TW202021610A - Application of peptide for treatment/prevention of hypertension and associated diseases capable of recovering blood pressure of individual to normal blood pressure range or regulating blood pressure - Google Patents

Abstract

The present invention discloses an application of peptide for treatment/prevention of hypertension and associated diseases, which means to administer an effective dosage of peptide or composition containing the peptide to an individual with hypertension for recovering the blood pressure of the individual to a normal blood pressure range or regulate the blood pressure of the individual to lower the blood pressure and thus achieve the effect of treatment or prevention of hypertension and associated diseases.

Description

Use of short peptides for the treatment/prevention of hypertension and related diseases

The present invention relates to the use of peptides, in particular to the use of a short peptide to treat/prevent hypertension and related diseases.

According to the theory, hypertension is a chronic disease that affects millions of people all over the world, and is a risk factor for myocardial infarction, heart failure, stroke, kidney damage and so on. In the current clinical treatment and prevention of hypertension, in addition to improving lifestyle and eating habits, for patients who have already suffered from hypertension, ACE inhibitors are often used as drugs for the treatment of hypertension, although ACE inhibitors can achieve Control blood pressure and protect other organs from being damaged by high blood pressure. However, synthetic ACE inhibitors such as captopril, enalapril or lisinopril have been studied and pointed out that long-term use will have adverse side effects on the human body, such as coughing, dizziness, Headache, kidney and liver damage, etc.

It can be seen that the long-term use of the drugs currently used in clinical practice will cause health concerns for the human body, causing some patients to resist drug treatment and face a high risk of cardiovascular disease. Therefore, if it can provide a treatment for hypertension and It can prevent the composition with good effect and can reduce the side effects on the human body, which will greatly help the prevention and treatment of hypertension and related diseases.

The main purpose of the present invention is to provide a short peptide for the treatment/prevention of hypertension and related diseases, which means that by administering an effective amount of a short peptide or a composition containing the short peptide to a patient An individual with hypertension can restore the individual's blood pressure to within the normal blood pressure range, or regulate the individual's blood pressure so that the blood pressure drops, so as to achieve the effect of treating or preventing hypertension and related diseases.

Another object of the present invention is to provide a short peptide for the preparation of a composition for inhibiting proteins related to cardiomyopathy. Specifically, the short peptide can inhibit the expression of proteins related to cardiomyopathy to reduce The risk of cardiomyopathy related diseases, especially heart diseases related to hypertension. In other words, by administering an effective amount of the short peptide or a composition containing the short peptide to an individual suffering from hypertension, it is possible to effectively treat or prevent cardiovascular diseases related to hypertension.

Among them, the protein system related to cardiomyopathy is phosphorylated p38, phosphorylated-ERK and phosphorylated-JUN, BNP, IL-6, Rac1, phosphorylated-JAK2, STAT3, MMP-2, TIMP1, CTGF, uPA, TLR-4, p-NFkB p65, TNF-α, BAD, apoptotic enzyme 3, activated Cleaved caspase 3, cytochrome C or at least two of the above proteins.

Another object of the present invention is to provide a use of a short peptide for the preparation of a mitochondrial regeneration promoter, which means that by administering an effective amount of the short peptide to an individual suffering from hypertension, it can maintain the The activity of mitochondria in individual cardiomyocytes is used to prevent or treat hypertension-related diseases.

In order to achieve the above objective, a short peptide is disclosed in the embodiment of the present invention, the amino acid sequence of which includes SEQ ID No. 1.

In an embodiment of the present invention, the amino acid sequence of the short peptide is SEQ ID No. 1.

In another embodiment of the present invention, the amino acid sequence of the short peptide is SEQ ID No.2.

The present invention discloses a short peptide whose amino acid sequence contains the sequence shown in SEQ ID No. 1. Specifically, the amino acid sequence of the peptide is SEQ ID No.: 1, SEQ ID No.: 2, or a sequence containing SEQ ID No.: 2.

