Cornu cervi pantotrichum active peptide and application thereof in anti-inflammation, anti-oxidation and anti-aging

Abstract

The invention discloses a pilose antler active peptide and application thereof in anti-inflammation, anti-oxidation and anti-aging, and belongs to the technical field of biology. According to the invention, the brand-new active peptide SPLASDLDL screened out from pilose antler for the first time is utilized, and competitive inhibition on TNFR1 is realized through the characteristic that the brand-new active peptide SPLASDLDL is specifically combined with a TNFR1 receptor. A constructed sertoli cell senescence model proves that the polypeptide has anti-inflammatory, anti-oxidation and anti-senescence effects. The invention successfully solves the technical problem that in the prior art, the competitive inhibition TNFR1 targeting peptide cannot be found from pilose antler and the action mechanism of the TNFR1 targeting peptide cannot be clarified, and provides the action mechanism and scientific basis for developing novel anti-inflammatory and anti-aging drugs derived from natural products.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a deer antler active peptide and its application in anti-inflammatory, antioxidant and anti-aging effects. Background Technology

[0002] The biological effects of TNF-α are achieved through binding to two receptors on the cell membrane: tumor necrosis factor receptor 1 (TNFR1) and tumor necrosis factor receptor 2 (TNFR2). TNFR1 is widely expressed on the surface of almost all somatic cells and is the main receptor mediating the pro-inflammatory and pro-apoptotic effects of TNF-α. When TNF-α binds to TNFR1, it recruits various intracellular adaptor proteins to form a signaling complex, which in turn activates downstream signaling pathways such as nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK). Activation of NF-κB can promote the expression of inflammatory factors such as interleukin-6 (IL-6) and IL-8, enhancing the inflammatory response. Therefore, competitively inhibiting the binding of TNFR1 to TNF-α can effectively block these harmful signaling pathways.

[0003] Deer antlers are the unossified, densely haired young antlers of male sika deer (Cervus nippon Temminck) or red deer (Cervus elaphus Linnaeus). Deer antlers are rich in proteins, polysaccharides, and polyamines, and possess a wide range of pharmacological activities, including anti-inflammatory, antioxidant, and anti-aging effects.

[0004] Although existing technologies have recognized that inhibiting the TNF-α / TNFR1 interaction can be an effective means of treating inflammatory diseases, and deer antler, as a traditional medicinal material, has been proven to have significant anti-inflammatory activity, there are still obvious limitations and technological gaps in current research. Existing technologies have failed to successfully screen out active peptides that can specifically target TNFR1 from deer antler, a natural resource. Summary of the Invention

[0005] The purpose of this invention is to provide a deer antler bioactive peptide and its application in anti-inflammatory, antioxidant, and anti-aging activities, thereby addressing the problems existing in the prior art. This invention utilizes SPLASDLDL, a novel bioactive peptide screened for the first time from deer antler, which competitively inhibits TNFR1 through its specific binding to the TNFR1 receptor. The anti-inflammatory, antioxidant, and anti-aging effects of this peptide were confirmed using a constructed testicular supporting cell aging model. This invention successfully solves the technical problem of prior art failing to discover a competitive inhibitory peptide targeting TNFR1 from deer antler and elucidate its mechanism of action, providing a mechanism of action and scientific basis for the development of novel anti-inflammatory and anti-aging drugs derived from natural products.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a deer antler active peptide, the amino acid sequence of which is shown in SEQ ID NO.14.

[0008] The present invention also provides the application of the above-mentioned deer antler active peptide in the preparation of anti-inflammatory drugs.

[0009] Furthermore, the drug exerts its anti-inflammatory effect by competitively inhibiting the binding of TNFR1 to TNF-α.

[0010] The present invention also provides the application of the above-mentioned deer antler active peptide in the preparation of antioxidant drugs.

[0011] Furthermore, the drug exerts its antioxidant effect by competitively inhibiting the binding of TNFR1 to TNF-α.

[0012] This invention also provides the application of the above-mentioned deer antler active peptides in the preparation of anti-aging drugs.

[0013] The present invention also provides an anti-inflammatory drug, wherein the drug uses the above-mentioned deer antler active peptide as an active ingredient.

[0014] Optionally, the drug may also contain a pharmaceutically acceptable carrier or excipient.

