Application of ALOX15B gene as target spot in screening medicine for treating pulmonary vascular remodeling and pulmonary arterial hypertension

Abstract

The invention discloses application of an ALOX15B gene serving as a target spot to screening of medicines for treating pulmonary vascular remodeling and pulmonary arterial hypertension, and belongs to the technical field of biological medicines. The invention discloses application of an ALOX15B gene or protein expression inhibitor in preparation of a medicine for treating pulmonary vascular remodeling and pulmonary arterial hypertension. By inhibiting expression of the ALOX15B gene or the encoding protein thereof, pulmonary vascular remodeling can be effectively reversed, and rising of pulmonary arterial pressure caused by oxygen deficit can be effectively reversed, so that the ALOX15B gene can be applied to treatment of pulmonary arterial hypertension related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the use of the ALOX15B gene as a target in screening drugs for treating pulmonary vascular remodeling and pulmonary arterial hypertension. Background Art

[0002] Pulmonary arterial hypertension is a malignant disease with a high incidence and poor prognosis, and its five-year survival rate is close to 60%. At present, the clinical treatment mainly uses antagonists of the endothelin pathway, the prostacyclin pathway, and the nitric oxide pathway, aiming to relieve symptoms by dilating blood vessels, but it cannot fundamentally relieve symptoms and improve its prognosis.

[0003] The pathogenesis of pulmonary arterial hypertension is as follows: after hypoxia or external stimulation, the pulmonary arterioles continuously contract, resulting in stenosis of the lumen, triggering pulmonary vascular remodeling, and then leading to an increase in pulmonary artery pressure. Pulmonary vascular remodeling mainly includes: neointima formation of pulmonary artery vessels, medial thickening, and adventitial fibrosis. Neointima formation is mainly caused by the proliferation and migration of endothelial cells, and medial thickening is mainly caused by the excessive proliferation of smooth muscle cells and the inhibition of apoptosis. Research shows that endothelial cells play an increasingly important role in pulmonary vascular remodeling. Endothelial cell dysfunction is the initiating factor leading to pulmonary artery vascular remodeling. Endothelial cells can also regulate the function of smooth muscle cells to cause smooth muscle proliferation, trigger inflammation, in-situ thrombosis, etc., and then lead to an increase in pulmonary artery pressure and trigger pulmonary vascular remodeling.

[0004] Studies have shown that ALOX15B (15-lipoxygenase type 2) is an important enzyme mainly involved in arachidonic acid metabolism, generating specific lipoxygenase products. Studies have shown that ALOX15B is highly expressed in tissues such as the skin, prostate, and lung, and its biological functions are closely related to cell differentiation, inflammatory responses, and lipid metabolism. ALOX15B plays an important role in inflammation and immune regulation by catalyzing the production of 15-hydroxyeicosatetraenoic acid (15-HETE) from arachidonic acid. In recent years, ALOX15B has been found to have a regulatory role in various diseases. In the field of dermatology, ALOX15B is related to the pathogenesis of psoriasis, and its expression level is significantly increased in psoriatic skin lesions, and it may participate in disease progression by regulating the differentiation and inflammatory responses of keratinocytes. In prostate cancer, the expression of ALOX15B is related to tumor invasiveness and prognosis, and its product 15-HETE may promote cancer cell proliferation and metastasis by affecting the tumor microenvironment. In addition, the role of ALOX15B in atherosclerosis has also attracted much attention, and the lipid oxidation mediated by it may be involved in the formation and stability of plaques. Studies have also shown that ALOX15B has a dual role in inflammatory diseases such as inflammatory bowel disease (IBD) and asthma. It may relieve symptoms through anti-inflammatory mechanisms, but may also exacerbate inflammatory responses under certain conditions. These findings suggest that ALOX15B is a potential therapeutic target, but its specific mechanism of action still needs further study. In summary, ALOX15B plays an important role in a variety of physiological and pathological processes, its functions are complex and tissue-specific, and future research is expected to provide new ideas for the diagnosis and treatment of related diseases. However, whether ALOX15B is involved in the process of pulmonary arterial hypertension and pulmonary vascular remodeling remains an unknown question. Therefore, in-depth exploration of the functions and exact mechanisms of ALOX15B in pulmonary arterial hypertension and pulmonary vascular remodeling may provide new targets and strategies for the clinical diagnosis and treatment of elevated pulmonary arterial hypertension and pulmonary vascular remodeling. Summary of the Invention

[0005] In view of the above deficiencies in the prior art, the present invention provides the use of the ALOX15B gene as a target in screening drugs for the treatment of pulmonary vascular remodeling and pulmonary arterial hypertension, and pulmonary vascular remodeling can be reversed and pulmonary arterial hypertension can be alleviated by inhibiting the expression of the ALOX15B gene or its encoded protein.