Since the short peptides disclosed in the present invention can regulate blood pressure and restore individual blood pressure from hypertension to the normal blood pressure range, it can be used as a composition for treating or preventing hypertension and related diseases, for example, by treating hypertension Blood pressure can effectively prevent hypertensive kidney disease, hypertensive heart disease, liver disease and other diseases.

Furthermore, it has been confirmed through animal experiments that the short peptides disclosed in the present invention can also effectively inhibit the expression of related proteins that cause cardiomyopathy and promote the regeneration of mitochondria in cardiomyocytes to protect cardiomyocytes from hypertension. It can cause pathological changes and can effectively improve the damage of cardiomyocytes and restore the damaged cardiomyocytes to a state similar to that of normal cardiomyocytes.

The short peptide disclosed in the present invention can be used as a compound, and in addition to the short peptide as the active ingredient, the composition must be matched with a pharmaceutically or food-acceptable carrier.

The short peptides disclosed in the present invention can be prepared by common methods in the technical field of the present invention, such as extraction or hydrolysis from organisms, and can also be chemically synthesized by peptides, or by recombinant microorganisms or genetically modified animals. It is prepared as a production platform, and it can be understood by those with ordinary knowledge in the technical field to which the invention belongs to, without affecting the normal physiological function of the short peptide disclosed in the present invention, obtained from the 5'end of the amino acid sequence or Additional peptide fragments for modification are added to the 3'end to improve the stability or characteristics of the short peptide disclosed in the present invention, and also achieve the effects of the present invention.

The IF peptide disclosed in the present invention refers to a peptide whose amino acid sequence is SEQ ID No. 1, and its description is shown in Table 1 below. Table 1: Description of IF peptides

The DF peptide disclosed in the present invention refers to the peptide whose amino acid sequence is SEQ ID No.: 2, and its description is shown in Table 2 below. Table 2: Description of DF peptides

In the following, in order to verify the efficacy of the technical features disclosed in the present invention, a number of examples and figures are given for further explanation as follows.

The animal experiments in the following examples are conducted by the Laboratory Animal Use and Care Guidelines (No. 85-23, 1996) according to the Animal Research Committee of China Medical University.

The SHR (spontaneously hypertensive rats) used in the following examples are rats that develop spontaneously hypertensive rats. At present, most studies use SHR rats as an animal model to simulate human hypertension.

Example 1: Preparation of short peptide

The IF peptide and DF peptide disclosed in the present invention were prepared by chemical synthesis, and the purity of the IF peptide and DF peptide was confirmed by HPLC. The results are shown in the first and second figures, showing that the chemical synthesis The peptide obtained by the method has high purity.

Example 2: Animal test

Take a plurality of male SHR rats and raise them at 22°C under a 12-hour light/dark cycle environment. The rats were randomly divided into 6 groups, each with 6 rats, and they were reared for 8 weeks at the age of 17 weeks according to the following group conditions. Among them: The first group: WHY rats, given phosphate buffer; the second group: SHR rats were given phosphate buffer; The third group: SHR rats were given IF peptides at a daily dose of 10 mg/kg; the fourth group: SHR rats were given DF peptides daily The dose was 10 mg/kg; the fifth group: SHR rats were given ACE inhibitor (Captopril) at a daily dose of 5 mg/kg.

After the aforementioned test structure, the blood pressure of each group of rats was measured with an instrument (Softron, BP-2010 series), and then each of the rats was sacrificed, and the heart tissue, muscle, and serum were taken for subsequent tests.

