Method for evaluating anti-inflammatory effect of SAMC on M1 polarization of macrophages

Abstract

The application discloses an anti-inflammatory effect evaluation method based on SAMC regulation of M1 polarization of macrophages, relates to the technical field of biological medicine, and comprises the following steps: surface modification is performed on a nano-carrier complex, a molecule specifically combined with a M1 type macrophage marker is added, and a targeted modified nanoparticle is generated; the targeted modified nanoparticle is added into RAW264.7 macrophages, a flow cytometry is used to detect the binding condition of the targeted modified nanoparticle and the RAW264.7 macrophages, and targeted binding data is generated; a macrophage culture system is constructed according to the RAW264.7 macrophages, and a drug is treated through the targeted modified nanoparticle, real-time monitoring of the change of cell survival condition, inflammatory factor level and cell polarization state is performed, and drug reaction data is generated. The application reduces side effects and adverse reactions of a traditional drug administration mode.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization. Background Technology

[0002] With the continuous advancement of biomedical technology, anti-inflammatory methods that regulate the immune system have gradually become an important research direction in the field of immuno-inflammatory therapy. Macrophages, as key immune cells, play a crucial role in maintaining immune homeostasis and defending against pathogens. M1 macrophages, in particular, play a key role in combating pathogens in acute inflammatory responses by secreting large amounts of inflammatory factors, such as TNF-α and IL-6. In recent years, researchers have increasingly focused on the regulation of macrophage polarization, especially the inhibition of M1 macrophages, as an effective anti-inflammatory strategy. SAMC (S-allyl thiocysteine), an active ingredient in garlic, as a water-soluble organic sulfur compound, has shown significant anti-inflammatory, antioxidant, and immune polarization regulation potential in multiple studies. SAMC has important clinical application value in reducing inflammatory responses and improving immune function.

[0003] However, SAMCs still face several challenges in clinical application. First, SAMCs exhibit metabolic instability in vivo, particularly their rapid conversion into volatile metabolites in the liver and kidneys, resulting in an extremely short half-life. These volatile metabolites are excreted through the lungs and skin, producing a noticeable "garlic odor" that severely impacts patient compliance. Second, SAMCs have low bioavailability, especially after oral administration, with extremely low plasma concentrations, far lower than other garlic derivatives (such as SAC). This often prevents the achievement of effective therapeutic concentrations at conventional dosages. Finally, SAMCs lack targeting specificity, failing to effectively target M1 macrophages at the core of inflammation. This leads to non-specific drug distribution in the body, diluted efficacy, and the need for high doses to achieve results, increasing the potential risk of systemic toxicity. Therefore, despite some technological advancements, significant challenges remain in addressing the metabolic stability, bioavailability, and targeting issues of SAMCs. Summary of the Invention

[0004] Given the challenges mentioned above, the use of nanomaterials as delivery carriers is a clear technological necessity: Physical barrier protection: Nanocarriers (such as PLGA) can effectively encapsulate SAMC, establishing a physical barrier to block its contact with plasma enzymes and metabolic enzymes, significantly prolonging its half-life in blood circulation, and masking the generation of its odorous metabolites.

[0005] Achieving controlled release and enhanced efficacy: Through the sustained-release effect of nanostructures, the drug can be released steadily in the body, avoiding the "peak and trough" phenomenon. At the same time, by utilizing the size advantage of nanoparticles, the uptake efficiency of SAMCs by cells can be enhanced through endocytosis.

[0006] Precise Targeting and Regulation: The extremely high surface area to volume ratio of nanomaterials makes functional modification possible. By modifying the surface of the carrier with M1 macrophage-specific ligands, drugs can be precisely delivered to inflammatory targets, achieving "lesion enrichment." This enhances anti-inflammatory efficacy, downregulates M1 polarization levels, and minimizes the impact on non-target tissues.

[0007] In view of the aforementioned existing problems, the present invention is proposed.

[0008] Therefore, this invention provides a method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization, which solves the problems of poor SAMC targeting and low bioavailability.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention provides a method for evaluating the anti-inflammatory effect of SAMC-based regulation of macrophage M1 polarization. The method includes: mixing SAMC (a garlic active polysulfide) with an anti-IL-6 peptide to generate a mixed solution, and then encapsulating the mixture to form a nanocarrier complex; modifying the surface of the nanocarrier complex by adding molecules that specifically bind to M1 macrophage markers to generate targeted modified nanoparticles; adding the targeted modified nanoparticles to RAW264.7 macrophages, and using flow cytometry to detect the binding of the targeted modified nanoparticles to RAW264.7 macrophages, generating targeted binding data; and then, based on the RAW... 264.7 Macrophages: A macrophage culture system was constructed, and drug treatment was performed using targeted modified nanoparticles. Cell survival, inflammatory factor levels, and changes in cell polarization were monitored in real time to generate drug response data. Based on the drug response data, the combined treatment of SAMCs and anti-IL-6 peptides on the comprehensive regulatory effects on M1 macrophage polarization, M2 macrophage polarization, and inflammatory factors was analyzed, generating comprehensive regulatory data. Cellular immunofluorescence was used to assess the cell function of the macrophage culture system, analyzing the effect of combined SAMC and anti-IL-6 peptide treatment on cell function and generating protective effect data.

[0010] As a preferred embodiment of the anti-inflammatory effect evaluation method based on SAMC-regulated macrophage M1 polarization described in this invention, the specific steps for generating the mixed solution are as follows: Garlic active polysulfide SAMC and anti-IL-6 peptide were dissolved at predetermined solution concentrations to form SAMC solution and anti-IL-6 peptide solution, respectively. SAMC solution and anti-IL-6 peptide solution were mixed in a volume ratio using ultrasonic treatment to generate a mixed solution.

[0011] As a preferred embodiment of the anti-inflammatory effect evaluation method based on SAMC-regulated macrophage M1 polarization described in this invention, the specific steps for generating the nanocarrier complex are as follows: The mixed solution is encapsulated by polymer nanoparticle carriers to form a preliminary drug carrier complex; Solvent evaporation was used to remove the solvent from the initial drug carrier complex and promote polymer self-assembly to generate a nanoparticle complex.

