Application of targeted Sirt1 medicine for preventing lung injury caused by nanoparticles

Abstract

The invention relates to the technical field of biological medicines, and discloses an application of a targeted Sirt1 medicine for preventing lung injury caused by nanoparticles, primary alveolar macrophages are cultured in vitro, cells are stimulated by using single-walled carbon nanotubes, and changes of Sirt1 protein and mRNA (messenger ribonucleic acid) levels thereof are analyzed by using Western Blotting and RT-qPCR (real-time quantitative polymerase chain reaction) technologies, so that the targeted Sirt1 medicine for preventing lung injury caused by nanoparticles is obtained. Meanwhile, SA-beta-gal staining and p16 immunofluorescent staining are adopted for evaluating the aging state of the AMs, so that data of Sirt1 in AMs aging regulation and control are obtained; and regulating and controlling AMs aging data based on the obtained Sirt1. According to the application of the targeted Sirt1 medicine for preventing the lung injury caused by the nanoparticles, primary alveolar macrophages are cultured in vitro, single-walled carbon nanotubes are used for stimulating cells, Western Blotting and RT-qPCR technologies are used for analyzing Sirt1 protein and mRNA level changes of the Sirt1 protein, SA-beta-gal dyeing and p16 immunofluorescence dyeing are adopted for evaluating the aging state of AMs, and through a series of experiments, the targeted Sirt1 medicine for preventing the lung injury caused by the nanoparticles can be used for treating the lung injury caused by the nanoparticles. The specific mechanism of Sirt1 in AMs aging regulation and control can be determined, and solid basic data can be provided for subsequent drug development.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically to the application of a targeted Sirt1 drug for preventing nanoparticle-induced lung injury. Background Art

[0002] Biomedicine is an interdisciplinary field that uses biotechnological means to develop new drugs, diagnostic tools, and treatment methods. It covers the whole process from basic scientific research to clinical application, including multiple branches such as molecular biology, cell biology, genetics, immunology, etc. However, traditional drug delivery systems often have problems such as low drug loading efficiency and uneven drug release, resulting in poor treatment effects. Moreover, existing treatment methods lack effective intervention means for nanoparticle-induced cell senescence and dysfunction. At the same time, the systemic administration method may lead to uneven distribution of drugs in lung tissue, affecting the treatment effect. Summary of the Invention

[0003] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Simplifications or omissions may be made in this part, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this part, the abstract, and the title. However, such simplifications or omissions shall not be used to limit the scope of the present invention.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] The application of a targeted Sirt1 drug for preventing nanoparticle-induced lung injury includes culturing primary alveolar macrophages in vitro, stimulating the cells with single-walled carbon nanotubes, analyzing the changes in Sirt1 protein and its mRNA levels using Western Blotting and RT-qPCR techniques, and simultaneously evaluating the senescence state of AMs by SA-β-gal staining and p16 immunofluorescence staining to obtain data on Sirt1 in regulating AMs senescence; based on the obtained data on Sirt1 regulating AMs senescence, virtual screening is carried out through computer-aided drug design software, selecting small molecule compounds with high affinity as candidate drugs, testing the effects of newly synthesized compounds on Sirt1 deacetylase activity in an in vitro cell model, and selecting Sirt1 activators; for the selected Sirt1 activators, they are applied to cell experiments through direct treatment to evaluate their effects on cell functions; based on the physiological characteristics of the target tissue, simulated physiological environment conditions are set, and the release rate of the drug in the drug delivery system with surface modification over time is monitored to evaluate the performance of the drug delivery system; cell experiments are used to evaluate the differences in the effects of Sirt1 activators in preventing nanoparticle-induced lung injury.

[0006] As a further aspect of the present invention: Primary alveolar macrophages are cultured in vitro, and the cells are stimulated with single-walled carbon nanotubes. The changes in the levels of Sirt1 protein and its mRNA are analyzed using Western Blotting and RT-qPCR techniques. At the same time, SA-β-gal staining and p16 immunofluorescence staining are used to evaluate the senescence state of AMs, so as to obtain data on the regulation of AMs senescence by Sirt1. The specific steps are as follows: Lung tissues are obtained from healthy donors, and primary AMs are isolated by digestion method; The isolated AMs are inoculated in high-glucose DMEM medium containing 10% fetal bovine serum and cultured under the conditions of 37°C and 5% CO2; After culturing to an appropriate density, the AMs are treated with 10 μg / ml SWCNT, and cell samples are collected at 6 hours, 12 hours, and 24 hours respectively; RT-qPCR technology is used to measure the level of Sirt1 mRNA; The amplification reaction is carried out using LC480SGIMM, and the conditions are 95°C for 5 minutes, followed by 45 cycles; The relative expression level of Sirt1 mRNA is calculated using the 2-ΔΔCt method, and the expression formula is:

[0007]

[0008] where, (ΔC t = C t,Sirt1 - C t,β-actin ), (ΔΔC t = ΔC t,实验组 - ΔC t,对照组 ), ΔC t is the difference in the C t value between the Sirt1 gene and the internal reference gene β-actin, and ΔΔC t is the difference in the ΔC t value between the experimental group and the control group. R is the relative expression level of Sirt1 mRNA;

[0009] The expression of Sirt1 protein is detected using Western Blotting technology; The signal is visualized using a Typhoon laser scanner, and the result is normalized to the signal intensity of the internal reference protein β-actin.

