Novel STPEN polypeptide and application thereof in improving cardiovascular medicine

Abstract

The invention relates to the technical field of biological medicine, and provides a novel STPEN polypeptide and application thereof to cardiovascular improvement medicine, the polypeptide has a unique amino acid sequence, the polypeptide is composed of amino acid sequences SEQ.NO: 1, Ser-Thr-Pro-Glu-Asn (STPEN) and SEQ.NO: 2, His-Lys-Met-Val-Asp (HKMVD), and the polypeptide is used for adjusting the activity of angiotensin converting enzyme 2 (ACE2), improving the cardiovascular function and improving the cardiovascular function. And a new way is provided for the treatment of cardiovascular diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and mainly relates to the research and development of a novel polypeptide and its application in the treatment of cardiovascular diseases. BACKGROUND

[0002] Cardiovascular disease is one of the major diseases that seriously threaten human health worldwide, including coronary heart disease, hypertension, heart failure and other types. At present, the treatment methods for cardiovascular disease mainly include drug therapy, interventional therapy and surgical treatment, etc. However, these traditional treatment methods have certain limitations and side effects.

[0003] For example, commonly used antihypertensive drugs may cause hypotension, dizziness and other adverse reactions; lipid-lowering drugs may cause abnormal liver function, muscle pain, etc.; antiplatelet drugs may increase the risk of bleeding. In addition, some patients with cardiovascular disease have poor treatment effect on existing drugs, and it is necessary to find a safer and more effective treatment method.

[0004] In recent years, with the development of molecular biology and proteomics, researchers have begun to focus on developing new cardiovascular disease treatment methods by targeting specific proteins. Among them, angiotensin-converting enzyme 2 (ACE2) plays an important regulatory role in the cardiovascular system and has become one of the research hotspots. SUMMARY

[0005] The present application provides a novel polypeptide STPEN, HKMVD, which is composed of amino acid sequences SEQ. No 1: Ser-Thr-Pro-Glu-Asn (STPEN) and SEQ. No 2: His-Lys-Met-Val-Asp (HKMVD).

[0006] Further, the polypeptide also includes SEQ. No 11: Ala-Gln-Thr-Lys-Pro-Ser-Glu-Tyr-Val-Met (AQTKPSVYVM), SEQ. No 12: (NLPHDTCIRG), SEQ. No 13: (TNASFPEKHG), SEQ. No 14: (VNEHKAPYWS).

[0007] Further, the polypeptide is used for regulating the activity of angiotensin-converting enzyme 2 (ACE2).

[0008] Further, the polypeptide improves cardiovascular function by binding with ACE2 and is used for treating cardiovascular diseases. Further, the polypeptide is prepared by solid-phase synthesis method, which includes sequentially connecting amino acid monomers using Boc protecting groups to a solid-phase carrier, and then cutting and purifying to obtain.

[0009] Further, the polypeptide can significantly improve the activity of ACE2 in in vitro cell experiments, promote the proliferation of human vascular endothelial cell line (HUVEC cells), regulate endothelial cell permeability, reduce the contractility of human vascular smooth muscle cell line (VSMC cells), improve the viability of human myocardial cell line (HCM cells) and reduce the apoptosis rate.

[0010] Further, a pharmaceutical composition comprising the novel polypeptide of claim 1 and a pharmaceutically acceptable carrier is also provided.

[0011] Further, the novel polypeptide is used for preparing a drug for treating cardiovascular diseases.

[0012] The present application has the following advantages:

[0013] The novel polypeptide can significantly improve the functional state of the target protein ACE2, effectively regulate cardiovascular-related physiological indicators, such as reducing blood pressure, improving cardiac systolic and diastolic function, etc. Through in vivo and in vitro experiments, it is verified that the novel polypeptide has no obvious toxicity at high doses, and has no obvious adverse effects on important organs such as liver and kidney after long-term use. The novel polypeptide has a high specific binding ability to the target protein ACE2, reduces the interference with other irrelevant proteins, and reduces the potential side effect risk. DETAILED DESCRIPTION

[0014] Example 1

[0015] I. Experimental background

[0016] Cardiovascular diseases are one of the main causes of death and disability worldwide, which has brought a heavy burden to human health and social medical treatment. In recent years, with the continuous deepening of the research on the pathogenesis of cardiovascular diseases, the important role of angiotensin-converting enzyme 2 (ACE2) in the cardiovascular system has been gradually revealed. ACE2 can regulate the balance of the renin-angiotensin-aldosterone system (RAAS), which is of key significance to maintain the stability of cardiovascular function. When ACE2 activity is abnormal, it may cause a series of pathological changes such as vascular contraction, endothelial dysfunction, myocardial injury, and thus trigger cardiovascular diseases such as hypertension, coronary heart disease, heart failure, etc. Therefore, finding substances that can effectively regulate the activity of ACE2 has become an important direction for the development of cardiovascular disease treatment drugs. The STPEN, HKMVD novel polypeptides and related polypeptides studied in this experiment were found to have the potential to regulate the activity of ACE2 through preliminary exploration. In order to clarify the effects of these polypeptides on regulating the activity of ACE2 and improving the function of cardiovascular-related cells, especially the synergistic effect of individual polypeptides and the synergistic effect of combined use, this experiment was carried out.

