CN111195254B

CN111195254B - LPA2 and use of agonists thereof

Info

Publication number: CN111195254B
Application number: CN202010118137.2A
Authority: CN
Inventors: 陈曦; 胡盛寿; 裴建秋; 丛祥凤; 蔡琳; 王芳; 白睿峰
Original assignee: Fuwai Hospital of CAMS and PUMC
Current assignee: Fuwai Hospital of CAMS and PUMC
Priority date: 2020-02-25
Filing date: 2020-02-25
Publication date: 2021-03-16
Anticipated expiration: 2040-02-25
Also published as: CN111195254A

Abstract

The invention discloses an LPA2 and application of an agonist thereof, and relates to the technical field of biomedicine. The invention discloses an LPA2 (lysophosphatidic acid receptor subtype 2) or an agonist thereof, which is used for treating diabetes and is selected from at least one of the following: (1) for the preparation of a medicament for the treatment or prevention of ischemic diseases; (2) for the preparation of a medicament for the treatment or prevention of a disease with symptoms of vascular leakage. The invention provides a new medicine and a new treatment idea for treating or preventing ischemic diseases and diseases caused by other vascular leakage.

Description

LPA2 and use of agonists thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to LPA2 and application of an agonist thereof.

Background

The heart disease is the leading cause of adult death worldwide, the number of cardiovascular diseases in China is about 2.9 hundred million, and the death rate of cardiovascular diseases accounts for more than 40% of the death rate of resident diseases, and is higher than that of tumors and other diseases in the top position. Acute Myocardial Infarction (AMI) is one of the most serious Acute cardiovascular diseases, and is Myocardial necrosis caused by Acute and persistent ischemia and hypoxia of coronary artery, which can cause severe arrhythmia, cardiogenic shock, heart failure, etc., and even death. Although current intervention means such as cardiovascular drug therapy, interventional therapy and surgical operation therapy can restore ischemic myocardial tissues to a certain extent by restoring blood flow in infarct and peripheral areas, a considerable part of patients still have unsatisfactory restoration effects in terms of long-term effects, and finally have heart failure. Increasing the blood supply to infarcted and peripheral myocardial tissues by promoting angiogenesis is another widely focused and non-negligible strategy for repair of damaged myocardium.

In the case of ischemic tissue, the body restores blood supply by itself through the neovasculature. Angiogenesis is to stimulate growth of small blood vessels in an ischemic area of the myocardium to realize self-bridging of the ischemic area of the myocardium, so that collateral circulation of the ischemic myocardium is established, and the blood flow condition of the ischemic area of the myocardium is improved. Angiogenesis is mainly composed of microangiogenesis, arteriolar formation and macroangiogenisis. In the early stage of myocardial ischemia or myocardial infarction, on one hand, the density of capillaries is increased, a large amount of capillaries are generated, and on the other hand, necrotic myocardial cells induce the expression of a plurality of cytokines and growth factors, and finally form normal arteries. The large amount of generated capillaries and newly generated arterioles form collateral circulation around the blocked and narrowed coronary arteries to generate new blood flow paths, thereby improving the myocardial ischemia state and playing an important role in myocardial regeneration. As early as the 70's of the 20 th century, Folkman, a medical professional, proposed the concept of angiogenesis, after which the study of angiogenesis became the leading science of treating ischemic diseases. However, natural angiogenic systems tend to be delayed and unable to cope with a wide range of tissue ischemic injuries. In addition, endothelial cells serve as the vascular intima and play an important role in the maintenance of vascular homeostasis. Impaired endothelial function can lead to changes in vascular permeability, and excessive increases in vascular permeability cause leakage of blood vessels, a significant cause of organ failure, leading to shock and death. Therefore, we need to further explore the mechanism of angiogenesis and early vascular homeostasis maintenance after AMI and to find potential therapeutic targets.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide LPA2 and application of agonists thereof.

The invention is realized by the following steps:

lysophosphatidic acid (LPA) is a water-soluble glycerophospholipid molecule of the simplest structure that exerts biological functions through its specific receptor. At least six lysophosphatidic acid receptors (LPA1-LPA6) have been identified as belonging to the family of G protein-coupled receptors, of which the lysophosphatidic acid receptor subtype 2 (LPA2) is one.

The inventor of the invention finds that LPA2 and an agonist thereof have the potential to treat or prevent ischemic diseases and diseases caused by vascular leakage for the first time, and puts forward the invention based on the potential.

In a first aspect, the present invention provides use of LPA2 or an agonist thereof, said use being selected from at least one of the following (1) to (2):

(1) for the preparation of a medicament for the treatment or prevention of ischemic diseases;

(2) for the preparation of a medicament for the treatment or prevention of a disease with symptoms of vascular leakage.

