Application of sciatin in preparation of product for improving cell viability and resisting aging

Abstract

The invention discloses an application of sciatin in preparation of a product for improving cell viability, resisting cell aging, resisting oxidation or inhibiting senescence-related secretory phenotype (SASP) inflammatory factors. According to the application disclosed by the invention, the anti-aging activity of the sciaflavone is evaluated by constructing an IMR-90 cell model for inducing aging by chemotherapy. Experimental results show that the vitality of senescent cells can be remarkably improved at the concentration of 10 mu M, and the enhancing effect is 40% or above. More importantly, the sciaflavones can significantly reduce the level of reactive oxygen species (ROS) in senescent cells, and the average inhibition level reaches 75.0%. Meanwhile, the sciaflavin has a regulating or inhibiting effect on secretion of SASP inflammatory factors IL-6 and IL-8 in senescent cells. The results jointly prove that the sciaflavones have the potential of relieving the cell senescence state and support the application of the sciaflavones in preparation of anti-aging products.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of Sciadopitysin in the preparation of products that improve cell vitality and anti-aging. Specifically, it relates to the effects of Sciadopitysin on improving cell activity, inhibiting reactive oxygen species (ROS), and inhibiting aging-related secretory phenotype (SASP) inflammatory factors and its application in the preparation of anti-aging products. Background Technology

[0002] Aging is a complex physiological process involving the combined effects of genes, cell function, metabolic disorders, and environmental factors, and is a major driving factor for a variety of age-related diseases.

[0003] Existing research indicates that one of the key characteristics of senescent cells is the senescence-associated secretory phenotype (SASP). SASP continuously drives the development of age-related diseases by influencing the microenvironment. SASP is complex, with interleukin-6 (IL-6) and interleukin-8 (IL-8) being important members. IL-6 can stimulate acute responses, hematopoiesis, and immune responses during infection and tissue damage, contributing to host defense. However, the persistent dysregulation of IL-6 synthesis has pathological effects on chronic inflammation and autoimmune diseases. IL-8 is an important chemokine that can activate immune cells such as neutrophils and lymphocytes to reach sites of inflammation, enhancing local inflammatory responses and helping to clear pathogens and damaged tissues. However, the sustained release of IL-8 can lead to chronic inflammation, affecting the function of normal cells and accelerating tissue aging. Furthermore, senescent cells are often accompanied by decreased mitochondrial function and reduced antioxidant enzyme activity, leading to a large accumulation of reactive oxygen species (ROS) within the cell, triggering oxidative stress and cell damage. The accumulation of ROS further activates pathways such as NF-κB and p53, which play a central role in cellular senescence and SASP. Therefore, improving cell vitality, inhibiting SASP inflammatory factors, and clearing excess intracellular ROS are currently key hot topics in anti-aging strategies.

[0004] Traditional Chinese medicine and natural products have unique advantages in the research of anti-aging products, making them one of the important directions for anti-aging drug development. Jinsong biflavonoids are biflavonoid compounds, chemical components of gymnosperms such as Ginkgo biloba, Taxus chinensis, and Podocarpus macrophyllus, and have been proven to possess various biological activities, including anti-inflammatory, tumor cell growth inhibition, free radical scavenging, and antioxidant effects. Existing technologies have reported that Jinsong biflavonoids can inhibit glucose-induced apoptosis in AC16 cardiomyocytes, suggesting its potential anti-aging activity. However, existing technologies lack systematic studies on the effects of Jinsong biflavonoids on core aging indicators (such as cell viability, SASP components, and ROS levels) in in vitro aging cell models, particularly failing to clarify its precise effects on improving aging cell viability and significantly inhibiting ROS accumulation.