Example 3: Blood pressure detection

The heart rate and blood pressure of each group of rats were measured, and the results are shown in Table 3. The results in Table 3 show that compared with the first group of rats, the second group of rats does have hypertension, which means that the second group of rats are hypertensive rats; the clinical drugs are administered In the fifth group of rats, its systolic blood pressure was slightly lower than that of the second group of rats, but its diastolic blood pressure and its mean arterial pressure were similar to those of the second group of rats, and there was no significant reduction effect; and the third group And the fourth group of rats were lower than the second group of rats in terms of systolic blood pressure, diastolic blood pressure, or mean arterial pressure, and the diastolic blood pressure reduction effect was significantly better than that of the fifth group of rats.

It can be seen from the above results that the DF peptide and the IF peptide system disclosed in the present invention respectively have the effect of lowering blood pressure, and can achieve the same or better effect than clinical ACE inhibitors in regulating blood pressure. In other words, the DF peptide and IF peptide disclosed in the present invention can indeed be used as a composition for treating or preventing hypertension and related diseases.

Table 3: Heart rate and blood pressure of rats in each group

Example 4: Heart function test

The heart system of each group of rats after sacrifice is shown in the third figure, and the body weight, heart weight, and tibia length of each group of rats were measured, and statistical analysis was performed. The results are shown in Table 4. Table 4: The results of measuring and calculating the body weight, heart weight, tibia length and the ratio of each group of rats

In addition, the rats in each group were detected by cardiac ultrasound, and the results are shown in the fourth figure. The long blue arrow indicates diastole and the short yellow arrow indicates cardiac contraction. In addition, the results of the cardiac ultrasound of each group of rats are detected and analyzed. , As shown in Table 5 below, where: IVSd is the end-diastolic ventricular septum thickness; LVIDd is the end-diastolic left ventricular inner size; LVPWd is the end-diastolic left ventricular posterior wall thickness; IVSs is the end-systolic ventricular septal thickness; LVIDs is The internal dimensions of the left ventricle at the end of systole; LVPWs is the thickness of the posterior wall of the left ventricle at the end of systole; EDV is the end-diastolic volume; ESV is the end-systolic volume; EF is the ventricular ejection rate; FS is the fraction of shortening, which means the left ventricle during each systole The degree of chamber shrinkage; SV is the cardiac output, which means the volume of blood discharged during each ventricular contraction; LVd Mass is the left ventricular end-diastolic mass; LVs is the left ventricular end-systolic mass.

Table 5: Analytical results of each rat's heart detected by cardiac ultrasound

From the results in Table 4 and Figure 3, it can be seen that compared with other groups of rats, the heart system of the second group of rats was significantly enlarged, and the third group and the third group of short peptides of the present invention were administered There was no significant difference between the heart size of the four groups of rats and the first group of rats. This shows that the short peptides disclosed in the present invention can effectively improve the symptoms of cardiac hypertrophy.

Furthermore, from the results in Table 5 and Figure 4, it can be seen that the cardiac function of the second group of rats is significantly lower than that of the first group of rats, indicating that SHR rats will cause cardiac function damage due to hypertension and cause cardiac Compared with the second group of rats, the occurrence of related diseases was significantly improved in the fifth group of rats administered with ACE inhibitors, showing that ACE inhibitors can indeed treat hypertension and improve the situation of impaired cardiac function ; Compared with the second group of rats, the visceral functions of the third and fourth groups of rats were restored to the state similar to the first group of rats, and compared to the fifth group of rats , The cardiac function of rats in the third and fourth groups was significantly better.

From the above results, it can be seen that the IF peptide and DF peptide system disclosed in the present invention can be used to treat or prevent heart disease caused by hypertension, and can restore the damaged heart function to nearly normal status. In other words, the IF peptides and DF peptides disclosed in the present invention can be used as pharmaceutical compositions for the treatment of hypertension and related diseases, respectively.

Example 5: Tissue staining section test

The rat hearts of each group were tissue sectioned, and H&E staining and plum three-color staining were performed respectively. The results are shown in the fifth and sixth figures.