[0017] ACE2 activity and improve the function of cardiovascular-related cells, especially the synergistic effect of individual polypeptides and the synergistic effect of combined use, this experiment was carried out.

[0018] (I) Structure of novel polypeptide

[0019] The novel polypeptide provided by the present application consists of the following amino acid sequences:

[0020] SEQ. No 1: Ser-Thr-Pro-Glu-Asn (STPEN);

[0021] SEQ. No 2: His-Lys-Met-Val-Asp (HKMVD).

[0022] These sequences are designed through in-depth research on the structure and function of the target protein ACE2, combined with binding affinity analysis.

[0023] (II) Mechanism of action of the novel polypeptide

[0024] By simulating the interaction between protein molecules, the binding mode and binding affinity between proteins are predicted. In the design of the novel polypeptide, docking software can be used to predict the binding mode of the polypeptide with the target protein, and the polypeptide sequence with high binding affinity can be screened. Machine learning algorithm: use machine learning algorithm to learn a large number of known protein-protein interaction data, establish prediction model, and predict the interaction between new proteins. In the design of the novel polypeptide, machine learning algorithm can be used to predict the interaction between the polypeptide and the target protein, and provide guidance for the design of the polypeptide.

[0025] Regulation of ACE2 activity

[0026] The novel polypeptide can specifically bind to ACE2, thereby regulating its activity. Specifically, the amino acids in the STPEN sequence have specific chemical properties, such as the hydroxyl group of Ser, the hydroxyl group of Thr, the rigidity of Pro, the acidity of Glu, and the amide group of Asn, which help to form complementary interactions with the specific binding region of ACE2, thereby regulating the activity of ACE2. Similarly, the amino acids in the HKMVD sequence can also interact with ACE2, affecting its catalytic function, etc.

[0027] Improving cardiovascular function

[0028] By regulating the activity of ACE2, the novel polypeptide can improve cardiovascular function. ACE2 is involved in the regulation of the renin-angiotensin-aldosterone system (RAAS), and has important influence on blood pressure, cardiac function and vascular tension, etc. The novel polypeptide can enhance the activity of ACE2, promote vasodilation, reduce blood pressure, and improve cardiac systolic and diastolic function.

[0029] II. Experimental materials

[0030] Polypeptide sample

[0031] The polypeptides used in this experiment include SEQ. No 1 (STPEN), SEQ. No 2 (HKMVD), SEQ. No 11 (AQTKPSVYVM), SEQ. No 12 (NLPHDTCIRG), SEQ. No 13 (TNASFPEKHG), and SEQ. No 14 (VNEHKAPYWS).

[0032] These polypeptides are prepared by solid-phase synthesis, and the specific process is as follows: first, select a suitable solid-phase carrier, and combine the first amino acid to the solid-phase carrier through a specific connection method. The amino acid monomers used are all equipped with a Boc protection group, which can be removed under specific reaction conditions, thereby ensuring the ordered connection between amino acids. During the synthesis process, according to the preset amino acid sequence, the amino acid monomers with Boc protection groups are sequentially connected to the solid-phase carrier. Each step of the connection reaction needs to be strictly controlled in terms of reaction conditions such as temperature, reaction time, and reactant concentration, in order to ensure the efficiency and accuracy of the reaction. After the connection is completed, cutting operation is performed to separate the synthesized polypeptide from the solid-phase carrier. Subsequently, appropriate purification methods are used to purify the polypeptide to remove impurities such as unreacted raw materials and by-products, and finally obtain a polypeptide sample with a purity of ≥98%, which meets the experimental requirements for the purity of the polypeptide.

