Small molecule peptides and uses thereof

Abstract

The application discloses a small molecule peptide and application, relates to the technical field of medicinal chemistry, aims at overcoming the defects of low safety and insufficient reliability in the prior art, and the amino acid sequence of the small molecule peptide is shown as SEQ ID NO:1. The application provides a safe and efficient new option for the prevention, relief and treatment of inflammation.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry. More specifically, this invention relates to a small molecule peptide and its applications. Background Technology

[0002] Inflammation is the body's defensive response to stimuli such as infection and injury. However, excessive or persistent inflammatory responses can lead to tissue damage and trigger various diseases, including infectious and autoimmune diseases, seriously threatening human health. Lipopolysaccharide (LPS), a key component of the cell wall of Gram-negative bacteria, is a common clinical trigger for inflammation. It can activate immune cells such as macrophages, induce excessive production of reactive oxygen species (ROS), downregulate the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), and simultaneously promote the release of nitric oxide (NO) and pro-inflammatory cytokines such as IL-α, IL-6, and IL-1β. It can also activate signaling pathways such as NF-κB and MAPK, ultimately amplifying the inflammatory cascade. The related pathological mechanisms have become a core direction in inflammation research.

[0003] Currently, commonly used anti-inflammatory drugs in clinical practice include nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, but long-term use can easily cause side effects such as gastrointestinal damage and immunosuppression, resulting in insufficient safety. Naturally derived small-molecule bioactive peptides have gradually become a research hotspot in the field of anti-inflammation due to their advantages such as good biocompatibility, low toxicity, and easy absorption. However, many anti-inflammatory peptides discovered in existing technologies suffer from unclear mechanisms of action, unstable anti-inflammatory activity, and insufficient targeting of LPS-induced inflammation. Furthermore, the effects of most peptide drugs are only limited to preliminary verification of anti-inflammatory activity, lacking elucidation of deeper mechanisms such as signal pathway regulation and metabolic network intervention, making it difficult to meet the clinical demand for safe and effective anti-inflammatory drugs.

[0004] Therefore, it is necessary to design a technical solution that can overcome the above-mentioned defects. Summary of the Invention

[0005] One object of the present invention is to provide a small molecule peptide and its application, offering a safe and efficient new option for the prevention, relief and treatment of inflammation.

[0006] To achieve these objectives and other advantages of the present invention, according to one aspect of the present invention, a small molecule peptide is provided, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] According to another aspect of the invention, the application of small molecule peptides is also provided for the preparation of medicaments for the prevention, relief and / or treatment of inflammation.

[0008] Furthermore, the inflammation is lipopolysaccharide-induced inflammation.

[0009] Furthermore, the dosage form of the drug is at least one of injection, tablet, oral liquid, granules or capsules.

[0010] According to another aspect of the invention, a pharmaceutical composition is also provided, characterized in that it comprises the aforementioned small molecule peptide.

[0011] The present invention has at least the following beneficial effects:

[0012] The small molecule peptide PYYFH of this invention possesses significant anti-inflammatory advantages and practical value. Firstly, it exhibits high safety, showing no toxicity to RAW264.7 cells within a concentration range of 50-1200 μg / mL, and good biocompatibility, avoiding the gastrointestinal damage and immunosuppression side effects of traditional anti-inflammatory drugs, making it suitable for long-term use. Secondly, it demonstrates strong anti-inflammatory targeting and a clear mechanism. By stably binding to the key inflammatory factor IL-6, it regulates the NF-κB and MAPK signaling pathways, enhancing SOD and CAT activity to improve oxidative stress, and inhibiting the release of NO and pro-inflammatory factors such as IL-α and IL-6. Furthermore, non-targeted metabolomics studies have revealed that its activity may regulate core metabolic pathways through Tyrosyl-Tyrosine and sn-Glycerol 3-phosphate, blocking LPS-induced inflammatory cascade responses from multiple dimensions, resulting in a stable and reliable anti-inflammatory effect. This small molecule peptide provides a safe and efficient new option for the prevention, relief, and treatment of LPS-induced inflammation, possessing significant clinical application prospects and promotional value.

[0013] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0014] Figure 1 The image shows the mass spectrum of the PYYFH peptide, with the horizontal axis representing the mass-to-charge ratio (m / z) and the vertical axis representing the relative intensity (%).

[0015] Figure 2 A 3D schematic diagram of the binding of PYYFH to IL-6 protein: IL-6 (interleukin-6) is shown in green, and PYYFH is a blue / cyan molecule; the magnified area on the right shows the key residues of the binding interface between the two, showing their binding mode and interaction sites.

[0016] Figure 3The bar chart shows the CAT activity of different treatment groups: the horizontal axis represents the treatment group (including Control, LPS and different concentration groups), and the vertical axis represents the CAT activity (unit U / mgprot). After LPS treatment, the CAT activity was significantly lower than that of the Control group. As the treatment concentration increased, the CAT activity gradually increased. The same letters (a, b, c, d, e) indicate that the difference between groups is not significant, and different letters indicate that the difference is significant.

[0017] Figure 4 A bar chart with two axes for IL-6 production in different treatment groups: the horizontal axis represents the treatment groups (including Control, LPS, and different concentration groups), and the left vertical axis represents IL-6 production (unit: pg / mL). IL-6 production increased significantly after LPS treatment, and gradually decreased with increasing treatment concentration. The same letters (a, b, c, d, e) indicate no significant difference between groups, while different letters indicate significant differences.

[0018] Figure 5 A bar chart showing NO concentration at different incubation times: the horizontal axis represents incubation time (in hours), and the vertical axis represents NO concentration (in μM). As the incubation time increased from 8 h to 24 h, nitric oxide production gradually increased, reaching its peak at 24 h. The same letters (a, b, c, d, e) indicate no significant difference between groups, while different letters indicate significant differences.

[0019] Figure 6 A bar chart showing NO production in different treatment groups: the horizontal axis represents the treatment group (including Control, LPS, and different concentration groups), and the vertical axis represents NO production (%). NO production increased significantly after LPS treatment, and gradually decreased with increasing treatment concentration. The same letters (a, b, c, d) indicate no significant difference between groups, while different letters indicate significant differences.

