CN110642937B - Polypeptide derivative, nanofiber and application thereof - Google Patents
Polypeptide derivative, nanofiber and application thereof Download PDFInfo
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- CN110642937B CN110642937B CN201910962002.1A CN201910962002A CN110642937B CN 110642937 B CN110642937 B CN 110642937B CN 201910962002 A CN201910962002 A CN 201910962002A CN 110642937 B CN110642937 B CN 110642937B
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Abstract
The invention provides a polypeptide derivative, a nanofiber and application thereof, wherein the polypeptide derivative can effectively simulate the biological activity of IGF-1 protein and is used as a substitute of the IGF-1 protein, and the polypeptide derivative has the advantages of simple production process, high yield and good storage stability. The sequence of the polypeptide derivative is X-Phe-Phe-Gly-Ser-Ser-Ser-Arg, wherein the end group X is Nap or Npx, Phe is in an L configuration or a D configuration at the same time, and the rest is in an L configuration. And heating and cooling the water mixture of the polypeptide derivative to form the nano-fiber. The polypeptide derivatives are capable of binding to the IGF-1 protein receptor and are useful for the treatment of muscle wasting and atherosclerosis.
Description
Technical Field
The invention relates to a polypeptide derivative, a nanofiber and application thereof.
Background
Insulin-like growth factor I (IGF-1) is a growth hormone that plays a crucial role in human development, growth, metabolism and homeostasis, and has been studied very extensively in the clinical and life science research fields.
Atherosclerosis is an important cause of cardiovascular diseases such as myocardial infarction, coronary heart disease, hypertension, stroke and the like, and is one of the main diseases harmful to human health. According to the latest data statistics of the world health organization WHO, the number of people WHO die from cardiovascular and cerebrovascular diseases accounts for 48 percent of the total number of non-infectious diseases, and is more than twice of the number of people WHO die from cancers. In most observational studies, low circulating levels of IGF-1 were associated with increased atherosclerotic complications. In a prospective study on the effects of IGF-1 levels, low circulating IGF-1 levels were associated with increased risk of ischemic heart disease during 15 years of follow-up in patients. In participants in the geriatric study of cervid, high free IGF-1 was associated with a reduction in carotid plaque and coronary artery disease, since free IGF-1 can easily cross endothelial cells, interact with its own receptors, and thereby regulate blood lipid balance. Therefore, restoration of IGF-1 physiological levels by exogenous IGF-1 supplementation is a promising approach for anti-atherosclerotic effects.
Muscular atrophy is a common chronic disease of middle-aged and old people, is mainly characterized by muscle strength decline and muscle quality decline, and is a pathological state of muscle protein excessive degradation caused by various factors such as genetic factors, long-term overdose of hormone drugs, body aging and the like. Muscle atrophy is a serious consequence of many chronic diseases and the aging process itself, as it directly leads to physical weakness, loss of independence and increased risk of death. Therefore, the development of professional drugs for treating muscle atrophy is increasingly demanded, and the development of professional drugs for treating muscle atrophy becomes an important research topic worldwide. However, to date, no effective drugs or treatment regimens other than exercise regimens have been developed to treat this disease. But the use of exercise regimens has been severely limited in the elderly, bedridden people or people with acute illnesses. In addition, existing methods of treating muscle atrophy have also typically relied on increasing the muscle mass by adding nutrients, adding appetite stimulants, or anabolic compounds (e.g., testosterone). However, these treatments are unsatisfactory and may cause side effects. IGF-1 plays an important role in maintaining muscle mass as an agonist of the insulin signaling pathway. The in vitro supplement of some extra IGF-1 can promote the synthesis of skeletal muscle protein and effectively prevent the occurrence of skeletal muscle deficiency.
The IGF-1 protein which is clinically applied or commercialized at present is natural or recombinant protein, has the problems of high production cost, difficult quality guarantee, harsh transportation and storage conditions and the like, and has no effect of directly injecting IGF-1 into damaged organs because the retention rate of the protein in tissues is low.
Chinese patent document CN109957000A discloses a polypeptide derivative with the sequence of Nap-FFGGYGSSSRRAPQT, which is used for simulating the biological activity of IGF-1 protein and can be used as a substitute of IGF-1 protein. The production process adopts FMOC-solid phase synthesis method, the product has definite chemical structure, high yield, obviously lower cost compared with natural or recombinant protein, and easy storage and transportation. Because the polypeptide organisms can be self-assembled in aqueous solution to form nano fibers, finally the hydrogel is formed, and the effects of improving the in vivo retention rate and further improving the biological activity can be achieved without the help of a carrier. However, the polypeptide chain length is 16 peptides, the synthesis process is complex and the yield is low; most importantly, the hydrogel formed by self-assembly is white opaque gel, has poor water solubility, and is not beneficial to long-term storage after being placed for 3-5 days.
In the process of implementing the invention, the inventor finds that at least the following problems exist in the prior art: provides a new polypeptide derivative which can effectively simulate the biological activity of IGF-1 protein, is used as a substitute of the IGF-1 protein, and has the advantages of simpler production process, higher yield and better storage stability.
Disclosure of Invention
In view of the above, the invention provides a polypeptide derivative, a nanofiber and an application thereof, wherein the polypeptide derivative can effectively simulate the biological activity of IGF-1 protein, is used as an IGF-1 protein substitute, and has the advantages of simple production process, high yield and good storage stability.
Specifically, the method comprises the following technical scheme:
according to the first aspect of the invention, the invention provides a polypeptide derivative, the sequence of the polypeptide derivative is X-Phe-Phe-Gly-Ser-Ser-Ser-Arg (hereinafter referred to as X-FFGSSSR), wherein the terminal group X is Nap or Npx, Phe is in L configuration or D configuration, the rest is in L configuration, and the terminal group structural formula is shown as follows:
alternatively, the first and second electrodes may be,
preferably, the sequence of the polypeptide derivative is Nap-Phe-Phe-Gly-Ser-Ser-Arg, and all amino acids are in L configuration.
As is common knowledge in the art, the following sequences are in the L configuration unless otherwise specified.
The polypeptide derivative is synthesized by adopting a known FMOC-short peptide solid phase synthesis method. Specifically, the preparation method of the polypeptide derivative comprises the following steps:
(1) the C-terminus of the Fmoc-amino acid is bound to the resin;
(2) removing and washing the Fmoc protecting group;
(3) coupling the C end of the next Fmoc-amino acid with the N end of the amino acid or polypeptide on the resin, and washing;
(4) repeating the steps (2) to (3) until the last amino acid coupling is finished, removing the Fmoc protecting group, and washing;
(5) coupling and washing the X-OH and the N end of the polypeptide on the resin;
(6) the polypeptide derivative is cut off from the resin to obtain a crude product;
(7) the crude product was purified using high performance liquid chromatography.
According to a second aspect of the invention, there is provided nanofibers of the polypeptide derivative formed by heating and cooling an aqueous mixture of the polypeptide derivative. When the concentration of the aqueous mixture of the polypeptide derivative reaches millimolar scale, a supramolecular hydrogel can be formed. As is common knowledge in the art, a supramolecular hydrogel is a gel formed by non-covalent interactions between small molecule compounds with molecular weights less than 2000, which aggregate, self-assemble to give a network structure and encapsulate water molecules. Experiments show that the X-FFGSSSR has good solubility and can form colorless and transparent hydrogel.
