CN105535932B - Medical application of three polypeptide fragments in preparation of anti-fibrosis drugs - Google Patents
Medical application of three polypeptide fragments in preparation of anti-fibrosis drugs Download PDFInfo
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- CN105535932B CN105535932B CN201610119073.1A CN201610119073A CN105535932B CN 105535932 B CN105535932 B CN 105535932B CN 201610119073 A CN201610119073 A CN 201610119073A CN 105535932 B CN105535932 B CN 105535932B
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
Abstract
The invention discloses three polypeptide fragments with anti-fibrosis effect, which are applied to preparing anti-fibrosis drugs, and the amino acid sequences of the three polypeptides are Pep69 KFYIHDSY, Pep70 PG L YYYFD and Pep71 PG L YYYFD.
Description
Technical Field
The invention relates to the field of medicines, in particular to application of three polypeptide fragments in preparing anti-fibrosis medicines.
Background
Fibrosis is the pathological process of excessive deposition of extracellular matrix in tissues and organ tissue remodeling, and its disease spectrum is large, including diseases involving multiple systems, such as systemic sclerosis, multifocal fibrosis, scleroderma, renal origin, and also including organ-tissue specific diseases such as lung, liver, kidney fibrosis, etc. transforming growth factor (TGF- β) is a key factor in fibrosis, responsible for initiating and maintaining fibroblast activation and myofibroblast differentiation, thus blocking TGF- β signaling pathway is the classical means for treating fibrotic diseases.
Recent studies have shown that adiponectin receptor activated by adiponectin can induce adenosine monophosphate activated protein kinase (AMPK) to block TGF- β classical signaling pathway and becomes an important pathway for treating fibrotic diseases, adiponectin is an endogenous bioactive protein secreted by adipocytes, has various effects of regulating in vivo energy balance, glycolipid metabolism, anti-inflammation, anti-arteriosclerosis and the like, is a cytokine with wide biological effects, consists of 244 amino acids, comprises an amino-terminal signal sequence, a collagen repeat sequence and a carbon-terminal globular domain, wherein the globular domain is a key site for adiponectin bioactivity, adiponectin receptor has two major subtypes in vivo, adipo r1 and adipo r2, has homology of 66.7%, is highly conserved between species, is a 7-transmembrane protein, adiponectin activates the signaling pathway by interacting with adipo r1, and activation of adiponectin receptor by TGF-28 induced TGF 2-smooth muscle kinase (ampa) and inhibits the development of fibrosis by ampa collagen production inhibitor (ampa) through interaction with adipo r1, and activation of adiponectin receptor via experiments, thus inhibiting the development of mouse fibrosis by ampa type SMA inhibitor (ampa) 5-SMA inhibitor, ampa mouse, mouse (mouse) and mouse (mouse) mouse (mouse) via mouse.
Disclosure of Invention
The invention aims to provide three polypeptide fragments which can be specifically combined with AdipoR1 and excite the receptor, induce an AMPK pathway to block a TGF- β classical signal pathway, and further inhibit the expression of α -SMA and CO L1A 1, thereby treating fibrotic diseases.
The invention uses a homologous modeling method to construct a three-dimensional structure model of AdipoR1, molecular docking research is utilized to analyze the interaction mode of the known active polypeptide and AdipoR1, the key amino acid of the AdipoR1 is confirmed, three polypeptide fragments with higher scores are virtually screened out by molecular docking, and in-vitro cell cycle arrest and anti-fibrosis activity tests show that the three polypeptide fragments can obviously inhibit the expression levels of α -SMA and CO L1A 1 genes/proteins, and the strong anti-fibrosis activity is prompted.
The three polypeptide fragments have sequences of Pep69 KFYIHDDY, Pep70 PG L YYYYFD and Pep71 PG L YYYFD, and can reduce the expression level of α -SMA and CO L1A 1 genes by blocking a TGF- β classical signal path, and further inhibit the protein expression of α -SMA and CO L1A 1, so that the purpose of treating fibrotic diseases is achieved.
Furthermore, the invention also provides application of the three polypeptide fragments in preparing anti-fibrosis drugs.