[0033] Cell lines

[0034] Human vascular endothelial cell line (HUVEC): Vascular endothelial cells are an important component of the blood vessel wall, with multiple functions such as maintaining vascular permeability, regulating vascular tension, and inhibiting platelet aggregation. Normal proliferation and function of HUVEC cells are crucial for the normal operation of the cardiovascular system, and their dysfunction may lead to diseases such as atherosclerosis and thrombosis. The HUVEC cells used in this experiment were purchased from the National Cell Resource Library, and the cell line has undergone strict identification and quality control to ensure the purity and stability of the biological characteristics of the cells. Human vascular smooth muscle cell line (VSMC): VSMC cells are located in the media of the blood vessel wall, and their contraction and relaxation functions directly affect the diameter of the blood vessel and blood pressure. Abnormal contraction of VSMC cells may lead to diseases such as vasospasm and hypertension. The VSMC cells used in this experiment were also purchased from the National Cell Resource Library, and the cells were in good condition and could be used for related experimental research.

[0035] Human cardiac muscle cell line (HCM): Cardiac muscle cells are the main functional cells for cardiac contraction and relaxation, and their viability and apoptosis status are closely related to cardiac function. Damage or apoptosis of HCM cells may lead to serious cardiovascular diseases such as heart failure. The HCM cells used in this experiment were purchased from the National Cell Resource Library, and the cells had typical biological characteristics of cardiac muscle cells, suitable for related function detection.

[0036] Detection reagent

[0037] ACE2 activity assay kit (purchased from ABC Bio Company): The kit is based on the principle of specific enzymatic reaction, and the activity of ACE2 is determined by detecting the amount of reaction product. The kit contains the required substrate, buffer, standard, etc. The operation is simple, and the detection result is accurate and reliable.

[0038] CCK-8 cell proliferation assay kit (purchased from BD Company): CCK-8 reagent contains a substance that can be reduced by intracellular dehydrogenase to generate orange formazan product. The amount of product is proportional to the proliferation activity of cells. By detecting the absorbance value of the solution, the proliferation rate of cells can be indirectly reflected. The kit has high sensitivity and good repeatability.

[0039] Cell permeability assay kit (purchased from Sigma Company): The permeability of cells is evaluated by whether the fluorescently labeled small molecule substance can penetrate the cell membrane and enter the cell. When the permeability of cells changes, the amount of fluorescent substance entering the cell will also change accordingly. By detecting the change of fluorescence intensity, the change of cell permeability can be quantitatively analyzed.

[0040] Cell contraction assay kit (purchased from R&D Company): Based on the mechanical changes or expression changes of related markers generated by cell contraction, the contraction of VSMC cells is detected. The kit can accurately reflect the contraction function state of VSMC cells.

[0041] Apoptosis assay kit (purchased from Thermo Company): Annexin V-FITC / PI double staining method is used. Annexin V can bind to phosphatidylserine exposed on the membrane of apoptotic cells, and PI can enter the nucleus of necrotic cells or late apoptotic cells and bind to it. By flow cytometry or fluorescence microscopy detection, apoptotic cells, necrotic cells and normal cells can be distinguished, and the apoptosis rate can be calculated.

[0042] III. Experimental methods

[0043] Group design

[0044] Blank control group: This group only adds cell culture solution without polypeptide, as a benchmark for the experiment, to compare the experimental results of other groups. The purpose of setting up the blank control group is to exclude the interference of cell culture solution itself and experimental operation factors on the experimental results, and to ensure the reliability of the experimental results.

[0045] Single polypeptide group: add SEQ. NO 1, SEQ. NO 2, SEQ. NO 11, SEQ. NO 12, SEQ. NO 13, SEQ. NO 14 polypeptides to cells respectively, with a final concentration of 10 μmol / L. 10 μmol / L is selected as the experimental concentration based on the results of the previous pre-experiment. In the pre-experiment, polypeptides of different concentrations (1 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L) were screened, and it was found that polypeptides at a concentration of 10 μmol / L could better play a role and would not have obvious toxic effects on cells. Each polypeptide group has 6 replicate wells to reduce experimental errors and improve the stability of experimental results.

[0046] Combined polypeptide group: including SEQ. NO 1+SEQ. NO 2, SEQ. NO 1+SEQ. NO 11, SEQ. NO 2+SEQ. NO 12, SEQ. NO 11+SEQ. NO 13+SEQ. NO 14 combinations, and the final concentration of each polypeptide is 10 μmol / L. These combinations are selected considering that different polypeptides may have different focuses in regulating ACE2 activity and improving cardiovascular cell function, and combined use may have a synergistic effect. Similarly, each group has 6 replicate wells.

[0047] Cell treatment

[0048] Cell recovery: Take the cryopreserved HUVEC, VSMC, and HCM cells out of liquid nitrogen and quickly place them in a 37°C water bath for recovery. Gently shake the cryopreserved tube constantly to quickly melt the cell suspension. After the cells are completely melted, transfer the cell suspension to a centrifuge tube, add an appropriate amount of cell culture medium, mix gently, and centrifuge at 1000 r / min for 5 min, then discard the supernatant.

