CN113712951A - Macrophagemin 1 in preparation of medicine for treating pulmonary hypertension and application thereof - Google Patents

Abstract

The invention discloses an application of macrophagin 1 in preparation of a medicine for treating pulmonary hypertension, and a novel pulmonary hypertension treatment scheme can be provided through the application, wherein macrophagin Maresin1 can reverse pulmonary hypertension, improve right heart dysfunction, and improve vascular remodeling by inhibiting the transformation of endothelial cells to interstitial cells and promoting the apoptosis of pulmonary vascular smooth muscle cells.

Description

Macrophagemin 1 in preparation of medicine for treating pulmonary hypertension and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a macrophagin 1 in preparation of a medicine for treating pulmonary hypertension and application thereof.

Background

Pulmonary Hypertension (PH) is a serious progressive pulmonary vascular disease characterized by increased pulmonary vascular resistance due to remodeling of the distal pulmonary arterioles, ultimately leading to right heart failure and death. The basic pathological features are pulmonary vascular remodeling, including endothelial dysfunction, increased proliferation and resistance to apoptosis of pulmonary artery endothelial cells and smooth muscle cells, production and accumulation of extracellular matrix, and increased expression and infiltration of peripheral pro-inflammatory cytokines and chemokines of blood vessels. In pulmonary hypertension caused by hypoxia, pulmonary arterial pressure can be elevated due to alveolar capillary bed destruction or chronic hypoxic vasoconstriction. Clinically, it is mainly manifested by a sustained elevated pulmonary artery pressure that produces an increase in pressure in the thin-walled right ventricle. The right ventricle adapts to the low pressure pulmonary circulation without being able to withstand the increased pressure, eventually causing right heart failure, which is also the most common cause of death in patients with pulmonary hypertension. The mechanisms underlying these pathologies are still poorly understood, and currently available therapies have limited efficacy in pathological vascular remodeling, studying new pathways involved in pulmonary vascular remodeling, and providing new therapeutic targets for pulmonary hypertension.

Specific pro-resolving mediators (SPMs) are lipid mediators produced by polyunsaturated fatty acids in the process of inflammation of the body, and have the effects of inhibiting neutrophil recruitment, reducing the release of inflammatory mediators, promoting phagocytes to clear apoptotic cells and promoting resolution of inflammation. Maresin1 (macrophagin 1) is an important endogenous candidate for regression from docosahexaenoic acid (DHA).

The existing macrophagin 1 is generally used as an anti-inflammatory drug in the treatment effect, and no relevant research is carried out on macrophagin 1 in the pulmonary hypertension treatment.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the macrophagin 1 in the preparation of the medicine for treating the pulmonary hypertension and the application thereof, and can provide a new pulmonary hypertension treatment scheme.

In order to achieve the purpose, the invention provides the following technical scheme: the application of macrophagin 1 in preparing a medicament for treating pulmonary hypertension.

Further, macrophagin 1 inhibits endothelial cell transformation to mesenchymal cells, reducing the source of pulmonary vascular smooth muscle cells.

Further, macrophagin 1 promotes apoptosis of pulmonary vascular smooth muscle cells to inhibit proliferation thereof, and improves pulmonary vascular remodeling.

A medicine for treating pulmonary hypertension comprises macrophagin 1 with effective dose and a carrier for carrying macrophagin 1.

Further, the administration mode of the medicine is intraperitoneal/intravenous injection.

The invention has the advantages that the macrophagin 1 can reverse pulmonary hypertension, improve right heart dysfunction and improve vascular remodeling by inhibiting the transformation of endothelial cells to interstitial cells and promoting the apoptosis of pulmonary artery smooth muscle cells.

Drawings

FIG. 1 is a schematic diagram of indexes successfully established in a pulmonary hypertension mouse model of the present invention, and (A) RVSP is monitored by invasive hemodynamics. (B) Right heart hypertrophy was assessed (Fulton index (RV/LV + S)). (C-F) evaluating the right heart function-related indicator with a small animal ultrasound machine, comprising: TAPSE, PATT/PET. (G) The HPLC tandem mass spectrum showed MaR1 retention times (m/z 359/177). Q1, M-H (parent ion) and Q3, diagnostic ions (daughter ions) in tandem mass spectrometry (MS/MS). (H) MS/MS spectrum and molecular structural formula of Maresin 1. (I) Concentration of Maresin1 in serum of each group of mice.

FIG. 2 is a schematic diagram showing the effect of Maresin1 on pulmonary artery pressure and right heart function in mice according to the present invention, and (A) experimental modeling protocol. (B) Hemodynamic monitoring RVSP. (C) Right heart hypertrophy (Fulton index (RV/LV + S)). (D-G) Small animal ultrasound machine detects right heart function related indicators including TAPSE, PAAT/PET.

FIG. 3 is a schematic diagram showing the effect of Maresin1 on pulmonary vascular remodeling in pulmonary hypertension mice, (A) observation of vascular morphology change by HE staining of distal pulmonary small vessels. (B) Vessel wall area/total vessel area (WA/TA). (C) Vessel wall thickness/total vessel thickness (WT/TT). (D) Masson staining observed perivascular collagen deposition. (E) Immunohistochemistry alpha-SMA observed changes in medial smooth muscle cells in pulmonary artery vessels. (F) Immunohistochemistry CD31 observed changes in endothelial cells in the pulmonary artery lining. (G) Quantitatively analyzing alpha-SMA positive cells of the vascular wall.