[0015] The present invention discloses the following technical effects:

[0016] This invention provides a novel deer antler bioactive peptide with competitive inhibition of TNFR1, its amino acid sequence being Ser-Pro-Leu-Ala-Ser-Asp-Leu-Asp-Leu (SPLASDLDL). Using surface plasmon resonance (SPR) technology, this invention confirmed the high affinity of this peptide for TNFR1, demonstrating its ability to competitively inhibit the binding of TNF-α to TNFR1. Furthermore, a constructed testicular Sertoli cell aging model confirmed the anti-inflammatory, antioxidant, and anti-aging effects of this peptide. This invention successfully solves the technical problem of prior art failing to discover competitive TNFR1-targeting peptides from deer antler and elucidate their mechanisms of action, providing a mechanism of action and scientific basis for the development of novel anti-inflammatory and anti-aging drugs derived from natural products. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The sensor response diagram of PAP after passing through the blank chip;

[0019] Figure 2 The response diagram of PAP through a chip sensor containing TNFR1 receptor;

[0020] Figure 3 Here is the chemical structural formula of the synthetic peptide SP;

[0021] Figure 4 Liquid chromatography for the synthesis of peptide SP;

[0022] Figure 5 Here is the chemical structural formula of the synthetic peptide PT;

[0023] Figure 6 Liquid chromatography for the synthesis of peptide PT;

[0024] Figure 7 To determine the binding kinetics of TNFR1 and PT using SPR technology;

[0025] Figure 8 Results for TNFR1 and PT affinity;

[0026] Figure 9 To determine the binding kinetics of TNFR1 and SP using SPR technology;

[0027] Figure 10 Results for the affinity of TNFR1 and SP;

[0028] Figure 11 The toxic effects of different concentrations of SP on TM4 cells;

[0029] Figure 12 Different concentrations of SP were used to improve the viability of D-gal-induced TM4 cells;

[0030] Figure 13 Effect of SP on D-gal-induced tight junction protein (ZO-1) in TM4 cells; scale bar is 100 μm;

[0031] Figure 14 The effect of SP on TNF-α induced by D-gal in TM4 cells;

[0032] Figure 15 The effect of SP on MDA, a marker of oxidative stress induced by D-gal, in TM4 cells;

[0033] Figure 16 The effect of SP on GSH-Px, a marker of oxidative stress induced by D-gal, in TM4 cells;

[0034] Figure 17The effect of SP on CAT, a marker of oxidative stress induced by D-gal, in TM4 cells;

[0035] Figure 18 The effect of SP on SOD, a marker of oxidative stress induced by D-gal, in TM4 cells;

[0036] Figure 19 The uptake of SP by cells at different incubation times; scale bar is 100 μm.

[0037] Figure 20 The effect of SP on the mRNA expression level of C-JUN in D-gal-induced TM4 cells;

[0038] Figure 21 The effect of SP on the mRNA expression level of JNK in D-gal-induced TM4 cells;

[0039] Figure 22 The effect of SP on the mRNA expression level of p38 in D-gal-induced TM4 cells;

[0040] Figure 23 The effect of SP on TNF-α mRNA expression level in D-gal-induced TM4 cells;

[0041] Figure 24 The effect of SP on the mRNA expression level of ZO-1 in D-gal-induced TM4 cells. Detailed Implementation

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0045] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0046] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0047] Example 1

[0048] 1. Materials

[0049] 1.1 Materials

[0050] The fresh sika deer antlers were purchased from Shuangyang Deer Farm in Changchun, Jilin Province, and identified by Professor Wei Jixiang of Jilin Agricultural University as sika deer antlers.

[0051] 1.2 Reagents

[0052] D-galactose was purchased from Shanghai Maclean Biotechnology Co., Ltd.; TNF-α inhibitor (Pomalidomide) was purchased from MCE; tocopherol was purchased from Meilun Biotechnology Co., Ltd.; CCK-8 reagent, SOD, MDA, CAT, and GSH-Px detection kits were all purchased from Beyotime Biotechnology Co., Ltd.; ready-to-use dialysis bags, TNF-α, FSH, LH, and sex hormone T detection kits were all purchased from Shanghai Youxuan Biotechnology Co., Ltd.; LC-MS grade methanol was purchased from Fisher Scientific; the primary antibody for ZO-1 was purchased from Wuhan Sanying Biotechnology Co., Ltd.; TNF-α (catalog number 26405-1-AP) was purchased from Thermo Fisher Scientific; and TNFR1 (catalog number 50496-M08H) was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0053] 1.3 Instruments

