An anti-inflammatory polypeptide from buthus martensii koch and screening method and application thereof

Abstract

The application discloses an anti-inflammatory polypeptide of Buthus martensii Karsch and a screening method and application thereof, and belongs to the technical field of traditional Chinese medicinal material polypeptides. The anti-inflammatory polypeptide of Buthus martensii Karsch is one of G4, G5 and G17. The nanoElute 2 nanoliter liquid phase separation, timsTOFPro2 mass spectrum analysis and BPS Novor library searching analysis are used to detect the polypeptide extract of Buthus martensii Karsch, screen the polypeptide of Buthus martensii Karsch, and further synthesize the candidate polypeptide screened above by using a solid-phase synthesis method; the effect of the synthesized candidate polypeptide on inhibition of microglial cell inflammation is evaluated by using a qPCR method, and then the anti-inflammatory polypeptide of Buthus martensii Karsch is screened. The anti-inflammatory polypeptides G4, G5 and G17 of Buthus martensii Karsch screened by the method, especially the G5, can significantly inhibit microglial cell inflammation in a concentration-dependent manner, provide a theoretical basis for medicinal development of the polypeptide of Buthus martensii Karsch, and reveal a potential anti-neuroinflammatory mechanism.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide technology of traditional Chinese medicine, specifically relating to an anti-inflammatory polypeptide of East Asian scorpion, its screening method and application. Background Technology

[0002] As a traditional Chinese medicine, the East Asian scorpion has the effects of attacking toxins and dispersing nodules, calming wind and relieving spasms, and clearing the meridians and relieving pain. It can be used to treat infantile convulsions, hemiplegia due to stroke, and spasms. Modern medical research focuses on the active ingredients of proteins and peptides. Studies have shown that peptides in the East Asian scorpion have analgesic, anti-inflammatory, antibacterial, neuroprotective, and cell proliferation-promoting effects.

[0003] Polypeptides are a class of small, bioactive molecules with significant value in drug development and biomedical research. Their structural diversity and specific functions make them promising candidates for applications in analgesia, anti-inflammation, antibacterial activity, neuroprotection, and cell proliferation promotion. Modern research also indicates that extracts from the East Asian scorpion (Scorpio spp.) are rich in proteins and peptides, potentially exerting anti-neuroinflammatory effects by inhibiting inflammatory factors. However, systematic screening of East Asian scorpion peptides and their specific mechanisms of action remain limited, particularly regarding the structure and function of individual peptides, which restricts their clinical application and exploration of molecular mechanisms. Summary of the Invention

[0004] To address the lack of research on the structure and function of single peptides in East Asian scorpion polypeptides in existing technologies, this invention provides an anti-inflammatory peptide from East Asian scorpion, its screening method, and its application, providing a theoretical basis for the pharmaceutical development of East Asian scorpion polypeptides and revealing its potential anti-neuroinflammatory mechanism.

[0005] This invention is achieved through the following technical solution:

[0006] In a first aspect, the present invention discloses an anti-inflammatory polypeptide of the East Asian scorpion, wherein the East Asian scorpion anti-inflammatory polypeptide is one of G4, G5 and G17;

[0007] The amino acid sequence of G4 is shown in SEQ ID NO.1;

[0008] The amino acid sequence of G5 is shown in SEQ ID NO.2;

[0009] The amino acid sequence of G17 is shown in SEQ ID NO.3.

[0010] In a second aspect, the present invention discloses a method for screening the anti-inflammatory peptides of the East Asian scorpion described above, comprising the following steps:

[0011] (1) Extraction of East Asian scorpion: Grind the East Asian scorpion slices evenly with liquid nitrogen, add lysis buffer to homogenize, extract protein by ultrasonic centrifugation, filter through aqueous phase membrane, desalt by SPE column, freeze dry to obtain East Asian scorpion polypeptide extract;

[0012] (2) The peptides were detected by nanoElute 2 nanoliter liquid chromatography, timsTOF Pro 2 mass spectrometry, and BPS Novor library search analysis. The peptides were screened according to Local Confidence ≥ 85% and Score ≥ 90. The bioactivity probability of the peptides was predicted by NCBI and the online website PeptideRanker, the toxicity of the peptides was predicted by ToxIBTL, and the sensitization was predicted by Algpred2.0 to obtain candidate peptides.

