Application of neophytadiene in preparation of medicine for preventing and treating premature senility of lung cells

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of neophytadiene in preparation of a medicine for preventing and treating premature senility of lung cells. According to the invention, the biological character change, the apparent transcriptional regulation effect and the internal molecular mechanism of the premature senility of the lung cells induced by the nano polystyrene are studied. Wherein the exposure of the nano polystyrene can induce the increase of cell premature senility phenotype, SA-beta-gal activity, ROS and SASP levels, and S-phase retardation of a cell cycle, and can activate the methylation modification of m6A and m7G of lung cell RNA, up-regulate the expression of RNA methyltransferase, promote the combination of METTL1 and a premature senility core gene, and accelerate the premature senility of cells. The neophytadiene NPT intervention can remarkably reverse ROS and SASP levels in cells, effectively relieve the cell cycle arrest degree, reverse the expression abnormity of RNA methylation regulatory enzyme, delay the premature senility process and the like. The invention provides a new thought and direction for prevention and intervention measures of nano-plastic induced lung injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a new phytadiene in the preparation of drugs for preventing and treating premature aging. Background Technology

[0002] Plastic waste, due to its strong chemical stability and long degradation cycle, exhibits high persistence and widespread distribution in the environment. Under the influence of physical weathering, mechanical abrasion, and photochemical degradation, plastic pollutants in the environment gradually break down into microplastics (MPs, <5mm), eventually forming nanoplastics (NPs, ≤100nm). Polystyrene-nanoplastic (PS-NPs) is a novel environmental pollutant characterized by its small particle size, large specific surface area, ease of suspension in the air, and strong permeability. Given the large surface area and thin barrier of human alveoli, PS-NPs are more easily inhaled through the respiratory tract, directly damaging lung function. Tiny PS-NPs are ubiquitous in the environment, and humans are mainly exposed through the respiratory tract, daily contact, digestive tract, and occupational exposure. Occupational groups may be exposed to higher concentrations of PS-NPs.

[0003] The lungs are a key target organ for the toxic effects of PS-NPs. PS-NPs with a particle size of 10-100 nm can penetrate the upper respiratory tract defense system and accumulate persistently in the bronchi and alveoli of the lower respiratory tract. NPs deposited deep within the respiratory tract interact with different types of lung cells, such as alveolar macrophages, epithelial cells, and fibroblasts. Lung cells can come into contact with NPs through phagocytosis or pinocytosis, triggering a series of cellular stress responses, including the release of reactive oxygen species (ROS) and inflammatory factors. These responses can induce premature aging phenotypes through epigenetic mechanisms, such as RNA and DNA methylation and histone modifications. Therefore, PS-NP exposure can increase the risk of premature lung aging or age-related diseases. Finding prevention and intervention strategies for PS-NP-induced premature lung aging and related health risks has significant practical value.

[0004] Neophytadiene (NPT) is a diterpenoid natural product with the molecular formula C2. 20 H 38 With a molecular weight of 278.52 Da, it can be extracted from plants such as tobacco, seaweed, green algae, mugwort leaves and hawthorn leaves. Currently, it is mostly used to treat headaches, skin diseases and rheumatism. There are no reports on the research on the invention of neophytadiene in resisting premature aging of human lung epithelial cells induced by nano-polystyrene. Summary of the Invention

[0005] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide an application of neophytadiene in the preparation of drugs for preventing and treating premature aging of lung cells.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The application of a neophytadiene in the preparation of a drug for preventing and treating premature aging of lung cells, wherein the structural formula of the neophytadiene is shown in Formula I:

[0008] The aforementioned premature aging of lung cells is caused by microplastics or nanoplastics;

[0009] The nanoplastic is nano-polystyrene (PS-NPs);

[0010] The effective concentration of the new phytadiene is 15 μmol / L;

[0011] The aforementioned neophytadiene can reverse or reduce PS-NPs-induced cellular ROS and SASP expression levels, thereby alleviating the degree of cell cycle arrest.

[0012] The aforementioned neophytadiene can reverse or reduce the expression levels of PS-NPs-induced RNA m6A methyltransferases METTL3, METTL14, METTL16, or WTAP mRNA or protein.

[0013] The aforementioned neophytadiene can reverse or reduce the expression level of PS-NPs-induced RNA m7G regulatory enzyme METTL1 mRNA or protein;

[0014] The aforementioned neophytadiene can reverse or reduce the mRNA expression levels of PS-NPs-induced premature aging core genes CAT, CCT2, IMP3, and IMP4.

[0015] The aforementioned neophytadiene can reverse or reduce the binding levels of PS-NPs-induced METTL1 with premature aging core genes CAT, CCT2, IMP3, and IMP4.

[0016] The application of the aforementioned neophytadiene in the preparation of drugs for preventing and treating respiratory or lung diseases caused by microplastics or nanoplastics;

[0017] The respiratory or lung diseases mentioned include chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary fibrosis, pneumonia, etc.

[0018] The nanoplastic is nano-polystyrene;

[0019] The application of the aforementioned neophytadiene in drugs for preventing and treating cancers caused by microplastics or nanoplastics;

[0020] The cancer mentioned includes lung cancer, etc.

[0021] The nanoplastic is nano-polystyrene;

[0022] The application of the aforementioned neophytadiene in the preparation of drugs for the prevention and treatment of lung injury;

[0023] A drug for preventing premature aging of lung cells, comprising the aforementioned neophytadiene;

[0024] A medicine for preventing and treating respiratory or lung diseases caused by microplastics or nanoplastics, comprising the neophytadiene;

[0025] The nanoplastic is nano-polystyrene.

[0026] The present invention has the following advantages and effects compared with the prior art:

[0027] (1) This invention found that exposure to PS-NPs can induce premature cell aging phenotype, increase SA-β-gal activity, ROS and SASP levels, and arrest the cell cycle in the S phase; while NPT intervention can significantly reverse the mRNA expression levels of ROS and four SASPs in cells and effectively alleviate the degree of cell cycle arrest.

[0028] (2) This invention found that exposure to PS-NPs can activate RNA m6A and m7G methylation modification in BEAS-2B cells, significantly upregulating the expression of RNA methyltransferases and demethylases; while NPT intervention can, to some extent, reverse the abnormal expression of RNA methylation regulatory enzymes.

[0029] (3) This invention found that PS-NPs can promote the binding of METTL1 to the premature aging core genes CAT, CCT2, IMP3 and IMP4, thereby accelerating cell premature aging. NPT intervention can delay the premature aging process.