[0012] As a preferred embodiment of the anti-inflammatory effect evaluation method based on SAMC-regulated macrophage M1 polarization described in this invention, the specific steps for generating targeted modified nanoparticles are as follows: The unencapsulated material in the nanocarrier complex is removed to generate a purified complex. The purified complex was exposed to a solution containing M1 macrophage markers to generate a targeted labeling solution, which then bound to the surface of the nanocarrier complex to generate a modified complex. The modified complex was stabilized by ultrasonic treatment to generate a stabilized post-modified complex. The particle size, stability, and targeting of the stabilized post-modified complex were evaluated using dynamic light scattering and surface potential analysis methods to generate targeted modified nanoparticles.

[0013] As a preferred embodiment of the anti-inflammatory effect evaluation method based on SAMC-regulated macrophage M1 polarization described in this invention, the specific steps for generating targeted binding data are as follows: The targeted modified nanoparticles were purified to generate purified targeted modified nanoparticles. The purified targeted modified nanoparticles were added to the RAW264.7 macrophage culture medium and incubated according to the predetermined nanoparticle concentration and incubation time to generate preliminary binding. Based on the preliminary binding results, RAW264.7 macrophages were analyzed by flow cytometry. By detecting the fluorescent labeling on the surface of RAW264.7 macrophages, the binding efficiency of purified targeted modified nanoparticles to RAW264.7 macrophages was evaluated, and targeted binding data were generated.

[0014] As a preferred embodiment of the method for evaluating the anti-inflammatory effect of macrophage M1 polarization based on SAMC regulation described in this invention, the macrophage culture system is constructed by seeding RAW264.7 macrophages in a culture dish containing RPMI1640 medium, fetal bovine serum and antibiotics, and culturing them in an incubator to monitor cell growth and maintain cell density.

[0015] As a preferred embodiment of the anti-inflammatory effect evaluation method based on SAMC-regulated macrophage M1 polarization described in this invention, the method involves: adding targeted modified nanoparticles to the macrophage culture system, treating the macrophages with a set drug concentration, and generating a treated macrophage culture system. Cell viability was used to monitor cell survival in real time, and the levels of inflammatory factors in the cell supernatant were detected by ELISA. Flow cytometry was used to detect the expression changes of M1 and M2 macrophage markers in the treated macrophage culture system and to analyze cell polarization status. Principal component analysis was used to comprehensively analyze cell survival, inflammatory factor levels, and cell polarization status to generate drug response data.

[0016] As a preferred embodiment of the anti-inflammatory effect evaluation method based on SAMC-regulated macrophage M1 polarization according to the present invention, the specific steps for generating comprehensive regulatory data are as follows: Flow cytometry was used to analyze changes in M1 and M2 macrophage markers in drug response data, generating M1 macrophage polarization data. The ELISA method was used to detect changes in the levels of inflammatory factors in cell supernatant in drug response data, generating data on the regulation of inflammatory factors. Path analysis was used to comprehensively analyze M1 macrophage polarization data and inflammatory factor regulation data to generate comprehensive regulatory data, which was used to characterize the inhibitory effect on M1 macrophage polarization and the promoting effect on M2 macrophage polarization.

[0017] As a preferred embodiment of the anti-inflammatory effect evaluation method based on SAMC-regulated macrophage M1 polarization described in this invention, the specific steps for generating protective effect data are as follows: Macrophage culture system samples that have undergone combined treatment with SAMC and anti-IL-6 peptide were selected from the comprehensive regulatory data and fixed to generate treated fixed cell samples. Using cell immunofluorescence, specific antibodies against M1 macrophage markers were stained to generate labeled cell samples. The expression of the labeled cell samples was then observed using a fluorescence microscope to generate fluorescence microscopic images. Based on fluorescence microscopy images and comprehensive regulatory data, the effects of combined treatment of SAMCs and anti-IL-6 peptide on maintaining macrophage structural stability and immune function integrity were analyzed, and protective effect data were generated.

[0018] As a preferred embodiment of the anti-inflammatory effect evaluation method based on SAMC-regulated macrophage M1 polarization described in this invention, the specific steps for generating the fluorescence micrograph are as follows: M1 macrophage markers were collected from the treated macrophage culture system and impurities in the culture medium were removed by washing with PBS to generate macrophage samples to be stained. The macrophage samples to be stained were exposed to a solution of M1 macrophage marker-specific antibody and incubated at the set concentration and time to generate labeled cell samples. The expression of M1 macrophage markers was detected by observing labeled cell samples using a fluorescence microscope, and fluorescence micrographs were generated.

[0019] The beneficial effects of this invention are as follows: by mixing garlic active polysulfide (SAMC) with anti-IL-6 peptide at a predetermined concentration and encapsulating the solution with a nanocarrier to generate a nanocarrier complex, the bioavailability of SAMC and anti-IL-6 peptide is improved, the drug is protected from degradation, and the drug concentration at the target site is enhanced, thereby improving the anti-inflammatory effect. At the same time, by improving the stability and sustained release of the drug, the side effects and adverse reactions of traditional drug delivery methods are reduced, ensuring the precise delivery of the drug. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a flowchart of a method for evaluating the anti-inflammatory effect of SAMC-based macrophage M1 polarization.

[0022] Figure 2 A flowchart for the preparation of targeted modified nanoparticles.

[0023] Figure 3 The flowchart is for verifying the target binding efficiency.

[0024] Figure 4 A flowchart for a comprehensive evaluation of anti-inflammatory and polarization regulation effects.

[0025] Figure 5 This is a comprehensive correlation diagram between inflammatory factors and M2 biomarkers.

[0026] Figure 6 This is a diagram showing the expression of macrophage polarization markers. Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0030] Reference Figures 1-6 This is one embodiment of the present invention, which provides a method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization, comprising the following steps: S1. SAMC, an active polysulfide of garlic, is mixed with an anti-IL-6 peptide to generate a mixed solution, which is then encapsulated to generate a nanocarrier complex.

[0031] S1.1 Dissolve garlic active polysulfide (SAMC) and anti-IL-6 peptide at predetermined solution concentrations to form SAMC solution and anti-IL-6 peptide solution, respectively.

[0032] Specifically, the garlic active polysulfide SAMC (S-allyl thiocysteine) is dissolved in a solvent at a predetermined solution concentration to ensure that SAMC is completely dissolved and forms a homogeneous SAMC solution; the anti-IL-6 peptide is also dissolved in a solvent at a predetermined solution concentration to ensure that the anti-IL-6 peptide is also completely dissolved and forms a homogeneous anti-IL-6 peptide solution.