From the results of the fifth and sixth graphs, it can be seen that the cardiomyocytes of the first group of rats are tightly arranged and structurally complete; the cardiomyocytes of the second group of rats are loosely arranged and have more interstitial spaces, and the myocardial tissue Contains a lot of collagen, showing that the cardiomyocyte cell line of the second group of rats is damaged by hypertension and has symptoms of fibrosis; compared with the second group of rats, whether it is the fifth group of clinical drugs administered In mice, either the third or fourth group of rats administered the short peptide disclosed in the present invention, the arrangement of cardiomyocytes is as neat as the first group of rats, and there is no collagen accumulation in the tissues.

It can be seen that the IF peptide and DF peptide disclosed in the present invention can not only be used to treat or prevent heart disease caused by hypertension, but also can repair damaged cardiomyocytes. Therefore, the IF peptide and DF peptide disclosed in the present invention The peptide can be used as a composition for treating or preventing hypertension and related diseases.

Example 6: Detection of blood biochemical value

The levels of uric acid, creatinine, AST (Aspartate Aminotransferase), ALT (Alanine transaminase) and creatine kinase (creatine kinase) in the serum of rats in each group were detected. The results are shown in Figures 7 to 11.

From the results in Figures 7 to 11, it can be seen that the rats in the second group of hypertensive mode were not given any drugs, so the serum levels of uric acid, creatinine, AST, ALT and creatine kinase were all higher than those in the first group. One group of rats is high, indicating that the heart, liver and kidney functions of the second group of rats are affected by hypertension and damaged, and even risk of myocardial infarction and kidney disease; and by administering the IF peptide disclosed in the present invention or The DF peptide is capable of reducing the indexes related to liver function in rat blood: ALT, AST, and the index related to heart function: the content of creatine kinase, showing that the IF peptide and DF peptide system disclosed in the present invention can Protect organs from being damaged by blood flow pressure caused by high blood pressure, greatly reducing the risk of individuals suffering from kidney disease, heart disease, myocardial infarction, and myocarditis.

It can be seen from the above results that the IF peptide and DF peptide system disclosed in the present invention can be used to treat or prevent diseases caused by hypertension, and can be used as a composition for treating or preventing liver damage, heart disease, and kidney disease.

Example 7: Detection of protein expression in heart cells (1)

The Western blot method was used to detect the protein expression in the heart cells of each group of rats, and after statistical analysis, the results are shown in Figures 12 to 27. Furthermore, the myocardial tissues of each group of rats were observed by TUNEL method and DAPI staining method. The results are shown in Figure 28. Then, the western blot method was used to detect the apoptosis and cell survival in each group of rat myocardial cells. Related protein performance, after quantitative statistics, the results are shown in Figure 29 to Figure 32.

From the results of the twelfth figure to the twentieth figure, it can be seen that the second group of rat lines will make the proteins related to cardiac hypertrophy: phosphorylated p38, phosphorylated-ERK and phosphorylated-JUN, BNP, IL-6, Rac1, The expression of phosphorylated-JAK2 and STAT3 increase, which can induce cardiac hypertrophy and related diseases; compared with the second group of rats, the third group of IF peptide or DF peptide disclosed in the present invention is administered And the fourth group of rats, which can reduce the protein expression related to cardiac hypertrophy, and can effectively prevent and treat heart diseases caused by hypertension.

From the results in Figure 21 to Figure 24, it can be seen that the second group of rat lines will increase the expression of proteins related to cardiac fibrosis: MMP-2, TIMP1, CTGF and uPA, which will increase the expression of cardiomyocytes. Toward fibrosis and induce heart disease; compared with the second group of rats, the third and fourth groups of rats administered with the IF peptide or DF peptide disclosed in the present invention can reduce the heart fiber To prevent and treat the heart disease caused by hypertension.

From the results in Figure 25 to Figure 27, it can be seen that the hypertension in the second group of rats is related to inflammation-related proteins in cardiomyocytes: TLR-4, p-NFkB p65 and TNF-α expression Increase, indicating that the heart will be damaged by high oxidative stress; compared with the second group of rats, the third and fourth groups of rats administered the IF peptide or DF peptide disclosed in the present invention can be Reduce the performance of the above-mentioned inflammation-related factors to achieve the effect of preventing heart disease caused by hypertension.

