WO2021068884A1 - Polypeptide derivative, nanofiber and application thereof - Google Patents

Abstract

Provided is a polypeptide derivative having a sequence of X-Phe-Phe-Gly-Ser-Ser-Ser-Arg, wherein the end group X is Nap or Npx, The Phes are in L configuration or D configuration simultaneously, and the rest are in L configuration, the polypeptide derivative can bind with an IGF-1 protein receptor and is used for treating muscle atrophy and atherosclerosis. The water mixture of the polypeptide derivative can form a nanofiber by heating and cooling method.

Description

Polypeptide derivatives, nanofibers and their applications Technical field

The invention relates to a polypeptide derivative, nanofiber and application thereof.

Background technique

Insulin-like growth factor I (IGF-1) is a growth hormone that plays a vital role in human development, growth, metabolism and homeostasis. It has been extensively studied in clinical and life science research fields.

Atherosclerosis is an important cause of cardiovascular diseases such as myocardial infarction, coronary heart disease, hypertension, and stroke, and has become one of the main diseases that endanger human health. According to the latest statistics from the World Health Organization WHO, the number of deaths from cardiovascular and cerebrovascular diseases accounts for 48% of the total number of non-communicable diseases, which is more than twice the number of deaths from cancer. In most observational studies, low circulating levels of IGF-1 are associated with increased atherosclerotic complications. In a prospective study on the effects of IGF-1 levels, low circulating IGF-1 levels were associated with an increased risk of ischemic heart disease during the 15-year follow-up of patients. Among the participants in the Rotterdam elderly study, high free IGF-1 is associated with the reduction of carotid plaque and coronary artery disease, because free IGF-1 can easily pass through endothelial cells and bind to its own receptors. And then regulate blood lipid balance. Therefore, restoring the physiological level of IGF-1 by supplementing exogenous IGF-1 is a promising method to play an anti-atherosclerotic effect.

Muscle atrophy is a common chronic disease of middle-aged and elderly people. The main manifestations are muscle strength loss and muscle mass decline. It is a pathology of excessive muscle protein degradation caused by genetic factors, long-term overdose of hormone drugs, and body aging. status. Muscle wasting is a serious consequence of many chronic diseases and the aging process itself, because it directly leads to physical weakness, loss of independence and increased risk of death. Therefore, there is an increasing demand for the development of specialized drugs for the treatment of muscle atrophy, which has become an important research topic worldwide. However, so far, in addition to exercise programs, no effective drugs and treatment programs have been developed to treat this disease. However, the use of exercise programs has been severely restricted in the elderly, bedridden people, or people suffering from acute illnesses. In addition, the existing methods of treating muscle atrophy usually rely on increasing nutrition, adding appetite stimulants or anabolic compounds (such as testosterone) to increase muscle mass. However, the effects of these treatments are not satisfactory and may cause side effects. As an agonist of the insulin signaling pathway, IGF-1 plays an important role in maintaining muscle mass. Supplementing some extra IGF-1 in vitro can promote the synthesis of skeletal muscle protein and effectively prevent the occurrence of skeletal muscle reduction.

The current clinical application or commercial IGF-1 protein is a natural or recombinant protein, which has problems such as high production cost, difficult quality assurance, and harsh transportation and storage conditions. At the same time, due to the low retention rate of the protein itself in the tissues, it is directly transmitted to the damaged organs. IGF-1 injection is not effective.

Chinese patent document CN109957000A discloses a polypeptide derivative with a sequence of Nap-FFGGYGSSSRRAPQT, which is used to simulate the biological activity of IGF-1 protein and can be used as a substitute for IGF-1 protein. The production process adopts the FMOC-solid phase synthesis method, and the product has a clear chemical structure, high yield, significantly lower cost than natural or recombinant protein, and is easy to store and transport. Since such polypeptide organisms can self-assemble in an aqueous solution to form nanofibers, and finally form a hydrogel, it can increase the retention rate in the body and further improve the biological activity without the help of a carrier. However, its polypeptide chain length is 16 peptides, the synthesis process is more complicated and the yield is low; the most important thing is that the self-assembled hydrogel is a white opaque gel, poor water solubility, and a small amount of precipitation after 3 to 5 days. Conducive to long-term preservation.

In the process of realizing the present invention, the inventors discovered that the prior art has at least the following problems: Provide a new polypeptide derivative that can effectively mimic the biological activity of the IGF-1 protein as a substitute for the IGF-1 protein , And the production process is relatively simple, the yield is high, and the storage stability is good.

Summary of the invention

In view of this, the present invention provides a polypeptide derivative, nanofiber and application thereof. The polypeptide derivative can effectively simulate the biological activity of the IGF-1 protein as a substitute for the IGF-1 protein, and the production process is relatively simple, The yield is higher and the storage stability is better.

Specifically, it includes the following technical solutions:

According to the first aspect of the present invention, the present invention provides a polypeptide derivative whose sequence is X-Phe-Phe-Gly-Ser-Ser-Ser-Arg (hereinafter referred to as X-FFGSSSR), Among them, the end group X is Nap or Npx, Phe is at the same time in the L configuration or the D configuration, and the rest are in the L configuration. The end group structural formula is as follows:

or,

Preferably, the sequence of the polypeptide derivative is Nap-Phe-Phe-Gly-Ser-Ser-Ser-Arg, and all amino acids are in the L configuration.

As common knowledge in the field, the following sequences are all in the L configuration unless otherwise specified.

The polypeptide derivatives are synthesized by the well-known FMOC-short peptide solid phase synthesis method. Specifically, the preparation method of the polypeptide derivative includes the following steps:

(1) The C-terminus of Fmoc-amino acid is combined with resin;

(2) Removal and washing of Fmoc protecting group;

(3) The C-terminus of the next Fmoc-amino acid is coupled to the N-terminus of the amino acid or polypeptide on the resin and washed;

(4) Repeat steps (2) to (3) until the coupling of the last amino acid is completed, remove the Fmoc protecting group, and wash;

(5) X-OH is coupled to the N-terminus of the polypeptide on the resin and washed;

(6) The polypeptide derivative is excised from the resin to obtain a crude product;

(7) Purify the crude product using high performance liquid chromatography.

According to the second aspect of the present invention, the present invention provides nanofibers of the polypeptide derivative, and the water mixture of the polypeptide derivative is heated and cooled to form the nanofibers. When the concentration of the water mixture of the polypeptide derivative reaches the millimolar level, a supramolecular hydrogel can be formed. As common knowledge in the art, supramolecular hydrogels are gels formed by small molecular compounds with a molecular weight of less than 2000 that aggregate with each other through non-covalent bonding, self-assemble to obtain a network structure and encapsulate water molecules. Tests have found that X-FFGSSSR has good solubility and can form a colorless and transparent hydrogel.

Further, the specific method of heating and cooling the water mixture of the polypeptide derivative to form nanofibers is: adding the polypeptide derivative to a PBS solution with a pH of 5.0 to 9.0, and using a sodium carbonate solution or a hydrochloric acid solution The pH value is adjusted to 6.0-7.0, heated to boiling to completely dissolve the compound, and cooled to room temperature to prepare a mixture of nanofibers containing polypeptide derivatives with a concentration of 10 nM to 1 μM.