FIG. 4 is a schematic diagram of the effect of Maresin1 on the transformation of pulmonary vascular endothelium into stroma, (A) co-localization of alpha-SMA and vWF staining by confocal laser observation. (B) Quantitative analysis of vWF + α -SMA + positive cells in α -SMA + cells.

FIG. 5 is a schematic diagram showing the effect of Maresin1 on apoptosis of pulmonary arterial hypertension pulmonary artery smooth muscle cells in mice according to the present invention, (A) Tunel staining to evaluate the distribution of apoptotic cells in arterioles localized by apoptosis of pulmonary artery smooth muscle cells in mice. (B) Statistical analysis of tunel positive cells in α -SMA + cells.

FIG. 6 is a schematic diagram showing the effect of Maresin1 on the expression of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax in the lung tissue of mouse pulmonary hypertension. (B) Data statistics map Bax/Bcl-2. (C) Expression of the pro-apoptotic protein clear-caspase 3. (D) Statistical data for clear-caspase 3 expression.

Detailed Description

The invention will be further described in detail with reference to the following examples, which are given in the accompanying drawings.

Pulmonary Hypertension (PH) is a progressive and fatal pulmonary vascular disease characterized by distal pulmonary arteriolar remodeling leading to gradual increase in pulmonary vascular resistance, ultimately leading to right heart failure and death. The basic pathological features are pulmonary vascular remodeling including endothelial dysfunction, increased proliferation and resistance to apoptosis of Pulmonary Artery Endothelial Cells (PAECs) and Pulmonary Artery Smooth Muscle Cells (PASMCs), increased production and accumulation of extracellular matrix, and increased expression of local pro-inflammatory cytokines and chemokines with subsequent leukocyte infiltration into the perivascular areas of the lung. Although current therapies improve quality of life and prognosis, the underlying mechanisms of pathology remain unclear and currently available therapeutics have limited efficacy in pathological vascular remodeling.

The pulmonary artery is divided into three layers, including an innermost monolayer of endothelial cells, a middle layer of smooth muscle cells, and an outer layer of fibroblasts. During the pathological development of pulmonary hypertension, three layers of cells all show abnormal activity. More and more studies report that in pulmonary hypertension, early vascular endothelial cell apoptosis is crucial in the development of pulmonary hypertension. Various environmental factors such as hypoxia can lead to injury of the endothelial cells of the pulmonary vessels, increased endothelial apoptosis and ultimately endothelial dysfunction. Endothelial cell dysfunction, produces large amounts of growth factors. For example, PDGFB, FGF2, TGF beta is highly expressed in lung tissues of patients with pulmonary hypertension, and pharmacological inhibition or knockout of these factors can effectively inhibit the development of pulmonary hypertension. In addition, increased expression of endothelial adhesion molecules, proinflammatory cytokines and chemokines, and leukocyte recruitment, also contribute to the development of pulmonary hypertension. The growth factor and the proinflammatory factor released by the endothelial cells can stimulate the proliferation of smooth muscle cells and fibroblasts of pulmonary arteries, and can further activate abnormal endothelial cells, so that the tube wall is thickened, the tube cavity is narrow, and the tube cavity can be seriously blocked. Following endothelial cell apoptosis, as the disease progresses, it may also induce endothelial cell hyperproliferation that produces an anti-apoptotic mesenchymal phenotype, a process known as endothelial mesenchymal transition (EndMT), leading to plexus-like lesions and vascular occlusion, resulting in severe pulmonary hypertension.

Thus, the control protocol was designed as follows:

selecting 6-8 weeks old C57BL/6, establishing a pulmonary hypertension classical animal model, randomly dividing the pulmonary hypertension classical animal model into 5 groups, and recording the day of molding as day 0:

example 1

Normoxic control (Ctr) mice were exposed to normoxic conditions for 6 weeks.

Example 2

Chronic hypoxia group (CH) mice were placed in a hypoxic chamber for 6 weeks of hypoxia.

Example 3

Hypoxia + sugen5416(SuHx) group mice sustained hypoxia for 6 weeks, 2.5mg/kg sugen5416 injected subcutaneously at 0, 7, 14.

Example 4

Hypoxia + Maresin1 group (CH + MaR1) mice continued to be hypoxic for 6 weeks, given Maresin1 treatment on day 21 of chronic hypoxia, i.p. 1 μ g/mouse, followed by 100ng/mouse addition every other day until day 42.

Example 5

Hypoxia + sugen5416+ Maresin1 group (SuHx + MaR1) mice continued hypoxia for 6 weeks, were treated with 2.5mg/kg of sugen5416, Maresin1 injected subcutaneously on day 21 of chronic hypoxia, i.p. with 1 ug/mouse, and then supplemented with 100ng/mouse every other day until day 42.

Example 6

For the experimental groups of examples 1-5, the body weights of the mice were recorded daily. After 21 days of modeling, the concentration of Maresin1 in plasma was determined by ultrasonic high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Small animal ultrasound and invasive hemodynamic monitoring assess Right Ventricular Systolic Pressure (RVSP) and right heart function index tricuspid annulus systolic displacement (tassel), Pulmonary Artery Acceleration Time (PAAT), pulmonary artery ejection time (PET), right heart hypertrophy index including right ventricle/(left ventricle + compartment) (right ventricle/left ventricle + compartment, RV/(LV + S)).