[0020] Figure 7 Cell fluorescence imaging images of different treatment groups are shown. The groups are Control, 1 μg / ml LPS, 200 μM PYYFH+LPS, 400 μM PYYFH+LPS, and 800 μM PYYFH+LPS, corresponding to (a), (b), (c), (d), and (e), respectively. The fluorescence of the LPS group was significantly enhanced, and the fluorescence intensity gradually decreased with increasing PYYFH concentration.

[0021] Figure 8The bar chart shows the SOD activity of different treatment groups: the horizontal axis represents the treatment group (including Control, LPS, and different concentration groups), and the vertical axis represents SOD activity (unit U / mgprot). SOD activity decreased significantly after LPS treatment, and gradually increased with increasing treatment concentration; the same letters (a, b, c, d, e) indicate no significant difference between groups, and different letters indicate significant differences.

[0022] Figure 9 The bar chart shows the TNF-α production of different treatment groups: the horizontal axis represents the treatment groups (including Control, LPS, and different concentration groups), and the vertical axis represents the TNF-α production (pg / mL). TNF-α production increased significantly after LPS treatment, and gradually decreased with increasing treatment concentration; the same letters (a, b, c, d, e) indicate no significant difference between groups, while different letters indicate significant differences.

[0023] Figure 10 Bar chart showing IL-1β production in different treatment groups: the horizontal axis represents the treatment group (including Control, LPS, and different concentration groups), and the vertical axis represents IL-1β production (pg / mL). IL-1β production significantly increased after LPS treatment, and gradually decreased with increasing treatment concentration; the same letters (a, b, c, d, e) indicate no significant difference between groups, while different letters indicate significant differences.

[0024] Figure 11 The bar chart shows cell viability at different peptide concentrations: the horizontal axis represents peptide concentration (μM), and the vertical axis represents cell viability (%). Cell viability remained at a high level in all concentration groups, with only a slight decrease at 1600 μM concentration; the same letters (a, b) indicate no significant difference between groups, while different letters indicate significant differences.

[0025] Figure 12 This image shows immunofluorescence staining of P65 protein, including Control, 1 μg / mL LPS, and different concentrations of PYYFH treatment groups. The images are divided into three columns: DAPI (nuclear staining), P65 (red fluorescence), and Merge. The LPS group showed significant P65 nuclear translocation (enhanced fluorescence). With increasing PYYFH concentration, P65 fluorescence gradually decreased, and the degree of nuclear translocation also declined.

[0026] Figure 13 The Western blot plot shows protein expression after LPS damage and treatment with different concentrations of PYYFH: NF-κB, MAPK pathway-related proteins (such as p65, p-JNK, etc.) and internal control β-Action were detected. Compared with the Model group, the expression levels of ordinary proteins and corresponding phosphorylated proteins in the PYYFH group were significantly reduced.

[0027] Figure 14 The scatter plot of VIPs for differentially regulated metabolites is shown in ((A) for the Control-Model group and (B) for the Control-PYYFH group): the horizontal axis represents the VIP score (reflecting the contribution of metabolite differences), and the vertical axis represents the metabolite name; red dots represent upregulated metabolites, and green dots represent downregulated metabolites.

[0028] Figure 15 The bubble chart is for metabolic pathway enrichment analysis. The horizontal axis represents the Rich Factor, the vertical axis represents the metabolic pathway, and the bubbles correspond to the P-values.

[0029] Figure 16 This is a correlation diagram for metabolic pathway enrichment analysis. The horizontal axis represents Impact, and the vertical axis represents the pathway. Points correspond to -log 10 (P) value. Detailed Implementation

[0030] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0031] It should be understood that terms such as "having," "comprising," and "including" used in the embodiments of this application do not exclude the presence or addition of one or more other elements or combinations thereof. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are only used to explain the relative positional relationship and movement of components in a specific posture. If the specific posture changes, the directional indication will also change accordingly. When an element is referred to as "fixed to" or "set on" another element, it can be directly on the other element or may have an intervening element present. When an element is referred to as "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element through an intervening element. Descriptions involving "first," "second," etc., in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features.

[0032] It should be noted that the technical solutions of the various embodiments of this application can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this application.

[0033] The embodiments of this application provide small molecule peptides, the amino acid sequences of which are shown in SEQ ID NO: 1.

[0034] Small peptides are polypeptide fragments formed by linking a small number of amino acid residues together via peptide bonds. They typically have the characteristics of small molecular weight, good biocompatibility, and easy absorption and utilization by the body. The amino acid sequence of this small peptide is the PYYFH sequence formed by sequentially linking proline (P), tyrosine (Y), phenylalanine (F), and histidine (H) (see SEQ ID NO:1 for details).

[0035] The small molecule peptide can be prepared by solid-phase synthesis or enzymatic hydrolysis. In solid-phase synthesis, the first amino acid, proline, is first immobilized on a solid support such as chloromethyl resin or Wang resin. Then, tyrosine, tyrosine, phenylalanine, and histidine are sequentially linked in sequence. Dicyclohexylcarbodiimide can be used as a condensing agent in each linking step. After linking, trifluoroacetic acid is used for deprotection. After all amino acids are linked, a lysis buffer is added to remove the small molecule peptide from the solid support. The lysis buffer can be a mixture of trifluoroacetic acid, water, and triisopropylsilane. Finally, the peptide is purified by high-performance liquid chromatography (HPLC). A C18 column can be used for purification, with acetonitrile and water as the mobile phase. The target peak is collected and freeze-dried to obtain a small molecule peptide with a purity that meets pharmaceutical requirements. The purity can be detected by mass spectrometry, which determines the presence and purity of the target peptide by detecting the mass-to-charge ratio.

[0036] The enzymatic preparation method uses walnut protein as raw material. First, the walnut protein is crushed and passed through a 60-mesh or 80-mesh sieve. An appropriate amount of deionized water is added to prepare a suspension with a protein concentration of 5% or 8%. The pH value of the system is adjusted, and alkaline protease or neutral protease is added for enzymatic hydrolysis. The amount of enzyme added can be 2% of the mass of the raw material. The enzymatic hydrolysis temperature is controlled at 50℃, and the enzymatic hydrolysis time can be 4 hours. After the enzymatic hydrolysis is completed, the system temperature is raised to 90℃ and kept at this temperature for 15 minutes to inactivate the enzyme activity. Then, the system is centrifuged, and the supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3kDa. The permeate is collected and further separated by gel filtration chromatography, then purified by high performance liquid chromatography, and finally freeze-dried to obtain the target small molecule peptide.

