBL in the system;

[0018] (3) Select substances that can upregulate the expression level of MBL as candidate drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 To prove that H2O2 can affect cell survival by causing DNA damage through oxidative stress, the cell survival rate of mouse embryonic fibroblasts NIH-3T3 treated with different concentrations of H2O2 was analyzed using a cell counting kit (Cell Counting Kit-8, CCK-8).

[0020] Figure 2 This is the result of MBL inhibiting the activity of β-galactosidase (SA-β-Gal), a senescence marker in mouse embryonic fibroblasts NIH-3T3;

[0021] Figure 3 This is a DAPI fluorescence staining image showing that MBL can maintain the normal morphology of the cell nucleus under H2O2-induced senescence conditions;

[0022] Figure 4 This is a graph showing that MBL inhibits the expression levels of senescence marker proteins P53 and P21 in mouse embryonic fibroblasts NIH-3T3;

[0023] Figure 5 The figure shows the expression level of SIRT1 mRNA in mouse embryonic fibroblasts NIH-3T3 enhanced by MBL;

[0024] Figure 6 is a graph showing the percentage of various growth phases of mouse embryonic fibroblasts NIH-3T3 under different treatment conditions, which illustrates the effect of MBL on the cell cycle of aging mouse embryonic fibroblasts, slowing down the cycle arrest of aging cells in the G1 phase;

[0025] Figure 7 This is a graph showing that MBL reduces the level of reactive oxygen species (ROS) in mouse embryonic fibroblasts NIH-3T3. DETAILED DESCRIPTION

[0026] The implementation process of the present invention is described in detail below in conjunction with specific embodiments.

[0027] Example

[0028] The non-therapeutic application of the mannan-binding lectin in this example in inhibiting cell senescence is described in detail as follows:

[0029] (1) Effects of different concentrations of H2O2 on the growth of mouse embryonic fibroblasts NIH-3T3.

[0030] 1.1. Collect cells in the logarithmic phase and adjust the concentration of the cell suspension to 1×10 4 Cells were seeded in 96-well plates and 100 μL of cell suspension was added to each well.

[0031] 1.2. Mouse embryonic fibroblast NIH-3T3 cells were cultured in DMEM containing 10% FBS in a 5% CO2, 37°C constant temperature incubator until the cell monolayer covered the bottom of the well, then the culture medium was removed and the cells were washed twice with PBS.

[0032] 1.3. Add different concentrations of H2O2 (0, 100, 200, 300, 400, 500, 600, 700 μM, final concentration), 0.1 mL / well, set up 3 replicate wells for each concentration, and set up 3 blank control wells (parallel to the experiment, wells with no cells but culture medium), continue to culture in a 37°C, 5% CO2 incubator for 3 hours, then discard. Wash three times with PBS or DMEM, add DMEM complete culture medium and continue to culture for three days.

[0033] 1.4. CCK-8 detection: add 10 μL of CCK-8 solution to each well under light-proof conditions, and then incubate in a constant temperature incubator for 1.5 to 2 hours. Use an enzyme marker to detect the absorbance value (OD value) of each well at a wavelength of 450 nm to detect the survival and proliferation of cells. It was found that 400 μM H2O2 can be used as a suitable induction concentration.

[0034] Figure 1 This is a graph showing the results of an experimental analysis of the cell survival rate of mouse embryonic fibroblast NIH-3T3 cells treated with different concentrations of H2O2.

[0035] (2) MBL inhibited the activity of senescence-associated beta-galactosidase (SA-β-Gal), a senescence marker, in mouse embryonic fibroblasts NIH-3T3 cells induced by H2O2.