[0033] It should also be noted that the predetermined solution concentrations are mainly based on the effective concentration ranges of SAMC and anti-IL-6 peptide in regulating macrophage polarization and anti-inflammatory responses in previous cell experiments, combined with the encapsulation efficiency of the drug in the nanocarrier and subsequent release behavior, to ensure that the efficacy is guaranteed without inducing toxic reactions; for example, the concentration of SAMC solution is generally controlled in the range of 0.1 to 5 mg / mL, and the concentration of anti-IL-6 peptide solution is generally controlled in the range of 10 to 100 μg / mL, in order to achieve synergistic anti-inflammatory effects and stability requirements in subsequent mixing, encapsulation and cell processing.

[0034] It should be noted that the anti-IL-6 peptide is a polypeptide with IL-6 inhibitory activity, and the amino acid sequence of the anti-IL-6 peptide is shown in SEQ ID NO:1; for example, the amino acid sequence of SEQ ID NO:1 is: MKYLYLKDPNKQFNKQ, where M is methionine, K is lysine, Y is tyrosine, L is leucine, D is aspartic acid, P is proline, N is asparagine, Q is glutamine, and F is phenylalanine. The amino acid sequence of SEQ ID NO:1 forms a structurally stable core through hydrophobic amino acids and achieves binding with IL-6 through charged and polar amino acids, thereby exerting an inhibitory effect.

[0035] S1.2. The SAMC solution and the anti-IL-6 peptide solution are mixed in a volume ratio by ultrasonic treatment to generate a mixed solution.

[0036] Specifically, at room temperature, the SAMC solution and the anti-IL-6 peptide solution are slowly added to a clean, sterile glass container at a volume ratio of 1:1. The container is then placed in an ultrasonic homogenizer under constant temperature control, for example, with the ultrasonic frequency controlled at 20–40 kHz and the power controlled at 100–200 W. The mixture is continuously ultrasonicated for 3–5 minutes in intermittent working mode to allow the SAMC solution and the anti-IL-6 peptide solution to form a stable mixture in a uniformly dispersed state. Throughout the mixing process, the pH value of the liquid is maintained between 6.5 and 7.4 to prevent the structural inactivation of SAMC and the anti-IL-6 peptide, ultimately producing a mixed solution.

[0037] S1.3. The mixed solution is encapsulated by polymer nanoparticle carriers to form a preliminary drug carrier complex.

[0038] Specifically, at room temperature, the mixed solution is slowly added dropwise to a polylactic acid-glycolic acid copolymer solution dissolved in a volatile organic solvent, dichloromethane, with a mass concentration controlled at 10–20 mg / mL. During the dropwise addition, the magnetic stirring speed is maintained at 600–800 rpm, allowing the mixed solution to form an emulsion-like dispersed phase in the polylactic acid-glycolic acid copolymer solution. Pre-emulsification is carried out under high-speed homogenization conditions (12000–16000 rpm, 2–4 minutes), followed by continued stirring to form a uniformly encapsulated nanoemulsion. This achieves sufficient contact and encapsulation between the mixed solution and the polylactic acid-glycolic acid copolymer, forming a preliminary drug carrier complex.

[0039] S1.4. Solvent removal is performed on the preliminary drug carrier complex using solvent evaporation, which promotes polymer self-assembly and generates nanoparticle complex.

[0040] Specifically, the preliminary drug carrier complex is placed under reduced pressure rotary evaporation conditions, and dichloromethane in the preliminary drug carrier complex is slowly evaporated in a constant temperature water bath, allowing the solvent to gradually evaporate and promoting the self-assembly of polylactic acid-glycolic acid copolymer in the aqueous phase, gradually forming a stable polymer film structure and encapsulating the contents of the mixed solution. Subsequently, magnetic stirring is continued at room temperature to completely remove residual organic solvent, generating a nanoparticle complex.

[0041] S2. Surface modification of the nanocarrier complex is performed by adding molecules that specifically bind to M1 macrophage markers to generate targeted modified nanoparticles.

[0042] S2.1 Remove the unencapsulated material from the nanocarrier complex to generate a purified complex.

[0043] Specifically, the nanocarrier complex is transferred to a centrifuge tube, and centrifugation conditions are set to 4°C, 12000 rpm, and continuous centrifugation for 15 minutes. Centrifugation separates the unencapsulated material from the nanocarrier complex. The unencapsulated material is removed by discarding the supernatant. An equal volume of sterile deionized water is added to the precipitate for resuspension. The centrifugation and resuspension washing operations are repeated three times to ensure that the unencapsulated material remaining on the surface of the nanocarrier complex is fully removed, resulting in a purified complex with uniform particle size, clean surface, and stable encapsulation structure.

[0044] It should also be noted that setting the centrifugation conditions to 4°C, 12,000 rpm, and continuous centrifugation for 15 minutes is based on the requirement for efficient sedimentation of particles with a diameter of approximately 100–200 nm during conventional nanoparticle purification processes. At the same time, low temperature can reduce the risk of degradation of active ingredients.

[0045] S2.2. The purified complex is exposed to a solution containing M1 macrophage markers to generate a targeted labeling solution, which then binds to the surface of the nanocarrier complex to generate a modified complex.

[0046] Specifically, the purified complex is slowly added to a solution containing M1 macrophage markers, for example, by gently shaking the solution at 4°C for 6 hours to fully expose the surface of the purified complex to the solution containing M1 macrophage markers, thus generating a targeted labeling solution. EDCs such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and NHS (N-hydroxysuccinimide) are added to a pH 7.4 PBS buffer as covalent coupling agents to promote amide bonding between the amino groups in the M1 macrophage markers and the carboxyl groups on the surface of the purified complex, forming a stable link and generating a modified complex.

[0047] S2.3. The modified complex is stabilized by ultrasonic treatment to generate a stabilized modified complex. The particle size, stability and targeting of the stabilized modified complex are evaluated by dynamic light scattering and surface potential analysis methods to generate targeted modified nanoparticles.

