Application of a small molecule tripeptide, Ser-Ser-Cys, in inhibiting phenotypic transformation of vascular smooth muscle cells

Abstract

This invention belongs to the field of biomedical technology and discloses the application of the small molecule tripeptide Ser-Ser-Cys (SSC) in inhibiting the phenotypic transformation of vascular smooth muscle cells (VSMCs). SSC is an endogenous differentially expressed tripeptide screened and identified from the serum of hypertensive non-atherosclerotic patients using non-targeted metabolomics technology. In vitro experiments show that SSC can effectively inhibit angiotensin II (Ang II)-induced abnormal proliferation, migration, and phenotypic transformation from contractile to synthetic VSMCs. Its mechanism of action is related to the regulation of the Keap1 / Nrf2 signaling pathway, improvement of cellular oxidative stress, and mitochondrial function. This invention provides a new approach for using SSC in the preparation of drugs or functional foods for the prevention and treatment of hypertension-related vascular remodeling.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the screening and application of bioactive peptides, and more specifically, to a small molecule tripeptide obtained through metabolomics screening and its application in regulating vascular smooth muscle cell function, particularly in inhibiting vascular smooth muscle cell phenotypic transformation and preventing hypertension-related vascular remodeling. Background Technology

[0002] Hypertension and its induced vascular remodeling are the core pathological basis for damage to target organs such as the heart, brain, and kidneys. In this process, the phenotypic transformation of vascular smooth muscle cells (VSMCs)—the shift from a "contractile" type that maintains vascular tone to a "synthetic" type characterized by proliferation, migration, and extracellular matrix secretion—is a key cellular event driving vascular wall thickening, decreased elasticity, and functional abnormalities. Angiotensin II (Ang II), a key pathological factor in hypertension, can drive the phenotypic transformation of VSMCs through multiple pathways, including inducing oxidative stress and inflammatory responses. Therefore, identifying bioactive substances that can effectively inhibit abnormal VSMC phenotypic transformation is of great significance for the prevention and treatment of hypertension-related vascular remodeling.

[0003] In recent years, research on the regulation of phenotypic switching in VSMCs has made some progress. For example, existing technologies (such as prior art document 1, CN106636120A) disclose an ArtRD protein and its gene identified from long non-coding RNA. This technology found that ArtRD protein expression in VSMCs can promote VSMC phenotypic switching and exacerbate vascular remodeling; therefore, it or its inhibitors could serve as potential research targets or therapeutic tools. This technology reveals a new protein involved in the regulation of phenotypic switching, but it focuses on factors that promote switching, and ArtRD is a newly discovered specific protein, whose application direction is fundamentally different from developing therapeutic substances that inhibit switching.

[0004] On the other hand, some natural products or their derivatives have also been found to regulate VSMC function. For example, prior art (such as prior art document 2, CN119868334A) discloses the application of δ-Viniferin (a resveratrol dehydrogenated dimer) in the preparation of drugs for treating metabolic cardiovascular diseases, mentioning that this compound can improve vascular function by inhibiting the expression of genes / proteins related to VSMC phenotypic transformation. This technology provides a strategy for using exogenous natural compounds to intervene in phenotypic transformation. However, δ-Viniferin belongs to structurally complex plant polyphenols, and its in vivo metabolism, bioavailability, and long-term safety still need further evaluation.

[0005] In summary, existing technologies still have significant shortcomings and room for improvement in suppressive interventions targeting phenotypic transitions in VSMCs:

[0006] 1. Limitations in target and direction of action: Existing research focuses on discovering new regulatory factors (such as ArtRD that promotes phenotypic conversion) or utilizing known pathways, but research on systematically screening and verifying small molecule bioactive peptides that inhibit phenotypic conversion directly from human endogenous metabolites is still insufficient.

[0007] 2. Limitations in the source and properties of active substances: Currently reported active ingredients with inhibitory effects (such as δ-Viniferin) are mostly exogenous complex molecules, whose pharmaceutical properties differ from endogenous substances. In contrast, endogenous small molecule peptides generally have better biocompatibility and potential metabolic safety, but mature and reproducible screening and validation protocols have not yet been developed for their specific functions and applications in VSMC phenotypic conversion.