[0036] 2.1. Culture of mouse embryonic fibroblasts and induction of aging model:

[0037] Mouse fibroblast NIH-3T3 cells (ATCC) were cultured in 10% fetal bovine serum (Gibco, USA) and high glucose medium (Sigma, USA) and placed in a humidified cell culture incubator at 37°C and 5% CO2. When the cell confluence reached 80%, the cells were digested with trypsin (Gibco, USA) and collected in a centrifuge tube. At a constant temperature of 37°C, the cells were treated with 400 μM H2O2 for 3 h and mixed by oscillation every 15 min to produce an oxidative stress response. The NIH-3T3 premature aging cell model was further obtained and 4 × 10 5 The cells were seeded at a density of 1000 cells / mL in 6-well plates and cultured for 3 days.

[0038] The senescence of NIH-3T3 cells was induced by using H2O2 to simulate reactive oxygen species stress. The initial stage of the experiment was defined as day 0. A total of 5 parallel groups were set up in the experiment. Each group was first treated with 4×10 5 The cells were inoculated in a 6-well plate at a density of 100 cells / mL. Five groups of cells were plated (each group had culture medium). After the cells adhered to the wall (about 7 to 8 hours), H2O2 was added to the 4th group (400 μM H2O2 group), and H2O2 and MBL were added to the 5th group (400 μM H2O2 and 5 μg / mL MBL co-treatment group). After 3 hours, the culture medium of the 4th and 5th groups was discarded, the culture medium of the 2nd group was aspirated and added to the 4th group, and the culture medium of the 3rd group was aspirated and added to the 5th group (MBL was added to the 5th group again), and then the cells of the 2nd and 3rd groups were discarded. The culture medium of the 4th and 5th groups was changed every 24 hours thereafter. Cells may secrete some cytokines that affect cell growth during the process of adherent growth. Replacing with the culture medium of the 2nd and 3rd groups can reduce the effect of cytokines that may be secreted during the process of cell adherent growth on the cells during the subsequent induction process. Group 1 was the blank control group, which was cultured using the standard culture method and the cell culture medium was changed every 24 hours. The experimental design of parallel experimental groups in the following experiments is the same as here.

[0039] It should be noted that MBL was added throughout the process; the concentration of MBL in the culture medium was maintained at 5 μg / mL. On the third day of culture, the cells were stained with a cell senescence β-galactosidase staining kit, and then photographed with an inverted microscope. Five areas were randomly selected, and three photos were taken in each area for statistical analysis.

[0040] 2.2. Results of 5 μg / mL MBL against H2O2-induced senescence:

[0041] On the first day of the experiment, mouse embryonic fibroblasts were treated with 400μM H2O2 for 3 hours and then cultured for 72 hours, which induced an increase in the activity of β-galactosidase (senescence-associated beta-galactosidase, SA-β-Gal) in mouse embryonic fibroblasts. In SA-β-Gal staining, 400μM H2O2 treatment caused 54% of NIH-3T3 cells to show senescence. NIH-3T3 cells that had been cultured with MBL were treated with 400μM H2O2 for 3 hours and then cultured for 72 hours without obvious cell senescence staining. The ratio of cells showing positive staining was 27%, which was not statistically different from the ratio of 19% in the control.

[0042] Figure 2This figure shows the results of MBL inhibiting the activity of senescence-associated beta-galactosidase (SA-β-Gal), a senescence marker in mouse embryonic fibroblasts NIH-3T3.

[0043] (3) DAPI fluorescence staining experiment showed that MBL can maintain the normal morphology of cell nucleus under 400μM H2O2-induced aging conditions.

[0044] 3.1. Culture of mouse embryonic fibroblasts and induction of aging model:

[0045] Mouse fibroblast NIH-3T3 cells were cultured in 10% fetal bovine serum high glucose medium and placed in a humidified cell culture incubator at 37°C and 5% CO2. When the cell confluence reached 80%, they were digested with trypsin and collected in a centrifuge tube. At a constant temperature of 37°C, the cells were treated with 400 μM H2O2 for 3 h and oscillated every 15 min to produce oxidative stress. The NIH-3T3 premature aging cell model was further obtained and 4 × 10 5 The cells were seeded at a density of 1000 cells / mL in 6-well plates and cultured for 3 days.