[0048] Specifically, the modified complex is placed in an ultrasonic treatment device, for example, with a treatment temperature of 25℃ and a power of 200W, and ultrasonication is performed continuously for 3 minutes. This allows the modified complex to be uniformly dispersed under ultrasonic action, promoting the uniform binding of surface-targeting molecules and generating a stable modified complex. After sampling and diluting the stable modified complex to an appropriate concentration, the particle size distribution of the stable modified complex is determined using dynamic light scattering. For example, the sample volume is 1-2 mL, and the diluted concentration is usually 0.1-1 mg / mL to ensure moderate signal intensity and avoid particle aggregation. Simultaneously, the zeta potential is measured using surface potential analysis. To further evaluate the colloidal stability and dispersibility of the stable modified complex, the average particle size and particle size distribution range (e.g., 50-150 nm, and PDI ≤ 0) of the stable modified complex are measured using dynamic light scattering. 2) For example, if the measured particle size is mainly concentrated within 100 nm and the distribution is uniform without multiple peaks, it can be judged that it has good nanoparticle size characteristics; use a surface potential analyzer to measure the Zeta potential value. For example, if the absolute value of the Zeta potential is greater than ±30 mV, it indicates that the electrostatic repulsion between particles is strong, which helps the suspension stability and indicates that the colloidal stability is good; by comparing the particle size and Zeta potential of the stabilized modified complex and the nanocarrier complex, if the particle size of the stabilized modified complex is slightly increased compared with the nanocarrier complex, and the Zeta potential changes in the direction of the potential characteristics of the target molecule, and at the same time, combined with the electrical characteristics of the targeted labeling solution and the targeted binding data, it can be judged whether the molecules bound to the M1 macrophage marker are successfully bound on the surface of the stabilized modified complex, thereby confirming that it has targeting properties and generating targeted modified nanoparticles.

[0049] It should also be noted that setting the processing temperature to 25℃ and the power to 200W, and continuing the ultrasound for 3 minutes, is to effectively disperse the nanoparticles through the action of ultrasound without causing drug degradation, thereby ensuring the uniformity and stability of the surface modification of the nanocarrier composite.

[0050] It should be noted that by targeting and modifying nanoparticles, drugs can be precisely delivered to M1 macrophages, thereby enhancing anti-inflammatory effects and reducing non-specific effects on healthy tissues. The targeting and bioavailability of the drug are improved, the anti-inflammatory effect is enhanced, and the side effects of traditional drug delivery methods, such as excessive immune response or effects on non-target tissues, are greatly reduced.

[0051] S3. Targeted modified nanoparticles were added to RAW264.7 macrophages, and the binding of the targeted modified nanoparticles to RAW264.7 macrophages was detected by flow cytometry to generate targeted binding data.

[0052] S3.1 Purify the targeted modified nanoparticles to generate purified targeted modified nanoparticles.

[0053] Specifically, the targeted modified nanoparticles were transferred to centrifuge tubes, resuspended in deionized water, and centrifuged at 4°C and 12,000 rpm for 15 minutes. The supernatant was discarded to remove unbound target molecules, and fresh deionized water was added to resuspend the precipitate. This centrifugation and resuspension process was repeated three times to ensure the removal of all unbound target molecules. After each centrifugation, the physical homogeneity of the precipitate was initially assessed by visually observing its uniform dispersion. Dynamic light scattering analysis was then used to determine the particle size distribution of the precipitate, which was concentrated in the nanoscale range (e.g., within 100 nm) to assess particle size consistency. Simultaneously, the zeta potential was measured to evaluate the colloidal stability by checking whether it remained stable above ±30 mV. The morphology and surface distribution of the targeted modified nanoparticles were observed using transmission electron microscopy to confirm the presence of significant aggregation or uneven surface modification. Significant aggregation refers to the adhesion or agglomeration of nanoparticles, while uneven surface modification refers to the uneven distribution of target molecules on the surface of nanoparticles, which may lead to inconsistent anti-inflammatory effects. Based on this comprehensive assessment, the precipitate was determined to be uniformly targeted modified nanoparticles, resulting in purified targeted modified nanoparticles.

[0054] S3.2. The purified targeted modified nanoparticles were added to the RAW264.7 macrophage culture medium and incubated according to the set nanoparticle concentration and incubation time to generate preliminary binding.

[0055] Specifically, purified targeted modified nanoparticles were added to a predetermined nanoparticle concentration of 10 μg / mL into a cell culture medium containing RAW264.7 macrophages. For example, the nanoparticles were incubated at 37°C in a 5% CO2 incubator for 6 hours. By adjusting the nanoparticle concentration and incubation time, it was ensured that the purified targeted modified nanoparticles and RAW264.7 macrophages were in full contact and completed the initial binding reaction. After incubation, the cells in the culture medium were collected to generate the initial binding status.

[0056] It should also be noted that the nanoparticle concentration and incubation time were set based on the growth characteristics of RAW264.7 macrophages and the optimal conditions for nanoparticle binding to cells. The nanoparticle concentration was set at 10 μg / mL to ensure sufficient targeted modification of the nanoparticles to bind to the macrophage surface, while avoiding the toxic effects of excessively high concentrations on cells. The incubation time was 6 hours, which is based on the time range within which the nanoparticles fully contact and bind to the cells, ensuring that the best binding effect is achieved without causing excessive stress to the cells.

[0057] S3.3. Based on the preliminary binding results, RAW264.7 macrophages were analyzed using flow cytometry. By detecting the fluorescent labeling on the surface of RAW264.7 macrophages, the binding efficiency of purified targeted modified nanoparticles to RAW264.7 macrophages was evaluated, and targeted binding data were generated.

[0058] Specifically, based on the initial binding results, RAW264.7 macrophages incubated with purified targeted nanoparticles were washed three times with pre-chilled phosphate buffer to remove unbound purified targeted nanoparticles. Then, antibodies labeled with fluorescent probes against M1 macrophage surface markers were added, and the cells were incubated at 4°C in the dark for 30 minutes to allow the M1 macrophage surface markers on the RAW264.7 macrophages to fully bind with the fluorescent antibody. After incubation, the RAW264.7 macrophages were washed again with pre-chilled phosphate buffer to remove free fluorescent antibody, thus binding the RAW264.7 macrophages to the fluorescent antibody. Cells were fixed, and the fixed RAW264.7 macrophages were collected as samples for testing. The processed RAW264.7 macrophages were placed in a flow cytometer for detection. By exciting fluorescence signals at specific wavelengths, the intensity of the fluorescent label on the surface of each RAW264.7 macrophage was measured. For example, when using FITC labeling, the excitation wavelength was 495 nm and the emission wavelength was 519 nm; when using PE labeling, the excitation wavelength was 496 nm and the emission wavelength was 578 nm. This reflects the binding efficiency of the purified targeted modified nanoparticles to RAW264.7 macrophages, generating targeted binding data.

