Polypeptide compound and application thereof in preparation of anti-hepatic fibrosis medicine

Abstract

The invention relates to a polypeptide compound and application thereof in preparation of anti-hepatic fibrosis medicines, and belongs to the technical field of biological medicines. According to the polypeptide compound, a polypeptide compound 1 is used as a parent peptide, amino acid mutation is carried out, and the amino acid sequence of the polypeptide compound 1 is shown as SEQ ID NO: 1. The polypeptide compound provided by the invention has better biological activity, high synthesis yield, good stability, easiness in large-scale production, low cost, low toxicity, larger safety window and smaller dosage, can hinder the combination of OGN and Collagen I, affects collagen to form collagenous fibers, and can better treat hepatic fibrosis and fibrosis diseases accompanied by hepatic diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to polypeptide compounds and their application in the preparation of drugs for anti-hepatic fibrosis. Background Art

[0003] Hepatic fibrosis is basically a wound healing response to various types of injuries, such as chronic liver injuries caused by non-alcoholic steatohepatitis, viral hepatitis, autoimmune hepatitis, non-alcoholic fatty liver disease, and cholestatic liver disease. Hepatic stellate cells are activated and transformed into myofibroblasts when injured, producing collagen, the main component of the extracellular matrix (ECM). The gradual accumulation of ECM will damage the normal liver structure and function, leading to liver cirrhosis and even hepatocellular carcinoma. Currently, there is still a lack of effective anti-hepatic fibrosis drugs in clinical practice. Developing effective anti-hepatic fibrosis drugs to prevent or reverse hepatic fibrosis is of great significance for reducing liver cirrhosis and liver cancer.

[0004] Polypeptide T1 is a polypeptide produced by the degradation of Osteoglycin, a member of the small leucine-rich proteoglycan (SLRP) family rich in leucine. There is currently no report on the anti-hepatic fibrosis aspect of polypeptide T1. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide polypeptide compounds and their application in the preparation of drugs for anti-hepatic fibrosis.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention provides that the polypeptide compound uses polypeptide compound 1 as the parent peptide, and through amino acid mutation, the amino acid sequence of polypeptide compound 1 is shown as SEQ ID NO: 1.

[0008] The present invention uses polypeptide compound 1 as the parent peptide, and through amino acid mutation, a series of polypeptide compounds are generated, which can interfere with the binding of the SLRP family member Osteoglycin (OGN) to Collagen I, thereby improving hepatic fibrosis.

[0009] Further, the carboxyl group at the C-terminus and the amino group at the N-terminus of the parent peptide are cyclized to form polypeptide compound 2.

[0010] Further, one amino acid in the parent peptide is mutated to amino acid Ala to form polypeptide compounds 3 to 14, and the amino acid sequences are shown as SEQ ID NOs: 2 to 13.

[0011] Furthermore, the amino acid Tyr at the 1st position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 3, and the amino acid sequence is as shown in SEQ ID NO: 2;

[0012] The amino acid Leu at the 2nd position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 4, and the amino acid sequence is as shown in SEQ ID NO: 3;

[0013] The amino acid Asp at the 3rd position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 5, and the amino acid sequence is as shown in SEQ ID NO: 4;

[0014] The amino acid His at the 4th position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 6, and the amino acid sequence is as shown in SEQ ID NO: 5;

[0015] The amino acid Asn at the 5th position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 7, and the amino acid sequence is as shown in SEQ ID NO: 6;

[0016] The amino acid Leu at the 7th position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 8, and the amino acid sequence is as shown in SEQ ID NO: 7;

[0017] The amino acid Glu at the 8th position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 9, and the amino acid sequence is as shown in SEQ ID NO: 8;

[0018] The amino acid Ser at the 9th position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 10, and the amino acid sequence is as shown in SEQ ID NO: 9;

[0019] The amino acid Val at the 10th position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 11, and the amino acid sequence is as shown in SEQ ID NO: 10;

[0020] The amino acid Pro at the 11th position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 12, and the amino acid sequence is as shown in SEQ ID NO: 11;

[0021] The amino acid Leu at the 12th position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 13, and the amino acid sequence is as shown in SEQ ID NO: 12;

[0022] The amino acid Asn at the 13th position at the N-terminus of the parent peptide is mutated to Ala, which is polypeptide compound 14, and the amino acid sequence is as shown in SEQ ID NO: 13.

