CN108159399B

CN108159399B - Application of thrombin aPC in medicine for preventing and treating diabetic cardiomyopathy

Info

Publication number: CN108159399B
Application number: CN201711484916.9A
Authority: CN
Inventors: 曾和松; 王洪杰; 王涛
Original assignee: Tongji Hospital Affiliated To Tongji Medical College Of Huazhong University Of Science & Technology
Current assignee: Tongji Hospital Affiliated To Tongji Medical College Of Huazhong University Of Science & Technology
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2020-07-24
Anticipated expiration: 2037-12-29
Also published as: CN108159399A

Abstract

The invention discloses an application of thrombin aPC in a medicine for preventing and treating diabetic cardiomyopathy caused by streptozotocin induction; in the invention, the thrombin aPC can inhibit ubiquitination of the YB1 of the myocardial cells in a high-sugar state, stabilize the expression of YB1 and reduce hypertrophy and fibrosis of the myocardial cells. The therapeutic research result shows that the thrombin aPC has the curative effects of obviously relieving the hypertrophy and fibrosis of myocardial cells, improving the myocardial contraction function and increasing the ejection fraction.

Description

Application of thrombin aPC in medicine for preventing and treating diabetic cardiomyopathy

Technical Field

The invention belongs to the technical field of new application of medicines, and particularly relates to an application of thrombin aPC in a medicine for preventing and treating diabetic cardiomyopathy.

Background

Diabetic Cardiomyopathy (DCM) is a structural and functional disorder of the heart caused by diabetes, independent of hypertension, coronary atherosclerotic heart disease, valvular heart disease, and other diseases known as heart disease. It is usually manifested as progressive left ventricular hypertrophy and dysfunction in relaxation and/or contraction, and its pathological features are cardiac myocyte hypertrophy, apoptosis, microangiopathy, interstitial fibrosis, etc. In 1972 Rubler et al first proposed the concept of diabetic cardiomyopathy, and for over 40 years, scholars at home and abroad conducted a great deal of basic and clinical research in the field, and confirmed that DCM is the main cause of high incidence of heart failure and high mortality of diabetic patients. DCM is currently identified as an independent complication of diabetes and is increasingly valued by clinicians and epidemiologists. The pathogenesis of DCM is very complex, involving metabolic disorders, oxidative stress, inflammatory reactions, autonomic dysfunction, insulin resistance, etc., and the specific mechanism of occurrence is still unclear. At present, no medicine for treating diabetic cardiomyopathy exists.

The protein c (pc) system is an important natural anticoagulant system for maintaining the stable environment in the body, and the activity of the system is mainly exerted by activated protein c (apc). Under the action of Thrombin-thrombomodulin (Thrombin-TM) complex, PC undergoes enzymolysis reaction, exposing the active center of serine hydrolase to form aPC. aPC binds to endothelial cell protein C receptor (EPCR) and performs biological functions by hydrolyzing the extracellular peptide of the G protein-coupled receptor-Protease Activated Receptor (PAR) to expose its own ligand to bind to the autoreceptor and transmit extracellular signals into the cell via the coupled G protein and other signaling molecules. In recent years, a large number of animals and clinical studies have confirmed that aPC and abnormal activation thereof are closely related to the development of various diseases such as sepsis, Disseminated Intravascular Coagulation (DIC), deep vein thrombosis, and purpura fulminans. The aPC not only regulates the coagulation and fibrinolysis balance of organisms, but also protects endothelial cells, improves microcirculation, slows down organ dysfunction and participates in the pathophysiological process of various diseases through an anti-inflammatory and anti-apoptosis mechanism. Many studies have shown that aPC can protect against cardiac ischemia reperfusion injury by anti-apoptotic, anti-inflammatory, modulating energy metabolism and autophagosome function. In addition, exogenous aPC has strong protective effect on diabetic nephropathy.