[0010] As a further aspect of the present invention: The RT-qPCR technology is used to measure the level of Sirt1 mRNA as follows: The operation includes extracting total RNA, reverse transcribing it into cDNA, and then performing quantitative PCR amplification; The primer design is as follows:

[0011] Sirt1 forward primer: 5'-TGGACAGGCTGTTGATGAGA-3'

[0012] Sirt1 reverse primer: 5'-TGTGGAAGGCTTCCTTCTTG-3'

[0013] Forward primer for the reference gene β-actin: 5'-CCTGTACGCCAACACAGTGC-3'

[0014] Reverse primer for the reference gene β-actin: 5'-ATACTCCTGCTTGCTGATCC-3.

[0015] As a further aspect of the present invention: The p16 immunofluorescence staining is used to evaluate the senescence state of AMs. The specific steps are as follows: The SA-β-gal staining is used to evaluate the senescence state of AMs. The fixed AMs are incubated overnight with the SA-β-gal staining solution containing 1 mg / ml X-gal as a substrate, and then the cells stained blue-green are observed under an optical microscope; The p16 immunofluorescence staining is used to further confirm the senescence of AMs. After the fixed AMs are permeabilized with 0.1% Triton X-100 for 30 minutes, blocked with normal goat serum for 30 minutes, incubated overnight with the anti-p16 antibody, and finally incubated with the secondary antibody conjugated with Alexa Fluor 594, and the cell nuclei are observed by DAPI staining; The proportion of senescent cells is calculated by an image analysis software, and the expression is:

[0016]

[0017] Wherein, P is the proportion of SA-β-gal positive cells, N SA-β-gal阳性细胞 is the number of SA-β-gal positive cells, N 总细胞数 is the total number of cells.

[0018] As a further aspect of the present invention: Based on the data of Sirt1 regulating the senescence of AMs obtained, virtual screening is carried out through computer-aided drug design software, and small molecule compounds with high affinity are selected as candidate drugs. The effects of the newly synthesized compounds on the deacetylase activity of Sirt1 are tested in an in vitro cell model, and Sirt1 activators are selected. The specific steps are as follows: Using the existing Sirt1 crystal structure PDB ID: 4I5I as a template, virtual screening is carried out through the molecular docking software AutoDock Vina; A small molecule library is selected from the chemical database ChEMBL, and the screening conditions are set; After preliminary screening, the top 10 small molecule compounds with the highest scores are selected as candidate drugs; The selected candidate drugs are added to the AMs cultured in vitro respectively, and different concentration gradients are set; The Sirt1 deacetylase activity detection kit is used to measure the deacetylase activity of Sirt1 in each group of cells; The Sirt1 activity is calculated by the change in fluorescence intensity, and the expression is:

[0019]

[0020] Among them, A is the relative activity of Sirt1 deacetylase, and F 实验组 is the fluorescence intensity of the experimental group, and F 空白对照组 is the fluorescence intensity of the blank control group, and F 阳性对照组 is the fluorescence intensity of the positive control group.

[0021] As a further aspect of the present invention: for the selected Sirt1 activator, it is applied to cell experiments by a direct treatment method to evaluate its effect on cell function. The specific steps are as follows: select Sirt1 activators with a concentration range from low to high to treat primary alveolar macrophages, and set the treatment time to 24 hours; after the treatment, use a CCK-8 kit to measure the cell viability to preliminarily judge the safety and effectiveness of the Sirt1 activator; evaluate the effect of the Sirt1 activator on cell function, and finally output a score. The expression is:

[0022]

[0023] Among them, E is the activator efficacy score, A is the cell viability adjustment coefficient, B is the cell cycle influencing factor, S is the relative expression level of Sirt1 protein, and C is the cell apoptosis index.

[0024] As a further aspect of the present invention: set the simulated physiological environment conditions based on the physiological characteristics of the target tissue, and monitor the release rate of the drug in the drug delivery system with surface modification over time to evaluate the performance of the drug delivery system. The specific steps are as follows: simulate the physiological environment of the lungs, set the simulated lung fluid as the release medium, select 37°C as the simulated body temperature, and maintain a constant temperature and an appropriate stirring speed through a thermostatic oscillator; place the drug delivery system with surface modification in a dialysis bag, and the cut-off molecular weight of the dialysis bag is less than the molecular weight of the Sirt1 activator. Immerse the dialysis bag in the preset simulated lung fluid, and regularly sample to measure the concentration of the Sirt1 activator in the release medium; use the ultraviolet-visible spectrophotometer method to measure the content of the Sirt1 activator in the sample; calculate the drug cumulative release percentage and the drug release rate.

[0025] As a further aspect of the present invention: the calculation of the drug cumulative release percentage and the drug release rate, the expression is:

[0026]

[0027] R = k·(1 - e -k·t );

[0028] Among them, CR% is the drug cumulative release percentage, and C t is the concentration of the Sirt1 activator released into the medium at time t, and Ctotal C is the total concentration of the Sirt1 activator in the initial drug-loading system, R is the drug release rate, k is the drug release rate constant, and t is the time;

[0029] The Higuchi equation was selected to establish the drug release kinetic model.