[0023] The amino acid at the 3rd position at the N-terminus of the parent peptide is mutated to the amino acid Glu, and the amino acid sequence is as shown in SEQ ID NO: 14, which is polypeptide compound 15;

[0024] Furthermore, the 4th amino acid His at the N-terminus of the parent peptide is mutated to the amino acid Lys or Arg, and the amino acid sequence is as shown in SEQ ID NO: 15 or SEQ ID NO: 16, which is polypeptide compound 16 or polypeptide compound 17;

[0025] The 5th amino acid Asn at the N-terminus of the parent peptide is mutated to the amino acid Ser or Thr, and the amino acid sequence is as shown in SEQ ID NO: 17 or SEQ ID NO: 18, which is polypeptide compound 18 or polypeptide compound 19;

[0026] The 6th amino acid Ala at the N-terminus of the parent peptide is mutated to Leu, and the amino acid sequence is as shown in SEQ ID NO: 19, which is polypeptide compound 20;

[0027] The 7th amino acid Leu at the N-terminus of the parent peptide is mutated to Val, and the amino acid sequence is as shown in SEQ ID NO: 20, which is polypeptide compound 21;

[0028] The 8th amino acid Glu at the N-terminus of the parent peptide is mutated to Asp, and the amino acid sequence is as shown in SEQ ID NO: 21, which is polypeptide compound 22;

[0029] The 9th amino acid Ser at the N-terminus of the parent peptide is mutated to Thr, and the amino acid sequence is as shown in SEQ ID NO: 22, which is polypeptide compound 23;

[0030] The 10th amino acid Val at the N-terminus of the parent peptide is mutated to Leu, and the amino acid sequence is as shown in SEQ ID NO: 23, which is polypeptide compound 24;

[0031] The 11th amino acid Pro at the N-terminus of the parent peptide is mutated to Ser, and the amino acid sequence is as shown in SEQ ID NO: 24, which is polypeptide compound 25;

[0032] The 12th amino acid Leu at the N-terminus of the parent peptide is mutated to Asp, and the amino acid sequence is as shown in SEQ ID NO: 25, which is polypeptide compound 26;

[0033] The 13th amino acid Asn at the N-terminus of the parent peptide is mutated to Asp, and the amino acid sequence is as shown in SEQ ID NO: 26, which is polypeptide compound 27.

[0034] In a second aspect, the present invention provides the use of the polypeptide compound in the preparation of a drug for anti-hepatic fibrosis.

[0035] Furthermore, the drug is a drug that inhibits the expression of collagen I and / or fibronectin.

[0036] Further, the drug is a drug that inhibits the binding of collagen I and osteoglycin.

[0037] Further, the drug is a drug that inhibits the activation of hepatic stellate cells.

[0038] Further, the drug is a drug that inhibits the aggregation of inflammatory cells in the liver.

[0039] In a third aspect, the present invention provides an anti-hepatic fibrosis drug, and the drug contains the polypeptide compound described above.

[0040] Further, the drug also contains a pharmaceutically acceptable pharmaceutical carrier and / or excipient.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] (1) The polypeptide compound of the present invention has better biological activity.

[0043] (2) The polypeptide compound of the present invention has a high synthesis yield, good stability, is easy to scale up production, and has low cost.

[0044] (3) The polypeptide compound of the invention has low toxicity, a larger safety window, and a smaller dosage.

[0045] (4) The polypeptide compound of the invention can hinder the binding of OGN and Collagen I, affect the formation of collagen fibers by collagen, and can better treat hepatic fibrosis and fibrotic diseases accompanying liver diseases. Description of the Drawings