[0005] Based on this, this invention systematically evaluates the effects of Jinsong Shuanghuangone on cellular senescence markers by constructing a chemotherapy-induced senescence model of human female fetal lung fibroblasts (IMR-90). The inventors unexpectedly discovered that Jinsong Shuanghuangone not only enhances cell viability, but more importantly, it significantly reduces elevated ROS levels in senescent cells and regulates the secretion of SASP inflammatory factors IL-6 and IL-8. This clearly demonstrates the significant efficacy of Jinsong Shuanghuangone in anti-cellular senescence, providing new and reliable experimental evidence for its application in the preparation of anti-aging products. Summary of the Invention

[0006] The purpose of this invention is to provide the application of Jinsong Shuanghuang flavonoids in the preparation of products that improve cell vitality and have anti-aging effects, aiming to solve the problem of the lack of systematic evaluation and significant activity discovery of the anti-aging activity of Jinsong Shuanghuang flavonoids in the prior art.

[0007] To achieve the above objectives, this invention provides the application of Jinsong Shuanghuangtong in the preparation of products that enhance cell vitality, resist cell aging, resist oxidation, or inhibit aging-related secretory phenotype (SASP) inflammatory factors.

[0008] Preferably, the anti-aging product is used to improve cell vitality, and is especially suitable for aging cells.

[0009] Preferably, the effective concentration of Jinsong Shuanghuang flavonoids in the anti-aging product is 1 μM to 10 μM.

[0010] Preferably, the anti-aging product has the effect of significantly reducing the level of reactive oxygen species (ROS) in senescent cells, and can achieve an average inhibition rate of 75.0% or more of the level of reactive oxygen species in senescent cells.

[0011] Preferably, the anti-aging product has the effect of regulating or inhibiting the secretion of SASP inflammatory factors such as IL-6 and IL-8 in senescent cells.

[0012] Preferably, the anti-aging product is a drug, health product, functional food, or cosmetic.

[0013] The beneficial effects of this invention are as follows: 1. Significantly enhances cell viability: At a concentration of 10 μM, Jin Song Shuang Huang Ke has a significant effect on enhancing the cell viability of senescent cells, alleviating the state of cell senescence, and reducing the number of senescent cell deaths.

[0014] 2. Extremely strong antioxidant activity: Jinsong biflavonoids can significantly reduce the ROS level in senescent cells, with an average inhibition level of up to 75.0%, indicating that it has excellent antioxidant activity and potential anti-aging activity.

[0015] 3. Regulation of SASP inflammatory factors: Jinsong Shuanghuangtong has a strong inhibitory effect on the secretion of IL-6 and IL-8, key inflammatory factors of SASP in senescent cells.

[0016] The above results collectively support the application of Jinsong Shuanghuangtong in the preparation of anti-aging products, and show that it has significant efficacy and novelty in combating cellular senescence caused by oxidative stress. Attached Figure Description

[0017] Figure 1 The results show the activity of sciadopitysin at 1 μM and 10 μM concentrations on normal proliferating and senescent cells. Figure 2 Images and quantitative calculation results of the effect of sciadopitysin on staining of β-galactosidase (SA-β-gal), a marker of cellular senescence. Figure 2 Image A shows the staining results. Figure 2 B is the graph of quantitative calculation results. The significance was calculated using the U test, and *** represents the P value < 0.001. Figure 3 The results show the inhibitory effect of Sciadopitysin on the SASP inflammatory factors IL-6 and IL-8. Figure 3 Figure A shows the detection results of the SASP inflammatory factor IL-6. Figure 3 B shows the detection results of the SASP inflammatory factor IL-8; Figure 4 The results of the ROS inhibition ability test of Jinsong Biflavonoids; Figure 4 A is a flow cytometry peak pattern of the inhibitory effect of Jinsong Biflavonoids on reactive oxygen species (ROS). Figure 4 B represents the quantitative calculation results of the inhibitory ability of Jinsong Biflavonoids against reactive oxygen species (ROS). Detailed Implementation

[0018] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0019] Example 1. Jin Song Biflavonoids can significantly improve the vitality of senescent cells. 1) Construction of cell models The senescent IMR-90 human female fetal lung fibroblast cell line (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu32) was treated with the chemotherapeutic drug etoposide. Culture conditions: IMR-90 cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C and 5% CO2. Senescence induction: 10 mM etoposide stock solution was diluted 1:200 to a working concentration of 50 μM and added to the above medium to induce cell senescence for 48 hours, followed by replacement with fresh medium to allow cells to recover for 72 hours. The normal proliferation control group was treated with 0.5% DMSO for 48 hours, with the remaining steps the same.

