CN113616637A

CN113616637A - Application of kaempferol in preparation of protein tyrosine phosphatase SHP-1 inhibitor

Info

Publication number: CN113616637A
Application number: CN202110920071.3A
Authority: CN
Inventors: 韩葳葳; 耿雅娇; 李婉南; 韩璐
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2021-08-11
Filing date: 2021-08-11
Publication date: 2021-11-09

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of kaempferol in preparation of an SHP-1 inhibitor and preparation of a medicine for treating SHP-1 related diseases. The invention provides an application of kaempferol in preparation of a protein tyrosine phosphatase SHP-1 inhibitor. In the invention, kaempferol can inhibit the activity of protein tyrosine phosphatase SHP-1 in vitro, has no toxic or side effect, and can be used for preparing the protein tyrosine phosphatase SHP-1 inhibitor. The kaempferol provided by the invention can inhibit the activity of protein tyrosine phosphatase SHP-1, so that the kaempferol can be used for preparing a medicament for treating diseases related to overhigh activity or abnormal overexpression of the protein tyrosine phosphatase SHP-1.

Description

Application of kaempferol in preparation of protein tyrosine phosphatase SHP-1 inhibitor

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of kaempferol in preparation of a protein tyrosine phosphatase SHP-1 inhibitor.

Background

Diabetes consists of a group of diseases characterized mainly by hyperglycemia, and is mainly classified into two types: the type I diabetes and the type II diabetes, wherein the type II diabetes accounts for 80 to 90 percent of the total disease incidence, and the main causes are that the tissues resist insulin and insulin secretion of islet beta cells is relatively insufficient. Insulin resistance means that the biological response of the target organ or tissue where insulin acts, such as liver, muscle, adipose tissue, etc., to a certain amount of insulin is below the expected normal level.

Reversible protein phosphorylation is a biological self-regulation mechanism, in the regulation process of cell signal transduction, protein phosphorylation and dephosphorylation are very important links, the phosphorylation of tyrosine residues of specific cell proteins is an important regulation mechanism of cell signal transduction, and the phosphorylation process dominates the fate of cells and regulates the functions of the cells. The study of tyrosine phosphorylation of proteins has become a focus of research in life sciences.

After years of research, SHP-1(SH 2-stabilizing tyrosine phosphatase 1) is a tyrosine phosphatase mainly expressed in hematopoietic cells, and is also called Hematopoietic Cell Phosphatase (HCP), SHPTP-1, SHP, PTP1C and PTPN 6. The cDNA was successfully cloned by sequential isolation and purification from research groups such as Washington university, etc., in the early 90 s of the 20 th century, with a total length of 641 amino acid residues and a molecular weight of 70510Da for SHP-1.

Research proves that SHP-1 in a human body can play a role in controlling blood sugar, compared with wild mice born at the same time, Ptpn6me-v/me-v mice with congenital functional deletion of SHP-1 protein strengthen insulin receptor signals of IRS-PI3K-Akt in liver and muscle and strengthen phosphorylation of CEACAM1, and have obvious glucose tolerance and insulin sensitivity, which indicates that the control capability of a diabetic patient on blood sugar can be possibly recovered by inhibiting the activity of SHP-1 protein in vivo. In addition, researchers have also found that SHP-1 inhibits the breakdown of insulin in the vicinity of the liver, explaining why insulin concentrations in obese patients are elevated due to metabolic disorders.

Therefore, screening specific inhibitors of SHP-1 is expected to improve the sensitivity of the body to insulin and effectively treat diseases such as type II diabetes, insulin resistance and obesity.

Disclosure of Invention

In view of the above, the present invention provides an application of kaempferol in the preparation of an inhibitor of protein tyrosine phosphatase SHP-1, wherein kaempferol can inhibit the activity of protein tyrosine phosphatase SHP-1 and can be used for preparing a medicine for treating diseases related to over-activity or over-expression of SHP-1.