HE and Masson staining assessed lung tissue pulmonary arteriole morphology and collagen deposition. Immunohistochemistry for α -SMA and CD31 assessed the extent of proliferation of pulmonary artery smooth muscle cells and endothelial cells. vWF and alpha-SMA immunofluorescence co-localization to assess the transformation of vascular endothelial cells into mesenchymal cells in small lung vessels. Tunel staining (TdT-media dUTP nic end labeling, Tunel) detects the degree of apoptosis of PASMCs. WB detected the expression of apoptosis-related proteins Bax, Bcl-2, clear-caspase 3 in lung tissue.

1. After the mice are exposed to the environment of chronic hypoxia and sugen5416 combined hypoxia for 3 weeks and a pulmonary arterial hypertension model is established, high performance liquid chromatography mass spectrometry and invasive haemodynamics results show that compared with a control group, the concentration of Maresin1 in the plasma of the CH group mice and SuHx group mice is reduced, RVSP is obviously increased, and TAPSE and PAAT/PET are obviously reduced.

2. After the mice are modeled for 42 days, noninvasive hemodynamics and animal ultrasound results show that the weight of CH group mice and SuHx group mice is reduced, RVSP is obviously increased, RV/(LV + S) is increased, TAPSE and PAAT/PET are obviously reduced, the weight of the mice is recovered after Maresin1 intervention treatment, RVSP and RV/(LV + S) are reduced, and TAPSE and PAAT/PET are increased. Maresin1 was shown to reverse pulmonary hypertension and improve right heart hypertrophy and right heart dysfunction.

The results of HE and Masson staining showed that the pulmonary artery of the control mice was morphologically normal, the wall of the vessel was thin, and the collagen accumulation in the vessel and its surroundings was low. The pulmonary distal arteriole wall of CH group mice and SuHx group mice is obviously thickened, the lumen is narrowed, and a large amount of collagen is generated and accumulated in blood vessels and the periphery of the blood vessels. The above abnormal pulmonary vascular remodeling is improved upon treatment with Maresin 1.

4. The immunohistochemical result shows that the expression levels of alpha-SMA and CD31 in the pulmonary artery hypertension model group are obviously increased, the inner layer and the middle layer of the blood vessel are abnormally proliferated, the wall of the blood vessel is thickened, and the lumen is narrowed. After Maresin1 treatment, the expression level of alpha-SMA and CD31 is obviously reduced, the tube wall is thinned, and the size of the tube cavity is restored to the level of a control group. Maresin1 can inhibit proliferation of smooth muscle cells and endothelial cells of distal pulmonary vessels and improve pulmonary vessel remodeling after intervention treatment.

5. Laser confocal results show that vWF of mice in a control group is expressed in a single-layer inner layer of pulmonary artery, and is not co-expressed with alpha-SMA, and the alpha-SMA is expressed at the outer edge of arterioles. In the pulmonary hypertension model group, vWF and alpha-SMA are co-expressed and the expression level is obviously increased. The co-expression of vWF and alpha-SMA in the Maresin1 treatment group is obviously reduced, and the expression amount is reduced.

The Tunel staining result shows that the pulmonary artery hypertension model group has increased expression of the vascular smooth muscle marker alpha-SMA compared with the control group, and pulmonary artery smooth muscle cells which are positive for Tunel staining have almost no expression. After Maresin1 treatment, the apoptosis of the alpha-SMA positive cells can be restored to a normal level, and the total expression of the alpha-SMA is reduced.

WB results show that in lung tissues of the pulmonary arterial hypertension model group, Bcl-2 expression is increased while Bax expression level is not changed greatly, the Bax/Bcl-2 ratio is reduced, and clear-caspase 3 protein expression is obviously reduced. Expression of Bax/Bcl-2 ratio and clear-caspase 3 was reversed following Maresin1 administration.

Example 7

Based on the conclusions of example 6, the following also provides a drug that can be used to treat pulmonary hypertension, the drug having an effective dose of macrophagin 1, i.e., Maresin 1. Maresin1 was carried by the vehicle and administered intraperitoneally or intravenously.

In conclusion, the scheme utilizes the macrophagin Maresin1 to inhibit proliferation and expression of distal pulmonary vascular smooth muscle cells alpha-SMA and endothelial cells CD31, improve pulmonary vascular remodeling, and obviously reduce co-expression and expression quantity of vWF and alpha-SMA. The macrophagin Maresin1 regulates and improves the Bax/Bcl-2 ratio and improves the clean-caspase 3 protein table to promote the apoptosis of the alpha-SMA positive cells, reduce the total expression of the alpha-SMA and restore the apoptosis of the alpha-SMA positive cells to a normal level. Maresin1 reverses pulmonary hypertension, improves right heart dysfunction, and improves vascular remodeling by inhibiting endothelial cell transformation into mesenchymal cells and promoting apoptosis of pulmonary smooth muscle cells. Therefore, the medicine has obvious effect on treating pulmonary hypertension symptoms.