[0006] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is:

[0007] The object of the present invention is to provide the use of the ALOX15B gene as a target in screening drugs for the treatment of pulmonary vascular remodeling and pulmonary arterial hypertension.

[0008] Furthermore, the drug can inhibit the expression of the ALOX15B gene or its encoded protein.

[0009] Another object of the present invention is to provide the use of an ALOX15B gene or protein expression inhibitor in the preparation of a medicament for treating pulmonary vascular remodeling and pulmonary arterial hypertension.

[0010] Further, the inhibitor includes a neutralizing antibody, siRNA, shRNA, small molecule compound or polypeptide.

[0011] Further, the pulmonary arterial hypertension is pulmonary arterial hypertension caused by hypoxia; the pulmonary vascular remodeling is pulmonary vascular remodeling caused by hypoxia.

[0012] Further, the inhibitor is used to inhibit the abnormal increase in pulmonary arterial pressure.

[0013] Further, the inhibitor can inhibit the thickening of the pulmonary arterial vascular wall.

[0014] Another object of the present invention is to provide a medicament for reversing pulmonary vascular remodeling, which includes the above-mentioned ALOX15B gene or protein expression inhibitor.

[0015] Another object of the present invention is to provide a medicament for treating pulmonary arterial hypertension, which includes the above-mentioned ALOX15B gene or protein expression inhibitor.

[0016] Another object of the present invention is to provide the use of a reagent for detecting the expression level of the ALOX15B gene in the preparation of a preparation for detecting the prognosis of pulmonary vascular remodeling and pulmonary arterial hypertension.

[0017] Further, the preparation is a kit.

[0018] Advantages of the present invention:

[0019] By inhibiting the expression of the ALOX15B gene or its encoded protein, the present invention can effectively reverse pulmonary vascular remodeling and effectively reverse the increase in pulmonary arterial pressure caused by hypoxia, indicating that it can be applied to the treatment of pulmonary arterial hypertension-related diseases. Description of the Drawings

[0020] Figure 1 To detect the expression of ALOX15B protein in the lung tissues, pulmonary artery endothelial cells and pulmonary artery smooth muscle cells of normal mice and hypoxic mice;

[0021] Figure 2 To detect the effect of ALOX15B knockout on the cardiopulmonary function caused by PAH;

[0022] Figure 3 To detect the effect of ALOX15B knockout on the cardiac function injury caused by PAH by ultrasonic measurement;

[0023] Figure 4To evaluate the effect of ALOX15B knockout on pulmonary vascular remodeling induced by PAH by H&E, MASSON, and EVG staining. Detailed implementation manners

[0024] The following describes the detailed implementation manners of the present invention to facilitate the understanding of the present invention by those skilled in the art of the present technology. However, it should be clear that the present invention is not limited to the scope of the detailed implementation manners. For those of ordinary skill in the art of the present technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0025] Example 1 Construction of a hypoxia model

[0026] 1. Construction of a hypoxia cell model

[0027] (1) Transfer mouse pulmonary artery endothelial cells (mPAECs) into a cell culture flask, culture them using ECM medium, digest them with trypsin during passage, evenly inoculate them into a new culture flask, and when their density is about 60%-70%, place the cells in a hypoxic incubator with the culture conditions of O 2 content of 1%, CO 2 content of 5%, and culture at 37°C for 48 hours to construct a hypoxic pulmonary artery endothelial cell model.

[0028] (2) Transfer mouse pulmonary artery smooth muscle cells (mPASMCs) into a cell culture flask, culture them using SMCM medium, digest them with trypsin during passage, evenly inoculate them into a new culture flask, and when their density is about 60%-70%, place the cells in a hypoxic incubator with the culture conditions of O 2 content of 1%, CO 2 content of 5%, and culture at 37°C for 48 hours to construct a hypoxic pulmonary artery smooth muscle cell model.