[0046] Figure 1 It is the Western Blot diagram and statistical bar chart of the expression of Collagen I and FN in the LX2 cell model in Example 2 of the present invention. Among them, A is the polypeptide compounds T1, Y1A, L2A, D3A and H4A; B is the polypeptide compounds T1, N5A, L7A, E8A and S9A; C is the polypeptide compounds T1, V10A, P11A, L12A and L13A; D is the polypeptide compounds T1, T1-1, T1-2, T1-3, T1-4 and T1-5; E is the polypeptide compounds T1, T1-6, T1-7, T1-8, T1-9 and T1-10; F is the polypeptide compounds T1, T1-11, T1-12, T1-13 and T1-14. *: indicates that the confidence level > 95%, and the difference between the two is significantly significant (P < 0.05); **: indicates that the confidence level > 99%, and the difference between the two is very significantly significant (P < 0.01); ***: indicates that the confidence level > 99.9%, and the difference between the two is extremely significantly significant (P < 0.001); ****: indicates that the confidence level > 99.99%, and the difference between the two is extremely significantly significant (P < 0.0001).

[0047] Figure 2 In Example 3 of the present invention, the blocking efficiency of the binding of OGN to Collagen I. Among them, A is polypeptide compounds T1, Y1A, L2A, D3A and H4A; B is polypeptide compounds T1, N5A, L7A, E8A and S9A; C is polypeptide compounds T1, V10A, P11A, L12A and L13A; D is polypeptide compounds T1, T1-1, T1-2, T1-3, T1-4 and T1-5; E is polypeptide compounds T1, T1-6, T1-7, T1-8, T1-9 and T1-10; F is polypeptide compounds T1, T1-11, T1-12, T1-13 and T1-14. *: indicates a confidence level > 95%, and the difference between the two is significant (P < 0.05); **: indicates a confidence level > 99%, and the difference between the two is very significant (P < 0.01); ***: indicates a confidence level > 99.9%, and the difference between the two is extremely significant (P < 0.001); ****: indicates a confidence level > 99.99%, and the difference between the two is extremely significant (P < 0.0001).

[0048] Figure 3 In Example 4 of the present invention, the bar chart of the content results of mouse serum ALT and AST. Among them, A is ALT; B is AST. * / #: indicates a confidence level > 95%, and the difference between the two is significant (P < 0.05); ** / ##: indicates a confidence level > 99%, and the difference between the two is very significant (P < 0.01); *** / : indicates a confidence level > 99.9%, and the difference between the two is extremely significant (P < 0.001); **** / #: indicates a confidence level > 99.99%, and the difference between the two is extremely significant (P < 0.0001).

[0049] Figure 4 In Example 4 of the present invention, the pathological section diagram of mouse liver H&E staining.

[0050] Figure 5 In Example 4 of the present invention, the pathological section diagram of mouse liver Sirius red staining.

[0051] Figure 6In Example 4 of the present invention, Western Blot and statistical bar graphs of the expression levels of α-SMA and Collagen I in mouse liver tissues. Among them, A is the Western Blot; B is the statistical bar graph. * / #: indicates a confidence level > 95%, and the difference between the two is significant (P < 0.05); ** / ##: indicates a confidence level > 99%, and the difference between the two is very significant (P < 0.01); *** / : indicates a confidence level > 99.9%, and the difference between the two is extremely significant (P < 0.001); **** / #: indicates a confidence level > 99.99%, and the difference between the two is extremely significant (P < 0.0001). Detailed implementation mode

[0052] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Other materials, reagents, etc. used in the examples can be obtained from commercial channels without special instructions.

[0053] All amino acids were purchased from NovaBiochem. Without special instructions, all other reagents were of analytical grade and purchased from Sigma. A Protein Technologies PRELUDE 6-channel polypeptide synthesizer was used. A Phenomenex Luna C18 preparative column (46 mm × 250 mm) was used to purify the polypeptide. The high-performance liquid chromatograph was a product of Waters. Mass spectrometry analysis was performed using an Agilent mass spectrometer.

[0054] Example 1 Synthesis of polypeptide compounds

[0055] 1. Synthesize polypeptide compound 1 (SEQ ID NO: 1), where the first amino acid Asp at the C-terminus is aminoacetylated to Asn, and the amino acid sequence is:

[0056] H2N-Tyr-Leu-Asp-His-Asn-Ala-Leu-Glu-Ser-Val-Pro-Leu-Asn-OH.

[0057] Based on the above amino acid sequence, the present invention adopted the Fmoc (fluorenylmethoxycarbonyl) solid-phase polypeptide synthesis method to synthesize polypeptide compound 1 from the carboxyl terminus to the amino terminus.