[0008] 3. Insufficient precision of screening strategies: Traditional discovery strategies are mostly based on hypothesis-driven or empirical screening, lacking a research path that starts from body fluid samples of specific disease populations (such as patients with hypertension but not atherosclerosis) and uses non-targeted metabolomics for unbiased systematic screening, thereby directly identifying endogenous small molecule peptides that are closely related to disease states and have functional potential.

[0009] Therefore, there is an urgent need in this field for a new technical solution that can efficiently and accurately screen small molecule active substances from disease-related endogenous molecular libraries that can effectively inhibit the pathological phenotypic transformation of VSMCs, and provide new intervention strategies and candidate substances for the prevention and treatment of hypertension vascular remodeling. Summary of the Invention

[0010] To address the shortcomings mentioned in the background section, such as insufficient attention to small peptides that endogenously inhibit vascular smooth muscle cell phenotypic transformation and a lack of systematic screening strategies in existing intervention methods, this invention proposes a small molecule tripeptide, Ser-Ser-Cys (SSC), obtained through non-targeted metabolomics screening, and its application in inhibiting vascular smooth muscle cell phenotypic transformation. This invention screens and identifies endogenous tripeptides with significantly differential expression by comparing serum metabolic profiles of hypertensive non-atherosclerotic patients and healthy controls. In vitro experiments demonstrate that SSC can effectively inhibit Ang II-induced abnormal proliferation, migration, and phenotypic transformation of vascular smooth muscle cells by regulating the Keap1 / Nrf2 oxidative stress signaling pathway. Compared to traditional empirical screening or exogenous complex compound intervention strategies, the SSC provided by this invention has advantages such as small molecular weight, well-defined structure, origin from the endogenous metabolic system, and good biocompatibility, providing a new potential active molecule and intervention strategy for the prevention and treatment of hypertension-related vascular remodeling.

[0011] To achieve the objectives of this invention, the invention includes the following technical solutions:

[0012] This invention discloses for the first time the application of the small molecule tripeptide Ser-Ser-Cys (SSC) in inhibiting phenotypic switching of vascular smooth muscle cells (VSMCs).

[0013] The small molecule tripeptide Ser-Ser-Cys (hereinafter referred to as SSC) is a small molecule peptide screened and identified using non-targeted metabolomics analysis technology. Specifically, this invention performs non-targeted metabolomics analysis on serum samples from hypertensive non-atherosclerotic patients and non-hypertensive, non-atherosclerotic controls to compare the differential expression characteristics of small molecule peptides between different populations, thereby screening out the significantly different small molecule tripeptide Ser-Ser-Cys.

[0014] This invention discloses for the first time the application of the small molecule tripeptide Ser-Ser-Cys (SSC) in the preparation of a drug for inhibiting phenotypic transformation of vascular smooth muscle cells (VSMCs).

[0015] The SSC is composed of serine-serine-cysteine, with the structural formula Ser-Ser-Cys. This peptide can be prepared by chemical synthesis with a purity of over 98%, meeting the requirements for research or pharmaceutical applications.

[0016] In some embodiments, when the SSC was applied to an Ang II-stimulated VSMC model, it was found that the SSC could effectively inhibit the Ang II-induced vascular smooth muscle cell phenotypic transformation process, specifically as follows:

[0017] Inhibits Ang II-induced abnormal proliferation and migration of VSMCs; reverses the phenotypic transformation of VSMCs from contractile to synthetic types; downregulates the expression of synthetic phenotype-related markers while upregulating the expression of contractile phenotype-related markers, thereby improving the functional state of vascular smooth muscle cells.

[0018] Furthermore, in the above application, the inhibition of vascular smooth muscle cell phenotype conversion inhibits the angiotensin II-induced phenotypic conversion of vascular smooth muscle cells from contractile to synthetic types.

[0019] Furthermore, in the above applications, the small molecule tripeptide Ser-Ser-Cys inhibits phenotypic transformation of vascular smooth muscle cells by regulating the Keap1 / Nrf2 signaling pathway. In some embodiments, SSC inhibits Ang II-induced excessive generation of reactive oxygen species (ROS) by regulating intracellular redox homeostasis, and promotes the activation of intracellular antioxidant defense systems through the Keap1 / Nrf2-mediated red oxygen-sensitive signaling pathway, thereby alleviating oxidative stress-related vascular smooth muscle cell dysfunction and participating in its phenotypic regulation.

[0020] Furthermore, in the above-described applications, the drug is used to prevent and treat hypertension-related vascular remodeling.