In order to solve the technical problem, the invention provides the application of kaempferol in preparing a protein tyrosine phosphatase SHP-1 inhibitor.

Preferably, the inhibitor is a competitive inhibitor.

Preferably, the inhibitor comprises a drug for the treatment of a disease associated with hyperactivity or abnormal overexpression of the protein tyrosine phosphatase SHP-1.

Preferably, the diseases associated with the high activity or abnormal overexpression of the protein tyrosine phosphatase SHP-1 comprise diabetes, obesity, leukemia or immunodeficiency.

Preferably, the diabetes is type ii diabetes.

Preferably, the dosage form of the medicament comprises an injection, a tablet or a capsule.

The invention provides an application of kaempferol in preparation of a protein tyrosine phosphatase SHP-1 inhibitor. In the in vitro test of the invention, kaempferol can inhibit the activity of protein tyrosine phosphatase SHP-1, has no toxic or side effect, and can be used for preparing the protein tyrosine phosphatase SHP-1 inhibitor. The kaempferol provided by the invention can inhibit the activity of protein tyrosine phosphatase SHP-1, so that the kaempferol can be used for preparing a medicament for treating diseases related to overhigh activity or abnormal overexpression of the protein tyrosine phosphatase SHP-1.

Drawings

FIG. 1 is a point-line graph showing the concentration and the inhibition ratio of kaempferol in a reaction solution;

FIG. 2 is 1/V₀And 1/[ S ]]A dot line graph of;

FIG. 3 is a dot line graph of [ I ] and κ;

FIG. 4 is a bar graph comparing blood glucose levels in each experimental group.

Detailed Description

The invention provides an application of kaempferol in preparation of a protein tyrosine phosphatase SHP-1 inhibitor. In the invention, the kaempferol has a structure shown in a formula I:

in the present invention, the inhibitor is preferably a competitive inhibitor; the half inhibitory concentration of the inhibitor is 10.8 mu mol/L, and the dissociation constant of kaempferol to protein tyrosine phosphatase SHP-1 is 8.0 mu M.

SHP-1 is a dephosphorylating enzyme which dephosphorylates phosphorylated proteins. The p-nitrophenyl phosphate disodium salt (pNPP) can be dephosphorylated by SHP-1 to p-nitrophenol, which is yellow in color. In the embodiment of the invention, the change of the SHP-1 activity is preferably indirectly detected by detecting the change of the light absorption value at 405nm of the solution to be detected, so as to determine the inhibition of the kaempferol on the SHP-1. In the present invention, the solution to be tested is preferably prepared according to the following preparation method:

dissolving kaempferol in dimethyl sulfoxide to obtain a kaempferol solution;

mixing MOPS buffer solution, SHP-1 and kaempferol solution and preheating;

and mixing the preheated solution and the p-nitrophenyl phosphate disodium salt for reaction, and terminating the reaction by using an aqueous solution of sodium bicarbonate to obtain a solution to be detected.

In the invention, kaempferol is dissolved in dimethyl sulfoxide to obtain a kaempferol solution. The present invention is not particularly limited as long as the dissolution can be completed. The invention has no special requirement on the concentration of the kaempferol solution, as long as the concentration of the kaempferol in the mixed solution of the MOPS buffer solution, the SHP-1 and the kaempferol solution can meet the required requirement. In the present invention, the kaempferol solution is preferably stored at 4 ℃ for later use.