[0037] The embodiments of this application also provide the application of small molecule peptides for the preparation of medicaments for the prevention, relief and / or treatment of inflammation.

[0038] Inflammation is a defensive physiological response of the body to stimuli such as infection and tissue damage. When this response is excessive or persistent, it can adversely affect normal tissues. This drug can be used to intervene in such abnormal inflammatory responses. Prevention refers to reducing the likelihood of inflammation by regularly using the drug before inflammation occurs or when the body is in a high-risk state for inflammation. Relief refers to alleviating the discomfort caused by inflammation when it has already occurred. Treatment refers to inhibiting the progression of inflammation and promoting the repair of damaged tissues through the action of the drug. The preparation of this drug can adopt conventional pharmaceutical formulation processes, using small molecule peptides as the core active ingredient, combined with pharmaceutically acceptable excipients, and going through a series of steps such as mixing, granulation, and molding to produce a drug product suitable for clinical use. In the process of exerting its anti-inflammatory effect, this small molecule peptide can reduce the activity of reactive oxygen species in macrophages, increase the levels of superoxide dismutase and catalase to improve the cellular oxidative stress state, and inhibit the production of nitric oxide and pro-inflammatory cytokines such as interleukin-α, interleukin-6, and interleukin-1β, thereby intervening in the occurrence and development of inflammatory responses from multiple aspects.

[0039] In existing technologies, drugs used for inflammation intervention are mostly nonsteroidal anti-inflammatory drugs (NSAIDs) or glucocorticoids. These drugs may require high doses to achieve the desired effect, and long-term use may have potential impacts on the gastrointestinal tract and immune system. This embodiment uses small molecule peptides as the active pharmaceutical ingredient. These small molecule peptides have good biocompatibility, minimal adverse effects on the body, and can intervene in the inflammatory process through targeted mechanisms such as regulating oxidative stress-related indicators and inflammatory factor levels. Compared with drugs in existing technologies, they are safer to use, applicable to a wider range of populations, and can meet the clinical need for mild and effective anti-inflammatory drugs.

[0040] In another embodiment, the inflammation is lipopolysaccharide (LPS)-induced inflammation. In an in vitro cellular model of RAW264.7 macrophage inflammation induced by LPS, the small peptide can stably bind to the key inflammatory factor IL-6 with a binding energy of -6.5 kcal / mol. This binding stability is maintained through multiple mechanisms, including hydrophobic interactions, hydrogen bonds, and salt bridges. Simultaneously, it can reduce the accumulation of intracellular reactive oxygen species, increase the activity of superoxide dismutase and catalase to improve cellular oxidative stress, inhibit excessive nitric oxide production, reduce the release of pro-inflammatory cytokines such as interleukin-α, interleukin-6, and interleukin-1β, and decrease the expression level of p65 protein. It also regulates the expression of proteins such as IκBα and p-IκBα in the NF-κB signaling pathway and p38, JNK, ERK, and their phosphorylated forms in the MAPK signaling pathway, thus blocking the inflammatory cascade at the molecular level. In an in vivo animal model of LPS-induced inflammation, small molecule peptides can influence core metabolic pathways such as glycerophospholipid metabolism and amino acid biosynthesis by regulating key metabolites such as Tyrosyl-Tyrosine and sn-Glycerol3-phosphate. At the same time, they can regulate the expression of inflammation-related proteins and the balance of metabolic networks in tissues, thereby exerting an overall anti-inflammatory effect. The anti-inflammatory effect can be fully confirmed by the improvement of inflammation-related indicators.

[0041] In another embodiment, the dosage form of the drug is at least one of the following: injection, tablet, oral liquid, granules, or capsules. The injection can be a sterile aqueous solution with a pH adjustable between 6.0 and 7.5, administered via intravenous or intramuscular injection, allowing the drug to rapidly enter the bloodstream and exert its effect. Tablets can be made by adding microcrystalline cellulose as a filler and sodium carboxymethyl starch as a disintegrant, and are produced through processes such as mixing, granulation, and tableting, with each tablet containing a small molecule peptide dose of up to 5 mg. Oral liquids can be made by adding sucrose as a sweetener and sodium benzoate as a preservative, resulting in a clear liquid dosage form suitable for children, the elderly, and other individuals with swallowing difficulties. Granules can be prepared using a spray-drying process, with lactose added as an excipient, and are dissolved in warm water before use. Capsules can be made by uniformly mixing small molecule peptides with suitable excipients and then encapsulating them in a hard capsule shell, facilitating precise dosage control.

[0042] Embodiments of this application also provide pharmaceutical compositions characterized by comprising the aforementioned small molecule peptides.

[0043] This pharmaceutical composition may contain the aforementioned small molecule peptide as the core active ingredient, and may be combined with one or more pharmaceutically acceptable excipients. These excipients can optimize the physicochemical properties of the composition and improve the stability and bioavailability of the active ingredient. Pharmaceutically acceptable excipients may include fillers, binders, disintegrants, lubricants, preservatives, solubilizers, etc., wherein the filler may be starch or mannitol, the binder may be povidone K30, the disintegrant may be crospovidone, and the lubricant may be magnesium stearate. The preparation process of this pharmaceutical composition may be as follows: first, the small molecule peptide and the selected excipients are separately pulverized, and then sieved. Then, the pulverized small molecule peptide and excipients are placed in a mixing device according to a preset ratio to ensure that the homogeneity of the mixture meets pharmaceutical requirements. After that, subsequent molding and processing steps are carried out according to the required dosage form.

[0044] The following specific examples further illustrate this.

[0045] Preparation methods of small molecule peptides:

[0046] Using walnut protein as raw material, the walnut protein was first pulverized and passed through an 80-mesh sieve. Substrate solutions of different concentrations were prepared, and 1 mol·L⁻¹ was used. -1 The pH value was adjusted to a suitable range using HCl or NaOH solution. The enzyme was then enzymatically hydrolyzed with alkaline protease at the optimal temperature for a certain period of time. The enzyme was inactivated at 90℃ for 10 min, and centrifuged at 4℃ and 8000 r / min for 20 min. The supernatant was collected and ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was collected and further separated by gel filtration chromatography, then purified by high performance liquid chromatography, and finally freeze-dried to obtain the target small molecule peptide.