[0054] Low-speed benchtop centrifuge (TDZ4K); electronic balance (FA1004N); optical microscope (Olympus BX51); Nikon image analysis system (NIS-ELEMNT BR); Q Exactive Plus LC-MS / MS (Thermo Fisher Scientific, USA); TurboIonSpray ion source (AB SCIEX, USA); UPLC (Ultra-High Performance Liquid Chromatography) system (Thermo Fisher Scientific, USA); H1850-R refrigerated centrifuge (Xiangyi); KQ-800DE ultrasonic cleaner (Shumei); FW-100 high-speed grinder (Beijing Zhongxing Weiye Instrument Co., Ltd.); DHG-9140A constant temperature drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.); Mili-QAdvantage A10 ultrapure water system (Millibert, USA); low-speed benchtop centrifuge (TD24K, Changsha Gaoke Xiangyi Centrifuge Instrument Co., Ltd.); electronic balance (FA1004N, Shanghai Precision Scientific Instrument Co., Ltd.); 12-channel semi-automatic peptide synthesizer (Shanghai Qiangyao Biotechnology Co., Ltd.).

[0055] 2. Method

[0056] 2.1 Screening of deer antler bioactive peptides that competitively inhibit TNFR1

[0057] 2.1.1 Preparation of deer antler polypeptide (PAP)

[0058] After thawing frozen fresh sika deer antlers, they were cut into thin slices approximately 1-2 mm thick. The slices were rinsed with pre-cooled distilled water at 4°C to remove surface blood and impurities. They were then placed in a vacuum freeze dryer and dehydrated at -50°C and 0.05 mBar for 12 hours. The thoroughly dried antler slices were removed, the surface hairs were removed, and the powder was pulverized using a high-speed grinder and passed through a 100-mesh sieve to obtain coarse antler powder. Soxhlet extraction was performed using anhydrous ethanol as the solvent for 6 consecutive hours to remove fats and pigments, yielding defatted antler powder. This powder was then sealed and stored at -20°C for later use.

[0059] Accurately weigh 0.5 g of defatted deer antler powder and add it to 30% ethanol solution at a liquid-to-solid ratio of 16 mL / g. Sonicate the solution at 9℃ for 30 min, then centrifuge at 4℃ and 4000 r / min for 15 min. Collect the supernatant as the deer antler protein extract. Add enzyme at a ratio of deer antler protein to pepsin of 100:3 (w / w) and enzymatically hydrolyze the extract in a 50℃ water bath for 4 h. After enzymatic hydrolysis, heat in a 100℃ water bath for 10 min to inactivate enzyme activity. After cooling, centrifuge at 4℃ and 3800 r / min for 20 min. Filter the supernatant and dialyze the resulting filtrate in ultrapure water at 4℃ for 12 h (changing the water every 4 h). Finally, freeze-dry the dialysate to obtain deer antler polypeptide (PAP), which is then sealed and stored at 4℃.

[0060] 2.1.2 Surface Plasmon Resonance (SPR) Technology

[0061] The freeze-dried antler peptide (PAP) sample was removed from its sealed storage environment at 4°C, while the receptor protein TNFR1 was removed from -20°C for later use. Six buffers required for the SPR experiment were prepared, including: analyte buffer (1% DMSOPBST: 1×PBS, 1% DMSO, 0.005% Tween 20), ligand buffer (HEPES: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20, pH 7.4), fixation buffer (10 mM Sodium Acetate, pH 4.5), activation buffer (0.4 M EDC + 0.1 M NHS), blocking buffer (1 M Ethanolamine hydrochloride), and regeneration buffer (10 mM Glycine-HCl, pH 2.0). The CM5 series sensor chip was removed and installed in the Biacore T200 instrument. The system was started, and the tubing was flushed and baseline equilibrated using deionized water.