[0058] (1) First step: Resin swelling

[0059] 1.5 g of Rink amide MBHA resin was swollen twice in N,N-dimethylformamide (DMF) and dichloromethane (DCM) (5 mL each), 15 min each time;

[0060] (2) Second step: Coupling of amino acids

[0061] Using Rink Amide MBHA resin as the carrier, 1-hydroxybenzotriazole (3×) and N,N-diisopropylcarbodiimide (3×) as coupling agents, and N,N-dimethylformamide (DMF) as the solvent, the condensation reaction is carried out successively to connect Fmoc-protected amino acids to obtain: H2N-Tyr-Leu-Asp-His-Asn-Ala-Leu-Glu-Ser-Val-Pro-Leu-Asn-RinkAmide MBHA peptide resin. Among them, the molar ratio of the feed amount of Fmoc-protected amino acid to the resin amount in each condensation reaction is 3:1, and the molar ratio of 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide to the amount of Fmoc-protected amino acid in each condensation reaction is 1:1. The deprotection solution is a 20% (v / v) piperidine DMF solution. After coupling, it is shrunk with pure methanol twice, 15 minutes each time, and dried by vacuum pumping to obtain the peptide resin.

[0062] (3) Third step: Peptide cleavage and deprotection

[0063] Add 15 mL of the cleavage solution (TFA / TIS / H2O = 95 / 2.5 / 2.5, v / v / v, TFA is trifluoroacetic acid, TIS is triisopropylsilane) to the peptide resin, charge argon for protection reaction in a peptide reactor, and react on a shaker for 240 minutes. After the reaction, inject the cleavage agent containing the peptide into a round-bottom flask containing ice ether. Let it stand until the precipitation is complete, centrifuge, pour off the supernatant, and dry the obtained precipitate with a vacuum freeze dryer to obtain the crude product of polypeptide compound 1.

[0064] (4) Fourth step: Purification

[0065] The crude polypeptide compound 1 obtained in the above third step was ultrasonically dissolved with 10 mL of a dissolution reagent, which was 95% (v / v) acetonitrile - 0.1% (w / v) TFA / water (i.e., water containing 0.1% TFA was mixed with acetonitrile, and the volume ratio of water containing 0.1% TFA to acetonitrile was 5:95). After dissolution and clarification, it was filtered through a 0.22 μm disposable syringe filter to obtain a filtrate. The filtrate was purified by semi - preparative HPLC (high - performance liquid chromatography) 2 times on a column filled with 20 mm reversed - phase C18 with a size of 21.2 mm×250 mm. 40% - 60% (v / v) acetonitrile - 0.1% (w / v) TFA / water (referring to the preparation method of the above dissolution reagent) was used as the mobile phase, and gradient elution was carried out at a flow rate of 10 mL / min for 60.0 min. The fractions containing polypeptide compound 1 were collected and dried with a vacuum freeze - dryer. A pure product of polypeptide compound 1 with an HPLC purity of 95% was obtained. The pure product of polypeptide compound 1 was analyzed by liquid chromatography - mass spectrometry, and it was found that the m / z value of the protonated molecular ion peak was: 1483.73, and the theoretical value was 1484.6.

[0066] Except for polypeptide compound 2, the rest of the polypeptides were synthesized based on the above steps.

[0067] 2. Synthesize polypeptide compound 2 with the amino acid sequence: H2N - Tyr - Leu - Asp - His - Asn - Ala - Leu - Glu - Ser - Val - Pro - Leu - Asn - OH (the head - to - tail amide bond forms a ring).

[0068] Based on the above amino acid sequence, the present invention adopted the Fmoc (fluorenylmethyloxycarbonyl) solid - phase polypeptide synthesis method, and synthesized polypeptide compound 2 in sequence from the carboxyl - terminal to the amino - terminal direction and obtained it through head - to - tail amide cyclization.

[0069] (1) The first step: resin swelling

[0070] 1.5 g of 2 - CTC resin was swollen twice in N,N - dimethylformamide (DMF) and dichloromethane (DCM) (5 mL each), 15 min each time.