[0021] Furthermore, in the above application, the small molecule tripeptide Ser-Ser-Cys is obtained by screening from the serum of hypertensive non-atherosclerotic patients using non-targeted metabolomics technology.

[0022] Furthermore, in the above applications, the small molecule tripeptide Ser-Ser-Cys is prepared by chemical synthesis and has a purity of not less than 98%.

[0023] Furthermore, in the above application, the concentration of the small molecule tripeptide Ser-Ser-Cys used is from 50 μg / mL to 500 μg / mL.

[0024] Furthermore, in the above application, the concentration of the small molecule tripeptide Ser-Ser-Cys is 100 μg / mL.

[0025] The present invention also discloses a pharmaceutical composition for inhibiting phenotypic transformation of vascular smooth muscle cells, comprising an effective dose of the small molecule tripeptide Ser-Ser-Cys and a pharmaceutically acceptable carrier.

[0026] Compared with the prior art, the present invention has the following outstanding advantages:

[0027] 1. More targeted screening strategy: Unbiased screening based on serum metabolomics of specific disease populations (hypertension without atherosclerosis) improves the efficiency and relevance of discovering endogenous active molecules closely related to vascular pathological states.

[0028] 2. Clearly defined active substance characteristics: SSC is an endogenous ultrashort chain tripeptide with a small molecular weight and simple structure. Compared with exogenous complex compounds, it may have better biocompatibility and metabolic safety.

[0029] 3. The mechanism of action has been preliminarily elucidated: Cell experiments have confirmed that SSCs can reduce oxidative stress through the Keap1 / Nrf2 pathway, thereby inhibiting phenotype conversion of VSMCs, providing experimental evidence for understanding its function.

[0030] 4. Clear application direction: It provides a new potential candidate molecule and intervention idea for the prevention and treatment of hypertension vascular remodeling, and has the potential to be further developed into related drugs or functional foods (additives). Attached Figure Description

[0031] Figure 1 A schematic diagram of the workflow for screening small molecule peptides Ser-Ser-Cys for non-targeted metabolomics analysis;

[0032] Figure 2 Schematic diagram of the SSC molecular structure;

[0033] Figure 3 : Schematic diagram of Ang II-induced phenotypic transformation in VSMCs;

[0034] Figure 4 Effects of SSC on Ang II-induced proliferation of VSMCs;

[0035] Figure 5 The effect of SSC on the migration ability of Ang II-induced VSMCs;

[0036] Figure 6 Effects of SSC on Ang II-induced cell cycle distribution in vascular smooth muscle cells;

[0037] Figure 7 : Schematic diagram of SSC's effect on oxidative stress / mitochondrial function;

[0038] Figure 8 Effects of SSC on Ang II-induced oxidative stress and mitochondrial function changes;

[0039] Figure 9 : Schematic diagram of SSC regulation of the Keap1 / Nrf2 pathway;

[0040] Figure 10 The effect of SSC on promoting Nrf2 nuclear translocation and activating the Keap1 / Nrf2 signaling pathway;

[0041] Figure 11 Effects of SSC on Ang II-induced expression and secretion of inflammatory factors. Detailed Implementation

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Terminology Explanation:

[0044] Non-targeted metabolomics refers to a method for systematically detecting and comparing small molecule metabolites in biological samples without pre-setting specific detection targets. It is used to screen small molecule substances that differ under different physiological or pathological conditions.

[0045] Phenotypic transformation of vascular smooth muscle cells: refers to the process by which vascular smooth muscle cells change from a contractile phenotype to a synthetic phenotype under pathological stimuli.

[0046] Ang II-induced model: refers to an in vitro experimental model that simulates abnormal cell function in hypertension-related pathological states by acting on vascular smooth muscle cells with angiotensin II.

[0047] Keap1 / Nrf2 signaling pathway: refers to an important signaling pathway involved in regulating cellular oxidative stress response, among which Nrf2 activation can induce the expression of antioxidant-related genes.

[0048] Example 1

[0049] Screening of small molecule tripeptide Ser-Ser-Cys (SSC) based on non-targeted metabolomics

[0050] This embodiment provides a method for screening endogenous small molecule peptides from the serum of hypertensive patients. For detailed procedures, please refer to [link to relevant documentation]. Figure 1 .

[0051] 1. Sample collection: Serum samples were collected from patients with hypertension but without atherosclerosis as the experimental group, and serum samples were collected from healthy individuals without hypertension and without atherosclerosis who were matched for age and sex as the control group.

