A polypeptide for enhancing immune killing of NK cells and inhibiting activation of HSCs and its application

Abstract

The present invention relates to the fields of medicine and pharmaceutical technology, and discloses a polypeptide for enhancing the immune killing of NK cells and inhibiting the activation of HSCs and its application. The polypeptide has the amino acid sequence of SEQ ID NO.1. The polypeptide obtained by the present invention can cross the vascular endothelium and serve as a medium connecting immune cells NK and hepatic fibrosis effector cells HSCs, which is beneficial to alleviating the symptoms of hepatic fibrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine and pharmacy, and particularly relates to a polypeptide for enhancing the immune killing of NK cells and inhibiting the activation of HSCs and its application. Background Art

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

[0003] Previous studies have shown that preventing the activation of HSCs (hepatic stellate cells) or inducing the 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 the active pro-apoptotic signals are provided by immune cells, especially natural killer (NK) cells. Therefore, as a medium connecting the key immune cell NK and the effector cell HSCs in the development of liver fibrosis, significantly slowing down and inhibiting the development process of liver fibrosis by affecting cell-cell interactions in both direct contact and signal transmission aspects is a new strategy for the research and development of liver fibrosis-specific drugs. Currently, there is no study on using small molecules to affect cell-cell interactions for immune drugs in liver fibrosis. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a polypeptide for enhancing the immune killing of NK cells and inhibiting the activation of HSCs and its application. The polypeptide obtained by the present invention can cross the vascular endothelium and serve as a medium connecting the immune cell NK and the liver fibrosis effector cell HSCs, which is beneficial to alleviating the symptoms of liver fibrosis.

[0005] The present invention provides a polypeptide for enhancing the immune killing of NK cells and inhibiting the activation of HSCs, and the polypeptide has the amino acid sequence of SEQ ID NO.1;

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

[0007] .

[0008] Furthermore, the polypeptide having the amino acid sequence of SEQ ID NO.1 is composed of a D-configured D GFFY amino acid sequence, a D-configured D TBP-3 targeting sequence, an L-configured competitive antagonist tetrapeptide LSKL, and a capping group.

[0009] In the present invention, the main chain of the polypeptide having the amino acid sequence of SEQ ID NO.1 is composed of a capping group, a D-configured D GFFY amino acid sequence, and a D-configured D TBP-3 targeting sequence. Among them, on the main chain of the polypeptide having the amino acid sequence of SEQ ID NO.1, there are a capping group, a D-configured D GFFY amino acid sequence and a D-configured D GFFY amino acid sequence connected in sequence.

[0010] Furthermore, the D-configured D GFFY amino acid sequence is D-configured glycine-D-configured phenylalanine-D-configured phenylalanine-D-configured tyrosine.

[0011] Furthermore, the capping group is 2-naphthoic acid (Nap).

[0012] Furthermore, the D-configured D TBP-3 targeting sequence is D-configured tyrosine-D-configured cystine-D-configured phenylalanine-D-configured histidine-D-configured tryptophan-D-configured histidine-D-configured arginine-D-configured leucine-D-configured aspartic acid-D-configured proline.

[0013] Furthermore, the L-configured competitive antagonist tetrapeptide LSKL is L-configured histidine-L-configured serine-L-configured lysine-L-configured histidine.

[0014] The present invention also provides the use of the polypeptide for enhancing NK cell immune killing and inhibiting HSCs activation in the preparation of a drug for alleviating liver fibrosis.

[0015] The present invention also provides a polypeptide that crosses vascular endothelium and selectively anchors to HSCs, and the polypeptide has the amino acid sequence of SEQ ID NO.2;

[0016] The structural formula of the polypeptide having the amino acid sequence of SEQ ID NO.2 is:

[0017] .

[0018] Furthermore, the polypeptide having the amino acid sequence of SEQ ID NO.2 is composed of a D-configuredD It consists of the GFFY amino acid sequence, cysteine, and the L-configuration VHPKQHR sequence.

[0019] Furthermore, the D GFFY amino acid sequence is D-glycine-D-phenylalanine-D-phenylalanine-D-tyrosine.

[0020] Furthermore, a near-infrared fluorescent group Cy5.5 is also connected to the sulfhydryl group of the cysteine.

[0021] Furthermore, the L-configuration VHPKQHR sequence is L-valine-L-histidine-L-proline-L-lysine-L-glutamic acid-L-histidine-L-arginine.

[0022] Furthermore, the present invention also provides the application of the polypeptide that crosses vascular endothelium and selectively anchors to HSCs in the preparation of a drug for alleviating liver fibrosis.

[0023] The present invention also provides a preparation method of a bioactive solution, and the preparation method includes: dispersing the polypeptide with the amino acid sequence of SEQ ID NO.1 and the polypeptide with the amino acid sequence of SEQ ID NO.2 in a PBS buffer solution, adjusting the pH value of the solution, heating, and then cooling to obtain a bioactive solution.

[0024] Furthermore, the pH value of the PBS buffer solution (phosphate buffer solution) is 5-9.

[0025] Furthermore, in the PBS buffer solution, the molar concentration ratio of the polypeptide with the amino acid sequence of SEQ ID NO.1 and the polypeptide with the amino acid sequence of SEQ ID NO.2 is 4:1.

[0026] Furthermore, the mass concentration of the polypeptide with the amino acid sequence of SEQ ID NO.1 in the PBS buffer solution is 3mg / 1.2mL - 3mg / 0.8mL.

[0027] Furthermore, the pH value of the adjusted solution is 6-8.

[0028] Furthermore, heat to a solution temperature of 90°C - 100°C.

[0029] Those skilled in the art should understand that heating is to promote the dissolution of the polypeptide in the solution, and the heating time ends when the polypeptide is completely dissolved.