[0030] As a further aspect of the present invention: The effect difference of the Sirt1 activator in preventing nanoparticle-induced lung injury was evaluated by cell experiments. The specific steps were as follows: primary alveolar macrophages were used for treatment, the changes in Sirt1 expression levels and the senescence state of AMs were evaluated, and the levels of profibrotic factors were detected; a control group was set up, and the Sirt1 activator was simply given without using the drug-loading system to compare the effect differences of different administration methods; at different time points after administration, cell samples and culture media were collected, and the levels of pro-inflammatory factors and profibrotic factors were detected by ELISA to evaluate the changes in biomarkers of inflammation and fibrosis; the relative expression levels of inflammatory factors and fibrotic factors were calculated, and the relative expression levels of inflammatory factors and fibrotic factors under different administration methods were analyzed and compared by data analysis to evaluate the effect of the Sirt1 activator.

[0031] As a further aspect of the present invention: The relative expression levels of inflammatory factors and fibrotic factors under different administration methods were analyzed and compared by data analysis as follows: Through t-test statistical analysis, the relative expression levels of inflammatory factors and fibrotic factors under different administration methods were analyzed and compared, and the treatment effect index was calculated.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] By culturing primary alveolar macrophages in vitro and stimulating the cells with single-walled carbon nanotubes, a deep understanding of the role of Sirt1 in regulating the senescence of AMs was achieved. Western Blotting and RT-qPCR techniques were used to analyze the changes in the levels of Sirt1 protein and its mRNA, and SA-β-gal staining and p16 immunofluorescence staining were combined to evaluate the senescence state of AMs. This not only revealed the role of Sirt1 in the cellular response to nanoparticle stimulation but also provided a scientific basis for the selection of subsequent drug targets. For the selected Sirt1 activator, it was applied to cell experiments through direct treatment to comprehensively evaluate its impact on cell function. The method ensured the safety and effectiveness of the candidate drug, and through the multi-faceted evaluation of cell function, it provided reliable theoretical support for the next in vivo experiment, achieving a comprehensive and accurate functional evaluation of the Sirt1 activator and providing a solid foundation for drug optimization. A simulated environment was designed according to the special physiological conditions of the lungs to test the drug release behavior of the drug delivery system. In this way, the stability and effectiveness of the drug delivery system in a simulated human environment could be accurately evaluated to ensure that the drug could exert its maximum efficacy at the right time and place. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic flow chart of the application of a Sirt1-targeted drug for preventing nanoparticle-induced lung injury. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention will be made in conjunction with the accompanying drawings of the specification.

[0036] Many specific details are set forth in the following description to facilitate a thorough understanding of the present invention, but the present invention may be practiced in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0037] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or alternative embodiments that exclude each other.

[0038] Example 1

[0039] Please refer to Figure 1 , which is the first embodiment of the present invention. This embodiment provides an application of a Sirt1-targeted drug for preventing nanoparticle-induced lung injury, including

[0040] S1. Primary alveolar macrophages were cultured in vitro and stimulated with single-walled carbon nanotubes. Western Blotting and RT-qPCR techniques were used to analyze the changes in the levels of Sirt1 protein and its mRNA. At the same time, SA-β-gal staining and p16 immunofluorescence staining were used to evaluate the senescence state of AMs, so as to obtain data on the regulation of AMs senescence by Sirt1.

[0041] Specifically, primary alveolar macrophages were cultured in vitro and stimulated with single-walled carbon nanotubes. Western Blotting and RT-qPCR techniques were used to analyze the changes in the levels of Sirt1 protein and its mRNA. At the same time, SA-β-gal staining and p16 immunofluorescence staining were used to evaluate the senescence state of AMs, so as to obtain data on the regulation of AMs senescence by Sirt1. The specific steps were as follows: lung tissues were obtained from healthy donors, and primary AMs were isolated by digestion method; the isolated AMs were inoculated into high-glucose DMEM medium containing 10% fetal bovine serum and cultured under the conditions of 37 °C and 5% CO2; after culturing to an appropriate density, the AMs were treated with 10 μg / ml SWCNT, and cell samples were collected at 6 hours, 12 hours, and 24 hours respectively; RT-qPCR technique was used to determine the level of Sirt1 mRNA; the amplification reaction was carried out using LC480 SGIMM, and the conditions were 95 °C for 5 minutes, followed by 45 cycles; the relative expression level of Sirt1 mRNA was calculated using the 2-ΔΔCt method, and the expression formula was:

[0042]

[0043] Among them, (ΔC t = C t,Sirt1 - C t,β-actin ), (ΔΔC t = ΔC t,实验组 - ΔC t,对照组 ), ΔC t is the difference in the C t value between the Sirt1 gene and the internal reference gene β-actin, and ΔΔC t is the difference in the ΔC t value between the experimental group and the control group. R is the relative expression level of Sirt1 mRNA;

[0044] Western Blotting technique was used to detect the expression of Sirt1 protein; a Typhoon laser scanner was used to visualize the signals, and the results were normalized to the signal intensity of the internal reference protein β-actin;