[0020] 2) Cell viability assay (CCK-8 assay) Cells were seeded in 96-well plates. After 24 hours of adherent growth, sciadopitysin was added for experiments. Drug treatment: Sciadopitysin was diluted in cell culture medium at working concentrations of 1 μM and 10 μM. The control group was treated with 0.1% DMSO. Incubation was performed for 72 hours. Detection: After incubation with CCK-8 working solution for 40 minutes, the signal intensity was measured at 450 nm using a microplate reader, with 620 nm as the reference wavelength.

[0021] 3) Results Analysis The results are as follows Figure 1 As shown, Jinsong Shuanghuangone exhibited cell viability-enhancing activity at both 1 μM and 10 μM concentrations. Particularly at the 10 μM concentration, its cell viability-enhancing effect was stronger, especially in senescent cells, where the activity enhancement effect exceeded 40%. Therefore, Jinsong Shuanghuangone can alleviate cellular senescence and reduce the number of dying senescent cells.

[0022] Example 2: Effects of Jinsong Biflavonoids on Cellular Senescence Markers (β-Galactosidase) The experimental working concentration of Jinsong Biflavonoids was chosen to be 10 μM.

[0023] 1) β-galactosidase staining A senescent cell model was constructed using the method described in Example 1, and the cells were seeded in 6-well plates. After 24 hours of adherent growth, 10 μM sciadopitysin was added for the experiment, while 0.1% DMSO was added to the control group. The experiment was conducted using a β-galactosidase staining kit (Solarbio, G1580) according to the kit instructions.

[0024] Fixation and washing: Aspirate the culture medium, wash once with PBS, and fix with 1 mL of β-Gal fixative for 15 minutes. Staining: Prepare the staining working solution according to the instructions, add it to the cells, and incubate overnight (12 hours) at 37°C. Observation and photography: Aspirate the staining solution, add 1 mL of PBS, and immediately observe and photograph under a regular optical microscope (5x objective lens). Figure 2 ).

[0025] Quantitative analysis: The β-galactosidase signal intensity of each image was calculated using the OpenCV (v4.8.1) library in Python (v3.8). The Mann-Whitney U test was performed using the stats module in the SciPy (v1.8.0) library to calculate the significance of differences between groups.

[0026] 2) Results Analysis The results are as follows Figure 2 As shown, after treatment with Jinsong Shuanghuangone, the positive signal of β-galactosidase in the normal proliferating group cells decreased significantly (P<0.001). In the senescent group, the mean value of the positive signal of β-galactosidase in the Jinsong Shuanghuangone-treated group was lower than that in the senescent control group, but the difference did not reach statistical significance (ns).

[0027] Example 3. Effects of Jinsong Shuangfantong on SASP inflammatory factors (IL-6 and IL-8) 1) Sample preparation Cells in each experimental group were treated with etoposide or DMSO, then the culture medium was replaced with fresh medium and treated with 10 μM Jinsong Biflavonoids for 72 hours. The culture medium supernatant was collected, centrifuged to remove cell debris, and then analyzed by ELISA.

[0028] 2) ELISA detection of SASP components (IL-6 and IL-8) Prepare standards and working solutions according to the instructions of Xinbosheng Company's Human IL-6 and IL-8 ELISA kits (EHC007, EHC008) and perform the operation. Add culture supernatant or standards to wells pre-coated with antibodies, incubate in the dark, and then add biotinylated antibody working solution, enzyme conjugate working solution, and chromogenic substrate (TMP) sequentially. Finally, add the reaction stop solution. Measure the absorbance at 450 nm using a microplate reader.

[0029] 3) Results Analysis The results are as follows Figure 3 A and Figure 3As shown in Figure B, in the senescent cell group treated with Jinsong Shuanghuangtong, the average levels of IL-6 and IL-8 decreased slightly, but the changes did not reach statistical significance (ns). In the normal cell group, IL-8 levels decreased significantly (P<0.05), while IL-6 levels showed no significant change. This indicates that Jinsong Shuanghuangtong has a certain regulatory effect on the secretion of IL-6 and IL-8.