[0071] (2) The second step: coupling of amino acids

[0072] Using 2 - CTC resin as the carrier, N,N - diisopropylethylamine (DIEA) as the coupling agent, and N,N - dimethylformamide (DMF) as the solvent, first add Fmoc - Asn - OH and shake well at 25 °C for 2 h for the condensation reaction to connect the Fmoc - protected amino acid. The molar ratio of Fmoc - Asn - OH to 2 - CTC resin is 3:1, the molar ratio of N,N - diisopropylethylamine (DIEA) to Fmoc - Asn - OH is 4:3, and the deprotection solution is a 20% (v / v) piperidine DMF solution to obtain the resin connected with Asn.

[0073] Then, using the resin linked to Asn as the carrier, 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylcarbodiimide (DIC) as coupling agents, and N,N-dimethylformamide (DMF) as the solvent, shake well at 25 °C for 2 h to continue the condensation reaction, and sequentially link the next Fmoc-protected amino acid. The molar ratio of the Fmoc-protected amino acid to the 2-CTC resin is 3:1, and the molar ratio of 1-hydroxybenzotriazole (HOBt), N,N-diisopropylcarbodiimide (DIC) to the Fmoc-protected amino acid is 1:1:1. Then repeat deprotection and coupling, and sequentially link the next Fmoc-protected amino acids until the target peptide is assembled. Shrink with methanol twice, 15 min each time, and vacuum dry to obtain the peptide resin.

[0074] (3) Step 3: Cleavage of the fully protected linear peptide from the resin

[0075] The peptide resin is oscillated in the cleavage solution at 25 °C for 2 h, the filtrate is collected by vacuum filtration, concentrated with a rotary evaporator to obtain a concentrated product, and the concentrated product is precipitated with pre-frozen ether, and the precipitate is collected by centrifugation. After washing the precipitate with ether, it is dried under vacuum to obtain the fully protected linear peptide. The cleavage solution is composed of 2,2,2-trifluoroethanol (TFE) and dichloromethane (DCM) with a volume ratio of 1:4.

[0076] (4) Step 4: Cyclization of the linear peptide

[0077] Dissolve benzotriazol-1-oxytris(dimethylamino)phosphonium hexafluorophosphate (PyBOP), sodium 1-hydroxy-7-azabenzotriazole (HOAt) and N,N-diisopropylethylamine (DIEA) in dichloromethane (DCM), and then slowly add the fully protected linear peptide dissolved in dichloromethane (DCM). The molar ratio of benzotriazol-1-oxytris(dimethylamino)phosphonium hexafluorophosphate (PyBOP) to the 2-CTC resin is 5:1, the molar ratio of sodium 1-hydroxy-7-azabenzotriazole (HOAt) to the 2-CTC resin is 5:1, and the molar ratio of N,N-diisopropylethylamine (DIEA) to the 2-CTC resin is 10:1. Make the final peptide concentration 1.0 mg / mL, bubble with nitrogen and stir, react overnight at 25 °C, and monitor the reaction process by HPLC. After the reaction is completed, add pure water for extraction and washing twice. Collect the organic phase, dry with anhydrous sodium sulfate, and evaporate the solvent to dryness to obtain the fully protected crude cyclic peptide.

[0078] (5) The crude cyclic peptide was subjected to polypeptide cleavage, deprotection, and purification according to the method for synthesizing polypeptide compound 1 to obtain a pure product of polypeptide compound 2 with an HPLC purity of 95%. The pure product of polypeptide compound 2 was analyzed by liquid chromatography-mass spectrometry, and it was found that the m / z value of the protonated molecular ion peak was: 1465.72, and the theoretical value was: 1465.72.

[0079] Based on the above synthesis steps, the following polypeptide compounds of the present invention were synthesized (Table 1).

[0080] Table 1 Structures of the polypeptide compounds synthesized in the examples of the present invention

[0081]

[0082]

[0083] In Table 1, Tyr is tyrosine, Leu is leucine, Asp is aspartic acid, His is histidine, Asn is asparagine, Ala is alanine, Glu is glutamic acid, Ser is serine, Val is valine, Pro is proline, Lys is lysine, Arg is arginine, and Thr is threonine.

