CN105061573B - The natural anti-inflammatory polypeptide cecropin-TY1 of Yao horsefly and its application - Google Patents

Abstract

The invention discloses a natural anti-inflammatory polypeptide cecropin-TY1 extracted from the salivary gland of Yaofly, which is composed of 39 amino acids, the carbon terminal is amidated, the molecular weight is 3970.22 Daltons, the isoelectric point is 11.17, and its amino acid sequence is SEQ ID: 1, and all amino acids in it are L‑type, the polypeptide has the characteristics of small molecular weight, obvious anti-inflammatory effect and low cytotoxicity, anti-inflammatory drugs, sepsis and endotoxin shock drugs caused by bacterial infections, veterinary drugs , animal feed and cosmetics have a wide range of application prospects, specifically in the application of inhibiting lipopolysaccharide-induced macrophages to produce nitric oxide; in inhibiting lipopolysaccharide-induced macrophages to produce inflammatory cytokines; in inhibiting lipid Applications in polysaccharide-induced inflammatory signaling pathways and in neutralizing endotoxin function.

Description

Yaofly natural anti-inflammatory polypeptide cecropin-TY1 and its application

technical field

The invention relates to a natural anti-inflammatory polypeptide cecropin-TY1 extracted from the salivary gland of Yaofly and its new application in anti-inflammation, which belongs to the technical field of biomedicine.

Background technique

In recent years, with the abuse of traditional antibiotics, microorganisms have become more and more resistant to traditional antibiotics, and microbial infection has become a problem that seriously threatens human health. These microorganisms, especially Gram-negative bacteria, can release lipopolysaccharide (LPS) during infection, which can cause sepsis and endotoxic shock. The main treatment methods for sepsis caused by microbial infection have been infusion, improving the body's oxygen supply, anti-infection therapy, and immunotherapy. At the same time, there are many shortcomings in these treatment methods. For example, anti-infection treatment will cause a large number of bacteria to be killed and release a large amount of endotoxin, which will aggravate sepsis. Therefore, there is a need for continued development of new agents for the treatment of sepsis.

Contents of the invention

In order to solve the above technical problems, the object of the present invention is to provide a natural anti-inflammatory polypeptide cecropin-TY1 of Yaofly and its application.

Technical scheme of the present invention is:

Cecropin antimicrobial peptides are a class of small molecular polypeptides with antibacterial functions, and part of cecropin antibacterial peptides also have anti-inflammatory functions and can neutralize endotoxins, that is, they can neutralize endotoxins released by bacterial infections while antibacterial toxins and has anti-inflammatory properties. According to reports, some researchers studied the successful blood-sucking mechanism of the blood-sucking insect Tabanus yao based on the interaction between host and ectoparasite, and discovered the cecropin-like polypeptide cecropin-TY1 from the salivary gland of Yao yao.

The natural anti-inflammatory polypeptide cecropin-TY1 of Yaofly according to the present invention is extracted from the salivary gland of Yaofly, and consists of 39 amino acids, the carbon terminal is amidated, the molecular weight is 3970.22 Daltons, the isoelectric point is 11.17, and its amino acid sequence is SEQID : ¹ , namely Gly ^{1 Try 2} Leu ³ Lys ⁴ Lys ⁵ Ile ⁶ Gly ⁷ Lys ⁸ Lys ^{9 Ile 10 Glu 11 Arg 12 Val 13 Gly 14 Gln 15 Asn 16 Val 17 Arg 18 Asn 19 Ala 20} Ala ²¹ Ile ²² Ser ²³ Thr ²⁴ Ile ²⁵ Pro ²⁶ Ile ²⁷ Ala ²⁸ Gln ²⁹ Gly ³⁰ Ala ³¹ Ala 32 Gly ^{33 Val 34} Ala ^{35 Gly 36 Ala 37} Leu ³⁸ Asn ³⁹ -NH ₂ , and all amino acids are L-form.

The application of the Yaofly natural anti-inflammatory polypeptide cecropin-TY1 in the preparation of anti-inflammatory drugs, drugs for sepsis and endotoxin shock caused by bacterial infection, veterinary drugs, animal feed and cosmetics.

