A biologically active solution and its application

Abstract

The present invention relates to the field of medicine and pharmaceutical technology, and discloses a bioactive solution and its application, wherein the bioactive solution comprises a polypeptide having an amino acid sequence of SEQ ID NO.1 and a polypeptide having an amino acid sequence of SEQ ID NO.2. The bioactive solution obtained by the present invention can cross the vascular endothelium and serve as a medium for connecting immune cells NK and liver fibrosis effector cells HSCs, which is beneficial to alleviate the symptoms of liver fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of medicine and pharmaceutical technology, and in particular to a bioactive solution and application thereof. Background Art

[0002] The interaction between different types of cells, chemokines, and signaling molecules forms a huge information network that jointly regulates and affects the development of liver fibrosis. With the deepening of our understanding of the immune system, supramolecular therapeutic strategies based on cell-to-cell interactions have gradually emerged in precision medicine. Classic cell-to-cell interactions are nothing more than two ways, namely direct contact and material signal transmission, which are closely related to the occurrence and development of various diseases. Influencing and reversing the development of diseases by regulating cell-to-cell interactions has become a new strategy for the development of nanomedicines. Cell-to-cell interactions in the body are highly dynamic. Along with cell proliferation, differentiation, and migration, cell-to-cell interactions and the extracellular microenvironment also affect the fate and function of cells. Immune checkpoint blockade, which is widely used in tumor immunotherapy, such as the well-known immune checkpoint TIGIT-PVR, is a new immune checkpoint after the T cell-based PD-1 / PD-L1.

[0003] Previous studies have shown that preventing the activation of HSCs (hepatic stellate cells) or inducing apoptosis of activated stellate cells has become a new target for the treatment of liver fibrosis. In particular, many HSCs survival signals are obtained from the extracellular matrix, while active pro-apoptotic signals are provided by immune cells, especially natural killer (NK) cells. Therefore, as a medium connecting the key immune cells NK and effector cells HSCs in the development of liver fibrosis, significantly slowing down and inhibiting the development of liver fibrosis by affecting cell-to-cell interactions in both direct contact and signal transduction is a new strategy for the development of liver fibrosis-specific drugs. So far, there has been no research on the use of small molecules to affect cell-to-cell interactions in immune drugs for liver fibrosis. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a bioactive solution and its application. The bioactive solution obtained by the present invention can cross the vascular endothelium and serve as a medium to connect immune cells NK and liver fibrosis effector cells HSCs, which is beneficial to alleviate the symptoms of liver fibrosis.

[0005] The present invention provides a bioactive solution, wherein the bioactive solution comprises a polypeptide having an amino acid sequence of SEQ ID NO.1 and a polypeptide having an amino acid sequence of SEQ ID NO.2;

[0006] The structural formula of the polypeptide having the amino acid sequence of SEQ ID NO.1 is:

[0007] (Formula 1);

[0008] The structural formula of the polypeptide having the amino acid sequence of SEQ ID NO.2 is:

[0009] (Formula 2).

[0010] Furthermore, the polypeptide having the amino acid sequence of SEQ ID NO.1 is composed of D D GFFY amino acid sequence, D configuration D TBP-3 targeting sequence, L-configuration competitive antagonistic tetrapeptide LSKL, and capping composition.

[0011] In the present invention, the main chain of the polypeptide of SEQ ID NO.1 amino acid sequence is composed of a capped, D-configuration D GFFY amino acid sequence, lysine and D configuration D TBP-3 targeting sequence, wherein the main chain of the polypeptide of SEQ ID NO.1 amino acid sequence is capped and D-configured D GFFY amino acid sequence, lysine and D configuration D The TBP-3 targeting sequences were connected sequentially.

[0012] Further, the D configuration D The amino acid sequence of GFFY is glycine-D-phenylalanine-D-phenylalanine-D-tyrosine.

[0013] Furthermore, the end capping is 2-naphthylacetic acid (Nap).

[0014] Further, the D configuration D The TBP-3 targeting sequence is glycine-glycine-D configuration, tyrosine-D configuration, threonine-D configuration, phenylalanine-D configuration, histidine-D configuration, tryptophan-D configuration, histidine-D configuration, arginine-D configuration, leucine-D configuration, asparagine-D configuration, proline.

[0015] Furthermore, the L-configuration competitive antagonistic tetrapeptide LSKL is L-configuration leucine-L-configuration serine-L-configuration lysine-L-configuration leucine.

[0016] Furthermore, the polypeptide having the amino acid sequence of SEQ ID NO.2 is composed of D D It consists of the GFFY amino acid sequence, glycine, cysteine, glycine, and the L-configuration VHPKQHR sequence.

[0017] Further, the D configuration D The amino acid sequence of GFFY is glycine-D-phenylalanine-D-phenylalanine-D-tyrosine.

[0018] Furthermore, maleimide is connected to the thiol group of the cysteine, and the maleimide is also connected to a near-infrared fluorescent group Cy5.5.

[0019] Furthermore, the L-configuration VHPKQHR sequence is L-configuration valine-L-configuration histidine-L-configuration proline-L-configuration lysine-L-configuration glutamine-L-configuration histidine-L-configuration arginine.

[0020] Furthermore, the molar concentration ratio of the polypeptide having the amino acid sequence of SEQ ID NO.1 to the polypeptide having the amino acid sequence of SEQ ID NO.2 in the biologically active solution is 4:1.

[0021] Furthermore, the mass concentration of the polypeptide having the amino acid sequence of SEQ ID NO.1 in the biologically active solution is 3 mg / 1.2 mL-3 mg / 0.8 mL.

[0022] The present invention also provides a method for preparing the bioactive solution, which comprises: dispersing a polypeptide having an amino acid sequence of SEQ ID NO.1 and a polypeptide having an amino acid sequence of SEQ ID NO.2 in a PBS buffer, adjusting the pH value of the solution, heating, and cooling to obtain a bioactive solution.

[0023] Furthermore, the pH value of the PBS buffer (phosphate buffer) is 5-9.

[0024] Furthermore, in PBS buffer, the molar concentration ratio of the polypeptide having the amino acid sequence of SEQ ID NO.1 to the polypeptide having the amino acid sequence of SEQ ID NO.2 is 4:1.