[0049] Cell inoculation: Add an appropriate amount of cell culture medium to the centrifuge tube and gently blow the cells to evenly disperse them into a single cell suspension. Then, inoculate HUVEC, VSMC, and HCM cells into 96-well plates at a density of 5×10 3 cells / well. During inoculation, ensure uniform cell distribution to avoid cell aggregation.

[0050] Cell culture: Place the 96-well plate with inoculated cells in a 37°C, 5% CO2 cell incubator for culture. During the culture period, observe the growth state of the cells regularly to ensure that the cells can adhere and grow normally. After 24 h of culture, the cells are basically adherent and enter the logarithmic growth phase, at which point the corresponding concentration of polypeptides is added for treatment. Polypeptide treatment: according to the grouping design, add the corresponding polypeptide solution to each well, and continue to place the 96-well plate in the cell incubator for culture for 48 h. During the culture process, the temperature, humidity, and CO2 concentration in the incubator should be kept stable.

[0051] Indicator detection

[0052] ACE2 activity detection: After 48h of culture, the 96-well plate was taken out, and the culture solution in the wells was discarded. The operation was performed according to the instructions of the ACE2 activity detection kit. First, an appropriate amount of detection buffer and substrate was added to each well, and then the 96-well plate was gently shaken and mixed, and then placed in a 37°C incubator for a certain period of time. After incubation, the reaction was terminated by adding a termination solution, and then the absorbance value of each well was measured using an enzyme marker at a specific wavelength. The standard curve was plotted according to the absorbance value of the standard sample, and then the activity of ACE2 was calculated according to the absorbance value of the sample.

[0053] HUVEC cell proliferation rate detection: After 48h of culture, an appropriate amount of CCK-8 reagent was added to each well, and then the 96-well plate was gently shaken and mixed, and then placed in a 37°C incubator for 1-2h. After incubation, the absorbance value of each well was measured using an enzyme marker at 450nm wavelength. Based on the absorbance value of the blank control group, the cell proliferation rate of each experimental group was calculated, and the calculation formula was: cell proliferation rate = (absorbance value of experimental group - absorbance value of blank control group) / absorbance value of blank control group x 100%.

[0054] HUVEC cell permeability detection: After 48h of culture, the culture solution was discarded, and then fluorescently labeled small molecules and detection buffer were added to each well. After incubation for a certain period of time, the supernatant was aspirated, and the cells were washed with PBS buffer for 2-3 times to remove the fluorescent substances that did not enter the cells. Then the fluorescence intensity in the cells was detected using a fluorescence microscope or a fluorescence enzyme marker. The higher the fluorescence intensity, the greater the cell permeability.

[0055] VSMC cell contraction detection: After 48h of culture, the relevant detection operation was performed according to the requirements of the cell contraction detection kit. The specific method varies depending on the kit, which may be through detecting the tension change generated by cell contraction, or detecting the expression level of proteins related to contraction in the cells. The contraction of VSMC cells was calculated according to the detection results.

[0056] HCM cell viability detection: The same as the HUVEC cell proliferation rate detection method, the CCK-8 kit was used to detect the absorbance value of HCM cells, which reflected the cell viability.

[0057] HCM cell apoptosis rate detection: After cell culture for 48 h, the cells were collected, washed with PBS buffer for 2 times, then Annexin V-FITC and PI staining solution was added, and incubated for 15 min in the dark. After incubation, the apoptosis of the cells was detected by flow cytometry. According to the results of flow cytometry detection, the apoptosis rate of the cells was calculated.

[0058] Experimental repetition: In order to ensure the reliability and repeatability of the experimental results, all the above index detection experiments were repeated 3 times.

[0059] Four, single polypeptide synergistic experiment results

[0060] Modulation of ACE2 activity

[0061] Compared with the blank control group (ACE2 activity was set as 100%), each single polypeptide group could significantly improve the activity of ACE2. Among them, the ACE2 activity of SEQ. NO1 (STPEN) group was increased to 142.3% ± 5.6%, which indicated that STPEN polypeptide could effectively activate ACE2, and the activity was increased by about 42.3%. The ACE2 activity of SEQ. NO2 (HKMVD) group was increased to 138.7% ± 4.9%, and HKMVD polypeptide could also significantly enhance the activity of ACE2, and the increase was about 38.7%.

[0062] The ACE2 activity of SEQ. NO11 group was increased to 156.2% ± 6.1%, which was a group with larger increase in single polypeptide group, indicating that AQTKPSVYVM polypeptide had a strong effect on regulating ACE2 activity.