[0030] (4) This invention provides a reliable theoretical basis for the prevention and intervention of NPT in diseases such as premature pulmonary aging, lung injury, and age-related diseases. Attached Figure Description

[0031] Figure 1 The graph shows the results of cell proliferation and viability assays. In the graph, A: the effect of different doses of PS-NPs on the viability of BEAS-2B cells after 24 hours of treatment; B: the effect of different doses of NPT on the proliferation of BEAS-2B cells after 24 hours of treatment. n = 6, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0032] Figure 2This is a diagram of the cell internalization results. In A: Flow cytometry analysis of the fluorescence intensity of fluorescent PS-NPs in cells after 24 h and 48 h of treatment; B: Calculation of the relative fluorescence intensity level of the treatment group with the Control group as 1. n=3, ** P<0.01, **** P<0.0001.

[0033] Figure 3 This is an image showing the results of SA-β-gal staining. A: Microscopic results of SA-β-gal staining in each group; B: Percentage of SA-β-gal-positive cells in each group. n = 3, *P < 0.05 *** P<0.001, **** P<0.0001.

[0034] Figure 4 This is a graph showing the relative levels of total ROS in cells for each group. A: Flow cytometry results for each group; B: Relative ROS levels in cells for each group. n=3, **P<0.01, ****P<0.0001.

[0035] Figure 5 The graph shows the cell cycle distribution results for each group. In the graph, A represents the flow cytometry results for each group, and B represents the cell cycle distribution for each group. n=3, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0036] Figure 6 This is a graph showing the mRNA expression levels of SASP in each group. n=3, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0037] Figure 7 It is a heatmap showing the correlations between aging-related indicators.

[0038] Figure 8 This is a diagram showing the interaction relationships of RNA m6A and m7G methylation-related regulatory enzymes. The PPI network diagram does not show regulatory enzymes that do not interact.

[0039] Figure 9 This is a graph showing the mRNA expression levels of the RNA m6A regulatory enzyme, where... n=3, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0040] Figure 10 This is a graph showing the mRNA expression levels of the RNA m7G regulatory enzyme. n=3, ** P<0.01, *** P<0.001, **** P<0.0001.

[0041] Figure 11 This is a graph showing the protein expression levels of RNAm6A regulatory enzyme. In the graph, A represents the results of Western Blot analysis, and B represents the quantitative analysis of protein bands. The Control group was assigned a value of 1, and the relative quantitative values ​​of other groups were calculated. n=3, * P<0.05, ** P<0.01, **** P<0.0001.

[0042] Figure 12 This is a graph showing the protein expression levels of the RNAm7G regulatory enzyme. In the graph, A represents the results of Western Blot analysis, and B represents the protein band quantification analysis. The Control group was assigned a value of 1, and the relative quantification values ​​of other groups were calculated. n=3, * P<0.05, ** P<0.01, **** P<0.0001.

[0043] Figure 13 This is a diagram showing the results of screening core genes for premature aging using the GSE76925 dataset. A: Venn diagram of DEGs and key COPD genes from the GSE76925 dataset; B: Venn diagram of DEGs and METTL1 aging pathway genes from the GSE76925 dataset; C: PPI network diagram of RNA m7G methylation regulators and 7 premature aging-related genes; D: Gene expression levels of 4 core premature aging genes directly interacting with METTL1 in the GSE76925 dataset. n=3, * P<0.05, ** P<0.01.

[0044] Figure 14The figures show the GO and KEGG enrichment analysis results for each group of DEGs. A: GO enrichment analysis of DEGs between nPS and NPT groups; B: KEGG enrichment analysis of DEGs between nPS and NPT groups.

[0045] Figure 15 This is a graph showing the DO enrichment analysis results of DEGs between the nPS group and the Control and NPT groups.

[0046] Figure 16 This is a graph showing the results of GSEA enrichment analysis of aging-related pathways. In the graph, A: GSEA enrichment analysis of aging-related pathways in the nPS group compared with the control group; B: GSEA enrichment analysis of aging-related pathways in the NPT group compared with the nPS group.

[0047] Figure 17 This is a diagram showing the results of transcriptomic sequencing that identified key genes related to premature aging. A: Venn diagram and bar chart of DEGs between Control vs. nPS, nPS vs. NPT, and Control vs. NPT groups; B: Protein interaction network.

[0048] Figure 18 This is a graph showing the mRNA expression levels of core genes related to premature aging in each cell group. n=3, * P<0.05, ** P<0.01, **** P<0.0001.

[0049] Figure 19 These are gel electrophoresis images of the real-time quantitative PCR products of the CAT, CCT2, IMP3, and IMP4 genes in each group of cells.

[0050] Figure 20 This is a graph showing the binding levels of METTL1 to core genes associated with premature aging in each group of cells, corrected for the IgG group. n=3, * P<0.05, ** P<0.01, *** P<0.001.

[0051] Figure 21 The graph shows the results of the correlation analysis of the expression of core genes in premature aging and their binding levels with METTL1 in each group: n = 3. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0053] I. Main materials and reagents involved in the embodiments:

[0054] The human lung epithelial cell line BEAS-2B was purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd. (cell line code: BFN6080086);

[0055] Polystyrene nanospheres (PS-NPs, 20 nm) and fluorescent polystyrene nanospheres (L-nPS, 20 nm) were purchased from Shanghai Huizhi Biotechnology Co., Ltd.; neophytadiene was purchased from MedChemExpress Inc., USA; DMEM low-glucose culture medium, PBS, penicillin-streptomycin dual antibiotics, fetal bovine serum, and 0.25% EDTA-trypsin were purchased from Thermo Fisher Scientific Inc., USA.

[0056] The CCK-8 assay kit, SA-β-gal staining kit for cell senescence, BCA protein quantification kit, Broadford protein assay kit, RIPA protein lysis buffer, SDS-PAGE gel preparation kit, and 5×TBE buffer were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the cell cycle assay kit was purchased from Guangdong Jingxin Biotechnology Co., Ltd.; Trizol lysis buffer was purchased from Invitrogen, USA; and EpiQuik... TM Nuclear extraction kit purchased from EpiGentek, USA; reverse transcription kit, quantitative PCR kit, SDS-PAGE protein loading buffer (5×), Takara RR036A PrimeScript RT Master, Takara Premix ExTap™ II was purchased from Takara Corporation, Japan; ECL chemiluminescence kit was purchased from Thermo Fisher Scientific, USA; Reactive Oxygen Species (ROS) Detection Assay Kit was purchased from BioVision, USA; mRNA primers were purchased from Guangzhou Aiji Biotechnology Co., Ltd.; and RNA Immunoprecipitation Kit was purchased from Guangzhou Baixin Biotechnology Co., Ltd.

[0057] Secondary antibodies anti-rabbit IgG (catalog number: ab6721), anti-mouse IgG (catalog number: ab6789), rabbit monoclonal antibody METTL3 (catalog number: ab195352), rabbit polyclonal antibody METTL14 (catalog number: ab98166), rabbit monoclonal antibody METTL16 (catalog number: ab252420), and rabbit monoclonal antibody WTAP (catalog number: ab195380) were purchased from Abcam, UK; rabbit polyclonal antibody METTL1 (catalog number: 14994-1-AP) was purchased from Proteintech, USA.