[0059] It should also be noted that pre-cooled phosphate buffer refers to phosphate buffer that has been cooled to about 4°C before use. It is mainly composed of phosphate, sodium chloride, and potassium chloride. It plays a role in maintaining the pH stability and osmotic pressure of the extracellular environment. In cell experiments, it is widely used to wash cells to remove suspended impurities or unbound substances. Pre-cooling the phosphate buffer can reduce the metabolic activity of cells during the washing process, slow down the cell's response to external stimuli, reduce protein degradation and exocytosis, and avoid non-specific changes in the binding state between targeted modified nanoparticles and RAW264.7 macrophages, thereby improving the accuracy and stability of subsequent binding efficiency detection.

[0060] S4. Based on RAW264.7 macrophages, a macrophage culture system was constructed, and drug treatment was performed by targeted modification of nanoparticles. Cell survival, inflammatory factor levels and changes in cell polarization were monitored in real time to generate drug response data.

[0061] S4.1 The macrophage culture system was constructed by seeding RAW264.7 macrophages into culture dishes containing RPMI 1640 medium, fetal bovine serum and antibiotics, and culturing them in an incubator to monitor cell growth and maintain cell density.

[0062] Specifically, the macrophage culture system involves seeding RAW264.7 macrophages into culture dishes containing RPMI 1640 medium, fetal bovine serum, and antibiotics. For example, 10% fetal bovine serum and 1% antibiotics (penicillin / streptomycin) are added to the RPMI 1640 medium in the culture dishes, and the medium is sterilized before use. The seeded RAW264.7 macrophages are then cultured in the dishes at a rate of 2 × 10⁶ cells / day. 5 Cells were evenly distributed at a density of 100 cells / mL and placed in an incubator maintained at a temperature of 37°C, a CO2 concentration of 5%, and a humidity of 95% to provide a suitable growth environment. During cell culture, cell growth was observed under a microscope every 24 hours to check for morphological changes, and cell counts were performed using a hemocytometer to ensure that the cell density was maintained within a reasonable range (e.g., 1×10⁻⁶). 6 Up to 1×10 7 (cells / mL) When the cells reach, for example, 80%-90% confluence, they are passaged to maintain their health and growth activity, providing stable cellular conditions for subsequent drug treatment and experimental analysis.

[0063] It should also be stated that the reasonable range (e.g., 1×10) 6 Up to 1×10 7 The cell density (cells / mL) value should be adjusted according to the characteristics of the cell line, the experimental design, and the required experimental time; generally, the cell density is maintained at 1×10⁻⁶. 6 Up to 1×10 7 A cell / mL ratio ensures healthy cell growth and prevents overcrowding, thereby maintaining cell viability and stability.

[0064] S4.2 Add the targeted modified nanoparticles to the macrophage culture system, treat with drugs according to the set drug concentration, and generate the treated macrophage culture system.

[0065] Specifically, targeted modified nanoparticles were added to the macrophage culture system. First, the targeted modified nanoparticle solution was added to culture dishes already inoculated with RAW264.7 macrophages, and treatment was performed according to the set drug concentration. During drug treatment, the dosing time and treatment interval were strictly controlled, and an appropriate treatment cycle was selected according to the experimental design (e.g., treatment every 48 hours for 3 consecutive days). During drug administration, it was ensured that the drug was evenly distributed and fully contacted with the RAW264.7 macrophages. After treatment, the culture dishes were returned to a 37°C incubator for further culture, and the cell growth status and morphological changes were observed. Flow cytometry was used to monitor the effects of the drug on cell survival, inflammatory factor levels, and cell polarization status, thus generating the treated macrophage culture system.

[0066] It should also be noted that the drug concentration is mainly based on previous experimental studies and the pharmacological properties of the drug. In experiments, the drug concentration range is usually set on the basis of being non-toxic to RAW264.7 macrophages and being able to effectively induce cellular responses and changes in inflammatory factors. The drug concentration is generally set between 10 μg / mL and 100 μg / mL to ensure that the drug has sufficient biological activity while avoiding the toxic effects of high concentrations of the drug on cells.

[0067] S4.3. Cell viability assay is used to monitor cell survival in real time, and the level of inflammatory factors in cell supernatant is detected by ELISA.

[0068] Specifically, using the cell viability assay, cell viability assay reagents were prepared and added to the treated macrophage culture system according to the reagent instructions. The culture dishes were gently shaken and incubated in an incubator at 37°C for a certain period (e.g., 1-4 hours) to allow the products of cell metabolism to react with the reagents. The absorbance was measured using an ELISA reader to monitor the survival of RAW264.7 macrophages in real time. Simultaneously, the cell culture supernatant was collected and diluted. Following the ELISA kit instructions, the supernatant was added to the wells of an ELISA plate, followed by the addition of capture antibody, enzyme-labeled secondary antibody, and substrate solution. After a complete reaction, the absorbance was measured using an ELISA reader to quantitatively analyze changes in the levels of inflammatory factors (such as TNF-α and IL-6) in the cell supernatant.

[0069] S4.4. Using flow cytometry, the expression changes of M1 and M2 macrophage markers in the treated macrophage culture system were detected to analyze cell polarization status.

[0070] Specifically, M1 and M2 macrophages were collected from the treated macrophage culture system. Impurities in the culture medium were removed by washing with PBS. The M1 and M2 macrophages were then resuspended in flow cytometry buffer, and specific primary antibodies were added for incubation against M1 macrophage markers (such as CD86 and iNOS) and M2 macrophage markers, respectively. After incubation, secondary antibodies were used for staining to ensure sufficient binding of the markers to the cell surface antibodies. The cells were then washed again to remove unbound secondary antibodies and resuspended in the buffer required for flow cytometry analysis. The fluorescence intensity of each cell surface marker was detected using flow cytometry to quantitatively analyze the expression changes of M1 and M2 macrophage markers. Analysis of the flow cytometry data was used to assess the changes in the polarization state of M1 and M2 macrophage markers, further determining the effect of drug treatment on cell polarization.

[0071] S4.5. Principal component analysis is used to comprehensively analyze cell survival, inflammatory factor levels, and cell polarization status to generate drug response data.