[0030] Furthermore, cool to room temperature.

[0031] Furthermore, the room temperature is 26°C - 28°C.

[0032] The present invention also provides the application of the bioactive solution in the preparation of a drug for alleviating liver fibrosis.

[0033] The embodiments of the present invention have the following technical effects:

[0034] 1. First, the polypeptide with the amino acid sequence of SEQ ID NO.1 of the present invention can selectively induce NK cells, and can induce NK cells to approach the hepatic stellate cells HSCs related to liver fibrosis, 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 polypeptide with the amino acid sequence of SEQ ID NO.1 in the present invention is conducive to the polypeptide crossing the vascular endothelium in the inflammatory state and entering the liver parenchyma, and the polypeptide structure does not change. The design of the polypeptide structure is the key technical point for the polypeptide of the present invention to achieve the alleviation of liver fibrosis symptoms.

[0035] 2. The polypeptide with the amino acid sequence of SEQ ID NO.1 of the present invention has high biocompatibility and basically does not have other effects on normal cells and the body.

[0036] 3. The polypeptide with the amino acid sequence of SEQ ID NO.1 of the present invention can effectively slow down the development process of liver fibrosis and maintain and reverse the fibrosis-related phenotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 It is the high performance liquid chromatography (HPLC) chart of the polypeptide PepA-TT in Example 1.

[0039] Figure 2 It is the mass spectrometry chart of the polypeptide PepA-TT in Example 1.

[0040] Figure 3 It is for the polypeptide in Example 1 Cy5.5 The high performance liquid chromatography (HPLC) chart of PepB-V.

[0041] Figure 4 It is for the polypeptide in Example 1 Cy5.5 The mass spectrometry chart of PepB-V.

[0042] Figure 5Microscopic morphology diagrams after co-assembly of two polypeptides with different molar concentrations in the bioactive solution in the examples and comparative examples. Among them, Figure 5 a in [figure number] is the microscopic morphology diagram of Comparative Example 1, Figure 5 b in [figure number] is the microscopic morphology diagram of Comparative Example 2, Figure 5 c in [figure number] is the microscopic morphology diagram of Comparative Example 3, Figure 5 d in [figure number] is the microscopic morphology diagram of Example 1.

[0043] Figure 6 Microscopic morphology diagrams after self-assembly of polypeptide PepA-TT and polypeptide Cy5.5 PepB-V in Example 1. Among them, Figure 6 a in [figure number] is the microscopic morphology diagram after self-assembly of polypeptide PepA-TT, Figure 6 b in [figure number] is the microscopic morphology diagram after self-assembly of polypeptide Cy5.5 PepB-V.

[0044] Figure 7 Microscopic morphology diagrams after co-assembly of polypeptide PepA-TT and polypeptide Cy5.5 PepB-V at the concentrations used in the cell-level experiments in Example 1 (the concentration of PepA-TT is 50 μM, the molar concentration of PepA-TT: Cy5.5 the molar concentration of PepB-V = 4:1). Among them, Figure 7 a in [figure number] is the microscopic morphology diagram after co-assembly, Figure 7 b in [figure number] is Figure 7 a partial enlarged view of a in [figure number].

[0045] Figure 8 Polypeptide vascular endothelial crossing ability tests were carried out using the bioactive solutions of the examples and comparative examples. Among them Figure 8 a in [figure number] is the microscopic diagram of Comparative Example 4, Figure 8 b in [figure number] is the microscopic diagram of endocytosis in Example 1, Figure 8 c in [figure number] is the microscopic diagram of exocytosis in Example 1, Figure 8 d in [figure number] is Figure 8 a partial enlarged view of a in [figure number], Figure 8 e in [figure number] is Figure 8 a partial enlarged view of b in [figure number], Figure 8 f in [figure number] is Figure 8 a partial enlarged view of c in [figure number].

[0046] Figure 9 Diagrams of the binding constants of polypeptides with target proteins TIGIT and VCAM-1 measured by microscale thermophoresis. Among them Figure 9 a in [figure number] is the binding constant diagram of target protein TIGIT with polypeptide PepA-TT, Figure 9 b in [figure number] is the binding constant diagram of target protein TIGIT with polypeptide Cy5.5 PepB-V,Figure 9 In c, it is the binding constant graph of the target protein TIGIT and the bioactive solution of Example 1. Figure 9 In d, it is the binding constant graph of the target protein VCAM-1 and the polypeptide PepA-TT. Figure 9 In e, it is the target protein VCAM-1 and the polypeptide Cy5.5 PepB-V binding constant graph. Figure 9 In f, it is the binding constant graph of the target protein VCAM-1 and the bioactive solution of Example 1.