[0052] 2. Non-targeted metabolomics detection: Non-targeted metabolomics analysis was performed on the two groups of serum samples. Liquid chromatography-mass spectrometry was used to obtain metabolite spectral data, including small molecule peptides.

[0053] 3. Data Analysis and Screening: Multivariate statistical analysis was performed on the obtained metabolomics data. First, orthogonal partial least squares discriminant analysis (OPLS-DA) model was used to distinguish between the two groups of samples. Then, the samples were screened based on variable importance projection values ​​(VIP>1.0), t-tests (p<0.05), and differential expression trends (downregulation in the hypertension group).

[0054] 4. Target peptide identification: From the differentially expressed molecules that met the above criteria, a small tripeptide with the amino acid sequence serine-serine-cysteine, namely Ser-Ser-Cys (SSC), was identified. This result indicates that SSC is an endogenous metabolic peptide in the human body, and its expression is significantly downregulated under hypertension, suggesting that it may be related to the dysregulation of vascular homeostasis.

[0055] Example 2

[0056] Chemical Synthesis and Characterization of Small Molecule Tripeptide SSC

[0057] This embodiment illustrates the preparation and quality control methods of SSC. A schematic diagram of its molecular structure can be found in [reference needed]. Figure 2 .

[0058] 1. Solid-phase synthesis: The standard Fmoc solid-phase peptide synthesis strategy is adopted, and cysteine ​​(C), serine (S) and serine (S) resins are sequentially linked, and finally cleaved to obtain crude peptides.

[0059] 2. Purification and identification: The crude peptide was purified by reversed-phase high-performance liquid chromatography. The main peak was collected and freeze-dried to obtain SSC pure product with a purity of ≥98%.

[0060] 3. Structure Confirmation: The molecular weight of the synthesized product was determined by mass spectrometry, and the result was consistent with the theoretical molecular weight, confirming its structure as Ser-Ser-Cys. The purified SSC was dissolved in PBS buffer or stored at -20℃ in cell culture medium for later use.

[0061] The above-mentioned small molecule tripeptide SSCs can also be purchased directly from commercial suppliers.

[0062] Example 3

[0063] Inhibitory effect of SSC on Ang II-induced vascular smooth muscle cell phenotypic transition

[0064] This embodiment verifies the protective function of the small molecule tripeptide SSC in a vascular smooth muscle cell (VSMC) pathological model, specifically its inhibitory effect on abnormal cell proliferation and migration. The experimental design and results diagram can be found in [link to diagram]. Figure 3 , Figure 4 (Proliferation) and Figure 5 (migrate).

[0065] 1. Experimental Materials and Methods

[0066] Cells: Primary cultured rat aortic vascular smooth muscle cells (VSMCs) were cultured in DMEM medium containing 10% fetal bovine serum, 1% smooth muscle cell growth factor, and 1% streptomycin / penicillin. The cells were routinely cultured at 37°C and 5% CO2. Cells in passage 3-5 and in good growth condition were selected for the experiment.

[0067] Main reagents: Angiotensin II (Ang II), small molecule tripeptide Ser-Ser-Cys (SSC, purity ≥98%, prepared in Example 2).

[0068] Experimental Grouping and Treatment: To comprehensively evaluate the role of SSC, the following four groups were established:

[0069] Control group: No stimulants or drugs were added.

[0070] Model group (Ang II): 1 μM Ang II stimulation was added to induce phenotypic transformation in VSMCs.

[0071] SSC group: Only 100 μg / mL SSC was added to assess its basal effect on normal VSMCs.

[0072] SSC+Ang II group: 1 μM Ang II and 100 μg / mL SSC were added simultaneously for co-treatment to evaluate the intervention effect of SSC on Ang II-induced damage.

[0073] All treatments were performed after the cell confluence reached 80%-90%, and the medium was changed to a low-serum medium. The treatment time lasted for 24 hours.

[0074] 2. Cell proliferation capacity detection

[0075] Cell viability was assessed using a CCK-8 assay kit to evaluate cell proliferation levels. Figure 4As shown, compared with the control group, the cell viability of the AngII model group was significantly increased, confirming that Ang II successfully induced abnormal proliferation of VSMCs. In contrast, the cell viability of the SSC+Ang II co-treatment group was significantly decreased compared with the model group, indicating that SSC can effectively inhibit Ang II-induced abnormal proliferation. There was no significant difference between the SSC-only treatment group and the control group, indicating that SSC itself has no toxic or inhibitory effect on the proliferation of normal VSMCs at the tested concentration.