[0084] Example 2 Activity evaluation of polypeptide compounds in LX-2 cells (human hepatic stellate cells) I. Experimental method

[0085] LX-2 cells treated with 10 ng / mL TGF-β or 20 μM of the polypeptide compound of the present invention for 24 h were collected, and LX-2 cells were lysed on ice with a lysis buffer containing protease inhibitors and phosphatase inhibitors, and proteins were extracted. The proteins were separated on 10% (w / v) SDS-PAGE in an electrophoresis buffer and then transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with bovine serum albumin (BSA) buffer, and incubated overnight at 4 °C with anti-Collagen I (type I collagen) antibody, anti-FN (fibronectin) antibody, and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody, washed 3 times with TBST, the signals were detected by chemiluminescence, and visualized with a chemiluminescence imaging system ChemiScope 3300Mini Imaging System.

[0086] II. Experimental results

[0087] As Figure 1 A~ Figure 1 shown in F, it can be clearly seen from the statistical chart of the experimental results that, compared with the GAPDH internal reference, the polypeptide compounds prepared in Example 1 of the present invention can all reduce the expression of Collagen I or FN to varying degrees.

[0088] The experimental results also show that the polypeptide compound of the present invention can inhibit the formation of fibrosis at the cellular level and also suggests that it can be used for liver fibrosis and fibrosis symptoms associated with liver diseases.

[0089] Example 3 Evaluation of the blocking efficiency of polypeptide compounds on the binding of OGN-Collagen I

[0090] I. Experimental method

[0091] 1. Coat a high-binding 96-well plate with 500 ng / mL Collagen I (coating conditions: 4°C, 12 h), and wash the wells 3 times with a washing buffer (TBST buffer containing 0.05% v / v Tween-20, pH 7.4) to remove unbound collagen.

[0092] 2. Add a blocking buffer (washing buffer containing 2% w / v BSA, pH 7.4) to the 96-well plate treated in step 1, and block at 37°C for 1.5 h to prevent non-specific binding, then wash the wells 3 times.

[0093] 3. Add different concentrations of the polypeptide compound prepared in Example 1 and 200 ng / mL OGN (osteoglycin) with His Tag to the 96-well plate treated in step 2, incubate at 37°C for 1 h, add an anti-His-HRP (His tag labeled with horseradish peroxidase) antibody and incubate for 1 h, then wash the wells 3 times.

[0094] 4. Add a 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution to the 96-well plate treated in step 3, incubate at 37°C for 20 min, and then add a stop solution.

[0095] 5. Read the absorbance (OD value) of each well on the 96-well plate treated in step 4 at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0096] II. Experimental results

[0097] As Figure 2 A~ Figure 2 shown in F, the polypeptide compounds prepared in Example 1 of the present invention can all block the binding of OGN to Collagen I to varying degrees, and among them, polypeptide compound 6 (H4A) and polypeptide compound 22 (T1-13) have the best effects.

[0098] Example 4 Pharmacodynamic evaluation of polypeptide compounds on a carbon tetrachloride (CCl4)-induced mouse liver fibrosis model

[0099] I. Experimental method

[0100] In this example, the polypeptide compounds 1 (T1), 6 (H4A), and 22 (T1-13) prepared in Example 1 were used as examples to evaluate the pharmacodynamics of the polypeptide compounds.

[0101] 1. Modeling method: Twenty male C57BL / 6J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) weighing 18 - 22 g and 8 weeks old were randomly divided into 4 groups, namely:

[0102] (1) Normal control group: Injected with corn oil (Oil) + normal saline, intraperitoneally, n = 5; The mice were given Oil three times a week for the first 6 weeks, and normal saline once a day for the 4th - 6th weeks.

[0103] (2) Model control group: Injected with CCl4 + normal saline, intraperitoneally, n = 5; The mice were given CCl4 three times a week for the first 6 weeks, and normal saline once a day for the 4th - 6th weeks.

[0104] (3) Drug administration group 1: Injected with CCl4 + 250 μg / kg polypeptide compound 1, intraperitoneally, n = 5; The mice were given CCl4 three times a week for the first 3 weeks; Compound 1 was given once a day for the 4th - 6th weeks.