[0047] Figure 10 It is the number and fluorescence intensity of the red fluorescent spots of the indicator polypeptide in the cytoplasm after incubating HUVEC cells in Example 1 for different times. Among them, Figure 10 In a, it is the fluorescence spot of the fluorophore Cy5.5 at 0 h during endocytosis. Figure 10 In b, it is the fluorescence spot of the nuclear dye DAPI at 0 h during endocytosis. Figure 10 In c, it is the fluorescence spot of all fluorescence channels at 0 h during endocytosis. Figure 10 In d, it is the fluorescence spot of the fluorophore Cy5.5 at 0 h during exocytosis. Figure 10 In e, it is the fluorescence spot of the nuclear dye DAPI at 0 h during exocytosis. Figure 10 In f, it is the fluorescence spot of all fluorescence channels at 0 h during exocytosis. Figure 10 In g, it is the fluorescence spot of the fluorophore Cy5.5 at 4 h during endocytosis. Figure 10 In h, it is the fluorescence spot of the nuclear dye DAPI at 4 h during endocytosis. Figure 10 In i, it is the fluorescence spot of all fluorescence channels at 4 h during endocytosis. Figure 10 In j, it is the fluorescence spot of the fluorophore Cy5.5 at 4 h during exocytosis. Figure 10 In k, it is the fluorescence spot of the nuclear dye DAPI at 4 h during exocytosis. Figure 10 In l, it is the fluorescence spot of all fluorescence channels at 4 h during exocytosis. Figure 10 In m, it is the fluorescence spot of the fluorophore Cy5.5 at 8 h during endocytosis. Figure 10 In n, it is the fluorescence spot of the nuclear dye DAPI at 8 h during endocytosis. Figure 10 In o, it is the fluorescence spot of all fluorescence channels at 8 h during endocytosis. Figure 10 In p, it is the fluorescence spot of the fluorophore Cy5.5 at 8 h during exocytosis. Figure 10 In q, it is the fluorescence spot of the nuclear dye DAPI at 8 h during exocytosis. Figure 10 In r, it is the fluorescence spot of all fluorescence channels at 8 h during exocytosis. Figure 10 In c1, it is Figure 10 The partial enlarged view of c. Figure 10 In f1, it isFigure 10 Partial enlarged view of f in Figure 10 where i1 in Figure 10 Partial enlarged view of i in Figure 10 where l1 in Figure 10 Partial enlarged view of l in Figure 10 where o1 in Figure 10 Partial enlarged view of o in Figure 10 where r1 in Figure 10 Partial enlarged view of r in

[0048] Figure 11 Fluorescent dots of NK cells and HSCs cells treated with the bioactive solutions prepared in Example 1 and Comparative Example 4 at different culture times, where Figure 11 a in is the fluorescent dot of the fluorophore Cy5.5 at 1 h of culture in Example 1, Figure 11 b in is the fluorescent dot of the nuclear dye DAPI at 1 h of culture in Example 1, Figure 11 c in is the fluorescent dot of the live cell dye CFSE pre-stained with NK at 1 h of culture in Example 1, Figure 11 d in is the fluorescent dot of all fluorescence channels at 1 h of culture in Example 1, Figure 11 e in is the fluorescent dot of the fluorophore Cy5.5 at 2 h of culture in Example 1, Figure 11 f in is the fluorescent dot of the nuclear dye DAPI at 2 h of culture in Example 1, Figure 11 g in is the fluorescent dot of the live cell dye CFSE pre-stained with NK at 2 h of culture in Example 1, Figure 11 h in is the fluorescent dot of all fluorescence channels at 2 h of culture in Example 1, Figure 11 i in is the fluorescent dot of the fluorophore Cy5.5 at 4 h of culture in Example 1, Figure 11 j in is the fluorescent dot of the nuclear dye DAPI at 4 h of culture in Example 1, Figure 11 k in is the fluorescent dot of the live cell dye CFSE pre-stained with NK at 4 h of culture in Example 1, Figure 11 l in is the fluorescent dot of all fluorescence channels at 4 h of culture in Example 1, Figure 11 m in is the fluorescent dot of the fluorophore Cy5.5 at 4 h of culture in Comparative Example 4, Figure 11 n in is the fluorescent dot of the nuclear dye DAPI at 4 h of culture in Comparative Example 4, Figure 11 o in is the fluorescent dot of the live cell dye CFSE pre-stained with NK at 4 h of culture in Comparative Example 4, Figure 11 p in is the fluorescent dot of all fluorescence channels at 4 h of culture in Comparative Example 4.

[0049] Figure 12 Are the test results of immunoblotting assay, where, "+" represents addition and "-" represents non-addition.

[0050] Figure 13 Effects of the examples and comparative examples on liver indices, where Figure 13 a in it is the content of alanine aminotransferase, Figure 13 b in it is the content of aspartate aminotransferase, Figure 13 c in it is the content of blood urea nitrogen, Figure 13 d in it is the content of total bilirubin. Specific implementation manners

[0051] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope protected by the present invention.

[0052] In a first aspect, in some embodiments of the present invention, a polypeptide for enhancing NK cell immune killing and inhibiting HSCs activation is provided, and the polypeptide has the amino acid sequence of SEQ ID NO.1;

[0053] The structural formula of the polypeptide having the amino acid sequence of SEQ ID NO.1 is:

[0054] .

[0055] In the present invention, the polypeptide having the amino acid sequence of SEQ ID NO.1 approaches hepatic stellate cells HSCs related to liver fibrosis by inducing NK cells, enhances NK immune killing activity while inhibiting the activation and abnormal proliferation of HSCs, thereby achieving the alleviation of liver fibrosis symptoms.

[0056] In some embodiments, the polypeptide having the amino acid sequence of SEQ ID NO.1 consists of the D GFFY amino acid sequence in D configuration, the D TBP-3 targeting sequence in D configuration, the competitive antagonist tetrapeptide LSKL in L configuration, and a capping group.

[0057] In some embodiments, the D GFFY amino acid sequence in D configuration is D-configured glycine-D-configured phenylalanine-D-configured phenylalanine-D-configured tyrosine.

[0058] In some embodiments, the capping group is 2-naphthoic acid (Nap).

[0059] In some embodiments, the DThe TBP-3 targeting sequence is D-configuration tyrosine-D-configuration cystine-D-configuration phenylalanine-D-configuration histidine-D-configuration tryptophan-D-configuration histidine-D-configuration arginine-D-configuration leucine-D-configuration aspartic acid-D-configuration proline.

[0060] In some embodiments, the L-configuration competitive antagonist tetrapeptide LSKL is L-configuration histidine-L-configuration serine-L-configuration lysine-L-configuration histidine.

[0061] In a second aspect, in some embodiments of the present invention, there is also provided an application of the polypeptide for enhancing NK cell immune killing and inhibiting HSCs activation in the preparation of a drug for alleviating liver fibrosis.