[0046] The senescence of NIH-3T3 cells was induced by simulating reactive oxygen stress with H2O2. The initial stage of the experiment was defined as day 0. A total of 5 parallel groups were set up in the experiment. The first group was set as the blank control group, which was cultured using the standard culture method and the cell culture medium was changed every 24 hours; the second and third groups were used to provide the culture medium for the fourth and fifth groups after H2O2 treatment; the fourth group was the group that was only treated with H2O2 to induce reactive oxygen stress; the fifth group was the group that was co-treated with 400μM H2O2 and MBL. The second and third groups were no longer involved in the experiment after the culture medium was changed. It should be noted that MBL was added throughout the process; the concentration of MBL in the culture medium was maintained at 5μg / mL. On the third day of culture, the cells of each group were digested, and the resuspended cells were seeded in the wells of a 24-well plate with a round cover glass. The cell density occupied about 1 / 2 of the bottom area of ​​the well of the 24-well plate and were cultured in an incubator.

[0047] 3.2 Perform DAPI fluorescence staining to observe the effect of 5 μg / ml MBL on the nuclear morphology of senescent cells:

[0048] After 24 hours of culture, take out the 24-well plate, add 0.5mL PBS to each well for washing, and wash 3 times. Discard PBS, add 0.5mL 4% paraformaldehyde to each well for fixation at room temperature for 10min. Discard 4% paraformaldehyde, add 0.5mL PBS to each well for washing, and wash 3 times. Discard PBS, add 0.5mL 0.5% Triton X-100 to each well for membrane permeation at room temperature for 5min. Discard 0.5% Triton X-100, add 0.5mL PBS to each well for washing, and wash 3 times. Discard PBS, add 300uL 1μg / mL DAPI to each well for staining at room temperature for 5min. Discard DAPI, add 0.5mL PBS to each well for washing, and wash 3 times. Prepare a slide, drop about 20μL of anti-fluorescence quenching sealing solution on the slide, and invert the round coverslip on the anti-fluorescence quenching sealing solution (the cell side faces the anti-fluorescence quenching sealing solution). Take the slide to the confocal fluorescence microscope for imaging observation and photography. Image J software was then used to analyze nuclear size and nuclear fluorescence intensity.

[0049] Figure 3 This is a DAPI fluorescence staining image showing that MBL can maintain the normal morphology of the cell nucleus under H2O2-induced aging conditions. The results show that MBL can maintain the normal size and morphology of the cell nucleus and the fluorescence intensity of the nucleus under oxidative stress conditions.

[0050] (4) Experimental study on down-regulating the expression levels of senescence markers P53 and P21 in mouse embryonic fibroblast NIH-3T3 cells.

[0051] 4.1. Cell culture and cell protein extraction:

[0052] The senescence of mouse embryonic fibroblast NIH-3T3 cells was simulated by H2O2 to simulate reactive oxygen species stress. NIH-3T3 cells were treated with 400 μM H2O2 in the culture medium for 3 hours to induce reactive oxygen species-induced senescence. At the beginning of the experiment, 4×10 5 The cells were seeded at a density of 1000 cells / mL in 6-well plates and cultured for 3 days.

[0053] The initial stage of the experiment was defined as Day 0. A total of 5 parallel groups were set up for the experiment. The first group was set as the blank control group, which was cultured using the standard culture method, and the cell culture medium was changed every 24 hours; the second and third groups were provided with replacement culture medium for the fourth and fifth groups after H2O2 treatment; the fourth group was the group that was only treated with H2O2 to induce reactive oxygen stress; the fifth group was the group that was co-treated with 400μM H2O2 and 5μg / mL MBL. The second and third groups no longer participated in the experiment after the culture medium was changed. It should be noted that MBL was added throughout the process; the concentration of MBL in the culture medium was maintained at 5μg / mL. On the third day of culture, the protein of the cells was extracted, and the expression of aging markers P53 and P21 was detected by western blot, and beta-actin was used as the internal reference protein;