The following, a detailed description of the above experimental procedure:

assessing whether a pulmonary hypertension model is successfully established

Mice were placed in a hypoxic chamber (oxygen concentration: 10% carbon dioxide concentration: 5%) for 3 weeks to induce a chronic hypoxic pulmonary hypertension model. Another group of mice were placed in a hypoxic chamber for 3 weeks of hypoxia and subcutaneously injected with sugen5416(2.5mg/kg) on days 0, 7, 14, inducing a hypoxia + sugen5416 pulmonary hypertension model. The control mice were kept in a normal oxygen environment. To assess whether the model was successfully established, mice were randomized into 3 groups: (1) normoxic control (Ctr): 3 weeks under normal oxygen environment; (2) hypoxic group (CH) sustained hypoxia for 3 weeks; (3) hypoxia + sugen5416(SuHx): hypoxia was continued for 3 weeks, with 2.5mg/kg of sugen5416 injected subcutaneously on days 0, 7, and 14. After the molding is finished for 21 days, the related indexes of the right ventricular systolic pressure and the right heart function are evaluated by a small animal ultrasonic instrument and hemodynamic detection. The eyes were then bled and the supernatant was collected, then the mice were sacrificed and lung tissue was collected and stored in a-80 ℃ freezer.

Evaluation of Maresin1 therapeutic Effect on pulmonary hypertension

Mice were placed in a hypoxic chamber (oxygen concentration: 10% carbon dioxide concentration: 5%) for 6 weeks to induce a chronic hypoxic pulmonary hypertension model. Another group of mice were placed in a hypoxic chamber for 6 weeks of hypoxia and subcutaneously injected with sugen5416(2.5mg/kg) on days 0, 7, and 14, inducing a hypoxic + sugen5416 pulmonary hypertension model. Control mice were housed in a normal oxygen environment. Maresin1 was purchased from Cayman Chemical, USA. Maresin1 purchased was dissolved in ethanol and stored at-80 ℃. Before use, ethanol was first blown off with a nitrogen blower and Maresin1 was then rapidly dissolved in sterile physiological saline. To evaluate the effect of Maresin1 on two mouse pulmonary hypertension models, mice were randomized into 5 groups: (1) normoxic control (Ctr) 6 weeks in normoxic environment; (2) hypoxic group (CH) hypoxia for 6 weeks; (3) hypoxia + sugen5416(SuHx) by continuous hypoxia for 6 weeks, followed by subcutaneous injection of 2.5mg/kg sugen5416 on days 0, 7, and 14; (4) hypoxia + Maresin1(CH + MaR1) continued for 6 weeks of hypoxia, given Maresin1(1 μ g/mouse) treatment by i.p. injection on day 21, followed by 100ng/mouse addition every other day until week 6; (5) hypoxia + sugen5416+ Maresin1(CH + MaR1) hypoxia was continued for 6 weeks with 2.5mg/kg of sugen5416 injected subcutaneously on days 0, 7, 14, and Maresin1(1 μ g/mouse) treatment administered intraperitoneally on day 21, followed by 100ng/mouse addition every other day until week 6. After 42 days of molding, the small animal ultrasound apparatus evaluates the right heart function related index, followed by invasive hemodynamic monitoring of right ventricular systolic pressure. Blood was collected from the eyeball, the supernatant was collected, the mice were sacrificed, and lung tissue was collected and fixed in paraformaldehyde or stored at-80 ℃ for further experiments.

High performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)

After the mice are modeled for 21 days, the mice are taken out from a hypoxia chamber, whole blood of the retroorbital plexus of the mice is collected before sacrifice, and then the whole blood is centrifuged to collect serum, and the serum is stored at minus 80 ℃ for standby. The concentration of Maresin1 in mouse serum was detected by hplc tandem mass spectrometry. Serum samples (1ml) were placed in ice methanol (4ml) with tritium-labeled Maresin1 in each sample. The samples were then placed at-20 ℃ for 45 minutes to allow protein to settle, then centrifuged and the supernatant collected. The total lipid medium in the serum is extracted by using a C18 solid phase chromatographic column of Waters company in the United states, and the content of Maresin1 in the serum is analyzed by ultra performance liquid chromatography-tandem mass spectrometry. Experiment water containing 0.01% acetic acid was selected as mobile phase a and methanol containing 0.01% acetic acid was used as mobile phase B. Data collection and analysis were performed using an ultra performance liquid chromatography system equipped with an AB Sciex instruments 6500Q-Trap mass spectrometer (SCHEX, USA) and Analyst 1.6 software (applied biosystems, USA). Quantification of Maresin1 was based on multiple reaction monitoring transition peak areas and linear calibration curves for each compound.

Echocardiography of small animals

After 42 days of molding, the mice were removed from the hypoxic chamber and allowed to acclimate for several hours in normoxic environment. Mice were induced to anesthesia with isoflurane (1.2-1.6% isoflurane, Shenzhen Riwold Life technologies, Inc., China) and maintained anesthesia with continued inhalation of isoflurane (1.5-3%). The mouse limbs and head were then fixed on a console, the hair on the chest was removed with depilatory cream, and the right heart function-related index of the mouse was examined by transthoracic ultrasound using vevo3100 mini-animal ultrasound (FUJIFILM Visual Sonics). Pulmonary Artery Acceleration Time (PAAT) and pulmonary artery ejection time (PET) were measured in the parasternal long axis of mice by pulsed doppler. Tricuspid annular systolic displacement (TAPSE) was measured using M-ultrasound in the mouse parasternal minor axis.

Invasive hemodynamic monitoring

After 42 days of mouse molding, the mice were taken out from the hypoxic chamber and acclimated in normoxic environment for several hours. The body weight was weighed and recorded. Injecting 2% sodium pentobarbital (60mg/kg) into the abdominal cavity for anesthesia, then fixing the four limbs and the head of a mouse on an operation plate, exposing a median incision of the neck, dissociating the external jugular vein by 0.8-1cm, ligating an upper end operation line, threading a lower end for standby, connecting a PE catheter with a Powerlab physiological signal recording system through a pressure sensor, wetting the catheter with heparin, inserting the right external jugular vein into the right ventricle to monitor the waveform and pressure of the right ventricle, recording and evaluating the systolic pressure of the right ventricle. The same procedure was used to insert the left carotid artery, and the waveform and pressure of the carotid artery was monitored and recorded to assess the stability of the systemic circulation in mice.