Further, the specific method for forming the nano-fiber by heating and cooling the water mixture of the polypeptide derivative comprises the following steps: adding the polypeptide derivative into a PBS (phosphate buffer solution) with the pH value of 5.0-9.0, adjusting the pH value of the solution to 6.0-7.0 by using a sodium carbonate solution or a hydrochloric acid solution, heating to boiling to completely dissolve the compound, and cooling to room temperature to obtain a mixture of 10 nM-1 MuM of the nanofiber containing the polypeptide derivative.
The inventors found that X-FFGSSSR has a shorter sequence structure than the polypeptide derivative Nap-FFGGYGSSSRRAPQT disclosed in the prior art, and accordingly, it is concluded from the common general knowledge that X-FFGSSSR binds to IGF-1 receptor protein less strongly than Nap-FFGGYGSSSRRAPQT, but surprisingly, nanofibers of X-FFGSSSR exhibit good biological activity, with outstanding results in mimicking IGF-1 for the treatment of muscle atrophy and atherosclerosis. Meanwhile, the X-FFGSSSR has a shorter sequence structure, so that the production process is shorter and simpler, the yield is higher, and the quality is more controllable; in addition, because the molar concentration required for exerting the same activity as that of Nap-FFGGYGSSSRRAPQT is the same, the mass required for exerting the same activity of X-FFGSSSR is less due to the fact that the sequence of the X-FFGSSSR is shorter and the molecular weight is lower, and therefore the cost is further greatly reduced; compared with Nap-FFGGYGSSSRRAPQT, the X-FFGSSSR has better solubility and more stable performance, and is beneficial to long-term storage.
According to a third aspect of the present invention, the present invention provides the use of said nanofibers for the preparation of a medicament for the treatment of muscle atrophy.
The inventor finds that the polypeptide derivative can be combined with IGF-1 protein receptor, activate insulin signal path, promote proliferation and differentiation of myoblasts, resist glucocorticoid-induced apoptosis 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 an L configuration, and the structural formula is shown as follows;
according to a fourth aspect of the present invention, the present invention provides an application of the nanofiber in the preparation of a drug for treating atherosclerosis, wherein the sequence of the polypeptide derivative is Npx-Phe-Gly-Ser-Arg (hereinafter referred to as Npx-FFGSSSR), wherein Phe is simultaneously in L configuration or D configuration, and the rest amino acids are in L configuration. The specific structural formula is as follows:
alternatively, the first and second electrodes may be,
the polypeptide derivative Npx-DFDFGSSSR and Npx-FFGSSSR can reduce lipid accumulation of macrophage, inhibit the transformation of vascular smooth muscle cell to macrophage-like phenotype, enhance the stability of plaque in vivo, and play an important role in delaying the occurrence of atherosclerosis.
The medicament is used for treating through intramuscular injection.
The technical scheme provided by the embodiment of the invention has the beneficial effects that at least:
1. the preparation process is simple, the chemical structure of the product is clear, the used raw materials are all amino acids necessary for human bodies every day, the polypeptide derivative can be prepared by a solid-phase synthesis method, and the preparation process is simple, high in yield, low in cost and good in biocompatibility;
2. the microstructure of the nanofiber can effectively resist the degradation of protease in vivo, the half-life period in vivo is obviously prolonged, and the tissue retention rate is improved;
3. the polypeptide derivative can be combined with IGF-1 protein receptor to activate insulin signal path;
4. the nanofiber formed by the polypeptide derivative Nap-FFGSSSR can promote the proliferation and differentiation of myoblasts, resist the apoptosis induced by glucocorticoid represented by dexamethasone and treat muscular atrophy;
5. the polypeptide derivative Npx-DFDWhen the nano-fiber formed by FGSSSR is used for treating atherosclerosis, the accumulation of lipid can be greatly reduced, the stability of plaque in vivo can be enhanced, and the occurrence and the development of atherosclerosis can be delayed.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a graph showing the effect of Nap-FFGSSSR in promoting proliferation of mouse myoblasts (C2C12) by detecting the number of positive cells by the EdU method.
FIG. 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.
FIG. 3 is a graph showing the evaluation of the activation of insulin signaling pathway by Nap-FFGSSSR by detecting the expression of T-AKT, P-AKT (T308) and P-AKT (S473) using Western Blot.
FIG. 4 is a graph showing changes in muscle mass in mice.
FIG. 5 is a transmission electron micrograph of the supramolecular hydrogels obtained in preparation examples 1-3 and comparative preparation example 4. Wherein A is the polypeptide derivative Npx-DFDTransmission electron microscope picture of FGSSSR supramolecular hydrogel (H1) prepared by heating and cooling method, B transmission electron microscope picture of supramolecular hydrogel (H2) prepared by heating and cooling method for polypeptide derivative Npx-FFGSSSR of preparation example 3, C transmission electron microscope picture of supramolecular hydrogel (H3) prepared by heating and cooling method for polypeptide derivative Nap-FFGSSSR of preparation example 1, and D transmission electron microscope picture of supramolecular hydrogel (H4) prepared by heating and cooling method for polypeptide derivative Nap-FFGSRSS of comparative preparation example 4. The scale is 100 nm.
FIG. 6 is a polypeptide derivative Npx-DFDFGSSSR, Npx-FFGSSSR, Nap-FFGSSSR, and determination of binding ability of Nap-FFGSRSS to IGF-1 receptor. Wherein A is Npx-DFDFGSSSR and IGF-1 receptor protein binding constant determination diagram, B is Npx-FFGSSSR and IGF-1 receptor protein binding constant determination diagram, C is Nap-FFGSSSR and IGF-1 receptor protein binding constant determination diagram, D is Nap-FFGSRSS and IGF-1 receptor protein binding constant determination diagram.
FIG. 7 shows lipid accumulation of peritoneal macrophages in ApoE knockout mice detected by lipid oil red O staining. Wherein A is lipid oil red O staining of peritoneal macrophages to evaluate the formation of foam cells, and B is gene expression of two major lipid transporters, ABCA1 and ABCG1, determined by Western Blot results.
FIG. 8 is a graph of aortic disease treatment using polypeptide derivatives in mice with atherosclerosis. A is a representative micrograph of aortic lesions measured by a lipid oil red O staining method, B is quantitative statistical data of each group in A, C is a photograph of aortic root cross section lesions measured by the oil red O staining method after the aortic root is subjected to tissue sectioning, and D is the quantitative statistical data of each group in C.
FIG. 9 is a graph showing the measurement of proinflammatory cytokine expression in mouse serum and aorta using enzyme-linked immunosorbent assay (ELISA) and real-time fluorescent quantitative polymerase chain reaction (RT-PCR). Wherein A is the expression quantity of TGF-alpha, IL-1 beta and IL-6 in the serum of the mouse measured by ELISA, and B is the expression quantity of TGF-alpha and IL-6 in the aorta of the mouse measured by RT-PCR method.