[0029] 2. Construction of a hypoxic pulmonary hypertension mouse model

[0030] Select adult male C57BJ / 6 mice at 6-8 weeks of age, subject them to hypoxia treatment for 28 days (oxygen concentration 10%, normal pressure), and subcutaneously inject Sugen5416 (20 mg / kg) on the first day of each week. Culture them together with the normoxia group (oxygen concentration 21%, normal pressure) for 28 days to construct a hypoxic pulmonary hypertension mouse model.

[0031] Example 2 Detection of the expression level of ALOX15B in the hypoxia model

[0032] 1. Expression level of ALOX15B in the hypoxic pulmonary hypertension mouse model

[0033] Weigh 20 mg of the tissue and operate on ice. Place it in a 2.0 mL round-bottom centrifuge tube. Add 500 - 600 μL of lysis buffer to the centrifuge tube. Use a homogenizer to fully homogenize the tissue without leaving tissue chunks. After homogenization, place it in a pre-cooled centrifuge at 4°C and centrifuge at 13,500 rpm for 15 minutes. After centrifugation, transfer the supernatant to a new 1.5 mL centrifuge tube (make marks according to the experimental groups). Quantify the protein concentration using the BCA method. After quantification, add 5x loading buffer according to the corresponding volume and mix at a ratio of 4:1. Heat at 100°C in a metal bath for 8 minutes to denature the protein. After denaturation, place the sample on ice to quickly cool it down. After cooling, aliquot it and store it at -20°C for subsequent Western blot detection. The results are shown in Figure 1 , Figure 1 where A in Figure 1 is the protein expression level of ALOX15B in the lung tissues of normal mice and hypoxic-induced mice detected by Western blot.

[0034] 2. Expression level of ALOX15B in the hypoxic cell model

[0035] Take out the cells after model establishment from the incubator and place them on ice. Discard the culture medium and wash them 3 times with 1x PBS, being as gentle as possible to avoid washing away the cells. Add an appropriate volume of RIPA lysis buffer according to the cell density. Scrape the cells with a cell scraper on ice and collect them into a 1.5 mL centrifuge tube. After collection, place it in a pre-cooled centrifuge at 4°C and centrifuge at 13,500 rpm for 15 minutes. After centrifugation, transfer the supernatant to a new 1.5 mL centrifuge tube (make marks according to the experimental groups). Quantify the protein concentration using the BCA method. After quantification, add 5x loading buffer according to the corresponding volume and mix at a ratio of 4:1. Heat at 100°C in a metal bath for 8 minutes to denature the protein. After denaturation, place the sample on ice to quickly cool it down. After cooling, aliquot it and store it at -20°C for subsequent Western blot detection. The results are shown in Figure 1 . Figure 1 where B in Figure 1 is the protein expression level of mouse pulmonary artery endothelial cells cultured under normal conditions and mouse pulmonary artery endothelial cells cultured under hypoxic conditions detected by Western blot; C is the protein expression level of mouse pulmonary artery smooth muscle cells cultured under normal conditions and mouse pulmonary artery smooth muscle cells cultured under hypoxic conditions detected by Western blot. (NOR represents culture under normoxic conditions, and HYP represents culture under hypoxic conditions).

[0036] As Figure 1As shown, the expression levels of ALOX15B in the lung tissues, pulmonary artery endothelial cells (mPAECs), and pulmonary artery smooth muscle cells (mPASMCs) of mice with hypoxia-induced pulmonary hypertension were detected by Western blot. It was found that the expression levels of ALOX15B were significantly upregulated in both the lung tissues and pulmonary artery endothelial cells of mice after hypoxia induction.

[0037] Example 3 Effect of ALOX15B gene knockout on pulmonary vascular remodeling caused by PAH

[0038] 1. For the constructed mouse model of pulmonary hypertension, the right ventricular systolic pressure (RVSP), right ventricular thickness (RVT), and right ventricular hypertrophy index (RV / LV+S) were measured respectively. Mice with ALOX15B gene deletion (ALOX15 - / - ) and wild-type (WT) mice (older than 8 weeks) were exposed to 10% O 2 for 4 weeks to establish a PAH model. The right ventricular systolic pressure (RVSP) was measured by right ventricular catheterization, and the right ventricular wall thickening was evaluated by hematoxylin and eosin (H&E) staining. The right ventricular hypertrophy index (RV / LV+S) was calculated to evaluate the degree of hypertrophy. The results are shown in Figure 2 .