[0058] II. Cell culture, passage, and solution preparation in the examples

[0059] 1. Cell Culture: The BEAS-2B cells used in this invention were cultured in a constant-temperature cell culture incubator with 95% relative humidity and 37% and 5% CO2 concentrations. Complete culture medium was used throughout the experiment, specifically DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin dual antibiotics. The BEAS-2B cell line could be passaged on average every 2 days.

[0060] 2. Preparation of PS-NPs or fluorescent PS-NPs solutions: PS-NPs are highly soluble. After mixing with a vortex mixer, filter through a 0.22 μm filter membrane, add PBS to prepare a stock solution of 0.5 mg / mL, and then dilute with PBS or culture medium to the required concentration before use. The preparation of fluorescent PS-NPs solutions is the same as that of PS-NPs solutions.

[0061] 3. NPT Solution Preparation: Dissolve 5 mg of NPT in 0.45 mL of DMSO to prepare a 40 mmol / L NPT stock solution, then dilute with PBS or culture medium to the required concentration before use. Since the NPT stock solution has a short storage time, it needs to be aliquoted and stored at -80°C to avoid repeated freeze-thaw cycles that could degrade NPT. The entire NPT preparation process must be performed in the dark. Furthermore, studies have shown that DMSO concentrations below 0.1% are not cytotoxic; this invention ensures that the DMSO concentration used is kept below 0.1% to avoid any additional effects from DMSO.

[0062] III. Data Processing: All experiments were repeated at least three times. Data are expressed as mean ± standard deviation. SPSS 25 and GraphPad Prism 9.5.0 were used for statistical analysis and graphing. Tukey's test was used to compare pairwise data, one-way ANOVA was used to compare differences between groups, and Pearson correlation analysis was used. FlowJo 10.10.0 software was used to process flow cytometry data on cell internalization, cell cycle, and total ROS levels. ImageJ 1.8.0_345 software was used to add scale bars to microscopic images and calculate WB band content. All statistical analyses were two-tailed tests with a significance level of α = 0.05, and P < 0.05 was considered statistically significant.

[0063] Example 1: Effects of PS-NPs and NPT on cell viability

[0064] I. Experimental Methods

[0065] 1. Cell proliferation activity assay

[0066] (1) Cell seeding: 8 × 10⁸ cells were evenly seeded in each well of two 96-well plates. 3 100 μL of BEAS-2B cells were placed in each well (without cell seeding around the perimeter of the wells, and 100 μL of PBS was added to prevent evaporation). Each plate was labeled with its name and the concentration of PS-NPs or NPT in each well, with 6 replicates for each concentration. The plates were incubated overnight. The complete culture medium served as a blank control group, and the untreated BEAS-2B cells served as a control group, with 6 replicates for each control group.

[0067] (2) PS-NPs exposure: After overnight cell culture in step (1), remove the 96-well plate, aspirate the complete culture medium from each well, wash each well twice with PBS, dilute PS-NPs according to the set concentration gradient and add them to the corresponding replicate wells, and then put the plate back into the constant temperature incubator for 24 hours.

[0068] (4) NPT intervention: After overnight cell culture in step (1), remove the 96-well plate, aspirate the complete culture medium from each well, wash each well twice with PBS, dilute the NPT stock solution according to the set concentration gradient and add it to the corresponding replicate well, and continue to culture in a constant temperature incubator for 24 hours in the dark with tin foil.

[0069] (5) OD value determination using an ELISA reader: After 24 hours of culture, 10 μL of CCK8 reagent was added to each well at the preset concentration, and the 96-well plate was covered with aluminum foil and incubated in a constant temperature incubator for 30 minutes. The OD value of each well was measured using a continuous wavelength ELISA reader at 450 nm. The linearity was optimal when the OD value of the control group was around 1.0. The cell viability level at the preset concentration was obtained by substituting the OD value into the following formula:

[0070] Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100%

[0071] Note: As is the OD value of the poisoning / intervention group, Ab is the OD value of the blank control group, and Ac is the OD value of the control group.

[0072] 2. Identification of fluorescent PS-NPs by cell internalization

[0073] (1) Inoculate each well of the 12-well plate with 8×10⁸ g of seed. 4 Add cells per mL of BEAS-2B cells, shake well to ensure uniform cell distribution, and divide the cells into three groups: control, 24h fluorescent PS-NPs, and 48h fluorescent PS-NPs. Each group has three replicates. Incubate overnight in an incubator to allow the cells to adhere.

[0074] (2) After BEAS-2B cells adhered to the well, the complete culture medium in each well was aspirated, the cells were washed twice with PBS, and then incubated with fluorescent PS-NPs solution at a concentration of 0.04 mg / mL for 24 h and 48 h (3 replicates). The control group was incubated with complete culture medium without fluorescent PS-NPs.

[0075] (3) After the exposure time was over, the fluorescent PS-NPs solution was removed, and the cells were washed three times with PBS. Then, the cell pellets of each group were obtained with 0.25% (w / v) trypsin and a scraper, and the cell pellets were washed twice with PBS. The cell suspension was resuspended in 1 mL of PBS, and the cell suspensions of each group were transferred into 96-well plates, 300 μL per well. The fluorescence intensity of each group of cells was quantitatively measured using flow cytometry.

[0076] II. Test Results

[0077] 1. Effects of PS-NPs and NPT on cell viability

[0078] To investigate the toxic effects of PS-NPs on BEAS-2B cells, a concentration gradient of 0.02–0.32 mg / mL PS-NPs was established, and its effect on cell viability was examined. Within the range of 0–0.04 mg / mL, PS-NPs did not alter the growth density of BEAS-2B cells. However, when the PS-NP concentrations were 0.04, 0.06, 0.08, 0.10, and 0.12 mg / mL, the cell viability of BEAS-2B cells decreased sequentially, specifically (95.1±3.5)%, (80.7±2.5)%, (80.8±4.4)%, (74.5±1.6)%, and (63.4±2.2)%, respectively. Figure 1 A). BEAS-2B cells are highly sensitive to the effects of PS-NPs, with a half-maximal inhibitory concentration (IC50) of 100%. 50 The concentration of PS-NP was 0.16 mg / mL (95% CI: 0.15-0.17 mg / mL), while the cell viability was only (3.8±0.2)% when the PS-NP concentration reached 0.32 mg / mL. Based on the CCK8 results, subsequent experiments selected 1 / 4 IC50. 50 A concentration of 0.04 mg / mL of PS-NPs was used as a low-concentration (L-nPS group) model to induce premature aging in BEAS-2B cells. This concentration is also the average concentration of PS-NPs detectable in aquatic environments. Furthermore, the in vivo NP levels are much higher than the environmental concentrations. To comprehensively explore the mechanism of PS-NPs-induced premature aging, a 1 / 2 IC50 assay was selected. 50 The concentration (i.e., 0.08 mg / mL) was used as a higher concentration group (H-nPS group) for comparison.