Evaluation of right ventricular hypertrophy

After invasive hemodynamic monitoring was complete, the chest of the mouse was opened and the heart and lungs were removed. After the heart was washed in PBS, the atria were dissected and removed, the Right Ventricle (RV) and left ventricle + ventricular septum (LV + S) were separated along the ventricular septum border, weighed and recorded, and RV/(LV + S) calculated.

Hematoxylin-eosin staining (HE staining) of mouse lung tissue

(1) Left lung distal fixation and embedding: after the mice were monitored by invasive hemodynamic monitoring, the chest was opened quickly. After the lungs were irrigated with PBS along the pulmonary artery, the distal portion of the left lung was fixed in 4% paraformaldehyde for 48 hours. After fixation was completed, the tissue was properly trimmed and placed in an embedding cassette, and the tissue was rinsed with running water for 30 minutes. Gradient alcohol dehydration and xylene clarification, then paraffin was poured into the embedding box. Sections (5 μm) were taken with a paraffin microtome while waiting for the paraffin to solidify.

(2) Dewaxing: the lung tissue sections were placed in an oven at 65 ℃ for 2 hours, then the sections were placed in xylene for 20 minutes and replaced with xylene for 10 minutes. Visually observing whether the wax is completely dehydrated or not

(3) Hydration: the slices were sequentially placed in 100%, 95%, 85%, 75% ethanol for 5 minutes each, according to concentration.

(4) Dyeing: after hydration of the sections, the sections were rinsed by immersion in PBS for 5 minutes each time for a total of 3 times. Hematoxylin staining (20ul,10 min) was added dropwise. After dyeing is completed, excess dyeing liquid is washed away by using distilled water. Adding into 1% hydrochloric acid ethanol for 3-5s for differentiation, immediately adding the slices into distilled water tank, adding into beaker containing tap water, and slowly rinsing with flowing water for 20 min.

(5) Eosin staining and dehydration of the sections: and adding eosin dye solution into the slices, reacting for 5 minutes, and then sequentially placing the slices into 75%, 85%, 95% and 100% alcohol tanks for 2 minutes respectively.

(6) Sealing and observing: and (3) putting the dehydrated lung tissue slices into dimethylbenzene for transparency, then dripping neutral gum on the sample, and covering a cover glass for mounting. Finally, the film is photographed under a microscope.

Immunohistochemistry

Materials: xylene, gradient alcohol, distilled water, sodium citrate, H2O2, phosphate buffer solution, donkey serum, DAB solution, hematoxylin, hydrochloric acid ethanol, neutral resin, a beaker, a glass jar and a cover glass.

The method comprises the following steps:

(1) dewaxing: paraffin sections of mouse lung tissue were placed in an oven at 65 ℃ for 2 hours, and then sections were placed in xylene for 20 minutes and replaced with xylene for 10 minutes. Visually observing whether the wax is completely removed.

(2) Hydration: the slices are sequentially put into 100 percent, 95 percent, 85 percent and 75 percent of ethanol according to the concentration for 5 minutes respectively, soaked in distilled water for 5 minutes, and the excessive liquid is sucked by filter paper.

(3) Removal of endogenous peroxidase: 3% H2O2 was added dropwise at 20ul and incubated for 5-10 min to remove peroxidase (endogenous) and washed 3 times with PBS.

(4) Antigen retrieval: putting the slices into a slice rack, putting the slice rack into a 1000ml beaker, pouring 400ml of citrate buffer solution, heating in a microwave oven until small bubbles are generated, closing the microwave oven at the moment, and cooling for 5 minutes. After 5 minutes, the heating in the microwave oven was continued, and when the small bubbles fused into large bubbles, the microwave oven was turned off, followed by cooling for 40 minutes.

(5) And (3) sealing: PBS was washed 3 times for 5 minutes each and excess liquid was blotted off on filter paper. 10% donkey serum was added dropwise, 20ul each, and blocked at room temperature for 1 hour.

(6) Incubating the primary antibody: after removing the blocking solution by aspiration, the α -SMA antibody or CD31 antibody was added dropwise, 20ul each, at a concentration of 1: incubation was carried out overnight at 100, 4 ℃.

(7) Dressing and breeding a secondary antibody: the following day, primary antibody was recovered and washed 3 times with PBS in EP tubes for 5 minutes each. Then, a secondary antibody (goat-rabbit resistant or goat-mouse resistant) was added dropwise at a concentration of 1:100, incubation at room temperature for 1 hour.

(8) The secondary antibody was aspirated and washed 3 times with PBS for 5 minutes each. And then dropwise adding DAB liquid, reacting for 5-10 minutes until the tissue turns yellow.

(9) Nuclear counterstaining: hematoxylin 20-30ul each, 10 minutes of reaction. Then putting into hydrochloric acid ethanol for 3-5s for differentiation.

(10) And (3) dehydrating: gradient of 75%, 85%, 95%, 100% alcohol for 5 min each.

(11) Sealing and observing: transparent in xylene, sealing with neutral resin, and taking a picture under an optical microscope.