Specifically:

The Yaofly natural anti-inflammatory polypeptide cecropin-TY1 of the present invention is used in inhibiting lipopolysaccharide-induced macrophages to produce nitric oxide; in inhibiting lipopolysaccharide-induced macrophages to produce inflammatory cytokines; in inhibiting lipopolysaccharide-induced macrophages to produce inflammatory cytokines; The application in the polysaccharide-induced inflammatory signaling pathway, including the application in inhibiting the activation of lipopolysaccharide-induced mitogen-activated protein kinase signaling pathway, and the application in inhibiting the activation of lipopolysaccharide-induced nuclear transcription factor NF-κB; Application in neutralizing endotoxin function.

By means of the above-mentioned scheme, the present invention has at least the following advantages: the natural anti-inflammatory polypeptide cecropin-TY1 extracted from the salivary gland of Yaofly described in the present invention has the characteristics of small molecular weight, simple synthesis, obvious anti-inflammatory effect, and low cytotoxicity. It has broad application prospects in fields such as medicine, cosmetics and aquaculture.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention and accompanying drawings are described in detail below.

Description of drawings

Figure 1 is a graph showing the transcription results of Cecropin-TY1 inhibiting inducible nitric oxide synthase of the present invention;

Fig. 2 is the result figure that Cecropin-TY1 of the present invention inhibits the production of nitrite;

Fig. 3 is the result figure that Cecropin-TY1 of the present invention inhibits the production of inflammatory cytokines;

Fig. 4 is a graph showing the results of Cecropin-TY1 inhibiting the activation of MAPKs of the present invention;

Fig. 5 is a result diagram showing that cecropin-TY1 of the present invention inhibits the activation of NF-κB;

Fig. 6 is the toxicity of cecropin-TY1 of the present invention to mouse macrophages;

Figure 7 is the endotoxin neutralizing activity of cecropin-TY1 of the present invention;

Fig. 8 is the circular dichroism chromatogram of cecropin-TY1 of the present invention in different solutions;

Fig. 9 is a schematic diagram of the solution structure simulation of cecropin-TY1 of the present invention.

Detailed ways

Below in conjunction with the accompanying drawings and specific examples, the specific implementation of the present invention will be further described in detail. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

1. The effect of anti-inflammatory polypeptide cecropin-TY1 from Yaofly saliva on the production of nitric oxide (NO) in macrophages induced by lipopolysaccharide (LPS).

(1), inhibit the transcription of inducible nitric oxide synthase (iNOS):

Inducible nitric oxide synthase (iNOS) is an essential synthetase for nitric oxide (NO) production. Firstly, the effect of cecropin-TY1 on the transcription level of nitric oxide synthase was detected. C57BL/6 mouse peritoneal macrophages were plated in 24-well cell culture plates (2.5×10 ⁵ cells/well), and treated with 2% fetal bovine serum, 100 U/mL ampicillin and 100 μg/mL streptomycin sulfate. RMPI-1640 medium (purchased from U.S. Gbico Company) was cultivated. After the cells adhered to the wall, as shown in Figure 1, 100ng/mL lipopolysaccharide (LPS, from Escherichia coli Escherichia coli 0111:B4, purchased from Escherichia coli) was added to the cells. From Sigma), 5, 10 and 20 μg/mL of cecropin-TY1 were added at the same time, and a variety of control groups were set up, including: only LPS group, only peptide group (10 μg/mL) and neither LPS nor Add cecropin-TY1 group. After co-incubation for 6 hours, the cells were collected, lysed with Trizol (purchased from Takara), RNA was extracted, and cDNA was synthesized with a reverse transcription kit (purchased from Takara), and the transcription level of iNOS in differently treated cells was detected by fluorescent quantitative PCR.

It can be seen from Figure 1 that cecropin-TY inhibited the transcription of iNOS in a dose-dependent manner. The iNOS transcription level of the cells treated only with LPS was set as 100%, but after adding 5, 10 and 20 μg/mL cecropin-TY1, the iNOS transcription level was only 41.2%, 31.3% and 15.8%, respectively. TY1 reduced the transcription of iNOS by 58.8%, 68.7% and 84.2%.

(2), inhibit the production of nitrite:

The accumulation level of nitrite in the cell culture medium can indirectly reflect the production of NO, and then the effect of cecropin-TY1 on the production of nitrite was detected. Spread the C57BL/6 mouse peritoneal macrophages in a 24-well cell culture plate (2.5×10 ⁵ cells/well) with 2% fetal bovine serum, 100 U/mL ampicillin and 100 μg/mL streptomycin sulfate The RMPI-1640 medium (purchased from U.S. Gbico Company) was cultivated, and after the cells adhered to the wall, as shown in Figure 2, 100ng/mL lipopolysaccharide (LPS, derived from Escherichia coli Escherichia coli 0111:B4, Purchased from Sigma), 5, 10 and 20 μg/mL cecropin-TY1 were added at the same time, and a variety of control groups were set up, including: only LPS group, only peptide group (10 μg/mL) and neither LPS nor No cecropin-TY1 group. After co-incubating for 24 hours, the culture supernatant was collected, and the accumulated level of nitrite in the cell culture supernatant after different treatments of the cells was detected with Griess reagent (purchased from Beyont).