The three polypeptide fragments can be used for preparing a medicament, and can be a single polypeptide fragment preparation, or a preparation formed by mixing the three polypeptide fragments in any proportion, or various preparations formed by mixing the three polypeptide fragments in any proportion and pharmaceutically acceptable carriers, such as injections, oral liquids, pills, tablets, capsules and the like, wherein the pharmaceutically acceptable carriers can be: the carrier materials include fillers, disintegrants, wetting agents, binders, effervescent agents, surfactants, lubricants, glidants, odorants, colorants, and other types of excipients for solid formulations.
The polypeptide fragment contains the following amino acid sequences of KFYIHSDY, PG L YYFD or PG L YYFD.
Drawings
FIG. 1 shows the effect of three polypeptide fragments on HSC-T6 cell proliferation, FIG. 2 shows the effect of three polypeptide fragments on HSC-T6 cell cycle, FIG. 3 shows the effect of three polypeptide fragments on α -SMA (A), CO L1A 1(B) and TGF- β 1(C) gene expression levels, and FIG. 4 shows the effect of three polypeptide fragments on α -SMA, CO L1A 1 and TGF- β 1 protein expression levels.
Detailed Description
The invention relates to three polypeptide fragments which can reduce the generation of α -SMA and CO L-1 induced by exogenous TGF- β by blocking TGF- β signal paths, thereby achieving the effect of anti-fibrosis.
The following are some of the anti-fibrosis tests and results of the present invention:
reagent and apparatus
The reagent comprises polypeptide fragments for detecting activity, namely Pep69 (KFYIHSDYDYDYDYDYDY), Pep70(PG L YYYYFD) and Pep71(PG L YYFD), positive control peptide ADP355 and negative control peptide Pep56(TETSQVAPA), cell strain HSC-T6 cells, and the reagent DMEM high-sugar culture mediumBase of(containing 0.1% FBS), mouse TGF- β 1, CCK-8 kit, TRIzol, and cDNA first strand synthesis kit.
Flow cytometry (BD FACSCalibur, BD, USA), plate reader (Model 550; Bio-Rad, Hercules, CA, USA), and fluorescence quantitative qPCR (Bio-Rad L analytes Inc., USA).
Effects of one or three polypeptide fragments on the proliferation and cycle of HSC-T6 cells:
1. cell culture
HSC-T6 cells were cultured in DMEM high-glucose complete medium containing 10% heat-inactivated fetal bovine serum at 37 ℃ with a volume fraction of 5% CO2And performing conventional culture under the condition of complete saturation humidity, replacing the culture medium every 48 hours, after the cells grow and cover 80% of the bottom of a culture flask, digesting and passaging the cells by using 0.25% trypsin and 0.02% EDTA, and selecting the cells in logarithmic phase in the experiment.
CCK-8 method for detecting influence of three polypeptides on proliferation of HSC-T6 cells
Taking rat hepatic stellate cell HSC-T6 in logarithmic growth phase, conventionally digesting, preparing single cell suspension, inoculating in 96-well culture plate, 1 × 104100 mul/well, arranging a cell blank control group and a pure culture solution background group, after the cells are plated for 24 hours, adding various polypeptide drugs with different concentrations (the final concentrations are respectively 0.05, 0.5, 1.0, 5.0, 10.0, 50 and 100 mul) or PBS into each well for continuous treatment for 24 hours, adding CCK-8 solution into each well, incubating for 4 hours at 37 ℃, measuring the absorbance OD value at 570nm wavelength on an enzyme-linked immunosorbent assay, and calculating the proliferation rate according to the following formula, wherein the proliferation rate is × 100 percent (experimental group OD value-background group OD value)/(control group OD value-background group OD value).
3. Flow cytometry to examine the Effect of three Polypeptides on the cell cycle of HSC-T6
Taking HSC-T6 cells in logarithmic growth phase, preparing single cell suspension, inoculating in 24-well culture plate, 1 × 104500. mu.l/well, 5% CO at 37 ℃2Culturing overnight under the condition, allowing cells in each well to adhere to the wall, adding different polypeptide drugs (final concentration: 50 μ M) or PBS for pretreatment for 1h, adding rat TGF- β 1 recombinant protein (final concentration: 5ng/M L) for stimulation for 24h, collecting HSC-T6 cells, centrifuging at 1000rpm for 5min, washing the cells with precooled PBS for 3 times, centrifuging to remove supernatant, adding precooled 75% ethanol 500 μ l for fixation, fixing at 4 ℃ overnight, centrifuging to remove fixative, and adopting pre-treatmentThe cells were washed 2 times with cold PBS and gently shaken to suspend the cells. Adding 15 μ l RNase A100 μ g/ml, mixing by vortex shaking, and standing at 4 deg.C in dark for 15 min. Adding 500 μ l PBS containing 50 μ g/ml PI, incubating for 10min at room temperature in dark, detecting the distribution of cells in each period by flow cytometry, wherein: the excitation light wavelength is 488nm, and the emission light wavelength is 630 nm.