[0105] (4) Drug administration group 2: Injected with CCl4 + 250 μg / kg polypeptide compound 6, intraperitoneally, n = 5; The mice were given CCl4 three times a week for the first 3 weeks; Polypeptide compound 6 was given once a day for the 4th - 6th weeks.

[0106] (5) Drug administration group 2: Injected with CCl4 + 250 μg / kg polypeptide compound 22, intraperitoneally, n = 5; The mice were given CCl4 three times a week for the first 3 weeks; Polypeptide compound 22 was given once a day for the 4th - 6th weeks.

[0107] The volume of each administration was calculated according to injection reagent / mouse body weight = 5.0 μL / g. The concentration of CCl4 given to the mice was 20% (v / v). The mice in the normal control group were given the same volume and frequency of injection of corn oil. CCl4 and Oil were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0108] 2. Pharmacodynamic evaluation

[0109] In the CCl4-induced liver fibrosis model, its manifestations are as follows: There is infiltration of inflammatory cells around the central vein area, hepatocyte swelling and degeneration, and a large amount of collagen fiber deposition in the portal area and hepatic lobule septum, etc. After 3 weeks of drug administration, the mice were sacrificed and samples were taken. Blood was collected from the retro-orbital venous plexus for serological index detection, and liver tissues were taken for pathological analysis.

[0110] 3. Experimental method

[0111] (1) Hematoxylin-eosin (H&E) staining: Take paraffin-embedded tissue sections, bake at 60 °C for 1 h to obtain dried paraffin sections, and perform dewaxing and hydration in the following steps: treat with xylene for 20 min, treat with xylene for 20 min, treat with absolute ethanol for 15 min, treat with absolute ethanol for 15 min, treat with 95% (v / v) ethanol for 10 min, treat with 90% (v / v) ethanol for 5 min, treat with 80% (v / v) ethanol for 5 min to obtain dewaxed and hydrated sections. Stain the dewaxed and hydrated sections in the following steps: treat with hematoxylin for 7 min, rinse thoroughly with tap water; differentiate with 1% (v / v) hydrochloric acid ethanol for 1 s, rinse thoroughly with tap water; stain with eosin for 15 s - 20 s, rinse with tap water to obtain stained sections. Dehydrate and clear the stained sections in the following steps: treat with 75% (v / v) ethanol for 1 s, treat with 85% (v / v) ethanol for 1 s, treat with 95% (v / v) ethanol for 1 s, treat with absolute ethanol for 1 s, treat with xylene for 1 s, treat with xylene for 1 s to obtain dehydrated and cleared sections. Seal the slides and air dry for 30 min, and seal with resin to obtain hematoxylin-eosin stained sections.

[0112] (2) Sirius red staining: Bake and dewax the paraffin-embedded tissue sections according to the hematoxylin-eosin staining method to obtain dewaxed and hydrated sections. Stain the dewaxed and hydrated sections: Let the dewaxed and hydrated sections stand in double-distilled water for 5.0 min, stain with Sirius red in the dark for 60 - 80 min, and rinse with 0.5% (v / v) glacial acetic acid for 5 s; dehydrate, clear, and seal the slides according to the hematoxylin-eosin staining method, and take pictures.

[0113] The H&E staining solution and Sirius red staining solution were purchased from Shanghai Sangon Biotech Co., Ltd.

[0114] II. Experimental results

[0115] Figure 3 A is a bar graph of the alanine aminotransferase (ALT) content in mouse serum, Figure 3 B is a bar graph of the aspartate aminotransferase (AST) content in mouse serum. After intraperitoneal injection of CCl4 to mice, severe liver injury occurred. After treatment with Compound 1, Compound 6, and Compound 22 drugs, the degree of liver injury was alleviated.

[0116] Figure 4 This is the pathological section diagram of mouse liver H&E staining in this example; Figure 5 This is the pathological section diagram of mouse liver Sirius red staining in this example. From Figure 4 and Figure 5The results showed that after intraperitoneal injection of CCl4 in mice, collagen deposition and fibrosis occurred in the liver. After treatment with Compound 1, Compound 6, and Compound 22, the aggregation of inflammatory cells and liver fibrosis in mice were significantly improved. It can be thus demonstrated that the polypeptide compounds of the present invention can significantly treat and improve collagen deposition, can well inhibit the accumulation of ECM, and treat and improve liver fibrosis.