[0062] In a third aspect, in some embodiments of the present invention, there is also provided a polypeptide that crosses vascular endothelial cells and selectively anchors to HSCs, and the polypeptide has the amino acid sequence of SEQ ID NO.2;

[0063] The structural formula of the polypeptide having the amino acid sequence of SEQ ID NO.2 is:

[0064] .

[0065] In the present invention, the polypeptide having the amino acid sequence of SEQ ID NO.2 can selectively connect with hepatic stellate cells HSCs, and is beneficial for NK cells to approach the hepatic stellate cells HSCs related to liver fibrosis, enhancing the NK immune killing activity while inhibiting the activation and abnormal proliferation of HSCs, thereby achieving the alleviation of liver fibrosis symptoms.

[0066] In some embodiments, the polypeptide having the amino acid sequence of SEQ ID NO.2 consists of the D GFFY amino acid sequence in D-configuration, cysteine, and the VHPKQHR sequence in L-configuration.

[0067] In the present invention, the D GFFY amino acid sequence in D-configuration and the VHPKQHR sequence in L-configuration are connected by cysteine.

[0068] In some embodiments, the D GFFY amino acid sequence in D-configuration is D-configuration glycine-D-configuration phenylalanine-D-configuration phenylalanine-D-configuration tyrosine.

[0069] In some embodiments, a near-infrared fluorescent group Cy5.5 is further connected to the sulfhydryl group of cysteine.

[0070] In some embodiments, the VHPKQHR sequence in the L-configuration is L-valine-L-histidine-L-proline-L-lysine-L-glutamic acid-L-histidine-L-arginine.

[0071] Fourthly, in some embodiments of the present invention, there is also provided an application of the polypeptide that crosses vascular endothelium and selectively anchors to HSCs in the preparation of a drug for alleviating liver fibrosis.

[0072] Fifthly, in some embodiments of the present invention, there is also provided a preparation method of a bioactive solution, and the preparation method includes: dispersing the polypeptide with the amino acid sequence of SEQ ID NO.1 and the polypeptide with the amino acid sequence of SEQ ID NO.2 in a PBS buffer solution, adjusting the pH value of the solution, heating, and then cooling to obtain a bioactive solution.

[0073] 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 to approach the hepatic stellate cells HSCs related to liver fibrosis, enhance the NK immune killing activity while inhibiting the activation and abnormal proliferation of HSCs, so as to alleviate the symptoms of liver fibrosis.

[0074] In some embodiments, the pH value of the PBS buffer solution (phosphate buffer solution) is 5-9.

[0075] In some embodiments, in the PBS buffer solution, the molar concentration ratio of the polypeptide with the amino acid sequence of SEQ ID NO.1 to the polypeptide with the amino acid sequence of SEQ ID NO.2 is 4:1.

[0076] In some embodiments, the mass concentration of the polypeptide with the amino acid sequence of SEQ ID NO.1 in the PBS buffer solution is 3mg / 1.2mL - 3mg / 0.8mL.

[0077] In some embodiments, the pH value of the adjusted solution is 6-8.

[0078] In some embodiments, heat to a solution temperature of 90°C - 100°C.

[0079] In some embodiments, cool to room temperature.

[0080] In some embodiments, the room temperature is 26°C - 28°C.

[0081] Sixthly, in some embodiments of the present invention, there is also provided an application of the bioactive solution in the preparation of a drug for alleviating liver fibrosis.

[0082] The following is elaborated with specific embodiments:

[0083] Example 1:

[0084] Method for preparing 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

[0085] Synthesized by Fmoc-solid phase synthesis method for short peptides. The specific steps are as follows:

[0086] (1) Weigh 0.5 mmol of 2-Cl-Trt resin into a solid-phase synthesizer, add 10 mL of anhydrous dichloromethane (DCM), place it on a shaker and shake for 10 min to fully swell the 2-Cl-Trt resin;

[0087] (2) Use an ear bulb to press out the DCM from the solid-phase synthesizer containing 2-Cl-Trt resin until it is completely removed;

[0088] (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 it to the above solid-phase synthesizer and react at room temperature for 1 h;

[0089] (4) Capping: Use an ear bulb to remove the reaction solution in the solid-phase synthesizer, then wash with 10 mL of anhydrous DCM, 1 min each time, for a total of 5 times. Add 20 mL of a solution prepared with a volume ratio of anhydrous DCM:DIEPA:methanol of 17:1:2 and react at room temperature for 20 min;

[0090] (5) Use an ear bulb to remove the reaction solution in the solid-phase synthesizer. First wash with anhydrous DCM, 10 mL of DCM each time, for 1 min each time, for a total of 5 times. Then wash with DMF (dimethylformamide), 10 mL of DMF each time, for 1 min each time, for a total of 5 times. Add 10 mL of a mixed solution of piperidine and DMF, where the volume ratio of piperidine to DMF is 2:8, react for 25 min, and then wash with DMF, 10 mL of DMF each time and 1 min for each wash, for a total of 5 times, and proceed to the next reaction;

[0091] (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;

[0092] (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;

[0093] (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.

[0094] (9) The purified tetrapeptide LSKL was connected to the amino group of lysine in the main chain by liquid phase synthesis. 1mmol of 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 continued 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 mixture was separated and purified by HPLC. A polypeptide having an amino acid sequence of SEQ ID NO.1 was obtained, which was denoted as PepA-TT and detected by high performance liquid chromatography-mass spectrometry. The results are shown in FIG. Figures 1 - 2 ;

[0095] 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.