After the kaempferol solution is obtained, the MOPS buffer solution, the SHP-1 and the kaempferol solution are mixed and preheated. In the invention, the pH value of the MOPS buffer solution is preferably 6.8-7.2, and more preferably 7; the mol concentration of the 3-morpholine propanesulfonic acid in the MOPS buffer solution is preferably 48-52 mmol/L, and more preferably 50 mmol/L; the molar concentration of Dithiothreitol (DTT) in the MOPS buffer solution is preferably 1.8-2.2 mmol/L, and more preferably 2 mmol/L; the mass concentration of Bovine Serum Albumin (BSA) in the MOPS buffer solution is preferably 1.8-2.2 mg/mL, and more preferably 2 mg/mL; the mol concentration of sodium chloride in the MOPS buffer solution is preferably 0.18-0.22 mol/L, and more preferably 0.2 mol/L. In the present invention, the concentration of kaempferol in the mixed solution of MOPS buffer, SHP-1 and kaempferol solution is preferably 5. mu. mol/L, 10. mu. mol/L, 50. mu. mol/L and 100. mu. mol/L.

The mixing is not particularly limited in the present invention as long as it can be mixed uniformly. In the invention, the preheating temperature is preferably 35-39 ℃, and more preferably 37 ℃; the time is preferably 4.5-5.5 min, and more preferably 5 min.

After preheating, the preheated solution and the p-nitrophenyl phosphate disodium salt are mixed for reaction, and the reaction is terminated by using a sodium bicarbonate aqueous solution to obtain a solution to be detected. In the invention, the molar concentration of the p-nitrophenyl phosphate disodium salt in the reaction solution is preferably 98-102 mmol/L, and more preferably 100 mmol/L; the volume fraction of the kaempferol solution in the reaction solution of the reaction is preferably 9.8-10.2%, and more preferably 10%. The reaction temperature in the invention is preferably 35-39 ℃, and more preferably 37 ℃; the time is preferably 18-22 min, and more preferably 20 min.

The molar concentration of the sodium bicarbonate aqueous solution is preferably 0.08-0.12 mol/L, and more preferably 0.1 mol/L. In the present invention, the volume of the sodium bicarbonate aqueous solution and the volume of the reaction solution are preferably 0.8 to 1.2:1, and more preferably 1: 1.

In the invention, the inhibition rate of kaempferol on SHP-1 is calculated according to formula 1:

wherein A is₀The light absorption value of a blank control group (without adding kaempferol); and A is the light absorption value of the solution to be detected added with the kaempferol solution.

In the present invention, the inhibitor preferably comprises a drug, preferably for the treatment of a disease associated with an excessive or abnormal overexpression of the protein tyrosine phosphatase SHP-1. In the present invention, the disease associated with the high activity or abnormal overexpression of the protein tyrosine phosphatase SHP-1 preferably comprises diabetes, obesity, leukemia or immunodeficiency. In the present invention, the diabetes is preferably type ii diabetes; the diabetes is preferably streptozotocin-induced.

In the present invention, the dosage form of the drug preferably includes an injection, a tablet or a capsule.

In the invention, when the medicine is used for treating diabetes, the medicine is preferably injected intravenously, and the dosage of the medicine is preferably 20-80 mg/kg, and more preferably 40-60 mg/kg based on the mass of kaempferol.

In order to further illustrate the present invention, the following technical solutions provided by the present invention are described in detail with reference to test examples, but they should not be construed as limiting the scope of the present invention.

Determination of median inhibitory concentration (IC50)

Dissolving kaempferol in dimethyl sulfoxide to prepare a kaempferol solution;

mixing 90 μ L of LMOPS buffer (50mmol/L, pH7.0, containing 2mmol/L DTT, 0.2mol/L NaCl, 2mg/ml BSA), 1 μ g of SHP-1 and 10 μ L of kaempferol solution, and preheating at 37 deg.C for 5 min; after preheating, mixing the preheated solution with 0.01mol p-NPP to obtain a reaction solution with the volume of 100 mu L, reacting for 20min at 37 ℃, and terminating the reaction by using 100 mu L of sodium bicarbonate water solution with the molar concentration of 0.1mol/L to obtain a solution to be detected;

the light absorption value of the solution to be tested is measured at the wavelength of 405nm, the inhibition rate of kaempferol with different concentrations on SHP-1 is calculated according to the formula 1, and the result is shown in Table 1.