[0025] Furthermore, the mass concentration of the polypeptide having the amino acid sequence of SEQ ID NO.1 in PBS buffer is 3 mg / 1.2 mL-3 mg / 0.8 mL.

[0026] Furthermore, the pH value of the solution is adjusted to 6-8.

[0027] Further, the solution is heated to a temperature of 90°C-100°C.

[0028] It should be understood by those skilled in the art that heating is to promote the dissolution of the polypeptide in the solution, and the heating time ends when the polypeptide is completely dissolved.

[0029] Furthermore, the cooling is to room temperature.

[0030] Furthermore, the room temperature is 26°C-28°C.

[0031] The present invention also provides application of the bioactive solution in preparing a medicine for alleviating liver fibrosis.

[0032] The embodiments of the present invention have the following technical effects:

[0033] 1. First, the polypeptide with the amino acid sequence of SEQ ID NO.1 in the bioactive solution of the present invention can selectively induce NK cells, while the polypeptide with the amino acid sequence of SEQ ID NO.2 can selectively connect with hepatic stellate cells HSCs. The two polypeptides in the bioactive solution of the present invention can induce NK cells and hepatic stellate cells HSCs related to liver fibrosis to approach each other, enhance the NK immune killing activity while inhibiting the activation and abnormal proliferation of HSCs, thereby alleviating the symptoms of liver fibrosis; in addition, the design of the self-structure of the two polypeptides in the bioactive solution of the present invention is conducive to realizing that the polypeptides can cross the vascular endothelium under the inflammatory state and enter the liver parenchyma, and the polypeptide structure does not change. The design of the two polypeptide structures is the key technical point for the bioactive solution of the present invention to achieve the alleviation of liver fibrosis symptoms.

[0034] 2. The two polypeptides of the present invention have high biocompatibility and basically have no other effects on normal cells and the body.

[0035] 3. The two polypeptides of the present invention can effectively slow down the development of liver fibrosis, maintain and reverse fibrosis-related phenotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 This is the high performance liquid chromatogram of the polypeptide PepA-TT in Example 1.

[0038] Figure 2 This is the mass spectrum of the polypeptide PepA-TT in Example 1.

[0039] Figure 3 The polypeptide in Example 1 Cy5.5 HPLC chromatogram of PepB-V.

[0040] Figure 4 The polypeptide in Example 1 Cy5.5 Mass spectrum of PepB-V.

[0041] Figure 5The microscopic morphology of the two polypeptides with different molar concentrations in the biologically active solution after co-assembly in the examples and comparative examples, wherein: Figure 5 a is a microscopic morphology diagram of Example 1. Figure 5 b is the microscopic morphology of Example 2. Figure 5 The figure c is the microscopic morphology of Example 3. Figure 5 Figure d is the microscopic morphology of Example 1.

[0042] Figure 6 The polypeptide PepA-TT and polypeptide Cy5.5 Microscopic morphology of PepB-V after self-assembly, where: Figure 6 Figure a is the microscopic morphology of the self-assembled polypeptide PepA-TT. Figure 6 b is a polypeptide Cy5.5 Microscopic morphology of PepB-V after self-assembly.

[0043] Figure 7 The polypeptide PepA-TT and polypeptide Cy5.5 PepB-V selected the concentration used in the cell-level experiment (PepA-TT concentration was 50 μM, PepA-TT molar concentration: Cy5.5 Microscopic morphology after co-assembly at PepB-V molar concentration = 4:1, where Figure 7 Figure a is the microscopic morphology after co-assembly. Figure 7 b is Figure 7 A partial enlarged view of a.

[0044] Figure 8 The bioactive solutions of the embodiments and comparative examples are used to test the endothelial cell-spanning ability of the polypeptides. Figure 8 a is a microscopic picture of Example 4. Figure 8 Middle b is a microscopic picture of endocytosis in Example 1, Figure 8 Figure c is a microscopic picture of exocytosis in Example 1. Figure 8 In D Figure 8 A partial enlarged view of a in the middle. Figure 8 The middle e is Figure 8 A partial enlarged view of b in the figure. Figure 8 f is Figure 8 A partial enlarged view of c.

[0045] Fig. 9 The binding constants of peptides with target proteins TIGIT and VCAM-1 were measured by microthermophoresis. Fig. 9 Figure a is the binding constant diagram of the target protein TIGIT and the peptide PepA-TT. Fig. 9 b is the target protein TIGIT and peptide Cy5.5 Binding constants of PepB-V, Fig. 9 Figure c is the binding constant diagram of the target protein TIGIT and the biologically active solution of Example 1. Fig. 9 Figure d in the figure is the binding constant diagram of the target protein VCAM-1 and the peptide PepA-TT. Fig. 9 The target protein VCAM-1 and peptide Cy5.5 Binding constants of PepB-V, Fig. 9 Figure f is a graph of the binding constants between the target protein VCAM-1 and the biologically active solution of Example 1.

[0046] Fig.10 The number and fluorescence intensity of the red fluorescent bright spots of the indicator polypeptide in the cytoplasm after incubation of HUVEC cells for different time periods in Example 1 as the incubation time elapses, wherein Fig.10 In the middle, a is the fluorescence point of the fluorescent group Cy5.5 at 0h during endocytosis. Fig.10 Middle b is the fluorescence point of the nuclear dye DAPI at 0h during endocytosis. Fig.10 The middle c is the fluorescence point of all fluorescence channels at 0h during endocytosis. Fig.10 The d in the middle is the fluorescence point of the fluorescent group Cy5.5 at 0h during exocytosis. Fig.10 The middle e is the fluorescence point of the nuclear dye DAPI at 0h during exocytosis. Fig.10 f in the middle is the fluorescence point of all fluorescence channels at 0h during exocytosis. Fig.10 The middle g is the fluorescence point of the fluorescent group Cy5.5 at 4h during endocytosis. Fig.10 The middle h is the fluorescence point of the nuclear dye DAPI at 4h during endocytosis. Fig.10 The i in the middle is the fluorescence point of all fluorescence channels at 4 hours during endocytosis. Fig.10 The middle j is the fluorescence point of the fluorescent group Cy5.5 at 4h during exocytosis. Fig.10 The k in the middle is the fluorescence point of the nuclear dye DAPI at 4h during exocytosis. Fig.10 The middle l is the fluorescence point of all fluorescence channels at 4h during exocytosis. Fig.10 In the middle, m is the fluorescence point of the fluorescent group Cy5.5 at 8h during endocytosis. Fig.10 The n in the middle is the fluorescence point of the nuclear dye DAPI at 8h during endocytosis. Fig.10 The o in the middle is the fluorescence point of all fluorescence channels at 8h during endocytosis. Fig.10 p in the middle is the fluorescence point of the fluorescent group Cy5.5 at 8h during exocytosis. Fig.10 The middle q is the fluorescence point of the nuclear dye DAPI at 8h during exocytosis. Fig.10 r in the middle is the fluorescence point of all fluorescence channels at 8h during exocytosis. Fig.10 Where c1 is Fig.10 A partial enlarged view of c in the middle. Fig.10 f1 is Fig.10 The local enlarged view of f in the middle, Fig.10 i1 is Fig.10 The partial enlarged picture of i in the figure, Fig.10 In l1 is Fig.10 A partial enlarged view of l in the figure. Fig.10 o1 is Fig.10 A partial enlarged view of o in the figure. Fig.10 r1 is Fig.10 A partial enlarged view of r in the figure.