The inventors found that X-FFGSSSR has a shorter sequence structure than the polypeptide derivative Nap-FFGGYGSSSRRAPQT disclosed in the prior art. Accordingly, it can be deduced from common knowledge that the X-FFGSSSR and IGF-1 receptor protein have a shorter sequence structure than the peptide derivative Nap-FFGGYGSSSRRAPQT disclosed in the prior art. The binding force is weaker than Nap-FFGGYGSSSRRAPQT, but unexpectedly, the nanofibers of X-FFGSSSR show good biological activity and are effective in simulating IGF-1 for the treatment of muscle atrophy and atherosclerosis. At the same time, because X-FFGSSSR has a shorter sequence structure, its production process is shorter and simpler, with higher yield, and more controllable quality; in addition, because of the molar concentration required for the same activity as Nap-FFGGYGSSSRRAPQT Similarly, due to the shorter X-FFGSSSR sequence and lower molecular weight, X-FFGSSSR requires less mass to exert the same activity, which further reduces the cost; and compared to Nap-FFGGYGSSSRRAPQT, the solubility of X-FFGSSSR Better and more stable performance, which is conducive to its long-term storage.

According to the third aspect of the present invention, the present invention provides the application of the nanofibers in the preparation of medicines for treating muscle atrophy.

The inventor found that the polypeptide derivatives can bind to the IGF-1 protein receptor, activate the insulin signal pathway, promote the proliferation and differentiation of myoblasts, resist cell apoptosis induced by glucocorticoids represented by dexamethasone, and treat Muscle atrophy.

Preferably, the sequence of the polypeptide derivative is Nap-Phe-Phe-Gly-Ser-Ser-Ser-Arg (hereinafter referred to as Nap-FFGSSSR), all amino acids are in the L configuration, and the structural formula is shown below;

According to the fourth aspect of the present invention, the present invention provides the use of the nanofibers in the preparation of drugs for the treatment of atherosclerosis, and the sequence of the polypeptide derivative is Npx-Phe-Phe-Gly-Ser-Ser-Ser -Arg (hereinafter referred to as Npx-FFGSSSR), where Phe is in the L configuration or the D configuration at the same time, and the rest of the amino acids are in the L configuration. The specific structural formula is as follows:

or,

The polypeptide derivatives Npx- ^D F ^D FGSSSR and Npx-FFGSSSR can reduce the lipid accumulation of macrophages, inhibit the transformation of vascular smooth muscle cells to a macrophage-like phenotype, enhance the stability of plaques in vivo, and delay arterial It plays an important role in the occurrence of atherosclerosis.

The drug is treated by intramuscular injection.

The beneficial effects of the technical solutions provided by the embodiments of the present invention include at least:

1. The preparation process of the present invention is simple, the chemical structure of the product is clear, the raw materials used are all amino acids necessary for the human body every day, and the polypeptide derivatives can be prepared by solid phase synthesis. The production process is simple, the yield is high, the cost is low, and the biological phase Good capacity;

2. The microstructure of nanofibers can effectively resist the degradation of proteases in the body, significantly prolong its half-life in vivo and increase the retention rate of tissues;

3. The polypeptide derivative can bind to the IGF-1 protein receptor and activate the insulin signaling pathway;

4. The nanofibers formed by the polypeptide derivative Nap-FFGSSSR can promote the proliferation and differentiation of myoblasts, resist apoptosis induced by glucocorticoids represented by dexamethasone, and treat muscle atrophy;

5, the polypeptide derivative nanofiber Npx- ^D F ^D FGSSSR formed for atherosclerosis hardening treatment, can greatly reduce the accumulation of lipids, enhance plaque stability in vivo, delay of atherosclerosis Occurrence and development.

Description of the drawings

In order to explain the technical solutions in the embodiments of the present invention more clearly, the following will briefly introduce the drawings needed in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained from these drawings without creative work.

Figure 1 is a graph showing the effect of Nap-FFGSSSR on promoting the proliferation of mouse myoblasts (C2C12) by detecting the number of positive cells using the EdU method.

Figure 2 is a graph showing the effect of Nap-FFGSSSR against Dex-induced apoptosis of mouse myoblasts (C2C12) by detecting the number of positive cells by the TUNEL method.

Figure 3 shows the use of Western Blot to detect the expression of T-AKT, P-AKT (T308) and P-AKT (S473) to evaluate the activation of the insulin signaling pathway by Nap-FFGSSSR.

Figure 4 is a graph showing changes in mouse muscle mass.

5 is a transmission electron micrograph of the supramolecular hydrogels obtained in Preparation Examples 1-3 and Comparative Preparation Example 4. Preparation Example 2 wherein A is a polypeptide derivative Npx- ^D F ^D FGSSSR prepared by the method of heating and cooling supramolecular hydrogels (H1) TEM FIG embodiment, B is a polypeptide derivative prepared in Example 3 by heating Npx-FFGSSSR The TEM image of the supramolecular hydrogel (H2) prepared by the cooling method. C is the TEM image of the supramolecular hydrogel (H3) prepared by the heating and cooling method of the polypeptide derivative Nap-FFGSSSR of Preparation Example 1. D is the transmission electron microscope image of the supramolecular hydrogel (H4) prepared by the heating and cooling method of the polypeptide derivative Nap-FFGSRSS of Comparative Preparation Example 4. The scale bar is 100nm.

FIG 6 is a polypeptide derivative Npx- ^D F ^D FGSSSR, measured Npx-FFGSSSR, Nap-FFGSSSR and binding ability Nap-FFGSRSS the IGF-1 receptor. Wherein A is a binding Npx- ^D F ^D FGSSSR the IGF-1 receptor protein assay FIG constant, B is Npx-FFGSSSR protein with IGF-1 receptor binding constants FIG measurement, C is Nap-FFGSSSR the IGF-1 receptor protein Binding constant determination diagram, D is the binding constant determination diagram of Nap-FFGSRSS and IGF-1 receptor protein.

Fig. 7 is the detection of lipid accumulation in peritoneal macrophages of ApoE knockout mice by using the lipid oil red O staining method. Among them, A is the lipid oil red O staining of peritoneal macrophages to assess the formation of foam cells, and B is the determination of the gene expression of the two main lipid transporters ABCA1 and ABCG1 through the results of Western Blot.

Figure 8 shows the use of polypeptide derivatives to treat aortic lesions in mice with atherosclerosis. Among them, A is the representative micrograph of aortic lesions measured by lipid oil red O staining method, B is the quantitative statistical data of each group in A, and C is the oil red O staining method after tissue sectioning of the aortic root Detect the photo of the lesion in the cross section of the aortic root, D is the quantitative statistical data of each group in C.

