CN105535932A - Medical application of three polypeptide fragments to preparation of anti-fibrosis drug - Google Patents

Abstract

The invention discloses three polypeptide fragments with anti-fibrosis effect, which are applied to the preparation of anti-fibrosis drugs. The amino acid sequences of these three polypeptides are: Pep69: KFYIHSDY, Pep70: PGLYYFD and Pep71: PGLYYFD.

Description

Medical application of three polypeptide fragments in the preparation of anti-fibrosis drugs

technical field

The invention relates to the field of medicines, in particular to the use of three polypeptide fragments in the preparation of anti-fibrosis medicines.

Background technique

Fibrosis is a pathological process of excessive deposition of extracellular matrix in tissues and remodeling of organ tissues. Its disease spectrum is large, including diseases involving multiple systems, such as systemic sclerosis, multifocal fibrosis, scleroderma, renal Multisystem fibrosis of origin, including organ-tissue-specific diseases such as lung, liver, and kidney fibrosis. Transforming growth factor (TGF-β) is a key factor in fibrosis, responsible for the initiation and maintenance of fibroblast activation and myofibroblast differentiation, thus blocking the TGF-β signaling pathway is a classic treatment for fibrotic diseases . The TGF-β signaling pathway involves many cytokines, and finding effective target cytokines is the key to the treatment of fibrotic diseases.

Recent studies have shown that after the adiponectin receptor is activated by adiponectin, it can induce adenosine monophosphate-activated protein kinase (AMPK) to block the classic TGF-β signaling pathway, which has become an important way for the treatment of fibrotic diseases. Adiponectin is an endogenous biologically active protein secreted by adipocytes. It has multiple functions such as regulating energy balance in the body, glucose and lipid metabolism, anti-inflammation, and anti-arteriosclerosis. It is a cytokine with a wide range of biological effects. Adiponectin in the human body consists of 244 amino acids, including an amino-terminal signal sequence, a collagen repeat sequence, and a carbon-terminal globular domain, wherein the globular domain is the key site of adiponectin's biological activity. There are two main subtypes of adiponectin receptors in the body, AdipoR1 and AdipoR2, with a homology of 66.7%, which are highly conserved among species, and both are seven transmembrane proteins. Adiponectin activates the AMPK signaling pathway by interacting with AdipoR1, and the activation of AMPK can reduce the production of TGF-β-induced α-smooth muscle actin (α-SMA) and type I collagen-α1 (COL1A1), thereby achieving anti-fibrotic role of transformation. Animal experiments have also proved that adiponectin can inhibit the process of fibrosis by interacting with AdipoR1, indicating that AdipoR1 agonists are a class of potential anti-fibrosis drugs.

Contents of the invention

The purpose of the present invention is to provide three polypeptide fragments that can specifically bind to AdipoR1 and activate the receptor, induce the AMPK pathway to block the classic TGF-β signaling pathway, and then inhibit the expression of α-SMA and COL1A1, thereby treating fibrotic diseases .

The present invention constructs a three-dimensional structure model of AdipoR1 by using a homology modeling method. The interaction mode between known active peptides and AdipoR1 was analyzed by molecular docking research, and the key amino acids of AdipoR1 were confirmed. Three high-scoring polypeptide fragments were screened out by molecular docking. The results of in vitro cell cycle arrest and anti-fibrosis activity tests showed that all three polypeptide fragments could significantly inhibit the expression levels of α-SMA and COL1A1 gene/protein, suggesting their strong anti-fibrosis activity.

The sequences of the three polypeptide fragments included in the present invention are respectively: Pep69: KFYIHSDY, Pep70: PGLYYFD and Pep71: PGLYYFD. All of them can block the TGF-β classic signaling pathway, down-regulate the gene expression level of α-SMA and COL1A1, and then inhibit the protein expression of α-SMA and COL1A1, so as to achieve the purpose of treating fibrotic diseases.