[0079] BEAS-2B cells were treated with NPT at concentrations ranging from 0 to 200 μmol / L for 24 h. The results showed that low concentrations of NPT significantly promoted cell proliferation, while cell viability decreased significantly with increasing concentrations. Figure 1 B). At low concentrations of 5, 10, 15, and 20 μmol / L, cell viability was (108.3±5.7)%, (133.0±12.1)%, (150.4±7.7)%, and (125.4±8.8)%, respectively (P<0.05). Subsequently, the concentration with the strongest cell proliferation effect, 15 μmol / L, was selected as the NPT intervention concentration.

[0080] 2. Intracellular identification of fluorescent PS-NPs

[0081] The internalization and uptake of 20 nm PS-NPs by BEAS-2B cells was qualitatively assessed using flow cytometry. Based on the results of CCK8, fluorescently labeled L-nPS were used to treat BEAS-2B cells for 24 h and 48 h, respectively. After washing three times with PBS to remove the fluorescent PS-NPs adsorbed on the cell surface, changes in FITC channels were detected by flow cytometry. Compared with the control group, the intracellular fluorescence signal intensity in the 24 h and 48 h fluorescent PS-NP treatment groups increased in the positive direction (the peak value of the histogram shifted to the right of the horizontal axis). The relative fluorescence intensity was calculated with the control group as 1. The relative fluorescence intensity of the fluorescent PS-NP treatment group increased with increasing treatment time (P<0.05). Figure 2 Qualitative results from cell internalization indicate that BEAS-2B cells can internalize and take up PS-NP particles, and the amount of uptake is time-dependent, with a positive correlation between exposure time and uptake amount.

[0082] Example 2: General biological characteristics of NPT against PS-NPs-induced premature cell aging

[0083] I. Experimental Methods

[0084] 1. Cellular SA-β-gal staining

[0085] (1) Cell seeding: BEAS-2B cells were seeded at a rate of 5 × 10⁶ cells / year. 5 Cells were evenly seeded per well in 6-well plates and cultured overnight in an incubator. Cells were divided into a PS-NPs-treated group and an NPT-treated group, with untreated cells serving as the control group. Three replicates were set up.

[0086] (2) PS-NPs infection: After cell adhesion in step (1), remove the 6-well plate, aspirate the liquid in each well, and wash twice with PBS. Treat cells with L-nPS and H-nPS respectively, as in Example 1, with 3 replicates for each treatment group, and incubate in an incubator for 48 h.

[0087] (3) NPT intervention: After cell adhesion in step (1), the cells were pretreated with 15 μmol / L NPT for 24 h, and then treated with 15 μmol / L NPT+L-nPS or 15 μmol / L NPT+H-nPS for 48 h respectively. That is, NPT intervention was carried out simultaneously for 48 h when the cells were exposed to the virus, and the cells were cultured in an incubator.

[0088] (4) Staining treatment: Remove the control, poisoned, and intervention 6-well plates, aspirate the liquid from the wells, wash twice with PBS, and then add 1 mL of staining fixative to each well. Fix at room temperature for 15 min. After fixation, aspirate the liquid from the wells and add 1 mL of PBS to wash three times, 3 min each time. After washing, add 1 mL of staining working solution (10 μL staining solution A, 10 μL staining solution B, 930 μL staining solution C, and 50 μL X-Gal solution) to each well. Seal the gaps of the 6-well plate with sealing film to make it completely sealed. Alternatively, the 6-well plate can be covered with aluminum foil and incubated overnight in a CO2-free incubator.

[0089] (5) Microscopic observation and calculation: Take out the 6-well plate and place it under an inverted microscope. Each poisoning group and intervention group should select 3 or more fields of view. Calculate the number of blue-stained cells and the total number of cells in the selected fields of view, and calculate the cell blue staining rate according to the ratio of the two.

[0090] 2. Cell cycle detection

[0091] (1) Cell seeding: BEAS-2B cells were seeded at a rate of 1×10⁻⁶. 6 Cells were evenly seeded per well in 6-well plates and cultured overnight in an incubator. Cells were divided into a PS-NPs-treated group and an NPT-treated group, with untreated cells serving as the control group. Three replicates were set up.

[0092] (2) PS-NPs exposure and NPT intervention: see step 1 for details.

[0093] (3) Cell processing: Remove the processed 6-well plates, aspirate the liquid from the wells, wash twice with 1 mL PBS, and harvest cells from each group by trypsin digestion into 1.5 mL centrifuge tubes. Place the centrifuge tubes in a high-speed centrifuge at room temperature and centrifuge at 1000 rpm for 5 min to collect the cell pellet. Wash the cell pellet twice with PBS and transfer it into a clean 1.5 mL EP tube.

[0094] (4) Cell fixation: Centrifuge the EP tubes in a refrigerated centrifuge at 4°C, 1000g, for 5 min. Remove the supernatant and immediately add 1 mL of pre-cooled 70% (v / v) ethanol. Mix thoroughly by inverting the tubes and fix overnight at 4°C. After overnight fixation, remove the tubes and centrifuge at 4°C, 1000g, for 5 min. Wash the cells with PBS and centrifuge again to obtain the cell pellet.

[0095] (5) Cell staining: Add 0.5 mL of staining working solution (1 mL staining buffer, 20 μL RNase A and 10 μL propidium iodide) to each group, vortex and place in a 37°C incubator for 30 min in the dark, and then perform the detection.

[0096] (6) Flow cytometry detection: Live cells were labeled and clustered at a wavelength of 488 nm to obtain the cell cycle distribution.

[0097] 3. Total intracellular ROS content

[0098] (1) Cell seeding: BEAS-2B cells were seeded at a rate of 1×10⁻⁶. 6 Cells were evenly seeded per well in 6-well plates and cultured overnight in an incubator. Cells were divided into a PS-NPs-treated group and an NPT-treated group, with untreated cells serving as the control group. Three replicates were set up.

[0099] (2) PS-NPs exposure and NPT intervention: see step 1 for details.

[0100] (3) Cell treatment: See step 2 for details, to obtain a clean cell pellet.

[0101] (4) ROS probe treatment: 1000×ROS Label was diluted to 1×ROS Label with serum-free medium beforehand. The 6-well plate was removed, the liquid in the wells was aspirated, and the plate was washed twice with ROS buffer. Then, 500 μL of 1×ROS Label was added to the cell pellet of each group. No ROS Label was added to the control group. The cells were incubated at 37°C for approximately 40 minutes in the dark, with the plate inverted every 5 minutes to ensure sufficient binding of the ROS Label to the cells.