Figure 9 shows the use of enzyme-linked immunosorbent assay (ELISA) and real-time fluorescent quantitative polymerase chain reaction (RT-PCR) to measure the expression of pro-inflammatory cytokines in mouse serum and aorta. Among them, A is the expression of TGF-α, IL-1β and IL-6 in mouse serum measured by ELISA, and B is the expression of TGF-α and IL-6 in mouse aorta measured by RT-PCR method.

Detailed ways

In order to make the technical solutions and advantages of the present invention clearer, the following further describes the embodiments of the present invention in detail with reference to the accompanying drawings.

According to the first aspect of the present invention, the present invention provides a polypeptide derivative whose sequence is X-Phe-Phe-Gly-Ser-Ser-Ser-Arg (hereinafter referred to as X-FFGSSSR), Wherein, the end group X is Nap or Npx, Phe is at the same time in the L configuration or the D configuration, and the rest are in the L configuration, and the end group structural formula is as follows:

or,

Preferably, the sequence of the polypeptide derivative is Nap-Phe-Phe-Gly-Ser-Ser-Ser-Arg, and all amino acids are in the L configuration.

The polypeptide derivatives are synthesized by the well-known FMOC-short peptide solid phase synthesis method. Specifically, the preparation method of the polypeptide derivative includes the following steps:

(1) The C-terminus of Fmoc-amino acid is combined with resin;

(2) Removal and washing of Fmoc protecting group;

(3) The C-terminus of the next Fmoc-amino acid is coupled to the N-terminus of the amino acid or polypeptide on the resin and washed;

(4) Repeat steps (2) to (3) until the coupling of the last amino acid is completed, remove the Fmoc protecting group, and wash;

(5) X-OH is coupled to the N-terminus of the polypeptide on the resin and washed;

(6) The polypeptide derivative is excised from the resin to obtain a crude product;

(7) Purify the crude product using high performance liquid chromatography.

According to the second aspect of the present invention, the present invention provides nanofibers of the polypeptide derivative, and the water mixture of the polypeptide derivative is heated and cooled to form the nanofibers. When the concentration of the water mixture of the polypeptide derivative reaches the millimolar level, a supramolecular hydrogel can be formed. As common knowledge in the art, supramolecular hydrogels are gels formed by small molecular compounds with a molecular weight of less than 2000 that aggregate with each other through non-covalent bonding, self-assemble to obtain a network structure and encapsulate water molecules.

Further, the specific method of heating and cooling the water mixture of the polypeptide derivative to form nanofibers is: adding the polypeptide derivative to a PBS solution with a pH of 5.0 to 9.0, and using a sodium carbonate solution or a hydrochloric acid solution The pH value is adjusted to 6.0-7.0, heated to boiling to completely dissolve the compound, and cooled to room temperature to prepare a mixture of nanofibers containing polypeptide derivatives with a concentration of 10 nM to 1 μM.

The inventor found that X-FFGSSSR has a shorter sequence structure than the polypeptide derivative Nap-FFGGYGSSSRRAPQT disclosed in the prior art. Correspondingly, according to common knowledge, it can be inferred that the polypeptide derivative X-FFGSSSR and IGF-1 receptor The binding power of protein is weaker than Nap-FFGGYGSSSRRAPQT, but unexpectedly, the supramolecular hydrogel of X-FFGSSSR exhibits good biological activity, and is effective in simulating IGF-1 for the treatment of muscle atrophy and atherosclerosis. . At the same time, because X-FFGSSSR has a shorter sequence structure, its production process is shorter and simpler, with higher yield, and more controllable quality; in addition, because of the molar concentration required for the same activity as Nap-FFGGYGSSSRRAPQT Similarly, due to the shorter X-FFGSSSR sequence and lower molecular weight, X-FFGSSSR requires less mass to exert the same activity, which further reduces the cost; and compared to Nap-FFGGYGSSSRRAPQT, the solubility of X-FFGSSSR Better and more stable performance, which is conducive to its long-term storage.

According to the third aspect of the present invention, the present invention provides the application of the nanofibers in the preparation of medicines for treating muscle atrophy.

Preferably, the sequence of the polypeptide derivative is Nap-Phe-Phe-Gly-Ser-Ser-Ser-Arg (hereinafter referred to as Nap-FFGSSSR), all amino acids are in the L configuration, and the structural formula is shown below;

The inventor found that the nanofibers of the polypeptide derivative Nap-FFGSSSR can bind to the IGF-1 protein receptor, activate the insulin signaling pathway, promote the proliferation and differentiation of myoblasts, and resist the induction of glucocorticoids represented by dexamethasone. Apoptosis, treatment of muscle atrophy.

According to the fourth aspect of the present invention, the present invention provides the use of the nanofibers in the preparation of drugs for the treatment of atherosclerosis, and the sequence of the polypeptide derivative is Npx-Phe-Phe-Gly-Ser-Ser-Ser -Arg (hereinafter referred to as Npx-FFGSSSR), where Phe is in the L configuration or the D configuration at the same time, and the rest of the amino acids are in the L configuration. The specific structural formula is as follows:

or,

The polypeptide derivatives Npx- ^D F ^D FGSSSR (D configuration) and Npx-FFGSSSR (L configuration) nanofibers can reduce the lipid accumulation of macrophages, and inhibit the transformation of vascular smooth muscle cells into a macrophage-like phenotype , Enhance the stability of plaque in the body, play an important role in delaying the occurrence of atherosclerosis.

The drug is treated by intramuscular injection.

The sources of the preparations involved in the following examples are as follows:

2-Cl-Trt resin was purchased from Tianjin Nankai Hecheng Technology Co., Ltd., with an activity of 1.2mmol/mL;

N,N-Diisopropylethylamine (hereinafter referred to as DIEPA), purchased from Adamas, with a purity of 99%;

Benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (hereinafter referred to as HBTU), purchased from Sigma-Aldrich (Sigma-Aldrich), purity 98%;

Trifluoroacetic acid (hereinafter referred to as TFA), purchased from Sigma-Aldrich, with a purity of 99%;

Triisopropylsilane (hereinafter referred to as TIS), purchased from Sigma-Aldrich, with a purity of 99%;

All amino acids were purchased from Gill Biochemical (Shanghai) Co., Ltd., with a purity of 98%;

EdU staining kit, TUNEL staining kit, hematoxylin-eosin staining solution, and lipid red oil O staining solution were all purchased from Sigma-Aldrich;

DMEM high glucose medium and fetal bovine serum were purchased from Tianjin Zhide Biotechnology Co., Ltd.;

Mouse myoblast C2C12, purchased from Shanghai Gefan Bio;

Female C57BL/6 mice aged 6-8 weeks and male ApoE gene-deficient mice aged 6-8 weeks were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.;

IGF-1 protein, purchased from Acro Biosystems, has a purity of 98%.