Furthermore, the present invention also provides the application of the three polypeptide fragments in the preparation of anti-fibrosis drugs.

Preparation of drugs from three polypeptide fragments can be a single polypeptide fragment preparation, or a preparation in which the three polypeptide fragments are mixed in any proportion, or a variety of preparations composed of three polypeptide fragments in any proportion and a pharmaceutically acceptable carrier. Preparations, such as injections, oral liquids, pills, tablets, capsules, etc., these pharmaceutically acceptable carriers can be: the carrier materials include fillers, disintegrating agents, wetting agents, binders, effervescent agents, Surfactants, lubricants, glidants, olfactory agents, colorants and other types of excipients for solid preparations.

The polypeptide fragment in the present invention contains the following amino acid sequence: KFYIHSDY, PGLYYFD or PGLYYFD.

Description of drawings

Figure 1: Effects of three polypeptide fragments on the proliferation of HSC-T6 cells; Figure 2: Effects of three polypeptide fragments on HSC-T6 cell cycle; Figure 3: Effects of three polypeptide fragments on α-SMA(A), COL1A1(B ) and TGF-β1 (C) gene expression levels; Figure 4: Effects of three polypeptide fragments on α-SMA, COL1A1 and TGF-β1 protein expression levels.

detailed description

The invention relates to three polypeptide fragments that block the TGF-β signaling pathway and reduce the production of α-SMA and COL-1 induced by exogenous TGF-β, so as to achieve the effect of anti-fibrosis.

Below is the part anti-fibrosis test and result of the present invention:

Reagents and Instruments

Reagents: Peptide fragments for detecting activity: Pep69 (KFYIHSDY), Pep70 (PGLYYFD) and Pep71 (PGLYYFD), positive control peptide: ADP355, negative control peptide Pep56 (TETSQVAPA); cell line: HSC-T6 cells; reagent: DMEM high glucose Medium (containing 0.1% FBS), mouse TGF-β1, CCK-8 kit, TRIzol, cDNA first strand synthesis kit.

Instruments: flow cytometer (BDFACSCalibur, BD, USA), microplate reader (Model 550; Bio-Rad, Hercules, CA, USA), fluorescence quantitative qPCR instrument (Bio-Rad Laboratories Inc., USA).

1. The effects of three polypeptide fragments on the proliferation and cycle of HSC-T6 cells:

1. Cell culture

HSC-T6 cells were routinely cultured in DMEM high-glucose complete medium containing 10% heat-inactivated fetal bovine serum at 37°C, with a volume fraction of 5% CO ₂ and fully saturated humidity. The medium was replaced every 48 hours, and the cell growth After 80% of the bottom of the culture flask was filled, it was digested and passaged with 0.25% trypsin combined with 0.02% EDTA, and the cells in the logarithmic growth phase were selected for the experiment.

2. CCK-8 method to detect the effect of three polypeptides on the proliferation of HSC-T6 cells

Rat hepatic stellate cells HSC-T6 in the logarithmic growth phase were routinely digested to prepare a single-cell suspension, and inoculated in a 96-well culture plate at 1×10 ⁴ /100 μl/well. A blank control group and simple Culture medium background group; after 24 hours of cell plating, each well was added with different concentrations of polypeptide drugs (final concentrations: 0.05, 0.5, 1.0, 5.0, 10.0, 50 and 100 μM) or PBS for 24 hours, and then added CCK-8 Solution, 10 μl/well, incubated at 37°C for 4h; placed on a microplate reader to measure the OD value of the absorbance at 570nm wavelength, and calculated the proliferation rate according to the following formula: proliferation rate=(OD value of the experimental group-OD value of the background group)/(control group OD value-OD value of background group)×100%.