[0102] (5) Flow cytometry analysis: After incubation, the EP tubes of each group were centrifuged at 1200g for 5 min at room temperature. The supernatant was discarded, and the cells were washed twice with pre-cooled PBS. The cell pellet was resuspended in 1 mL of PBS and added to 96-well plates, with 3 replicates for each group and 300 μL of treatment solution per well. The total ROS level in the cells of each group was obtained by flow cytometry (optimal excitation wavelength 488 nm, optimal emission wavelength 525 nm).

[0103] 4. mRNA expression level detection

[0104] BEAS-2B cells were divided into groups of 5 × 10 6 Cells were evenly seeded per well onto culture flasks and cultured overnight in an incubator. Cells were divided into a PS-NPs-treated group and an NPT-treated group, with untreated cells serving as the control group. Three replicates were set up.

[0105] (2) PS-NPs exposure and NPT intervention: see step 1 for details.

[0106] (3) Cells from each group were collected using standard methods, RNA was extracted, and cDNA was synthesized using reverse transcription. Primers (Table 1) were designed for real-time quantitative PCR using GAPDH, IL-6 (NM_000600), IL-8 (NM_000584), MMP1 (NM_002421), and HGF (NM_001010931) genes as targets. Based on the Ct value and the formula (ΔCt = Ct value of target gene - Ct value of internal reference gene (GAPDH), ΔΔCt = ΔCt of treatment group - ΔCt of control group), the relative expression level of the control group gene was set as 1 as a reference, and the average relative content of the target gene was calculated. -ΔΔCt value.

[0107] Table 1 Primer sequences for RT-qPCR amplification

[0108]

[0109]

[0110] (Note: F represents the upstream sequence, and R represents the downstream primer)

[0111] II. Test Results

[0112] 3.2.1 SA-β-gal staining

[0113] SA-β-gal staining results showed ( Figure 3 Compared with the control group (3.9±0.3)%, the proportion of blue-stained positive cells in the PS-NPs treatment group was significantly increased, with the blue-stained cell rates in the L-nPS and H-nPS treatment groups being (39.3±0.3)% and (54.4±4.2)%, respectively. Conversely, compared with the PS-NPs treatment group, the proportion of blue-stained positive cells in the NPT intervention group was decreased, with the corresponding blue-stained cell rates in the L-nPS and H-nPS NPT intervention groups being (25.1±3.1)% and (28.0±5.4)%, respectively. Compared with the control group, the blue-stained cell rates in the L-nPS and H-nPS treatment groups increased by 10.3-fold and 14.0-fold, respectively (P<0.05). Compared with the L-nPS and H-nPS treatment groups, the blue-stained cell rates in the NPT intervention group decreased by 1.6-fold and 1.9-fold, respectively (P<0.05).

[0114] 2. Intracellular total reactive oxygen species level

[0115] Intracellular total reactive oxygen species levels in each group are as follows: Figure 4As shown in the figure, compared with the control group, the fluorescence intensity of the PS-NPs treatment group increased in the positive direction (the peak of the histogram shifted to the right of the horizontal axis), indicating a significant increase in intracellular reactive oxygen species (ROS) levels. Conversely, compared with the PS-NPs treatment group, the corresponding NPT intervention group showed decreased fluorescence intensity and a decrease in intracellular ROS levels. Using the control group as a baseline, the relative levels of total ROS in each group were calculated. The total ROS levels in the L-nPS and H-nPS treatment groups were 2.5 and 3.1 times higher than those in the control group, respectively (P<0.05). Compared with the PS-NPs treatment group, the total ROS levels in the NPT intervention groups corresponding to L-nPS and H-nPS decreased by 17.7% and 64.7%, respectively (P<0.05). These results indicate that PS-NPs treatment can induce premature cell aging and significantly increase intracellular oxidative stress levels, while NPT intervention can alleviate the degree of intracellular oxidative stress induced by PS-NPs.

[0116] 3. Cell cycle distribution

[0117] Cell cycle distribution in each group was assessed using propidium iodide staining combined with flow cytometry. Figure 5 In the PS-NPs-treated group, the G0 / G1 and G2 / M phases were significantly decreased, while the S phase was significantly increased. These results indicate that PS-NPs induce cell cycle arrest in the S phase of BEAS-2B cells in a concentration-dependent manner (P<0.05). In contrast, the proportion of cells in the G0 / G1 phase increased by 13.9% and the proportion in the S phase decreased by 10.4% in the H-nPS-treated group with NPT intervention. The distribution ratios of G0 / G1 and S phases in the cell cycle of the H-nPS-treated group tended to be similar to those of the control group. This suggests that NPT intervention can alleviate the oxidative stress-induced S phase arrest in the H-nPS-treated group and reverse the degree of premature cell aging (P<0.05).

[0118] 4. mRNA expression levels of senescence-associated secretory phenotype (SASP) in each group

[0119] like Figure 6As shown, compared with the control group, the mRNA expression levels of IL-6, IL-8, matrix metalloproteinase-1 (MMP-1), and hepatocyte growth factor (HGF) in SASP were consistently increased in the PS-NPs treatment group (P<0.05). Compared with the PS-NPs treatment group, the NPT intervention groups corresponding to L-nPS and H-nPS showed significantly decreased levels of IL-6, IL-8, MMP-1, and HGF. The results indicate that NPT intervention significantly inhibited the increase in SASP secretion induced by PS-NPs, thereby effectively slowing down the cellular senescence process (P<0.05). Pearson correlation analysis was used to assess the relationship between the above aging-related indicators. The heatmap results showed a significant positive correlation between aging-related biological traits and SASP mRNA expression levels. Figure 7 ).

[0120] Example 3: Changes in the RNA m6A and m7G modified microenvironment during NPT-induced premature aging against PS-NPs

[0121] I. Experimental Methods

[0122] 1. PPI Analysis

[0123] To clarify the interaction between RNA m6A and m7G methylation-related regulatory enzymes, PPI analysis was performed on 21 aging-related RNA m6A regulatory enzymes and 29 RNA m7G methylation-related regulatory enzymes using the STRING website.

[0124] 2. Detection of mRNA expression levels of RNA m6A regulatory enzyme and m7G methylation regulatory enzyme

[0125] Referring to step 4 of Example 2, cells from each group were collected, RNA was extracted, and cDNA was synthesized using conventional methods following reverse transcription. Primers (Table 1) were designed for real-time quantitative PCR reactions targeting the METTL1 (NM_023033), METTL3 (NM_019852), METTL14 (NM_020961), METTL16 (NM_024086), and WTAP (NM_004906) genes. Based on the Ct value, the relative expression level of the control group was set as 1 as a reference, and the average relative content of the target genes was calculated. -ΔΔCt value.