[0047] Single-factor experiments were conducted with the following conditions: hydrolysis time (1 h, 2 h, 3 h, 4 h, 5 h); hydrolysis temperature (30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃); substrate concentration (2%, 4%, 6%, 8%, 10%); and hydrolysis pH (5, 6, 7, 8, 9). When investigating different single-factor experimental conditions, fixed experimental conditions were set: hydrolysis time 3 h, hydrolysis temperature 50 ℃, substrate concentration 6%, and hydrolysis pH 7. Based on the results of the single-factor experiments, the three most influential factors on the hydrolysate—hydrolysis temperature, pH, and substrate concentration—were selected as independent variables. Response surface methodology was used, with DPPH free radical scavenging rate as the response value, to determine the optimal conditions for walnut hydrolysate, and verification experiments were performed.

[0048] Based on the Box-Behnken central composite design principle, the optimal parameters for achieving the highest DPPH radical scavenging rate were calculated and analyzed using Design Expert software: enzymatic hydrolysis temperature 50℃, enzymatic hydrolysis pH 8, substrate concentration 8%, and enzymatic hydrolysis time 3h. Under these conditions, the DPPH radical scavenging rate of the hydrolysate was 68.07±1.84%.

[0049] Experiment 1: Molecular simulation to detect the binding status of walnut peptide PYYFH to the inflammatory protein IL-6

[0050] The peptide fragment information of PYYFH was analyzed by secondary spectrum analysis, and the peptide structure was confirmed based on mass-to-charge ratio and relative intensity. After pretreatment of IL-6 protein with PyMOLv2.5.4 software, molecular docking was performed. The protein structure was displayed using the Cartoon model, and the PYYFH and interacting amino acid residues were presented using the stick model. The interaction type was analyzed.

[0051] Figure 1 In the secondary spectrum, the mass-to-charge ratio (m / z) on the horizontal axis shows clear fragment ion peaks in the range of 100-600. Peaks such as y1+158 (corresponding to the C-terminal histidine fragment) and b2+261.12 (corresponding to the N-terminal proline-tyrosine fragment) are sharp and have relatively high intensity. The mass spectrometry characteristics of the PYYFH peptide structure are completely matched with those of the fragment ion peaks, confirming that the peptide has not been degraded and its structure is intact. Figure 2 The 3D molecular docking diagram shows that the IL-6 protein is represented by a Cartoon model, and PYYFH is embedded in the active pocket of IL-6 in a stick-like model. Gray dashed lines represent hydrophobic interactions, blue solid lines represent hydrogen bonds, green dashed lines represent π-π stacking, orange dashed lines represent π-cation interactions, yellow dashed lines represent salt bridges, and yellow spheres represent valence charges. There are a total of 8 pairs of hydrophobic interactions, 6 pairs of hydrogen bonds, 1 pair of salt bridges, 0 pairs of π-π stacking, and 0 pairs of π-cation interactions between the IL-6 protein and the peptide PYYFH.

[0052] PYYFH binds to IL-6 protein at a rate of -6.5 kcal / mol, exhibiting strong binding affinity. Furthermore, it forms multiple stable binding modes through hydrophobic interactions, hydrogen bonds, and salt bridges. This stable binding characteristic can block the interaction between IL-6 and its downstream receptors, inhibiting IL-6-mediated inflammatory signaling and laying a crucial molecular foundation for PYYFH's anti-inflammatory effects.

[0053] Experiment 2: Protective effect of PYYFH peptide on inflammation in RAW264.7 cells

[0054] RAW264.7 cells were treated with PYYFH at concentrations of 0, 50, 100, 200, 400, 800, 1200, 1600, and 1800 μg / mL to assess cell viability and cytotoxicity. Cells were divided into a Control group, a Model group (treated with 1 μg / mL LPS for 24 h), and a PYYFH intervention group (pretreated with 200, 400, and 800 μg / mL PYYFH followed by LPS). ROS levels were detected using a ROS fluorescent probe, and SOD and CAT activities, as well as the levels of NO, IL-α, IL-1β, and IL-6, were detected using a kit.

[0055] Cytotoxicity diagram ( Figure 11 The results showed that the cell viability in the 50-1200 μg / mL PYYFH treatment group was 101.82%-107.06%, which was not significantly different from the control group (100%). P >0.05), while the cell survival rate decreased slightly in the 1600 μg / mL and above concentration groups, indicating that PYYFH in the concentration range of 25-1200 μg / mL has no toxic effect on RAW264.7 cells. LPS damage time map ( Figure 5 In the study, as the culture time increased, the intracellular NO content gradually increased, reaching a peak at 24 h in the Model group, which was significantly higher than that at 8, 12, 16, and 20 h. P <0.01), confirming that 24 hours is the optimal injury time for the LPS-induced RAW264.7 cell inflammation model. ROS fluorescence image ( Figure 7 In the control group, the green fluorescence intensity was the weakest, while the fluorescence intensity of the model group was significantly enhanced. The fluorescence intensity of the low, medium, and high dose PYYFH groups decreased sequentially, showing a concentration-dependent decreasing trend. SOD activity ( Figure 8 ) and CAT activity map ( Figure 3 In the Model group, SOD and CAT activities were significantly lower than those in the Control group. P <0.01), the activities of SOD and CAT in all PYYFH dose groups significantly increased, and the SOD and CAT activities in the high-dose group were close to the levels of the control group. P >0.05). NO and inflammatory factor map ( Figure 4 , Figure 6 , Figure 9 , Figure 10 In the Model group, the levels of NO, IL-α, IL-1β, and IL-6 were significantly higher than those in the Control group. P <0.01), all the above indicators in each PYYFH dose group decreased in a concentration-dependent manner, and the high-dose group showed a significant difference compared with the Model group. P <0.01).

[0056] PYYFH showed no significant toxicity to RAW264.7 cells within a concentration range of 25-1200 μg / mL and exhibited good biocompatibility. In an LPS-induced inflammation model of RAW264.7 cells, PYYFH reduced intracellular ROS accumulation and alleviated oxidative stress damage in a concentration-dependent manner; simultaneously, it significantly increased the activity of antioxidant enzymes such as SOD and CAT, enhancing cellular antioxidant capacity and resisting LPS-induced oxidative stress responses. Furthermore, PYYFH effectively inhibited excessive NO production and reduced the release of pro-inflammatory cytokines such as IL-α, IL-1β, and IL-6. By blocking the LPS-induced inflammatory cascade response at two key stages—oxidative stress regulation and inflammatory factor inhibition—PYYFH exerted a significant protective effect against cellular inflammation, and this protective effect was significantly concentration-dependent, with the highest dose showing the best results.