Detailed Description
In order to make the technical solutions and advantages of the present invention clearer, the following will describe embodiments of the present invention in further detail with reference to the accompanying drawings.
According to the first aspect of the invention, the invention provides a polypeptide derivative, the sequence of the polypeptide derivative is X-Phe-Phe-Gly-Ser-Ser-Ser-Arg (hereinafter referred to as X-FFGSSSR), wherein the terminal group X is Nap or Npx, Phe is in L configuration or D configuration, the rest is in L configuration, and the terminal group structural formula is shown as follows:
alternatively, the first and second electrodes may be,
preferably, the sequence of the polypeptide derivative is Nap-Phe-Phe-Gly-Ser-Ser-Arg, and all amino acids are in L configuration.
The polypeptide derivative is synthesized by adopting a known FMOC-short peptide solid phase synthesis method. Specifically, the preparation method of the polypeptide derivative comprises the following steps:
(1) the C-terminus of the Fmoc-amino acid is bound to the resin;
(2) removing and washing the Fmoc protecting group;
(3) coupling the C end of the next Fmoc-amino acid with the N end of the amino acid or polypeptide on the resin, and washing;
(4) repeating the steps (2) to (3) until the last amino acid coupling is finished, removing the Fmoc protecting group, and washing;
(5) coupling and washing the X-OH and the N end of the polypeptide on the resin;
(6) the polypeptide derivative is cut off from the resin to obtain a crude product;
(7) the crude product was purified using high performance liquid chromatography.
According to a second aspect of the invention, there is provided nanofibers of the polypeptide derivative formed by heating and cooling an aqueous mixture of the polypeptide derivative. When the concentration of the aqueous mixture of the polypeptide derivative reaches millimolar scale, a supramolecular hydrogel can be formed. As is common knowledge in the art, a supramolecular hydrogel is a gel formed by non-covalent interactions between small molecule compounds with molecular weights less than 2000, which aggregate, self-assemble to give a network structure and encapsulate water molecules.
Further, the specific method for forming the nano-fiber by heating and cooling the water mixture of the polypeptide derivative comprises the following steps: adding the polypeptide derivative into a PBS (phosphate buffer solution) with the pH value of 5.0-9.0, adjusting the pH value of the solution to 6.0-7.0 by using a sodium carbonate solution or a hydrochloric acid solution, heating to boiling to completely dissolve the compound, and cooling to room temperature to obtain a mixture of 10 nM-1 MuM of the nanofiber containing the polypeptide derivative.
The inventors have found that the X-FFGSSSR has a shorter sequence structure than the polypeptide derivative Nap-FFGGYGSSSRRAPQT disclosed in the prior art, and accordingly, it is concluded from the common general knowledge that the binding force of the polypeptide derivative X-FFGSSSR to IGF-1 receptor protein is weaker than that of Nap-FFGGYGSSSRRAPQT, but surprisingly, the supramolecular hydrogel of X-FFGSSSR exhibits good biological activity and is superior in mimicking IGF-1 for the treatment of muscle atrophy and atherosclerosis. Meanwhile, the X-FFGSSSR has a shorter sequence structure, so that the production process is shorter and simpler, the yield is higher, and the quality is more controllable; in addition, because the molar concentration required for exerting the same activity as that of Nap-FFGGYGSSSRRAPQT is the same, the mass required for exerting the same activity of X-FFGSSSR is less due to the fact that the sequence of the X-FFGSSSR is shorter and the molecular weight is lower, and therefore the cost is further greatly reduced; compared with Nap-FFGGYGSSSRRAPQT, the X-FFGSSSR has better solubility and more stable performance, and is beneficial to long-term storage.
According to a third aspect of the present invention, the present invention provides the use of said nanofibers for the preparation of a medicament for the treatment of 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 an L configuration, and the structural formula is shown as follows;
the inventor finds that the nanofiber of the polypeptide derivative Nap-FFGSSSR can be combined with an IGF-1 protein receptor, activate an insulin signaling pathway, promote the proliferation and differentiation of myoblasts, resist the glucocorticoid-induced apoptosis represented by dexamethasone and treat muscle atrophy.
According to a fourth aspect of the present invention, the present invention provides an application of the nanofiber in the preparation of a drug for treating atherosclerosis, wherein the sequence of the polypeptide derivative is Npx-Phe-Gly-Ser-Arg (hereinafter referred to as Npx-FFGSSSR), wherein Phe is simultaneously in L configuration or D configuration, and the rest amino acids are in L configuration. The specific structural formula is as follows:
alternatively, the first and second electrodes may be,
the polypeptide derivative Npx-DFDThe nano-fibers of FGSSSR (D configuration) and Npx-FFGSSSR (L configuration) can reduce the lipid accumulation of macrophages, inhibit the conversion of vascular smooth muscle cells to macrophage-like phenotype, enhance the stability of plaque in vivo and play an important role in delaying the occurrence of atherosclerosis.
The medicament is used for treating through intramuscular injection.
The sources of the formulations referred to in the following examples are as follows:
the 2-Cl-Trt resin is purchased from Tianjin Nankai and science and technology Limited and has the activity of 1.2 mmol/mL;
n, N-diisopropylethylamine (DIEPA below) was purchased from Adamas, Inc. (Adamas) with a purity of 99%;
benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate (hereinafter HBTU) was purchased from Sigma Aldrich (Sigma-Aldrich) with a purity of 98%;
trifluoroacetic acid (hereinafter indicated as TFA), purchased from Sigma Aldrich (Sigma-Aldrich), 99% pure;
triisopropylsilane (hereinafter referred to as TIS), purchased from Sigma Aldrich (Sigma-Aldrich) with a purity of 99%;
all amino acids were purchased from gill biochemical (shanghai) ltd with a purity of 98%;
EdU staining kit, TUNEL staining kit, hematoxylin-eosin staining solution, and lipid Red oil O staining solution were purchased from Sigma Aldrich (Sigma-Aldrich);
DMEM high-sugar medium and fetal bovine serum were purchased from Tianjin Zhi Biotech limited;
mouse myoblast C2C12, purchased from santa haiensis;
6-8 week old female C57BL/6 mice, 6-8 week old male ApoE gene deficient mice were purchased from Beijing Wittingle laboratory animal technology, Inc.;
IGF-1 protein, purchased from Acro biosystems, 98% pure.