[0076] 3. Cell migration ability detection

[0077] Migration ability was assessed using cell scratch healing assays and Transwell assays. Figure 5 As shown, Ang II stimulation significantly accelerated the healing rate of scratches, indicating enhanced cell migration. However, under SSC+Ang II co-treatment, the scratch healing rate was significantly delayed, with effects similar to the control group, demonstrating that SSC can effectively inhibit Ang II-induced abnormal migration of VSMCs.

[0078] Conclusion: The results of this embodiment show that the small molecule tripeptide SSC can significantly inhibit Ang II-induced abnormal proliferation and migration of vascular smooth muscle cells, that is, it has a clear inhibitory effect on the pathological phenotypic transformation of VSMCs.

[0079] Example 4

[0080] SSC inhibits abnormal proliferation of vascular smooth muscle cells by arresting cell cycle progression.

[0081] This embodiment aims to elucidate the cellular mechanism by which the small molecule tripeptide SSC inhibits the abnormal proliferation of vascular smooth muscle cells (VSMCs) from the perspective of cell cycle dynamics. The experimental design is based on the grouping and model established in Example 3, exploring the effect of SSC on Ang II-interfered cell cycle distribution.

[0082] 1. Experimental grouping and cell treatment

[0083] Primary rat aortic VSMCs were cultured according to the method described in Example 3. After the cells reached an appropriate density, they were grouped and treated as follows:

[0084] Control group: Normal culture, no additives.

[0085] Ang II model group: 1 μM Ang II stimulation was added.

[0086] SSC group: Add 100 μg / mL SSC.

[0087] SSC+Ang II group: 1 μM Ang II and 100 μg / mL SSC were added simultaneously.

[0088] All groups were treated in low-serum medium for 24 hours to focus on early changes in the cell cycle.

[0089] 2. Cell cycle distribution detection

[0090] After treatment, collect cells from each group and perform the following operations:

[0091] Wash cells with pre-cooled PBS.

[0092] Cells were fixed with 70% ethanol at 4°C for more than 2 hours.

[0093] After fixation, centrifuge to remove ethanol, then resuspend and wash with PBS.

[0094] Add propidium iodide (PI) staining solution containing RNase A, and avoid incubation for 30 minutes.

[0095] The DNA content of each sample was detected by flow cytometry, and the percentage of cells distributed in each phase of the cell cycle (G0 / G1 phase, S phase, G2 / M phase) was analyzed by software.

[0096] 3. Results Analysis

[0097] Flow cytometry results clearly showed the differences in cell cycle distribution among the VSMCs in each group:

[0098] Compared with the control group, the cell cycle distribution in the Ang II model group was significantly altered, characterized by a significant increase in the proportion of cells in the DNA synthesis phase (S phase), a corresponding decrease in the proportion of cells in the G0 / G1 phase, and an increase in the proportion of cells in the S phase. Figure 6 As shown in the figure. This directly confirms that Ang II stimulation can drive more VSMCs into the DNA replication and synthesis stage, which is a key step leading to their abnormal proliferation.

[0099] In the SSC+Ang II co-treatment group, the aforementioned abnormal cell cycle progression was effectively corrected. Compared with the model group, the proportion of cells in the S phase in this group decreased significantly, while the proportion of cells in the quiescent / pre-synthetic (G0 / G1) phase increased significantly, and its distribution curve was closer to that of the normal control group.

[0100] There was no statistically significant difference in cell cycle distribution between the SSC-treated group and the control group, indicating that SSC itself does not interfere with the cell cycle of normal VSMCs.

[0101] Conclusion: This example reveals the mechanism of action of SSC from a cell kinetics perspective. The results show that Ang II promotes abnormal proliferation of VSMCs by driving more cells into the S phase; while the small-molecule tripeptide SSC can reverse this process, arresting cells in the G0 / G1 phase, thereby effectively inhibiting the pathological progression of the cell cycle. This provides a direct cellular mechanism explanation for the SSC-induced proliferation inhibition observed in Example 3, further confirming the important role of SSC in regulating VSMC homeostasis.

[0102] Example 5

[0103] SSC exerts a protective effect by improving oxidative stress and mitochondrial function.