[0062] The TNFR1 receptor protein was diluted to 20 μg / mL with immobilization buffer and injected into the Fc2 sample channel of the CM5 chip at a flow rate of 10 μL / min. The reference channel Fc1 was not immobilized with any ligands and underwent the same activation and blocking treatment. This channel will generate and acquire Blank signals in real time in all subsequent binding experiments to subtract system background factors such as buffer refractive index changes and non-specific binding. After immobilization, the temperature was set to 22℃, the analyte injection flow rate to 20 μL / min, the binding time to 100 s, and the dissociation time to 180 s. The system was switched to the preset multi-cycle kinetic method. After each cycle, regeneration buffer was injected at a flow rate of 200 μL / min for 30 s. During the experiment, the system synchronously flowed analyte solutions of the same concentration (PAP) sequentially through Fc1 (Blank channel) and Fc2 (TNFR1 immobilization channel) in ascending order of concentration. The system automatically records and processes the response values ​​of both channels in real time, ultimately generating a Blank curve (Fc1 channel) for each analyte concentration point, and a binding response curve for the Fc2 channel. By subtracting the corresponding Fc1 Blank curve from the Fc2 curve, the pure specific binding signal can be obtained.

[0063] The specific binding sensor data acquired by the SPR instrument, after background subtraction via the reference channel, were imported into statistical software for analysis. A 1:1 Langmuir binding model was selected to globally fit the binding and dissociation curves of different concentrations of PAP analytes, directly calculating and reporting the kinetic parameters.

[0064] 2.1.3 Liquid Chromatography-Mass Spectrometry (LC-MS)

[0065] The lyophilized powder of deer antler polypeptide (PAP), which was confirmed by SPR assay to have binding activity with the TNFR1 receptor, was extracted and processed using standard chemical methods to prepare a sample for analysis.

[0066] Following the analytical method described in WIPO publication WO 03 / 006492, liquid chromatography-mass spectrometry (LC-MS) was used. The sample was injected into the LC-MS system, and the components were separated by liquid chromatography. The separated components were then directly introduced into the mass spectrometer for ionization and mass analysis. The mass spectrometry system acquired mass spectrometric data of peptide ions (including precursor ion and fragment ion information). The obtained mass spectrometric data were compared with a protein sequence database to identify the amino acid sequence of the specific polypeptide contained in the natural extract (PAP).

[0067] 2.1.4 Synthesis of deer antler polypeptides

[0068] Peptides were synthesized using solid-phase synthesis, and the crude peptides were purified using high-performance liquid chromatography (HPLC). The target peptide solution was then collected and concentrated in a lyophilizer, and freeze-dried into a white powder.

[0069] 2.1.5 Competitive Binding Inhibition Experiment

[0070] Extract the receptor protein TNFR1 and ligand TNF-α for later use. Prepare the required buffer solution and prepare different concentrations (0-200 µM) of deer antler peptide PAP. Install the CM5 sensor chip into the instrument.

[0071] TNF-α was immobilized in the Fc2 channel of a CM5 chip, while the Fc1 channel remained unimmobilized. In four separate Eppendorf tubes, 100 µM of TNFR1 protein was mixed with different concentrations (0, 50, 100, 200 µM) of synthetic PAP peptides and incubated for 30 min to form pre-complexes. Subsequently, a multi-cycle method was used, with a temperature of 22 °C, a flow rate of 20 µL / min, a binding time of 100 s, and a dissociation time of 180 s. TNFR1-PAP complexes with different inhibitor concentrations were sequentially injected as analytes into the Fc2 channel of the chip immobilized with TNF-α. Simultaneously, a 100 µM TNFR1 protein solution without inhibitor was injected as a control. The system monitored and recorded the binding response signals of TNF-α to TNFR1 in different solutions in real time.

[0072] The raw data processing used the maximum binding response of TNF-α to TNFR1 without inhibitors as 100%, and calculated the relative binding rate (%) at each inhibitor concentration. Then, using a four-parameter logistic model, the dose-response curve was fitted with the logarithm of the inhibitor concentration (µM) on the x-axis and the relative binding rate on the y-axis to calculate the half-maximal inhibitory concentration (IC50) at which the inhibition rate reached 50%. 50 .