[0096] (1) Weigh 0.5 mmol of 2-Cl-Trt resin into a solid-phase synthesizer, add 10 mL of anhydrous dichloromethane (DCM), place it on a shaker and shake for 10 min to fully swell the 2-Cl-Trt resin;

[0097] (2) Use an ear bulb to press out the DCM from the solid-phase synthesizer containing 2-Cl-Trt resin until it is completely removed;

[0098] (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 it to the above solid-phase synthesizer and react at room temperature for 1 h;

[0099] (4) Capping: Use an ear bulb to remove the reaction solution in the solid-phase synthesizer, then wash it with 10 mL of anhydrous DCM for 1 min each time, for a total of 5 times. Add 20 mL of a prepared solution with a volume ratio of anhydrous DCM:DIEPA:methanol of 17:1:2 and react at room temperature for 20 min;

[0100] (5) Use an ear bulb to remove the reaction solution in the solid-phase synthesizer. First, wash it with anhydrous DCM, with 10 mL of DCM used each time and a washing time of 1 min, for a total of 5 times. Then wash it with DMF (dimethylformamide), with 10 mL of DMF used each time and a washing time of 1 min, for a total of 5 times. Add 10 mL of a mixed solution of piperidine and DMF, where the volume ratio of piperidine to DMF is 2:8, and react for 25 min. Then wash it with DMF, with 10 mL of DMF used each time and a washing time of 1 min, for a total of 5 times, and proceed to the next reaction;

[0101] (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;

[0102] (7) Repeat the methods of steps (5) and (6) to sequentially add L-configured and D-configured amino acids and capping groups (2-naphthoic acid) according to the main-chain amino acid sequence of SEQ ID NO.2; then wash it 5 times with DMF and 5 times with dichloromethane, and proceed to the next step;

[0103] (8) Add 10 mL of a solution composed of 95% TFA (trifluoroacetic acid), 2.5% TIS (triisopropylsilane), and 2.5% H2O (water) by volume percentage to the above solid-phase synthesizer, react for 0.5 h, cleave the product from the 2-Cl-Trt resin, concentrate it under vacuum to remove the solvent to obtain the crude product, and then separate and purify it by HPLC; those skilled in the art can also complete it by preparing a TFA solution with a volume percentage concentration of 1% of TFA by mixing TFA and DCM at a volume ratio of 1:99, taking 3 mL of this TFA solution each time and adding it to the above solid-phase synthesizer for a total of ten times, with a reaction time of 1 min each time.

[0104] (9) Next, add the pure main chain product and Cy5.5-MAL (Cy5.5-maleimide) in a mass ratio of 1:1 to the system and vortex for 30 seconds. Adjust the pH to 7.5 using sodium carbonate (0.01 M) and leave it overnight at 4 °C. Finally, obtain the polypeptide Cy5.5 PepB-V. Detect the polypeptide with the amino acid sequence of SEQ ID NO.2 by high-performance liquid chromatography-mass spectrometry, and the results are shown in Figures 3 - 4 ;

[0105] Add 3 mg of PepA-TT polypeptide, Cy5.5 0.75 mg of PepB-V polypeptide, add 1 mL of PBS buffer solution (pH = 7.4) to the system. Among them, in the PBS buffer solution, the molar concentration ratio of PepA-TT polypeptide to Cy5.5 PepB-V polypeptide is 4:1. Adjust its pH value to 7.4 using sodium carbonate solution, and vortex for 3 - 5 seconds to completely dissolve it to obtain the bioactive solution before assembly. Then carry out a heating-cooling reaction, heat the system to boiling, and after 3 - 5 s, let it cool to room temperature to obtain the bioactive solution after co-assembly.

[0106] Comparative Example 1:

[0107] The preparation method is the same as that of Example 1, but the added masses of PepA-TT polypeptide and Cy5.5 PepB-V polypeptide are different. Among them, in the PBS buffer solution, the molar concentration ratio of PepA-TT polypeptide to Cy5.5 PepB-V polypeptide is 1:1.

[0108] Comparative Example 2:

[0109] The preparation method is the same as that of the Example, but the added masses of PepA-TT polypeptide and Cy5.5 PepB-V polypeptide are different. Among them, in the PBS buffer solution, the molar concentration ratio of PepA-TT polypeptide to Cy5.5The molar concentration ratio of the PepB-V polypeptide is 2:1.

[0110] Comparative Example 3:

[0111] The preparation method is the same as that of the example, but the added mass of the PepA-TT polypeptide and Cy5.5 the PepB-V polypeptide is different. Among them, in the PBS buffer solution, the molar concentration ratio of the PepA-TT polypeptide to Cy5.5 the PepB-V polypeptide is 3:1.

[0112] Comparative Example 4:

[0113] Weigh 8 g of NaCl (sodium chloride), 0.2 g of KCl (potassium chloride), 1.44 g of Na2HPO4 (disodium hydrogen phosphate), and 0.24 g of KH2PO4 (potassium dihydrogen phosphate), dissolve them in 800 mL of distilled water, adjust the pH value of the solution to 7.4 with HCl (hydrochloric acid), and finally make up the volume to 1 L with distilled water to obtain the PBS solution. After sterilizing with an autoclave, store it in a refrigerator at 4°C.

[0114] The bioactive solutions obtained from the examples and comparative examples were tested experimentally:

[0115] Results and analysis:

[0116] Weigh 3 mg of the polypeptide PepA-TT prepared in Example 1 and the polypeptide Cy5.5 PepB-V with different molar concentration ratios to it. 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 to 5 seconds to completely dissolve it to obtain the bioactive solution before assembly. Then carry out the heating-cooling reaction, heat the system to boiling, and place it at room temperature to cool after 3 to 5 seconds to obtain the bioactive solution after co-assembly. The bioactive solutions obtained from Example 1 and Comparative Examples 1-3 were observed for their microscopic morphology after assembly with a transmission electron microscope, and the results are shown in Figure 5 . Figure 5 It shows that the polypeptides PepA-TT and Cy5.5 PepB-V with different molar concentration ratios will present completely different microscopic structures after co-assembly. As the molar concentration of the PepA-TT polypeptide in the system gradually increases, the nanostructures presented after co-assembly gradually change from the state where nanofibers and nanoparticles exist relatively independently to a more ordered higher-level structure. When the polypeptides PepA-TT and Cy5.5When the molar concentration ratio of PepB-V is 4:1, the co-assembly presents a nanofiber network similar to a blood vessel network and is loaded with fine nanoparticles, indicating that the system reaches a more stable assembly state at this ratio. Therefore, further preferably, the concentration of polypeptide PepA-TT in PBS buffer and the Cy5.5 molar concentration of polypeptide PepB-V in PBS buffer have a ratio of 4:1.