[0030] Example 4. Antioxidant effect of Jinsong Biflavonoids (Reactive Oxygen Species (ROS) detection) 1) Flow cytometry detection of reactive oxygen species (ROS) After treatment with etoposide or DMSO, cells in each experimental group were replaced with fresh culture medium and then treated with 10 μM Jinsong Biflavonoids in the culture medium for 72 hours.

[0031] H2DCFDA (2',7'-Dichlorodihydrofluorescein diacetate, MedChemExpress, Shanghai, China) was used to detect intracellular reactive oxygen species (ROS). H2DCFDA was dissolved in DMSO to prepare a 10 mM stock solution. For experiments, H2DCFDA was diluted to 10 μM in serum-free medium as the working solution. Cells from each experimental group were seeded in 6-well plates and treated with etoposide to induce senescence or with DMSO to obtain cells for the experiments. Each well was treated with fresh DMEM complete medium and different drugs for 72 hours.

[0032] The staining process was performed as follows: Cells were digested with trypsin, centrifuged, and then resuspended in PBS and washed twice to remove culture medium components; 1 ml of H2DCFDA staining working solution was added to each tube, gently mixed, and then incubated at 37°C for 30 min; the cells were resuspended in PBS and washed twice to remove unbound dye; the processed cell samples were placed on ice and flow cytometry (BD LSR Fortessa) was used within 45 min to detect fluorescence intensity, and the results were analyzed using BD FACSDiva 9.0 software.

[0033] Peak plots were generated using Flowjo 10.9, and the mean and standard deviation of fluorescence signals for each group were calculated. Based on the number of cells detected in each group, the mean and standard deviation of fluorescence signals, bar charts were generated using the pyplot module in the matplotlib (v3.7.5) package in Python. Independent samples t-tests were performed using the stats module in the scipy (v1.8.0) library.

[0034] 2) Results Analysis As cells age, mitochondrial function declines, and the activity of antioxidant enzymes (such as superoxide dismutase and glutathione peroxidase) also decreases significantly. This leads to an increased accumulation of reactive oxygen species (ROS) within cells, thereby increasing the risk of oxidative stress and cell damage. ROS can also activate multiple signaling pathways, such as NF-κB and p53, which play a central role in cellular senescence, SASP, and age-related diseases. Therefore, increased ROS is an important characteristic of senescent cells. The results of this invention show that ROS levels in senescent cells are significantly increased, averaging nearly 5-fold. Compared with the senescent control group, 10 μM Jin Song Bihuangdan significantly reduced ROS levels in senescent cells (P<0.001), with an average inhibition level of 75.0% (…). Figure 4 (A and 4B).

[0035] This result indicates that Jinsong Shuanghuangone possesses significant antioxidant activity in senescent cells. In normal cells, Jinsong Shuanghuangone did not show a clear ROS-inhibiting effect. This is because the antioxidant system of normal cells is relatively intact and can effectively scavenge ROS. Senescent cells, however, have weaker antioxidant capacity and are therefore more sensitive to the antioxidant effects of Jinsong Shuanghuangone. These results collectively support the significant anti-aging activity of Jinsong Shuanghuangone.

Claims

1. Application of Jinsong Biflavonoids in the preparation of products that enhance cell vitality and have anti-aging effects.

2. The application according to claim 1, wherein the product is for improving cell vitality, especially improving the vitality of senescent cells.

3. According to claim 1, the product is an antioxidant product that can significantly reduce the level of reactive oxygen species in aging cells.

4. According to claim 3, the product contains Jinsong Biflavonoids, which can achieve an average inhibition rate of 75.0% or higher of reactive oxygen species levels in senescent cells.

5. The application according to claim 1, wherein the anti-aging product includes products for regulating or inhibiting aging-related secretory inflammatory factors.

6. The application according to claim 5, wherein the inflammatory factors include interleukin-6 and interleukin-8.

7. The application according to claim 1, wherein the product is a drug, health product, functional food, or cosmetic.

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