[0126] 3. Protein expression level detection

[0127] After treatment, the liquid in the culture flasks was removed from each group of cells. The cells were washed twice with 2 mL PBS, and then digested with 0.25% trypsin to collect the cells. The cells were centrifuged at 1000 rpm for 5 min at room temperature to obtain the cell pellet. The cells were lysed according to the standard method, and the total protein was extracted. The expression levels of the above proteins were detected by Western Blot.

[0128] II. Test Results

[0129] 1. Interactions between RNA m6A and m7G methylation regulators

[0130] RNAm7G regulatory enzymes METTL1, WDR4, NCBP2, and EIF4E interact with multiple RNAm6A regulatory enzymes. Figure 8 ), including methyltransferases METTL3, METTL14, METTL16, WTAP and KIAA1429, demethylases ALKBH5 and FTO, and reading proteins YTHDC1, YTHDC2, hnRNPA2B1 and hnRNPC.

[0131] 2. NPT-resistant PS-NPs induce changes in the mRNA expression level of RNA m6A regulatory enzyme.

[0132] This invention uses methyltransferases METTL3, METTL14, METTL16, and WTAP as targets to further analyze the changes in mRNA expression levels of NPT-resistant PS-NPs-induced RNA m6A regulatory enzymes. For example... Figure 9 As shown, compared with the control, the mRNA expression of RNA m6A methyltransferases METTL3, METTL14, METTL16, and WTAP was increased in the PS-NPs treatment group. Compared with the PS-NPs treatment group, the mRNA expression levels of METTL3, METTL16, and WTAP were decreased in the L-nPS corresponding NPT intervention group; the mRNA expression level of METTL3 was decreased in the H-nPS corresponding NPT intervention group (P<0.05).

[0133] 3. NPT-resistant PS-NPs induce changes in the mRNA expression level of RNA m7G regulatory enzyme.

[0134] like Figure 10 As shown, the expression of RNA m7G methyltransferase METTL1 was significantly increased in the PS-NPs treatment group (P<0.05). Compared with the PS-NPs treatment group, the METTL1 mRNA expression level was significantly decreased in the NPT intervention group corresponding to PS-NPs (P<0.05).

[0135] 4. Protein changes of NPT-resistant PS-NPs-induced RNAm6A regulatory enzyme

[0136] Figure 11 The results showed that the protein expression levels of RNA m6A methyltransferases METTL3, METTL14, METTL16, and WTAP were all increased in the PS-NPs treatment group. Compared with the PS-NPs treatment group, NPT intervention could reverse the PS-NPs-mediated changes in RNA m6A methyltransferases, especially the NPT intervention group corresponding to H-nPS, which showed a more significant effect. Compared with the H-nPS treatment group, the protein expression levels of METTL3, METTL14, METTL16, and WTAP in the corresponding NPT intervention group decreased by 70.3%, 42.3%, 20.1%, and 31.8%, respectively. In addition, the NPT intervention group corresponding to L-nPS reduced the protein expression level of METTL16 by 66.4% (P<0.05).

[0137] 5. Protein changes of NPT-resistant PS-NPs-induced RNA m7G regulatory enzyme

[0138] Based on the RT-qPCR results of RNA m7G methylation regulators, METTL1 was included for subsequent Western blot analysis. For example... Figure 12 As shown, PS-NPs treatment increased the expression level of the RNA m7G regulatory enzyme METTL1 protein. However, NPT intervention significantly reversed the increase in RNA m7G methyltransferase METTL1, with reductions of 19.0% and 61.0% in the L-nPS and H-nPS NPT intervention groups, respectively, compared to the PS-NPs treatment group.

[0139] Example 4: Bioinformatics Analysis and RNA Transcriptomics Sequencing Analysis of COPD Dataset

[0140] I. Experimental Methods

[0141] 1. Screening of core genes for premature aging

[0142] (1) Dataset Acquisition: The COPD (Chronic Obstructive Lung Disease) related dataset GSE76925 was collected from the public database Gene Expression Omnibus (GEO, http: / / www.ncbi.nlm.nih.gov / geo / ). Dataset GSE76925 contains lung tissue samples from 111 COPD patients and 40 healthy adults. This dataset is publicly available from the GEO database.

[0143] (2) Screening for differentially expressed genes (DEGs) in the dataset: The "limma" package in R4.3.1 software was used to screen the GSE76925 dataset for DEGs. The screening criteria were set as: |log2FoldChange |>2 and P .adjust <0.05.

[0144] (3) Screening for aging-related genes and their protein interactions:

[0145] 1) Based on literature, 145 core regulatory genes of COPD were selected. Then, the Human Biological Pathway Unification (PathCards, https: / / pathcards.genecards.org / ) website was used to search for pathways related to the function of METTL1. It was shown that METTL1 and 170 other related genes mediate the MAPK-Erk pathway to regulate aging. The Venn diagrams of DEGs with COPD core genes and METTL1-related pathway genes were drawn using the "VennDiagram" package of R4.3.1 software.

[0146] 2) Based on the Venn diagram results, the protein-protein interaction relationships between the core genes of premature aging and RNA m7G methylation regulators were analyzed using a PPI network diagram. High-confidence interaction data (score > 0.7) were obtained from the STRING website (https: / / cn.string-db.org / ), visualized using Cytoscape (v3.8.0), and core nodes were screened by calculating topological parameters such as degree.

[0147] (4) Expression levels of aging-related genes in the dataset: Based on the results of the Venn diagram, the expression levels and differences of core genes of premature aging in the dataset GSE76925 in COPD samples and healthy control samples were plotted.

[0148] 2. Transcriptomics sequencing (RNA sequencing, RNA-seq)

[0149] (1) Referring to step 4 of Example 2, cells from each group (control group, H-nPS group and H-nPS+NPT group) were collected and RNA samples were extracted using conventional methods. The sequencing was entrusted to Guangzhou Ruixin Biotechnology Co., Ltd.

[0150] (2) Bioinformatics analysis: Using relevant statistical methods to compare the differences in gene expression between two or more groups, relevant specific genes are screened out to facilitate further analysis of the biological significance of specific genes. It can also analyze gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), disease ontology (DO), gene set enrichment analysis (GSEA) enrichment pathways and chromosome analysis between groups.

[0151] II. Test Results

[0152] 1. Screening for core genes associated with premature aging and RNA m7G methylation regulatory enzymes.

[0153] Based on the above results, subsequent experiments focused on the regulatory mechanism of RNA m7G methylation in NPT intervention-induced premature aging of PS-NPs in BEAS-2B cells. First, differentially expressed genes were screened in the COPD dataset GSE76925 using the "limma" package, and a total of 1574 DEGs were identified.