[0047] Fig.11 The NK cells and HSCs cells were treated with the bioactive solutions prepared in Example 1 and Comparative Example 4, and the fluorescence points at different culture times were as follows: Fig.11 a in the figure is the fluorescent spot of the fluorescent group Cy5.5 when cultured for 1 hour in Example 1. Fig.11 b is the fluorescence point of the nuclear dye DAPI when cultured for 1 hour in Example 1. Fig.11 Figure c in the figure is the fluorescent spot of CFSE, a live cell dye of NK pre-stained after 1 hour of culture in Example 1. Fig.11 In the figure d, the fluorescence points of all fluorescence channels are shown in Example 1 after 1 hour of culture. Fig.11 e in the figure is the fluorescent spot of the fluorescent group Cy5.5 when cultured for 2 hours in Example 1. Fig.11 Figure f in the figure is the fluorescence point of the nuclear dye DAPI when cultured for 2 hours in Example 1. Fig.11 Figure g in the middle is the fluorescent spot of CFSE, a live cell dye of NK pre-stained after culturing for 2 hours in Example 1. Fig.11 h in the middle is the fluorescence points of all fluorescence channels when cultured for 2 hours in Example 1, Fig.11 i is the fluorescent spot of the fluorescent group Cy5.5 when cultured for 4 hours in Example 1, Fig.11 In the figure j, the fluorescence point of the cell nucleus dye DAPI is shown in Example 1 after culturing for 4 hours. Fig.11 k in the figure is the fluorescence point of NK live cell dye CFSE prestained after culturing for 4 hours in Example 1. Fig.11 Figure 1 is the fluorescence points of all fluorescence channels when cultured for 4 hours in Example 1. Fig.11 The middle m is the fluorescence point of the fluorescent group Cy5.5 when cultured for 4 hours in comparative example 4, Fig.11 The n in the middle is the fluorescence point of the nuclear dye DAPI when cultured for 4 hours in Example 4. Fig.11 o is the fluorescence point of NK live cell dye CFSE pre-stained in Example 4 after culturing for 4 hours. Fig.11 In the middle, p is the fluorescence points of all fluorescence channels when the comparative example 4 was cultured for 4 hours.

[0048] Fig.12 It is the test result of Western blot test, where "+" represents addition and "-" represents no addition.

[0049] Fig.13 is the effect of the embodiments and comparative examples on liver indexes, wherein Fig.13 a is the alanine aminotransferase content, Fig.13 b is the aspartate aminotransferase content, Fig.13 Where c is the blood urea nitrogen content, Fig.13 Where d is the total bilirubin content. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0051] In a first aspect, some embodiments of the present invention provide a bioactive solution, wherein the bioactive solution includes a polypeptide having an amino acid sequence of SEQ ID NO.1 and a polypeptide having an amino acid sequence of SEQ ID NO.2;

[0052] The structural formula of the polypeptide having the amino acid sequence of SEQ ID NO.1 is:

[0053] ;

[0054] The structural formula of the polypeptide having the amino acid sequence of SEQ ID NO.2 is:

[0055] .

[0056] In the bioactive solution of the present invention, the co-assembly system of the two polypeptides can completely cross the vascular endothelium, further induce NK cells and hepatic stellate cells HSCs related to liver fibrosis to approach each other, enhance the NK immune killing activity while inhibiting the activation and abnormal proliferation of HSCs, thereby alleviating the symptoms of liver fibrosis.

[0057] In some embodiments, the polypeptide having the amino acid sequence of SEQ ID NO.1 is composed of D D GFFY amino acid sequence, D configuration D TBP-3 targeting sequence, L-configuration competitive antagonistic tetrapeptide LSKL, and capping composition.

[0058] In some embodiments, the D-configuration D The amino acid sequence of GFFY is glycine-D-phenylalanine-D-phenylalanine-D-tyrosine.

[0059] In the present invention, D-configuration D The GFFY amino acid sequence can drive the polypeptide having the amino acid sequence of SEQ ID NO.1 to self-assemble.

[0060] In some embodiments, the end-capping agent is 2-naphthylacetic acid.

[0061] In some embodiments, the D-configuration D The TBP-3 targeting sequence is glycine-glycine-D configuration, tyrosine-D configuration, threonine-D configuration, phenylalanine-D configuration, histidine-D configuration, tryptophan-D configuration, histidine-D configuration, arginine-D configuration, leucine-D configuration, asparagine-D configuration, proline.

[0062] In some embodiments, the L-configuration competitive antagonistic tetrapeptide LSKL is L-configuration leucine-L-configuration serine-L-configuration lysine-L-configuration leucine.

[0063] In some embodiments, the polypeptide having the amino acid sequence of SEQ ID NO.2 is composed of D D It consists of the GFFY amino acid sequence, glycine, cysteine, glycine, and the L-configuration VHPKQHR sequence.

[0064] In the present invention, D-configuration D The GFFY amino acid sequence and the L-configuration VHPKQHR sequence are linked via cysteine.

[0065] In some embodiments, the D-configuration D The amino acid sequence of GFFY is glycine-D-phenylalanine-D-phenylalanine-D-tyrosine.