From the results of Figure 28 to Figure 32, it can be seen that based on the second group of rat strains, the proteins related to cardiac apoptosis: BAD, apoptotic enzyme 3, and activated apoptotic enzyme 3 (Cleaved The expression of caspase 3) and cytochrome C increased, and the protein related to cell survival: Beclin decreased, which means that high blood pressure will gradually apoptosis of cardiomyocytes and increase the incidence of heart disease; compared with the second group of rats The third and fourth groups of rats administered with the IF peptide or DF peptide disclosed in the present invention can treat the symptoms of hypertension and regulate blood pressure, thereby enabling the expression of protein related to cardiac apoptosis Reduce and increase the expression of protein related to cell survival to achieve the effect of preventing and treating heart disease caused by hypertension.

Example 8: Detection of protein expression in heart cells (2)

The Western blot method was used to detect the expression of proteins related to mitochondrial regeneration in rat cardiomyocytes of each group: SIRTI, FOXO3a and CREB, and the results shown in Figure 33 to Figure 35 were obtained after quantitative statistics.

The results from Figure 33 to Figure 35 show that the mitochondrial system in the cardiomyocytes of the second group of rats suffering from hypertension is in a state of damage, which means that the mitochondria of cardiomyocytes synthesize ATP. Decreased ability, unable to supply enough energy to the heart cells, which will affect the function of the heart and increase the possibility of heart-related diseases; compared with the second group of rats, the IF peptide disclosed in the present invention was administered Or the rats in the third and fourth groups of DF peptide can not only treat the symptoms of hypertension, regulate blood pressure and reduce inflammation, but also regulate the path of mitochondrial regeneration and keep the mitochondria of cardiomyocytes active. Continuously synthesize enough ATP to myocardial cells to reduce the possibility of heart-related diseases.