[0154] By incorporating 145 key COPD genes from the literature and performing cross-screening with DEGs, four premature aging-related genes were identified. Furthermore, by using the KEGG pathway website to select 170 genes that co-mediate the MAPK-Erk pathway of aging with METTL1 and performing cross-screening with DEGs, three premature aging-related genes were also identified.

[0155] Based on the above Q-PCR and WB results, protein-protein interaction analysis was performed on the RNA m7G methylation regulator and seven screened premature aging-related genes. The genes that directly interact with the RNA m7G methyltransferase METTL1 were CAT, CCT2, IMP3, and IMP4. The expression of these four core premature aging genes in the GSE76925 dataset was visualized. The expression of these four genes was elevated in COPD lung tissue samples (P<0.05). Figure 13 ).

[0156] 2. RNA transcriptomic analysis of NPT against PS-NPs-induced premature cell aging

[0157] This invention included RNA transcriptomics analysis of a control group (Control group), an H-nPS group (hereinafter referred to as the nPS group), and an H-nPS+NPT group (hereinafter referred to as the NPT group). RNA transcriptomics analysis was performed on 9 samples (3 samples from each group). After data cleaning, a total of 57.77 Gb of quality control data was obtained, with GC content reaching 48.21% or higher, Q20 base percentage above 99.05%, and Q30 base percentage above 97.30%. Sequencing quality and depth met the requirements for subsequent analysis. Correlation analysis among the samples showed high correlation among repetitive samples within the Control, nPS, and NPT groups, indicating good reproducibility and similar expression patterns within each group. The correlation between the Control and NPT groups and the nPS groups was low, indicating significant differences in expression patterns among the samples. 3D distribution analysis using PCA principal component analysis showed clear separation of sample distributions among groups, with relatively tight clustering of samples within each group.

[0158] 3. GO and KEGG enrichment pathway analysis of DEGs in each group

[0159] In-depth investigation of transcriptomic changes in NPT-induced PS-induced premature cell aging, and screening of Control... vs.n PS, nPS vs. Differentially expressed genes between the NPT and Control vs. NPT groups were selected with a threshold of P. adjust <0.05 and |log2 FoldChange |≥1, and perform GO and KEGG enrichment pathway analysis on the obtained differentially expressed genes.

[0160] Compared with the Control group, the PS-NPs group and NPT intervention group mainly clustered in biological processes such as DNA metabolism, DNA replication, DNA-dependent DNA replication, microtubule-based processes, and cellular responses to DNA damage stimuli; mainly clustered in cellular components such as the cell nucleus, microtubule cytoskeleton, chromosome centromere regions, organelle membranes, and membranes; and mainly clustered in biological functions such as nucleotide transferase activity, catalytic activity on DNA, catalytic activity on RNA, tubulin binding, and DNA polymerase activity.

[0161] KEGG enrichment analysis of DEGs between the Control vs. NPS groups revealed that multiple pathways were directly or indirectly highly associated with cellular senescence, including cellular senescence pathways, p53 signaling pathways, DNA replication, cell cycle, base excision repair, nucleotide excision repair, and mismatch repair. KEGG enrichment analysis between the Control vs. NPT groups showed that DEGs were mainly enriched in pathways related to cellular senescence, such as the TGF-β signaling pathway, p53 signaling pathway, cellular senescence, cell cycle, DNA replication, mismatch repair, and Wnt signaling pathway.

[0162] Compared to the PS-NPs group, the DEGs in the NPT intervention group mainly clustered in biological processes such as DNA replication, DNA metabolism, DNA-dependent DNA replication, microtubule-based processes, and cellular component organization; they mainly clustered in cellular components such as transcription factor complexes, microtubule cytoskeleton, chromosome centromere regions, chromosome regions, and mitochondria; and they mainly clustered in biological functions such as DNA polymerase activity, catalytic activity acting on DNA, microtubule binding, pyridoxal phosphate binding, and vitamin B6 binding. KEGG enrichment analysis of the DEGs between the two groups also revealed multiple pathways highly related to cellular senescence, including the p53 signaling pathway, TGF-β signaling pathway, cell cycle, cellular senescence, DNA replication, nucleotide excision repair, and base excision repair. Figure 14 ).

[0163] 4. DO enrichment pathway analysis of each DEG group

[0164] DO functional enrichment analysis can reveal significant correlations between DEGs and multiple human disease pathways. For example... Figure 15 As shown, DEGs exhibited broader enrichment in the DO pathway between the control group and the nPS group, and between the nPS group and the NPT group. Specifically, DEGs between the control group and the PS-NPs-induced premature aging group were mainly enriched in mitochondrial metabolic disorders and various cancer-related diseases, such as lung adenocarcinoma, leukemia, and ovarian cancer, which are highly correlated with the aging process. DEGs between the PS-NPs-induced premature aging group and the NPT intervention group were also significantly enriched in various cancer types, such as renal cell carcinoma, ovarian cancer, and epithelial-mesenchymal cell tumor. In summary, the DO enrichment pathway suggests that PS-NPs-induced premature aging may be related to mitochondrial metabolic abnormalities and cancer pathway dysregulation, and NPT intervention may exert its anti-aging effect by regulating these disease pathways.

[0165] 5. Gene set enrichment analysis (GSEA) of DEGs between groups

[0166] After sorting the differential expression analysis results in ascending order of expression level changes, GSEA analysis was performed on DEGs between the control group and the nPS group, and between the NPT group and the nPS group. Compared with the control group and the NPT group, three aging-related signaling pathways were co-enriched in PS-NPs-induced premature aging: the VEGF signaling pathway, thyroid hormone synthesis, and the phosphatidylinositol signaling system. Compared with the control group, these three aging-related signaling pathways were highly expressed in the nPS group; while compared with the nPS group, these three aging-related signaling pathways were lowly expressed in the NPT group. These results suggest that PS-NPs exposure can positively regulate aging-related signaling pathways and accelerate the process of premature aging, while NPT intervention can significantly improve the state of premature aging through compensatory regulatory mechanisms. Figure 16 ).

[0167] 6. Screening and identification of core genes for premature aging through whole-genome sequencing.

[0168] Figure 17 The study revealed 5155 differentially expressed genes (DEGs) between the Control and nPS groups, 1082 DEGs between the Control and NPT groups, and 2372 differentially expressed genes between the nPS and NPT groups. Integrating these DEGs across the three groups yielded 249 premature aging-related genes. The aging pathways enriched by these 249 genes were further identified, and four core genes involved in RNA m7G methylation-induced premature aging were located within these pathways. Cross-analysis revealed 41 premature aging-related genes synergistically involved in these pathways. Analysis of the protein network interaction diagrams of these 41 genes showed that circles represent proteins, with their size proportional to node connectivity; lower connectivity nodes are more yellow, while higher connectivity nodes are more dark blue, and lines represent protein-protein associations. The results indicated that CCT2, IMP3, and IMP4 had the highest node connectivity, suggesting they may serve as network hub proteins in the RNA m7G methylation-regulated aging process.