Tissue immunofluorescence

Tissue immunofluorescence allows visualization of the localization of proteins in tissues. When immunofluorescence is performed on a paraffin section of lung tissue, the tissue needs to be dewaxed and antigen repaired first, and then immunofluorescence of the tissue needs to be performed.

Materials: xylene, gradient alcohol, distilled water, sodium citrate, H2O2, phosphate buffer solution, donkey serum, DAB solution, hematoxylin, hydrochloric acid ethanol, neutral resin, a beaker, a glass jar and a cover glass.

The method comprises the following steps:

(1) dewaxing: the paraffin section of the mouse lung tissue is placed in an oven at 65 ℃ for 2 hours, xylene is used for 20 minutes, and the xylene is replaced for 10 minutes. Visually observing whether the wax is completely removed.

(2) Hydration: the slices are sequentially put into 100 percent, 95 percent, 85 percent and 75 percent of ethanol according to the concentration for 5 minutes respectively, soaked in distilled water for 5 minutes, and wiped by filter paper.

(3) 0.5% Triton-100 was added and allowed to permeate for 10 minutes at room temperature and washed 3 times with PBS.

(4) Antigen retrieval: the slices were placed in a slicing rack, the slicing rack was placed in a 1000ml beaker, poured into 400ml of citrate buffer, heated in a microwave oven until small bubbles were generated, and then cooled for 40 minutes.

(5) And (3) sealing: PBS was washed 3 times for 5 minutes each and excess liquid was blotted off on filter paper. 10% donkey serum was added dropwise, 20ul each, and blocked at room temperature for 1 hour.

(6) Incubating the primary antibody: the blocking solution was aspirated off, a sufficient amount of primary antibody (1% donkey serum diluted primary antibody) was added dropwise to each sample, and incubated overnight in a wet box at 4 ℃. Primary antibodies used for tissue immunofluorescence were as follows: α -SMA, vWF (1: 100).

(7) Incubation of secondary antibody: the following day, primary antibody was recovered and washed 3 times with PBS in EP tubes for 5 minutes each. A secondary antibody (1: 200) was added dropwise at a sufficient concentration and incubated at room temperature for 1 hour in the dark.

(8) Nuclear counterstaining: the secondary antibody was aspirated and washed 3 times with PBS for 5 minutes each. DAPI was added dropwise to the glass slides to counterstain nuclei, and incubated for 3-5 minutes in the dark.

(9) Sealing and observing: PBS was rinsed 3 times for 5 minutes each. And sealing the chip by using a sealing liquid containing an anti-fluorescence quencher, and observing and acquiring images under a fluorescence microscope or a confocal microscope.

Tunel (TdT-meditedUTPnickeldlabelling) apoptosis assay

The Tunel cell apoptosis detection kit is used for detecting the breakage condition of the nuclear DNA of tissue cells in the early apoptosis process. Since normal or proliferating cells have little DNA fragmentation and thus no 3-OH formation, they are rarely stained. The method specifically and accurately locates the cells in apoptosis.

The method comprises the following steps:

(1) dewaxing: the paraffin section of the mouse lung tissue is placed in an oven at 65 ℃ for 2 hours, xylene is used for 20 minutes, and the xylene is replaced for 10 minutes. Visually observing whether the wax is completely removed.

(2) Hydration: the slices are sequentially put into 100 percent, 95 percent, 85 percent and 75 percent of ethanol according to the concentration for 5 minutes respectively, soaked in distilled water for 5 minutes, and wiped by filter paper.

(3) 0.5% Triton-100 was added and allowed to permeate for 10 minutes at room temperature and washed 3 times with PBS.

(4) A tunel reaction mixture was prepared.

(5) 50ul of the tube reaction mixture was dropped onto the specimen and reacted for 1 hour at 37 ℃ in a dark and wet box. (6) The mixture was aspirated off and washed 3 times with PBS for 5 minutes each.

(7) DAPI was added dropwise to the glass slides to counterstain nuclei, and incubated for 3-5 minutes in the dark.

(8) PBS was rinsed 3 times for 5 minutes each.

(9) And sealing the chip by using a sealing liquid containing an anti-fluorescence quencher, and observing and acquiring images under a fluorescence microscope or a confocal microscope.

Masson staining

Masson staining was used to observe the pathological collagen deposition and degree of pre-fibrosis. The principle is that Masson staining gives collagen fibers a blue color (stained with aniline blue) and muscle fibers a red color (stained with acid fuchsin), which is related to the size and tissue permeability of the anionic dye molecules.

The method comprises the following steps:

(1) dewaxing: the mouse lung tissue paraffin section is placed in a 65 ℃ oven for 2 hours, xylene is carried out for 20 minutes, and the section is replaced

Xylene for 10 minutes. Visually observing whether the wax is completely removed.

(2) Hydration: placing the slices into 100%, 95%, 85%, and 75% ethanol in sequence according to concentration for 5 min, and steaming

Soaking in distilled water for 5 min, and wiping with filter paper.

(3) Adding hematoxylin staining solution dropwise for reaction for 5-10 min, washing with slow flowing water, and differentiating with 1% hydrochloric acid. (4) Washing with running water for several minutes, and compounding staining solution with massson for 5 minutes.

(5) Slightly washed by distilled water, and treated with 1% phosphotungstic acid solution for about 5 minutes.

(6) And re-dyeing with aniline blue solution for 5 minutes.

(7) 1% glacial acetic acid water for 1 min.

(8) And (3) dehydrating: gradient of 75%, 85%, 95%, 100% alcohol for 5 min each.