It can be seen from Figure 2 that cecropin-TY inhibited the production of nitrite in a dose-dependent manner. The accumulation level of nitrite in the LPS-only treatment group was 20.4 μM. After adding 5, 10 and 20 μg/mL cecropin-TY1, the accumulation level of nitrite was only 12.0, 7.3 and 5.0 μM, respectively, and cecropin-TY1 decreased 40.0, 63.7 and 75.0% nitrite production.

2. The effect of Yaofly saliva anti-inflammatory peptide cecropin-TY1 on the production of inflammatory cytokines in macrophages induced by lipopolysaccharide (LPS).

Spread the C57BL/6 mouse peritoneal macrophages in a 24-well cell culture plate (2.5×10 ⁵ cells/well) with 2% fetal bovine serum, 100 U/mL ampicillin and 100 μg/mL streptomycin sulfate The RMPI-1640 medium (purchased from U.S. Gbico Company) was cultivated. After the cells adhered to the wall, as shown in Figure 3, 100ng/mL lipopolysaccharide (LPS, from Escherichia coli Escherichia coli0111:B4, purchased from From Sigma), 5, 10 and 20 μg/mL of cecropin-TY1 were added at the same time, and a variety of control groups were set up, including: only LPS group, only peptide group (10 μg/mL) and neither LPS nor Add cecropin-TY1 group. After co-incubating for 6 hours, the cell culture supernatant was collected, and the cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin- Cumulative levels of 6 (IL-6). Cells were collected at the same time, cells were lysed with Trizol (purchased from Takara), RNA was extracted, cDNA was synthesized with a reverse transcription kit (purchased from Takara), and inflammatory cytokines TNF-α and IL-1β in cells treated with different treatments were detected by fluorescent quantitative PCR and IL-6 transcript levels.

As shown in Figure 3, cecropin-TY1 inhibited the transcription and production of LPS-induced inflammatory cytokines TNF-α, IL-1β and IL-6 in a dose-dependent manner. At a concentration of 20mg/mL, cecropin-TY1 inhibited the transcription of 60.3% of TNF-a, 67.5% of IL-1β and 94.1% of IL-6, at the same time, 20mg/mL of cecropin-TY1 reduced the transcription of 72.1% Production of TNF-α, 63.4% IL-1β and 76.2% IL-6.

3. The effect of Cecropin-TY1 on inflammatory signaling pathways.

C57BL/6 mouse peritoneal macrophages were plated in a 6-well cell culture plate (1×10 ⁶ cells/well), treated with 2% fetal bovine serum, 100 U/mL ampicillin and 100 μg/mL streptomycin sulfate RMPI-1640 medium (purchased from U.S. Gbico Company) was cultivated. After the cells adhered to the wall, as shown in Figure 4 and Figure 5, 100ng/mL lipopolysaccharide (LPS, from Escherichia coli Escherichia coli0111: B4, purchased from Sigma), while adding 5, 10 and 20 μg/mL cecropin-TY1, respectively, and set up a variety of control groups, including: only adding LPS group, only adding peptide group (10 μg/mL) and neither LPS without cecropin-TY1 group. After incubating for 30 minutes, the cells were collected, and the cells were lysed with RIPA lysate (purchased from Biyuntian), and the cell protein was collected, and the protein was quantified using the BCA protein quantification kit (purchased from Biyuntian). The cleaved protein was electrophoresed on a 10% polyacrylamide gel at 100 volts for 90 minutes according to the loading amount of 40 micrograms, and then the protein was electrotransferred (300 mA, 90 minutes) to the hybridization membrane, and then electrophoresis was carried out at room temperature for 5 minutes. Block with % bovine serum albumin for 2 hours.