[0117] Weigh 3 mg of polypeptide PepA-TT prepared in Example 1 and the polypeptide Cy5.5 PepB-V 0.6 mg prepared in Example 1 respectively. Add 1 mL of 1×PBS buffer (pH = 7.4) solution to each, adjust its pH value to 7.4 with sodium carbonate solution, and ultrasonically dissolve it to obtain a PepA-TT solution with a concentration of 1096 μM and a Cy5.5 PepB-V solution with a concentration of 274 μM, so that it meets the concentration for biological transmission electron microscopy experiments. Then, perform a heating-cooling reaction. Heat the system to boiling, and after 3 seconds - 5 seconds, place it at room temperature to cool, that is, obtain the assembled PepA-TT bioactive solution and the assembled peptide Cy5.5 PepB-V bioactive solution. Observe the microscopic morphology of the above-mentioned assembled polypeptide PepA-TT and polypeptide Cy5.5 PepB-V bioactive solution with a transmission electron microscope. The results are as Figure 6 shown. Figure 6 It shows that the nanostructure presented by the assembled polypeptide PepA-TT is a network formed by entangled slender nanofibers, while the assembled polypeptide Cy5.5 PepB-V forms nanoparticles with different diameters.

[0118] Next, weigh 0.7 mg of polypeptide PepA-TT prepared in Example 1 and the polypeptide Cy5.5 PepB-V 0.15 mg prepared in Example 1. Add 5 mL of 1×PBS buffer (pH = 7.4) solution to the system, adjust its pH value to 7.4 with sodium carbonate solution, and ultrasonically dissolve it to obtain a solution with a concentration of 50 μM of polypeptide PepA-TT (PepA-TT molar concentration: Cy5.5 PepB-V molar concentration = 4:1). After the heating-cooling reaction, obtain the bioactive solution of the co-assembly system. Observe the microscopic morphology of the above-mentioned co-assembled bioactive solution with a transmission electron microscope. The results are as Figure 7 shown. Figure 7 It shows that in the bioactive solution at a concentration of 50 μM (PepA-TT molar concentration: Cy5.5The nanostructure presented at a PepB-V molar concentration of 4:1) is still a vascular nanofiber network, and individual fibers are longitudinally arranged and aggregated into bundles. Those skilled in the art should understand that when the polypeptide concentration in the experiments at the cellular level is changed, the polypeptides in the prior art will undergo structural changes due to poor structural stability. However, in the polypeptide structure of the present invention, in the experiments at the cellular level, the polypeptides in the bioactive solution at the selected drug addition concentration still have highly ordered nanostructures. It can be seen that the polypeptide structure of the present invention is stable and can be verified from Figure 7 it.

[0119] To explore the ability of the polypeptide of the present invention to cross the vascular endothelium, we conducted a biological transmission electron microscopy experiment. Human umbilical vein endothelial HUVEC cells were seeded in a 10-cm diameter culture dish at a density of 1×10 6 cells / dish. After the cells adhered, the culture medium in the dish was removed. The co-assembly system of Example 1, that is, a bioactive solution with a polypeptide PepA-TT concentration of 50 μM (PepA-TT molar concentration: Cy5.5 PepB-V molar concentration = 4:1), was added to the system, and the PBS buffer solution of Comparative Example 4 was used as a blank control. After incubation for 8 hours, the culture medium in the dish was replaced with fresh drug-free culture medium and incubated for another 6 hours. The HUVEC cells after incubation for 8 hours and after replacing the culture medium and incubating for another 6 hours were respectively collected into 1.5-mL EP tubes, and cell pellets were obtained by centrifugation. The cell pellets were gently scratched into small cell clusters with a needle tip, and a fixing solution (2.5% glutaraldehyde solution) was slowly added along the tube wall and fixed overnight at 4°C. The fixing solution in the tube was removed by centrifugation again, and the sample was rinsed 3 times with PBS buffer solution at pH 7.0 for 15 minutes each time. The sample was fixed with 1% osmium tetroxide solution for 1-2 hours. Then, the osmium tetroxide waste solution was carefully removed, and the sample was rinsed 3 times with PBS buffer solution at pH 7.0 for 15 minutes each time. The sample was dehydrated with ethanol solutions of gradient concentrations (including six concentrations of 30%, 50%, 70%, 80%, 90% and 95%), each concentration was treated for 15 minutes, then treated with 100% ethanol for 20 minutes, and finally transitioned to pure acetone treatment for 20 minutes. The sample was treated with a mixture of Spurr embedding agent and acetone (V / V = 1 / 1) for 1 hour, treated with a mixture of Spurr embedding agent and acetone (V / V = 3 / 1) for 3 hours, and treated with pure embedding agent overnight. The infiltrated sample was embedded and heated at 70°C overnight to obtain the embedded sample. The sample was sectioned in an ultramicrotome to obtain sections of 70 nm - 90 nm. The sections were stained with lead citrate solution, uranyl acetate and 50% ethanol saturated solution for 5 - 10 minutes each, and then could be observed in a Hitachi Hitachi-7800 transmission electron microscope. The results are as Figure 8 shown.