[0104] This embodiment aims to explore the downstream cellular mechanisms by which SSCs exert their protective effects, focusing on their role in improving Ang II-induced oxidative stress and mitochondrial dysfunction. See the schematic diagram of the relevant mechanisms and some results below. Figure 7 and Figure 8 .

[0105] 1. Experimental Grouping

[0106] Similar to Example 3, control group, Ang II model group, SSC group, and SSC+Ang II group were set up, and the treatment conditions were the same.

[0107] 2. Detection of intracellular reactive oxygen species (ROS) levels

[0108] Intracellular total ROS levels were detected using the DCFH-DA fluorescent probe. After treatment, the probe was loaded, and fluorescence intensity was quantitatively analyzed using a fluorescence microplate reader or fluorescence microscope. Results are as follows: Figure 7 As shown, the fluorescence intensity in the Ang II model group was significantly enhanced compared to the control group, indicating a large accumulation of intracellular ROS. In the SSC+Ang II co-treatment group, the ROS fluorescence intensity was significantly reduced compared to the model group, recovering to levels close to the control group. There was no difference between the SSC-treated group and the control group. These results demonstrate that SSC can effectively alleviate Ang II-induced oxidative stress in VSMCs.

[0109] 3. Mitochondrial function assessment

[0110] Mitochondrial membrane potential detection: The JC-1 fluorescent probe was used. Under normal conditions with a high mitochondrial membrane potential, JC-1 forms a polymer and emits red fluorescence; when the membrane potential decreases, JC-1 remains a monomer and emits green fluorescence. The red / green fluorescence ratio was calculated by flow cytometry or fluorescence microscopy. The experiment showed that Ang II treatment significantly decreased the red / green fluorescence ratio, indicating mitochondrial membrane potential depolarization and impaired function. Co-treatment with SSC and Ang II partially restored this ratio, suggesting that SSC helps maintain the stability of the mitochondrial membrane potential.

[0111] Mitochondrial superoxide detection: using MitoSOX TM A red-specific fluorescent probe. This probe targets mitochondria and emits red fluorescence after being oxidized by superoxide. Detection results showed that Ang II stimulation significantly increased superoxide levels in mitochondria. Co-treatment with SSCs significantly attenuated this fluorescence signal, indicating that SSCs can specifically reduce oxidative stress in mitochondria.

[0112] Conclusion: This embodiment demonstrates that the small molecule tripeptide SSC can effectively antagonize Ang II-induced excessive ROS production in vascular smooth muscle cells and improve mitochondrial function (including stabilizing membrane potential and reducing mitochondrial superoxide). Its antioxidant effect is one of the important mechanisms by which it inhibits cell phenotypic transformation (e.g., Figure 7 (As shown in the summary).

[0113] Example 6

[0114] SSC regulates oxidative stress by activating the Keap1 / Nrf2 signaling pathway.

[0115] This embodiment reveals in detail the core functional pathway of SSC, corresponding to Figure 9 .

[0116] Nrf2 nuclear translocation detection: Intracellular localization of Nrf2 was observed using immunofluorescence techniques. For example... Figure 10 As shown, Nrf2 is partially located in the nucleus in normal cells; Ang II inhibits its nuclear translocation; while SSC treatment significantly promotes the accumulation of Nrf2 in the nucleus. Furthermore, the distribution changes of Nrf2 in the nucleus and cytoplasm were detected using a nuclear / cytoplasmic protein separation method. The results showed that Ang II treatment reduced the level of Nrf2 in the nucleus, while SSC treatment significantly increased the level of Nrf2 in the nucleus.

[0117] Downstream target gene expression detection: The expression of Nrf2 downstream antioxidant genes (such as CAT, GCLC, and FTH1) at the protein and mRNA levels was detected by Western blotting and RT-qPCR, respectively. The results showed that SSC significantly upregulated the expression of these antioxidant proteins and genes.

[0118] Mechanism Conclusion: Based on the above results, it is demonstrated that SSCs activate the cell's antioxidant defense system by regulating the Keap1 / Nrf2 signaling pathway, thereby antagonizing Ang II-induced oxidative stress and ultimately inhibiting phenotypic switching in VSMCs (see pathway summary). Figure 9 ).

[0119] Example 7

[0120] SSC inhibits Ang II-induced expression / secretion of inflammatory factors.