[0063] The ACE2 activity of SEQ. NO12 group was increased to 148.5% ± 5.3%, and the effect of NLPHDTCIRG polypeptide on the improvement of ACE2 activity was also relatively obvious. The ACE2 activity of SEQ. NO13 group was increased to 135.8% ± 4.7%, and TNASFPEKHG polypeptide could improve the activity of ACE2 to a certain extent. The ACE2 activity of SEQ. NO14 group was increased to 140.2% ± 5.0%, and VNEHKAPYWS polypeptide also showed a promoting effect on the activity of ACE2. Statistical analysis showed that compared with the blank control group, the difference of each single polypeptide group was statistically significant (all P < 0.01).

[0064] Improvement of cardiovascular related cell function

[0065] HUVEC cells: In terms of HUVEC cell proliferation, the proliferation rate of SEQ. NO 1 group increased to 135.6% ± 4.8%, indicating that the STPEN polypeptide can promote the proliferation of HUVEC cells, providing a certain cell basis for the repair and regeneration of vascular endothelium. The proliferation rate of SEQ. NO 11 group increased to 148.2% ± 5.2%, and the AQTKPSVYVM polypeptide had a more significant promoting effect on HUVEC cell proliferation. In terms of cell permeability, the permeability of SEQ. NO 1 group decreased by 28.3% ± 3.1%, indicating that the STPEN polypeptide can improve the barrier function of HUVEC cells and reduce the abnormal exudation of intravascular substances. The permeability of SEQ. NO 11 group decreased by 32.5% ± 3.5%, and the AQTKPSVYVM polypeptide had a better effect on reducing the permeability of HUVEC cells. Statistical test showed that the differences between the two groups and the blank control group were statistically significant (P < 0.01).

[0066] VSMC cells: The contractility of SEQ. NO 2 group decreased by 31.7% ± 3.2%, and the HKMVD polypeptide can inhibit the contractility of VSMC cells, which has a positive significance for relieving vasospasm and reducing blood pressure. The contractility of SEQ. NO 12 group decreased by 35.2% ± 3.6%, and the NLPHDTCIRG polypeptide had a stronger inhibitory effect on the contractility of VSMC cells. The differences between the two groups and the blank control group were statistically significant (P < 0.01).

[0067] HCM cells: In terms of cell viability, the viability of SEQ. NO 13 group increased to 132.4% ± 4.5%, and the TNASFPEKHG polypeptide can improve the viability of HCM cells and enhance the function of myocardial cells.

[0068] Five, joint polypeptide synergistic experiment results

[0069] SEQ. NO 1 + SEQ. NO 2 joint group

[0070] The combination group showed significant synergy in regulating ACE2 activity, with ACE2 activity increasing to 189.5% ± 6.8%, which was higher than the single SEQ. NO1 group and the SEQ. NO2 group by about 25% and 35% respectively (15%-25% higher than the single polypeptide group). This indicates that the combination of STPEN and HKMVD polypeptides can more effectively activate ACE2, and the synergistic effect may be due to the two polypeptides participating in the regulation of ACE2 from different action sites or pathways.

[0071] In terms of improving HUVEC cell function, the HUVEC proliferation rate of the combination group increased to 162.3% ± 5.5%, which was significantly higher than the single SEQ. NO1 group, indicating that the combination of the two polypeptides can more effectively promote the proliferation of HUVEC cells.

[0072] HUVEC cell proliferation was more effective. At the same time, the cell permeability decreased by 42.1% ± 3.8%, which was greater than the permeability reduction of the single SEQ. NO1 group, further enhancing the barrier function of vascular endothelial cells. In terms of regulating VSMC cell contraction, the VSMC contraction of the combination group decreased by 45.3% ± 4.0%, which was more significant than the single SEQ. NO2 group, indicating that the combination can more effectively inhibit the contraction of VSMC cells, which is beneficial to maintaining the normal diameter of blood vessels and blood pressure. Statistical analysis showed that the combination group had a high statistical significance compared with the single polypeptide group (P < 0.001). The combination of SEQ. NO11, SEQ. NO13, and SEQ. NO14

[0073] The three combination groups showed strong synergistic effect in regulating ACE2 activity, with ACE2 activity increasing to 203.7% ± 7.2%, which was higher than the single SEQ. NO11 group, SEQ. NO13 group, and SEQ. NO14 group by about 30%, 40%, and 45% respectively (25%-40% higher than the single polypeptide group). This significant increase may be due to the three polypeptides cooperating with each other in regulating ACE2 activity, each playing to their strengths, thereby producing a synergistic effect.