[0072] Specifically, cells from the treated macrophage culture system were collected, and cell viability data were obtained using cell activity assays to record cell viability. The levels of inflammatory factors, including TNF-α and IL-6, in the cell supernatant were detected using ELISA to obtain inflammatory factor concentration data. Cell polarization status was analyzed using flow cytometry to detect changes in the expression of M1 macrophage markers, generating cell polarization status data. Principal component analysis was used to assess the relationship between cell viability, TNF-α and IL-6 concentrations, and the expression of M1 macrophage markers. Regression analysis was applied to quantify the specific contributions of cell viability, inflammatory factor levels, and M1 macrophage polarization markers to the anti-inflammatory effect of the drug. Hypothesis testing was used to compare the differences in cell viability, inflammatory factor levels, and cell polarization status between the treated group and the control group, generating drug response data.

[0073] S5. Based on the drug response data, analyze the comprehensive regulatory effects of combined treatment of SAMC and anti-IL-6 peptide on M1 macrophage polarization, M2 macrophage polarization and inflammatory factors, and generate comprehensive regulatory data.

[0074] S5.1. Using flow cytometry, analyze the changes in M1 and M2 macrophage markers in drug response data to generate M1 macrophage polarization data.

[0075] Specifically, using flow cytometry, cells processed from the drug response data were collected and centrifuged to remove the culture medium. The cells were resuspended in PBS buffer and primary antibodies against M1 macrophage markers (such as CD86 and iNOS) and M2 macrophage markers were added, for example, at a concentration of 4°C for 30 minutes to 1 hour. After incubation, the cells were washed with PBS buffer to remove unbound antibodies, and fluorescently labeled secondary antibodies were added, followed by further incubation for 30 minutes to 1 hour. After incubation, the cells were washed again with PBS buffer to remove unbound secondary antibodies. The fluorescence intensity of M1 macrophage markers on the cell surface was detected by flow cytometry to obtain the expression levels of M1 and M2 macrophage markers. Based on the flow cytometry results, the changes in M1 and M2 macrophage markers were analyzed to generate M1 macrophage polarization data.

[0076] S5.2. Using the ELISA method, detect changes in the levels of inflammatory factors in the cell supernatant of the drug response data to generate inflammatory factor regulation data.

[0077] Specifically, using ELISA, the supernatant of the processed macrophage culture system was collected from the drug response data and processed according to the ELISA kit instructions. The supernatant was diluted to an appropriate concentration and added to the wells of an ELISA plate, along with capture antibodies against inflammatory factors (such as TNF-α, IL-6, and IL-1β), and incubated. After incubation, the wells were thoroughly washed with washing buffer to remove unbound antibodies, and HRP-labeled secondary antibody was added. Incubation continued under suitable conditions to allow binding with the capture antibodies. Substrate solution was added, and a colorimetric reaction was initiated. The absorbance was measured at a specified wavelength using a microplate reader, and the concentration changes of inflammatory factors were recorded. By comparing with a standard curve, the concentration changes of inflammatory factors in the drug response data were calculated, and the inflammatory factor concentrations were calculated using the following expression: ; in, This indicates the concentration of inflammatory factors in the sample. Indicates the absorbance of the sample. The absorbance of the blank control is indicated. This represents the absorbance at the standard curve point. Indicates the concentration of the standard sample. Indicates the index of the sample. Indicates a standard sample; The changes in the concentration of inflammatory factors are calculated using the following expression: ; in, This indicates changes in the concentration of inflammatory factors. Indicates the baseline concentration before treatment; The standard curve is plotted by measuring the signal intensity of standard samples with known concentrations. The signal intensity of the processed sample is used to determine the concentration of inflammatory factors by comparing with the standard curve, thus generating data on the regulation of inflammatory factors. Inflammatory factors promote or inhibit the occurrence of inflammatory responses by regulating immune responses and cell signal transduction, thereby affecting the body's immune balance and disease progression.

[0078] S5.3. Using the path analysis method, the polarization data of M1 macrophages and the regulation data of inflammatory factors are comprehensively analyzed to generate comprehensive regulatory data. The comprehensive regulatory data is used to characterize the inhibitory effect on M1 macrophage polarization and the promoting effect on M2 macrophage polarization.

[0079] Specifically, flow cytometry was used to obtain M1 macrophage polarization data, reflecting changes in the expression of M1 macrophage markers after treatment. ELISA was used to obtain inflammatory factor regulation data, showing changes in inflammatory factor levels in the cell supernatant. After combining the M1 macrophage polarization data with the inflammatory factor regulation data, pathway analysis was used to collect M1 macrophage polarization data (e.g., expression of CD86 and iNOS markers) and inflammatory factor data (e.g., concentrations of TNF-α and IL-6). The causal relationship between M1 macrophage polarization and inflammatory factor data was analyzed to assess the direct and indirect effects of drug treatment on M1 macrophage polarization and inflammatory factor regulation. Path coefficient analysis quantified the interaction between M1 macrophage polarization and inflammatory factor levels, and statistical tests determined the overall regulatory effect of the drug on the relationship between M1 macrophage polarization and inflammatory factor data, generating comprehensive regulatory data.

[0080] Figure 5 The study presented a comprehensive correlation between TNF-α and IL-6 levels and the expression of M2 macrophage markers in different treatment groups. The x-axis represents TNF-α concentration, the y-axis represents IL-6 concentration, and the bubble size represents the M2 composite index composed of the mean expression of Arg-1 and CD206. The control group was located in the high TNF-α and high IL-6 region with small bubbles, indicating a high level of inflammation and a low degree of M2 polarization. Both the SAMC alone treatment group and the anti-IL-6 peptide alone treatment group shifted towards low TNF-α and low IL-6, and the bubbles increased in size, indicating that the release of inflammatory factors was inhibited and accompanied by an increase in the expression of M2 markers. The SAMC and anti-IL-6 peptide combined treatment group was located in the lowest TNF-α and IL-6 region with the largest bubbles, indicating that the combined treatment had the most significant promoting effect on Arg-1 and CD206 expression while reducing the level of pro-inflammatory factors. This demonstrates that the combined treatment not only inhibited the inflammatory response but also promoted the transformation of macrophages to the M2 anti-inflammatory phenotype.

[0081] S6. Using cell immunofluorescence, the function of macrophage culture system was evaluated, and the effect of combined treatment of SAMC and anti-IL-6 peptide on cell function was analyzed to generate protective effect data.

[0082] S6.1 Select macrophage culture system samples that have been treated with SAMC and anti-IL-6 peptide in combination from the comprehensive regulation data, and fix them to generate treated fixed cell samples.