Preparation example 1:
polypeptide derivative Nap-FFGSSSR and synthesis and preparation of nano-fiber thereof
(1) Solid phase synthesis of Nap-FFGSSSR
The method comprises the following specific steps:
1) weighing 0.5mmol of 2-Cl-Trt resin in a solid phase synthesizer, adding 10mL of anhydrous dichloromethane (hereinafter represented by DCM), placing on a shaking table, shaking for 5min, and fully swelling the 2-Cl-Trt resin;
2) removing DCM from the solid phase synthesizer containing 2-Cl-Trt resin by washing the ear with an ear bulb;
3) dissolving 0.75mmol of Fmoc-Arg in 10mL of anhydrous DCM, adding 0.75mmol of DIPEA, then transferring to the solid phase synthesizer, supplementing 0.75mmol of DIPEA, and reacting for 1h at room temperature;
4) and (3) sealing: removing reaction liquid in the solid phase synthesizer by using an aurilave, washing with 10mL of anhydrous DCM for 1min each time for 5 times, adding 20mL of prepared solution with the volume ratio of anhydrous DCM to DIPEA to methanol being 17: 1: 2, and reacting at room temperature for 10 min;
5) removing reaction liquid in the solid phase synthesizer by using an aurilave, washing by using anhydrous DCM for 5 times, washing by using N, N-dimethylformamide (hereinafter referred to as DMF) for 10mL each time for 1min, washing for 5 times, adding 10mL of DMF containing 20% by volume of piperidine for reaction for 25min, reacting by using 10mL of DMF containing 20% by volume of piperidine for 5min, washing by using DMF for 1min, washing for 5 times, and carrying out next reaction by using 10mL of DMF for 10mL each time for 1 min;
6) adding Fmoc-Ser 1mmol, HBTU 1.5mmol, DIPEA 2mmol and 10ml DMF, adding the prepared solution into the solid phase synthesizer, and reacting for 2 h;
7) repeating the steps 5) and 6), sequentially adding Fmoc-Ser, Fmoc-Gly, Fmoc-Phe and 2-naphthylacetic acid, washing with DMF for 5 times, washing with DCM for 5 times, and carrying out the next reaction;
8) as 95% TFA, 2.5% TIS, 2.5% H2Adding 10mL of solution consisting of O in volume percent into the solid phase synthesizer, reacting for half an hour (or preparing a TFA solution with the volume percent concentration of 1% by volume by using a TFA/DCM volume ratio of 1: 99), adding 3mL of the TFA solution into the solid phase synthesizer each time, adding ten times, wherein the reaction time is 1min each time), cutting the product from the 2-Cl-Trt resin, concentrating in vacuum, removing the solvent to obtain a crude product, and then separating and purifying by using HPLC (high performance liquid chromatography) to obtain the polypeptide derivative Nap-FFGSGSS with the yield of 75%.
(2) Preparation of nanofibers of polypeptide derivatives
Weighing 1.0mg of purified polypeptide derivative Nap-FFGSSSR, placing the polypeptide derivative Nap-FFGSSSR in a 2mL glass bottle, adding 500 μ L of PBS (pH 7.4), adjusting the pH value to 7.4 by using sodium carbonate solution, heating to boil to completely dissolve the compound, and cooling to room temperature to obtain colorless transparent invertible hydrogel. Whether or not the gel is formed is determined by methods known in the art, such as by inverting the bottle, leaving a hydrogel at the bottom of the bottle and a liquid if flowable. The hydrogel is colorless and transparent, can be placed at normal temperature for more than 1 month, and shows good solubility and storage stability.
Weighing 1.1mg of purified polypeptide derivative Nap-FFGSSSR, placing the purified polypeptide derivative Nap-FFGSSSR in a 2mL glass bottle, adding 1mL of PBS (pH 7.4), adjusting the pH value to 7.4 by using a sodium carbonate solution, heating to boiling to completely dissolve the compound, sucking 1 mu L of the compound by using a microsampler, adding the mixture into 999 mu L of the PBS solution, and heating to boiling to completely dissolve the compound to obtain 1 mu M Nap-FFGSSSR solution; and (3) sucking 10 mu L of Nap-FFGSSSR solution with the concentration of 1 mu M by using a microsampler, adding the Nap-FFGSSSR solution into 990 mu L of PBS solution, and heating to boil to completely dissolve the compound to obtain 10nM Nap-FFGSSSR solution. The electron microscope photo shows that the internal structures of the Nap-FFGSSSR solution with the concentration of 1 mu M and 10nM are all nanofibers.
Comparative preparation example 1
Sterile 1 XPBS
Weighing 8g NaCl, 0.2g KCl and 1.44g Na2HPO4And 0.24g KH2PO4Dissolving in 800ml distilled water, adjusting pH value of the solution to 7.4 with HCl, and adding distilled water to constant volume to 1L. Sterilizing with autoclave, and storing in refrigerator at room temperature or 4 deg.C.
Comparative preparation example 2
0.47mg of purified polypeptide derivative Ac-SSSR was weighed into a 2mL glass vial, 1mL of PBS solution (pH 7.4) was added, the pH was adjusted to 7.4 with sodium carbonate solution, the solution was boiled to completely dissolve the compound, and after cooling down the vial was inverted to find a fluid solution without hydrogel formation, to obtain a 1mM Ac-SSSR solution.
Comparative preparation example 3
1.8mg of IGF-1 protein powder was weighed out and placed in a 2mL glass bottle, and 500 μ L of PBS solution (pH 7.4) was added thereto, and the pH thereof was adjusted 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 hydrogel of polypeptide derivative Nap-FFGSSSR at cell level
(1) Experiment for promoting proliferation of mouse myoblast (C2C12)
1) Heating the water bath to 37 deg.C in advance, preheating culture medium and serum in the water bath, and simultaneously turning on ultra-clean bench ultraviolet lamp to irradiate for half an hour;
2) the frozen mouse myoblast C2C12 was removed from the liquid nitrogen tank, placed quickly in a 37 ℃ water bath to thaw the cells, and then quickly transferred to a clean bench for the following operations: carefully transferring the solution containing the cells into a centrifuge tube containing a culture medium by using a pipette, centrifuging for 3min, removing a supernatant, resuspending the cell in a DMEM culture medium containing 10% fetal calf serum, transferring the cell into a culture dish, and then putting the cell into an incubator at 37 ℃ for culture;
3) observing the cell state on the next day, and carrying out the following experiment after the cell state is good and first generation;
4) adding 2mL pancreatin, gently shaking, uniformly covering, tapping the bottom of the culture dish, digesting at 37 ℃ for 3min, observing most cell suspension under a microscope, blowing and beating 5-10 times by using a 1mL gun, adding 2mL culture medium, uniformly blowing and beating, collecting cell culture solution, sucking into a centrifuge tube, centrifuging at the rotating speed of 1000rpm for 3min, then discarding supernatant solution, adding DMEM culture medium containing 10% fetal calf serum, uniformly blowing and beating by using a gun, counting by using a cell counting plate to obtain the cell suspension containing 5 multiplied by 10 per milliliter7And (4) cells.
5) The cells were resuspended in a 24-well plate with a glass slide placed on the bottom, and 1mL of DMEM medium containing 10% fetal bovine serum was added to 5 ten thousand cells per well, and cultured overnight in an incubator at 37 ℃.
6) mu.L of a stock solution of dexamethasone (hereinafter referred to as Dex) at a concentration of 1mM was added to 990. mu.L of DMEM medium containing 10% fetal bovine serum to prepare 1ml of a medium containing 10. mu.M Dex. mu.L of 1mM Dex stock solution and 10. mu.L of 1mM Ac-SSSR solution were added to 980. mu.L of DMEM medium containing 10% fetal bovine serum to prepare 1ml of a medium containing 10. mu.MDex and 10nM Ac-SSSR. The same similar preparation method resulted in a medium containing 10. mu.M Dex and 10nM Nap-FFGSSSR, a medium containing 10. mu.M Dex and 10nM IGF-1. To the control group, 1ml of DMEM medium containing no Dex and any polypeptide derivative and containing only 10% fetal bovine serum was added.