TABLE 1 inhibition of SHP-1 by kaempferol at different concentrations

A point line graph is drawn with the concentration of kaempferol in the reaction solution as abscissa and the inhibition ratio of kaempferol to SHP-1 as ordinate according to the data of Table 1, as shown in FIG. 1. As is clear from FIG. 1, the half maximal inhibitory concentration of kaempferol against SHP-1 was 10.8. mu. mol/L.

Determination of the type of suppression

Inhibiting Concentration (IC) according to the above-mentioned half₅₀) The difference lies in that when the concentration of kaempferol in the prepared reaction solution is 0 mu mol/L, the initial concentration of p-NPP is 0.25mmol/L, 0.5mmol/L, 1mmol/L, 2mmol/L, 4mmol/L and 10 mmol/L; when the concentration of kaempferol in the reaction solution is 20 mu mol/L, the initial concentrations of p-NPP are 0.25mmol/L, 0.5mmol/L, 1mmol/L, 2mmol/L, 4mmol/L and 10 mmol/L; when the concentration of kaempferol in the reaction solution was 50. mu. mol/L, the initial concentrations of p-NPP were 0.25mmol/L, 0.5mmol/L, 1mmol/L, 2mmol/L, 4mmol/L and 10 mmol/L.

Obtaining an initial rate (V) of the enzymatic reaction from the result of the detection₀) And at 1/V₀As ordinate, 1/[ S ]]A dot line graph is plotted for the abscissa, as shown in fig. 2. Wherein [ S ]]Is the initial substrate concentration, i.e.the initial concentration of p-NPP.

As can be seen from FIG. 2, the L-B equation for different inhibitor concentrations, which was plotted on the Y-axis, demonstrates that the inhibition of SHP-1 by kaempferol is a competitive inhibition.

Determination of dissociation constants

κ was calculated according to equation 2, and the results are shown in table 3:

where Km is the Michaelis constant and Vm is the maximum rate of the enzymatic reaction, [ I]Is the inhibitor concentration, K_iIs the suppression constant.

TABLE 3 dissociation constants and inhibitor concentration data

[I](μmol/L)	0	125	200
				κ	0	152	265

Plotting [ I ] as the abscissa and κ as the ordinate yields a straight line, as shown in FIG. 3. As can be seen from FIG. 3, the dissociation constant of kaempferol for SHP-1 was determined to be 8.0. mu. mol/L at the intersection with the X-axis.

Hypoglycemic effect of kaempferol on Streptozotocin (STZ) -induced type II diabetic mice

The experimental mouse is a Kunming mouse, is provided by Jilin university experimental animal center, is male, is 8 weeks old, and has the weight of 18-22 g; and feeding the animals in a clean-grade conventional animal laboratory at 22-25 ℃ for 12/12 hours day and night.

Drugs and reagents for this experiment:

high-sugar and high-fat feed: 10% of lard, 20% of cane sugar, 2.5% of cholesterol, 1% of sodium cholate and 66.5% of basic feed.

Normal feed: a base binder.

Citric acid-sodium citrate buffer (pH4.2-4.4): 2.1g of citric acid is dissolved in 100mL of ultrapure water to prepare solution A, 2.94g of sodium citrate is dissolved in 100mL of ultrapure water to prepare solution B, and 11.4mLB solution and 8.6mLA solution are mixed to prepare a citric acid buffer solution.

Streptozotocin (STZ): 50mg of STZ was weighed and dissolved in 10mL of a citric acid buffer solution with a molar concentration of 0.1mol/L at a concentration of 5mg/mL, and prepared immediately before use by dark-shielded operation on ice.