3. The effect of three polypeptides on the HSC-T6 cell cycle detected by flow cytometry

Take HSC-T6 cells in the logarithmic growth phase, prepare a single cell suspension, inoculate in a 24-well culture plate, 1×10 ⁴ /500 μl/well, and culture overnight at 37°C and 5% CO ₂ to make the cells in each well adhere The walls were pretreated with different polypeptide drugs (final concentration: 50 μM) or PBS for 1 hour, and then rat TGF-β1 recombinant protein (final concentration: 5 ng/mL) was added to stimulate for 24 hours. Collect HSC-T6 cells, centrifuge at 1000rpm for 5 minutes, wash the cells 3 times with pre-cooled PBS, and discard the supernatant by centrifugation. Add 500 μl of pre-cooled 75% ethanol to fix, and fix overnight at 4°C. Centrifuge to remove the fixative, wash the cells twice with pre-cooled PBS, and shake gently to suspend the cells. Add 15 μl of 100 μg/ml RNaseA, mix well by vortexing, and place at 4°C in the dark for 15 minutes. Add 500 μl PBS containing 50 μg/ml PI, incubate at room temperature in the dark for 10 minutes, and detect the distribution of cells in each cycle by flow cytometry, wherein: excitation light wave=488nm, emission light wavelength=630nm.

Judgment of results: All three polypeptide fragments can inhibit the proliferation of rat hepatic stellate cell HSC-T6; at the same time, on the basis of inhibiting cell proliferation, the three polypeptide fragments also have a significant cycle arrest effect on HSC-T6 cells, Among them, Pep70 has the strongest inhibitory activity, suggesting that the polypeptide has anti-fibrotic activity in vitro. The results are shown in Figures 1-2.

In this experiment, the effects of the three polypeptide fragments on the proliferation and cell cycle of HSC-T6 cells were respectively determined.

It can be seen from Figure 1 that all three polypeptide fragments can inhibit the proliferation of HSC-T6 cells in a dose-dependent manner, and the inhibitory effect is the strongest when the concentration reaches 100 μM. Among the three polypeptide fragments, Pep70 has the most obvious inhibitory effect on the proliferation of HSC-T6 cells, and the inhibitory rate reaches 18.3% at a dose of 100 μM, which is significantly different from the normal group (P<0.05).

It can be seen from Figure 2 that all three polypeptide fragments can block the cell cycle of HSC-T6 cells. As the concentration increases, the proportion of cells in G0/G1 phase gradually increases, while the proportion of cells in G2/M phase decreases. Compared with the model control group, the proportion of HSC-T6 cells in the G2/M phase of the Pep69 and Pep70 treatment groups decreased significantly, and there was a significant difference (p<0.05).

2. In vitro anti-fibrosis activity of three polypeptide fragments

1. Establishment of cell fibrosis model in vitro

HSC-T6 cells in good growth state were taken respectively, and seeded in 24-well plates at 1×10 ⁴ /500 μl/well. When the confluence of the cells reached 80%, the culture medium containing 0.1% FBS was replaced, starved for 16 hours to synchronize the cells, and stimulated with rat TGF-β1 recombinant protein (final concentration: 5 ng/ml) for 24 hours. The medium was discarded, and the total cellular RNA was extracted with Trizol, and the gene expression levels of α-SMA and COL1A1 were detected by Q-PCR to determine whether the modeling was successful.

2. Determination of anti-fibrotic activity in vitro

2.1 Effects of three polypeptide fragments on the expression levels of α-SMA and COL1A1 genes

Take well-growing HSC-T6 cells and inoculate them on a 24-well plate (density: 1×10 ⁴ /500 μl/well). When the cell growth confluence reaches 80%, replace it with a medium containing 0.1% FBS for 16 hours. . Grouping: (1) blank group: adding corresponding volume of PBS to treat; (2) model group, adding corresponding volume of PBS+TGF-β1 stimulation; (3) positive control group, adding ADP355 at concentrations of 10, 50 and 100 μM respectively + TGF-β1 stimulation; (4) experimental group, adding Pep69, Pep70, Pep71 with concentrations of 10, 50 and 100 μM respectively, and adding TGF-β1 stimulation; (5) negative control group, adding concentrations of 10, 50 and 100 μM respectively Stimulation with 100 μM of Pep56+TGF-β1. In a 24-well plate, 3 replicate wells were set up for each group, and the corresponding polypeptide or solvent was pretreated for 1 h, and the TGF-β1 stimulation group was stimulated by adding exogenous rat TGF-β1 recombinant protein (final concentration: 5ng/ml) 24h. The medium was discarded, Trizol was added to extract the total RNA of the cells, and the gene expression of α-SMA and COL1A1 was detected by Q-PCR.