[0045] RT-qPCR technique was used to determine the level of Sirt1 mRNA, specifically as follows:

[0046] The operations include extracting total RNA, reverse transcribing it into cDNA, and then performing quantitative PCR amplification;

[0047] The primers were designed as follows:

[0048] Forward primer for Sirt1: 5'-TGGACAGGCTGTTGATGAGA-3'

[0049] Reverse primer for Sirt1: 5'-TGTGGAAGGCTTCCTTCTTG-3'

[0050] Forward primer for the internal reference gene β-actin: 5'-CCTGTACGCCAACACAGTGC-3'

[0051] Reverse primer for the internal reference gene β-actin: 5'-ATACTCCTGCTTGCTGATCC-3;

[0052] The senescence state of AMs was evaluated by p16 immunofluorescence staining. The specific steps were as follows.

[0053] The senescence state of AMs was evaluated by SA-β-gal staining. The fixed AMs were incubated overnight with the SA-β-gal staining solution containing 1 mg / ml X-gal as the substrate, and then the cells stained blue-green were observed under an optical microscope. To further confirm the senescence of AMs by p16 immunofluorescence staining, after the fixed AMs were permeabilized with 0.1% Triton X-100 for 30 minutes, blocked with normal goat serum for 30 minutes, incubated overnight with the anti-p16 antibody, and finally incubated with the secondary antibody conjugated with Alexa Fluor 594, the cell nuclei were observed by DAPI staining. The proportion of senescent cells was calculated by an image analysis software, and the expression was:

[0054]

[0055] Among them, P is the proportion of SA-β-gal positive cells, N SA-β-gal阳性细胞 is the number of SA-β-gal positive cells, N 总细胞数 is the total number of cells.

[0056] It should be noted that during the entire experimental process, the culture conditions must be strictly controlled to ensure that the cells are in the best growth state. The cell samples were collected at three time points: 6 hours, 12 hours, and 24 hours, in order to comprehensively understand the changing trend of Sirt1 expression over time, so as to more accurately evaluate its role in regulating the senescence of AMs. β-actin was used as the internal reference gene because its expression is relatively stable under different conditions and can effectively correct the differences between samples. The role of Sirt1 in the senescence of AMs was comprehensively evaluated through a variety of technical means (Western Blotting, RT-qPCR, SA-β-gal staining, and p16 immunofluorescence staining), which increased the reliability and accuracy of the results.

[0057] S2. Based on the data obtained on the regulation of AMs senescence by Sirt1, virtual screening was carried out using computer-aided drug design software, and small molecule compounds with high affinity were selected as candidate drugs. The effects of the newly synthesized compounds on the deacetylase activity of Sirt1 were tested in an in vitro cell model, and Sirt1 activators were selected.

[0058] Specifically, based on the data obtained on the regulation of AMs senescence by Sirt1, virtual screening was carried out using computer-aided drug design software, and small molecule compounds with high affinity were selected as candidate drugs. The effects of the newly synthesized compounds on the deacetylase activity of Sirt1 were tested in an in vitro cell model, and Sirt1 activators were selected. The specific steps were as follows: Using the existing Sirt1 crystal structure PDB ID: 4I5I as a template, virtual screening was carried out using the molecular docking software AutoDock Vina; a small molecule library was selected from the chemical database ChEMBL, and the screening conditions were set; after preliminary screening, the top 10 small molecule compounds with the highest scores were selected as candidate drugs; the selected candidate drugs were added to the AMs cultured in vitro respectively, and different concentration gradients were set; an Sirt1 deacetylase activity detection kit was used to measure the deacetylase activity of Sirt1 in each group of cells; the Sirt1 activity was calculated through the change in fluorescence intensity, and the expression was:

[0059]

[0060] Among them, A is the relative activity of Sirt1 deacetylase, F 实验组 is the fluorescence intensity of the experimental group, F 空白对照组 is the fluorescence intensity of the blank control group, F 阳性对照组 is the fluorescence intensity of the positive control group.

[0061] It should be noted that through virtual screening using computer-aided drug design software, a large number of small molecule compounds can be quickly screened out, greatly reducing the experimental time and cost. Strict screening conditions are set, including but not limited to the affinity for the Sirt1 binding site, molecular weight, polar surface area, etc., to ensure that the screened compounds have high potential. Although virtual screening provides potential candidate drugs, they ultimately need to be verified in an in vitro cell model to confirm their effects on Sirt1 deacetylase activity.

[0062] S3. For the selected Sirt1 activators, apply them to cell experiments through direct treatment and evaluate their effects on cell functions.

[0063] Specifically, for the selected Sirt1 activators, apply them to cell experiments through direct treatment and evaluate their effects on cell functions. The specific steps are as follows: select primary alveolar macrophages and treat them with Sirt1 activators at a concentration range from low to high, and set the treatment time to 24 hours; after the treatment, use a CCK-8 kit to measure cell viability to preliminarily judge the safety and effectiveness of the Sirt1 activators; evaluate the effects of the Sirt1 activators on cell functions and finally output a score, with the expression:

[0064]

[0065] Among them, E is the activator efficacy score, A is the cell viability adjustment coefficient, B is the cell cycle influence factor, S is the relative expression level of Sirt1 protein, and C is the cell apoptosis index.