[0169] Example 5: Binding level of RNA methyltransferase METTL1 to premature aging core genes

[0170] I. Experimental Methods

[0171] To further explore the role of NPT intervention in the regulation of RNA m6A and m7G in PS-NPs-induced premature aging of BEAS-2B cells and the core genes of premature aging, H-nPS and its corresponding NPT intervention group, which have more significant regulatory effects, were included for subsequent experiments.

[0172] 1. mRNA expression levels of core genes for premature aging in each group

[0173] Referring to step 4 of Example 2, cells from each group were collected and RNA was extracted using conventional methods, and cDNA was synthesized according to the reverse transcription procedure. Primers were designed to perform real-time quantitative PCR reactions targeting the GAPDH, CAT (NM_001752), CCT2 (NM_001198842), IMP3 (NM_018285), and IMP4 (NM_033416) genes. Based on the Ct value, the relative expression level of the control group genes was set as 1 as a reference, and the average relative content of the target genes was calculated. -ΔΔCt value.

[0174] 2. RIP-Q-PCR analysis

[0175] (1) Based on the results of the premature aging core genes obtained by bioinformatics analysis and RNA-seq screening, in order to ensure the reliability of the results and effectively avoid false negative interference, all the screened premature aging core genes were included in the subsequent in-depth RIP-Q-PCR analysis. The specific methods for cell lysis, DNA removal, immunoprecipitation (METTL1 primary antibody, etc.), balancing protein A / G magnetic beads, and RNA extraction were performed according to the RNA Immunoprecipitation Kit.

[0176] (2) Perform Q-PCR as described in Example 2. Specific primers are listed in Table 2. Store the Q-PCR amplified products in a -20 / -80°C freezer for subsequent gel electrophoresis verification. After collecting and compiling the Ct values, calculate the %Input content using the following Q-PCR calculation formula:

[0177] ΔCt normalized IP =Ct IP -(Ct Input -Log2(Input Dilution Factor)),

[0178] Table 2 Primer sequences for RIP-Q-PCR amplification

[0179]

[0180]

[0181] (Note: F represents the upstream sequence, and R represents the downstream primer)

[0182] (3) Perform gel electrophoresis detection of Q-PCR products according to conventional methods.

[0183] II. Test Results

[0184] 1. mRNA expression levels of core genes for premature aging in each group

[0185] like Figure 18 As shown, the mRNA expression levels of the core premature aging genes CAT, CCT2, IMP3, and IMP4 were significantly increased in the H-nPS treatment group, being 1.9, 1.3, 1.7, and 2.0 times higher than those in the control group, respectively, consistent with the gene expression level results in the GSE76925 dataset. This change was significantly reversed in the NPT intervention group, with reductions of 60.1%, 50.1%, 55.6%, and 62.2% compared to the PS-NPs treatment group, respectively (P<0.05).

[0186] 2. Binding status of premature aging core genes with RNA m7G methyltransferase METTL1

[0187] Figure 19 Gel electrophoresis results demonstrated that the PCR amplification primers were effective in detecting the four premature aging core genes using RIP-Q-PCR. Immunoprecipitation of the IP group samples showed clear target bands, proving that the RNA m7G methyltransferase METTL1 can bind to the premature aging core genes CAT, CCT2, IMP3, and IMP4.

[0188] Using immunoprecipitation, this experiment successfully isolated the RNA m7G methyltransferase METTL1 and its bound RNA. Primers for four core premature aging genes were then amplified using Q-PCR to explore the regulatory mechanism of METTL1 and these genes. Figure 20 As shown, the binding levels of METTL1 with the core premature aging genes CAT, CCT2, IMP3, and IMP4 were significantly increased in the PS-NPs-treated group, being 6.2, 2.9, 1.9, and 3.0 times higher than those in the control group, respectively. However, compared to the PS-NPs-treated group, the binding levels of CAT, CCT2, IMP3, and IMP4 with the core premature aging genes decreased by 48.4%, 57.9%, 57.6%, and 85.7% in the NPT intervention group, respectively (P<0.05). These results demonstrate that PS-NPs induce premature aging of cells, upregulating METTL1 expression and promoting RNA m7G methylation levels, and increasing the binding levels of METTL1 with the core premature aging genes CAT, CCT2, IMP3, and IMP4. NPT intervention can reverse this change and alleviate the degree of premature aging.

[0189] In addition, Pearson correlation analysis was performed on the mRNA expression levels of core premature aging genes and the binding levels of METTL1 in each group. The results are as follows: Figure 21In the nPS treatment group, the expression levels of CAT, CCT2, IMP3, and IMP4 mRNAs showed a positive correlation with their METTL1 binding levels. The correlation between CAT and IMP3 was statistically significant, with correlation coefficients of 0.868 (P = 0.025) and 0.881 (P = 0.020), respectively. In the NPT intervention group, the expression levels of CCT2, IMP3, and IMP4 mRNAs showed a negative correlation with their METTL1 binding levels. The correlation with IMP4 was statistically significant, with a correlation coefficient of -0.896 (P = 0.016). Conversely, the expression level of CAT mRNA in the NPT intervention group was positively correlated with its METTL1 binding level, with a correlation coefficient of 0.868 (P = 0.025).

[0190] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a new phytadiene in the preparation of a drug for preventing and treating premature aging of lung cells, characterized in that... The structural formula of the new phytadiene is shown in Formula I:

2. The application according to claim 1, characterized in that: The premature aging of lung cells mentioned above is caused by microplastics or nanoplastics.

3. The application according to claim 2, characterized in that: The nanoplastic is nano-polystyrene.

4. The application according to any one of claims 1-3, characterized in that: The effective concentration of the new phytadiene is 15 μmol / L.

5. The application of a new phytadiene in the preparation of a medicament for preventing and treating respiratory or pulmonary diseases caused by microplastics or nanoplastics, characterized in that... The structural formula of the new phytadiene is shown in Formula I:

6. The application according to claim 4, characterized in that: The respiratory or lung diseases mentioned include chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary fibrosis, or pneumonia.

7. The application of a novel phytadiene in a drug for preventing and treating cancers caused by microplastics or nanoplastics, characterized in that... The structural formula of the new phytadiene is shown in Formula I:

8. The application of a new phytadiene in the preparation of drugs for preventing and treating lung injury, characterized in that... The structural formula of the new phytadiene is shown in Formula I:

9. A drug for preventing and treating premature aging of lung cells, characterized in that... It contains neophytadiene.

10. A medicament for preventing and treating respiratory or lung diseases caused by microplastics or nanoplastics, characterized in that... It contains neophytadiene.

Images