[0073] 2.2 Study on the effect of peptides in improving D-galactose-induced testicular Sertoli cell (TM4) damage

[0074] 2.2.1 Primary isolation, purification, and culture of testicular Sertoli cells (TM4 cells)

[0075] Ten Kunming mice aged 16-22 days were used. Testicular tissue was extracted under aseptic conditions, and the capsule and blood vessels were carefully dissected. The tissue was repeatedly rinsed with Hank's balanced salt solution. The tissue was minced to 1 mm and washed twice by centrifugation with D-Hank's balanced salt solution to remove blood cells. The tissue sample was placed in a digestion solution containing protease (20 μg / mL DNAse) and digested in a 37°C water bath with shaking for 30 min. After digestion, pre-chilled Hank's solution containing 2 mg / mL BSA was added to terminate the reaction. The supernatant was discarded after centrifugation at 1000 r / min to remove interstitial cells and other impurities. Subsequently, the precipitated tissue was digested a second time using 0.2% collagenase, 15 μg / mL DNAse, and 0.15% hyaluronidase, and digested again in a 37°C water bath with shaking for 45 min. The reaction was terminated with fetal bovine serum and pre-cooled D-Hank's solution. The cell suspension was filtered through a 200-mesh filter to remove fascia tissue and washed three times with D-Hank's solution (1000 r / min, centrifuged for 3 min). Finally, the cell pellet was resuspended in 4 mL of 1×Hank's solution.

[0076] The uniformly pipetted cell suspension was mixed with an equal volume of 1 / 8 HBSS hypotonic solution and centrifuged at 1000 r / min for 8 min at 25°C. The supernatant was discarded to further remove residual impurities. Subsequently, the cell pellet was resuspended in 8 mL of 1×Hank's solution, centrifuged again, and washed once with DMEM / F12 medium.

[0077] The isolated cells were counted under an inverted microscope, and the cell density was adjusted to 2-4 × 10⁻⁴. 6 Cells were seeded at a density of 10000 / mL into 50 mL culture flasks and cultured in DMEM / F12 medium supplemented with 10-15% FBS, 0.2 mol / L glutamine, 100,000 units / mL penicillin-streptomycin, and 20 mmol / L HEPES. Cells were cultured at 37°C, 5% CO2, and saturated humidity.

[0078] To remove most of the spermatogonia, after culturing for 24 hours, the original culture medium was discarded, and the cells were hypotonic with 50 mmol / L Tris-HCl solution at pH 7.1 for 3-5 min. After that, the cells were rinsed with HBSS, replaced with fresh culture medium, and cultured again.

[0079] 2.2.2 Cell viability assay

[0080] TM4 cells were seeded in 96-well plates and divided into a blank control group (complete culture medium), a model group (cell senescence model induced by 40 mg / mL D-gal), a drug treatment group (24 h after D-gal modeling, 500 μg / mL positive control VE, 100 μM TNF-α inhibitor, and different concentrations of deer antler polypeptide were added respectively), and a toxicity assay group (different concentrations of deer antler polypeptide were added respectively). Each group had three replicates for each concentration. After 24 h of culture, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37℃ with 5% CO2 in the dark for 40 min. After incubation, the absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated.

[0081] 2.2.3 Immunofluorescence assay

[0082] After cell culture and drug treatment, cells were thoroughly washed with 0.5% Triton X-100 for 15 min, followed by blocking with 10% sheep serum for 1 hour. Cells were incubated with ZO-1 antibody overnight at 4°C, and then washed with PBS. Secondary antibody diluted 1:400 with PBS was added to the cells and incubated for 2 hours at room temperature. Finally, cells were treated with 4′,6-diamidinyl-2-phenylindole (DAPI) for 5 min, and imaging was performed using a fluorescence microscope.

[0083] 2.2.4 ELISA assay for detecting inflammatory factors and antioxidant markers

[0084] TM4 cells were seeded in 6-well plates. After model establishment and drug administration, the cells were centrifuged at 3000 rpm for 10 min to separate them from the supernatant. The supernatant was stored at -80℃ for later use. The cells were resuspended in cell lysis buffer to lyse the cells, and centrifuged at 3000 rpm for 10 min to separate the supernatant. The supernatant was then stored at -80℃ for later use. The levels of TNF-α, MDA, SOD, CAT, and GSH-Px in the cells were measured according to the instructions of the assay kit.

[0085] 2.2.5 Cell uptake experiment

[0086] The solid-phase synthesized peptide was coupled with the fluorescent dye 5-carboxyfluorescein (5-FITC). After coupling, the product (hereinafter referred to as SP-FITC) was obtained in lyophilized powder or crystalline form, with an appearance that met the requirements, and its counterion was trifluoroacetate.