Establishing a diabetes mouse model:

65 Kunming mice were randomly divided into two groups, 10 in the normal group and 55 in the model group. Normal group is fed with normal feed, and modeling group is fed with high-sugar and high-fat feed. After feeding for 4 weeks, fasting each group of mice for 8 hours without water supply, and injecting citric acid-sodium citrate buffer solution with the molar concentration of 0.1mol/L and the pH value of 4.2 into the abdominal cavity of the normal group of mice; the model mice were injected intraperitoneally with STZ solution (35mg/kg) for 5 consecutive days. After 2 weeks the mice of the model group had fasting for 8 hours, Fasting Blood Glucose (FBG) was measured, and mice with values >11.1mmol/L were successfully modeled as type II diabetes model (T2DM) mice.

And (3) detecting blood sugar and body weight:

in order to observe the change of the blood sugar and the body weight of the mouse in the experimental process, after the mouse has an empty stomach for 8 hours, blood is taken from the tail vein of the mouse, the blood sugar value is measured by a blood glucose monitor, the body weight value of the mouse is weighed by an electronic balance, the result is recorded, the change is counted by measuring once per week.

Grouping experiments:

type ii diabetes model (T2DM) mice were randomized into 5 groups by blood glucose and body weight, i.e.: the group of type II diabetes mellitus model, metformin group (positive drug group), kaempferol low dose group (20mg/Kg), kaempferol medium dose group (40mg/Kg) and kaempferol high dose group (80mg/Kg) were fed with 10 of high-sugar and high-fat feed. The normal group was 10 normal mice fed with normal diet. Each group of mice was supplied with distilled water.

Each group of mice was gavaged at the same time daily for 8 weeks. Mice were observed daily for changes in general status, food intake, water intake, and urine output. The fasting blood glucose level of the mice was measured weekly in accordance with the above method, and the results are shown in Table 4.

TABLE 4 blood glucose values of mice of each experimental group

A histogram is plotted from the data of table 4, as shown in fig. 4. As can be seen from table 4 and fig. 4, fasting blood glucose of the type ii diabetes model group mice showed a gradual increase trend, and the trend was significant, compared to the normal group. During the 8-week administration period, the fasting blood glucose of mice in the kaempferol administration group and the metformin administration group showed a gradual decrease trend compared with the II-type diabetes model group, and the trend was obvious. After 8 weeks of administration, the fasting blood glucose value (28.10 +/-2.34) of the mice in the type II diabetes model group is obviously higher than that of the mice in the normal group (6.43 +/-1.08); compared with a II type diabetes model group, the fasting blood glucose value (20.12 +/-1.53) of the kaempferol low-dose administration group (20mg/Kg) has P <0.05, which shows that the kaempferol low-dose administration group has significant difference; compared with a II type diabetes model group, the fasting blood glucose value (16.75 +/-2.01) of the kaempferol medium dosage group (40mg/Kg) is less than 0.01, which shows that the kaempferol medium dosage group has very significant difference; the fasting blood glucose value (14.80 +/-1.06) of the kaempferol high-dose administration group (80mg/Kg) is less than 0.01 compared with the II type diabetes model group, which shows that the kaempferol high-dose administration group has very significant difference, and the kaempferol high-dose administration group has no significant difference compared with the metformin administration group (13.88 +/-1.36). The results of this experiment demonstrate that kaempferol can lower blood glucose in type ii diabetic mice induced by streptozotocin.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims

1. Application of kaempferol in preparation of protein tyrosine phosphatase SHP-1 inhibitor is provided.

2. The use of claim 1, wherein the inhibitor is a competitive inhibitor.

3. The use of claim 1, wherein the inhibitor comprises a medicament for the treatment of a disease associated with hyperactivity or overexpression of the protein tyrosine phosphatase SHP-1.

4. The use of claim 3, wherein the disease associated with the hyperactivity or overexpression of protein tyrosine phosphatase SHP-1 comprises diabetes, obesity, leukemia or immunodeficiency.

5. The use of claim 4, wherein the diabetes is type II diabetes.

6. The use of claim 3, wherein the pharmaceutical dosage form comprises an injection, a tablet or a capsule.