The research of the invention shows that LPA2 or an agonist thereof can maintain vascular homeostasis in the early stage of acute myocardial infarction, and reduce the occurrence of acute shock and even death caused by leakage caused by vascular permeability change. In addition, the traditional Chinese medicine composition can also promote angiogenesis, repair damaged cardiac muscle, obviously reduce the myocardial infarction area (12.8%), obviously improve the cardiac function after myocardial infarction (LVEF is increased by nearly 50%), and has obvious effect.

Therefore, LPA2 and the agonist thereof can be used in the field of preparing drugs or preparations for treating or preventing ischemic diseases and diseases caused by vascular leakage and the like, and provide a new drug and a new treatment idea for ischemic diseases and diseases with vascular leakage symptoms.

In an alternative embodiment, the ischemic disease is ischemic heart disease.

In an alternative embodiment, the ischemic heart disease is acute myocardial infarction or ischemic heart failure.

In an alternative embodiment, the ischemic disease is a vascular disease.

In an alternative embodiment, the vascular disorder is ischemic disease of the lower extremities.

In alternative embodiments, the disease with symptoms of vascular leakage comprises shock or sepsis due to organ failure caused by vascular leakage.

In alternative embodiments, LPA2 or an agonist thereof acts to treat or prevent ischemic disease or vascular leakage-induced disease by one or more of the following means (a) - (j):

(a) promoting endothelial cell proliferation;

(b) inhibiting endothelial cell apoptosis;

(c) improving endothelial cell viability;

(d) repairing endothelial cell damage;

(e) improving endothelial cell tube forming ability;

(g) increase myocardial vascular density;

(h) preventing fibrosis of infarcted parts;

(i) increasing gastrocnemius vascular density;

(j) maintain vascular homeostasis and reduce vascular leakage.

The present study further shows that LPA2 or an agonist thereof has the following effects: by promoting endothelial cell proliferation, inhibiting endothelial cell apoptosis, improving endothelial cell viability, repairing endothelial cell injury, increasing the number of inter-myocardial vessels, increasing inter-myocardial vessel density, preventing fibrosis of infarcted portions, increasing the number of inter-gastrocnemius vessels, and increasing vascular permeability, LPA2 or an agonist thereof can be used for treating or preventing ischemic diseases or diseases with vascular leakage symptoms.

In an alternative embodiment, the agonist is a specific agonist of LPA 2;

in an alternative embodiment, the agonist is lysophosphatidic acid.

The present study shows that the use of lysophosphatidic acid can promote endothelial cell proliferation.

It is noted that, in light of the present disclosure, one skilled in the art would readily envision the use of other specific agonists for treating or preventing ischemic diseases or diseases caused by vascular leakage.

In an alternative embodiment, the specific agonist is DBIBB.

DBIBB, English name 2- (N- (4- (1, 3-dioxo-1H-benzo [ de ] isoquinolin-2(3H) -yl) butyl) sulfomethyl) benzoic acid, has a molecular formula of C23H20N2O6S, a molecular weight of 452.5, and a chemical formula as follows:

the research of the invention shows that the specific agonist of LPA2, such as DBIBB, can produce positive effects on the vascular homeostasis after the myocardial infarction and the cardiac function, such as improving the cardiac function, reducing the infarct area, improving the myocardial vascular density or obviously reducing the vascular permeability and the like.

It is noted that based on the present disclosure, one skilled in the art could readily envision other LPA2 specific agonists for the treatment or prevention of ischemic diseases or diseases caused by vascular leakage.

In yet another aspect, the present invention also provides another use of LPA2 or an agonist thereof, selected from at least one of the following (a) - (j):

(a) for preparing a preparation or medicament for promoting endothelial cell proliferation;

(b) used for preparing a preparation or a medicament for inhibiting endothelial cell apoptosis;

(c) for preparing a preparation or medicament for improving the viability of endothelial cells;

(d) used for preparing a preparation or a medicament for repairing endothelial cell injury;

(e) for preparing a preparation or a medicament for improving endothelial cell tube forming ability;

(f) for preparing a preparation or medicament for increasing the number of blood vessels between the heart muscle;

(g) for preparing a preparation or medicament for increasing myocardial vascular density;

(h) for preparing a preparation or a medicament for preventing fibrosis of an infarcted part in an acute myocardial infarction state;

(i) used for preparing a preparation or a medicament for improving the blood vessel density of gastrocnemius under the condition of lower limb ischemic diseases;

(j) can be used for preparing preparation or medicine for improving blood vessel homeostasis and inhibiting blood vessel leakage.

In an alternative embodiment, the agonist is a specific agonist of LPA 2.

In an alternative embodiment, the specific agonist is DBIBB.

In still another aspect, the present invention provides a medicament having any one of the following uses (1) to (2):

(2) for the preparation of a medicament for the treatment or prevention of a disease with symptoms of vascular leakage;

the medicine contains: a therapeutically effective amount of LPA2 or an agonist thereof.

In an alternative embodiment, the agonist is a specific agonist of LPA 2.

In an alternative embodiment, the specific agonist is DBIBB.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a graph showing the effect of a deletion of lysophosphatidic acid receptor subtype 2 (LPA2) in the present example on survival and cardiac function in adult mice 4 weeks post-myocardial infarction.