[0054] 4.2. 5 μg / mL MBL inhibited the accumulation of senescence marker proteins in H2O2-treated mouse embryonic fibroblasts NIH-3T3:

[0055] On the first day of the experiment, mouse embryonic fibroblasts were treated with 400 μM H2O2 for 3 hours and then cultured for 72 hours. The cell proteins were collected and the expressions of cell senescence protein markers P53 and P21 were compared using western blot.

[0056] Figure 4 This is a graph showing that MBL inhibits the expression levels of aging marker proteins P53 and P21 in mouse embryonic fibroblasts NIH-3T3.

[0057] The results showed that after 3 hours of treatment with 400 μM H2O2, the expression of senescence marker proteins P53 and P21 in mouse embryonic fibroblasts was significantly accumulated after 72 hours of continuous culture. The addition of MBL inhibited the accumulation of senescence marker proteins P53 and P21 induced by 400 μM H2O2.

[0058] (5) Experiments in which MBL enhanced the expression level of SIRT1 mRNA in mouse embryonic fibroblast NIH-3T3 cells.

[0059] 5.1. Culture of mouse embryonic fibroblasts and induction of aging model:

[0060] Mouse fibroblast NIH-3T3 cells were cultured in 10% fetal bovine serum high glucose medium and placed in a humidified cell culture incubator at 37°C and 5% CO2. When the cell confluence reached 80%, they were digested with trypsin and collected in a centrifuge tube. At a constant temperature of 37°C, the cells were treated with 400 μM H2O2 for 3 h and oscillated every 15 min to produce oxidative stress. The NIH-3T3 premature aging cell model was further obtained and 4 × 10 5 The cells were seeded at a density of 1000 cells / mL in 6-well plates and cultured for 3 days. The senescence of NIH-3T3 cells was induced by using H2O2 to simulate reactive oxygen species stress.

[0061] The initial stage of the experiment was defined as day 0. A total of 5 parallel groups were set up in the experiment. The first group was set as the blank control group, which was cultured using the standard culture method and the cell culture medium was changed every 24 hours; the second and third groups were provided with replacement culture medium for the fourth and fifth groups after H2O2 treatment; the fourth group was the group that was only treated with H2O2 to induce reactive oxygen stress; the fifth group was the group that was co-treated with 400μM H2O2 and 5μg / mL MBL. The second and third groups were no longer involved in the experiment after the culture medium was changed. It should be noted that MBL was added throughout the process; the concentration of MBL in the culture medium was maintained at 5μg / mL.

[0062] 5.2. Real-time fluorescence quantitative PCR detection of SIRT1 mRNA expression level:

[0063] The total RNA of each group of cells was extracted by RNAiso Plus lysis, and cDNA was synthesized using a reverse transcription kit. The above cDNA was used for qRT-PCR reaction. The reaction system was: TB Green Premix Ex TaqTMII 5μL, forward primer 0.2μL, reverse primer 0.2μL, cDNA 1μL and Sterile Purified Water 3.6μL. The reaction conditions were: 95℃ pre-denaturation for 30s, 95℃ denaturation for 5s, 60℃ annealing for 30s, 40 cycles, and melting curve analysis was performed to observe whether there was a single melting curve characteristic peak to determine the specificity of the primer reaction. Three replicate wells were set for each sample. The 2-ΔΔCt method was used to perform relative quantitative analysis with GAPDH as the internal reference. The primer sequences used are as follows:

[0064] SIRTUIN1-F:5'-GCTGACGACTTCGACGACG-3';

[0065] SIRTUIN1-R: 5'-TCGGTCAACAGGAGGTTGTCT-3';

[0066] β-actin-F: 5'-AGATGACCCAGATCATGTTTGAG-3';

[0067] β-actin-R: 5'-AGAGCATAGCCCTCGTAGAT-3';

[0068] Figure 5 The figure shows that MBL enhances the expression level of SIRT1 mRNA in mouse embryonic fibroblasts NIH-3T3. The results show that compared with the control group, the expression level of SIRT1 mRNA in the 5μg / mL MBL treatment group increased.