And (5) judging a result: the three polypeptide fragments can inhibit the proliferation of rat hepatic stellate cell HSC-T6; meanwhile, on the basis of inhibiting cell proliferation, the three polypeptide fragments have obvious cycle blocking effect on HSC-T6 cells, wherein Pep70 has the strongest inhibition activity, which indicates the in vitro anti-fiber activity of the polypeptide. The results are shown in FIGS. 1-2.
The effect of three polypeptide fragments on the proliferation and cell cycle of HSC-T6 cells was determined separately in this experiment.
As can be seen from FIG. 1, the three polypeptide fragments all inhibited the proliferation of HSC-T6 cells dose-dependently, and the inhibition was strongest when the concentration reached 100. mu.M. Of the three polypeptide fragments, Pep70 has the most obvious inhibition effect on HSC-T6 cell proliferation, and the inhibition rate reaches 18.3% under the condition of 100 mu M of dosage, and has a significant difference (P is less than 0.05) compared with a normal group.
As can be seen from FIG. 2, the three polypeptide fragments all have a blocking effect on the cell cycle of HSC-T6 cells, and the proportion of G0/G1 cells gradually increases and the proportion of G2/M cells decreases with increasing concentration. Compared with the model control group, the proportion of HSC-T6 cells in the G2/M phase of the Pep69 and Pep70 treated group is obviously reduced and has a significant difference (p < 0.05).
In vitro anti-fibrosis activity of two and three polypeptide fragments
1. Establishment of in vitro cell fibrosis model
Respectively taking HSC-T6 cells with good growth state, and adding 1 × 104Inoculating 500 μ l/well into 24-well plate, changing culture medium containing 0.1% FBS when cell growth fusion degree reaches 80%, starving for 16h, synchronizing cells, stimulating rat TGF- β 1 recombinant protein (final concentration: 5ng/ml) for 24h, removing culture medium, extracting total RNA of cells by Trizol, detecting gene expression levels of α -SMA and CO L1A 1 by Q-PCR,it is determined whether the modeling was successful.
2. Determination of in vitro anti-fibrotic Activity
2.1 Effect of three polypeptide fragments on α -SMA and CO L1A 1 Gene expression levels
HSC-T6 cells with good growth status were seeded on 24-well plates (density: 1 × 10)4500 mul/well), when the cell growth fusion degree reaches 80%, treating with a culture medium containing 0.1% FBS for 16h, grouping, (1) blank group adding PBS with corresponding volume, (2) model group adding PBS + TGF- β with corresponding volume for stimulation, (3) positive control group adding ADP355+ TGF- β 1 with concentrations of 10, 50 and 100 mul for stimulation respectively, (4) experimental group adding Pep69, Pep70 and Pep71 with concentrations of 10, 50 and 100 mul and adding TGF- β for stimulation respectively, and (5) negative control group adding Pep56+ TGF- β with concentrations of 10, 50 and 100 mul for stimulation respectively, setting 3 multiple wells in 24 well plate according to the concentration, giving corresponding polypeptide or solvent pretreatment 1h, TGF- β stimulation group adding exogenous TGF- β 1 recombinant protein (final concentration: 5ng/ml) to rat- β stimulation group, removing culture medium 24h, and using PCR to detect total expression of total RNA of PCR and total gene L A.
2.2 Effect of three polypeptide fragments on the expression level of α -SMA and CO L1A 1 proteins
Taking HSC-T6 cells with good growth state, inoculating, grouping and processing according to the medium density of 2.1, adding exogenous rat TGF- β 1 recombinant protein (final concentration: 5ng/ml) to stimulate for 24h, sucking and removing the culture medium, gently washing for 2 times by adopting a cold HBSS solution, adding cell lysate to ice, standing for 10min, shaking to fully lyse the cells, and detecting the protein expression conditions of α -SMA and CO L1A 1 by adopting Western blot.