The first picture is the result of IF peptide analysis by HPLC. The second figure is the result of HPLC analysis of DF peptide. The third picture is the heart taken out after the sacrifice of each group of rats. The fourth image is to observe the images of the hearts of rats in each group with cardiac ultrasound. The fifth figure shows the results of H&E staining of rat heart tissue sections in each group. The sixth picture shows the results of the three-color staining of the heart tissue sections of rats in each group. The seventh figure is the result of detecting the content of uric acid in the serum of rats in each group. The eighth figure is the result of detecting the serum creatinine content of rats in each group. The ninth figure is the result of detecting the content of AST in the serum of rats in each group. The tenth figure is the result of detecting the ALT content in the serum of rats in each group. The eleventh figure is the result of detecting the content of serum creatine kinase in each group of rats. The twelfth figure is the results of the TUNEL method and DAPI staining method to detect the heart tissue of each group of rats. The thirteenth picture is the western ink spot method to detect and quantitatively analyze the expression of p-ERK in the myocardial tissue of each group of rats. The fourteenth picture shows the detection and quantitative analysis of the expression of p-JUK in the myocardial tissue of each group of rats by the Western blot method. The fifteenth picture shows the detection and quantitative analysis of the expression of p-p38 in the myocardial tissue of each group of rats by the Western blot method. The sixteenth picture is the detection and quantitative analysis of the expression of BNP in the myocardial tissue of each group of rats by the Western blot method. The seventeenth picture shows the detection and quantitative analysis of IL-6 expression in the myocardial tissue of each group of rats by Western ink dot method. The eighteenth figure is to detect and quantitatively analyze the expression of Rac1 in the myocardial tissue of each group of rats by the western ink dot method. The nineteenth picture is the detection and quantitative analysis of the expression of p-JAK2 in the myocardial tissue of each group of rats by the western ink spot method. The twentieth figure is a Western blot method to detect and quantitatively analyze the expression of STAT3 in the myocardial tissue of each group of rats. The twenty-first picture is a Western blot method to detect and quantitatively analyze the expression of MMP-2 in the myocardial tissue of each group of rats. The 22nd picture is the western ink spot method to detect and quantitatively analyze the expression of TIMP1 in the myocardial tissue of each group of rats. The twenty-third figure is a Western blot method to detect and quantitatively analyze the expression of CTGF in the myocardial tissue of each group of rats. The twenty-fourth picture is a Western blot method to detect and quantitatively analyze the expression of uPA in the myocardial tissue of each group of rats. The twenty-fifth image is a Western blot method to detect and quantitatively analyze the expression of TLR-4 in the myocardial tissue of each group of rats. The twenty-sixth image is a Western blot method to detect and quantitatively analyze the expression of p-NFkB p65 in the myocardial tissue of each group of rats. The twenty-seventh figure is a Western blot method to detect and quantitatively analyze the expression of TNF-α in the myocardial tissue of each group of rats. The twenty-eighth image is a Western blot method to detect and quantitatively analyze the expression of BAD in the myocardial tissue of each group of rats. The twenty-ninth picture shows the detection and quantitative analysis of the expression level of apoptotic enzyme 3 in the myocardial tissue of each group of rats by the Western blot method. The thirtieth figure is the western ink dot method to detect and quantitatively analyze the expression of activated apoptotic enzyme 3 in the myocardial tissue of each group of rats. The thirty-first figure is a Western blot method to detect and quantitatively analyze the expression of cytochrome C in the myocardial tissue of each group of rats. The thirty-second picture is the Western blot method to detect and quantitatively analyze the expression of Beclin in the myocardial tissue of each group of rats. The thirty-third figure is a Western blot method to detect and quantitatively analyze the SIRTI expression in the myocardial tissue of each group of rats. The thirty-fourth image is a Western blot method to detect and quantitatively analyze the expression of FOXO3a in the myocardial tissue of each group of rats. The thirty-fifth picture is the Western blot method to detect and quantitatively analyze the expression of CREB in the myocardial tissue of each group of rats.

Claims (10)

A use of a short peptide for preparing a composition for treating or preventing hypertension and related diseases, wherein the amino acid of the short peptide comprises SEQ ID No. 1. The use according to item 1 of the scope of patent application, wherein the amino acid sequence of the short peptide is SEQ ID No. 1. The use according to item 1 of the scope of patent application, wherein the amino acid sequence of the short peptide is SEQ ID No.2. According to the application described in item 1 of the scope of patent application, the diseases related to hypertension are selected from the group consisting of hypertensive kidney disease, hypertensive heart disease and liver disease. A use of a short peptide for the preparation of a composition for inhibiting a protein related to cardiomyopathy, wherein the amino acid series of the short peptide comprises SEQ ID No. 1; and the protein series related to a cardiomyopathy is Phosphorylated p38, phosphorylated-ERK and phosphorylated-JUN, BNP, IL-6, Rac1, phosphorylated-JAK2, STAT3, MMP-2, TIMP1, CTGF, uPA, TLR-4, p-NFkB p65, TNF- α, BAD, apoptotic enzyme 3, activated Cleaved caspase 3 or cytochrome C. The use according to item 5 of the scope of patent application, wherein the amino acid sequence of the short peptide is SEQ ID No. 1. According to the application described in item 5 of the scope of patent application, the amino acid sequence of the short peptide is SEQ ID No.2. A use of a short peptide for preparing a mitochondrial regeneration promoter, wherein the amino acid of the short peptide contains SEQ ID No. 1. The use according to item 8 of the scope of patent application, wherein the amino acid sequence of the short peptide is SEQ ID No. 1. The use according to item 8 of the scope of patent application, wherein the amino acid sequence of the short peptide is SEQ ID No.2.

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