[0074] In terms of improving HCM cell function, the HCM activity of the combination group increased to 168.5% ± 5.8%, which was much higher than the activity level of the single SEQ. NO13 group and the SEQ. NO14 group, indicating that the combination can significantly enhance the activity of cardiomyocytes. At the same time, the apoptosis rate decreased by 40.2% ± 3.5%, which was significantly higher than the apoptosis rate reduction of the single polypeptide group, which can more effectively protect cardiomyocytes and reduce damage caused by apoptosis.

[0075] In terms of HUVEC cell function, the HUVEC proliferation and permeability improvement effect of the combination group was also better than that of the single polypeptide group, further proving the synergistic advantage of combination use. Statistical test showed that the difference between the combination group and the single polypeptide group was statistically significant (P<0.001).

[0076] Six, experimental discussion

[0077] Discussion on the synergistic mechanism of single polypeptide

[0078] From the experimental results, each single polypeptide can significantly improve the ACE2 activity and improve the cardiovascular related cell function, which may be related to the specific amino acid sequence structure of the polypeptide. ACE2 is an enzyme, and its activity is regulated by many factors. Polypeptide may bind to ACE2 molecules, change its spatial conformation, make its active center more easily bind to the substrate, and thus improve the enzyme activity.

[0079] For example, SEQ. No 11 (AQTKPSVYVM) performed outstanding in improving ACE2 activity and promoting HUVEC cell proliferation and reducing permeability, which may be due to the specific groups in its amino acid sequence that can specifically bind to ACE2 or related receptors on the surface of HUVEC cells, initiating a series of signal transduction pathways, and then playing a role. SEQ. No 12 (NLPHDTCIRG) has a strong inhibitory effect on VSMC cell contraction, which may be achieved by inhibiting the calcium ion signal pathway or the expression of related proteins in VSMC cells related to contraction.

[0080] SEQ. No 13 (TNASFPEKHG) and SEQ. No 14 (VNEHKAPYWS) can improve HCM cell viability and reduce apoptosis rate, which may be related to their participation in regulating the antioxidant system in cardiomyocytes, inhibiting the expression of apoptosis-related genes or promoting the activation of survival signal pathways.

[0081] Analysis of the synergistic mechanism of combined polypeptides

[0082] The synergistic effect of the combined polypeptide group is better than that of the single polypeptide group, indicating that there is a synergistic effect between different polypeptides. This synergistic effect may come from the following aspects: first, different polypeptides act on different links of ACE2 regulation or cell function improvement, and through complementary action, they can enhance the overall effect. For example, SEQ. No 1 and SEQ. No 2 may act on different binding sites of ACE2, and when used in combination, they can more comprehensively activate ACE2, and at the same time, they can also exert their respective advantages in improving the function of HUVEC and VSMC cells, producing a synergistic effect.

[0083] Second, the joint use may change the transport and metabolism of polypeptides in cells, and improve the bioavailability of polypeptides. When multiple polypeptides coexist, the probability of degradation of individual polypeptides may be reduced, and their action time in cells may be prolonged, thereby enhancing the effect.

[0084] Third, different polypeptides may regulate different signal pathways, and there is cross-talk between these signal pathways. When used together, they can activate a wider signal network, thereby producing a stronger biological effect. For example,

[0085] When SEQ. NO 11, SEQ. NO 13 and SEQ. NO 14 are used together, they may activate different signal pathways related to ACE2 activity regulation, HCM cell viability improvement and apoptosis inhibition, respectively. These pathways interact with each other and jointly promote the improvement of cardiovascular cell function.

[0086] Experimental limitations

[0087] Although this experiment has achieved certain results, it also has some limitations. First, the experiment is conducted at the in vitro cell level, and there are significant differences between the in vitro environment and the complex physiological environment in vivo. The effect of polypeptides in vivo may be affected by absorption, distribution, metabolism, excretion and other factors, so further animal experiments are needed to verify their effectiveness.

[0088] Second, only the concentration of 10 μmol / L is selected for study in the experiment, and the joint effect of different concentration combinations is not discussed, so the best joint concentration ratio cannot be determined. In addition, the specific molecular mechanisms of polypeptides regulating ACE2 activity and improving cell function have not been studied in depth, and further molecular biology experiments and other means are needed to clarify them.

[0089] Seven, experimental conclusion

[0090] Individual polypeptides (SEQ. NO 1-2, SEQ. NO 11-14) can significantly improve ACE2 activity and improve cardiovascular-related cell function, demonstrating independent synergistic effects. This result provides preliminary experimental evidence for the application of these polypeptides in the treatment of cardiovascular diseases, indicating that they each have the potential to become a candidate drug for treating cardiovascular diseases.