[0087] TM4 cells were fed at a rate of 1×10 5 Cells were seeded at a density of 1 cell / mL in culture dishes, with 1 mL of cell suspension added to each well. The dishes were incubated at 37°C and 5% CO2 for 24 hours to allow complete cell adhesion. The culture dishes were then removed from the incubator, and the original culture medium was discarded. Preheated (37°C) sterile PBS buffer was added, and the cells were gently washed three times to remove residual serum and metabolic waste. Subsequently, 0.5 mL of complete culture medium containing 10 μM SP-FITC was added to each well, and the cells were incubated for different times (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h) to observe cell uptake of SP-FITC at different time points. After incubation, the drug-containing culture medium was discarded, and pre-cooled (4°C) PBS was immediately added to terminate the uptake process. The cells were washed three times again to thoroughly remove fluorescent peptides that failed to enter the cells or only adhered to the cell membrane surface. The PBS was discarded, and 1 mL of 4% paraformaldehyde solution was added to each well. The cells were fixed at room temperature for 20 min. After fixation, the cells were washed three times with cold PBS. Finally, by exciting FITC (green fluorescence, excitation wavelength -488 nm), the distribution location of the taken-up SP-FITC in the cell and its relationship with the cell nucleus can be clearly distinguished.

[0088] 2.2.6 RT-qPCR assay to detect the effect of peptides on D-gal-induced TM4 cell mRNA

[0089] Total RNA was extracted from tissues using the TransZol Up Plus RNA Kit (product number: ER501-01), followed by reverse transcription, amplification, and real-time quantitative polymerase chain reaction (RT-qPCR). The primer sequences for RT-qPCR are detailed in Table 1. Relative expression levels were determined by 2... -ΔΔCt Formula quantification.

[0090] Table 1. Primer sequences related to RT-qPCR

[0091]

[0092] 2.3 Data Analysis

[0093] Experimental data are reported as mean ± standard deviation (SD). Statistically significant differences were assessed using one-way ANOVA (p < 0.05).

[0094] 3. Results

[0095] 3.1 Ligand fishing to screen for TNFR1 ligand in deer antler polypeptide (PAP)

[0096] 3.1.1 Screening for potential TNFR1 ligands using surface plasmon resonance (SPR) and LC-MS / MS (liquid chromatography-tandem mass spectrometry).

[0097] Using TNFR1 as the target protein, it was immobilized on the surface of a CM5 sensor chip, and affinity incubated with total antler peptides (PAP). Real-time SPR response curves showed that the response signal of PAP to TNFR1 was significantly higher than that of the blank buffer group, suggesting the presence of a component in PAP that can specifically bind to TNFR1. Figure 1 and 2 ); then the TNFR1-binding fraction was eluted and enriched, and after desalting, it was analyzed by LC-MS / MS.

[0098] LC-MS / MS analysis was performed using a C18 reversed-phase column (2.1 × 150 mm, 1.8 μm) with a mobile phase of 0.1% formic acid aqueous solution-acetonitrile. Mass spectrometry was performed in positive ion scanning mode (m / z range 300-1500). The top 30 peptides with the highest binding intensity were identified from the bound components through database searching and peptide matching. Based on the proteins from which the peptides originated, PTTKTYFPHF (SEQ ID NO. 13, hereinafter referred to as PT) and SPLASDLDL (SEQ ID NO. 14, hereinafter referred to as SP) were ultimately selected for subsequent activity validation.

[0099] 3.1.2 Solid-phase synthesis of peptides

[0100] The structural formula of the target peptide SP was determined using reversed-phase high-performance liquid chromatography (RP-HPLC) at a detection wavelength of 220 nm as follows: Figure 3 As shown, the retention time of the main peak in the liquid chromatography was 8.447 min, and the peak area accounted for 96% of the total peak area. Figure 4 The structural formula of the target peptide PT is as follows: Figure 5 As shown, the retention time of the main peak in the liquid chromatography was 8.447 min, and the peak area accounted for 97.2166% of the total peak area. Figure 6 The purity of the synthesized peptides was all above 95%, meeting the experimental requirements.

