Role of frizzled 2 as a target in treating pressure-overload myocardial injury

Abstract

The application discloses a role of Frizzled 2 as a target point in treating pressure overload myocardial injury. The application simulates pressure overload myocardial injury in mice by using a transverse aortic constriction (TAC) operation, and in the animal model experiment of pressure overload myocardial injury, myocardial injury of the mice can be relieved and heart function of the mice can be improved after Frizzled 2 is knocked down by using an adeno-associated virus, and therefore, the result of the application shows that the adeno-associated virus for knocking down FZD2 can be used for treating patients with pressure overload myocardial injury, can delay the progress of myocardial injury, and can improve the life quality of the patients.

Description

Technical Field

[0001] The present invention relates to the role of Frizzled 2 as a target in treating pressure overload myocardial injury, and belongs to the technical field of biomedicine. Background Art

[0002] Pressure overload, caused by factors such as hypertension and valvular disease, is the most common cause of myocardial injury. Currently, in addition to treatment targeting the underlying cause, cardioprotective drugs are often used to reverse myocardial remodeling caused by pressure overload. However, current cardioprotective drugs cannot completely alleviate the myocardial cell damage caused by pressure overload, necessitating the discovery of new targets for cardioprotection.

[0003] Frizzled 2 (FZD2) is a protein that plays an important role in the body and is a member of the Frizzled family. The Frizzled family is a family of proteins closely associated with cell signaling and development, playing key roles in physiological processes such as cell polarity, embryonic development, tissue repair, and cancer. FZD2 is expressed on human cell membranes and, as a seven-transmembrane receptor, recognizes and interacts with Wnt proteins. The Wnt signaling pathway is a key component of the cell signaling network and is crucial for embryonic development, tissue repair, and tissue maintenance in adulthood. As a key component of the Wnt signaling pathway, FZD2 regulates processes such as cell proliferation, differentiation, and migration. Research has shown that FZD2 plays a role in various physiological and pathological conditions. During normal embryonic development, FZD2 participates in the formation of the embryonic body axis and organ development. Furthermore, FZD2 plays an important role in the development of the nervous system, skeleton, and cardiovascular system. However, the potential of FZD2 as a therapeutic target for pressure-overload myocardial injury has not yet been fully explored, and the prognosis of pressure-overload myocardial injury remains worrying. Summary of the Invention

[0004] The purpose of the present invention is to address the shortcomings of traditional myocardial injury treatments, such as cardiotonic, diuretic, and anti-vascular therapies. The present invention provides a target for Frizzled 2 in the treatment of pressure-overload myocardial injury. Animal experiments have shown that knocking down Frizzled 2 can alleviate myocardial damage and improve cardiac function in mice.

[0005] In order to achieve the above object, the present invention provides the use of Frizzled 2 in the preparation of a medicament for treating pressure overload myocardial injury.

[0006] Preferably, the active ingredient of the drug is an agent that inhibits the expression or activity of the Frizzled 2 gene or protein.

[0007] Preferably, the agent is RNAi, microRNA, shRNA, siRNA, an antibody to Frizzled 2 protein or an activity inhibitor of Frizzled 2 protein that specifically inhibits Frizzled 2 gene expression, or a plasmid vector or adeno-associated virus vector containing the RNAi, microRNA, shRNA or siRNA.

[0008] Preferably, the agent is an adeno-associated viral vector, which can specifically inhibit Frizzled 2 gene expression.

[0009] Preferably, the adeno-associated viral vector comprises shRNA targeting Frizzled 2.

[0010] Preferably, the adeno-associated virus vector is AAV9 virus.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] Adeno-associated virus (AAV) has become one of the most important gene vectors in the field of gene therapy due to its advantages such as long-term expression, low toxicity, low immunogenicity, and high tissue specificity. To address the shortcomings of traditional myocardial injury treatments such as cardiotonic, diuretic, and anti-vascular therapies, the present invention establishes a pressure-overload myocardial injury mouse model through transverse aortic constriction (TAC). In vivo animal experimental results show that knocking down FZD2 using AAV can be used to treat patients with pressure-overload myocardial injury, delaying the progression of myocardial injury and improving patients' quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The results of FZD2 knockdown were detected by Western blotting.

[0014] Figure 2 Comparison of ejection fraction among the four groups; ns: no statistical difference; *: P < 0.05; **: P < 0.01; ***: P < 0.001);

[0015] Figure 3 Comparison of heart weight / tibia ratio among the four groups; ns: no statistical difference; *: P < 0.05; **: P < 0.01; ***: P < 0.001. DETAILED DESCRIPTION

[0016] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0018] Example

[0019] Transverse aortic constriction (TAC) was performed to simulate pressure-overload myocardial injury in mice. Adult male C57BL / 6J mice (8 weeks old, 20-25 g) were anesthetized and intubated. Under anesthesia and mechanical ventilation, the thoracic area was trimmed and disinfected. The skin was incised at the second and third intercostal spaces on the left side of the sternum. Subcutaneous muscle was separated with hemostats, the second rib was cut, and the chest wall was expanded with a chest expander. The aortic arch was isolated with microforceps, and a 27-gauge needle was placed transversely across the aortic arch. Silk suture was then tied, and the needle was gently withdrawn, successfully inducing TAC. In the SHAM group, only suture was threaded without ligation; the remaining procedures were the same as in the TAC group. The mice were randomly divided into a control adeno-associated virus (AAV-Con)-injected group and a FZD2 knockdown adeno-associated virus (AAV-shFZD2)-injected group. AAV was injected via the tail vein on the day of surgery, and cardiac function was subsequently monitored by echocardiography.