[0066] It should be noted that in this process, cells are treated with Sirt1 activators within a selected concentration range, and the effects of the activators on cell functions are comprehensively evaluated through various means such as using a CCK-8 kit to measure cell viability, flow cytometry to analyze cell cycle distribution and apoptosis. This method ensures the safety and effectiveness of the candidate drugs, and through the multi-faceted evaluation of cell functions, it provides reliable theoretical support for the next in vivo experiment, ultimately achieving a comprehensive and accurate functional evaluation of Sirt1 activators and providing a solid foundation for drug optimization.

[0067] S4. Set simulated physiological environment conditions based on the physiological characteristics of the target tissue and monitor the release rate of the drug in the drug delivery system with surface modification over time to evaluate the performance of the drug delivery system.

[0068] Specifically, the simulated physiological environment conditions are set based on the physiological characteristics of the target tissue, and the release rate of the drug in the drug delivery system after surface modification is monitored over time to evaluate the performance of the drug delivery system. The specific steps are as follows: simulate the physiological environment of the lungs, set the simulated lung fluid as the release medium, select 37 °C as the simulated body temperature, and maintain a constant temperature and an appropriate stirring speed through a thermostatic oscillator; place the surface-modified drug delivery system in a dialysis bag with a cut-off molecular weight smaller than that of the Sirt1 activator, immerse the dialysis bag in the preset simulated lung fluid, and regularly sample to measure the concentration of the Sirt1 activator in the release medium; use the ultraviolet-visible spectrophotometer method to measure the content of the Sirt1 activator in the sample; calculate the cumulative drug release percentage and the drug release rate;

[0069] Calculate the cumulative drug release percentage and the drug release rate, and the expressions are as follows:

[0070]

[0071] R = k·(1 - e -k·t );

[0072] where CR% is the cumulative drug release percentage, C t is the concentration of the Sirt1 activator released into the medium at time t, C total is the total concentration of the Sirt1 activator in the initial drug delivery system, R is the drug release rate, k is the drug release rate constant, and t is the time;

[0073] Select the Higuchi equation to establish the drug release kinetic model.

[0074] It should be noted that setting the simulated lung fluid as the release medium and selecting 37 °C as the simulated body temperature ensure that the experimental conditions are as close as possible to the actual human situation. The cut-off molecular weight of the dialysis bag is smaller than that of the Sirt1 activator, ensuring that the drug will not pass through the dialysis bag into the release medium, so as to accurately measure the drug release rate. Through the calculation of the cumulative release percentage and the drug release rate, the performance of the drug delivery system can be quantitatively evaluated, providing data support for subsequent optimization.

[0075] S5. Evaluate the difference in the effect of the Sirt1 activator in preventing nanoparticle-induced lung injury using cell experiments.

[0076] Specifically, cell experiments were used to evaluate the differential effects of Sirt1 activators in preventing nanoparticle-induced lung injury. The specific steps were as follows: primary alveolar macrophages were used for treatment, the changes in Sirt1 expression levels and the senescence status of AMs were evaluated, and the levels of profibrotic factors were detected; a control group was set up, where only the Sirt1 activator was given without using a drug delivery system to compare the differential effects of different administration methods; at different time points after administration, cell samples and culture media were collected, and the levels of pro-inflammatory factors and profibrotic factors were detected by ELISA to evaluate the changes in biomarkers of inflammation and fibrosis; the relative expression levels of inflammatory factors and fibrogenic factors were calculated, and the relative expression levels of inflammatory factors and fibrogenic factors under different administration methods were analyzed and compared by data analysis to evaluate the effects of Sirt1 activators; the relative expression levels of inflammatory factors and fibrogenic factors under different administration methods were compared, and data analysis and comparison were carried out as follows: through t-test statistical analysis, the relative expression levels of inflammatory factors and fibrogenic factors under different administration methods were compared, and the treatment effect index was calculated.

[0077] It should be noted that through the analysis of the changes in Sirt1 expression levels and the senescence status of AMs, as well as the detection of the levels of profibrotic factors, researchers can gain an in-depth understanding of how Sirt1 activators affect cell function, especially the mechanism of action in response to nanoparticle stimulation. This step provides a theoretical basis for subsequent optimization of drug design. At different time points after administration, cell samples and culture media were collected, and the levels of pro-inflammatory factors and profibrotic factors were detected by ELISA to evaluate the changes in biomarkers of inflammation and fibrosis. The quantitative analysis method allows researchers to objectively measure the effects of drugs, avoiding biases caused by subjective judgment. This step, through systematic cell experiments and rigorous statistical analysis, not only verifies the potential preventive effect of Sirt1 activators on nanoparticle-induced lung injury but also clarifies the efficacy differences under different administration methods. The methodological rigor enhances the credibility of the research results and also provides a valuable reference framework for future research.