[0057] Experiment 3: Immunofluorescence co-localization investigation of the effect of PYYFH on inflammation in RAW264.7

[0058] RAW264.7 cells were divided into four groups: Control group (no exogenous stimulation), Model group (treated with 1 μg / mL LPS for 24 h), 200 μg / mL PYYFH group, 400 μg / mL PYYFH group, and 800 μg / mL PYYFH group. All PYYFH groups were pretreated and then treated with LPS for 24 h. After cell treatment, immunofluorescence staining was performed. The cell nuclei were labeled with DAPI, and NF-κBp65 protein was labeled with p65-specific antibody. The localization and fluorescence intensity of p65 protein were observed by fluorescence microscopy.

[0059] RAW264.7 cells were grown at a rate of 2 × 10⁻⁶. 4Individual cells / well were seeded in a laser confocal microscopy culture dish and cultured for 24 h before being divided into the following groups: Control group (no treatment), Model group (1 μg / mL LPS treatment for 24 h), low-dose PYYFH group (200 μg / mL PYYFH pretreatment for 2 h + 1 μg / mL LPS treatment for 24 h), medium-dose PYYFH group (400 μg / mL PYYFH pretreatment for 2 h + 1 μg / mL LPS treatment for 24 h), and high-dose PYYFH group (800 μg / mL PYYFH pretreatment for 2 h + 1 μg / mL LPS treatment for 24 h). After cell treatment, fix with 4% paraformaldehyde at room temperature for 15 min, wash three times with PBS (5 min each time); permeabilize with 0.3% Triton X-100 at room temperature for 10 min, wash with PBS; block with 5% bovine serum albumin (BSA) at room temperature for 30 min, remove the blocking solution, add rabbit anti-mouse NF-κB p65 primary antibody (1:200 dilution), and incubate overnight at 4°C; the next day, wash three times with PBS, add Alexa Fluor 594-labeled goat anti-rabbit secondary antibody (1:500 dilution), and incubate at room temperature in the dark for 1 h; after washing with PBS, add DAPI staining solution (1 μg / mL) and incubate at room temperature in the dark for 5 min to stain the cell nuclei; finally, mount with anti-fluorescence quenching mounting medium, and image using a laser confocal microscope (LSM 880). Excitation wavelength: DAPI 350 nm, Alexa Fluor 594 590 nm; emission wavelength: DAPI 460 nm, Alexa Fluor 594 590 nm. 594 is 617nm. Five fields of view were randomly selected in each group. The fluorescence intensity of p65 protein in the cell nucleus was quantitatively analyzed using ImageJ software. The average fluorescence intensity value was calculated. One-way ANOVA was used for comparison between groups.

[0060] Laser confocal imaging ( Figure 12In the control group, the blue fluorescence stained with DAPI clearly marked the location of the cell nucleus, while the red fluorescence stained with Alexa Fluor 594 represented the expression and localization of p65 protein. In the control group, the red fluorescence intensity was weak and mainly distributed in the cytoplasm, with very little red fluorescence in the cell nucleus, indicating that under normal conditions, p65 protein is mostly located in the cytoplasm and the NF-κB pathway is not activated. In the model group, the red fluorescence intensity was significantly enhanced, and the red fluorescence overlapped greatly with the blue fluorescence. The red fluorescence in the cell nucleus was dense, suggesting that p65 protein underwent nuclear translocation after LPS stimulation, and the NF-κB pathway was activated. The red fluorescence intensity in the cell nucleus was weakened in the low-dose PYYFH group, further decreased in the medium-dose group, and the red fluorescence intensity was the weakest in the high-dose group, with sparse distribution of red fluorescence in the cell nucleus, similar to the control group. This indicates that as the concentration of PYYFH increases, the nuclear expression of p65 protein decreases in a concentration-dependent manner, and p65 nuclear translocation is significantly inhibited. Furthermore, the area of ​​the overlapping region between red and blue fluorescence in the Merge plot gradually decreased with increasing PYYFH concentration, further confirming the inhibitory effect of PYYFH on p65 nuclear translocation.

[0061] LPS stimulation induces nuclear translocation of NF-κB p65 protein in RAW264.7 cells, activating the NF-κB signaling pathway and subsequently initiating the transcriptional expression of inflammation-related genes, triggering an inflammatory response. PYYFH can, in a concentration-dependent manner, inhibit LPS-induced nuclear expression and translocation of p65 protein, reducing p65 protein accumulation in the nucleus, thereby blocking the activation of the NF-κB signaling pathway, inhibiting the transcriptional synthesis of downstream inflammatory factors, and ultimately improving the LPS-induced inflammatory state in RAW264.7 cells. Among the controlled groups, 800 μg / mL PYYFH showed the most significant inhibitory effect on p65 nuclear translocation, with no significant difference compared to the control group, indicating that at this concentration, PYYFH can effectively block the activation of the NF-κB pathway, providing important molecular mechanism support for its anti-inflammatory effect.

[0062] Experiment 4: Western Blot investigation of the effects of PYYFH on NF-κB and MAPK signaling pathway proteins in RAW264.7 cell inflammation.

[0063] RAW264.7 cells were divided into four groups: Control group, Model group (treated with 1 μg / mL LPS for 24 h), 200 μg / mL PYYFH group, 400 μg / mL PYYFH group, and 800 μg / mL PYYFH group. All PYYFH groups were pretreated with LPS and then treated for 24 h. Cells were washed three times with ice-cold PBS, and then lysed on ice for 30 min with RIPA lysis buffer containing 1 mm PMSF. Total protein was obtained from the cells, and protein concentration was determined using a dioctanine acid (BCA) protein assay kit. Total protein was separated by electrophoresis using a 12% SDS-PAGE gel, then transferred to an active PVDF membrane. The membrane was blocked for 1 hour with 5% skim milk in a TBS solution containing 0.1% Tween-20. It was then incubated overnight at 4°C with primary antibodies (NF-KB p65, anti-P-38, anti-P-ERK, anti-P-JNK, anti-P-4IkB-α, anti-IkB-α, anti-P-P38, anti-ERK, anti-JNK). The membrane was then incubated with secondary antibody (conjugated with goat anti-rabbit IgG) at room temperature for 1 hour. The blot was visualized using a chemiluminescence detection kit.