Preparation Example 1:

Synthesis and preparation of peptide derivative Nap-FFGSSSR and its nanofibers

(1) Solid-phase synthesis of Nap-FFGSSSR

Specific steps are as follows:

1) Weigh 0.5mmol 2-Cl-Trt resin in a solid phase synthesizer, add 10mL of anhydrous dichloromethane (hereinafter referred to as DCM), place it on a shaker and shake for 5 minutes to fully swell the 2-Cl-Trt resin ；

2) Use an ear wash ball to remove DCM from the solid phase synthesizer equipped with 2-Cl-Trt resin;

3) Dissolve 0.75mmol of Fmoc-Arg in 10mL of anhydrous DCM, add 0.75mmol of DIPEA, then transfer to the above solid phase synthesizer, add 0.75mmol of DIPEA, and react at room temperature for 1h;

4) Sealing: Remove the reaction liquid in the solid phase synthesizer with ear wash ball, and then wash with 10 mL of anhydrous DCM, each time for 1 min, wash 5 times in total, add the prepared volume ratio of anhydrous DCM: DIPEA: methanol = 20 mL of 17:1:2 solution, react for 10 min at room temperature;

5) Remove the reaction liquid in the solid phase synthesizer with an ear-washing ball, first wash with anhydrous DCM, each DCM dosage is 10mL, washing time 1min, wash 5 times, and then use N,N-dimethylformamide ( The following uses DMF to express) washing, each DMF dosage is 10mL, washing time is 1min, wash 5 times, add 10mL DMF containing 20% piperidine by volume, react for 25min, and then use 10mL with 20% piperidine by volume DMF of pyridine was reacted for 5 minutes, and then washed with DMF. Each time the amount of DMF was 10 mL, the washing time was 1 minute, and the washing time was 5 times, and then proceed to the next reaction;

6) Added Fmoc-Ser 1mmol, HBTU 1.5mmol, DIPEA 2mmol and 10mL DMF, add the prepared solution to the above solid phase synthesizer, and react for 2h;

7) Repeat steps 5) and 6) to add Fmoc-Ser, Fmoc-Ser, Fmoc-Gly, Fmoc-Phe, Fmoc-Phe, 2-naphthalene acetic acid, and then wash 5 times with DMF and 5 times with DCM. Proceed to the next step;

8) Add 10 mL of a solution composed of 95% TFA, 2.5% TIS, and 2.5% H ₂ O by volume to the solid phase synthesizer, and react for half an hour (or the volume ratio of TFA to DCM is 1:99, and the volume A TFA solution with a percentage concentration of 1%, add 3 mL of the TFA solution to the above solid phase synthesizer each time, add ten times, each reaction time is 1 min), cut the product from the 2-Cl-Trt resin It was concentrated in vacuo and the solvent was removed to obtain a crude product, which was separated and purified by HPLC to obtain the polypeptide derivative Nap-FFGSSSR with a yield of 75%.

(2) Preparation of nanofibers of polypeptide derivatives

Weigh 1.0 mg of the purified peptide derivative Nap-FFGSSSR, place it in a 2mL glass bottle, add 500μL PBS solution (pH=7.4), adjust the pH value to 7.4 with sodium carbonate solution, and heat to boiling to complete the compound After dissolving and cooling to room temperature, a colorless, transparent and invertible hydrogel is obtained. Whether the gel is formed or not is judged by methods known in the art, such as the method of inverting the bottle, the hydrogel remaining at the bottom of the bottle is hydrogel, and if it is flowing, it is liquid. The hydrogel is colorless and transparent, can be stored at room temperature for more than one month, and shows that its solubility and storage stability are good.

Weigh 1.1 mg of purified peptide derivative Nap-FFGSSSR, place it in a 2 mL glass bottle, add 1 mL of PBS solution (pH = 7.4), adjust its pH to 7.4 with sodium carbonate solution, and heat to boiling to complete the compound Dissolve, use a micro sampler to add 1 μL to 999 μL of PBS solution, and then heat to boiling to completely dissolve the compound to obtain 1 μM Nap-FFGSSSR solution; use a micro sampler to suck 10 μL of 1 μM Nap-FFGSSSR solution and add to 990 μL In the PBS solution, heated to boiling to completely dissolve the compound to obtain a 10nM Nap-FFGSSSR solution. Electron micrographs showed that the internal structure of Nap-FFGSSSR solutions with concentrations of 1μM and 10nM were all nanofibers.

Comparative preparation example 1

Sterile 1×PBS

Weigh 8g NaCl, 0.2g KCl, 1.44g Na ₂ HPO ₄ and 0.24g KH ₂ PO ₄ , dissolve them in 800ml distilled water, adjust the pH of the solution to 7.4 with HCl, and finally add distilled water to make the volume to 1L. After sterilization in an autoclave, store in a refrigerator at room temperature or 4℃.

Comparative preparation example 2

Weigh 0.47mg of the purified polypeptide derivative Ac-SSSR, place it in a 2mL glass bottle, add 1mL PBS solution (pH=7.4), adjust the pH value to 7.4 with sodium carbonate solution, and heat to boiling to make the compound complete After dissolving and cooling down, the bottle was turned upside down and found that there was no hydrogel but a fluid solution, and an Ac-SSSR solution with a concentration of 1 mM was obtained.

Comparative Preparation Example 3

Weigh 1.8 mg of IGF-1 protein powder, place it in a 2 mL glass bottle, add 500 μL of PBS solution (pH = 7.4), adjust its pH to 7.4 with sodium carbonate solution to obtain an IGF-1 protein solution with a concentration of 2 mM .

Cell Example 1:

Activity test of the hydrogel of peptide derivative Nap-FFGSSSR at the cell level

(1) Promoting the proliferation of mouse myoblasts (C2C12)

1) Heat the water bath to 37℃ in advance, put the culture medium, serum, etc. into the water bath to preheat, and turn on the ultra-clean UV lamp for half an hour at the same time;

2) Take out the cryopreserved mouse myoblasts C2C12 from the liquid nitrogen tank, quickly place them in a 37°C water bath to thaw the cells, and then quickly transfer them to the ultra-clean table to perform the following operations: The liquid container was carefully transferred to a centrifuge tube containing culture medium, centrifuged for 3 minutes, the supernatant was removed, resuspended in DMEM medium containing 10% fetal bovine serum, transferred to a petri dish, and then placed in a 37°C incubator bring up;

3) Observe the cell status on the second day. After the cell status is good, perform the following experiment after passing one generation;

4) Add 2 mL of pancreatin, shake gently to cover evenly, tap the bottom of the petri dish, digest for 3 minutes at 37°C, observe most of the cell suspension under a microscope, pipette with 1 mL gun for 5-10 times, add 2 mL of medium, pipette evenly, and collect The cell culture solution was sucked into a centrifuge tube, centrifuged at 1000 rpm for 3 minutes, then the supernatant solution was discarded, and the DMEM medium containing 10% fetal bovine serum was added and blown evenly with a gun. The count was 5 per milliliter with a cell counter. ×10 ⁷ cells.

5) Resuspend the cells in a 24-well plate with a glass slide at the bottom, 50,000 cells per well, 1 mL of DMEM medium containing 10% fetal bovine serum, and culture overnight in an incubator at 37°C.