[0101] 3.1.3 Results of competitive detection of synthetic peptides and TNF-α

[0102] To verify the competitive binding ability of the screened synthetic peptides PT and SP to the natural ligand of TNFR1, tumor necrosis factor α (TNF-α), a competitive binding experiment was conducted using SPR. TNFR1 was immobilized on a CM5 chip, and TNF-α was co-incubated with gradient concentrations of synthetic peptides before being introduced into the chip. The binding response signal between TNF-α and TNFR1 was then detected.

[0103] The results showed that with increasing PT and SP concentrations, the binding response signal of TNF-α to TNFR1 decreased in a concentration-dependent manner; the SPR binding curve ( Figure 7 and Figure 9 The data showed that the dissociation constant of PT with TNFR1 was 5.1 μM, while that of SP with TNFR1 was 3.1 μM. In biomolecular interactions, a smaller KD (dissociation constant) value indicates a tighter intermolecular binding and stronger affinity; therefore, SP has a stronger competitive binding ability than PT. Kinetic curves ( Figure 8 and Figure 10 The study further revealed the binding and dissociation processes of peptides at different concentrations with TNFR1, with the response signal increasing with increasing peptide concentration. As the concentration of the synthetic peptide SP increased, the binding reaction between TNF-α and TNFR1 significantly decreased, showing a negative correlation. When the concentration of the synthetic peptide SP reached 200 μM (IC50), the binding response further decreased. 50 When TNF-α is used, it inhibits the binding of TNF-α to TNFR1 by half, suggesting that the peptide has TNFR1-targeting antagonistic activity.

[0104] 3.2 Mechanism of action of peptides in improving D-galactose-induced testicular Sertoli cell (TM4) damage

[0105] 3.2.1 Effects of peptides on the cell viability of D-gal-induced TM4 cells

[0106] In a D-gal-induced TM4 cell senescence model, cell viability was assessed using the CCK-8 assay to evaluate the protective effect of SP. Figure 11As shown, SP treatment at 16.125–1000 μg / mL was not cytotoxic to TM4 cells compared to the Control group. Figure 12 As shown, compared with the model group, different concentrations of SP all improved the D-gal-induced decrease in TM4 cell viability in a dose-dependent manner. Finally, 125, 250, and 500 μg / mL SP were selected as low, medium, and high dose groups for further experiments.

[0107] 3.2.2 Effects of peptides on D-gal-induced tight junction protein (ZO-1) in TM4 cells

[0108] ZO-1 is a key tight junction protein for maintaining barrier integrity. Immunofluorescence staining was used to observe the expression and distribution of ZO-1 in TM4 cells. Figure 13 As shown, ZO-1 expression was normal in the Control group, mainly distributed continuously along the cell membrane, presenting a clear and complete network structure. D-gal treatment led to a significant reduction in ZO-1 expression and disordered distribution, manifested as weakened fluorescence signal, discontinuous distribution, and blurred structure, indicating impaired tight junctions. Compared with the model group, all treatment groups improved ZO-1 expression and localization to varying degrees. Among them, the high-concentration SP treatment group showed the strongest ZO-1 fluorescence signal, which increased in a concentration-dependent manner. These results demonstrate that SP can upregulate ZO-1 expression and promote its proper distribution, thereby protecting the integrity of tight junctions in TM4 cells.

[0109] 3.2.3 Effects of peptides on inflammatory factors and antioxidant capacity of D-gal-induced TM4 cells

[0110] TNF-α is a core indicator reflecting the degree of inflammatory response. Results showed that TNF-α levels were significantly increased in the model group compared to the control group; compared to the D-gal group, TNF-α levels were significantly decreased in the positive drug treatment group (D-gal+VE) and the D-gal+TNF group (****, P<0.0001), while TNF-α levels decreased in a dose-dependent manner in all D-gal+SP dose groups (high, medium, and low). SP can inhibit TNF-α and effectively alleviate D-gal-induced inflammatory responses. Figure 14 ).

[0111] MDA is a characteristic product of lipid peroxidation, and its level directly reflects the degree of oxidative damage. The results showed that compared with the control group, the model group had a significantly higher MDA level, indicating severe lipid peroxidation damage in the model group; compared with the D-gal group, the D-gal+VE group and the D-gal+TNF group had lower MDA levels, and the high-dose D-gal+SP group had a significantly lower MDA level, indicating that SP can alleviate D-gal-induced oxidative damage. Figure 15 ).