7) And (3) sucking a DMEM culture medium containing 10% fetal calf serum the next day, adding 1mL of the culture medium prepared in advance in the step 6 into each well, incubating in an incubator at 37 ℃ for 24h, staining the cells according to the instruction of an EdU staining kit, randomly selecting 3 visual fields in each group by using a confocal microscope for photographing, finally counting the EdU positive cells by using Image J software, and counting the difference among the groups.
FIG. 1 is a graph showing the effect of the Nap-FFGSSSR hydrogel in promoting the proliferation of mouse myoblasts (C2C12) by detecting the number of positive cells by the EdU method. In the figure, a Control histogram represents the number of EdU positive cells stained after C2C12 is cultured in a cell culture medium without Nap-FFGSSSR for 24 hours and then is incubated for 3 hours by EdU, other histograms represent the number of EdU positive cells stained after C2C12 is respectively cultured in a culture medium containing dexamethasone (Dex), Dex + Ac-SSSR, Dex + Nap-FFGSSSR and Dex + IGF-1 for 24 hours and then is incubated for 3 hours by EdU, the obvious reduction of the number of the EdU positive cells in the Dex group indicates that the EdU positive cells can really inhibit the proliferation of the C2C12 cells, and the number of the EdU positive cells in the Dex + Nap-FFGSSSR and Dex + IGF-1 groups is far greater than that in the Control group when the cells are in a normal growth state, which indicates that the Nap-FFGSSSR has the function of promoting the proliferation of C2C 12. The number of EdU positive cells in the Dex + Ac-SSSR group was slightly more than that in the Dex group, but was much less than that in the Control group, the Dex + Nap-FFGSSSR group and the Dex + IGF-1 group, indicating that Ac-SSSR has a weak function of promoting the proliferation of C2C 12.
(2) Experiment for resisting apoptosis of mouse myoblast (C2C12)
1) After repeating the above steps 1) -4) of the cell proliferation experiment, the cells were resuspended in 24-well plates, 5 ten thousand cells per well and 1mL of DMEM medium containing 10% fetal bovine serum, and cultured overnight in an incubator at 37 ℃.
2) mu.L of a stock solution of dexamethasone (hereinafter referred to as Dex) at a concentration of 1mM was added to 990. mu.L of DMEM medium containing 10% fetal bovine serum to prepare 1ml of a medium containing 10. mu.M Dex. mu.L of 1mM Dex stock solution and 10. mu.L of 1mM Ac-SSSR solution were added to 980. mu.L of DMEM medium containing 10% fetal bovine serum to prepare 1ml of a medium containing 10. mu.M Dex and 10nM Ac-SSSR. The same similar preparation method resulted in a medium containing 10. mu.M Dex and 10nM Nap-FFGSSSR, a medium containing 10. mu.M Dex and 10nM IGF-1. To the control group, 1ml of DMEM medium containing no Dex and any polypeptide derivative and containing only 10% fetal bovine serum was added.
3) And (3) sucking a DMEM culture medium containing 10% fetal calf serum the next day, adding 1mL of the culture medium prepared in advance in the step 2 into each well, incubating in an incubator at 37 ℃ for 24h, staining cells according to the instruction of the TUNEL staining kit, randomly selecting 3 visual fields in each group by using a confocal microscope for photographing, counting TUNEL positive cells by using Image J software, and counting the difference among the groups.
FIG. 2 is a graph showing the effect of Nap-FFGSSSR hydrogel on the resistance to Dex-induced apoptosis of mouse myoblasts (C2C12) by detecting the number of positive cells by the TUNEL method. In the figure, a Control histogram represents the number of TUNEL positive cells stained after C2C12 is cultured in a cell culture medium without Nap-FFGSSSR for 24h and then cultured by TUNEL for 1h, other histograms represent the number of TUNEL positive cells stained after C2C12 is cultured in a culture medium containing dexamethasone (Dex), Dex + Ac-SSSR, Dex + Nap-FFGSSSR and Dex + IGF-1 for 24h and then cultured by TUNEL for 1h, and the number of the TUNEL positive cells in a Dex group is obviously increased, which shows that the cells can really induce C2C12 apoptosis, and the number of the TUNEL positive cells in the Dex + Nap-FFGSSSR and Dex + IGF-1 groups is equivalent to that of the Control group with cells in a normal growth state, which indicates that the Nap-FFGSSSR has the function of resisting Dex-induced C2C12 apoptosis. While the number of TUNEL positive cells in the Dex + Ac-SSSR group was slightly less than that in the Dex group, but much more than in the Control group, Dex + Nap-FFGSSSR and Dex + IGF-1 groups, suggesting that Ac-SSSR has weak function against Dex-induced apoptosis of C2C 12.
(3) Experiment of polypeptide hydrogel activating insulin signal path for treating muscular atrophy
1) After repeating the above steps 1) -4) of the cell proliferation experiment, the cells were resuspended in 6-well plates, 10 ten thousand cells per well and 1mL of DMEM medium containing 10% fetal bovine serum, and cultured overnight in an incubator at 37 ℃.
2) mu.L of a stock solution of dexamethasone (hereinafter referred to as Dex) at a concentration of 1mM was added to 990. mu.L of DMEM medium containing 10% fetal bovine serum to prepare 1ml of a medium containing 10. mu.M Dex. mu.L of 1mM Dex stock solution and 10. mu.L of 1mM Ac-SSSR solution were added to 980. mu.L of DMEM medium containing 10% fetal bovine serum to prepare 1ml of a medium containing 10. mu.M Dex and 10nM Ac-SSSR. The same similar preparation method resulted in a medium containing 10. mu.M Dex and 10nM Nap-FFGSSSR, a medium containing 10. mu.M Dex and 10nM IGF-1. To the control group, 1ml of DMEM medium containing no Dex and any polypeptide derivative and containing only 10% fetal bovine serum was added.
3) Sucking out DMEM medium containing 10% fetal calf serum the next day, adding each component into 1mL of the prepared medium in step 2 in each well, incubating in an incubator at 37 ℃ for 24h, extracting total protein, performing SDS-PAGE electrophoresis, transferring membranes, adding primary antibodies of T-AKT, P-AKT (T308) and P-AKT (S473), incubating at 4 ℃ overnight, adding secondary antibodies, incubating for 1h, adding color developing solution, exposing with an exposure apparatus, and taking pictures. Quantitative calculations were performed using Image J software to assess the extent of AKT phosphorylation and activation of the insulin signaling pathway.