The Cold Shock protein YB-1(Ybox binding protein-1) is a highly conserved nucleic acid binding protein, a member of the Cold Shock Domain (Cold Shock Domain) protein superfamily, exists in the cytoplasm and nucleus of mammals, has various cell efficacies, and is involved in gene transcription, translation regulation, DNA damage induction and repair, anti-cancer drug resistance, environmental stimulation and other cell reactions. YB-1 consists of an alanine/proline rich N-terminus, a cold shock domain mediating DNA and RNA binding, and a C-terminal pull chain structure with alternating positively or negatively charged amino acids, which has a very complex role in gene expression and can directly regulate gene expression by binding to DNA or alter DNA conformation to indirectly affect the binding of other transcription factors. In addition, YB-1 may also affect the function by binding to the translation of RNA-binding regulatory genes and to other proteins. YB-1 can induce or inhibit the expression of genes, including cytokines and chemokines and their receptors, these functional parts being regulated by the cytoplasmic translocation of YB-1 to the nucleus in cells. It is noteworthy that YB-1 can be secreted non-routinely from monocytes or mesangial cells, that YB-1 secretion is associated with 301/304 lysine residues at its C-terminus, and that secreted proteins have chemokine-like effects or function as ligands for binding to some receptors (e.g., NOTCH-3). Research shows that YB-1 participates in and regulates heart diseases, for example, David JJ and the like find that YB-1 can regulate the embryonic gene expression of myocardial cells and is relevant to the long-term prognosis of patients through the heart tissue biopsy of patients after heart transplantation, and research of Kamalov G and the like finds that YB-1 can influence the prognosis of myocardial infarction by regulating the proliferation and migration of myofibroblasts in tissues after the heart infarction of mice. However, no research reports about the relationship between the occurrence and development of YB-1 and DCM yet.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a new application of the thrombin aPC in preparing a medicine with heart function protection in treating or preventing diabetic cardiomyopathy, and particularly has an obvious treatment effect in streptozotocin-induced diabetic cardiomyopathy.

In order to achieve the purpose, the invention adopts the following technical scheme:

an application of thrombin aPC in preparing a medicine for treating or preventing diabetic cardiomyopathy caused by streptozotocin induction.

Use of the coagulation protease aPC for the preparation of a product comprising: 1) a medicament for treating and/or preventing diabetic cardiomyopathy; 2) a drug (product) which inhibits the ubiquitination of the myocardial cells YB1 in a high-sugar state, thereby stabilizing the expression of YB 1; 3) the medicine (product) for relieving myocardial cell hypertrophy and fibrosis and improving myocardial contraction function.

In the invention, the thrombin aPC and other medicines for treating the diabetic cardiomyopathy are combined to be used as active ingredients for preparing medicines for treating or preventing the diabetic cardiomyopathy.

In the invention, the diabetic cardiomyopathy is diabetic cardiomyopathy induced by streptozotocin.

In the invention, the dosage form of the medicine is oral preparation or injection.

In addition, the invention also claims a medicament for preventing and/or treating diabetic cardiomyopathy, wherein the active ingredient comprises the thrombin aPC.

Compared with the prior art, the invention has the following advantages and effects:

in the invention, the thrombin aPC can inhibit ubiquitination of the YB1 of the myocardial cells in a high-sugar state, stabilize the expression of YB1 and reduce hypertrophy and fibrosis of the myocardial cells. The therapeutic research result shows that the thrombin aPC has the curative effects of obviously relieving the hypertrophy and fibrosis of myocardial cells, improving the myocardial contraction function and increasing the ejection fraction.