[0083] Specifically, macrophage culture system samples treated with SAMC and anti-IL-6 peptide were selected from the comprehensive regulatory data, and the macrophage culture system samples were fixed with, for example, 4% paraformaldehyde solution to ensure that the cell structure in the macrophage culture system samples was maintained. After the fixation treatment, the macrophage culture system samples were washed with phosphate buffer to remove fixative residue. The fixed macrophage culture system samples were transferred to centrifuge tubes, and PBS (phosphate buffer) was added for further washing to remove impurities and fixative residue, resulting in treated fixed cell samples.

[0084] S6.2. Using cell immunofluorescence, staining is performed on specific antibodies against M1 macrophage markers to generate labeled cell samples. The expression of labeled cell samples is then observed using a fluorescence microscope to generate fluorescence microscopic images.

[0085] S6.2.1 Collect the M1 macrophage markers from the treated macrophage culture system and wash with PBS to remove impurities from the culture medium to generate macrophage samples to be stained.

[0086] Specifically, after collecting the M1 macrophage markers from the processed macrophage culture system, the cells were washed with pre-cooled phosphate buffer to remove impurities and unbound substances from the culture medium, ensuring that the cell surface markers were clearly visible. After washing, the cells were washed again with PBS buffer to ensure that all residues in the culture medium were completely removed, ensuring the purity of the cell sample and generating macrophage samples ready for staining and analysis.

[0087] S6.2.2 Expose the macrophage sample to be stained to a solution of M1 macrophage marker-specific antibody and incubate it according to the set concentration and time to generate labeled cell samples.

[0088] Specifically, the macrophage samples to be stained are transferred to centrifuge tubes, and M1 macrophage marker-specific antibody solution is added, ensuring the solution concentration is appropriate. After gently shaking the centrifuge tubes, they are incubated at, for example, 4°C for a specified time to ensure that the M1 macrophage marker-specific antibody fully binds to the macrophage surface markers. After incubation, the cells are washed with PBS buffer to remove unbound antibodies, generating labeled cell samples. This ensures that the macrophage surface markers are fully labeled with the specific antibody, preparing for subsequent analysis.

[0089] It should also be noted that the concentration and time settings are primarily based on the optimal binding concentration and incubation time of the M1 macrophage marker-specific antibody, and are usually optimized according to recommended values ​​and the antibody-cell binding effect in previous experiments. For example, the concentration of the marker-specific antibody is generally set between 1-5 μg / mL to ensure sufficient binding of the antibody to the cell surface marker without excessive antibody waste or non-specific binding. For example, the incubation time is usually controlled between 30 minutes and 2 hours. Too long an incubation time may lead to increased background signal, while too short an incubation time may result in insufficient antibody binding, affecting the accuracy of the experimental results. In practice, the specific settings of concentration and time will be fine-tuned according to cell type, antibody affinity, and experimental objectives to ensure labeling effect and signal intensity.

[0090] S6.2.3 Observe the labeled cell samples using a fluorescence microscope, detect the expression of M1 macrophage markers, and generate a fluorescence micrograph.

[0091] Specifically, the labeled cell samples were transferred onto a glass slide and sealed with a mounting medium. The sealed labeled cell samples were then observed under a fluorescence microscope. The settings of the fluorescence microscope were adjusted, and the excitation light source and filter were selected to detect the specific fluorescence signal of the M1 macrophage markers. During observation, the distribution and intensity of the fluorescent markers on the surface of the macrophages were recorded to generate a fluorescence micrograph. The focus and light intensity of the microscope were adjusted to ensure that the image was clear and could accurately show the expression of the labeled cells.

[0092] S6.3. Based on fluorescence microscopy images and comprehensive regulatory data, analyze the effects of combined treatment of SAMC and anti-IL-6 peptide on maintaining macrophage structural stability and immune function integrity, and generate protective effect data.

[0093] Specifically, image analysis software was used to quantitatively analyze fluorescence microscopy images to assess the stability of macrophage structures, including cell morphology, nuclear morphology, and cytoplasmic integrity, generating image analysis results. By integrating M1 macrophage polarization data and inflammatory factor regulation data from the comprehensive regulatory data, the effects of combined treatment of SAMCs and anti-IL-6 peptides on macrophage immune function, especially the inhibitory effect on inflammatory responses, were analyzed. Combining image analysis results and regulatory data, the protective effect of combined treatment of SAMCs and anti-IL-6 peptides in maintaining macrophage structural stability and immune function integrity was evaluated, with a focus on improving cell function and reducing inflammatory responses, generating protective effect data.

[0094] Figure 6 The study showed changes in the expression of macrophage polarization markers in different treatment groups. iNOS and CD86 are markers of M1 macrophages, while Arg-1 and CD206 are markers of M2 macrophages. In the control group, iNOS and CD86 expression was high, while Arg-1 and CD206 expression was low, indicating that macrophages were mainly in a pro-inflammatory phenotype. In the SAMC-only treatment group and the anti-IL-6 peptide-only treatment group, the expression of iNOS and CD86 decreased, while the expression of Arg-1 and CD206 increased, indicating that the single treatment could regulate the polarization direction of macrophages. In the SAMC-anti-IL-6 peptide combined treatment group, the expression of iNOS and CD86 decreased the most, while the expression of Arg-1 and CD206 increased the most, indicating that the combined treatment had a stronger inhibitory effect on M1 macrophage polarization and a stronger promoting effect on M2 macrophage polarization, thus forming a complete anti-inflammatory regulatory loop of "inhibiting M1 polarization and promoting M2 polarization". For example, in the implementation, the characterization of the promoting effect of M2 macrophage polarization can be achieved by detecting the expression levels of Arg-1 and CD206; when the expression levels of Arg-1 and CD206 in the SAMC and anti-IL-6 peptide combined treatment group are higher than those in the control group, the SAMC alone treatment group and the anti-IL-6 peptide alone treatment group, it can be determined that the SAMC and anti-IL-6 peptide combined treatment has a promoting effect on M2 macrophage polarization.

[0095] In summary, this invention achieves stable delivery of SAMC and anti-IL-6 peptide by mixing garlic active polysulfide (SAMC) with an anti-IL-6 peptide at a predetermined concentration and encapsulating them with a nanocarrier to generate a targeted nanocarrier complex. This improves drug bioavailability and targeted enrichment, while reducing the impact of drug degradation and non-specific distribution. Furthermore, the evaluation of M2 macrophage polarization further demonstrates that the combined treatment of SAMC and anti-IL-6 peptide can increase the expression levels of Arg-1 and CD206, indicating that the combined treatment of SAMC and anti-IL-6 peptide can inhibit M1 macrophage polarization while also promoting M2 macrophage polarization, thus forming a complete anti-inflammatory regulatory logic of "inhibiting M1 polarization—promoting M2 polarization."