(1), inhibit the activation of mitogen-activated protein kinase (MAPKs) signaling pathway

The primary antibodies related to the MAPKs signaling pathway: P-ERK, ERK, P-JNK, JNK, P-p38 and p38 (purchased from CST, USA), and the internal reference GAPDH (purchased from Shanghai Dingguo Biotechnology Co., Ltd.) were used for 5 % bovine serum albumin was diluted according to the method of 1:2000, and the blocked hybridization membrane was incubated with the diluted primary antibody at 4°C overnight, and Tris buffer containing 0.1% Tween-20 (TBST, 2.42g/L Trisbase , 8g/L NaCl, 0.1% Tween 20, pH 7.6) washed 3 times for 5 minutes each time, and then diluted with 5% bovine serum albumin and the secondary antibody labeled with horseradish peroxidase (1:5000, After incubating at room temperature for 1 hour, wash with TBST for 10 minutes each time, and then use a luminescent substrate (purchased from China Tiangen Biochemical Technology Co., Ltd.) to expose to the hint development.

As shown in Figure 4, cecropin-TY1 inhibited the phosphorylation of ERK, JNK, and p38 in a dose-dependent manner, and inhibited the activation of LPS-induced MAPKs signaling pathway.

(2), cecropin-TY1 inhibits the activation of nuclear transcription factor kappa B (NF-κB)

Primary antibodies related to NF-κB signaling pathway: P-IκBα, IκBα, P-p65, and p65 (purchased from CST Company, USA), and internal reference β-actin (purchased from Shanghai Dingguo Biotechnology Co., Ltd.) were used with 5% The bovine serum albumin was diluted according to the method of 1:2000, and the blocked hybridization membrane was incubated with the diluted primary antibody at 4°C overnight, and the Tris buffer containing 0.1% Tween-20 (TBST, 2.42g/L Trisbase, 8g/L NaCl, 0.1% Tween 20, pH 7.6) washed 3 times, 5 minutes each time, and then diluted with 5% bovine serum albumin, the secondary antibody labeled with horseradish peroxidase (1:5000, purchased After incubating at room temperature for 1 hour, wash with TBST 3 times for 10 minutes each time, and then use a luminescent substrate (purchased from China Tiangen Biochemical Technology Co., Ltd.) to expose to the hint development.

As shown in Figure 5, cecropin-TY1 inhibited the phosphorylation of IκBα and p65 in a dose-dependent manner, inhibiting the activation of LPS-induced NF-κB signaling pathway.

4. Determination of cytotoxicity of cecropin-TY1.

C57BL/6 mouse peritoneal macrophages and RAW264.7 cells were plated in 96-well culture plates (2×10 ⁴ cells/well) with 2% fetal bovine serum, 100 U/mL ampicillin and 100 μg/mL Cultured in RMPI-1640 medium (100 μL/well, purchased from Gbico, USA) in mL streptomycin sulfate, after the cells adhered to the wall, a series of 2-fold serially diluted cecropin-TY1 as shown in Figure 6 was added for co-cultivation After 24 hours, the cell proliferation and toxicity detection reagent CCK-8 (10 μL/well, purchased from Beijing Wobison Technology Co., Ltd.) was added to each well, and after incubation for 4 hours, the light absorption at 450 nm was detected, and the cell viability without adding polypeptide was defined as 100%, and calculate the cell viability after treatment with different concentrations of cecropin-TY1.

As shown in Figure 6, cecropin-TY1 had no detectable cytotoxicity to mouse macrophages at concentrations as high as 100 μg/mL. It shows that its anti-inflammatory activity is not achieved by affecting the activity of macrophages.

5. Determination of cecropin-TY1 neutralizing endotoxin function.

The polypeptide was dissolved in 10 mM, pH6.0 phosphate buffer, and different concentrations of cecropin-TY1 (0, 12.5, 25, 50, 100 μg/mL) were mixed with LPS (1 μg/mL, purchased from Sigma) in a In a pyrogen-free test tube, after co-incubating at 37°C for 30 minutes, add 100 μL of Limulus reagent (purchased from Xiamen Limulus Reagent Co., Ltd.) to every 100 μL of polypeptide-LPS mixture, incubate at 37°C for 10 minutes, and then add the The substrate was incubated at 37°C for 6 minutes, and the light absorbance at 545 nm was measured on a microplate reader, and then the neutralization ability of different concentrations of cecropin-TY1 was calculated, wherein the LPS without peptide was defined as 0 neutralization tube.

As shown in Figure 7, cecropin-TY1 neutralized the endotoxin activity of LPS in a dose-dependent manner. 65.1% endotoxin activity.