[0078] In summary, by culturing primary alveolar macrophages in vitro and stimulating the cells with single-walled carbon nanotubes, a deep understanding of the role of Sirt1 in regulating the senescence of AMs was achieved. The changes in the levels of Sirt1 protein and its mRNA were analyzed using Western Blotting and RT-qPCR techniques, and the senescence status of AMs was evaluated by combining SA-β-gal staining and p16 immunofluorescence staining. This not only revealed the role of Sirt1 in the cellular response to nanoparticle stimulation but also provided a scientific basis for the selection of subsequent drug targets. For the selected Sirt1 activator, it was applied to cell experiments through direct treatment to comprehensively evaluate its effects on cell functions. The method ensured the safety and effectiveness of the candidate drug, and through the multi-faceted evaluation of cell functions, it provided reliable theoretical support for the next in vivo experiment, achieving a comprehensive and accurate functional evaluation of the Sirt1 activator and providing a solid foundation for drug optimization. A simulated environment was designed according to the special physiological conditions of the lungs to test the drug release behavior of the drug delivery system. In this way, the drug delivery system in the simulated human body can be accurately evaluated.

[0079] Example 2

[0080] Please refer to Table 1, which is the second embodiment of the present invention. To further verify the technical solution of the present invention, experimental simulation data on the application of a targeted Sirt1 drug for preventing nanoparticle-induced lung injury are given.

[0081] To verify the effectiveness of a novel Sirt1 activator in preventing nanoparticle-induced lung injury, a systematic in vitro experiment was conducted. First, lung tissues were obtained from healthy donors, and primary alveolar macrophages were isolated by digestion method. These cells were seeded in high-glucose DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2 until an appropriate density was reached. Then, the AMs were treated with 10 μg / ml of single-walled carbon nanotubes (SWCNT), and cell samples were collected at 6 hours, 12 hours, and 24 hours respectively.

[0082] The changes in the level of Sirt1 mRNA were determined using RT-qPCR technique. The specific operations included extracting total RNA, reverse-transcribing it into cDNA, and then performing quantitative PCR amplification. Specific primers were used to amplify the Sirt1 gene and the internal reference gene β-actin, and the amplification reaction was carried out using an LC480 SGIMM instrument. The conditions were set as pre-denaturation at 95°C for 5 minutes, followed by 45 cycles, and the relative expression level of Sirt1 mRNA was calculated using the 2-ΔΔCt method.

[0083] The senescence status of AMs was evaluated by SA-β-gal staining and p16 immunofluorescence staining. The fixed AMs were incubated overnight with SA-β-gal staining solution containing 1 mg / ml X-gal as a substrate, and the cells with blue-green staining were observed under an optical microscope. In addition, after permeabilization with 0.1% Triton X-100 and blocking with normal goat serum, they were incubated overnight with anti-p16 antibody, and finally incubated with a secondary antibody conjugated to Alexa Fluor 594. The cell nuclei were observed by DAPI staining. The proportion of SA-β-gal positive cells was calculated by image analysis software to evaluate cell senescence.

[0084] Virtual screening was performed using the computer-aided drug design software AutoDock Vina, and small molecule compounds with high affinity were selected as candidate drugs. After preliminary screening, the top 10 small molecule compounds with the highest scores were selected as candidate drugs, and the effects of the newly synthesized compounds on Sirt1 deacetylase activity were tested in an in vitro cell model. The Sirt1 deacetylase activity detection kit was used to measure the Sirt1 deacetylase activity in each group of cells, and the Sirt1 activity was calculated based on the change in fluorescence intensity.

[0085] The selected Sirt1 activators were directly applied to cell experiments to evaluate their effects on cell function. Primary alveolar macrophages were treated with Sirt1 activators at a concentration range from low to high, and the treatment time was set to 24 hours. After the treatment, the CCK-8 kit was used to measure cell viability to evaluate the safety and effectiveness of the Sirt1 activators. At the same time, flow cytometry was used to analyze the cell cycle distribution and apoptosis to further understand the effects of the activators on the cell life process.

[0086] The physiological environment of the lung was simulated, and simulated lung fluid was set as the release medium. 37°C was selected as the simulated body temperature, and the constant temperature and appropriate stirring speed were maintained by a thermostatic oscillator. The surface-modified drug delivery system was placed in a dialysis bag and immersed in the preset simulated lung fluid. Samples were taken regularly to measure the concentration of the Sirt1 activator in the release medium. The content of the Sirt1 activator in the samples was measured by the ultraviolet-visible spectrophotometer method, and the cumulative drug release percentage and drug release rate were calculated. A drug release kinetic model was established according to the Higuchi equation to ensure that the drug could exert the maximum efficacy at the right time and place.

[0087] Finally, cell experiments were used to evaluate the differences in the effects of Sirt1 activators in preventing nanoparticle-induced lung injury. A control group was set up, that is, only Sirt1 activators were given without using a drug delivery system, to compare the differences in the effects of different administration methods. At different time points after administration, cell samples and culture media were collected, and the levels of pro-inflammatory factors and profibrotic factors were detected by ELISA to evaluate the changes in biomarkers of inflammation and fibrosis. The relative expression levels of inflammatory factors and fibrotic factors were calculated, and the relative expression levels of these factors under different administration methods were analyzed and compared to evaluate the effects of Sirt1 activators.