6) Take 10 μL of dexamethasone (hereinafter referred to as Dex) mother liquor with a concentration of 1 mM and add 990 μL of DMEM medium containing 10% fetal bovine serum to prepare 1 ml of medium containing 10 μM Dex. Take 10 μL of Dex mother solution with a concentration of 1 mM and 10 μL of Ac-SSSR solution with a concentration of 1 μM and add 980 μL of DMEM medium containing 10% fetal bovine serum to prepare 1 ml of medium containing 10 μM Dex and 10 nM Ac-SSSR. The same preparation method is similar to obtain the medium containing 10μM Dex and 10nM Nap-FFGSSSR, and the medium containing 10μM Dex and 10nM IGF-1. Add 1 ml of DMEM medium without Dex and any peptide derivatives and containing only 10% fetal bovine serum to the control group.

7) Aspirate the DMEM medium containing 10% fetal bovine serum on the next day, add 1mL per well of the medium pre-configured in step 6 to each group, incubate in a 37°C incubator for 24 hours and then follow the EdU staining kit In the instructions, the cells were stained, and then 3 fields of view were randomly selected in each group to take pictures with a confocal microscope. Finally, the EdU-positive cells were counted with Image J software, and the differences between the groups were counted.

Figure 1 is a graph showing the effect of Nap-FFGSSSR hydrogel on promoting the proliferation of mouse myoblasts (C2C12) by using the EdU method to detect the number of positive cells. The Control histogram in the figure represents the number of EdU-positive cells stained after C2C12 was cultured in cell culture medium without Nap-FFGSSSR for 24h and then incubated with EdU for 3h. The other histograms represent C2C12 containing dexamethasone (Dex) and Dex+Ac. -SSSR, Dex+Nap-FFGSSSR, Dex+IGF-1 culture medium for 24h and then incubated with EdU for 3h, the number of EdU positive cells stained, the number of EdU positive cells in the Dex group was significantly reduced, indicating that it can indeed inhibit the proliferation of C2C12 cells, Dex The number of EdU positive cells in the +Nap-FFGSSSR and Dex+IGF-1 groups was much greater than that in the Control group where the cells were in a normal growth state, indicating that Nap-FFGSSSR has the function of promoting the proliferation of C2C12. The number of EdU-positive cells in the Dex+Ac-SSSR group was slightly more than that in the Dex group, but far less than in the Control group, Dex+Nap-FFGSSSR and Dex+IGF-1 groups, indicating that Ac-SSSR had a weak ability to promote the proliferation of C2C12 Features.

(2) Anti-apoptotic experiment of mouse myoblasts (C2C12)

1) After repeating steps 1)-4) of the above cell proliferation experiment, resuspend the cells in a 24-well plate with 50,000 cells per well and 1 mL of DMEM medium containing 10% fetal bovine serum in a 37°C incubator Cultivate overnight.

2) Take 10 μL of dexamethasone (hereinafter referred to as Dex) mother liquor with a concentration of 1 mM and add 990 μL of DMEM medium containing 10% fetal bovine serum to prepare 1 ml of medium containing 10 μM Dex. Take 10 μL of Dex mother solution with a concentration of 1 mM and 10 μL of Ac-SSSR solution with a concentration of 1 μM and add 980 μL of DMEM medium containing 10% fetal bovine serum to prepare 1 ml of medium containing 10 μM Dex and 10 nM Ac-SSSR. The same preparation method is similar to obtain the medium containing 10μM Dex and 10nM Nap-FFGSSSR, and the medium containing 10μM Dex and 10nM IGF-1. Add 1 ml of DMEM medium without Dex and any peptide derivatives and containing only 10% fetal bovine serum to the control group.

3) Aspirate the DMEM medium containing 10% fetal bovine serum on the second day, add 1mL per well of the medium pre-configured in step 2 to each group, and incubate in a 37°C incubator for 24 hours according to the TUNEL staining kit In the instructions, the cells were stained, and then 3 fields of view were randomly selected in each group with a confocal microscope to take pictures. Finally, the TUNEL positive cells were counted with Image J software, and the differences between the groups were counted.

Figure 2 is a graph showing the effect of Nap-FFGSSSR hydrogel on resisting Dex-induced apoptosis of mouse myoblasts (C2C12) by detecting the number of positive cells using the TUNEL method. The Control histogram in the figure represents the number of TUNEL positive cells stained after C2C12 was cultured in cell culture medium without Nap-FFGSSSR for 24 hours and then incubated with TUNEL for 1 hour. The other histograms represent C2C12 containing dexamethasone (Dex) and Dex+Ac. -SSSR, Dex+Nap-FFGSSSR, Dex+IGF-1 culture medium for 24h and then incubated with TUNEL for 1h, the number of TUNEL-positive cells stained, the number of TUNEL-positive cells in the Dex group increased significantly, indicating that it can indeed induce C2C12 cell apoptosis. The number of TUNEL-positive cells in the Dex+Nap-FFGSSSR and Dex+IGF-1 groups was equivalent to that in the Control group with cells in a normal growth state, indicating that Nap-FFGSSSR has the function of resisting Dex-induced C2C12 apoptosis. The number of TUNEL positive cells in the Dex+Ac-SSSR group is slightly less than that in the Dex group, but far more than the Control group, Dex+Nap-FFGSSSR and Dex+IGF-1 groups, indicating that Ac-SSSR has weak resistance to Dex-induced The function of C2C12 apoptosis.

(3) The experiment of activating the insulin signaling pathway by peptide hydrogel for the treatment of muscle atrophy

1) After repeating steps 1)-4) of the above cell proliferation experiment, resuspend the cells in a 6-well plate with 100,000 cells per well and 1 mL of DMEM medium containing 10% fetal bovine serum in a 37°C incubator Cultivate overnight.

2) Take 10 μL of dexamethasone (hereinafter referred to as Dex) mother liquor with a concentration of 1 mM and add 990 μL of DMEM medium containing 10% fetal bovine serum to prepare 1 ml of medium containing 10 μM Dex. Take 10 μL of Dex mother solution with a concentration of 1 mM and 10 μL of Ac-SSSR solution with a concentration of 1 μM and add 980 μL of DMEM medium containing 10% fetal bovine serum to prepare 1 ml of medium containing 10 μM Dex and 10 nM Ac-SSSR. The same preparation method is similar to obtain the medium containing 10μM Dex and 10nM Nap-FFGSSSR, and the medium containing 10μM Dex and 10nM IGF-1. Add 1 ml of DMEM medium without Dex and any peptide derivatives and containing only 10% fetal bovine serum to the control group.

3) Aspirate the DMEM medium containing 10% fetal bovine serum on the second day, add 1 mL per well of the medium pre-configured in step 2 to each group, incubate in a 37°C incubator for 24 hours, extract total protein, and perform SDS -After PAGE electrophoresis and transfer, add the primary antibodies of T-AKT, P-AKT (T308) and P-AKT (S473) and incubate overnight at 4°C, then add the secondary antibody and incubate for 1 hour, add the color developing solution, and use the exposure instrument for exposure After taking a picture, save the picture. Use Image J software to perform quantitative calculations to evaluate the phosphorylation degree of AKT and the activation of the insulin signaling pathway.