[0064] Protein band images ( Figure 13 The results showed that the β-Actin bands were uniformly bright across all groups, indicating balanced protein loading and reliable experimental results. Among the NF-κB signaling pathway-related protein bands, the brightness of the IκBα, p-IκBα, and p65 protein (nuclear factor κB repressor protein α, phosphorylated nuclear factor κB repressor protein α, and nuclear factor κB p65 subunit) bands in the Model group was significantly higher than that in the Control group. In the PYYFH intervention groups at each dose, the brightness of these protein bands gradually decreased with increasing concentration, and the protein expression levels were significantly lower compared to the Model group. P >0.05%. Among the MAPK signaling pathway-related protein bands, the band brightness of p-p38, p-JNK, and p-ERK proteins (phosphorylated p38 protein, phosphorylated JNK protein, and phosphorylated ERK protein) in the Model group was significantly higher than that in the Control group, while the brightness of the total p38, JNK, and ERK protein bands did not differ significantly among the groups. P >0.05); The intensity of p-p38, p-JNK, and p-ERK protein bands in each PYYFH dose group gradually decreased with increasing concentration, and the high-dose group showed a significant difference compared to the Model group ( P The total protein expression level remained unchanged (<0.01), indicating that PYYFH mainly inhibits the phosphorylation activation of key proteins in the MAPK pathway, rather than affecting the synthesis of total protein.

[0065] LPS stimulation significantly activated the NF-κB and MAPK signaling pathways in RAW264.7 cells, manifested as upregulation of IκBα, p-IκBα, and p65 protein expression in the NF-κB pathway and increased phosphorylation levels of p38, JNK, and ERK proteins in the MAPK pathway. PYYFH inhibited NF-κB signaling pathway activation and reduced the transcription of inflammation-related genes by concentration-dependently reducing the expression of IκBα, p-IκBα, and p65 proteins. Simultaneously, PYYFH specifically inhibited the phosphorylation of p38, JNK, and ERK proteins, blocking MAPK signaling pathway transduction and thus inhibiting the production of downstream pro-inflammatory factors. Furthermore, PYYFH had no effect on total protein expression in the MAPK pathway, indicating that its target is the phosphorylation modification stage of proteins and possesses high specificity. In conclusion, PYYFH exerts a significant anti-inflammatory effect by synergistically regulating the two key inflammatory signaling pathways of NF-κB and MAPK.

[0066] Experiment 5: Non-targeted metabolomics of LPS-damaged RAW264.7 cells by UPLC-Q / TOF-MS

[0067] Cells were grouped as in Experiment 3. Cell samples were collected and metabolites were extracted. Metabolite detection was performed using UPLC-Q / TOF-MS technology. The differences in metabolites among groups were analyzed using the OPLS-DA model. Differential metabolites with high VIP values ​​were screened, and KEGG enrichment analysis was performed on the differential metabolites to explore related metabolic pathways.

[0068] For differentially derived metabolites obtained by comparing the Control group with the model group, see [link to relevant documentation]. Figure 14(A), differential metabolites (ordinate) include: Oxooctanoylcarnitine (Car8:1-O), Lauryl sulfate, 3-[(5,6-Dimethylthieno[2,3-d]pyrimidin-4-yl)amino)propanoic acid (DMTP-β-Ala), Methyl 4-(trifluoromethyl)benzoylacetate, sn-Glycerol-3-phosphate, Histamine, Glycerophosphoethanolamine, 1-(9Z-tetradecenoyl)-sn-glycero-3-phosphocholine (LysoPC 14:1(9Z)), 1,3-dihydroxy-N-methylacridone, 3,4,8,10-tetrahydroxy-2-(hydroxymethyl)-9-methoxy-3,4,4a,10b-t etrahydro-2H-pyrano[3,2-c]isochromen-6-one (bergenin), 6,6'-Dihydroxy-5,5'-dimethoxybiphenyl-3-carboxylic acid (DHDMB-CA), Glochidic Acid, N-Arachidonoyl dopamine, 2-Amino-2-methyloctanoic acid, LPE(22:4), (6E,12E)-8,9,16,18-tetrahydroxy-4-methyleicosa-6,12-dienoicacid (20:4(6E,12 E)(8,9,16,18-tetraOH,4-Me)), 1-(2-Hydroxyethyl)-2,2,6,6-tetramethyl-4-piperidinol, Lysyl-Leu cine, CPI-637 (full name: (R)-4-Methyl-6-(1-methyl-3-(1-methyl-1H-pyrazol-4-yl)-1H-indazol-5-yl)-4,5 -dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one), 4-(1,3-Dioxoisoindolin-2-yl)-2-hydroxybenzoic acid, namely octenylcarnitine (or 8:1-O-carnitine), lauryl sulfate, 3-((5,6-dimethylthiophene[2,3-d]pyrimidin-4-yl)amino)propionic acid, methyl 4-(trifluoromethyl)benzoylacetate, glycerol-3-phosphate (or glycerol 3-phosphate), histamine, glycerol phosphoryl ethanolamine, 1-(9Z)-tetradecano-9-enoyl-sn-glycerol-3-phosphate choline, 1,3-dihydroxy-N-methylacridone, bergenin, 6,6'-dihydroxy-5,5'-dimethoxybiphenyl-3-carboxylic acid, arachidic acid, N-arachidonicyl dopamine (NADA), 2-amino-2-methyloctanoic acid, lysophosphatidylethanolamine (22:4) ), (6E,12E)-8,9,16,18-tetrahydroxy-4-methyleicosicosano-6,12-dienoic acid, 1-(2-hydroxyethyl)-2,2,6,6-tetramethyl-4-piperidinol, lysine-leucine, (R)-4-methyl-6-[1-methyl-3-(1-methyl-1H-pyrazol-4-yl)-1H-indazol-5-yl]-4,5-dihydro-1H-benzo[b][1,4]diazazo-2(3H)-one, 4-(1,3-dioxoisoindoline-2-yl)-2-hydroxybenzoic acid. Among the metabolites mentioned above, Histamine, N-Arachidonoyl Dopamine (NADA), LPE (22:4) (lysophosphatidylethanolamine (22:4)), sn-Glycerol 3-phosphate, and Oxooctanoylcarnitine are associated with inflammation. In inflammatory states, cellular metabolic patterns undergo significant alterations (the "Warburg effect"). Glycerol 3-phosphate is a crucial link between carbohydrate and lipid metabolism, and lipid metabolism products (such as prostaglandins and leukotrienes) are potent inflammatory mediators. Therefore, changes in its metabolic flux are an important component of the inflammatory response. Figure 14 (A) It can be seen that sn-Glycerol 3-phosphate is upregulated in the model group, indicating that LPS may induce inflammation by regulating sn-Glycerol 3-phosphate.