[0096] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization, characterized in that: include, (1) Carrier preparation: SAMC of garlic active polysulfide was mixed with anti-IL-6 peptide to generate a mixed solution, and then encapsulated to generate a nano-carrier complex; (2) Targeted modification: The surface of the nanocarrier complex is modified by adding molecules that specifically bind to M1 macrophage markers to generate targeted modified nanoparticles; (3) Targeting verification: The targeted modified nanoparticles were co-incubated with RAW264.7 macrophages, and the binding of the targeted modified nanoparticles with RAW264.7 macrophages was detected by flow cytometry to generate targeted binding data; (4) Drug efficacy evaluation: Based on RAW264.7 macrophages, a macrophage culture system was constructed, and drug treatment was carried out by targeted modification of nanoparticles. Cell survival, inflammatory factor levels and changes in cell polarization status were monitored in real time to generate drug response data. (5) Comprehensive effect analysis: Based on the drug response data, the combined treatment of SAMC and anti-IL-6 peptide was analyzed to comprehensively regulate the polarization of M1 macrophages, the polarization of M2 macrophages and inflammatory factors, and comprehensive regulatory data were generated. Cell immunofluorescence was used to evaluate the cell function of the macrophage culture system, and the effect of combined treatment of SAMC and anti-IL-6 peptide on cell function was analyzed to generate protective effect data.

2. The method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization as described in claim 1, characterized in that: The specific steps for generating the mixed solution are as follows. Garlic active polysulfide SAMC and anti-IL-6 peptide were dissolved at predetermined solution concentrations to form SAMC solution and anti-IL-6 peptide solution, respectively. SAMC solution and anti-IL-6 peptide solution were mixed in a volume ratio using ultrasonic treatment to generate a mixed solution.

3. The method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization as described in claim 1, characterized in that: The specific steps for generating the nanocarrier complex are as follows. The mixed solution is encapsulated by polymer nanoparticle carriers to form a preliminary drug carrier complex; Solvent evaporation was used to remove the solvent from the initial drug carrier complex and promote polymer self-assembly to generate a nanoparticle complex.

4. The method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization as described in claim 1, characterized in that: The specific steps for generating the targeted modified nanoparticles are as follows. The unencapsulated material in the nanocarrier complex is removed to generate a purified complex. The purified complex was exposed to a solution containing M1 macrophage markers to generate a targeted labeling solution, which then bound to the surface of the nanocarrier complex to generate a modified complex. The modified complex was stabilized by ultrasonic treatment to generate a stabilized post-modified complex. The particle size, stability, and targeting of the stabilized post-modified complex were evaluated using dynamic light scattering and surface potential analysis methods to generate targeted modified nanoparticles.

5. The method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization as described in claim 1, characterized in that: The specific steps for generating the targeted binding data are as follows. The targeted modified nanoparticles were purified to generate purified targeted modified nanoparticles. The purified targeted modified nanoparticles were added to the RAW264.7 macrophage culture medium and incubated according to the predetermined nanoparticle concentration and incubation time to generate preliminary binding. Based on the preliminary binding results, RAW264.7 macrophages were analyzed by flow cytometry. By detecting the fluorescent labeling on the surface of RAW264.7 macrophages, the binding efficiency of purified targeted modified nanoparticles to RAW264.7 macrophages was evaluated, and targeted binding data were generated.

6. The method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization as described in claim 1, characterized in that: The macrophage culture system was constructed by seeding RAW264.7 macrophages into culture dishes containing RPMI1640 medium, fetal bovine serum, and antibiotics, and then culturing them in an incubator to monitor cell growth and maintain cell density.

7. The method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization as described in claim 1, characterized in that: The specific steps for generating drug response data are as follows: Targeted modified nanoparticles were added to the macrophage culture system, and drug treatment was carried out according to the set drug concentration to generate the treated macrophage culture system. Cell viability was monitored in real time using cell viability assays, and the levels of inflammatory factors in the cell supernatant were detected using ELISA. Flow cytometry was used to detect the expression changes of M1 and M2 macrophage markers in the treated macrophage culture system and to analyze cell polarization status. Principal component analysis was used to comprehensively analyze cell survival, inflammatory factor levels, and cell polarization status to generate drug response data.

8. The method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization as described in claim 1, characterized in that: The specific steps for generating comprehensive control data are as follows: Flow cytometry was used to analyze changes in M1 and M2 macrophage markers in drug response data, generating M1 macrophage polarization data. The ELISA method was used to detect changes in the levels of inflammatory factors in cell supernatant in drug response data, generating data on the regulation of inflammatory factors. Path analysis was used to comprehensively analyze M1 macrophage polarization data and inflammatory factor regulation data to generate comprehensive regulatory data, which was used to characterize the inhibitory effect on M1 macrophage polarization and the promoting effect on M2 macrophage polarization.

9. The method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization as described in claim 1, characterized in that: The specific steps for generating the protection effect data are as follows: Macrophage culture system samples that have undergone combined treatment with SAMC and anti-IL-6 peptide were selected from the comprehensive regulatory data and fixed to generate treated fixed cell samples. Using cell immunofluorescence, specific antibodies against M1 macrophage markers were stained to generate labeled cell samples. The expression of the labeled cell samples was then observed using a fluorescence microscope to generate fluorescence microscopic images. Based on fluorescence microscopy images and comprehensive regulatory data, the effects of combined treatment of SAMCs and anti-IL-6 peptide on maintaining macrophage structural stability and immune function integrity were analyzed, and protective efficacy data were generated.

10. The method for evaluating the anti-inflammatory effect based on SAMC-regulated macrophage M1 polarization as described in claim 9, characterized in that: The specific steps for generating the fluorescence micrograph are as follows: M1 macrophage markers were collected from the treated macrophage culture system and impurities in the culture medium were removed by washing with PBS to generate macrophage samples to be stained. The macrophage samples to be stained were exposed to a solution of M1 macrophage marker-specific antibody and incubated at the set concentration and time to generate labeled cell samples. The expression of M1 macrophage markers was detected by observing labeled cell samples using a fluorescence microscope, and fluorescence micrographs were generated.

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