The activation of the insulin signaling pathway by Nap-FFGSSSR hydrogel was evaluated by detecting the expression of T-AKT, P-AKT (T308) and P-AKT (S473) using Western Blot. The results are shown in FIG. 3, and whether the corresponding substances are added is shown by plus (+) signs and minus (-) signs, and it can be seen from the figure that three genes of Control group with cells in normal growth state are normally expressed in the group without adding four substances of Dex, Ac-SSSR, Nap-FFGSSSR and IGF-1, once Dex is added, the expression level of the two genes of P-AKT (T308) and P-AKT (S473) is obviously reduced, and after Nap-FFGSSSR and IGF-1 are added, the expression level of the two genes starts to be up-regulated again, which indicates that Nap-FFGSS can activate insulin signaling pathway, and after Ac-SSSR is added, the expression level of the two genes is slightly increased, which indicates that Ac-SSSR can partially activate insulin signaling pathway.
Animal example 1:
experiment for treating muscular atrophy in mice
1) Establishing a model: a female C57BL/6 mouse with the age of 6-8 weeks is selected, the weight is 20g +/-3 g, Dex is injected according to the dose of 0.2mg/kg/day every day for 28 consecutive days, and a muscle atrophy mouse model is constructed.
2) Administration treatment and statistics: after successful modeling, mice were randomly divided into 5 groups of 6 mice each, and leg portions of mice of different groups were injected once a week with 100 μ L of PBS buffer solution and a solution containing 10 μ M of Ac-SSSR, Nap-FFGSSSR, IGF-1 protein. The control group was injected with PBS buffer. After 4 weeks, the mice were monitored for changes in muscle mass before and after treatment using a dual energy X-ray detector. The change of muscle mass of mice in each group is counted, and the difference between the groups is analyzed statistically.
FIG. 4 is a graph showing changes in muscle mass in mice. From the figure, it can be seen that the muscle mass of the mice in Dex group is obviously reduced, and the muscle mass of the mice in Nap-FFGSSSR and IGF-1 group is equivalent to that of normal mice, indicating that the Nap-FFGSSSR can treat the muscle atrophy of the mice. The muscle mass of the mice in the Ac-SSSR group was slightly increased, indicating that Ac-SSSR had a slight therapeutic effect on muscle atrophy in the mice.
Preparation example 2:
polypeptide derivative Npx-DFDFGSSSR and synthesis and preparation of hydrogel thereof
(1)Npx-DFDSolid phase synthesis of FGSSSR
The method comprises the following specific steps:
1) weighing 0.5mmol of 2-Cl-Trt resin in a solid phase synthesizer, adding 10mL of anhydrous DCM, placing on a shaking table, shaking for 5min, and fully swelling the 2-Cl-Trt resin;
2) removing DCM from the solid phase synthesizer containing 2-Cl-Trt resin by washing the ear with an ear bulb;
3) dissolving 0.75mmol of Fmoc-Arg in 10mL of anhydrous DCM, adding 0.75mmol of DIPEA, then transferring to the solid phase synthesizer, supplementing 0.75mmol of DIPEA, and reacting for 1h at room temperature;
4) and (3) sealing: removing reaction liquid in the solid phase synthesizer by using an aurilave, washing with 10mL of anhydrous DCM for 1min each time for 5 times, adding 20mL of prepared solution with the volume ratio of anhydrous DCM to DIPEA to methanol being 17: 1: 2, and reacting at room temperature for 10 min;
5) removing reaction liquid in the solid phase synthesizer by using an aurilave, washing by using anhydrous DCM for 5 times, washing by using 10mL of DCM for each time, washing for 1min, washing by using DMF for 5 times, washing by using 10mL of DMF for each time, washing for 1min, washing for 5 times, adding 10mL of DMF containing 20% of piperidine by volume, reacting for 25min, reacting for 5min by using 10mL of DMF containing 20% of piperidine by volume, washing by using DMF for 5 times, washing by using 10mL of DMF for each time, washing for 1min, and washing for 5 times, and carrying out the next reaction;
6) adding Fmoc-Ser 1mmol, HBTU 1.5mmol, DIPEA 2mmol and 10mL DMF, adding the prepared solution into the solid phase synthesizer, and reacting for 2 h;
7) repeating the steps 5) and 6), sequentially adding Fmoc-Ser, Fmoc-Gly, Fmoc-D-Phe and (+) -alpha-methyl-6-methoxy-2-naphthylacetic acid, washing with DMF for 5 times, washing with DCM for 5 times, and carrying out the next reaction;
8) as 95% TFA, 2.5% TIS, 2.5% H2Adding 10mL of solution consisting of O in volume percent into the solid phase synthesizer, reacting for half an hour (or preparing a TFA solution with the volume percent concentration of 1% by taking the volume ratio of TFA to DCM as 1: 99), adding 3mL of the TFA solution into the solid phase synthesizer each time, adding ten times, wherein the reaction time is 1min each time), cutting the product from the 2-Cl-Trt resin, concentrating in vacuum, removing the solvent to obtain a crude product, and then separating and purifying by HPLC to obtain the polypeptide derivative Npx-DFDFGSSSR, yield about 80%.
(2) Preparation of nanofibers of polypeptide derivatives
Weighing 1.0mg of the purified polypeptide derivative Npx-DFDFGSSSR, placed in a 2mL glass bottle, added 500 μ L PBS solution (pH 7.4), adjusted to pH 7.4 with sodium carbonate solution, heated to boiling to completely dissolve the compound, cooled to room temperature to obtain a colorless transparent invertible hydrogel. The method of inverting the bottle is adopted to judge whether the gel is formed or not, the hydrogel is remained at the bottom of the bottle, and the liquid is formed if the hydrogel is flowing. The hydrogel has good solubility and can be placed at normal temperature for more than 1 month. The hydrogel is colorless and transparent, can be placed at normal temperature for more than 1 month, and shows good solubility and storage stability.
Preparation example 3:
synthesis and preparation of polypeptide derivative Npx-FFGSSSR and nanofiber thereof
The procedure was as in preparation example 2, except that Fmoc-D-Phe added in step 7) of (1) solid phase synthesis was changed to Fmoc-Phe.
Comparative preparation example 4:
polypeptide derivative Nap-FFGSRSS and synthesis and preparation of nano-fiber thereof
The procedure was as in preparation example 2 except that Fmoc-Arg added in step 3) of (1) solid phase synthesis was changed to Fmoc-Ser, Fmoc-Gly, Fmoc-D-Phe, (+) -alpha-methyl-6-methoxy-2-naphthylacetic acid and that Fmoc-Arg, Fmoc-Ser, Fmoc-Gly, Fmoc-Phe, 2-naphthylacetic acid were added in this order in step 7).
The microstructures of the hydrogels formed from the polypeptide derivatives obtained in preparation examples 1 to 3 and comparative preparation example 4 were observed by negative staining technique. First, 10 μ L of hydrogel was pipetted with a microsampler and added to the carbon-coated copper grid. Half a minute later the hydrogel was wiped dry with filter paper, the copper mesh was rinsed twice with water, and then the water was wiped dry with filter paper. After 10. mu.L of uranium acetate was sucked up with a microsampler and dyed for 1min, the dye solution was sucked up with filter paper, and the copper mesh was dried overnight in a desiccator. . The microscopic morphology of the hydrogel was observed by transmission microscopy the next day, and the results are shown in FIG. 5, in which A, B, C, D corresponds to Npx-DFDFGSSSR (H1), Npx-FFGSSSR (H2), Nap-FFGSSSR (H3) and Nap-FFGSRSS (H4). It can be seen that all four hydrogels have a large number of uniform nanofibers intertwined to form a three-dimensional network structure, but the diameters of the fibers are about 26.8nm, 14.6nm, 9.8nm and 17.3nm, respectively.