[0066] In some embodiments, maleimide is connected to the thiol group of cysteine, and maleimide is also connected to a near-infrared fluorescent group Cy5.5.

[0067] In some embodiments, the L-configuration VHPKQHR sequence is L-configuration valine-L-configuration histidine-L-configuration proline-L-configuration lysine-L-configuration glutamine-L-configuration histidine-L-configuration arginine.

[0068] In some embodiments, the molar concentration ratio of the polypeptide having the amino acid sequence of SEQ ID NO.1 to the polypeptide having the amino acid sequence of SEQ ID NO.2 in the biologically active solution is 4:1.

[0069] In some embodiments, the mass concentration of the polypeptide having the amino acid sequence of SEQ ID NO.1 in the biologically active solution is 3 mg / 1.2 mL-3 mg / 0.8 mL.

[0070] In a second aspect, some embodiments of the present invention also provide a method for preparing the bioactive solution, which comprises: dispersing a polypeptide having an amino acid sequence of SEQ ID NO.1 and a polypeptide having an amino acid sequence of SEQ ID NO.2 in a PBS buffer, adjusting the pH value of the solution, heating, cooling, and obtaining a bioactive solution.

[0071] In some embodiments, the pH value of the PBS buffer is 5-9.

[0072] In some embodiments, in PBS buffer, the molar concentration ratio of the polypeptide having the amino acid sequence of SEQ ID NO.1 to the polypeptide having the amino acid sequence of SEQ ID NO.2 is 4:1.

[0073] In some embodiments, the mass concentration of the polypeptide having the amino acid sequence of SEQ ID NO.1 in PBS buffer is 3 mg / 1.2 mL-3 mg / 0.8 mL.

[0074] In some embodiments, the pH value of the solution is adjusted to 6-8.

[0075] In some embodiments, the solution is heated to a temperature of 90°C-100°C.

[0076] In some embodiments, the cooling is to room temperature.

[0077] In a third aspect, some embodiments of the present invention also provide use of the bioactive solution in the preparation of a drug for alleviating liver fibrosis.

[0078] The following is described in conjunction with specific embodiments:

[0079] Embodiment 1:

[0080] Preparation method of polypeptide having amino acid sequence of SEQ ID NO.1: Nap-G D F D F D YK(LSKL)GG D Y D T D F D H D W D H D R D L D N D Synthesis of P

[0081] The Fmoc-short peptide solid phase synthesis method was used for synthesis. The specific steps are:

[0082] (1) Weigh 0.5 mmol of 2-Cl-Trt resin into a solid phase synthesizer, add 10 mL of anhydrous dichloromethane (DCM), and shake on a shaker for 10 min to allow the 2-Cl-Trt resin to fully swell;

[0083] (2) Use an ear bulb to remove DCM from the solid phase synthesizer containing 2-Cl-Trt resin;

[0084] (3) Dissolve 0.5 mmol of Fmoc-protected amino acid (Fmoc-D-Phe-OH) in 10 mL of anhydrous DCM, add 1 mmol of DIEPA (N,N-diisopropylethylamine), and then transfer to the above-mentioned solid phase synthesizer and react at room temperature for 1 h.

[0085] (4) Blocking: Use an ear bulb to remove the reaction solution in the solid phase synthesizer, then wash with 10 mL of anhydrous DCM for 1 min each time, for a total of 5 washes, add 20 mL of a solution with a volume ratio of anhydrous DCM: DIEPA: methanol of 17:1:2, and react at room temperature for 20 min;

[0086] (5) Remove the reaction solution in the solid phase synthesizer with an ear bulb, first wash with anhydrous DCM, 10 mL of DCM each time, washing time 1 min, wash 5 times in total, then wash with DMF (dimethylformamide), 10 mL of DMF each time, washing time 1 min, wash 5 times in total, add 10 mL of a mixed solution of piperidine and DMF, wherein the volume ratio of piperidine to DMF is 2:8, react for 25 min, then wash with DMF, 10 mL of DMF each time, washing time 1 min, wash 5 times in total, and proceed to the next step of reaction;

[0087] (6) Add 1 mmol of the second Fmoc-protected amino acid (Fmoc-D-His(Boc)-OH), 1.5 mmol of HBTU (benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate), 2 mmol of DIEPA and 10 mL of DMF, add the prepared solution to the above solid phase synthesizer and react for 2 h;

[0088] (7) Repeat steps (5) and (6) to sequentially add L-configuration and D-configuration amino acids and a capping group (2-naphthylacetic acid) according to the amino acid sequence of SEQ ID NO.1; then wash with DMF 5 times and with dichloromethane 5 times, and proceed to the next step;

[0089] (8) 10 mL of a solution consisting of 95% TFA (trifluoroacetic acid), 2.5% TIS (triisopropylsilane), and 2.5% H2O (water) by volume was added to the solid phase synthesizer and reacted for 0.5 h. The product was cut from the 2-Cl-Trt resin and concentrated in vacuo to remove the solvent to obtain a crude product, which was then separated and purified by HPLC. A person skilled in the art can also prepare a TFA solution having a TFA volume percentage concentration of 1% by TFA and DCM in a volume ratio of 1:99, and add 3 mL of the TFA solution to the solid phase synthesizer ten times, with each reaction time of 1 min.

[0090] (9) The purified tetrapeptide LSKL was connected to the amino group of lysine in the main chain by a liquid phase synthesis method. 1mmol of the tetrapeptide LSKL, 1.1mmol NHS (N-hydroxysuccinimide), and 1.2mmol DCC (1,3-dicyclohexylcarbodiimide) were dissolved in a small amount of DMF until clear, and the exposed carboxyl group of LSKL was activated. The reaction was carried out for more than 4 hours, and the reaction liquid was filtered. 1mmol of the main chain pure product and 1mmol of DIEPA were added to the system, and the pH value was adjusted to 8-10. After reacting overnight, the system was separated and purified by HPLC. A polypeptide having an amino acid sequence of SEQ ID NO.1 was obtained, which was recorded as PepA-TT. The amino acid sequence of the obtained PepA-TT is shown in SEQ ID NO.1. The detection was performed by high performance liquid chromatography-mass spectrometry. The results are shown in FIG. Figure 1-Figure 2 ;

[0091] Preparation method of a polypeptide having an amino acid sequence of SEQ ID NO.2: The Fmoc-short peptide solid phase synthesis method of a polypeptide having an amino acid sequence of SEQ ID NO.1 can also be applied to the synthesis of an amino acid sequence of SEQ ID NO.2, which is recorded as Cy5.5 PepB-V.