[0121] This embodiment further verifies the regulatory role of SSC on the inflammatory response under Ang II-induced conditions, corresponding to Figure 11 .

[0122] 1. Experimental Grouping

[0123] Similar to Example 3, control group, Ang II model group, SSC group, and SSC+Ang II group were set up, and the treatment conditions were the same.

[0124] 2. Detection of inflammatory factor secretion

[0125] Culture supernatants of vascular smooth muscle cells under different treatment conditions were collected, and the secretion levels of inflammatory factors IL-1β and IL-6 were detected using enzyme-linked immunosorbent assay (ELISA). Figure 9 As shown, compared with the normal control group, AngII treatment significantly promoted the secretion of IL-1β and IL-6; while after SSC treatment, the secretion levels of the above inflammatory factors were significantly reduced.

[0126] 3. Detection of inflammation-related gene expression

[0127] Total RNA was further extracted from cells, and the mRNA expression levels of IL-1β and IL-6 were detected by RT-qPCR. The results showed that Ang II significantly upregulated the transcriptional levels of inflammation-related genes, while SSC treatment effectively inhibited the mRNA expression of IL-1β and IL-6.

[0128] Mechanism Conclusion: The above results indicate that SSCs can inhibit the production and secretion of Ang II-induced inflammatory factors IL-1β and IL-6, suggesting a role in alleviating inflammatory responses. This anti-inflammatory effect is consistent with the role of SSCs in regulating cellular oxidative stress, jointly participating in the inhibition of pathological phenotypic transformation of vascular smooth muscle cells (see results below). Figure 9 ).

[0129] The function and mechanism of the small molecule tripeptide SSC were systematically verified through seven examples. First, endogenous tripeptide SSC was screened and identified based on serum metabolomics from hypertensive patients, and high-purity samples were obtained through chemical synthesis. Cell experiments showed that SSC significantly inhibited Ang II-induced abnormal proliferation and migration of vascular smooth muscle cells and arrested the cell cycle at the G0 / G1 phase. Mechanistically, SSC effectively reduced intracellular reactive oxygen species levels, improved mitochondrial function, and activated the Keap1 / Nrf2 antioxidant signaling pathway by promoting Nrf2 nuclear translocation. Simultaneously, SSC also inhibited the expression and secretion of Ang II-induced inflammatory factors (such as IL-1β and IL-6), thereby alleviating cellular inflammatory responses. These multiple effects worked synergistically to ultimately inhibit the pathological phenotypic transformation of vascular smooth muscle cells.

[0130] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. Application of small molecule tripeptide Ser-Ser-Cys (SSC) in inhibiting phenotypic switching of vascular smooth muscle cells (VSMCs).

2. The application according to claim 1, characterized in that, The application refers to the use of the small molecule tripeptide Ser-Ser-Cys (SSC) in the preparation of drugs or functional foods for inhibiting phenotypic transformation of vascular smooth muscle cells (VSMCs).

3. The application according to claim 2, characterized in that, The inhibition of vascular smooth muscle cell phenotype conversion is described as inhibiting the angiotensin II-induced phenotypic conversion of vascular smooth muscle cells from contractile to synthetic types.

4. The application according to claim 2, characterized in that, The small molecule tripeptide Ser-Ser-Cys inhibits phenotypic transformation of vascular smooth muscle cells by regulating the Keap1 / Nrf2 signaling pathway.

5. The application according to claim 2, characterized in that, The drug is used to prevent and treat hypertension-related vascular remodeling.

6. The application according to claim 2, characterized in that, The small molecule tripeptide Ser-Ser-Cys was obtained from the serum of hypertensive non-atherosclerotic patients using non-targeted metabolomics technology.

7. The application according to claim 2, characterized in that, The small molecule tripeptide Ser-Ser-Cys is prepared by chemical synthesis and has a purity of not less than 98%.

8. The application according to claim 2, characterized in that, In applications, the concentration of the small molecule tripeptide Ser-Ser-Cys used is from 50 μg / mL to 500 μg / mL.

9. The application according to claim 8, characterized in that, The concentration of the small molecule tripeptide Ser-Ser-Cys used is 100 μg / mL.

10. A pharmaceutical composition for inhibiting phenotypic transformation of vascular smooth muscle cells, characterized in that, It contains an effective dose of the small molecule tripeptide Ser-Ser-Cys and a pharmaceutically acceptable carrier.

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