Weighing 1.1mg of the purified polypeptide derivative Npx-DFDFGSSSR, put into 2mL glass bottle, add 1mL PBS solution (pH 7.4), adjust its pH value to 7.4 with sodium carbonate solution, heat to boil and dissolve the compound completely, suck 1 uL with microsampler and add to 999 uL PBS solution, heat to boil and dissolve the compound completely to get 1 uM Npx-DFDFGSSSR solution; 10 μ L of Npx-one with a concentration of 1 μ M was aspirated with a microsamplerDFDFGSSSR solution is added into 990 μ L PBS solution, and then heated to boiling to completely dissolve the compound, thus obtaining Npx-DFDFGSSSR solution. The electron microscope photographs show that the internal structures of both solutions are nanofibers. A solution of Npx-FFGSSSR was prepared at 1. mu.M and 10nM in the same manner. Two kinds of pictures are shown by electron microscopeThe internal structure of the solution is all nano-fiber.
Determination of polypeptide derivative Npx-DFDBinding constants of FGSSSR (H1) to IGF-1 receptor protein was labeled with fluorescent dye NT-647 using a single molecule NT protein labeling kit. PBS buffer containing 0.05% Tween-20 (pH 7.4) was used as the detection buffer. The concentration of the fluorescently labeled IGF-1 receptor protein was kept constant at 10. mu.M, and Npx-DFDFGSSSR (H1) formed hydrogels with concentrations diluted from 2 μ M to 0.054nM in multiples, resulting in a series of concentration-gradient solutions. The fluorescent protein solution was then mixed with solutions of different concentrations at a 1:1 volume ratio. After 1 minute of incubation, the samples were loaded into nt.115 standard glass capillaries and analyzed using nt.115 monomer system. KD values were calculated using the nanotemperer 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, and the results are shown in FIG. 6, in which A, B, C, D corresponds to Npx-DFDFGSSSR (H1), Npx-FFGSSSR (H2), Nap-FFGSSSR (H3), Nap-FFGSRSS (H4). Npx-DFDThe binding constants of FGSSSR (H1), Npx-FFGSSSR (H2) and Nap-FFGSSSR (H3) and IGF-1 receptor protein are 145.36nM, 172.54nM and 229.75nM respectively, which shows Npx-DFDFGSSSR (H1), Npx-FFGSSSR (H2) and Nap-FFGSSSR (H3) have specific binding interaction with IGF-1 receptor protein, and Npx-DFDFGSSSR has the strongest binding ability to IGF-1 receptor protein, Npx-FFGSSSR (H2) and Nap-FFGSSSR (H3) the second. Nap-FFGSRSS (H4) has no binding constant to IGF-1 receptor protein, indicating that it is unable to bind.
Cell example 2:
(1) experiment for inhibiting conversion of mouse peritoneal macrophages to foam cells
1) 3ml of 4% sulfur colloid sterilized at high temperature is injected into the abdominal cavity of the mouse at one time for treatment for 4 to 5 days;
2) after euthanasia, the abdominal crux is dissected, and 10ml of sterilized PBS is injected into the abdominal cavity twice to obtain PBS solution containing macrophages;
3) centrifuging at 1000rpm for 3min, discarding supernatant, suspending cells with RPMI1640 culture medium, and plating;
4) after 1h, the cells (resuspended monocytes and blood cells) were washed with sterile PBS and the adherent macrophages were allowed to stabilize for two days before treatment.
5) 10 μ L of Npx-one with a concentration of 1 μ MDFDFGSSSR solution was added to 990. mu.L of RPMI1640 medium containing 10% fetal bovine serum to prepare 1ml of 10nM Npx-DFDFGSSSR (H1). Culture media containing 10nM of Npx-FFGSSSR (H2), Nap-FFGSSSR (H3), Nap-FFGSRSS (H4), or IGF-1 protein were prepared in the same manner.
6) Taking the glass sheets out of the absolute ethyl alcohol by using a pair of dissecting forceps, baking the glass sheets on an alcohol lamp without baking the glass sheets to be broken, and then paving the glass sheets one by one in each hole of a 24-hole plate; using 3ml RPMI1640 culture medium containing 10% fetal calf serum to blow and beat abdominal cavity macrophage uniformly, then adding into the 24-well plate with spread sheet, respectively adding culture medium prepared in step 5 into different groups of wells, adding 1ml RPMI1640 culture medium containing 10% fetal calf serum and not containing any polypeptide derivative into control (C) group, and placing into CO at 37 deg.C2An incubator;
7) after culturing for 16h, sucking away the culture medium in a 24-well plate, adding 1ml of 1 XPBS to wash the cells twice, sucking away the PBS, adding 500 mu l of 4% paraformaldehyde, and fixing for 30min at room temperature; after absorbing paraformaldehyde, washing the cells twice by using PBS, then adding 600 mu l of oil red O working solution, and dyeing for 45-60min at room temperature; washing the cells for 3-4 times by using ultrapure water, washing off redundant staining solution, and then adding 450 mu l of hematoxylin staining solution for staining for 30 s; placing the cells in ultrapure water, standing for 6min to change the hematoxylin-stained cell nucleus from purple to blue; dropping a proper amount of mounting agent on the glass slide, carefully taking out the glass slide by using a dissecting forceps, slightly sucking the liquid on the filter paper, and carefully covering the side of the glass slide with cells on the mounting agent to avoid bubbles; placing the wafer on a clean experiment table for air drying, and then taking and storing images under an upright microscope; macrophages containing lipid droplet numbers >20 were defined as foam cells and the foam cell formation ratio was calculated. The results are shown in FIG. 7A.
(2) Experiments to increase expression levels of lipid transporters ABCA1 and ABCG1
1) After repeating steps 1) -5) of the above cell experiment, the cells were resuspended in 6-well plates, 10 ten thousand cells per well and 1mL of RPMI1640 medium containing 10% fetal bovine serum, and cultured overnight in an incubator at 37 ℃.
2) The next day, the RPMI1640 medium containing 10% fetal bovine serum was aspirated, and 10nM of Npx-DFDFGSSSR (H1), 10nM Npx-FFGSSSR (H2), 10nM Nap-FFGSSSR (H3), 10nM Nap-FFGSRSS (H4) and IGF-1 protein RPMI1640 medium containing 10% fetal calf serum each well 1mL, control (C) group was 1mL RPMI1640 medium containing no polypeptide derivative and only 10% fetal calf serum, total protein was extracted after incubation in 37 ℃ incubator 24H, SDS-PAGE was performed, membrane transfer was performed, primary antibody of ABCA1 and ABCG1 was added for overnight incubation at 4 ℃, secondary antibody was added for incubation 1H, color developing solution was added, and pictures were taken after exposure with exposure apparatus. Quantitative calculations were performed using Image J software to assess the expression level of lipid transporters. The results are shown in FIG. 7B.