By using Western Blot to detect the expression of T-AKT, P-AKT (T308) and P-AKT (S473) to evaluate the activation of the insulin signaling pathway by Nap-FFGSSSR hydrogel. The result is shown in Figure 3. The plus sign (+) and minus sign (-) are used to indicate whether to add the corresponding substances. It can be seen from the figure that Dex, Ac-SSSR, Nap-FFGSSSR and IGF-1 are not added. The group of the four substances is the control group where the cells are in a normal growth state. All three genes are expressed normally. Once Dex is added, the expression of the two genes P-AKT (T308) and P-AKT (S473) will be significantly reduced. After adding Nap-FFGSSSR and IGF-1, the expression of these two genes began to increase again, indicating that Nap-FFGSSSR can activate the insulin signaling pathway. After adding Ac-SSSR, the expression of these two genes increased slightly, indicating that Ac-SSSR The insulin signaling pathway can be partially activated.

Animal Example 1:

Experiment on the treatment of muscle atrophy in mice

1) Model establishment: 6-8 weeks old female C57BL/6 mice with a body weight of 20g±3g were injected with Dex at a dose of 0.2mg/kg/day every day for 28 consecutive days to construct a mouse model of muscle atrophy.

2) Administration treatment and statistics: After the modeling is successful, the mice are randomly divided into 5 groups with 6 mice in each group. The mice in different groups are injected with PBS buffer solution and 10μM Ac- into the muscles of the legs once a week. SSSR, Nap-FFGSSSR, IGF-1 protein solution, the injection volume is 100μL. Among them, the control group was injected with PBS buffer solution. Four weeks later, a dual-energy X-ray detector was used to monitor the changes in the muscle mass of the mice before and after treatment. The changes in muscle mass of the mice in each group were counted, and the differences between the groups were statistically analyzed.

Figure 4 is a graph showing changes in mouse muscle mass. It can be seen from the figure that the muscle mass of the Dex group mice was significantly decreased, and the muscle mass of the Nap-FFGSSSR and IGF-1 groups was comparable to that of normal mice, indicating that Nap-FFGSSSR can treat muscle atrophy in mice. The muscle mass of the mice in the Ac-SSSR group increased slightly, indicating that Ac-SSSR had a slight therapeutic effect on the muscle atrophy of the mice.

Preparation Example 2:

Synthesis and preparation of peptide derivative Npx- ^D F ^{D FGSSSR and its hydrogel}

(1) Solid-phase synthesis of ^{Npx- D} F ^{D FGSSSR}

Specific steps are as follows:

1) Weigh 0.5mmol of 2-Cl-Trt resin in a solid phase synthesizer, add 10 mL of anhydrous DCM, place on a shaker and shake for 5 minutes to fully swell the 2-Cl-Trt resin;

2) Use an ear wash ball to remove DCM from the solid phase synthesizer equipped with 2-Cl-Trt resin;

3) Dissolve 0.75mmol of Fmoc-Arg in 10mL of anhydrous DCM, add 0.75mmol of DIPEA, then transfer to the above solid phase synthesizer, add 0.75mmol of DIPEA, and react at room temperature for 1h;

4) Sealing: Remove the reaction liquid in the solid phase synthesizer with ear wash ball, and then wash with 10 mL of anhydrous DCM, each time for 1 min, wash 5 times in total, add the prepared volume ratio of anhydrous DCM: DIPEA: methanol = 20 mL of 17:1:2 solution, react for 10 min at room temperature;

5) Remove the reaction liquid in the solid phase synthesizer with ear wash ball, first wash with anhydrous DCM, wash 5 times with 10 mL of DCM each time, wash time 1 min, and then wash with DMF, wash with DMF 10 mL each time, wash Wash 5 times in total for 1 min, add 10 mL DMF containing 20% piperidine by volume, react for 25 min, then react with 10 mL DMF containing 20% piperidine by volume for 5 min, then wash with DMF, each time with DMF Use 10mL, wash time 1min, wash 5 times in total, proceed to the next reaction;

6) Added Fmoc-Ser 1mmol, HBTU 1.5mmol, DIPEA 2mmol and 10mL DMF, add the prepared solution to the above solid phase synthesizer, and react for 2h;

7) Repeat steps 5) and 6) to add Fmoc-Ser, Fmoc-Ser, Fmoc-Gly, Fmoc-D-Phe, Fmoc-D-Phe, (+)-α-methyl-6-methoxy 2-naphthylacetic acid, then washed 5 times with DMF and 5 times with DCM, and proceed to the next step;

8) Add 10 mL of a solution composed of 95% TFA, 2.5% TIS, and 2.5% H ₂ O by volume to the solid phase synthesizer, and react for half an hour (or the volume ratio of TFA to DCM is 1:99, and the volume A TFA solution with a percentage concentration of 1%, add 3 mL of the TFA solution to the above solid phase synthesizer each time, add ten times, each reaction time is 1 min), cut the product from the 2-Cl-Trt resin , concentrated in vacuo, and the solvent was removed to give crude product, followed by HPLC separation and purification, to obtain a polypeptide derivative Npx- ^D F ^D FGSSSR, a yield of about 80%.

(2) Preparation of nanofibers of polypeptide derivatives

Said polypeptide derivative Npx- ^D F ^D FGSSSR after taking 1.0mg purified, placed in 2mL vial, 500μL PBS was added (pH = 7.4), with sodium carbonate solution to adjust its pH to 7.4, heated to boiling The compound is completely dissolved, and after cooling to room temperature, a colorless and transparent invertible hydrogel is obtained. Whether it is gelled or not is judged by the method of inverting the bottle. If it is left at the bottom of the bottle, it is a hydrogel, if it is flowing, it is a liquid. The hydrogel has good solubility and can be stored at room temperature for more than 1 month. The hydrogel is colorless and transparent, can be stored at room temperature for more than one month, and shows that its solubility and storage stability are good.

Preparation Example 3:

Synthesis and preparation of peptide derivative Npx-FFGSSSR and its nanofibers

The steps are the same as those in Preparation Example 2, except that the Fmoc-D-Phe added in step 7) of (1) solid phase synthesis is changed to Fmoc-Phe.

Comparative preparation example 4:

Synthesis and preparation of peptide derivative Nap-FFGSRSS and its nanofibers

The steps are the same as those in Preparation Example 2, except that (1) the Fmoc-Arg added in step 3) of the solid phase synthesis is changed to Fmoc-Ser, and step 7) is sequentially added with Fmoc-Ser, Fmoc-Ser, Fmoc-Gly, Fmoc-D-Phe, Fmoc-D-Phe, (+)-α-methyl-6-methoxy-2-naphthaleneacetic acid, instead of adding Fmoc-Arg, Fmoc-Ser, Fmoc-Gly, Fmoc- Phe, Fmoc-Phe, 2-naphthalene acetic acid.