2.2 Effects of three polypeptide fragments on the expression levels of α-SMA and COL1A1 proteins

Take the HSC-T6 cells in a good growth state, inoculate, group and process according to the medium density of 2.1, and after adding exogenous rat TGF-β1 recombinant protein (final concentration: 5ng/ml) to stimulate for 24 hours, discard the medium , gently washed twice with cold HBSS solution, added cell lysate and placed on ice for 10 min, shaken to fully lyse the cells, and Western blot was used to detect the protein expression of α-SMA and COL1A1.

Judgment of the results: Intervention of exogenous rat TGF-β1 recombinant protein for 24 hours can significantly up-regulate the expression of α-SMA, COL1A1 and TGF-β1 genes, suggesting that the in vitro cell fibrosis model was successfully established. All three polypeptide fragments have significant anti-fibrotic activity in vitro, and can dose-dependently inhibit the gene and protein expression of fibrosis factors α-SMA, COL1A1 and TGF-β1 up-regulated by exogenous TGF-β1, thereby inhibiting myofibroblasts Cell differentiation and blocking of TGF-β1-induced collagen accumulation, ultimately achieving anti-fibrotic effects. The results are shown in Figures 3-4.

It can be seen from Figure 3 that the three polypeptide fragments can significantly inhibit the gene expression of fibrosis factors α-SMA, COL1A1 and TGF-β1 induced by exogenous TGF-β1, and there is a certain dose-effect relationship, among which Pep70 The anti-fiber activity is the most significant. When its concentration reaches 100 μM, the inhibition rates of α-SMA, COL1A1 and TGF-β1 are: 78.1%, 82.5% and 85.0%, respectively, and the inhibitory effect is slightly stronger than that of the positive control peptide (ADP355). .

It can be seen from Figure 4 that, on the basis of down-regulating the gene levels of α-SMA, COL1A1 and TGF-β1, the three polypeptide fragments also showed obvious inhibitory effects on the proteins corresponding to the three fibrosis factors, and the inhibition rates were : 24.0%, 52.0%, 52.0% (α-SMA), 12.5%, 20.8%, 29.2% (COL1A1) and 61.0%, 66.1%, 69.5% (TGF-β1), compared with the model control group, there is a significant Difference (p<0.05). It was shown that the three polypeptide fragments had significant inhibitory effects on fibrosis induced by exogenous TGF-β1.

Claims (5)

1. three polypeptide fragments are preparing the purposes of anti-fibrosis medicine, and aminoacid sequence is respectively Pep69:KFYIHSDY, Pep70:PGLYYFD and Pep71:PGLYYFD.

2. three polypeptide fragments as claimed in claim 1 are preparing the purposes in anti-fibrosis medicine, it is characterized in that: described pharmaceutical pack containing in these three polypeptide fragments, two or three.

3. an anti-fibrosis medicine, is characterized in that containing the medicine described in claim 2 and one or more pharmaceutically acceptable carriers.

4. anti-fibrosis medicine as described in claim 3, is characterized in that described pharmaceutically acceptable carrier refers to: diluent, filler, excipient, binding agent, wetting agent, disintegrating agent, surfactant, absorption enhancer, lubricant or absorption carrier.

5. anti-fibrosis medicine as described in claim 3, is characterized in that the dosage form of this medicine is: tablet, pill, capsule, suspending agent, Emulsion, injection or dry powder doses.