[0092] (1) Weigh 0.5 mmol of 2-Cl-Trt resin into a solid phase synthesizer, add 10 mL of anhydrous dichloromethane (DCM), and shake on a shaker for 10 min to allow the 2-Cl-Trt resin to fully swell;

[0093] (2) Use an ear bulb to remove DCM from the solid phase synthesizer containing 2-Cl-Trt resin;

[0094] (3) Dissolve 0.5 mmol of Fmoc-protected amino acid (Fmoc-D-Phe-OH) in 10 mL of anhydrous DCM, add 1 mmol of DIEPA (N,N-diisopropylethylamine), and then transfer to the above-mentioned solid phase synthesizer and react at room temperature for 1 h.

[0095] (4) Blocking: Use an ear bulb to remove the reaction solution in the solid phase synthesizer, then wash with 10 mL of anhydrous DCM for 1 min each time, for a total of 5 washes, add 20 mL of a solution with a volume ratio of anhydrous DCM: DIEPA: methanol of 17:1:2, and react at room temperature for 20 min;

[0096] (5) Remove the reaction solution in the solid phase synthesizer with an ear bulb, first wash with anhydrous DCM, 10 mL of DCM each time, washing time 1 min, wash 5 times in total, then wash with DMF (dimethylformamide), 10 mL of DMF each time, washing time 1 min, wash 5 times in total, add 10 mL of a mixed solution of piperidine and DMF, wherein the volume ratio of piperidine to DMF is 2:8, react for 25 min, then wash with DMF, 10 mL of DMF each time, washing time 1 min, wash 5 times in total, and proceed to the next step of reaction;

[0097] (6) Add 1 mmol of the second Fmoc-protected amino acid (Fmoc-D-His(Boc)-OH), 1.5 mmol of HBTU (benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate), 2 mmol of DIEPA and 10 mL of DMF, add the prepared solution to the above solid phase synthesizer and react for 2 h;

[0098] (7) Repeat steps (5) and (6) to sequentially add L-configuration and D-configuration amino acids and a capping group (2-naphthylacetic acid) according to the main chain amino acid sequence of SEQ ID NO.2; then wash with DMF 5 times and with dichloromethane 5 times, and proceed to the next step;

[0099] (8) 10 mL of a solution consisting of 95% TFA (trifluoroacetic acid), 2.5% TIS (triisopropylsilane), and 2.5% H2O (water) by volume was added to the solid phase synthesizer and reacted for 0.5 h. The product was cut from the 2-Cl-Trt resin and concentrated in vacuo to remove the solvent to obtain a crude product, which was then separated and purified by HPLC. A person skilled in the art can also prepare a TFA solution having a TFA volume percentage concentration of 1% by TFA and DCM in a volume ratio of 1:99, and add 3 mL of the TFA solution to the solid phase synthesizer ten times, with each reaction time of 1 min.

[0100] (9) Then, the main chain pure product and Cy5.5-MAL (Cy5.5-maleimide) were added to the system at a mass ratio of 1:1, and vortexed for 30 seconds. The pH was adjusted to 7.5 using sodium carbonate (0.01 M) and placed at 4°C overnight. Finally, the peptide was obtained by freeze drying. Cy5.5PepB-V. The polypeptide having the amino acid sequence of SEQ ID NO.2 was detected by high performance liquid chromatography-mass spectrometry. The results are shown in Figure 3-Figure 4 ;

[0101] Add PepA-TT peptide 3mg, Cy5.5 0.75 mg of PepB-V peptide was added to the system, and 1 mL of PBS buffer solution (pH = 7.4) was added to the system. In the PBS buffer solution, PepA-TT peptide and Cy5.5 The molar concentration ratio of PepB-V polypeptide is 4:1. The pH value is adjusted to 7.4 using sodium carbonate solution, and Vortex is used for 3-5 seconds to completely dissolve it to obtain the biologically active solution before assembly. Then, a heating-cooling reaction is performed, the system is heated to boiling, and then cooled to room temperature after 3-5 seconds to obtain the biologically active solution after co-assembly.

[0102] Comparative Example 1:

[0103] The preparation method is the same as in Example 1, but PepA-TT polypeptide and Cy5.5 The added masses of PepB-V peptides were different, among which, in PBS buffer, PepA-TT peptides and Cy5.5 The molar concentration ratio of PepB-V peptides was 1:1.

[0104] Comparative Example 2:

[0105] The preparation method is the same as in Example 1, but PepA-TT polypeptide and Cy5.5 The added masses of PepB-V peptides were different, among which, in PBS buffer, PepA-TT peptides and Cy5.5 The molar concentration ratio of PepB-V polypeptide was 2:1.

[0106] Comparative Example 3:

[0107] The preparation method is the same as in Example 1, but PepA-TT polypeptide and Cy5.5 The added masses of PepB-V peptides were different, among which, in PBS buffer, PepA-TT peptides and Cy5.5 The molar concentration ratio of PepB-V polypeptide was 3:1.

[0108] Comparative Example 4:

[0109] Weigh 8g NaCl (sodium chloride), 0.2g KCl (potassium chloride), 1.44g Na2HPO4 (disodium hydrogen phosphate) and 0.24g KH2PO4 (potassium hydrogen phosphate), dissolve in 800mL distilled water, adjust the pH value of the solution to 7.4 with HCl (hydrochloric acid), and finally add distilled water to make up to 1L to obtain PBS solution. After sterilization in an autoclave, store in a refrigerator at 4°C.