(9) Sealing and observing: transparent in xylene, sealing with neutral resin, and taking a picture under an optical microscope.

Western blotting detection

(1) Preparing lung tissue protein homogenate: after lung tissue was rinsed with PBS and dried, it was cut into pieces with scissors and placed in lysis solution. The lysis solution comprises: PMSF and RIPA lysates, phosphatase inhibitors. The lung tissue sample is prepared into protein homogenate by a tissue grinding instrument, the sample is centrifuged for 20min at 12000Xg and 4 ℃ after being treated by ultrasonic, and supernatant fluid is taken.

(2) Measurement of protein concentration: protein concentrations were determined using the BCA protein assay kit and subsequently the protein concentrations were configured consistently for each sample.

(3) Electrophoresis: after the protein is denatured, the protein is added into 10% SDS-PAGE electrophoresis gel, and the pressure is adjusted according to different target proteins.

(4) Film transfer: transferring the protein on the electrophoresis gel to a PVDF membrane, and adjusting the membrane transferring time according to the molecular weight of the protein.

(5) And (3) sealing: PVDF membrane was blocked with 10% skim milk at room temperature for 2 h.

(6) Antibody incubation: PVDF membrane was incubated overnight at 4 ℃ in primary antibody solution. The primary antibody concentration is 1: 1000-1: 2000. the following day the PVDF membrane was rinsed 3 times with TBST for 10 minutes each, and placed in secondary antibody solution (goat anti-rabbit or goat anti-mouse) to incubate for 2 hours at room temperature at a concentration of 1: 3000.

(7) exposure: after the PVDF membrane is rinsed, the exposure liquid is dripped, and the membrane is exposed by using Quant LAS 4000 mini. The exposure pictures were analyzed using alphaease fc software.

The above test procedure also leads to the following conclusions:

1. maresin1 concentration reduction in plasma in two pulmonary hypertension mouse models

Many studies have shown that chronic hypoxia or sugen5416 in combination with chronic hypoxia successfully induced pulmonary hypertension in mice. Previous studies also determined that mice exposed to hypoxic conditions for 3 weeks induced pulmonary hypertension and distal pulmonary arteriolar remodeling. In the present experiment, two high pressure models of PAH were used: chronic hypoxia or sugen5416 (subcutaneous injection on days 0, 7, 14, 2.5mg/kg) was combined with chronic hypoxia-induced pulmonary hypertension in mice for 21 days of molding. The RVSP and FultonIndex verified whether the models were successfully built or not was monitored using invasive hemodynamics. Further, arteriolar ultrasound results found that the right heart function of both PAH high-pressure mouse models was significantly impaired, manifested by shortened tase and PAAT/PET. Finally, the high performance liquid chromatography mass spectrometry detects the concentration of Maresin1 in the plasma, and the result shows that the concentration of Maresin1 in the plasma of the pulmonary hypertension mouse is obviously reduced compared with that of a control group. The above experimental data show that: in the pulmonary hypertension model group of mice, there was a significant increase in RVSP and right heart hypertrophy, right heart dysfunction, accompanied by a significant decrease in Maresin1 concentration in plasma (figure 1). These results indicate that the model was successfully established for 21 days, and that Maresin1 concentration in plasma was reduced in both pulmonary hypertension models.

Maresin1 improves pulmonary artery pressure and right heart dysfunction in mice

To determine the effect of Maresin1 on pulmonary hypertension, mice were exposed to chronic hypoxic conditions for 3 weeks with sugen5416, i.e., after the model of pulmonary hypertension was successfully established, and were then administered therapeutically to Maresin1 for 3 weeks, with hypoxia continuing for the duration of treatment for a total molding time of 6 weeks (fig. 2A). After 6 weeks, RVSP was monitored to assess the occurrence and progression of PAH. Experimental data showed that the pulmonary hypertension group RVSP was significantly elevated, and Maresin1 treatment was able to significantly reduce CH and SuHx, inducing an increase in pulmonary artery pressure (fig. 2B). Further the FultonIndex results showed that Maresin1 decreased RV/(LV + S), improving right heart hypertrophy (fig. 2C). Finally, the influence of Maresin1 on the right heart dysfunction induced by PAH is further identified, the small animal ultrasound apparatus evaluates the right heart function related indexes of the pulmonary hypertension mice, and the Maresin1 is found to increase TAPSE and PAAT/PET (figures 2D-E) and improve the right heart dysfunction. The above experimental data show that: in the pulmonary hypertension of mice, the right ventricular systolic pressure is obviously increased, the right heart is hypertrophied, and the right heart dysfunction is improved, and Maresin1 can obviously reduce the right ventricular systolic pressure and the right heart hypertrophy and can improve the right heart dysfunction at the same time. These results confirm the protective effect of Maresin1 on pulmonary hypertension.

