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Application of Chinese soft-shelled turtle cathelicidin-ps-cath4 peptide in the preparation of anti-inflammatory drugs

A ps-cath4, Chinese soft-shelled turtle technology, applied in the field of biomedicine, can solve the problems of no reports, and achieve the effects of simple structure, low cytotoxicity and hemolytic activity, and good stability

Active Publication Date: 2017-12-26
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese soft-shelled turtle belongs to the genus Sino soft-shelled turtle of the family Turtleidae of the order Turtle, and there is no report on the research and application of the cathelicidin host defense peptide of the Chinese soft-shelled turtle.

Method used

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  • Application of Chinese soft-shelled turtle cathelicidin-ps-cath4 peptide in the preparation of anti-inflammatory drugs
  • Application of Chinese soft-shelled turtle cathelicidin-ps-cath4 peptide in the preparation of anti-inflammatory drugs
  • Application of Chinese soft-shelled turtle cathelicidin-ps-cath4 peptide in the preparation of anti-inflammatory drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Ps-CATH4 peptide inhibits LPS-induced inflammatory cytokine and NO synthase (INOS) gene expression

[0058] Mouse macrophages RAW264.7 were treated with DMEM medium containing 10% fetal bovine serum at 37˚C CO 2 Cultivate in an incubator. When the cells grow to a good logarithmic growth phase, they are digested with 0.25% trypsin, discarded, and blown down adherent cells with DMEM medium with 10% fetal bovine serum. Count the cells after pipetting and adjust the cell concentration to 1x10 6 / ml. Add the evenly pipetted cells to a 24-well plate, 1ml per well, at 37˚C CO 2 Culture overnight in an incubator to make the cells adhere to the wall, then aspirate the supernatant and discard it, add 960ul of fresh DMEM medium containing 10% fetal bovine serum, add 20ul of a certain concentration of LPS to the positive control group and the dosing group, and then add 20ul of Ps-CATH4 diluted with DMEM and filtered and sterilized with different concentrations of Ps-CATH4...

Embodiment 2

[0060] Example 2 Ps-CATH4 peptide regulates the secretion of LPS-induced cellular inflammatory factors and NO in vitro

[0061] Mouse macrophages RAW264.7 were cultured in DMEM medium containing 10% fetal bovine serum to the logarithmic growth phase. After trypsinization, they were seeded in 96-well plates, 1x10 per well 6 One. CO at 37 ˚C 2 Incubate overnight in an incubator to make the cells adherent. After the cells adhere to the wall, discard the supernatant, add 180ul of fresh DMEM medium containing 10% fetal bovine serum to each well, and then add 10ul of Ps-CATH4 of different concentrations and 10ul of LPS of a certain concentration to each well, add blank 20ul serum-free DMEM medium, treated for 24h. The samples treated for 24 hours were used to collect the supernatant to detect the release of NO and inflammatory factors with Griess kit and ELISA kit, and 5 replicates were set for each experimental group. The Griess kit was purchased from Biyuntian Biological Reagent Co...

Embodiment 3

[0062] Example 3 Western blot method to detect the influence of Ps-CATH4 on MAPK inflammation signal pathway

[0063] Mouse macrophages RAW264.7 were cultured in DMEM medium containing 10% fetal bovine serum to the logarithmic growth phase. After digestion with trypsin, the cells were digested with 3x10 6 Pieces / ml were inoculated into 6-well plates, 2ml per well. CO at 37˚C 2 Incubate overnight in an incubator to make the cells adhere to the wall, then discard the upper cell culture medium, and use phosphate buffered saline (PBS: 0.8g NaCl; 0.2g KCl; 2.9gNa 2 HPO 4 .12H 2 O; 0.27g KH 2 PO 4 Dissolve in 800ml deionized water, stir to dissolve, make the volume to 1L, adjust the pH to 7.4 with concentrated HCl, and autoclave. ) Wash the cells 2-3 times, change to serum-free DMEM medium and starve for 16h. Add 20ul samples of different concentrations to each well (make the final concentration of each well 5, 10, 20ug / ml), add 20ul serum-free DMEM to the blank, and after treatment fo...

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Abstract

The invention discloses application of Chinese softshell turtle Cathelicidin-Ps-CATH4 peptide in preparation of an anti-inflammatory drug, and belongs to the technical field of biomedicine. Ps-CATH4 is linear peptide, and contains 32 amino acid residues; RT-PCR displays that the peptide has an inhibition effect on expression of critical inflammatory factors induced by lipopolysaccharide and an inducible NO synthase gene; and on the protein level, the peptide has a negative regulation effect on LPS-induced inflammatory factors and NO release, has an ascending effect on expression of inflammatory factors, and is mainly used for inhibiting release of the inflammatory factors by inhibiting activation of a TLR4 acceptor and a downstream MAPK signal channel. The peptide is simple in structure, does not contain a disulfide bond or a cyclic structure; chemical synthesis and genetic engineering preparation are facilitated; the primary medicinal evaluation shows low cytotoxicity and hemolytic activity; and the peptide is good in stability on salt, blood serum, temperature and time, and is applied to preparation of the anti-inflammatory drug.

Description

Technical field [0001] The invention provides a Chinese soft-shelled turtle ( Pelodiscus sinensis ) Cathelicidin-Ps-CATH4 and its coding gene, amino acid sequence and application in the preparation of anti-inflammatory drugs belong to the field of biomedical technology. Background technique [0002] Cathelicidin is a family of multifunctional immunomodulatory peptides composed of the N-terminal signal peptide region, the middle conserved cathelin domain and the C-terminal highly specific mature peptide region. It is found in almost all kinds of animals (mammals, birds, Amphibians and fish) have been found. Like defensins, Cathelicidin is a host defense peptide unique to most vertebrates, including humans, and constitutes a key component of vertebrate natural immune response. The bridge between natural immunity and specific immunity plays an extremely important role in the innate immune system of animals. In addition to its broad-spectrum antibacterial activity, Cathelicidin also...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46C12N15/12A61K38/17A61P29/00
CPCA61K38/00C07K14/46
Inventor 于海宁王义鹏乔雪鲁一灵王晨
Owner DALIAN UNIV OF TECH
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