[0020] 1. Adeno-associated virus preparation and injection:

[0021] Adeno-associated virus was commissioned to Heyuan Bio to prepare the shRNA fragment targeting mouse FZD2 (5'-GAAAGAAGGCTGCACTATACT-3', SEQ ID NO: 1) and constructed into the pcAAV-cTNT-MCS-WPRE vector. U6 was used to initiate shRNA expression, and the polyA tail was used to terminate expression. After the vector was constructed, it was transfected into HEK-293T cells together with the packaging system plasmid for viral packaging. The rAAV9 containing the adeno-associated virus targeting mouse FZD2 shRNA was injected into the tail vein at a dose of 1x 10 11 vg (vector genome) was administered to mice that had undergone TAC surgery or SHAM. The negative control (rAAV9eGFP) expressed enhanced green fluorescent protein (eGFP) alone. Two weeks later, the knockdown efficiency of FZD2 was assessed by Western blotting. Figure 1 shown.

[0022] 2. Experimental animals and animal model preparation:

[0023] Select SPF-grade male C57BL / 6J mice (purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.) with an age of 8 weeks and a weight of 20-25g, and prepare tracheal intubation items, fixing tape, etc.; place the mouse in an anesthesia closed box, and after the muscle strength disappears, quickly fix it on its back on the operating board; aim the cold light source at the mouse's neck, and gently pull out the mouse's tongue with tweezers. Under the illumination of the cold light source, you can see the tracheal opening with strong refractive index opening and closing in line with the breathing frequency. Quickly insert the tracheal tube into the airway. During this period, if the mouse shows signs of awakening, it should quickly inhale isoflurane again; connect to the anesthesia ventilator. If the intubation is successful, you can see the mouse's chest cavity fluctuate with the ventilator frequency; use scissors to cut the hair on the mouse's chest area and disinfect it with iodine; cut the skin between the second and third ribs on the left side of the sternum, use hemostatic forceps to separate the subcutaneous muscles, expose the ribs, cut the second rib along the left side of the sternum, and expand the ribs. A chest device is placed in it and the chest wall is stretched open; blood can be absorbed with a sterile cotton swab or a small cotton ball to expose the surgical field of view; the thymus is carefully separated along the middle of the bilateral thymus with microtweezers to expose the ascending aorta and its branches; the aortic arch and excess tissue outside the bifurcation are continued to be separated with microtweezers to fully expose the aortic arch and the first and second branches; with the help of homemade tools, No. 5 silk thread is passed through the brachiocephalic trunk and the left common carotid artery, a 27G needle is placed horizontally on the aortic arch, and the silk thread is tied to the 27G needle; the needle is gently withdrawn; after confirming that there is no active bleeding, the chest wall and muscles are sutured layer by layer, and the chest wall is gently squeezed at the end of chest closure to try to expel the gas in the chest cavity, and finally the skin is sutured; the skin is disinfected with iodine tincture, connected to a small ventilator without anesthesia, and placed back in the cage after the mouse is fully awake. A heater can be used to help increase the temperature around the mouse to accelerate the mouse's awakening; the SHAM group only threaded the thread without ligation, and the rest of the process was the same as the surgical group.

[0024] 3. Experimental Grouping

[0025] There were 4 groups in the experiment: SHAM+AAV-Con group; SHAM+AAV-shFZD2 group; TAC+AAV-Con group; TAC+AAV-shFZD2 group. The virus titer was 1×10 11 VG / mouse.

[0026] 4. Echocardiography Evaluation of Mouse Cardiac Function

[0027] Echocardiography was performed at 8 weeks, with a probe frequency of 30 MHz. Specifically, after the animals were anesthetized with isoflurane, M-mode images were recorded when the heart rate of the mice was maintained at 450-550 beats / min. B-Mode images of the parasternal long axis section and the apical four-chamber section were collected. The parasternal left ventricular short axis was taken, and the left ventricular short axis section was shown by 2D ultrasound. The left ventricular movement was recorded by M-mode ultrasound at the level of the papillary muscle, and the left ventricular ejection fraction (LVEF) was mainly observed. The changes in cardiac morphology and function of the mice in each group were compared. All measured values ​​are the average of 5 consecutive cardiac cycles. The results showed that the cardiac function indicators of the mice in the TAC+AAV-Con group were significantly lower than those in the SHAM+AAV-Con group, and the cardiac function of the mice in the TAC+AAV-shFZD2 group was improved. The LVEF of the mice in the TAC+AAV-shFZD2 group was 18.98% higher than that of the mice in the TAC+AAV-Con group (P<0.05) (see Figure 2 ); the heart weight / tibia ratio of mice in the TAC+AAV-shFZD2 group was lower than that in the TAC+AAV-Con group (P<0.05) (see Figure 3 ).

[0028] In summary, knockdown of Frizzled 2 can alleviate myocardial damage in mice and improve cardiac function in mice. Therefore, the results of the present invention indicate that knockdown of FZD2 using adeno-associated virus can be used to treat patients with pressure overload myocardial injury, delay the progression of myocardial injury, and improve the quality of life of patients.

[0029] The above description is only a preferred embodiment of the present invention and does not constitute any formal or substantial limitation to the present invention. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Use of an agent for inhibiting the expression or activity of Frizzled 2 gene or protein in the preparation of a drug for treating pressure overload myocardial injury, characterized in that: The reagent is shRNA that specifically inhibits the expression of Frizzled 2 gene or an adeno-associated virus vector containing the shRNA. The sequence of the shRNA is shown in SEQ ID NO: 1.

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