[0039] (1) The specific process of hematoxylin-eosin staining is as follows:

[0040] Take paraffin sections of mouse lung tissues and place them in an oven at 60 °C for 3 hours to dissolve the paraffin. After dissolution, proceed in the following order: xylene I for 10 minutes, xylene II for 10 minutes, xylene III for 10 minutes, 100% ethanol for 5 minutes, 100% ethanol for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, distilled water for 1 minute, hematoxylin for 5 minutes, rinse slightly with running water, differentiate with 1% hydrochloric acid alcohol for 5 seconds, blue with running water for 15 minutes, stain with 0.5% eosin for 1 minute, rinse slightly with distilled water, 80% ethanol for 5 minutes, 90% ethanol for 5 minutes, 100% ethanol for 5 minutes, xylene I for 10 minutes, xylene II for 10 minutes, xylene III for 10 minutes, and mount with neutral gum.

[0041] (2) The specific process of pulmonary artery pressure measurement is as follows:

[0042] The mice were anesthetized and fixed according to their body weight. The hair on the right neck was removed, and the epidermis was cut open with surgical scissors. The sternocleidomastoid muscle was found and the muscle fiber tissue was dissected along the medial margin to fully expose the external jugular vein. The distal end was ligated with surgical thread, and a small "V" incision was made with surgical scissors. The pressure probe catheter was slowly inserted into the right ventricle along the incision, and the pressure transducer was turned on to continuously monitor the changes in the right ventricular pressure of the mice, thereby indirectly reflecting the condition of the pulmonary artery. After the detection, the chest cavity was opened, the mouse heart was separated, and the right ventricle / left ventricle + interventricular septum (RV / LV+S) was calculated by weighing on an analytical balance to evaluate the degree of right ventricular hypertrophy.

[0043] As Figure 2 shown, after 4 weeks of Su / Hx treatment of ALOX15B gene knockout mice, the effect of ALOX15B knockout on the symptoms of pulmonary hypertension in mice was determined by RVSP, RVT, and RV / LV+S. It was found that compared with the normal group of mice, the RVSP of the pulmonary artery in the knockout mice was significantly decreased (Figures B and C). Histological analysis of myocardial tissue (HE staining) showed that the free wall of the right ventricle in the HYP-WT group was significantly thickened, and the right ventricular hypertrophy index was significantly increased. In the Su / Hx-ALOX15 - / - group, these pathological changes were basically reversed (Figures D-F).

[0044] 2. The effects of ALOX15B on the cardiac function of PAH mice were evaluated by detecting RVT, TAPSE, and PAT / PET of the heart by ultrasound. The results are shown in Figure 3 . The process of ultrasound measurement is as follows:

[0045] Before ultrasound, preparations were made. The hair on the chest and abdomen of the mice was removed with depilatory cream in advance, and at the same time, gaseous anesthetic (isoflurane), gas anesthesia machine, ultrasound coupling agent, mouse fixator, etc. were prepared. During ultrasound, a Vevo3000 or INNO6 was used to measure the ultrasound indexes of the mouse heart. The main indexes to be observed included the thickness of the anterior wall of the right ventricle (RVT), tricuspid annular systolic displacement (TAPSE), the ratio of pulmonary artery acceleration time to ejection time (PAT / PET), and the velocity time integral of pulmonary artery ejection (VTI). During the measurement, the mice were anesthetized and fixed on a foam board, and gaseous anesthesia was continuously administered. The ultrasound probe observed the overall shape of the mouse heart on the long axis of the heart, and the RVT of the mice was measured under M-mode; the mouse heart was observed on the short axis, and the pulmonary artery ejection was observed under color Doppler mode to measure PAT, PET, and VTI; the apical four-chamber image was observed with the apex position facing the heart direction, and the TAPSE of the mice was measured under M-mode. After the measurement was completed, the data was saved.

[0046] As Figure 3As shown, it was found that the thickening of RVT in ALOX15B gene knockout mice was alleviated (Figures A and B), TAPSE was significantly restored (Figures C and D), and PAT / PET was significantly increased (Figures E and F), indicating that the symptoms of PAH in mice were significantly improved.