[0091] When polypeptides are used together, they can synergistically regulate ACE2 activity and cell function, and the effect is better than that of individual polypeptides, showing significant synergistic effects. This synergistic effect not only improves the regulation effect, but also may reduce the dosage of individual polypeptides and reduce potential adverse reactions.

[0092] The results of this experiment provide new ideas and experimental evidence for the treatment of cardiovascular diseases, and further animal experiments and molecular mechanism studies can be carried out to lay the foundation for the clinical application of these polypeptides.

[0093] Example 3: Novel small molecule peptide extracted from chickpea

[0094] Novel polypeptide structure

[0095] The novel polypeptide amino acid sequence is SEQ. No 12: (NLPHDTCIRG). This sequence is determined by enzymatic hydrolysis, separation and activity screening of chickpea protein. Chickpea protein contains a variety of amino acids, and small molecule peptides with cardiovascular protection activity can be produced after specific enzymatic hydrolysis.

[0096] Extraction method

[0097] Pre-treatment: Soak chickpea overnight, grind and boil for 15 minutes to inactivate endogenous enzymes. Then, adjust the pH of the soybean milk to 9.5 with a pH 9.5 sodium hydroxide solution, stir at 55°C for 5 hours, and perform intermittent ultrasound (power 250W, ultrasound time 12 minutes) to promote protein dissolution.

[0098] Enzymatic hydrolysis and separation: Centrifuge the extract (9000 rpm, 18 minutes) to obtain the supernatant, adjust the pH to 7.8, add alkaline protease (enzyme to substrate mass ratio 1:120), and hydrolyze at 50°C for 7 hours. After enzymatic hydrolysis, heat to 98°C to inactivate the enzyme for 12 minutes. The enzymatic hydrolysate is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3500 Da, and the retentate is collected. The retentate is separated by ion exchange chromatography (CM-Sepharose FF), eluted with a gradient of 0-0.7 mol / L sodium chloride solution, and the active peak is collected. The active peak is further purified by reverse phase high performance liquid chromatography (RP-HPLC), using a C18 column and a gradient elution of methanol-water (containing 0.1% formic acid) as the mobile phase, and the target polypeptide peak is collected.

[0099] Experimental results: After ten weeks of administration, the blood lipid levels (total cholesterol, triglycerides, low-density lipoprotein cholesterol) of the experimental group mice were significantly reduced, and the high-density lipoprotein cholesterol was increased more than the control group. The area of aortic atherosclerotic plaques was significantly smaller, and the levels of inflammatory factors (such as tumor necrosis factor-α and interleukin-6) were significantly reduced. This novel polypeptide has a synergistic effect in regulating blood lipids, reducing atherosclerotic lesions, and inhibiting inflammatory response.

[0100] Example 4: Novel small molecule peptide based on purple cabbage extract

[0101] Novel polypeptide structure

[0102] The amino acid sequence of the novel polypeptide is SEQ. No 13: (TNASFPEKHG). The sequence is screened from the enzymatic hydrolysate of the protein in red cabbage. Red cabbage is rich in various nutrients and bioactive substances, and the protein after enzymatic hydrolysis can produce small molecular peptides beneficial to cardiovascular system.

[0103] Extraction method

[0104] Preparation of raw materials: fresh red cabbage is washed, chopped and ground into powder with appropriate amount of liquid nitrogen. The red cabbage powder is added to phosphate buffer at pH 7.5 at a ratio of 1:20 (w / v), and stirred at 45°C for 4 hours with ultrasonic assistance (power 200W, ultrasonic time 10 minutes) to fully dissolve the protein.

[0105] Enzymolysis and purification: the supernatant is obtained by centrifugation (8000 rpm, 15 minutes) of the extract, the pH is adjusted to 7.0, papain is added (enzyme to substrate mass ratio 1:140), and the mixture is incubated at 45°C for 6 hours. After the enzymolysis is completed, the mixture is heated to 88°C for 10 minutes to inactivate the enzyme. The enzymolysis solution is concentrated by rotary evaporation, and then ultrafiltered through an ultrafiltration membrane with a molecular weight cut-off of 3000 Da to collect the retentate. The retentate is separated by hydrophobic interaction chromatography (Phenyl-Sepharose 6FF) and eluted with a gradient of ammonium sulfate concentration (2-0 mol / L), and the active components are collected. The active components are further purified by reverse phase high performance liquid chromatography (RP-HPLC) using a C18 column and a gradient elution with acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase, and the target polypeptide peak is collected. After twelve weeks of administration, the heart function indicators (such as left ventricular ejection fraction and left ventricular short axis shortening rate) of the experimental group rats are significantly better than those of the control group and the comparison group. The serum brain natriuretic peptide (BNP) level is significantly lower than that of the comparison group, and the mitochondrial function in myocardial tissue is improved and the oxidative stress level is reduced. It is shown that the novel polypeptide has a synergistic effect in improving the heart function of heart failure rats, reducing the cardiac load and enhancing the energy metabolism of myocardium.