[0013] (3) The candidate peptides obtained in step (2) were synthesized using solid-phase synthesis.

[0014] (4) The effect of the candidate peptides synthesized in step (3) on microglial cell inflammation was evaluated by qPCR, and anti-inflammatory peptides of East Asian scorpion with anti-inflammatory properties were screened out.

[0015] Furthermore, the lysis buffer in step (1) contains 10% aqueous solution of glacial acetic acid (V:V); the pore size of the aqueous filter membrane is 0.22 μM.

[0016] Further, the desalting treatment method in step (1) is as follows: activate the desalting column with 100% acetonitrile, equilibrate the column with 0.1% TFA aqueous solution, load 10% glacial acetic acid aqueous solution containing peptides onto the column, then wash the residual salt with 0.1% TFA aqueous solution, and finally elute with 70% acetonitrile, collect the eluent, and complete the desalting treatment.

[0017] Further, the chromatographic conditions for nanoElute 2 nanoliter liquid phase separation in step (2) are as follows: column: C18 column (20cm×75 μm, 1.3 μm); mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid acetonitrile solution, flow rate: 300 nL / min, gradient elution; elution program: 0~45min 2~22% mobile phase B, 45~50min 22~37% mobile phase B, 50~55min 37~80% mobile phase B, 55~60min 80% mobile phase B;

[0018] In step (2), the mass spectrometry conditions for the timsTOF Pro 2 mass spectrometry analysis were as follows: timsTOF Pro 2 mass spectrometer, CaptiveSpray nano-spray ion source, ion spray voltage of 1.5 kV, full scan range of m / z 100-1700, and ion mobility of 0.60-1.60 V·s / cm.2 The standard 10 ddaPASEF scanning protocol is run in dda-PASEF mode.

[0019] Furthermore, the microglia mentioned in step (4) are BV2 cell lines.

[0020] In a third aspect, the present invention discloses the application of the above-mentioned East Asian scorpion anti-inflammatory polypeptide in the preparation of anti-inflammatory drugs, wherein the East Asian scorpion anti-inflammatory polypeptide is one of G4, G5 and G17.

[0021] Furthermore, the anti-inflammatory drug is an antineuritis drug.

[0022] Furthermore, the East Asian scorpion anti-inflammatory polypeptide is G5.

[0023] The beneficial effects achieved by this invention are as follows:

[0024] The anti-inflammatory peptides G4, G5, and G17 obtained by the method of this invention, especially G5, can significantly inhibit the expression of inflammatory factors in a concentration-dependent manner, providing a theoretical basis for the pharmaceutical development of Scorpion peptides and revealing their potential anti-neuroinflammatory mechanism, which can be used as anti-neuritis drugs. Attached Figure Description

[0025] Figure 1 The figure shows the results of 25 candidate peptides inhibiting the expression of the inflammatory cytokine IL-1β in microglia;

[0026] Figure 2 The figure shows the results of 25 candidate peptides inhibiting the expression of the inflammatory factor IL-6 in microglia;

[0027] Figure 3 The figure shows the results of different concentrations of the East Asian scorpion anti-inflammatory peptide G4 inhibiting the expression of inflammatory factors IL-1β and IL-6 in microglia. (a) represents IL-1β, and (b) represents IL-6.

[0028] Figure 4 The figures show the results of different concentrations of the East Asian scorpion anti-inflammatory peptide G5 inhibiting the expression of inflammatory factors IL-1β and IL-6 in microglia. (a) represents IL-1β, and (b) represents IL-6.