The results judge that exogenous rat TGF- β 1 recombinant protein intervenes for 24h, the expression of α -SMA, CO L1A 1 and TGF- β 1 genes can be obviously up-regulated, the establishment of an in-vitro cell fibrosis model is prompted to be successful, three polypeptide fragments have obvious in-vitro anti-fibrosis activity, the gene and protein expression of exogenous TGF- β 1 up-regulated fibrosis factors α -SMA, CO L1A 1 and TGF- β 1 can be inhibited dose-dependently, myofibroblast differentiation is further inhibited, collagen accumulation induced by TGF- β 1 is blocked, and the anti-fibrosis effect is finally realized, and the results are shown in a figure 3-4.
As can be seen from FIG. 3, the three polypeptide fragments can obviously inhibit the gene expression of exogenous TGF- β 1-induced fibrosis factors α -SMA, CO L1A 1 and TGF- β 1, and have a certain dose-effect relationship, wherein the anti-fibrosis activity of Pep70 is most obvious, when the concentration of the polypeptide fragments reaches 100 mu M, the inhibition rates of the polypeptide fragments on α -SMA, CO L1A 1 and TGF- β 1 are respectively 78.1%, 82.5% and 85.0%, and the inhibition effect is slightly stronger than that of positive control peptide (ADP 355).
As can be seen from FIG. 4, on the basis of the down-regulation of α -SMA, CO L1A 1 and TGF- β 1 gene levels, the three polypeptide fragments also express obvious inhibition effects on the proteins corresponding to the three fibrosis factors, wherein the inhibition rates are respectively 24.0%, 52.0% (α -SMA), 12.5%, 20.8%, 29.2% (CO L1A 1) and 61.0%, 66.1%, 69.5% (TGF- β 1), and compared with a model control group, the inhibition rates have significant differences (p is less than 0.05), which shows that the three polypeptide fragments have significant inhibition effects on fibrosis induced by exogenous TGF- β 1.
Claims (5)
1. The application of the two polypeptide fragments in preparing the anti-hepatic fibrosis medicine is that the amino acid sequences of the polypeptide fragments are Pep 69: KFYIHSDY and Pep 70: PG L YYFD respectively.
2. The use of two polypeptide fragments according to claim 1 for the preparation of a medicament against liver fibrosis, characterized in that: the drug comprises one or both of the two polypeptide fragments.
3. The anti-hepatic fibrosis medicine is characterized by comprising one or two of polypeptide fragments Pep69 and Pep70 and one or more pharmaceutically acceptable carriers, wherein the amino acid sequences of Pep69 and Pep70 are Pep69, KFYIHSDY and Pep70, PG L YYYFD respectively.
4. The anti-hepatic fibrosis drug of claim 3, wherein the pharmaceutically acceptable carrier is: diluents, fillers, excipients, binders, wetting agents, disintegrants, surfactants, absorption enhancers, lubricants or adsorption carriers.
5. The anti-hepatic fibrosis drug of claim 3, wherein the drug is in the form of: tablets, pills, capsules, suspensions, emulsions, injections or dry powders.
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| CN109142606B (en) * | 2018-08-23 | 2020-11-24 | 广西壮族自治区中医药研究院 | In vitro screening and chemical component analysis of active site of Kadsura heteroclita for reversing hepatic fibrosis |
| CN109810176B (en) * | 2019-01-29 | 2020-07-28 | 中山大学 | Adiponectin receptor-1 and receptor-2 dual agonist peptides for the treatment of non-alcoholic steatohepatitis and liver fibrosis |
| CN113577241A (en) * | 2020-12-24 | 2021-11-02 | 南开大学 | Design and screening method of small blocking peptide and application of small blocking peptide in synthesizing medicament for treating fibrotic diseases |
| WO2022178841A1 (en) * | 2021-02-26 | 2022-09-01 | 深圳市图微安创科技开发有限公司 | Combination of adiponectin receptor agonist and elastin receptor inhibitor for prevention or treatment of non-alcoholic fatty liver disease |
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| A potent peptide as adiponectin receptor 1 agonist to against fibrosis;Ma L et al.;《J Enzyme Inhib Med Chem》;20171231;624-631 * |
| Adiponectin receptor binding proteins--recent advances in elucidating adiponectin signalling pathways;Buechler C et al.;《FEBS Lett》;20101031;4280-4286 * |
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