In the figure: a is a mouse animal experimental planning chart;

b is a survival curve of the mice at 4 weeks after myocardial infarction, and the survival rate of the mice with the LPA2 deficiency is obviously lower than that of the wild mice; in the figure: the solid line represents the survival curve of Wild Type (WT) mice and the dotted line represents the survival curve of LPA2 deleted (KO) mice, i.e. loss of LPA2 can cause increased mortality in early post-myocardial infarction in mice;

C-D is the effect of the deletion of LPA2 (expressed as KO) on adult mouse myocardial function in the examples of the present invention; the heart function of the mice with LPA2 deletion is obviously reduced compared with wild mice after 8 weeks of operation (C is a left ventricular short axis M-type ultrasonic representation picture, sham represents a pseudo operation group, and MI represents a myocardial infarction operation group; D is the Left Ventricular Ejection Fraction (LVEF) and the left ventricular short axis shortening rate (LVFS) of the mice with LPA2 deletion at 8 weeks of operation (8wpM), and the two are important indexes for evaluating the heart function);

E-F is the influence of LPA2 deletion on myocardial infarction area of adult mice 8 weeks after myocardial infarction in the invention example (in the figure, E is a representative graph showing infarct area by Masson dyeing; F is the calculation result of infarct area and infarct wall thickness, and the calculation method of infarct area is (infarct inner diameter + infarct outer diameter)/(left ventricular inner diameter + left ventricular outer diameter);

G-H is the Tianlang scarlet staining of the heart 8 weeks after the myocardial infarction of the mouse in the embodiment of the invention, shows the degree of myocardial fibrosis, and the result shows that the degree of myocardial fibrosis of the LPA 2-deleted mouse is more severe than that of a wild type mouse (G is a Tianlang scarlet staining representative graph; H is a statistical result); the results show that the LPA2 deletion can cause the death rate of mice after myocardial infarction to be increased, the cardiac function to be reduced and the malignant reconstruction of the heart to be aggravated.

FIG. 2 is a graph showing the effect of a deletion of LPA2 in an example of the present invention on the density of neovascularization in the peri-infarct zone following myocardial infarction in adult mice; the results show that the blood vessel density in the myocardium of LPA 2-deficient mice (KO) is significantly reduced compared with wild mice after myocardial infarction; in the figure: a is a representative image of the vascular endothelial cell marker vWF immunofluorescence staining, and DAPI is cell nucleus staining; b, a representative image of the blood vessel endothelial cell marker CD31 immunofluorescence staining, and DAPI is a staining result of cell nucleus staining; inflammatory cells such as mononuclear macrophages and neutrophil infiltration also have a tendency to be increased compared with wild mice, CD68 in C is a macrophage marker, and Ly6G in D is a neutrophil marker.

FIG. 3 is a graph showing the effect of LPA2 deletion in an example of the present invention on cardiac regeneration after neonatal myocardial infarction; the result shows that LPA2 deletion can block the regeneration of suckling mice, and the density of blood vessels between cardiac muscles is obviously reduced; in the figure: a is Masson staining for comparing the heart regeneration condition, B is a heart function M-type ultrasonic representation picture, C is a statistical result of infarct size, D is a statistical result of Left Ventricular Ejection Fraction (LVEF) heart function, E is blood vessel CD31 marker molecule immunohistochemical staining, and F is a statistical value of CD31 immunohistochemical staining.

Fig. 4 shows the effect of LPA2 deletion on angiogenesis capacity in the present invention, and the results show that LPA2 deletion can affect the blood flow recovery of lower limbs of mice by reducing angiogenesis capacity, and in vitro cell experiments also show that the endothelial cell tube forming capacity and migration capacity after LPA2 deletion are both significantly reduced compared with wild endothelial cells; in the figure: a is a representative diagram of blood flow conditions of different time points after lower limb ischemia of a Doppler detection mouse and a statistical diagram of the blood flow conditions; b is a CD31 staining representative graph and a blood vessel density statistical graph of the blood vessel density between gastrocnemius muscles 14 days after operation; c is a representative graph of in vitro tube formation experiments and statistics of tube formation number and length of primary endothelial cells isolated from Wild (WT) and LPA2 deletion (KO); d is a representative graph and a statistical graph of WT and KO primary endothelial cell migration experiments.