Maresin1 improves aberrant pulmonary vascular remodeling

Maresin1 was found to improve pulmonary artery pressure and right heart dysfunction in previous experiments. To further determine the protective effect of Maresin1 on pulmonary hypertension, it was hypothesized that Maresin1 protected pulmonary hypertension by ameliorating abnormal pulmonary vascular remodeling. Histological staining was used to assess relevant indicators of pulmonary vascular remodeling. And the HE staining result shows that the pulmonary artery hypertension group mice have obviously thickened pulmonary distal arteriole walls and narrow lumens. After treatment with Maresin1, the vessel wall was thinned and the lumen morphology was similar to the control group (fig. 3A-C). Masson staining showed increased and accumulated perivascular collagen production in CH and SuHx mice, whereas collagen accumulation was decreased in Maresin1 treated group (fig. 3D). The pulmonary artery is mainly composed of pulmonary artery endothelial cells and pulmonary artery smooth muscle cells, and the abnormal proliferation of the pulmonary artery endothelial cells and the pulmonary artery smooth muscle cells is the main pathological feature of the pulmonary vascular remodeling of the PAH. To determine the effect of Maresin1 on the proliferation of PAH endothelial cells and smooth muscle cell abnormalities, two-layer vascular localization analysis using immunohistochemistry revealed that both α -SMA and CD31 expression increased in pulmonary vessels of mice in both CH and SuHx groups, indicating abnormal proliferation of smooth muscle cells and endothelial cells in pulmonary hypertension group, and decreased α -SMA and CD31 expression in pulmonary arterioles following Maresin1 intervention, which inhibited smooth muscle cell and endothelial cell proliferation and luminal thinning (fig. 3E-G). The above experimental results show that: maresin1 improved abnormal pulmonary small vessel remodeling in both mouse pulmonary hypertension models.

Maresin1 inhibits endothelial to mesenchymal transition

The main pathological feature of pulmonary hypertension is pulmonary vascular remodeling, and in the process of pulmonary vascular remodeling, transformation of endothelial cells into mesenchymal cells (EndMT) is one of the main pathological features, wherein the transformation of endothelial cells into mesenchymal cells leads to thickening of pulmonary vascular walls and narrowing of lumens, and severe lumen occlusion can be caused. Vascular endothelial cells were evaluated for expression in chronic hypoxia and SuHx-induced pulmonary hypertension models and whether edmt was experienced, and the effect of Maresin1 on this process was observed. Co-expression of the endothelial cell marker vWF and the smooth muscle cell marker alpha-SMA in the pulmonary arterioles was examined using confocal laser. In the pulmonary arteries of control mice, vWF expression was associated with the inner monolayer and not co-localized with α -SMA, which was expressed at the outer edge of the arterioles. In pulmonary arteries of hypoxic and SuHx group mice, co-expression of vWF and α -SMA was observed with a significant increase in expression. After Maresin1 treatment, the co-expression of vWF- α -SMA was significantly reduced (fig. 4 AB). The above results show that: in a mouse pulmonary hypertension model, therapeutic administration of Maresin1 can inhibit the transformation of endothelial cells to interstitial cells, inhibit the proliferation of endothelium and smooth muscle, thin the wall of the pulmonary blood vessel, and obviously improve the pulmonary vascular remodeling.

Maresin1 promoting pulmonary arterial alpha-SMA positive cell apoptosis of pulmonary arterial hypertension mice

Apoptosis-tolerant α -SMA-positive cells over-proliferate, leading to thickening of the distal pulmonary artery vessel wall and narrowing of the lumen, which is a significant cause of pulmonary arterial hypertension pulmonary artery occlusion. To investigate whether Maresin1 improved vascular remodeling by promoting apoptosis in apoptosis-resistant α -SMA positive cells, the distribution of apoptotic cells within the α -SMA localized arterioles was observed. Consistent with the above results, cells showing simultaneous positive Tunel staining for the smooth muscle marker α -SMA were barely expressed in both the chronic hypoxia and sugen5416 combined hypoxia-induced pulmonary hypertension models in mice. A significant increase was achieved following Maresin1 treatment, which restored normal levels of α -SMA positive cells and reduced total α -SMA expression (fig. 5 AB). At the same time, the expression of apoptosis-related proteins in mouse lung tissue was further examined. The result of protein immunoblotting shows that in the lung tissue of the pulmonary hypertension mouse, the expression of the anti-apoptotic protein Bcl-2 is obviously increased, and the expression level of the pro-apoptotic protein Bax is not greatly changed, so that the Bax/Bcl-2 ratio is reduced (FIG. 6 AB). Caspase-3 expression downstream of the Bax/Bcl-2 signaling pathway is also reduced. After Maresin1 treatment, the expression of the anti-apoptotic protein Bcl-2 was down-regulated, the Bax/Bcl-2 ratio was up-regulated, and the expression of the pro-apoptotic protein cleaned-caspase 3 was up-regulated (FIG. 6 CD). The results show that in two mouse pulmonary artery hypertension models, Maresin1 can reduce the expression of mouse pulmonary artery alpha-SMA and promote the apoptosis of alpha-SMA positive cells, thereby inhibiting the excessive proliferation of pulmonary artery smooth muscle and improving the pulmonary vascular remodeling.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

Claims (5)

1. The application of macrophagin 1 in preparing a medicament for treating pulmonary hypertension.

2. The use of macrophagin 1 in the preparation of a medicament for treating pulmonary hypertension according to claim 1, wherein macrophagin 1 inhibits endothelial cell to mesenchymal cell transformation and decreases the source of pulmonary vascular smooth muscle cells.

3. The use of macrophagin 1 according to claim 1 or 2 for the preparation of a medicament for treating pulmonary hypertension, wherein macrophagin 1 promotes apoptosis of pulmonary vascular smooth muscle cells, inhibits proliferation thereof, and improves pulmonary vascular remodeling.

4. The medicine for treating pulmonary hypertension is characterized by comprising an effective dose of macrophagin 1 and a carrier for carrying the macrophagin 1.

5. The medicament of claim 4, wherein the medicament is administered by intraperitoneal/intravenous injection.

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