Drawings

FIG. 1 is a flowchart of an animal experiment in example 1 of the present invention;

FIG. 2A is a graph showing HE staining of cardiac tissue and measurement of cell area in example 1 of the present invention;

FIG. 2B is a WGA staining and cell area statistics of cardiac tissue in accordance with example 1 of the present invention;

FIG. 2C is a graph showing the results of Masson staining of mouse heart tissue in example 1 of the present invention;

FIG. 3A is a graph showing the expression and histogram of the marker β -MHC in myocardial hypertrophy in example 1 of the present invention;

FIG. 3B is a graph showing the expression and histogram of TGF- β, a marker of myocardial fibrosis, in accordance with example 1 of the present invention;

FIG. 4A is a typical echocardiogram, from left to right, of mice in a control group, a model group and a thrombin aPC administration group in accordance with example 1 of the present invention;

FIG. 4B is a graph showing the statistical effect of the thrombin aPC of the present invention on the cardiac Ejection Fraction (EF) and the short axis shortening rate (FS) and the thickness of the posterior wall (L VPW) in STZ-induced diabetic cardiomyopathy mice;

FIG. 5 is a diagram of cardiac function testing of cardiac catheters according to example 1 of the present invention;

FIG. 6A is a graph showing representative immunohistochemistry expression of YB1 in heart tissue of mice in a control group, a model group and a thrombin aPC administration group according to example 1 of the present invention;

FIG. 6B shows the expression and histogram statistics of YB1 protein in mouse heart tissue of Westernblotting detection control group, model group and thrombin aPC administration group in example 1 of the present invention;

FIG. 7 is a graph showing the effect of thrombin aPC on myocardial cell YB1 expression in example 2 of the present invention;

FIG. 8 is a graph showing the effect of thrombin protease aPC on ubiquitination of YB1 protein in cardiomyocytes as verified by the co-immunoprecipitation method in example 3 of the present invention.

The specific implementation mode is as follows:

the technical solution of the present invention will be further described in detail with reference to specific examples. It will be understood that the examples are for the purpose of further illustrating the subject invention and should not be construed in any way as limiting the scope of the invention.

Example 1

Therapeutic effect of thrombin aPC on heart insufficiency of diabetic cardiomyopathy mice

Male C57B L/J mice (8 weeks old) were randomly divided into 3 groups, a control group, a diabetic group and an aPC-treated group, the diabetic group and the aPC-treated group were subjected to 5 consecutive days of intraperitoneal injection of STZ solution using multiple small dose streptozotocin (STZ, citric acid buffer dissolved in 0.05M pH4.5, 60mg/kg) administration, the control group was subjected to intraperitoneal injection of citric acid buffer (FIG. 1), after STZ22 weeks, mice were tested using cardiac ultrasonography for post-diabetic left ventricular ejection fraction (L VEF), left ventricular short axis shortening (left ventricular tachycardia/aspiration, L VFS), left ventricular flow interval thickness (left ventricular diastole/contraction) and ventricular flow interval (VPstereoscopic aspiration) parameters (VP64 Wtrauma) and VPWvenous outflow parametersThe left ventricular end diastolic pressure (L V end-diastolic pressure, PED), the left ventricular end systolic pressure (L V end-sylitic pressure, PES), and the maximum rate of decrease of the left ventricular pressure (dp/dt)_min) And the maximum rate of rise of left ventricular pressure (dP/dt)_max) And (3) harvesting the blood plasma of the mouse, preserving each organ sample by adopting different methods according to needs, recording the weight and the organ weight, performing immunohistochemical staining of a heart pathological section HE, WGA, Masson, YB-1 and the like, and detecting indexes such as β -MHC, TGF- β, YB-1 and the like in the heart specimen of the mouse by WesternBlot.