[0112] Superoxide dismutase (SOD) is a key antioxidant enzyme that scavenges superoxide anions, and its activity reflects the body's basic antioxidant capacity. Results showed that compared to the Control group, SOD activity was significantly reduced in the D-gal group, indicating impaired antioxidant system function in the model group. Compared to the D-gal group, SOD activity rebounded in the D-gal+VE and D-gal+TNF groups, and SOD activity was significantly increased in the high-dose D-gal+SP group, indicating that SP can improve D-gal-induced SOD activity inhibition. Figure 16 ).

[0113] GSH-Px participates in the scavenging of intracellular peroxides and is an important component of the antioxidant system. Results showed that compared with the control group, GSH-Px activity was significantly reduced in the D-gal group, indicating a decreased peroxide scavenging capacity in the model group; compared with the D-gal group, GSH-Px activity rebounded in the D-gal+VE group, and GSH-Px activity was significantly increased in the high-dose D-gal+SP group, while the medium and low-dose groups showed varying degrees of activity recovery. This result indicates that SP can improve D-gal-induced oxidative damage (…). Figure 17 ).

[0114] CAT is responsible for catalyzing the decomposition of hydrogen peroxide, and its activity reflects the body's ability to scavenge reactive oxygen species. Results showed that compared to the Control group, CAT activity was significantly reduced in the D-gal group, indicating a decreased hydrogen peroxide scavenging capacity in the model group. Compared to the D-gal group, CAT activity significantly increased in the D-gal+VE and D-gal+TNF groups (****, P<0.0001), and CAT activity was further increased in the high-dose D-gal+SP group. This result indicates that SP can enhance the antioxidant activity of D-gal-induced TM4 (…). Figure 18 ).

[0115] 3.2.4 Uptake of peptides by TM4 cells

[0116] The results of the cellular uptake fluorescence imaging experiment of SP-conjugated FITC fluorescent label (SP-FITC) are as follows: Figure 19 As shown in the figure, at 0h, the field of view appears dark green with almost no obvious fluorescence signal, indicating that the cell uptake is minimal at this time. At 2h, the fluorescence signal gradually increases, and clearer fluorescent spots can be observed inside the cells; at 8h, the fluorescence signal reaches the strongest level among all time points, and the green fluorescence in the cell area is more concentrated and brighter, indicating that the fluorescence signal becomes stronger with prolonged incubation time, and the cell uptake of SP-FITC increases continuously in a time-dependent manner.

[0117] 3.2.5 Effect of peptides on mRNA expression in D-gal-induced TM4 cells

[0118] RT-qPCR was used to detect the mRNA expression of key molecules in the TNF-α / p38MAPK pathway (c-JUN, JNK, p38, TNF-α) and the cell junction protein ZO-1 (GAPDH as internal control). Compared with the control group, the D-gal group showed significantly upregulated c-JUN, JNK, p38, and TNF-α (P<0.001), and significantly downregulated ZO-1 mRNA expression (P<0.001), suggesting that D-gal activates the TNF-α / p38MAPK pathway and inhibits cell junction-related transcription. Compared with the D-gal group, each dose of D-gal+SP dose-dependently downregulated the mRNA expression levels of c-JUN, JNK, p38, and TNF-α (P<0.05), while upregulating ZO-1 levels (P<0.05). These results indicate that SP can inhibit TNF-α and improve D-gal-induced TM4 inflammatory damage and cell junction expression. Figures 20-24 ).

[0119] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A pilose antler active peptide, characterized in that, The amino acid sequence of the antler active peptide is shown as SEQ ID NO.

14.

2. Use of the antler active peptide of claim 1 in the preparation of an anti-inflammatory drug.

3. Use according to claim 2, characterized in that, The drug exerts an anti-inflammatory effect by competitively inhibiting the binding of TNFR1 to TNF-α.

4. Use of the antler active peptide of claim 1 in the preparation of an antioxidant drug.

5. Use according to claim 4, characterized in that, The drug exerts an antioxidant effect by competitively inhibiting the binding of TNFR1 to TNF-α.

6. Use of the antler active peptide of claim 1 in the preparation of an anti-aging drug.

7. An anti-inflammatory medicament, characterized by, The drug contains the antler active peptide of claim 1 as an effective component.

8. The medicament according to claim 7, characterized in that, The drug also contains a pharmaceutically acceptable carrier or excipient.

Images