[0029] Figure 5 The figure shows the results of different concentrations of the East Asian scorpion anti-inflammatory peptide G17 inhibiting the expression of microglial inflammatory factors IL-1β and IL-6. (a) is IL-1β and (b) is IL-6. Detailed Implementation

[0030] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. This example is merely a detailed implementation scheme and specific operation process based on the present invention, and is not intended to limit the scope of protection of the present invention. Any modifications or improvements made within the principles of the present invention should be included within the scope of protection.

[0031] Example 1

[0032] Polypeptide extract from East Asian pincers:

[0033] 100 mg of East Asian scorpion slices were ground evenly with liquid nitrogen, and then lysed with lysis buffer (10% glacial acetic acid aqueous solution (V:V)). The mixture was ground into a homogenate, and the protein was extracted by ultrasonic centrifugation. The extract was filtered through a 0.22 μm aqueous phase filter membrane, desalted by SPE column, and lyophilized to obtain East Asian scorpion polypeptide extract.

[0034] The desalting process is as follows: activate the desalting column with 100% acetonitrile, equilibrate the column with 0.1% TFA aqueous solution, load 10% glacial acetic acid aqueous solution (V:V) containing peptides onto the column, wash the residual salts with 0.1% TFA aqueous solution, and finally elute with 70% acetonitrile. Collect the eluent to complete the desalting process.

[0035] Example 2

[0036] The peptides were detected using nanoElute 2 nanoliter liquid chromatography, timsTOF Pro 2 mass spectrometry, and BPS Novor library search analysis. Peptides were screened based on Local Confidence ≥ 85% and Score ≥ 90. The bioactivity probability of peptides was predicted using NCBI and the online website PeptideRanker, the toxicity of peptides was predicted using ToxIBTL, and the sensitization was predicted using Algpred 2.0 to further screen peptides and obtain candidate peptides.

[0037] The chromatographic conditions for nanoElute 2-nano liquid chromatography separation were as follows: column: C18 column; mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid acetonitrile solution, flow rate: 300 nL / min, gradient elution; elution program: 0~45 min 2~22% mobile phase B, 45~50 min 22~37% mobile phase B, 50~55 min 37~80% mobile phase B, 55~60 min 80% mobile phase B.

[0038] The mass spectrometry conditions for the timsTOF Pro 2 mass spectrometry analysis were as follows: timsTOF Pro 2 mass spectrometer, CaptiveSpray nano-spray ion source, ion spray voltage of 1.5 kV, full scan range of m / z 100-1700, and ion mobility of 0.60-1.60 V·s / cm. 2The standard 10 ddaPASEF scanning protocol is run in dda-PASEF mode;

[0039] The parameters for the BPS Novor database search are set as follows: Enzyme, Non-specific cleavage; First-orderion mass deviation, ±20ppm; Secondary ion mass deviation, ±0.02Da; Missingcutting number, 2;

[0040] A total of 121 candidate peptides were obtained by simultaneously meeting the criteria of Local Confidence ≥ 85% and Score ≥ 90. Further comparison using NCBI identified 39 free peptides. Using online platforms (PeptideRanker to predict peptide bioactivity probability, ToxIBTL to predict peptide toxicity, and Algpred 2.0 to predict sensitization), 25 candidate peptides were identified. Information on these 25 candidate peptides is shown in Table 1 below.

[0041] Table 1. Information on candidate peptides obtained from screening in Example 2

[0042] .