[0117] From the Western Blot images ( Figure 6 A) and the statistical bar graphs ( Figure 6 B) of the expression of α-SMA (α-smooth muscle actin) and Collagen I in mouse liver tissues, it can be seen that after treatment with Compound 1, Compound 6, and Compound 22, the expression levels of Collagen I and α-SMA in the mouse liver were significantly improved in terms of treatment. It can be thus demonstrated that the polypeptide compounds of the present invention can inhibit the expression of Collagen I and α-SMA in the liver and treat and improve liver fibrosis.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A polypeptide compound, characterized in that, The polypeptide compound uses polypeptide compound 1 as the parent peptide and undergoes amino acid mutations. The amino acid sequence of polypeptide compound 1 is shown as SEQ ID NO:

1.

2. The polypeptide compound according to claim 1, characterized in that, The carboxyl group at the C-terminus and the amino group at the N-terminus of the parent peptide form a ring, resulting in polypeptide compound 2.

3. The polypeptide compound according to claim 1, wherein One amino acid in the parent peptide is mutated to amino acid Ala, resulting in polypeptide compounds 3 - 14, and the amino acid sequences are shown as SEQ ID NOs: 2 - 13.

4. The polypeptide compound according to claim 1, characterized in that, The third amino acid at the N-terminus of the parent peptide is mutated to amino acid Glu, and the amino acid sequence is shown as SEQ ID NO: 14, resulting in polypeptide compound 15; The fourth amino acid His at the N-terminus of the parent peptide is mutated to amino acid Lys or Arg, and the amino acid sequences are shown as SEQ ID NO: 15 or SEQ ID NO: 16, resulting in polypeptide compound 16 or polypeptide compound 17; The fifth amino acid Asn at the N-terminus of the parent peptide is mutated to amino acid Ser or Thr, and the amino acid sequences are shown as SEQ ID NO: 17 or SEQ ID NO: 18, resulting in polypeptide compound 18 or polypeptide compound 19; The sixth amino acid Ala at the N-terminus of the parent peptide is mutated to Leu, and the amino acid sequence is shown as SEQ ID NO: 19, resulting in polypeptide compound 20; The seventh amino acid Leu at the N-terminus of the parent peptide is mutated to Val, and the amino acid sequence is shown as SEQ ID NO: 20, resulting in polypeptide compound 21; The eighth amino acid Glu at the N-terminus of the parent peptide is mutated to Asp, and the amino acid sequence is shown as SEQ ID NO: 21, resulting in polypeptide compound 22; The ninth amino acid Ser at the N-terminus of the parent peptide is mutated to Thr, and the amino acid sequence is shown as SEQ ID NO: 22, resulting in polypeptide compound 23; The tenth amino acid Val at the N-terminus of the parent peptide is mutated to Leu, and the amino acid sequence is shown as SEQ ID NO: 23, resulting in polypeptide compound 24; The eleventh amino acid Pro at the N-terminus of the parent peptide is mutated to Ser, and the amino acid sequence is shown as SEQ ID NO: 24, resulting in polypeptide compound 25; The twelfth amino acid Leu at the N-terminus of the parent peptide is mutated to Asp, and the amino acid sequence is shown as SEQ ID NO: 25, resulting in polypeptide compound 26; The thirteenth amino acid Asn at the N-terminus of the parent peptide is mutated to Asp, and the amino acid sequence is shown as SEQ ID NO: 26, resulting in polypeptide compound 27.

5. Use of the polypeptide compound according to any one of claims 1 - 4 in the preparation of a drug for anti - liver fibrosis.

6. The application according to claim 5, characterized in that, The drug is a drug that inhibits the expression of collagen I and / or fibronectin.

7. The application according to claim 5, characterized in that The drug is a drug that inhibits the binding of collagen I and osteoglycin.

8. The application according to claim 5, characterized in that The drug is a drug that inhibits the activation of hepatic stellate cells.

9. The application according to claim 5, characterized in that The drug is a drug that inhibits the aggregation of liver inflammatory cells.

10. A drug for anti - liver fibrosis, characterized in that, The drug contains the polypeptide compound according to any one of claims 1 - 4.

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