The negative staining technique was used to observe the microstructure of the hydrogel formed by the polypeptide derivatives obtained in Preparation Examples 1-3 and Comparative Preparation Example 4. First, use a micro-sampler to draw 10 μL of hydrogel and add it to the carbon-coated copper grid. After half a minute, dry the hydrogel with filter paper, rinse the copper mesh twice with water, and then dry the water with filter paper. After dyeing with a micro-sampler of 10 μL of uranyl acetate for 1 min, blot the dye solution with filter paper, and place the copper mesh in a desiccator to dry overnight. . On the second day, the microscopic morphology of the hydrogel was observed with a transmission microscope. The results are shown in Fig. 5, where A, B, C, and D correspond to Npx- ^D F ^D FGSSSR(H1), Npx-FFGSSSR(H2), Nap -Electron microscope samples of the hydrogel formed by FFGSSSR (H3) and Nap-FFGSRSS (H4). It can be seen that the four kinds of hydrogels have a large number of uniform nanofibers entangled to form a three-dimensional network structure, but the fiber diameters are about 26.8nm, 14.6nm, 9.8nm, and 17.3nm, respectively.

Said polypeptide derivative Npx- ^D F ^D FGSSSR after taking 1.1mg purified, placed in 2mL vial, was added 1mL PBS solution (pH = 7.4), with sodium carbonate solution to adjust its pH to 7.4, heated to boiling the compound is completely dissolved, with the added micro sampler suction 1μL to 999μL of PBS solution, and then heated to boiling to completely dissolve the compound to obtain Npx- ^D F ^D FGSSSR solution of [mu] M; suction 10μL microsampler concentrations of the Npx 1μM - ^D F ^D FGSSSR solution was added to 990μL of PBS solution, and then heated to boiling to completely dissolve the compound to obtain Npx- ^D F ^D FGSSSR solution of 10nM. Electron micrographs showed that the internal structure of the two solutions were nanofibers. Prepare 1μM, 10nM Npx-FFGSSSR solution according to the same method. Electron micrographs showed that the internal structure of the two solutions were nanofibers.

Determination of binding constants polypeptide derivative Npx- ^D F ^D FGSSSR (H1) and the IGF-1 receptor protein using microscale thermophoresis methods, using a single molecule NT Protein labeling kit, with fluorescent dye labeled NT-647 IGF-1 receptor protein. PBS buffer containing 0.05% Tween-20 (pH 7.4) was used as the detection buffer. Holding IGF-1 receptor protein labeled with a fluorescent constant concentration of 10μM, the concentration of hydrogel Npx- ^D F ^{D FGSSSR} (H1) is formed from the start times greater than 2μM diluted to 0.054nM, to give a range of concentrations Gradient solution. Then mix the fluorescent protein solution with solutions of different concentrations in a 1:1 volume ratio. After incubating for 1 minute, the sample was loaded into NT.115 standard glass capillary, and the NT.115 monomer system was used for analysis. The KD value is calculated using NanoTemper software package. The same method was used to determine the binding constants of Npx-FFGSSSR (H2), Nap-FFGSSSR (H3), Nap-FFGSRSS (H4) and IGF-1 receptor protein. The results are shown in Figure 6, where A, B, C, D corresponds to the test results of Npx- ^D F ^D FGSSSR(H1), Npx-FFGSSSR(H2), Nap-FFGSSSR(H3), Nap-FFGSRSS(H4). Npx- illustrated ^{D F D FGSSSR (H1),} Npx-FFGSSSR (H2), Nap-FFGSSSR (H3) in FIG IGF-1 binding with the receptor protein are constants 145.36nM, 172.54nM and 229.75nM, described Npx ^{- D F D FGSSSR (H1)} , Npx-FFGSSSR (H2) and Nap-FFGSSSR (H3) are each specific for binding to IGF-1 receptor protein, and Npx- ^D F ^D FGSSSR and IGF-1 by The binding capacity of body protein is the strongest, Npx-FFGSSSR (H2) is second, and Nap-FFGSSSR (H3) is the weakest. Nap-FFGSRSS (H4) has no binding constant with IGF-1 receptor protein, indicating that it cannot bind.

Cell Example 2:

(1) Experiment to inhibit the transformation of mouse peritoneal macrophages into foam cells

1) One-time injection of 3ml of high-temperature sterilized 4% sulfur gum into the abdominal cavity of mice for 4-5 days;

2) After euthanasia, the abdominal skin was cut open, and 10ml sterilized PBS was injected into the abdominal cavity twice to obtain a PBS solution containing macrophages;

3) Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, resuspend the cells in RPMI 1640 medium, and plate;

4) Wash the cells with sterile PBS (to remove the suspended monocytes and blood cells) after 1 hour, and treat the adherent macrophages after they are stable for two days.

5) Take 10μL concentration Npx- ^D F ^D FGSSSR 1μM solution of 990μL was added RPMI1640 medium containing 10% fetal bovine serum, the medium was prepared containing 1ml ^{10nM Npx- D F D FGSSSR (H1} ) of. The same method was used to prepare a medium containing 10 nM Npx-FFGSSSR (H2), Nap-FFGSSSR (H3), Nap-FFGSRSS (H4) or IGF-1 protein.

6) Use dissecting tweezers to remove the glass slides from the absolute ethanol, and bake them on an alcohol lamp. Do not break the glass slides, and then spread one piece in each hole of the 24-well plate; use 3ml containing 10% fetal bovine serum RPMI1640 medium will blow the peritoneal macrophages evenly, and then add them to the spread 24-well plate, add the medium prepared in step 5 to the wells of different groups, and add 1ml to the control (C) group RPMI1640 medium containing 10% fetal bovine serum without any peptide derivatives, and then placed in a 37°C CO ₂ incubator;

7) After culturing for 16 hours, aspirate the medium in the 24-well plate, add 1ml 1×PBS to wash the cells twice, after aspirate PBS, add 500μl 4% paraformaldehyde, fix at room temperature for 30 minutes; after aspirate paraformaldehyde, wash with PBS Cells were added twice, and then 600μl of Oil Red O working solution was added and stained for 45-60min at room temperature; the cells were washed 3-4 times with ultrapure water to wash off the excess staining solution, and then 450μl of hematoxylin staining solution was added for staining for 30s; Leave it in ultrapure water for 6 minutes, the nucleus stained with hematoxylin turns from purple to blue; drop an appropriate amount of mounting medium onto the glass slide, then use dissecting tweezers to carefully take out the glass slide, and gently lift the top onto the filter paper Soak up the liquid, and then carefully cover the cell side of the glass slide on the mounting plate, taking care to avoid air bubbles; place the slide on a clean bench to air dry, take the image under an upright microscope and save it; Macrophages with lipid droplets> 20 were defined as foam cells, and the foam cell formation ratio was calculated. The result is shown in Figure 7A.

(2) Experiments to increase the expression levels of lipid transporters ABCA1 and ABCG1

1) After repeating steps 1)-5) of the above cell experiment, resuspend the cells in a 6-well plate with 100,000 cells per well and 1 mL of RPMI1640 medium containing 10% fetal bovine serum, overnight in a 37°C incubator bring up.