FIG. 5 is a search experiment for early injury after myocardial infarction in mice caused by LPA2 deletion in the present example. The results indicate that the loss of LPA2 increases vascular permeability, leads to an increased range of early injury after myocardial infarction of mice, and acute heart failure, possibly causes the early acute death of the mice; in the figure: a is the experimental detection time arrangement; b is a left ventricular short axis M-shaped ultrasonic representative graph and statistical values of Left Ventricular Ejection Fraction (LVEF) and left ventricular short axis shortening rate (LVFS) 2 days after myocardial infarction; c is TTC staining 1 day after myocardial infarction, wherein a dark color area is a viable cardiac muscle, a white part is an anoxic injury area, and the statistical value of the injury area is obtained; d is the change of the content of peripheral blood Lactate Dehydrogenase (LDH), and the higher the LDH content is, the more serious the myocardial damage is; e is myocardial tissue apoptosis detection TUNEL staining 3 days after myocardial infarction, and the statistical result shows that the cardiac tissue apoptosis of KO mice after myocardial infarction is increased; f is a representation of myocardial leakage of mice after 3 days of postoperation tail vein injection with 70kD FITC-Dextran, and a fluorescence intensity statistic. When the vascular permeability is increased, the content of FITC-Dextran between myocardial tissues is increased; g is a detection statistical chart of heart vessel leakage of the mice after myocardial infarction displayed by Evans blue; H-I is the apoptosis and activity detection result of the isolated primary wild and LPA2 deleted endothelial cells under the stimulation of hypoxia: h, the graph shows that Annexin V/PI staining flow-type detection cell apoptosis shows that the KO type endothelial cell apoptosis ratio is obviously increased compared with a wild type cell apoptosis ratio; figure I shows that KO-type endothelial cells are also significantly less active than WT-type endothelial cells.

FIG. 6 is a graph showing the effect of LPA stimulation on endothelial cell proliferation in an example of the present invention. The results show that the absence of LPA2 abolished the proliferative effect of endothelial cells on LPA stimulation (cell proliferation on the ordinate, different concentrations of LPA on the abscissa; and & represent differences compared to the KO group in the absence of LPA and at the same concentration of LPA, respectively, both with p < 0.0001).

FIG. 7 shows the effect of Ad-Lpar 2-mediated LPA2 overexpression treatment of adenovirus carrying Lpar2 gene on the post-myocardial function and infarct size of mice and the effect on the angiogenesis of heart tissues in the examples of the present invention; the result shows that the over-expression of LPA2 can reduce the malignant reconstruction of mice after myocardial infarction and improve the cardiac function; in the figure: a is a schematic diagram and a statistical value of the heart infarction condition 4 weeks after infarction; b is an M-type ultrasonic schematic diagram and a statistical value of 4 weeks of cardiac function detection after myocardial infarction; c is an immunofluorescence staining schematic diagram and a statistical value of myocardial intermyocardial vascular density CD31 7 days after myocardial infarction; d is a schematic diagram and a statistical value of the immunofluorescence staining of the myocardial intermyocardial blood vessel density CD31 after 4 weeks of myocardial infarction.

FIG. 8 shows the effect of DBIBB treatment, a LPA 2-specific agonist, on the post-myocardial function and infarct size of mice in an example of the invention, as well as the early survival and vascular permeability changes in cardiac tissue after DBIBB treatment; the result shows that the LPA2 agonist DBIBB can reduce malignant reconstruction of mice after myocardial infarction, improve cardiac function, reduce vascular permeability in early stage after myocardial infarction, maintain vascular homeostasis and improve the condition of increased mortality caused by cardiac failure in early stage; in the figure: a is the early survival curve after myocardial infarction of DBIBB and a control group; b is a schematic diagram and a statistical value of the 4-week post-infarction condition of the heart; c is a schematic diagram and a statistical value of ultrasonic detection of cardiac function 4 weeks after myocardial infarction respectively; d is a schematic diagram and a statistical value of the immunofluorescence staining of the blood vessel density CD31 between the cardiac muscles 4 weeks after myocardial infarction; e is myocardial tissue apoptosis detection TUNEL staining 3 days after myocardial infarction, and shows that DBIBB treatment can protect the cardiac tissue and reduce myocardial cell apoptosis; f is a FITC-Dextran leakage representation diagram and a fluorescence intensity statistical diagram, and shows that DBIBB treatment can improve the vascular permeability after myocardial infarction and reduce heart injury.

FIG. 9 shows the results of the relative changes in vascular permeability of the organs after LPS-induced sepsis damage in mice due to the deletion of LPA2 in example 8 of the present invention, where: a is Heart tissue (Heart), B is Liver tissue (Liver), C is Spleen tissue (Spleen), D is Lung tissue (Lung), and E is Kidney tissue (Kidney).

In fig. 1-9: ns indicates no difference, represents p <0.05, represents p <0.01, and represents p < 0.001.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Effect of LPA2 deletion on post-myocardial function and infarct size in mice