The massive increase of foam cells is an important signal for the development of atherosclerosis, so we can find the foam cells by staining. As shown in FIG. 7A, the IGF-1 protein and the polypeptide derivative Npx were addedDFDThe content of foam cells in the group of FGSSSR (H1), Npx-FFGSSSR (H2) and Nap-FFGSSSR (H3) is significantly lower than that in the group of control (C), so that IGF-1 protein and the three polypeptide derivatives have the function of reducing macrophage lipid accumulation. The content of foam cells in the group added with the polypeptide derivative Nap-FFGSRSS (H4) is not much different from that in the group added with the polypeptide derivative control (C), which indicates that the content cannot reduce the lipid accumulation of macrophages. And we found that the number of foam cells in the H1 group is much smaller than that in the IGF-1 protein group, indicating that the ability to reduce macrophage lipid accumulation is stronger than that of the IGF-1 protein. As can be seen from FIG. 7B, in the case of addition of the polypeptide derivative Npx-DFDThe expression quantity of two proteins, ABCA1 and ABCG1, in the group of FGSSSR (H1) or Npx-FFGSSSR (H2) is obviously improved, H1 is obviously better than that of IGF-1 protein group, and H2 and IGF-1 protein group have equivalent effects. N is a radical ofThe protein expression level of the ap-FFGSSSR (H3) group is also improved, but is obviously weaker than that of the IGF-1 protein group. The protein expression level of Nap-FFGSRSS (H4) group is not increased, and the effect is equivalent to that of control (C) group.
Animal example 2:
experiment for treating atherosclerosis of mice with ApoE gene knockout
1) Establishing a model: selecting atherosclerosis-promoting model mice and male ApoE gene-deficient mice, randomly grouping (15 mice in each group), feeding high-fat food containing 21% of fat and 0.5% of cholesterol, feeding for 16 weeks, and constructing the atherosclerosis model of the mice.
2) Administration treatment and statistics: mice were injected intramuscularly with PBS buffer and Npx containing 1. mu.M once a week for 16 weeks of model constructionDFDFGSSSR (H1), Npx-FFGSSSR (H2), Nap-FFGSSSR (H3), Nap-FFGSRSS (H4) and IGF-1 protein, and the injection amount is 100. mu.L. In which PBS buffer solution was injected, control (group C).
3) Tissue section staining: at the end of the experiment, all mice were euthanized with an excess of 2,2, 2-tribromoethanol (640mg/kg) and then aortic, blood and peritoneal macrophages were collected. Aortic root cross-section sections were prepared and stained with lipid oil red O to detect sinus lesions. The results are shown in FIG. 8.
As can be seen from FIG. 8, pass Npx-DFDThe aortic plaque of the FGSSSR (H1) treated mice was significantly reduced, and the effect was superior to that of IGF-1 proteome. The ratio of Npx-FFGSSSR (H2) and Nap-FFGSSSR (H3) group to control (C) group can also reduce the number of specks, but the effect is weaker than Npx-DFDFGSSSR (H1) and IGF-1 protein. Nap-FFGSRSS (H4) failed to reduce aortic plaque.
The cytokine content of the collected mouse serum was measured by ELISA method, and the result is shown in FIG. 9A. The expression of proinflammatory cytokines in the aorta was determined by RT-PCR and the results are shown in FIG. 9B. The Nap-FFGSRSS (H4) group is found to express a higher level of inflammatory factors, similar to the control (C) group. And Npx-DFDFGSSSR (H1) group, Npx-FFGSSSR (H2) group, Nap-FFGSSSR (H3) group, TNF alpha, IL-1 beta and IL-6 of IGF-1 groupThe factor content is reduced. Wherein Npx-DFDThe FGSSSR (H1) group has the lowest content, which indicates that the FGSSSR (H1) group can effectively reduce the expression of proinflammatory cytokines, thereby inhibiting the generation and the development of inflammation and treating atherosclerosis.
In conclusion, the anti-atherosclerotic effect of the polypeptide derivatives is: npx-DFDFGSSSR (H1) is obviously superior to IGF-1 protein, Npx-FFGSSSR (H2) has equivalent effect with IGF-1 protein, Nap-FFGSSSR (H3) is slightly inferior to IGF-1 protein, Nap-FFGSRSS (H4) has almost no effect and has equivalent effect with control (C) group.
The above description is only for facilitating the understanding of the technical solutions of the present invention by those skilled in the art, and is not intended to limit the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A polypeptide derivative is 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,
when X is Nap, all amino acids are in L configuration;
when X is Npx, Phe is simultaneously in L configuration or D configuration, and the rest are in L configuration.
2. The polypeptide derivative of claim 1, wherein the sequence of said polypeptide derivative is Nap-Phe-Gly-Ser-Arg, and wherein all amino acids are in L-configuration.
3. A nanofiber comprising the polypeptide derivative of claim 1, wherein said nanofiber is formed by heating and cooling an aqueous mixture of said polypeptide derivative.
4. The nanofiber according to claim 3, wherein the specific method for forming the nanofiber by heating and cooling the aqueous mixture of the polypeptide derivative is as follows: adding the polypeptide derivative into a PBS (phosphate buffer solution) with the pH value of 5.0-9.0, adjusting the pH value of the solution to 6.0-7.0 by using a sodium carbonate solution or a hydrochloric acid solution, heating to boiling to completely dissolve the compound, and cooling to room temperature to obtain a mixture of 10 nM-1 MuM nanofiber containing the polypeptide derivative.
5. Use of the nanofibres according to claim 3 or 4 for the preparation of a medicament for the treatment of muscle wasting, wherein the sequence of the polypeptide derivative is Nap-Phe-Gly-Ser-Arg, all amino acids being in L configuration.
6. Use of the nanofibers according to claims 3 or 4 in the preparation of a medicament for the treatment of atherosclerosis, wherein the sequence of said polypeptide derivative is Npx-Phe-Phe-Gly-Ser-Ser-Ser-Arg, wherein Phe is simultaneously in L or D configuration and the remaining amino acids are in L configuration.
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| FR2943346B1 (en) * | 2009-03-19 | 2011-05-06 | Univ Paris Curie | PEPTIDES MODULATING THE ACTIVITY OF IGF-1 AND THEIR APPLICATIONS |
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| CN107778367B (en) * | 2017-11-14 | 2020-09-29 | 南开大学 | Polypeptide, application thereof and medicine containing polypeptide |
| CN109957000B (en) * | 2017-12-14 | 2022-08-02 | 南开大学 | Polypeptide derivative for promoting cell proliferation and preparation method and application thereof |
| CN110642937B (en) * | 2019-10-11 | 2021-04-06 | 南开大学 | Polypeptide derivative, nanofiber and application thereof |
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2019
- 2019-10-11 CN CN201910962002.1A patent/CN110642937B/en active Active
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2020
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| CN110642937A (en) | 2020-01-03 |
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