[0110] The bioactive solutions obtained from the examples and comparative examples were tested experimentally:

[0111] Results and analysis:

[0112] Weigh 3 mg of the polypeptide PepA-TT prepared in Example 1 and polypeptides with different molar concentration ratios thereof. Cy5.5 PepB-V, add 1 mL of 1×PBS buffer solution (pH=7.4) to the system, adjust its pH value to 7.4 with sodium carbonate solution, and Vortex for 3-5 seconds to completely dissolve it to obtain the bioactive solution before assembly. Then, a heating-cooling reaction is performed, and the system is heated to boiling. After 3-5 seconds, it is placed at room temperature to cool to obtain the bioactive solution after co-assembly. The bioactive solutions obtained in Example 1 and Comparative Examples 1-3 are observed by transmission electron microscopy for their microscopic morphology after assembly. The results are shown in FIG. Figure 5 . Figure 5 It shows that the peptide PepA-TT and peptide Cy5.5 PepB-V will present a completely different microstructure after co-assembly. As the molar concentration of the PepA-TT peptide in the system gradually increases, the nanostructure presented after co-assembly gradually changes from the relatively independent existence of nanofibers and nanoparticles to a more ordered higher-order structure. Cy5.5 When the molar concentration ratio of PepB-V is 4:1, the co-assembly presents a nanofiber network shaped like a vascular network and loaded with fine nanoparticles, indicating that the system has reached a more stable assembly state at this ratio. Therefore, it is further preferred that the concentration of the polypeptide PepA-TT in the PBS buffer is 4:1. Cy5.5 The molar concentration ratio of PepB-V in PBS buffer was 4:1.

[0113] Weigh 3 mg of the polypeptide PepA-TT prepared in Example 1 and 3 mg of the polypeptide prepared in Example 1 respectively. Cy5.5 PepB-V 0.6 mg, each was added with 1 mL of 1× PBS buffer solution (pH = 7.4), and the pH value was adjusted to 7.4 with sodium carbonate solution. After ultrasonic dissolution, a PepA-TT solution with a concentration of 1096 μM and a PepA-TT solution with a concentration of 274 μM were obtained. Cy5.5 PepB-V solution was prepared to meet the concentration required for biological transmission electron microscopy experiments, followed by a heating-cooling reaction, where the system was heated to boiling, and then cooled at room temperature after 3-5 seconds to obtain the assembled PepA-TT bioactive solution and the assembled peptide. Cy5.5 PepB-V biologically active solution. Transmission electron microscopy was used to observe the assembled peptides PepA-TT and peptide Cy5.5The microscopic morphology of the PepB-V bioactive solution is shown in the following figure. Figure 6 shown. Figure 6 The results showed that the nanostructure of the assembled peptide PepA-TT was a network of entangled long nanofibers. Cy5.5 PepB-V formed nanoparticles with different diameters.

[0114] Next, weigh 0.7 mg of the polypeptide PepA-TT prepared in Example 1 and 0.7 mg of the polypeptide Cy5.5 PepB-V 0.15 mg, add 1×PBS buffer (pH=7.4) solution 5 mL to the system, adjust its pH value to 7.4 with sodium carbonate solution, and dissolve it completely by ultrasound to obtain a solution with a peptide PepA-TT concentration of 50 μM (PepA-TT molar concentration: Cy5.5 PepB-V molar concentration = 4:1), heating-cooling reaction to obtain the bioactive solution of the co-assembled system. The microscopic morphology of the bioactive solution after the co-assembly was observed by transmission electron microscopy. The results are as follows Figure 7 shown. Figure 7 It is shown that in the bioactive solution at a concentration of 50 μM (PepA-TT molar concentration: Cy5.5 The nanostructure presented by the PepB-V molar concentration = 4:1 is still a vascular nanofiber network, with individual fibers arranged longitudinally and aggregated into bundles. It should be understood by those skilled in the art that when the polypeptide concentration in the cell-level experiment is changed, the polypeptide in the prior art will undergo structural changes due to poor structural stability. In the polypeptide structure of the present invention, in the cell-level experiment, the polypeptide in the biologically active solution at the selected drug addition concentration still has a highly ordered nanostructure, which shows that the polypeptide structure of the present invention is stable and can be seen from Figure 7 Verification in.

[0115] In order to explore the endothelial transendothelial ability of the polypeptide of the present invention, we conducted a biological transmission electron microscopy experiment. 6 Human umbilical vein endothelial HUVECs were seeded in a culture dish with a diameter of 10 cm at a density of 1 cell / dish, and the culture medium in the dish was removed after the cells adhered to the wall. The co-assembly system of Example 1 was added to the system, i.e., the concentration of the polypeptide PepA-TT was 50 μM (PepA-TT molar concentration: Cy5.5PepB-V molar concentration = 4:1) biologically active solution, with the PBS buffer of comparative example 4 as a blank control. After incubation for 8 hours, the medium in the dish was replaced with fresh drug-free medium and incubated for another 6 hours. HUVEC cells were collected into 1.5mL EP tubes after incubation for 8 hours and after replacing the medium for another 6 hours, respectively, and the cell pellets were obtained by centrifugation. The cells were gently scratched into tiny cell clusters with a needle tip, and the fixative (2.5% glutaraldehyde solution) was slowly added along the tube wall, and fixed at 4°C overnight. Centrifuge again to remove the fixative in the tube, rinse the sample with pH 7.0 PBS buffer 3 times for 15 minutes each time, and fix the sample with 1% osmium acid solution for 1-2 hours. Then, carefully remove the osmium acid waste liquid and rinse the sample with pH 7.0 PBS buffer 3 times for 15 minutes each time. The samples were dehydrated with ethanol solutions of gradient concentrations (including six concentrations: 30%, 50%, 70%, 80%, 90% and 95%), treated for 15 minutes at each concentration, then treated with 100% ethanol for 20 minutes, and finally transitioned to pure acetone for 20 minutes. The samples were treated with a mixture of Spurr embedding agent and acetone (V / V=1 / 1) for 1 hour, with a mixture of Spurr embedding agent and acetone (V / V=3 / 1) for 3 hours, and with pure embedding agent overnight. The infiltrated samples were embedded and heated at 70°C overnight to obtain embedded samples. The samples were sliced ​​in an ultrathin microtome to obtain 70nm-90nm slices. The slices were stained with lead citrate solution, uranyl acetate and 50% ethanol saturated solution for 5-10 minutes each, and then observed in a Hitachi-7800 transmission electron microscope. The results are shown in the figure. Figure 8 shown.