[0047] 3. Evaluation of the degree of pulmonary artery vascular remodeling in mice by lung tissue staining

[0048] (1) The thickening of the pulmonary artery wall in each group was evaluated by H&E staining, and the process was the same as the above-mentioned H&E staining.

[0049] (2) The collagen deposition in the pulmonary artery was quantitatively determined by MASSON staining. The specific process was as follows:

[0050] The tissue sections were dewaxed to water; stained with Weigert iron hematoxylin for 5 - 10 minutes and rinsed slightly with running water; differentiated with 1% hydrochloric acid alcohol for several seconds and rinsed with running water for several minutes, stained with ponceau acid fuchsin for 10 minutes and rinsed slightly with running water; treated with phosphomolybdic acid for 5 minutes, poured off the staining solution without washing with water; counterstained with aniline blue for 5 minutes, poured off the staining solution without washing with water; rinsed the sections with 1% glacial acetic acid until no blue color was removed; rinsed slightly with 95% alcohol, decolorized with absolute alcohol, cleared with xylene, and sealed with neutral gum.

[0051] (3) Elastic fiber staining was used to detect the proliferation and injury of the pulmonary artery. The specific process was as follows:

[0052] The tissue sections were dewaxed to water; stained with potassium permanganate for 5 minutes and rinsed slightly with water; bleached with oxalic acid solution for 3 minutes until the specimen was colorless and rinsed with a thin stream of water; rinsed slightly with 95% alcohol, immersed in Elastin staining solution and reacted at room temperature for 8 - 24 hours; differentiated with 95% alcohol and quickly rinsed until no excess staining solution was removed, rinsed with a thin stream of water for 3 - 5 minutes; stained with Van Gieson staining solution for 1 minute, poured off the staining solution without washing with water; differentiated quickly with 95% alcohol for several seconds, dehydrated with absolute alcohol, cleared with xylene, and sealed with neutral gum.

[0053] The test results are shown in Figure 4 , Figure 4 where NOR - WT represents wild - type mice cultured under normoxic conditions, Su / Hx - WT represents wild - type mice cultured under hypoxic conditions, NOR - ALOX15 - / - represents ALOX15 knockout mice cultured under normoxic conditions, and Su / Hx - ALOX15 - / - represents wild - type mice cultured under hypoxic conditions.

[0054] As Figure 4As shown, H&E, MASSON, and EVG staining were used to detect the degree of pulmonary artery vascular remodeling in PAH mice. It was found that the thickening of blood vessel walls was alleviated in ALOX15B gene knockout mice (Figs. A and B), the degree of perivascular collagenization was significantly reduced (Figs. C and D), and the hyperplasia and fracture damage of elastic fibers in the media of blood vessels were significantly reduced (Figs. E and F). This further demonstrated that the PAH symptoms in mice after knocking out the ALOX15B gene were significantly improved, and at the same time, the process of pulmonary vascular remodeling was reversed.

[0055] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Use of ALOX15B gene as a target in screening drugs for the treatment of pulmonary vascular remodeling and pulmonary hypertension.

2. The use according to claim 1, characterized in that The drug can inhibit the expression of the ALOX15B gene or the protein encoded by it.

3. Use of ALOX15B gene or protein expression inhibitors in the preparation of drugs for treating pulmonary vascular remodeling and pulmonary hypertension.

4. The use according to claim 3, characterized in that Inhibitors include neutralizing antibodies, siRNA, shRNA, small molecule compounds or peptides.

5. The use according to claim 3, characterized in that: Pulmonary hypertension is pulmonary arterial hypertension caused by hypoxia; pulmonary vascular remodeling is pulmonary vascular remodeling caused by hypoxia.

6. A drug for reversing pulmonary vascular remodeling, characterized in that: The invention comprises the ALOX15B gene or protein expression inhibitor as described in claim 3.

7. A drug for treating pulmonary artery disease, characterized in that: The invention comprises the ALOX15B gene or protein expression inhibitor as described in claim 3.

8. Use of a reagent for detecting the expression level of the ALOX15B gene in the preparation of a preparation for detecting pulmonary vascular remodeling and the prognostic effect of pulmonary hypertension.

9. The use according to claim 8, characterized in that The preparation is a kit.

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