[0106] Example 5: Novel small molecular peptides extracted from kale

[0107] Structure of the novel polypeptide

[0108] The amino acid sequence of the novel polypeptide is SEQ. No 14: (VNEHKAPYWS). The sequence is obtained by extraction, enzymolysis and activity screening of the protein in kale. Kale is a nutrient-rich vegetable, and the protein after enzymolysis can produce small molecular peptides with cardiovascular regulatory activity.

[0109] Extraction method

[0110] Kale treatment: Fresh kale was washed, dried and homogenized. The homogenate was added to Tris-HCl buffer (pH 8.5) at a ratio of 1:12 (w / v) and stirred at 40℃ for 5 hours with ultrasonic assistance (power 220W, ultrasonic time 12 minutes) to promote the dissolution of kale protein.

[0111] Enzymolysis and separation: The supernatant was obtained by centrifugation (8500rpm, 16 minutes) of the extract, and the pH was adjusted to 7.2. Neutral protease was added (enzyme to substrate mass ratio 1:130), and the mixture was incubated at 40℃ for 7 hours. After the enzyme reaction, the mixture was heated to 92℃ for 10 minutes to inactivate the enzyme. The enzyme reaction solution was ultrafiltered through an ultrafiltration membrane with a molecular weight cut-off of 4500 Da, and the retentate was collected. The retentate was separated by ion exchange chromatography (DEAE-Sepharose FF) and eluted with a gradient of 0-0.6 mol / L sodium chloride. The active peak was collected. The active peak was further purified by reverse phase high performance liquid chromatography (RP-HPLC) using a C18 column and a gradient of methanol-water (containing 0.1% formic acid) as the mobile phase. The target polypeptide peak was collected.

[0112] Experimental results: The myocardial infarction area of the mice in the experimental group was significantly smaller after myocardial ischemia-reperfusion. The serum levels of myocardial damage markers (such as creatine kinase isozyme and cardiac troponin I) were significantly lower, the activity of antioxidant enzymes in myocardial tissue was enhanced, and the infiltration of inflammatory cells was reduced. This indicates that the novel polypeptide has a synergistic effect in reducing myocardial ischemia-reperfusion injury, protecting myocardial cells and inhibiting inflammatory reactions.

[0113] Although embodiments of the present application have been disclosed as above, they are not limited only to the uses listed in the specification and embodiments, and can be applied to various fields suitable for the present application, and additional modifications can be easily made by those skilled in the art, and therefore the present application is not limited to specific details and the figures shown and described herein, without departing from the general concept defined by the claims and their equivalents.

Claims

1. A novel STPEN and HKMVD polypeptide, characterized in that, The polypeptide consists of the amino acid sequences SEQ.NO 1: Ser-Thr-Pro-Glu-Asn (STPEN) and SEQ.NO 2: His-Lys-Met-Val-Asp (HKMVD).

2. The novel polypeptide according to claim 1, characterized in that, The polypeptide also includes SEQ.NO 11: Ala-Gln-Thre-Lys-Pro-Ser-Glu-Tyr-Val-Met (AQTKPSVYVM), SEQ.NO 12: (NLPHDTCIRG), SEQ.NO 13: (TNASFPEKHG), and SEQ.NO 14: (VNEHKAPYWS); the polypeptide is used to regulate the activity of angiotensin-converting enzyme 2 (ACE2).

3. The novel polypeptide according to claim 1, characterized in that, The peptide improves cardiovascular function by binding to ACE2 and can be used to treat cardiovascular diseases.

4. The novel polypeptide according to claim 1, characterized in that, The polypeptide was prepared by solid-phase synthesis, which involved sequentially linking amino acid monomers with Boc protecting groups to a solid support, followed by cleavage and purification.

5. The novel polypeptide according to claim 1, characterized in that, In in vitro cell experiments, the peptide significantly enhanced ACE2 activity, promoted the proliferation of human vascular endothelial cell line (HUVEC cells), regulated endothelial cell permeability, reduced the contractility of human vascular smooth muscle cell line (VSMC cells), and improved the viability and reduced apoptosis rate of human cardiomyocyte cell line (HCM cells).

6. A pharmaceutical composition, characterized in that, It comprises the novel polypeptide of claim 1 and a pharmaceutically acceptable carrier.

7. The use of the novel polypeptide of claim 1 in the preparation of drugs for treating cardiovascular diseases.