[0120] Figure 8It is shown that a large number of nanofiber structures can be seen in the cytoplasm of HUVEC cells after endocytosis. After exocytosis, the formation of secretory vesicles on the cell membrane and the obvious reduction and excretion of fine fibers in the cytoplasm can be observed. From the above results, it can be seen that endocytosis and exocytosis of vascular endothelial cells do not affect the morphology of the co-assembly system in Example 1, and the complete crossing of vascular endothelium can be achieved.

[0121] 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. Then, it was mixed and pipetted evenly with 100 μL of 10 μM rhTIGIT protein and rhVCAM-1 protein (buffer: 1×PBS pH 7.4, Tween-20 (0.05%)), and the mixture was placed in the dark for 30 minutes for reaction. After the reaction was completed, the excess dye was completely washed away through a molecular sieve, and a quantitative buffer was added to make the solution flow out under the action of gravity, and the fluorescently labeled protein was collected. According to the method for preparing the bioactive solution in Example 1, the two polypeptides prepared in Example 1 and the co-assembly system in Example 1 (PepA-TT molar concentration: Cy5.5 PepB-V molar concentration = 4:1) were prepared into bioactive solutions with different concentrations. Bioactive solutions of polypeptides 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 solutions were aspirated with a capillary and loaded onto the machine for detection. Finally, the binding ability of different polypeptides to PD-L1 protein was measured by a microscale thermophoresis instrument (Monolith NT.115), and the results are as Figure 9 shown.

[0122] Figure 9 Among them, different polypeptides have different binding constants for TIGIT protein and VCAM-1 protein. The polypeptides PepA-TT and Cy5.5 PepB-V obtained in Example 1 and the bioactive solution (PepA-TT molar concentration: Cy5.5 PepB-V molar concentration = 4:1) were respectively mixed with TIGIT protein and VCAM-1 protein, and the protein binding ability was measured by a microscale thermophoresis instrument. When the K D value is smaller, it represents stronger affinity. For the binding ability to TIGIT protein, the polypeptide PepA-TT in the present invention has a strong ability due to the presence of the TIGIT affinity sequence. The K D value of the polypeptide PepA-TT is 2.19 μM, compared with the polypeptide Cy5.5 PepB-V lacking the TIGIT affinity sequence, the KD Compared with the value of 44.8 μM, the affinity for TIGIT protein was increased by 19.5 times. Similarly, for the binding ability to VCAM-1, the polypeptide in the present invention Cy5.5 PepB-V has a strong ability, and the polypeptide PepA-TT K D reached a value of 8.72 μM, which is 5.8 times that of the polypeptide PepA-TT prepared in Example 1. In addition, the bioactive solution in the present invention has good binding abilities to both TIGIT protein and VCAM-1 protein. It can be seen that the co-assembly system in Example 1 (molar concentration of PepA-TT: Cy5.5 molar concentration of PepB-V = 4:1) has good binding abilities to the two target proteins, laying a foundation for its cell selectivity and functional specificity.

[0123] At a density of 5×10 4 human umbilical vein endothelial cells HUVEC per well were seeded on a 24-well plate with a round cell slide. After the cells adhered to the surface of the cell slide, the medium in the wells was removed. Bioactive solutions with a polypeptide PepA-TT concentration of 50 μM (molar concentration of PepA-TT: Cy5.5 molar concentration of PepB-V = 4:1) were prepared in the wells according to the method for preparing the bioactive solution in Example 1, using the PBS solution in Comparative Example 1 as a blank control. Incubate at different time points to observe the endocytosis of HUVEC, and then replace the medium in the wells with fresh drug-free medium and continue to incubate at different time points to explore the exocytosis of HUVEC, so as to verify the vascular endothelial crossing ability of the polypeptide described in the present invention. After the above steps were completed, 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 0.5 μg / mL DAPI (4',6-diamidino-2-phenylindole) at room temperature for 4 minutes. All the above operations need to be carried out in the dark. After staining, images were taken at the same voltage by a laser scanning confocal microscope (Carl Zeiss LSM 900) to detect the vascular endothelial crossing of the polypeptide, as Figure 10 shown.

[0124] From Figure 10It can be seen that after incubating HUVEC cells with the bioactive solution prepared in Example 1 for different times, as the incubation time progresses, the number and fluorescence intensity of the red fluorescent spots indicating the polypeptide in the cytoplasm gradually increase and are distributed closely around the cell nucleus. The above results indicate 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 medium in the wells with fresh medium, the red fluorescent spots in the cytoplasm gradually decrease as exocytosis proceeds, and the small amount of fluorescent spots remaining in the cytoplasm are also distributed away from the cell nucleus. The above results show that the polypeptide in Example 1 enters vascular endothelial cells by endocytosis and is further transported extracellularly by receptor-mediated exocytosis, creating conditions for further enhancing the interaction between effector cells.

[0125] Next, human hepatic stellate cells LX-2 were seeded in 24-well plates at a density of 5×10 4 cells / well. After the cells adhered, the medium in the wells was removed. Bioactive solutions with a concentration of 50 μM of the polypeptide PepA-TT prepared in Example 1 (molar concentration of PepA-TT: Cy5.5 molar concentration of PepB-V = 4:1) were added to the wells respectively, using the PBS solution of Comparative Example 1 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 added to each well for co-culture according to the cell ratio of NK:HSCs = 5:1. After incubating the cells at different time points, the solution in the wells was aspirated, and the cells were washed 3 times with PBS buffer, 1 minute each time. At room temperature, the cells attached to the coverslips were fixed with 4% paraformaldehyde in PBS buffer for 10 minutes. Then, the solution in the wells was aspirated, the cells were washed 3 times with PBS buffer, 1 minute each time, and then stained with 0.5 μg / mL DAPI at room temperature for 4 minutes. All operations needed to be carried out in the dark. CFSE (carboxyfluorescein succinimidyl ester) is a live cell dye. After staining, images were taken at the same voltage by a laser scanning confocal microscope (Carl Zeiss LSM 900) to detect the effect of the polypeptide on the adhesion ability of NK cells, as Figure 11 shown.