[0069] Metabolites obtained by comparing the control group with the PYYFH intervention group are shown in [reference]. Figure 14(B), Differential metabolites (vertical axis) include: 2-Amino-2-methyloctanoic acid, Oxooctanoylcarnitine (Car8:1-O), Lauryl sulfate, CPI-637 (full name (R)-4-Methyl-6-(1-methyl-3-(1-methyl-1H-pyrazol-4-yl)-1H-indazol-5-yl)-4,5-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one), Tyrosyl-Tyrosine, (3beta,23E)-3-Hydroxy-27-norcyclart-23-en-25-oic acid, 3-methyl-N-(2-methylpropyl)butanamide, N-acetyl-3-mercapto-DL-valine, 1-(2-Hydroxyethyl)-2,2,6,6-tetramethyl-4-piperidinol, L-prolyl-L-tyrosine, Octenedioylcarnitine (Car8:1-0 2), 4-(1-Dioxoisoindolin-2-yl)-2-hydroxybenzoicacid, 1-Tetracosanoyl-sn-glycero-3-phosphocholine, Uridine, 5-(2-(4-Chlorophenyl)hydrazono)-2,2-dimethyl-1,3-cyclohexanedione, 11-Amin oundecanoicacid, Scopine, Car(18:1), 1-(pyridin-2-yl)-1H-pyrazole-4-carboxylicacid, Ethiofencarb, i.e., 2-amino-2-methyloctanoic acid, oxyoctanoic acid carnitine (Car(8:1-O)), lauryl sulfate, (R)-4-methyl-6-[1-methyl-3-(1-methyl-1H-pyrazol-4-yl)-1H-indazole-5-yl] ]-4,5-dihydro-1H-benzo[b][1,4]diazazo-2(3H)-one, tyrosyl-tyrosine, (3β,23E)-3-hydroxy-27-nor-atun-23-en-25-acid, 3-methyl-N-(2-methylpropyl)butyramide, N-acetyl-3-mercapto-DL-valine, 1-(2-hydroxyethyl)-2,2,6,6-tetramethyl-4-piperidinol, L-prolyl-L-tyrosine, octenylcarnitine (Car(8:1-02)), 4-(1,3-Dioxoisoindoline-2-yl)-2-hydroxybenzoic acid, 1-tetracosyl-sn-glycerol-3-phosphorylcholine, uridine, 5-(2-(4-chlorophenyl)hydrazine)-2,2-dimethyl-1,3-cyclohexanedione, 11-aminoundecanoic acid, scopolamine, octadecanoylcarnitine, 1-(2-pyridyl)-1H-pyrazole-4-carboxylic acid, ethylthiocarboxamide.

[0070] Among the metabolites mentioned above, lauryl sulfate, 1-tetracosanoyl-sn-glycero-3-phosphochol, N-acetyl-3-mercapto-DL-valine, Car(18:1), Octenedioylcarnitine (Car(8:1-02)), and Tyrosyl-Tyrosine are associated with inflammation. Tyrosine kinase-mediated inflammatory signaling regulation: Tyrosyl-tyrosine is a substrate or product of tyrosine kinases (such as Src family kinases), and tyrosine kinases are important signaling molecules for the activation of inflammatory cells (such as macrophages and neutrophils). In inflammatory states, tyrosine kinases are activated, phosphorylating key transcription factors such as NF-κB and MAPK, and promoting the expression of pro-inflammatory factors (such as TNF-α and IL-6). Changes in tyrosyl-tyrosine levels may reflect the activity state of tyrosine kinases, thereby affecting the transmission of inflammatory signals. Markers and regulation of oxidative stress: Tyrosine is easily oxidized by reactive oxygen species (ROS) to generate tyrosyl radicals, which in turn form tyrosyl-tyrosine dimers. During inflammation, large amounts of ROS are produced, leading to the accumulation of tyrosyl-tyrosine, which may further exacerbate oxidative stress, forming a positive feedback loop of "oxidation-inflammation." Furthermore, tyrosyl-tyrosine can serve as a marker of oxidative stress; elevated levels indicate increased oxidative damage in the inflammatory microenvironment. Association with metabolic reprogramming: In inflammation-related metabolic reprogramming, tyrosine metabolism is enhanced. Tyrosyl-tyrosine, as an intermediate product, may participate in regulating energy metabolism (e.g., by affecting mitochondrial function) or amino acid metabolism, indirectly affecting the survival and function of inflammatory cells. In summary, Tyrosyl-Tyrosine is closely related to the occurrence and development of inflammation by regulating tyrosine kinase signaling, participating in oxidative stress, and metabolic reprogramming, and is one of the key metabolites connecting inflammatory signaling pathways and metabolic abnormalities. It can be seen that Tyrosyl-Tyrosine is upregulated in the PYYFH intervention group, indicating that PYYFH may improve inflammation by regulating Tyrosyl-Tyrosine.