[0116] Figure 8 It is shown that a large number of nanofiber structures can be seen in the cytoplasm of HUVEC cells after endocytosis, and after exocytosis, the formation of secretory vesicles on the cell membrane and the significant reduction and discharge of fine fibers in the cytoplasm can be seen. The above results show that the endocytosis and exocytosis of vascular endothelial cells will not affect the morphology of the co-assembly system of Example 1, and the complete crossing of the vascular endothelium can be achieved.

[0117] According to the steps provided by the Monolith NT™ Protein Labeling Kit BLUE-NHS protein labeling kit, 5 μL of "Dye NT-495 NHS" with DMSO (dimethyl sulfoxide) as the solvent was added to PBS and diluted 20 times, and then it was mixed with 100 μL of 10 μM rhTIGIT protein and rhVCAM-1 protein (buffer: 1×PBS pH 7.4, Tween-20 (0.05%)) and blown evenly, and then the mixture was placed in the dark to react for 30 minutes. After the reaction is completed, the excess dye is completely washed out through the molecular sieve, and a quantitative buffer is added to allow the solution to flow out under gravity, and the fluorescently labeled protein is collected. According to the method for preparing a biologically active solution in Example 1, Example 1, the two polypeptides prepared, and Example 1 co-assembly system (PepA-TT molar concentration: Cy5.5 The bioactive solutions of different concentrations were prepared with PepB-V molar concentration = 4:1. The bioactive solutions of peptides with different concentration gradients were incubated with equal volumes of fluorescently labeled rhTIGIT protein and rhVCAM-1 protein at room temperature for 5 minutes, and the capillary aspirated solutions were tested on the machine. Finally, the binding ability of different peptides to PD-L1 protein was measured by microcalorimetry (Monolith NT.115). The results are shown in Figure 2. Fig. 9 shown.

[0118] Fig. 9 Different peptides have different binding constants for TIGIT protein and VCAM-1 protein. The peptides PepA-TT and peptides Cy5.5 PepB-V and bioactive solution (PepA-TT molar concentration: Cy5.5 PepB-V (molar concentration = 4:1) was mixed with TIGIT protein and VCAM-1 protein, and the protein binding capacity was determined by microthermophoresis. D The smaller the value, the stronger its affinity. Regarding the binding ability to TIGIT protein, the polypeptide PepA-TT in the present invention has a stronger ability due to the presence of TIGIT affinity sequence. The K D The value was 2.19 μM, which was comparable to the peptide lacking the TIGIT affinity sequence. Cy5.5 K of PepB-V D The affinity of the polypeptide of the present invention to TIGIT protein was increased by 19.5 times compared with the value of 44.8 μM. Cy5.5 PepB-V has strong ability, and the peptide PepA-TT K DThe value reached 8.72 μM, which is 5.8 times that of the polypeptide PepA-TT prepared in Example 1. In addition, the bioactive solution of the present invention has good binding ability for both TIGIT protein and VCAM-1 protein. It can be seen that the co-assembly system in Example 1 (PepA-TT molar concentration: Cy5.5 PepB-V molar concentration = 4:1) has good binding ability to both target proteins, laying the foundation for its cell selectivity and functional specificity.

[0119] According to 5×10 4 Human umbilical vein endothelial HUVECs were seeded on a 24-well plate covered with a circular cell slide at a density of 10 cells / well. After the cells attached to the surface of the cell slide, the culture medium in the well was removed. The method for preparing the bioactive solution according to Example 1 was added to the wells to prepare a polypeptide PepA-TT concentration of 50 μM (PepA-TT molar concentration: Cy5.5 PepB-V molar concentration = 4:1) biologically active solution, and the PBS solution of comparative example 1 was used as a blank control. Incubate at different time points to observe the endocytosis of HUVEC, and then replace the culture medium in the wells with fresh drug-free culture medium and continue to incubate at different time points to explore the exocytosis of HUVEC to verify the endothelial crossing ability of the polypeptide of the present invention. After the above steps are completed, the solution in the wells is aspirated, the cells are washed 3 times with PBS buffer for 1 minute each time, and then stained with 0.5 μg / mL DAPI (4',6-diamidino-2-phenylindole) at room temperature for 4 minutes. All the above operations must be protected from light. After staining, images are taken at the same voltage using a laser scanning confocal microscope (Carl Zeiss LSM 900) to detect the endothelial crossing of the polypeptide, such as Fig.10 shown.

[0120] from Fig.10 It can be seen that after incubating HUVEC cells with the bioactive solution prepared in Example 1 for different times, the number and fluorescence intensity of the red fluorescent bright spots indicating the polypeptide in the cytoplasm gradually increased with the incubation time, and were closely distributed around the nucleus. The above results show that HUVEC cells expressing the adhesion receptor VCAM-1 can endocytose the co-assembly system of Experimental Example 1 into the cytoplasm through the receptor-mediated endocytosis pathway. However, after replacing the drug-containing culture medium in the well with fresh culture medium, the red fluorescent bright spots in the cytoplasm gradually decreased with the progress of exocytosis, and the small amount of fluorescent bright spots remaining in the cytoplasm were also distributed away from the nucleus. The above results show that after the polypeptide in Example 1 enters the vascular endothelial cells through endocytosis, it is further transported to the outside of the cell through receptor-mediated exocytosis, creating conditions for further enhancing the interaction between effector cells.

[0121] Next, follow the 5×104 Human hepatic stellate cells LX-2 were seeded on a 24-well plate at a density of 10 cells / well, and the medium in the wells was removed after the cells adhered to the wall. The method for preparing the bioactive solution according to Example 1 was added to the wells respectively, and the concentration of the polypeptide PepA-TT prepared in Example 1 was 50 μM (PepA-TT molar concentration: Cy5.5 PepB-V molar concentration = 4:1) biological active solution, and the PBS solution of comparative example 1 was used as a blank control. At the same time, human NK cells NK92-MI were added to the wells according to a certain cell number ratio to explore the effect of the polypeptide of the present invention on the interaction between the two effector cells and whether it can enhance the adhesion ability of NK cells to HSCs. 2.5×10 5 NK cells were co-cultured. After incubating the cells for different time points, the solution in the wells was aspirated and the cells were washed 3 times with PBS buffer for 1 minute each time. The cells attached to the coverslips were fixed with 4% paraformaldehyde in PBS buffer for 10 minutes at room temperature. Then, the solution in the wells was aspirated and the cells were washed 3 times with PBS buffer for 1 minute each time, and then stained with 0.5μg / mL DAPI for 4 minutes at room temperature. All operations must be protected from light. CFSE (hydroxyfluorescein diacetate succinimidyl ester) is a live cell dye. After staining, images were taken at the same voltage using a laser scanning confocal microscope (Carl Zeiss LSM 900) to detect the effect of the polypeptide on the adhesion ability of NK cells, such as Fig.11 shown.