[0126] From Figure 11It can be seen that when the NK cells co-cultured with HSCs were treated with the bioactive solution prepared in Example 1 at different time points, the number of pre-stained green fluorescent NK cells adhered to the surface of LX-2 cells gradually increased. Taking the PBS buffer group of Comparative Example 1 as a blank control, after incubating for 4 hours in the same way, Example 1 could significantly improve the adhesion ability of NK cells to HSCs.

[0127] The effect of the polypeptide of the present invention on the liver fibrosis-related phenotypes of HSCs was detected by immunoblotting assay. LX-2 cells were seeded on a cell culture dish with a diameter of 10 mm. After culturing for 24 hours until the cells adhered and grew to about 80% density, PepA-TT prepared by the method of adding the polypeptide bioactive solution prepared according to Example 1 was added. Cy5.5 PepB-V and the co-assembled polypeptide bioactive solution were used, and the PBS solution described in Comparative Example 1 was used as a blank control. The concentration of PepA-TT was 50 μM. Cy5.5 The concentration of PepB-V was 12.5 μM. The cells were incubated for 24 hours, and the culture medium was removed. Then the cells were washed 3 times with PBS buffer. The cells were collected and lysed in a cell lysis buffer containing protease inhibitors. After quantitative analysis by BCA, equal amounts (30 μg) of samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% BSA (bovine serum albumin) to eliminate non-specific interference. The membrane was incubated with the corresponding primary antibody overnight at 4 °C, and then co-incubated with the HRP (horseradish peroxidase)-labeled secondary antibody of ImmunoWay for 1.5 hours. Finally, the membrane was detected by Tanon 5500 using Immobilon Western HRP substrate to detect the ability of the polypeptide to reverse the TGF-β1-induced liver fibrosis-related phenotypes of LX-2. GAPDH was used as an internal reference protein, and Collegen III, Desmin, and α-SMA were fibrosis-related indicators. The results are as Figure 12 shown (where "+" represents addition and "-" represents non-addition).

[0128] From Figure 12 it can be seen that taking the PBS group as a blank control group, under the condition that the internal reference protein bands in each group were basically consistent, after incubating the individual polypeptide PepA-TT, polypeptide Cy5.5 PepB-V and Example 1 for 24 hours, the liver fibrosis-related phenotypes of LX-2 cells were significantly reduced, and the fibrosis degree induced by TGF-β1 could be effectively reversed, indicating that the polypeptide PepA-TT, polypeptide Cy5.5 PepB-V and Example 1 had a significant in vitro inhibitory effect on the fibrosis of LX-2 cells, providing a basis for further in vivo experiments.

[0129] We used a CCL4 (carbon tetrachloride)-induced mouse liver fibrosis model to explore the application of the polypeptide described in the present invention in anti-liver fibrosis in vivo. CCL4 was dissolved in mineral oil to prepare an injection solution with a final concentration of 25%, and it was administered by intraperitoneal injection at a dose of 2 mL / kg, twice a week for four weeks. The control group of mice was intraperitoneally injected with the same amount and frequency of mineral oil and fed with normal feed. The drug was administered by tail vein injection, and the administration concentration was selected as 10 mg / kg, once on the 1st, 4th, and 7th days, for a total of three administrations. Serum of each group of mice was collected 24 hours after the last administration for biochemical analysis of liver injury-related indicators, such as Figure 13 shown.

[0130] The main test indicators include indicators representing liver parenchymal injury: aspartate aminotransferase (AST) and alanine aminotransferase (ALT), important indicators reflecting liver function: total bilirubin (TBIL), and indicators evaluating kidney performance: blood urea nitrogen (BUN). It can be seen from Figure 13 that after three administrations, the liver injury-related indicators of the mice treated by tail vein injection with polypeptide PepA-TT, Cy5.5 PepB-V, and the co-assembled system of Example 1 were reversed to varying degrees compared with those of the model group mice.

[0131] Therefore, the polypeptide provided by the present invention can cross the vascular endothelium and enhance cell-cell interaction, and inhibit the proliferation and activation of HSCs from two aspects of direct contact and immune killing by enhancing the adhesion and killing of NK cells, thus slowing down the process of liver fibrosis.

[0132] It should be noted that the terms used in the present invention are only for describing specific embodiments and do not limit the scope of the present application. As shown in the specification of the present invention, unless the context clearly indicates an exception, words such as "a", "an", "one", and / or "the" are not specifically singular and may also include the plural. The term "comprising", "including", or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or also includes elements inherent to such a process, method, or device. Without further limitation, an element defined by the statement "comprising one..." does not exclude the existence of additional identical elements in the process, method, or device comprising the element.

[0133] It should also be noted that the orientation or positional relationship indicated by terms such as "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is 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. Therefore, it should not be construed as a limitation to the present invention. Unless otherwise clearly specified and defined, terms such as "installed", "connected", "coupled" 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, an electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, and it can be the communication inside 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.

[0134] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide for enhancing NK cell immune killing and inhibiting HSCs activation, characterized in that: The polypeptide has an amino acid sequence of SEQ ID NO.1; The structural formula of the polypeptide having the amino acid sequence of SEQ ID NO.1 is: 。 2. Use of a polypeptide for enhancing NK cell immune killing and inhibiting HSCs activation as claimed in claim 1 in the preparation of a drug for alleviating liver fibrosis.

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