2) The next aspirated RPMI1640 medium containing 10% fetal bovine serum, a solution containing ^{10nM Npx- D F D FGSSSR (H1} ), 10nM Npx-FFGSSSR (H2), 10nM Nap-FFGSSSR (H3), 10nM Nap- FFGSRSS (H4) and IGF-1 protein contain 10% fetal bovine serum RPMI1640 medium per well 1mL, control (C) group is added 1ml does not contain any peptide derivatives and only contains 10% fetal bovine serum RPMI1640 medium, incubate in a 37℃ incubator for 24 hours, extract total protein, perform SDS-PAGE electrophoresis, add ABCA1 and ABCG1 primary antibodies after transfer, and incubate overnight at 4℃, then add secondary antibodies and incubate for 1 hour before adding color developing solution. The exposure meter takes a picture and saves the picture after exposure. Image J software was used for quantitative calculation to evaluate the expression level of lipid transporters. The result is shown in Figure 7B.

The large increase in foam cells is an important signal for the occurrence of atherosclerosis, so we can find it by staining foam cells. 7A, the addition of IGF-1 protein, polypeptide derivative ^{Npx- D F D FGSSSR (H1)} , the content of Npx-FFGSSSR (H2) and Nap-FFGSSSR (H3) in the group is significantly lower than in the foam cells Control (C) group, it can be seen that IGF-1 protein and these three polypeptide derivatives have the function of reducing lipid accumulation in macrophages. However, the content of foam cells in the group with the peptide derivative Nap-FFGSRSS (H4) is not much different from that in the Control (C) group, indicating that it cannot reduce the lipid accumulation of macrophages. And we found that the number of foam cells in the H1 group was much smaller than that of the IGF-1 protein group, indicating that its ability to reduce lipid accumulation in macrophages was stronger than that of the IGF-1 protein. As it can be seen from FIG. 7B, addition of a polypeptide derivative Npx- ^D F ^D FGSSSR (H1) or Npx-FFGSSSR (H2) in the group ABCA1 and ABCG1 expression of these two proteins is significantly increased, and significantly H1 Better than IGF-1 protein group, H2 and IGF-1 protein group have similar effects. Although the protein expression of Nap-FFGSSSR (H3) group also increased, it was significantly weaker than that of IGF-1 protein group. The protein expression level of Nap-FFGSRSS (H4) group did not increase, which was equivalent to the effect of Control (C) group.

Animal Example 2:

An experiment on the treatment of atherosclerosis in ApoE knockout mice

1) Model establishment: select atherosclerosis-promoting model mice, male ApoE gene-deficient mice, and randomly divide them into groups (15 mice per group), and feed them with high-fat food containing 21% fat and 0.5% cholesterol, respectively, and feed treatment At 16 weeks, a mouse model of atherosclerosis was constructed.

2) administration of a therapeutically and Statistics: 16 weeks to build the model in mice weekly intramuscular injection and a PBS buffer solution Npx- ^D F ^D FGSSSR (H1) containing 1μM of, Npx-FFGSSSR (H2), Nap-FFGSSSR (H3), Nap-FFGSRSS (H4) and IGF-1 protein solution, the injection volume is 100μL. Among them, the control (C) group was injected with PBS buffer solution.

3) Tissue section staining: At the end of the experiment, all mice were euthanized with an overdose of 2,2,2-tribromoethanol (640mg/kg), and then the aorta, blood and peritoneal macrophages were collected. A cross-sectional section of the aortic root was prepared and stained with lipid oil red O to detect sinus lesions. The result is shown in Figure 8.

As can be seen from Figure 8, mice aortic plaque through Npx- ^D F ^D FGSSSR (H1) treatment significantly reduced, more effective than IGF-1 protein group. Npx-FFGSSSR (H2) and Nap-FFGSSSR (H3) group and Control (C) than the group, although the number can be reduced plaque, but the effect was weaker than Npx- ^D F ^D FGSSSR (H1) and IGF-1 protein. Nap-FFGSRSS (H4) cannot reduce aortic plaque.

The cytokine content of the collected mouse serum was determined by the ELISA method, and the result is shown in Figure 9A. The expression of pro-inflammatory cytokines in the aorta was measured by RT-PCR, and the results are shown in Figure 9B. It can be found that the Nap-FFGSRSS (H4) group has a higher expression of inflammatory factors, which is similar to the Control (C) group. And Npx- ^D F ^D FGSSSR (H1) group, Npx-FFGSSSR (H2) group and Nap-FFGSSSR (H3) group, TNFα IGF-1 group, IL-1β, IL-6 cytokine levels are three The drop. Wherein Npx- ^D F ^D FGSSSR (H1) minimum group content, which can effectively reduce described the expression of pro-inflammatory cytokines, thereby suppressing the occurrence and development of inflammation, the treatment of atherosclerosis.

In summary, the anti-atherosclerotic effect of polypeptide derivatives ^{are: Npx- D F D FGSSSR (H1} ) significantly better than IGF-1 protein, Npx-FFGSSSR (H2) and IGF-1 protein quite effective, NAP- FFGSSSR (H3) is slightly inferior to IGF-1 protein, while Nap-FFGSRSS (H4) has almost no effect, which is equivalent to the effect of Control (C) group.

The above description is only for the convenience of those skilled in the art to understand the technical solutions of the present invention, and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (7)

1. A polypeptide derivative, characterized in that the sequence of the polypeptide derivative is X-Phe-Phe-Gly-Ser-Ser-Ser-Arg, wherein the end group X is Nap or Npx, and Phe is in the L configuration at the same time Or D configuration, the rest are L configuration.
2. The polypeptide derivative of claim 1, wherein the sequence of the polypeptide derivative is Nap-Phe-Phe-Gly-Ser-Ser-Ser-Arg, and all amino acids are in the L configuration.
3. The nanofiber comprising the polypeptide derivative of claim 1, wherein the water mixture of the polypeptide derivative is heated and cooled to form the nanofiber.
4. The nanofiber of claim 3, wherein the specific method of heating and cooling the water mixture of the polypeptide derivative to form the nanofiber is: adding the polypeptide derivative to a pH=5.0-9.0 In the PBS solution, adjust the pH value to 6.0-7.0 with sodium carbonate solution or hydrochloric acid solution, heat to boiling to completely dissolve the compound, and cool to room temperature to prepare a mixture of nanofibers containing polypeptide derivatives with a concentration of 10 nM to 1 μM .
5. The use of the nanofibers of claim 3 or 4 in the preparation of drugs for treating muscle atrophy.
6. The application according to claim 5, wherein the sequence of the polypeptide derivative is Nap-Phe-Phe-Gly-Ser-Ser-Ser-Arg, and all amino acids are in the L configuration.
7. The application of the supramolecular hydrogel of claim 3 or 4 in the preparation of a drug for the treatment of atherosclerosis, characterized in that the sequence of the polypeptide derivative is Npx-Phe-Phe-Gly-Ser-Ser-Ser -Arg, where Phe is in L configuration or D configuration at the same time, and the rest of the amino acids are in L configuration.

Images