(1) Establishment of myocardial infarction model of adult mouse

After the mice were weighed and the body weights were recorded, they were anesthetized by intraperitoneal injection of a tribromoethanol solution at a concentration of 400mg/kg (about 0.2mL for about 25g mice);

sterilizing the left chest of the mouse by using 75% alcohol, and removing hair near the surgical field by using depilatory cream; performing tracheal intubation and connecting a breathing machine;

cutting skin on the outer part of the left chest along the parallel direction of the left rib, separating each layer of muscle along the shape walking direction of the muscle to expose the rib, and opening the muscle by using an opener to expose the rib layer in the center of the visual field; separating muscle layer with fine straight forceps in 3 rd-4 th intercostal space, and spreading the space between upper and lower costal space with mouse spreader to expose heart and lung organs;

carefully pulling the pericardium by using fine straight forceps and curved forceps; ligating blood vessels at 1-2 mm of the lower margin of auricle with 7-0 silk thread with needle, wherein the blood vessels are knotted to show rapid ischemia and whitening of the far-end left ventricular myocardium, which is used as the basis for successful ligation;

withdrawing the spreader to a subcutaneous muscle layer, sewing the opened upper and lower ribs with 7-0 threads, covering the muscle layer by layer, and sewing the skin with 5-0 threads; the incision and surrounding skin were cleaned with 75% alcohol, the mice were laid flat on a warm heating pad, and returned to the rearing cage after the mice were awake.

(2) Experimental grouping and processing

a. Wild mouse sham surgery group: a wild mouse of the SPF-grade BALb/c strain is taken, a 7-0 silk thread with a needle is passed through a left anterior descending branch without knotting in the process of constructing a myocardial infarction model according to example 1, and the rest of the myocardial infarction groups are operated.

Lpa 2-deficient mouse sham group: LPA 2-deficient mice were harvested and the myocardial infarction model constructed as described in example 1, with 7-0 needled silk passed through the left anterior descending branch without knot, and the other groups were operated on concentric infarcts.

c. Wild mouse myocardial infarction group: a myocardial infarction model is constructed according to the method by taking a SPF-grade BALB/c strain wild mouse (purchased from Beijing Wittingerihua laboratory animal technology Co., Ltd.).

Lpa 2-deficient mouse myocardial infarction group: LPA 2-deficient mice (mice donated by Jerold Chun, national breed, of Sanford Burnham Prebys medical research center) were used to construct myocardial infarction models.

(3) Experimental methods

Utilizing LPA2 deletion mice and mouse myocardial infarction models, setting a sham operation control group, carrying out cardiac ultrasonic detection on cardiac function 8 weeks after myocardial infarction, carrying out cardiac material taking after ultrasonic treatment, fixing paraformaldehyde for 48 hours, embedding dehydrated paraffin, and determining infarct area by adopting Masson staining. The specific method of Masson staining comprises the following steps: paraffin embedded tissue sections about 3-5 μm thick; baking the slices on a 68 ℃ slice baking machine for 1 hour, and dewaxing; soaking in 2.5% potassium bichromate solution for over 16 hr; flushing tap water for 5 minutes; acid fuchsin dyeing for 5 minutes; separating the color of the phosphotungstic acid for 1-2 minutes; washing with water, dripping aniline blue for dyeing for about 30 seconds, and immediately washing with water; the slices are dehydrated and transparent, sealed by neutral gum, dried in a fume hood and photographed.

(4) Results

Mice were scored for survival 4 weeks after myocardial infarction, and cardiac ultrasounds were performed 8 weeks after surgery, followed by material selection. As can be seen from fig. 1B, mortality of LPA 2-deficient mice was significantly higher after the myocardial infarction than wild-type mice, and fig. 1C and D show that cardiac function was also significantly reduced compared to wild-type mice 8 weeks post-surgery. Masson staining in E and F in figure 1 shows that LPA 2-deficient mice had a larger left ventricular infarct size and thinner infarct wall. The G and H sirius red staining in figure 1 shows increased fibrosis. This is consistent with its reduced cardiac function results. Similarly, vWF and CD31 staining, which are markers for vascular endothelial cells, showed a decrease in the number of blood vessels between the myocardium in the peripheral region of the myocardial infarction in LPA 2-deficient mice (fig. 2).

Example 2

Effect of LPA2 deletion on myocardial regeneration in suckling mice

(1) Establishment of neonatal suckling mouse myocardial infarction model

The second day after birth (P2) of newborn suckling mice was operated. The suckling mice were removed from the nest, isolated from the parents and anesthetized on ice (approximately 2-4 minutes);

fixing the mouse in a lateral decubitus position, and cutting the skin outside the left chest along the parallel direction of the left rib to expose the rib layer in the center of the visual field; cutting off the 2 nd-3 rd intercostal space with a pair of microshear, and opening the space between the upper and lower costal spaces with fine curved forceps to expose heart and lung organs;

carefully pulling the pericardium by using fine straight forceps and curved forceps; the 7-0 silk thread with a needle is used for ligating the blood vessel at the lower margin of the auricle, and after knotting, the rapid ischemia and whitening of the far-end left ventricular myocardium can be seen, which is taken as the basis for successful ligation;

after the ligation is finished, sewing the opened upper and lower ribs by 7-0 silk threads with needles, and sewing the skin by 7-0 silk threads with needles; the incision and surrounding skin were cleaned with 75% alcohol, and the suckling mice were laid flat on a warm heating pad, and returned to the rearing cage after they were awake.

(2) Experimental grouping and processing

The same as in example 1.