The results showed that HE and WGA staining indicated that thrombin aPC could improve cardiomyocyte hypertrophy in STZ-induced diabetic cardiomyopathy mice (fig. 2A and 2B), while Masson staining assay indicated that thrombin aPC could inhibit myocardial fibrosis in STZ-induced diabetic cardiomyopathy mice (fig. 2C), Western Blot assay indicated that thrombin aPC could inhibit the expression of the cardiac hypertrophy marker β -MHC and the fibrosis marker TGF- β (fig. 3A and 3B), Western Blot and immunohistochemistry assay indicated that thrombin aPC could increase the STZ-induced decrease in cardiac tissue cold shock protein YB1 in diabetic cardiomyopathy mice (fig. 6A and 6B), and cardiac ultrasound results indicated that ejection fraction and short axis shortening rate of diabetic cardiomyopathy mice decreased, while thrombin aPC could significantly improve myocardial damage in STZ-induced mice (fig. 4A and 4B), consistent with the results of cardiac ductal assay (fig. 5), indicating that thrombin injury in cardiomyopathy mice significantly improved cardiac function.

The experimental conclusion proves that the thrombin aPC provided by the invention has the effect of treating diabetic cardiomyopathy cardiac insufficiency.

Example 2

Coagulation protease aPC (alpha-beta-glucosidase) for inhibiting degradation of myocardial cell YB1 protein

H9C2 cells were randomly grouped into groups: PBS control group, proteasome inhibitor MG132 treatment group, thrombin aPC intervention group. According to the above groups, after 1 hour of intervention with CHX (10MG/ml), the protein synthesis inhibitor was administered for PBS, MG132, and

thrombin aPC

0,1,3,6, and 12 hours, cell proteins were collected, and after quantification by BCA method, expression of YB-1 and internal reference protein GAPDH was detected by 20 ug/well Western Blot.

The result shows that the Western Blot result shows that the thrombin aPC can obviously inhibit the degradation of the YB1 protein of the myocardial cells (figure 7).

The experimental conclusion proves that the thrombin aPC provided by the invention can stabilize the expression of the YB1 protein of the myocardial cells.

Example 3

Blood coagulation protease aPC inhibits ubiquitination level of myocardial cell YB1 protein

H9C2 cells are treated by high sugar for 0,1,3,6,12 and 24 hours, cell protein is collected, and the co-immunoprecipitation experiment detects the ubiquitination level of YB1 protein.

H9C2 cells were divided into 3 groups: control, high-sugar treated, high-sugar plus thrombin aPC treated. According to the groups, after the intervention of the high-sugar and thrombin aPC treatment group on thrombin aPC for 1 hour, the intervention of the high-sugar treatment group and the high-sugar and thrombin aPC treatment group on high sugar for 3 hours respectively, cellular protein is collected, and a co-immunoprecipitation experiment is carried out to detect the ubiquitination level of YB1 protein in each group.

All cell experiments stimulated by adding high sugar increase the isotonic mannitol group, and the influence of osmotic pressure change caused by high sugar on the experiment result is eliminated.

The results show that the co-immunoprecipitation experiment result shows that the ubiquitination level of H9C2 cell YB1 induced by high sugar reaches the highest level in 3 hours, and the thrombin aPC can inhibit ubiquitination of myocardial cell YB1 protein induced by high sugar (figure 8).

The experimental conclusion proves that the blood coagulation protease aPC can inhibit the ubiquitination of the YB1 protein of the myocardial cells, thereby stabilizing the expression of YB 1.

It should be noted that the above-described embodiments may enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. Thus, it will be appreciated by those skilled in the art that the invention may be modified and equivalents may be substituted; all technical solutions and modifications thereof which do not depart from the spirit and technical essence of the present invention should be covered by the scope of the present patent.

Claims

1. An application of thrombin aPC in preparing the product for relieving the hypertrophy and fibrosis of myocardial cells of diabetes and improving the myocardial contraction function.

2. The use according to claim 1, wherein the thrombin protease aPC is used in combination with other antidiabetic cardiomyopathy medicaments as an active ingredient in the manufacture of a medicament for treating or preventing diabetic cardiomyopathy.

3. The use according to claim 2, wherein the diabetic cardiomyopathy is streptozotocin-induced diabetic cardiomyopathy.

4. The use according to any one of claims 2 to 3, wherein the medicament is in the form of an oral preparation or an injection.