[0088] The experimental data table is as follows:

[0089] Table 1 Comparative data table of the prior art and the present invention

[0090]

[0091]

[0092] Through the comparative analysis of the table content, it can be clearly seen that the present invention shows significant advantages and creative effects in many aspects:

[0093] In the present invention, the relative expression level of Sirt1 mRNA increased by 30%, which indicates that the novel Sirt1 activator can more effectively promote the expression of the Sirt1 gene, enhance the resistance of cells to nanoparticle-induced damage, not only proves the effectiveness of the activator, but also implies that it may delay cell senescence by upregulating Sirt1, thus providing a new treatment approach.

[0094] In the present invention, the proportion of SA-β-gal positive cells decreased by 5%, indicating that the novel activator can effectively reduce cell senescence. A lower proportion of senescent cells means that cell viability is better maintained, which is crucial for the prevention and treatment of lung injury caused by nanoparticles.

[0095] In the present invention, the relative expression levels of pro-inflammatory factors and profibrotic factors decreased by about 42% and 33% respectively, far lower than the prior art level, indicating that the novel Sirt1 activator can not only inhibit the inflammatory response, but also significantly reduce the risk of fibrosis, which is of great significance for reducing lung injury.

[0096] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. An application of a Sirt1-targeted drug for preventing lung injury caused by nanoparticles, characterized in that: include, Primary alveolar macrophages were cultured in vitro and stimulated with single-walled carbon nanotubes. Western Blotting and RT-qPCR were used to analyze the changes in Sirt1 protein and mRNA levels. SA-β-gal staining and p16 immunofluorescence staining were used to evaluate the aging state of AMs to obtain data on Sirt1 in regulating AMs aging. Based on the data obtained on Sirt1 regulation of AMs aging, virtual screening was performed using computer-aided drug design software to select small molecule compounds with high affinity as candidate drugs. The effects of the newly synthesized compounds on Sirt1 deacetylase activity were tested in in vitro cell models to select Sirt1 activators. The selected Sirt1 activators were directly applied to cell experiments to evaluate their effects on cell function. Simulate physiological environmental conditions based on the physiological characteristics of the target tissue, monitor the release rate of drugs in the surface-modified drug-carrying system over time, and evaluate the performance of the drug delivery system; Cell experiments were used to evaluate the differences in the effects of Sirt1 activators in preventing nanoparticle-induced lung injury.

2. The use of a Sirt1-targeted drug for preventing nanoparticle-induced lung injury according to claim 1, characterized in that: The method involves culturing primary alveolar macrophages in vitro, stimulating the cells with single-walled carbon nanotubes, analyzing the changes in Sirt1 protein and mRNA levels using Western Blotting and RT-qPCR techniques, and evaluating the aging state of AMs using SA-β-gal staining and p16 immunofluorescence staining to obtain data on Sirt1 in regulating AMs aging. The specific steps are: Lung tissue was obtained from healthy donors, and primary AMs were isolated by digestion; The isolated AMs were inoculated in high-glucose DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2; After culturing to an appropriate density, AMs were treated with 10 μg / ml SWCNTs, and cell samples were collected at 6 h, 12 h, and 24 h; Sirt1 mRNA levels were measured using RT-qPCR technology; Amplification reaction was performed using LC480SGIMM at 95°C for 5 min, followed by 45 cycles; The 2-ΔΔCt method was used to calculate the relative expression of Sirt1 mRNA, and the expression was: Among them, (ΔC t =C t,Sirt1 -C t,β-actin ), (ΔΔC t =ΔC t,实验组 -ΔC t,对照组 ), ΔC t is the C sequence of Sirt1 gene and internal reference gene β-actin t The difference between the values, ΔΔC t is the ΔC between the experimental group and the control group t The difference between the values, R is the relative expression of Sirt1 mRNA; Western Blotting technique was used to detect Sirt1 protein expression; Signals were visualized using a Typhoon laser scanner and the results were normalized to the signal intensity of the endogenous reference protein β-actin.

3. The use of a Sirt1-targeted drug for preventing nanoparticle-induced lung injury according to claim 1, characterized in that: The Sirt1 mRNA level was determined using RT-qPCR technology, as follows: The procedure includes extracting total RNA, reverse transcribing it into cDNA, and then performing quantitative PCR amplification; Primers were designed as follows: Sirt1 forward primer: 5'-TGGACAGGCTGTTGATGAGA-3' Sirt1 reverse primer: 5'-TGTGGAAGGCTTCCTTCTTG-3' Internal reference gene β-actin forward primer: 5'-CCTGTACGCCAACACAGTGC-3' Internal reference gene β-actin reverse primer: 5'-ATACTCCTGCTTGCTGATCC-3'.

4. The use of a Sirt1-targeted drug for preventing nanoparticle-induced lung injury according to claim 1, characterized in that: The p16 immunofluorescence staining is used to evaluate the aging state of AMs, and the specific steps are as follows: SA-β-gal staining was used to assess the senescence status of AMs. Fixed AMs were incubated overnight with SA-β-gal staining solution containing 1 mg / ml X-gal as a substrate, and the blue-green stained cells were observed under a light microscope; p16 immunofluorescence staining was used to further confirm AMs senescence. Fixed AMs were permeabilized with 0.1% Triton X-100 for 30 min, blocked with normal goat serum for 30 min, incubated with anti-p16 antibody overnight, and finally incubated with Alexa Fluor 594-conjugated secondary antibody. DAPI staining was used to observe the cell nucleus. The proportion of senescent cells was calculated by image analysis software, and the expression was: Among them, P is the proportion of SA-β-gal positive cells, N SA-β-gal阳性细胞 N is the number of SA-β-gal positive cells. 总细胞数 is the total number of cells.