6. Determination of the secondary structure of cecropin-TY1 solution.

The circular dichroism spectrum of the sample in different solution environments was detected by a circular dichroism spectrometer JASCO J-810 (JASCO, Tokyo, Japan) to analyze the composition of the secondary structure of cecropin-TY1 in different solutions. The cecropin-TY1 sample was dissolved in water and membrane simulated environment (TFE/H ₂ O, LPS/H ₂ O, SDS/H ₂ O solution), and the dissolved concentration was 0.2 mg/mL. Set the test temperature to 25°C, scan the wavelength range of 190-260nm, the wavelength interval is 0.2nm, the sample cell length is 0.1cm, and the bandwidth is 1nm. Each sample is scanned 3 times continuously. The secondary structure of cecropin-TY1 in different solution environments was estimated by circular dichroism spectrum according to Yang formula.

As shown in Figure 8, in H ₂ O, cecropin-TY1 exhibits the maximum negative absorption at 199nm, indicating that the main conformation at this time is random coil. When in the membrane simulation environment (TFE/H ₂ O, LPS/H ₂ O, SDS/H ₂ O solution), cecropin-TY1 exhibits double negative absorption at 208 and 222 nm, indicating that the main secondary conformation at this time is α- spiral. The results showed that a part of the random coil conformation was converted to the α-helical conformation in the membrane simulation environment.

7. Determination of the tertiary structure of cecropin-TY1 solution.

Use Easymodeler version 2.0 to calculate the three-dimensional solution structure of cecropin-TY1, and analyze its surface charge distribution. Using papiliocin from butterfly (59% similarity, PDB entry code 2LA2) as a template for homology simulation, the structure was optimized and visualized with MODELLER and PYMOL software (http://www.pymol.org).

As shown in Figure 9, cecropin-TY1 exhibits a helix-hinge-helix conformation, that is, two α-helical domains (red region, Leu ³ -Thr ²⁴ and Ile ²⁷ -Leu ³⁸ ) are separated by a hinge domain (green region, Ile ²⁵ -Pro ²⁶ ) connection (Fig. 9A). Surface charge analysis revealed that part of the surface region of cecropin-TY1 was positively charged (Fig. 9B, blue area).

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements can be made without departing from the technical principle of the present invention. and modifications, these improvements and modifications should also be considered as the protection scope of the present invention.

SEQUENCE LISTING

<110> Soochow University

<120> Yaofly natural anti-inflammatory polypeptide cecropin-TY1 and its application

<130> 2015

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 39

<212> PRT

<213> Tabanua Yao

<400> 1

Gly Try Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln Asn Val Arg

1 5 10 15

Asn Ala Ala Ile Ser Thr Ile Pro Ile Ala Gln Gly Ala Ala Gly Val Ala Gly Ala

20 25 30 35

Leu Asn

Claims (8)

1. The application of a natural anti-inflammatory polypeptide cecropin-TY1 of Yaofly in the preparation of anti-inflammatory drugs, the amino acid sequence of the natural anti-inflammatory polypeptide cecropin-TY1 of Yaofly is SEQ ID: 1. 2. the application of a kind of natural anti-inflammatory polypeptide cecropin-TY1 of Yao afly in the preparation of sepsis and endotoxic shock medicine caused by bacterial infection, the amino acid sequence of said natural anti-inflammatory polypeptide cecropin-TY1 of Yao afly is SEQ ID: 1. 3. The application according to any one of claims 1-2, characterized in that: the natural anti-inflammatory polypeptide cecropin-TY1 of Yaofly is used in inhibiting lipopolysaccharide-induced macrophages from producing nitric oxide. 4. The application according to any one of claims 1-2, characterized in that: the application of Yaofly's natural anti-inflammatory polypeptide cecropin-TY1 in inhibiting the production of inflammatory cytokines by lipopolysaccharide-induced macrophages. 5. The application according to any one of claims 1-2, characterized in that: the application of Yaofly's natural anti-inflammatory polypeptide cecropin-TY1 in inhibiting the inflammatory signaling pathway induced by lipopolysaccharide. 6. The application according to claim 5, characterized in that: the application of the natural anti-inflammatory polypeptide cecropin-TY1 of Yaofly in inhibiting the activation of lipopolysaccharide-induced mitogen-activated protein kinase signaling pathway. 7. The application according to claim 5, characterized in that: the application of Yaofly's natural anti-inflammatory polypeptide cecropin-TY1 in inhibiting lipopolysaccharide-induced activation of nuclear transcription factor NF-κB. 8. The application according to any one of claims 1-2, characterized in that: the application of Yaofly's natural anti-inflammatory polypeptide cecropin-TY1 in neutralizing endotoxin function.