[0043] Example 3

[0044] The 25 candidate peptides screened in Example 2 were synthesized using a solid-phase synthesis method:

[0045] The solid-phase synthesis method described above is as follows:

[0046] (1) Solvent treatment: DMF and methanol were soaked overnight in G3 pore molecular sieve before use to remove impurities and water;

[0047] (2) Full swelling of resin: Weigh 2.0 g blank Wang resin into a clean and dry reaction tube, add 15 mL DMF, and activate at room temperature for about 30 min;

[0048] (3) Adding the first amino acid: At room temperature, filter out the solvent from step (2) through a sand core filter, add 1 mmol of 5 times the molar excess of the first C-terminal amino acid, 5 times the molar excess of DMAP, 5 times the molar excess of DIC, and DMF as the solvent. React at room temperature for 3 hours. After the reaction is complete, wash with DMF 5 times, 5-6 mL each time. Then add a pyridine and acetic anhydride mixture with a volume ratio of 1:1 and react for 30 minutes. After the reaction is complete, wash with DMF 5 times, 5-6 mL each time.

[0049] (4) Removal of Fmoc protecting group: Remove the solvent in step (3) by filtration, add 10 mL of 20% piperidine DMF solution to the resin, stir with N2 for 10 min and filter out the solution, add another 10 mL of 20% piperidine DMF solution, stir with N2 for 5 min and filter out the solution again. Repeat this operation twice, then stir with DMF 4 times and wash with methanol 2 times, 5-6 mL each time.

[0050] (5) Detection of ninhydrin removal effect: Take 15 mg of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN and phenol solution, heat at 105-110 ℃ for 5 min, and the reaction is positive when it turns dark blue, indicating that the removal is complete and the next step can be carried out; if it is colorless, it means that the protecting group has not been completely removed and the above deprotection operation needs to be repeated.

[0051] (6) Removal of the second amino acid and Fmoc protecting group: Weigh 3 times the molar excess of the second C-terminal amino acid, 3 times the molar excess of HBTU, and 3 times the molar excess of HOBT into a reaction tube. Add an appropriate amount of DMF solution to completely dissolve them, then add 10 times the molar excess of DIEA. React at room temperature for 40 min, and wash with DMF 5 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If it turns colorless, add 10 mL of 20% piperidine DMF solution to remove Fmoc. Repeat this process twice, for 10 min and 5 min respectively. Then wash with DMF 4 times and methanol 2 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If it turns blue, proceed to the next step of the reaction.

[0052] (7) Repeat step (6) in this manner until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then dry the mixture.

[0053] (8) Resin shedding and pure product separation detection: The peptide was cut with trifluoroacetic acid cutting solution (95% TFA: 2% TIS: 2% EDT: 1% H2O) for 2 h. The reaction solution was filtered to obtain a trifluoroacetic acid solution of the peptide. The lysis solution was dried as much as possible with nitrogen gas, then precipitated with ether, centrifuged, and then washed with ether 4 times to obtain a white solid. After being dissolved in pure water, it was desalted and purified by HPLC, and then lyophilized to precipitate crystals to obtain the target peptide.

[0054] The 25 candidate peptides listed in Table 1 were all synthesized using the methods described in steps (1) to (7) above.

[0055] Example 4

[0056] Effects of different East Asian scorpion peptides on the expression of inflammatory factors in microglia

[0057] The culture medium described below is DMEM medium containing 10% fetal bovine serum and 1 wt% penicillin antibiotics;

[0058] (1) Culture and passage of microglia (BV2): BV2 cells were placed in culture medium and cultured in a 37 ℃, 5% CO2 cell culture incubator. The cell culture medium was changed every 3 days. When the cells reached about 80% confluence, they were passaged and the logarithmic growth phase cells were used for subsequent experiments.

[0059] (2) BV2 cells in the logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 5 Cells were seeded at a density of 1 μM in 6-well plates. After 24 h, the expression of inflammatory factors IL-1β and IL-6 was stimulated with LPS. Cells were then co-treated with 10 ng / mL LPS for 20 h using 25 candidate peptides at a concentration of 1 µM. Total RNA was extracted from the cells using the Trizol Total RNA Extraction Kit, and the concentration and purity of RNA were determined using Nanodrop 100. Reverse transcription was performed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit, and amplification was performed using the ChamQ Blue Universal SYBR qPCR Master Mix kit. Table 1 shows the effects of the 25 candidate peptides on the expression of inflammatory factors IL-1β and IL-6 in BV2 cells. Figure 1 and Figure 2 As shown:

[0060] Depend on Figure 1 and Figure 2 It was found that different candidate peptides had significantly different effects on microglial cell activity. Compared with the control group, LPS stimulation of BV2 cells significantly increased the gene expression levels of inflammatory factors IL-1β and IL-6. Some of the 25 candidate peptides inhibited the expression of inflammatory factor genes. Among them, peptides G4, G5, and G17 showed significant inhibitory effects on IL-1β expression, while peptides G4, G5, G6, G8, G10, G12, G13, G14, G15, G16, G17, G18, G20, G21, G23, and G25 showed significant inhibitory effects on IL-6 expression. The three East Asian scorpion peptides (G4, G5, and G17) that showed the most significant inhibition of inflammatory factors in BV2 cells were selected as East Asian scorpion anti-inflammatory peptides for subsequent experiments.

[0061] The three East Asian scorpion peptides that most significantly inhibited BV2 cell inflammatory factors were G4, G5, and G17. The amino acid sequence of G4 (SEQ ID NO.1) is DDDLEEPFLKKT, the amino acid sequence of G5 (SEQ ID NO.2) is ALANLQKDKKQ, and the amino acid sequence of G17 (SEQ ID NO.3) is APLGQRVR.

[0062] Example 5

[0063] Effects of different concentrations of East Asian scorpion anti-inflammatory peptides (G4, G5, G17) on the expression of inflammatory factors in microglia

[0064] (1) The culture and passage of microglia (BV2) were the same as in Example 4;

[0065] (2) BV2 cells in the logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 5 Cells were seeded at a density of 0.5 g / mL in 6-well plates. After 24 h, the expression of inflammatory factors IL-1β and IL-6 was stimulated with LPS. Cells were then co-treated with three anti-inflammatory peptides from *Scorpio scorpio* (0.1, 0.5, 1, and 5 µM) and 10 ng / mL LPS for 20 h. Total RNA was extracted from the cells using the Trizol Total RNA Extraction Kit, and the concentration and purity of RNA were determined using Nanodrop 100. Reverse transcription was performed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit, and amplification was performed using the ChamQ Blue Universal SYBR qPCR Master Mix kit. The effects of three different concentrations of *Scorpio scorpio* anti-inflammatory peptides (G4, G5, and G17) on the expression of inflammatory factors IL-1β and IL-6 in BV2 cells were as follows: Figure 3 , Figure 4 and Figure 5 As shown in the figure: (a) represents IL-1β, and (b) represents IL-6;

[0066] Depend on Figures 3-5 It can be seen that different concentrations of East Asian scorpion anti-inflammatory peptides have significant differences in their effects on the expression of inflammatory factors in BV2 cells. The inhibitory effects of the three East Asian scorpion anti-inflammatory peptides on inflammatory factors are all concentration-dependent, with East Asian scorpion peptide G5 showing a much higher effect than the other two East Asian scorpion peptides.

Claims

1. An anti-inflammatory polypeptide from the East Asian scorpion, characterized in that, The aforementioned East Asian scorpion anti-inflammatory polypeptide is one of G4, G5, and G17; The amino acid sequence of G4 is shown in SEQ ID NO.1; The amino acid sequence of G5 is shown in SEQ ID NO.2; The amino acid sequence of G17 is shown in SEQ ID NO.

3.

2. The application of the East Asian scorpion anti-inflammatory polypeptide according to claim 1 in the preparation of anti-inflammatory drugs, characterized in that, The aforementioned East Asian scorpion anti-inflammatory polypeptide is one of G4, G5, and G17; the aforementioned anti-inflammatory drug is an antineuritis drug.

3. The application of the East Asian scorpion anti-inflammatory polypeptide according to claim 2 in the preparation of anti-inflammatory drugs, characterized in that, The anti-inflammatory polypeptide from the East Asian scorpion is G5.

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