5. The use of a Sirt1-targeted drug for preventing nanoparticle-induced lung injury according to claim 1, characterized in that: Based on the obtained data on Sirt1 regulating AMs aging, virtual screening is performed using computer-aided drug design software to select small molecule compounds with high affinity as candidate drugs, and the effects of the newly synthesized compounds on Sirt1 deacetylase activity are tested in an in vitro cell model to select Sirt1 activators. The specific steps are: Using the existing Sirt1 crystal structure PDB ID: 4I5I as a template, virtual screening was performed using the molecular docking software AutoDock Vina; Select a small molecule library from the chemical database ChEMBL and set the screening conditions; After the initial screening, the top 10 small molecule compounds with the highest scores were selected as drug candidates; The selected candidate drugs were added to AMs cultured in vitro and different concentration gradients were set; The Sirt1 deacetylase activity detection kit was used to measure the deacetylase activity of Sirt1 in each group of cells; Sirt1 activity was calculated by the change in fluorescence intensity, and the expression was: Among them, A is the relative activity of Sirt1 deacetylase, F 实验组 is the fluorescence intensity of the experimental group, F 空白对照组 is the fluorescence intensity of the blank control group, F 阳性对照组 is the fluorescence intensity of the positive control group.

6. The use of a Sirt1-targeted drug for preventing nanoparticle-induced lung injury according to claim 1, characterized in that: The selected Sirt1 activator is applied to a cell experiment by direct treatment to evaluate its effect on cell function. The specific steps are: Sirt1 activators were selected to treat primary alveolar macrophages at concentrations ranging from low to high, and the treatment time was set to 24 hours; After treatment, the cell survival rate was measured using a CCK-8 kit to preliminarily determine the safety and effectiveness of the Sirt1 activator; Evaluate the effect of Sirt1 activators on cell function and finally output a score expressed as: Among them, E is the activator efficacy score, A is the cell survival rate adjustment coefficient, B is the cell cycle influencing factor, S is the relative expression of Sirt1 protein, and C is the cell apoptosis index.

7. The use of a Sirt1-targeted drug for preventing lung injury caused by nanoparticles according to claim 1, characterized in that: The method of setting simulated physiological environment conditions based on the physiological characteristics of the target tissue, monitoring the release rate of the drug in the surface-modified drug-carrying system over time, and evaluating the performance of the drug delivery system comprises the following specific steps: To simulate the physiological environment of the lungs, simulated lung fluid was used as the release medium, 37°C was selected as the simulated body temperature, and a constant temperature and appropriate stirring speed were maintained by a thermostatic oscillator; The surface-modified drug-carrying system is placed in a dialysis bag, the molecular weight cutoff of which is smaller than the molecular weight of the Sirt1 activator, and the dialysis bag is immersed in a preset simulated lung fluid, and samples are taken regularly to determine the concentration of the Sirt1 activator in the release medium; The Sirt1 activator content in the samples was determined using a UV-visible spectrophotometer method; The cumulative drug release percentage and drug release rate were calculated.

8. The use of a Sirt1-targeted drug for preventing nanoparticle-induced lung injury according to claim 7, characterized in that: The calculation of the cumulative drug release percentage and drug release rate is expressed as: R=k·(1-e -k·t ); Among them, CR% is the cumulative release percentage of the drug, C t is the concentration of Sirt1 activator released into the medium at time t, C total is the total concentration of Sirt1 activator in the initial drug-loading system, R is the drug release rate, k is the drug release rate constant, and t is the time; The Higuchi equation was selected to establish the drug release kinetics model.

9. The use of a Sirt1-targeted drug for preventing lung injury caused by nanoparticles according to claim 1, characterized in that: The cell experiment is used to evaluate the difference in the effect of Sirt1 activators in preventing lung injury caused by nanoparticles, and the specific steps are as follows: Primary alveolar macrophages were used for treatment to evaluate the changes in Sirt1 expression levels and the senescence status of AMs, and to detect the levels of profibrotic factors; A control group was set up to simply administer the Sirt1 activator without using a drug delivery system to compare the effects of different administration methods; At different time points after administration, cell samples and culture medium were collected, and the levels of pro-inflammatory and pro-fibrotic factors were detected by ELISA to evaluate the changes in biomarkers of inflammation and fibrosis; The relative expression levels of inflammatory factors and fibrosis factors were calculated, and data analysis and comparison were performed on the relative expression levels of inflammatory factors and fibrosis factors under different administration methods to evaluate the effect of Sirt1 activator.

10. The use of a Sirt1-targeted drug for preventing nanoparticle-induced lung injury according to claim 9, characterized in that: The relative expression levels of inflammatory factors and fibrosis factors under different administration methods were compared, and data analysis and comparison were performed, as follows: The relative expression levels of inflammatory factors and fibrosis factors under different administration methods were compared by t-test statistical analysis, and the treatment effect index was calculated.

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