(3) Experimental methods

Utilizing LPA2 deletion milk mice and milk mouse myocardial infarction models, setting a sham operation control group, carrying out cardiac ultrasonic detection on cardiac function 21 days after myocardial infarction, carrying out cardiac material drawing after ultrasonic, fixing paraformaldehyde for 24 hours, embedding dehydrated paraffin, and determining infarction area by adopting Masson staining. Masson staining protocol example 1 was referenced.

(4) Results of the experiment

As can be seen from fig. 3, the mice with LPA2 deficiency were obtained 21 days after the myocardial infarction, and Masson staining showed that the myocardial regeneration ability of LPA 2-deficient mice was significantly decreased, compared to the mice with wild myocardial regeneration and repair, the mice with LPA 2-deficient mice had fibrous scar tissue replacement (a in fig. 3), and the infarct size was significantly larger than that of the wild mice (C in fig. 3). Functionally, LPA 2-deficient suckling mice had decreased cardiac function (B and D in fig. 3) and decreased left ventricular ejection fraction due to fibrosis at the infarct site. And the LPA 2-deficient suckling mice were obtained 21 days after surgery, and CD31 staining showed that the number of blood vessels between cardiac muscles was significantly reduced compared to wild type mice (F in fig. 3 is CD31 immunohistochemical staining, and F in fig. 3 is a statistical graph of the number of blood vessels).

Example 3

Effect of LPA2 deletion on mouse angiogenesis

(1) Establishment of lower limb ischemia model of adult mouse

After the adult mice are anesthetized, the hair at the left and right lower limbs is removed, the femoral artery of the left lower limb is dissociated under a stereoscope, and the branched front section of the femoral artery is ligated by a 7-0 thread.

(2) Experimental grouping and processing

a. Wild type mouse surgery group; lpa 2-deficient mice surgery group. Control was taken from its own right hind limb.

(3) Experimental methods

Mice with wild-type and LPA2 deficiency and mice lower limb ischemia models were doppler tested for lower limb blood flow before, after (0 day), 4, 7 and 14 days, respectively.

(4) Results

In fig. 4, a shows that blood flow recovery after lower limb ischemia of LPA 2-deleted mice was inhibited compared to wild-type mice, and the CD31 immunohistochemical staining of the lower gastrocnemius muscle obtained by ligation of the selected material showed that the number of blood vessels between gastrocnemius muscles of LPA 2-deleted mice was significantly less than that of wild-type mice (B in fig. 4). In vitro culture of isolated wild-type and LPA 2-deficient primary thoraco-abdominal aortic endothelial cells showed that LPA2 deficiency reduced endothelial cell vascularization and migration capacity (C and D in fig. 4).

Example 4

Effect of LPA2 deletion on early survival of mice after myocardial infarction by modulating vascular permeability

(1) Establishment of myocardial infarction model of adult mouse

The procedure is as in example 1.

(2) Experimental grouping and processing

a. Control sham group: the SPF-grade BALb/c strain wild and LPA 2-deleted mice were selected, and in the process of constructing a myocardial infarction model according to example 1, 7-0 silk with needles was passed through the left anterior descending branch without knotting, and the other groups of concentric peduncles were operated.

b. Operation group: a myocardial infarction model is constructed according to the method by taking SPF-grade BALb/c strain wild mice (purchased from Beijing Wittingeri laboratory animal technology Co., Ltd.) and LPA2 deletion type mice.

(3) Experimental methods

Using LPA2 deletion mouse and mouse myocardial infarction model, setting a pseudo-operation control group, using LPA2 deletion mouse and mouse myocardial infarction model, setting a pseudo-operation control group, performing ultrasonic detection on cardiac function respectively 2 days after myocardial infarction to determine whether heart failure occurs, injecting 70kD FITC-Dextran or Evans blue into caudal vein 3 days after myocardial infarction, detecting infiltration degree in FITC-Dextran tissue by frozen section, extracting Evans blue by formamide, determining OD_620nmAnd OD_740nmAnd the difference value is used for detecting the vascular permeability of the heart tissue of the mouse and carrying out heart ultrasonic detection on the heart function 2 days after the myocardial infarction. And judging the ischemic injury area of the heart by adopting TTC staining. The TTC dyeing method comprises the following steps: heart tissue sections were prepared to 1% concentration TTC, stained in the dark for 15-30 minutes, and photographed.

(4) Results

Cardiac ultrasound was performed 2 and 7 days after the myocardial infarction of the mice (a in fig. 5). It can be seen from B in fig. 5 that the LPA 2-deficient mice had significantly reduced cardiac function 2 days after the myocardial infarction, and TTC staining showed a significant increase in the range of cardiac injury (C in fig. 5), increased cardiac injury (D in fig. 5), and increased apoptosis of myocardial tissue (E in fig. 5). FITC-Dextran and Evans blue showed that the absence of LPA2 caused an increase in vascular permeability (F and G in FIG. 5). Therefore, we speculate that LPA2 loss is the cause of increased vascular permeability in the early stage after myocardial infarction, which leads to cardiac tissue edema, acute heart failure and acute death increase. It is shown that LPA2 plays an important role in maintaining vascular homeostasis after myocardial infarction, i.e., the presence of LPA2 helps to reduce shock and death due to tissue organ failure caused by vascular leakage.