[0122] from Fig.11 It can be seen that the number of NK cells pre-stained with green fluorescence adhering to the surface of LX-2 cells gradually increased at different time points of co-culture of NK cells and HSCs treated with the bioactive solution prepared in Example 1. The PBS buffer group of Comparative Example 1 was used as a blank control. After the same incubation for 4 hours, Example 1 could significantly improve the adhesion ability of NK cells to HSCs.

[0123] The effect of the polypeptide of the present invention on the liver fibrosis-related phenotype of HSCs was detected by immunoblotting. LX-2 cells were seeded on a cell culture dish with a diameter of 10 mm and cultured for 24 hours until the cells adhered to the wall and grew to a density of about 80%. PepA-TT, Cy5.5 PepB-V and co-assembled polypeptide bioactive solutions, with the PBS solution described in Comparative Example 1 as a blank control, the PepA-TT concentration was 50 μM, Cy5.5The concentration of PepB-V was 12.5 μM. The cells were incubated for 24 hours and the culture medium was removed. The cells were then washed three times with PBS buffer. The cells were collected and lysed in a cell lysis buffer containing protease inhibitors. After quantitative analysis with BCA, equal amounts (30 μg) of samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% BSA (bovine serum albumin) to eliminate nonspecific interference. The membrane was incubated with the corresponding primary antibody at 4°C overnight and then co-incubated with ImmunoWay's HRP (horseradish peroxidase)-labeled secondary antibody for 1.5 hours. Finally, Tanon 5500 was used to detect the membrane through Immobilon Western HRP substrate to detect the ability of the peptide to reverse the TGF-β1-induced LX-2 liver fibrosis-related phenotype. GAPDH was used as the internal reference protein, and Collegen III, Desmin, and α-SMA were liver fibrosis-related indicators. The results are shown in the figure. Fig.12 as shown (where “+” represents addition and “-” represents non-addition).

[0124] from Fig.12 It can be seen that, with the PBS group as the blank control group, under the condition that the internal reference protein bands of each group were basically consistent, after incubation for 24 hours in Example 1, the fibrosis-related phenotypes of LX-2 cells were significantly reduced, and the degree of fibrosis induced by TGF-β1 could be effectively reversed, indicating that Example 1 had a significant in vitro inhibitory effect on LX-2 cell fibrosis, providing a basis for further in vivo experiments.

[0125] We used CCL4 (carbon tetrachloride) to induce a mouse liver fibrosis model to explore the application of the polypeptide of the present invention in vivo anti-liver fibrosis. CCL4 was dissolved in mineral oil to prepare an injection solution with a final concentration of 25%. The solution was injected intraperitoneally at a dose of 2 mL / kg, twice a week for four weeks. The mice in the control group were injected intraperitoneally with an equal amount of mineral oil and fed with normal feed. The drug was administered by tail vein injection at a concentration of 10 mg / kg. The drug was administered once on the 1st, 4th, and 7th days, for a total of three times. The serum of mice in each group was collected 24 hours after the last dose for blood biochemical analysis of liver damage-related indicators, such as Fig.13 shown.

[0126] The main test indicators include aspartate aminotransferase (AST) and alanine aminotransferase (ALT) which represent liver parenchymal damage, total bilirubin (TBIL) which is an important indicator of liver function, and blood urea nitrogen (BUN) which is an indicator for evaluating kidney performance. Fig.13 It can be seen that after three doses, the peptide PepA-TT, Cy5.5The liver injury-related indicators of mice treated with PepB-V and the co-assembled system of Example 1 via tail vein administration were reversed to varying degrees compared with the model group mice.

[0127] Therefore, the polypeptide provided by the present invention can cross the vascular endothelium and enhance cell-to-cell interaction, and inhibit the proliferation and activation of HSCs from both direct contact and immune killing by enhancing the adhesion and killing of NK cells, thereby slowing down the progression of liver fibrosis.

[0128] It should be noted that the terms used in the present invention are only for describing specific embodiments, rather than limiting the scope of the present application. As shown in the present specification, unless the context clearly indicates an exception, the words "one", "a", "a kind of" and / or "the" do not specifically refer to the singular, but may also include the plural. The terms "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method or device including a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method or device. In the absence of more restrictions, the elements defined by the sentence "include one..." do not exclude the presence of other identical elements in the process, method or device including the elements.

[0129] It should also be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention. Unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", etc. should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be a connection between the two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0130] 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. A biologically active solution, characterized in that The biologically active solution includes a polypeptide having a structure of Formula 1 and a polypeptide having a structure of Formula 2; The polypeptide of formula 1 is: ； The polypeptide of formula 2 is: 。 2. A biologically active solution according to claim 1, characterized in that: The molar concentration ratio of the polypeptide of formula 1 to the polypeptide of formula 2 in the biologically active solution is 4:

1.

3. A biologically active solution according to claim 1, characterized in that: The mass concentration of the polypeptide of formula 1 in the biologically active solution is 3 mg / 1.2 mL-3 mg / 0.8 mL.

4. Use of the bioactive solution according to any one of claims 1 to 3 in the preparation of a medicament for alleviating liver fibrosis.

5. The use according to claim 4, characterized in that The preparation method of the bioactive solution comprises: dispersing the polypeptide of formula 1 and the polypeptide of formula 2 in a PBS buffer, adjusting the pH value of the solution, heating, and cooling to obtain the bioactive solution.

6. The use according to claim 5, characterized in that: The pH value of the PBS buffer is 5-9; The pH value of the adjusted solution is 6-8.

7. The use according to claim 5, characterized in that: In PBS buffer, the molar concentration ratio of the polypeptide of Formula 1 to the polypeptide of Formula 2 is 4:

1.

8. The use according to claim 5, characterized in that: The mass concentration of the polypeptide of formula 1 in PBS buffer is 3 mg / 1.2 mL-3 mg / 0.8 mL.

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