[0069] (6) MBL regulates the cell cycle of H2O2-induced aged mouse embryonic fibroblasts NIH-3T3.

[0070] 6.1. Preparation of cell samples with different treatment methods:

[0071] Mouse fibroblast NIH-3T3 cells were cultured in 10% fetal bovine serum high glucose medium and placed in a humidified cell culture incubator at 37°C and 5% CO2. When the cell confluence reached 80%, they were digested with trypsin and collected in a centrifuge tube. At a constant temperature of 37°C, the cells were treated with 400 μM H2O2 for 3 h and oscillated every 15 min to produce oxidative stress. The NIH-3T3 premature aging cell model was further obtained and 4 × 10 5 The cells were seeded at a density of 1000 cells / mL in 6-well plates and cultured for 3 days.

[0072] The senescence of NIH-3T3 cells was induced by simulating reactive oxygen stress with H2O2. The initial stage of the experiment was defined as day 0. A total of 5 parallel groups were set up in the experiment. The first group was set as the blank control group, which was cultured using the standard culture method and the cell culture medium was changed every 24 hours; the second and third groups were used to provide the culture medium for the fourth and fifth groups after H2O2 treatment; the fourth group was the group that was only treated with H2O2 to induce reactive oxygen stress; the fifth group was the group that was co-treated with 400μM H2O2 and 5μg / mL MBL. The second and third groups were no longer involved in the experiment after the culture medium was changed. It should be noted that MBL was added throughout the process; the concentration of MBL in the culture medium was maintained at 5μg / mL. On the third day of culture, the samples were processed with a cell cycle detection kit, and then the red fluorescence was detected at an excitation wavelength of 488nm using a flow cytometer, and the light scattering was detected at the same time.

[0073] 6.2. Results of cell cycle regulation of 5μg / mL MBL against H2O2-induced senescence:

[0074] On the first day of the experiment, mouse embryonic fibroblasts were treated with 400μM H2O2 for 3 hours and then cultured for 72 hours. Compared with the control group and the 400μM H2O2-induced senescence group, the percentage of cells in the GO / G1 phase was significantly lower in the presence of 5μg / ml MBL, while the percentage of cells in the S and G2 / M phases was higher.

[0075] In the presence of 5 μg / ml MBL, the cell cycle entered the S and G2 / M phases that enable cell proliferation, thereby enhancing the ability of cell proliferation.

[0076] Figure 6 The figure is a graph showing the percentage of various growth phases of mouse embryonic fibroblasts NIH-3T3 under different treatment conditions, which illustrates the effect of MBL on the cell cycle of aged mouse embryonic fibroblasts and slows down the cell cycle arrest of senescent cells in the G1 phase.

[0077] (7) MBL regulates the reactive oxygen species (ROS) levels in H2O2-induced aged mouse embryonic fibroblasts NIH-3T3.

[0078] 7.1. Preparation of cell samples with different treatment methods:

[0079] Mouse fibroblast NIH-3T3 cells were cultured in 10% fetal bovine serum high glucose medium and placed in a humidified cell culture incubator at 37°C and 5% CO2. When the cell confluence reached 80%, they were digested with trypsin and collected in a centrifuge tube. At a constant temperature of 37°C, the cells were treated with 400 μM H2O2 for 3 h and oscillated every 15 min to produce oxidative stress. The NIH-3T3 premature aging cell model was further obtained and 4 × 10 5 The cells were seeded at a density of 1000 cells / mL in 6-well plates and cultured for 3 days. The senescence of NIH-3T3 cells was induced by using H2O2 to simulate reactive oxygen species stress.