Example 5

Effect of LPA2 deletion on vascular endothelial cell proliferation

(1) Thoracoabdominal aortic dissection in mice

A. Killing mouse cervical vertebra by dislocation, soaking and sterilizing in 75% ethanol for 1min, spreading a sterile towel on an ultra-clean bench, and fixing the mouse on an operation table;

B. cutting the thoracoabdominal median incision layer by layer to expose the whole thoracoabdominal cavity, finding out aorta on the left side of the spine, dissecting the aortic arch under a 10-fold microscope, carefully separating the arterial tunica and peripheral adipose tissues, cutting off the branch blood vessels of the aortic arch in the thoracoabdominal cavity, completely taking out the thoracic aorta and the abdominal aorta, and immediately putting the thoracic aorta and the abdominal aorta into a sterile PBS culture dish;

C. repeatedly washing blood vessel in sterile container containing PBS solution for 3-4 times, placing into another sterile culture dish, longitudinally dissecting blood vessel, cutting into blood vessel implant blocks of 1mm × 1mm with microscope, attaching the inner surface of blood vessel onto 6-well plate with a diameter of about 1cm²Placing 1 planting block.

D.37℃、5％CO₂Standing in incubator for 5-10min until the blood vessel implant block is attached to the bottom of the plate, adding ECM complete culture medium containing 20% FBS, and continuously adding CO₂Culturing in an incubator.

E.60 hours right observation of cell growth, vessel implantation removal, 1 liquid change. F. Changing the solution 1 time every 2 days, and subculturing after the cells are fused into a monolayer and spread to the bottom of the hole. And (5) passing to the third generation, and identifying the purity of the endothelial cells by using flow type for subsequent experiments.

(2) Experimental grouping and processing

Culturing the thoraco-abdominal aorta endothelial cells of the mice with wild type and LPA2 deletion respectively. The grouping is as follows: the control group, LPA stimulation group, wherein the LPA stimulation group was further provided with different concentrations of four groups of 1. mu.M, 5. mu.M, 10. mu.M and 20. mu.M, respectively. And normal culture and anoxic culture.

And (3) treatment: media containing different concentrations of LPA were prepared separately and 100. mu.l was added to each well of 96-well plate. The hypoxic group was cultured in an hypoxic box for 6 hours.

(3) Experimental methods

And digesting and transferring the cells to a 96-well plate when the cells cover about 90 percent, culturing in an incubator, changing the culture medium into a serum-free basic culture medium after the cells are attached to the wall, and performing starvation treatment for 24 hours to synchronize the cells. OD determination after 3-4 hours of incubation with CCK-8_450nmBaseline levels of cells. Then replacing culture medium with LPA with no or different concentration, culturing for 48 hr, or placing into an anoxic box, culturing for 6 hr, adding CCK-8, culturing for 3-4 hr, and determining OD_450nmAnd represents the cell number after stimulation by different concentrations of LPA and the activity of different types of endothelial cells in hypoxic culture.

(4) Results

As shown in a of fig. 6, wild-type endothelial cell proliferation increased with increasing LPA concentration, whereas LPA 2-deficient endothelial cells were non-responsive to LPA stimulation, showing no apparent proliferation. At the same time, LPA2 deletion decreased endothelial cell activity (B in fig. 6).

Example 6

Influence of Lpar2 gene overexpression on vascular homeostasis and cardiac function of mice after myocardial infarction

(1) Establishment of myocardial infarction model of adult mouse

The procedure is as in example 1.

(2) Experimental grouping and processing

a. Wild type mice were divided into two groups, and injected with Ad-Lpar2 (adenoviral vector expressing LPA2) and control Ad-GFP (adenoviral vector expressing GFP), respectively.

b. Grouping the operations: heart peduncle operation: refer to example 1. The sham operation group: a wild mouse of the SPF-grade BALb/c strain is taken, a 7-0 silk thread with a needle is passed through a left anterior descending branch without knotting in the process of constructing a myocardial infarction model according to example 1, and the rest of the myocardial infarction groups are operated.

c. The virus injection mode is as follows: the wild mouse is taken, virus is injected from the left ventricular cavity before the operation, and ligation is carried out after the blood circulation is carried out for 1 min.

(3) Experimental methods

A mouse myocardial infarction model is used, a sham operation control group is set, and virus is injected 1min before ligation. The heart function was examined by ultrasound for 4 weeks, followed by subsequent sampling and infarct size detection by Masson staining. And collecting materials at 7 days and 4 weeks after operation to detect angiogenesis.