[0071] KEGG enrichment analysis was performed on the differentially expressed metabolites between the control group and the PYYFH intervention group, and the results were obtained. Figure 15 and Figure 16 . Figure 15 This is a bubble chart. Figure 16This diagram illustrates the connections between various metabolic pathways, encompassing over forty different types, including: Biosynthesis of amino acids, Neuroactive ligand-receptor interaction, Renal cell carcinoma, Pathways in cancer, Inflammatory mediator regulation of TRP channels, Inositol phosphate metabolism, Synaptic vesicle cycle, Neomycin, kanamycin and gentamicin biosynthesis, Glycolysis / Gluconeogenesis, Citrate cycle (TCA cycle), Glyoxylate and dicarboxylate metabolism, Pyrimidine metabolism, Taste transduction, and Etherlipidemia. Metabolism (ether lipid metabolism pathway), Starch and sucrose metabolism, Choline metabolism in cancer, Central carbon metabolism in cancer, Glutathione metabolism, Glycerophospholipid metabolism, Carbon metabolism, Fatyacid biosynthesis, Purine metabolism, Arginine and proline biosynthesis, Histidine metabolism, Alanine...Aspartate and glutamate metabolism pathway, fructose and mannose metabolism pathway, pantothenate and CoA biosynthesis pathway, aminoacyl-tRNA biosynthesis pathway, galactose metabolism pathway, lysine biosynthesis pathway, valine, leucine and isoleucine biosynthesis pathway, ascorbate and aldarate metabolism pathway, beta-alanine metabolism pathway, N-glycan biosynthesis pathway, and glycine metabolism pathway.The pathways encompassing glycine, serine, and threonine metabolism, thiamine metabolism, tryptophan metabolism, ubiquinone and other terpenoid-quinone biosynthesis, pentose phosphate pathway, pentose and glucuronate interconversions, selenium compound metabolism, pyrimidine and purine biosynthesis, cysteine ​​and methionine metabolism, taurine and hypotaurine metabolism, and amino sugar and nucleotide sugar metabolism, among others, cover multiple dimensions of cellular metabolism, including amino acid synthesis, energy metabolism, signal transduction, substance synthesis, and cancer-related metabolism, comprehensively reflecting the metabolic changes in cells under inflammatory conditions.

[0072] In the bubble chart, the redder the bubble, the stronger the statistical significance of pathway enrichment; the larger the bubble, the more differential metabolites are contained in the pathway; the higher the value of the "enrichment factor" on the horizontal axis, the more prominent the proportion of differential metabolites in the pathway. As can be seen from the figure, the bubbles corresponding to Glycerophospholipid metabolism and Biosynthesis of amino acids are not only the reddest and largest in area, but also have high enrichment factors, making them the pathways with the most concentrated differential metabolites and the strongest statistical significance. The enrichment levels of pathways such as Carbon metabolism, Glycolysis / Gluconeogenesis, Citrate cycle (TCA cycle), and Fatty acid biosynthesis are relatively lower. On the other hand, the bubbles of pathways such as Renal cell carcinoma, Pathways in cancer, Taste transduction, and Synaptic vesicle cycle are lighter in color and smaller in area, and have relatively lower enrichment factors and significance, indicating that the distribution of differential metabolites in these pathways is less and their participation in inflammatory metabolic disorders is relatively limited.

[0073] The higher the "influence value" on the horizontal axis of the correlation diagram, the more critical the pathway's role in the overall cellular metabolism; the depth of the color of the dot corresponds to the significance of the pathway enrichment. From this analysis, the glycerophospholipid metabolism pathway remains prominent, ranking highly in both impact value and enrichment significance. Additionally, the starch and sucrose metabolism pathway also exhibits high impact values ​​and strong enrichment significance, indicating that these two pathways play a more central role in the metabolic network. The citric acid cycle (TCA cycle), pyruvate metabolism, biosynthesis of amino acids, and carbon metabolism pathways have moderate impact values ​​and significance. Meanwhile, the neuroactive ligand-receptor interaction pathway, inositol phosphate metabolism pathway, neomycin, kanamycin, and gentamicin biosynthesis pathway, and Taste... The low impact values ​​of pathways such as transduction (taste transduction pathway) indicate that their functional importance in the overall metabolic network is relatively weak, but they still participate in inflammation-related regulatory processes through their own metabolic changes.

[0074] Combining the information from the two sub-graphs reveals that LPS-induced inflammation extensively disrupts cellular metabolism. Differential metabolites are distributed across all forty-plus pathways, involving multiple key aspects such as cellular synthesis, energy supply, signal transduction, and cancer-related metabolism, fully demonstrating the comprehensive impact of inflammation on the metabolic system. Glycerophospholipid metabolism and the Biosynthesis of amino acids pathway are the core targets of inflammatory metabolic disorders. These pathways not only have a high concentration of differential metabolites but also possess the highest functional importance within the metabolic network, representing a crucial breakthrough in regulating inflammatory metabolic imbalances. The anti-inflammatory effect of PYYFH does not target a single pathway but rather works by synergistically regulating these two core pathways, as well as the Carbon metabolism pathway and Starch and sucrose pathways. It can regulate other pathways involved in inflammatory metabolism, such as starch and sucrose metabolism pathways and glycolysis / gluconeogenesis pathways, while also moderately regulating other pathways involved in inflammatory metabolism. This comprehensively reverses the metabolic imbalance caused by inflammation, fully demonstrating its anti-inflammatory characteristics of "multi-target and multi-pathway synergy". It also provides strong metabolomics support for the stability and reliability of its anti-inflammatory effects.

[0075] LPS-induced inflammation in RAW264.7 cells is accompanied by significant metabolic network disruption, manifested as abnormal expression of multiple inflammation- and oxidative stress-related metabolites, as well as imbalances in core metabolic pathways such as amino acid biosynthesis and glycerophospholipid metabolism. PYYFH intervention significantly reversed LPS-induced metabolic disturbances by upregulating Tyrosyl-Tyrosine and regulating sn-Glycerol 3-phosphate expression, targeting two key pathways: amino acid biosynthesis and glycerophospholipid metabolism. Regulation of the amino acid biosynthesis pathway promotes the synthesis of anti-inflammatory amino acids, enhancing cellular antioxidant and anti-inflammatory capabilities; intervention in the glycerophospholipid metabolism pathway improves cell membrane structural integrity and reduces the release of inflammatory mediators. Furthermore, PYYFH also has a regulatory effect on pathways such as neuroactive ligand-receptor interactions, indicating that its anti-inflammatory effect involves the synergistic regulation of multiple metabolic pathways, rather than single-target intervention. This experiment reveals the potential metabolic mechanism by which PYYFH improves LPS-induced cellular inflammation from a metabolomics perspective.

[0076] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A small molecule peptide, characterized in that, The amino acid sequence of the small molecule peptide is shown in SEQ ID NO:

1.

2. The application of the small molecule peptide as described in claim 1, characterized in that, Used to prepare a medicine for the prevention, relief and / or treatment of inflammation; said inflammation being lipopolysaccharide-induced inflammation.

3. The application of the small molecule peptide as described in claim 2, characterized in that, The dosage form of the drug is at least one of the following: injection, tablet, oral liquid, granules, or capsule.

4. A pharmaceutical composition, characterized in that, Includes the small molecule peptides described in claim 1.

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