[0080] The initial stage of the experiment was defined as day 0. A total of 5 parallel groups were set up in the experiment. The first group was set as the blank control group, which was cultured using the standard culture method and the cell culture medium was changed every 24 hours; the second and third groups were provided with replacement culture medium for the fourth and fifth groups after H2O2 treatment; the fourth group was the group that was only treated with H2O2 to induce reactive oxygen stress; the fifth group was the group that was co-treated with 400μM H2O2 and 5μg / mL MBL. The second and third groups were no longer involved in the experiment after the culture medium was changed. It should be noted that MBL was added throughout the process; the concentration of MBL in the culture medium was maintained at 5μg / mL. On the third day of culture, the samples were treated with a reactive oxygen detection kit, and then the intensity of fluorescence before and after stimulation was detected by flow cytometry using an excitation wavelength of 488nm and an emission wavelength of 525nm.

[0081] 7.2. Results of 5 μg / mL MBL regulation on ROS in H2O2-induced senescent cells:

[0082] On the first day of the experiment, mouse embryonic fibroblasts were treated with 400μM H2O2 for 3 hours and then cultured for 72 hours. When cells age, mitochondria are damaged and the amount of ROS produced increases. Compared with the 400μM H2O2-induced aging group, the level of ROS in cells was significantly reduced in the presence of 5μg / ml MBL. The reason is that in the presence of 5μg / ml MBL, the mitochondrial function of aging cells is improved, which can reduce the level of ROS in aging cells.

[0083] Figure 7 This is a graph showing that MBL reduces the level of reactive oxygen species (ROS) in mouse embryonic fibroblasts NIH-3T3.

[0084] Based on the above examples, mannose binding aggregation can be used to prepare drugs for preventing or treating cell senescence and related diseases, especially showing the application potential of MBL in inhibiting cell senescence induced by DNA damage caused by oxidative stress. Related diseases are age-related neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease and amyotrophic lateral sclerosis (ALS).

[0085] Based on the research results of the above examples, MBL can also be used to screen candidate drugs that can prevent or treat cell senescence and related diseases or conditions. The relevant screening steps are as follows:

[0086] (1) treating a system expressing or containing MBL with a test substance;

[0087] (2) detecting the expression level of MBL in the system;

[0088] (3) Select substances that can upregulate the expression level of MBL as candidate drugs.

Claims

1. A non-therapeutic use of mannan-binding lectin in inhibiting cell senescence, characterized in that: The cell senescence is induced by DNA damage, and the DNA damage is caused by H2O2 through oxidative stress; the cells are mouse embryonic fibroblasts.

2. The use according to claim 1, characterized in that The embryonic fibroblasts are NIH-3T3.

3. The use according to claim 1, characterized in that After cell senescence, by upregulating the content or expression level of mannan-binding lectin in the cell growth environment, it inhibits the activity of senescence-related β-galactosidase, maintains the normal morphology of the cell nucleus, inhibits the accumulation of senescence marker proteins P53 and P21, enhances the mRNA expression level of the cell longevity gene SIRT1, inhibits cell arrest in the G1 / G0 phase, and thus enhances cell proliferation ability.

4. The use according to claim 1, characterized in that After cell senescence, the level of reactive oxygen species in cells is reduced by upregulating the content or expression level of mannan-binding lectin in the cell growth environment.

5. The use according to any one of claims 1 to 4, characterized in that: The application is to prepare medicine for preventing or treating cell senescence.

6. The use according to any one of claims 1 to 4, characterized in that: The application is to screen candidate drugs for preventing or treating cell senescence, and the screening of the candidate drugs comprises the following steps: (1) Treating a system expressing or containing MBL with the substance to be tested; (2) detecting the expression level of MBL in the system; (3) Select substances that can upregulate the expression level of MBL as candidate drugs.

Images