(4) Results

As can be seen from fig. 7, after myocardial infarction, the LPA2 overexpression mouse myocardial function test 4 weeks after myocardial infarction shows that LPA2 overexpression can significantly reduce the myocardial infarction area (a in fig. 7) of mice and significantly improve the cardiac function (B in fig. 7) and the density of blood vessels between myocardium (C and D in fig. 7).

Example 7

Effect of LPA 2-specific agonist DBIBB on vascular homeostasis and cardiac function in mice after myocardial infarction

(1) Establishment of myocardial infarction model of adult mouse

The procedure is as in example 1.

(2) Experimental grouping and processing

a. Wild type mice were divided into two groups, injected with LPA2 specific agonist DBIBB and Control-Control: DMSO.

Lpa 2-specific agonist DBIBB administration: wild type mice were injected 3 times with LPA2 agonist DBIBB at 3mg/kg, before, 24 hours and 48 hours post-surgery, respectively.

(3) Experimental methods

Mouse myocardial infarction model is utilized, a sham operation control group is set, mouse survival is counted, 70kD FITC-Dextran is injected into tail vein 3 days after operation, and the infiltration degree in FITC-Dextran tissue is detected by using frozen sections for detecting the vascular permeability of mouse heart tissue. After 4 weeks, the heart function is detected by ultrasonic wave, then materials are taken, and the infarct size is detected by Masson staining.

(4) Results

3 days after the myocardial infarction, the permeability detection dye FITC-Dextran or Evans blue was injected 30 minutes before the material was drawn. As can be seen from fig. 8, the survival rate of the DBIBB-treated mice was improved after the myocardial infarction (a in fig. 8) and the infarct size was reduced (B in fig. 8) compared to the control group. Cardiac function testing 4 weeks after infarction showed that DBIBB treated mice also significantly improved cardiac function (C in fig. 8) and also increased inter-myocardial vascular density (D in fig. 8). The wild type mice treated with DBIBB had a reduced level of apoptosis in heart tissue (E in fig. 8) and significantly reduced vascular permeability (green fluorescence indicates dye leaking from the blood vessels into the interstitial space of the tissue, indicating how much of the dye leaked out indicates vascular permeability) compared to the control mice (F in fig. 8).

Example 8

The LPA2 deletion has an effect on the vascular permeability of organs induced by LPS (lipopolysaccharide), an endotoxin, a commonly used induction factor in experimental animal models for the treatment of mouse sepsis.

(1) Establishment of adult mouse sepsis model

Adult mice were weighed and were intraperitoneally injected with LPS at a dose of 15mg/kg (approximately 0.25mL in 25g mice).

(2) Experimental grouping and processing

SPF grade Balb/c wild-type mice and LPA2 knockout mice are taken, and two groups of mice are respectively provided with a PBS control group and an LPS model group.

(3) Experimental methods

Injecting Evans Blue into the tail vein of a live mouse 24h after LPS injection, taking the viscera (heart, liver, spleen, lung and kidney) 30min later, weighing, placing in 1mL formamide solution, performing water bath at 55 deg.C for 48h, taking the supernatant, centrifuging, and measuring absorbance (OD)_620nmAnd OD_740nm)。

(4) Results

In FIG. 9, A-E shows that vascular permeability of each organ of LPA 2-deficient mice was significantly increased after LPS-induced sepsis compared to wild-type mice, indicating disruption of systemic vascular homeostasis.

In summary, the research results of the embodiments of the present invention show that: the lysophosphatidic acid or lysophosphatidic acid receptor 2 and the agonist thereof can promote endothelial cell proliferation and angiogenesis so as to repair damaged cardiac muscle, can obviously reduce the myocardial infarction area (12.8 percent), and obviously improve the cardiac function after myocardial infarction (the LVEF is increased by nearly 50 percent), and has obvious effect. In addition, lysophosphatidic acid or lysophosphatidic acid receptor 2 and agonists thereof can protect vascular homeostasis in pathological conditions and reduce diseases of shock and death due to organ failure caused by vascular leakage. Therefore, the lysophosphatidic acid or the lysophosphatidic acid receptor 2 and the agonist thereof can be used for preparing a medicine or a preparation for treating or preventing ischemic diseases such as ischemic heart diseases or ischemic diseases of lower limbs, or diseases with vascular leakage symptoms such as sepsis or shock death caused by organ failure possibly caused by vascular leakage, and the like, and provides a new medicine and a new treatment idea for treating or preventing heart diseases such as ischemic heart diseases or other vascular diseases such as vascular leakage.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

Use of LPA2 or an agonist thereof, wherein the agonist is DBIBB, selected from at least one of the following (1) to (2):

(1) for the preparation of a medicament for the treatment or prevention of ischemic diseases; the ischemic disease is ischemic heart disease or vascular disease, the ischemic heart disease is acute myocardial infarction or ischemic heart failure, and the vascular disease is lower limb ischemic disease;

(2) for the preparation of a medicament for the treatment or prevention of a disease with symptoms of vascular leakage; the disease with vascular leak symptoms is sepsis.