CN115671124A

CN115671124A - Application of aucubin in preparation of hypoxic pulmonary hypertension vascular remodeling drug

Info

Publication number: CN115671124A
Application number: CN202211029755.5A
Authority: CN
Inventors: 邓堂; 金桂云; 吕鹏飞; 钟士杰; 魏胜超; 廖勇; 刘华江; 张名林; 彭锦; 王迅开
Original assignee: First Affiliated Hospital Of Hainan Medical University
Current assignee: First Affiliated Hospital Of Hainan Medical University
Priority date: 2022-08-25
Filing date: 2022-08-25
Publication date: 2023-02-03
Anticipated expiration: 2042-08-25
Also published as: CN115671124B

Abstract

The invention discloses an application of aucubin in preparation of a hypoxic pulmonary artery high-pressure blood vessel remodeling medicine, and relates to the field of new application of medicines. The in vitro cell experiment result shows that aucubin can effectively inhibit the proliferation of the PASMCs, weaken the migration capacity of cells and promote the increase of apoptosis; in vivo experiments show that aucubin can significantly reduce pulmonary artery pressure and right ventricular systolic pressure in a rat hypoxic pulmonary hypertension model induced by chronic hypoxia, reduce right ventricular hypertrophy index, and improve right ventricular hypertrophy condition, and in addition, the pulmonary artery pathological tissue results also prove that aucubin can effectively inhibit pathological changes such as pulmonary artery blood vessel wall thickening, narrow and small vessel lumen after intervening in an HPH rat model, promote the vessel wall to be obviously thinned, enlarge the vessel lumen, and show that the aucubin can significantly improve the vascular remodeling of the HPH rat model.

Description

Application of aucubin in preparation of hypoxic pulmonary hypertension vascular remodeling drug

Technical Field

The invention relates to the field of new application of medicines, in particular to application of aucubin in preparation of a hypoxic pulmonary hypertension vascular remodeling medicine.

Background

Pulmonary Arterial Hypertension (PAH) refers to the increase in pulmonary arterial pressure (the mean pulmonary arterial pressure measured by the right heart catheter is not less than 25mmHg in the resting state at sea level) caused by various reasons, and is a chronic cardiovascular and pulmonary vascular disease which is difficult to cure, and has a complex pathogenesis and poor prognosis. Among them, hypoxic Pulmonary Hypertension (HPH) caused by hypoxia belongs to the third category of PAH classification, which is a clinical syndrome mainly manifested by dyspnea and hypodynamia after activity, and its clinical features are consistent with those of PAH. The disease is mainly characterized by long-term hypoxia leading to pulmonary vascular remodeling, progressive increase in cardiopulmonary circulation resistance, and ultimately right heart failure. Epidemiological investigations on HPH are rare on a global basis, but clinically common Chronic Obstructive Pulmonary Disease (COPD), restrictive pulmonary disease, and high altitude heart disease of the disease all cause HPH, wherein COPD is the most common cause of HPH, and the prevalence rate of concomitant HPH is about 20% -91%. Under long-term hypoxia stimulation, the balance of oxidation/antioxidation in vivo is broken, oxidative stress is enhanced, the smooth muscle layer of the pulmonary artery is thickened, and the pulmonary artery stenosis and remodeling are the most important pathophysiological changes of HPH, however, the key mechanism for causing the pulmonary artery remodeling is not clear, and no drug treatment method is approved by FDA (American food and drug administration) aiming at the disease. Long-term oxygen therapy is the first recommended scheme for HPH in chinese guidelines for pulmonary artery hypertension diagnosis and treatment (2021 edition), but this method is time-consuming and difficult to tolerate in many patients, and oxygen therapy can only improve partial alveolar hypoxia, and has a limited effect on pulmonary artery pressure reduction. Therefore, how to develop a hypoxic pulmonary hypertension vascular remodeling drug is still a problem to be solved urgently.

The Aucubin (Aucubin) is a chemical name of beta-D-glucopyranoside, belongs to iridoid glycoside compounds, and is white powder or crystal, is easily soluble in water and methanol, soluble in ethanol, and insoluble in diethyl ether, chloroform, benzene and petroleum ether. The aucubin exists in traditional Chinese medicines such as eucommia ulmoides, plantain herb, rehmannia glutinosa and the like, can be extracted from natural plants, has wide sources, is cheap and easy to obtain, is convenient for industrial extraction, and has almost no toxic or side effect on cells and animals in a therapeutic dose. At present, documents (CN 102068450B-aucubin in the preparation of antithrombotic drugs and CN 104116753B-aucubin in the preparation of idiopathic pulmonary fibrosis drugs) report the application of aucubin in the preparation of antithrombotic drugs or idiopathic pulmonary fibrosis drugs, so that the clinical research of aucubin and the development of new drugs by using aucubin become possible, but related application research of aucubin in the preparation of hypoxic pulmonary hypertension vascular remodeling drugs is not found.

Disclosure of Invention

In view of the above, the invention provides an application of aucubin in preparing a drug for remodeling hypoxic pulmonary hypertension vessels.

Preferably, the aucubin is added with pharmaceutical auxiliary materials to prepare the injection.

Preferably, the concentration of aucubin in the injection is 1mg/mL.

By adopting the application of aucubin in preparing the hypoxic pulmonary hypertension vascular remodeling medicine, on one hand, the in vitro cell experiment result proves that the aucubin effectively inhibits the proliferation of the PASMCs, weakens the migration capacity of cells and promotes the obvious increase of the apoptosis; on the other hand, in vivo experiments show that aucubin can significantly reduce pulmonary artery pressure and right ventricular systolic pressure in a rat pulmonary artery hypertension model induced by chronic hypoxia, reduce right ventricular hypertrophy index and improve right ventricular hypertrophy conditions. In addition, pathological tissue results also prove that the aucubin can effectively inhibit the thickening of the pulmonary artery blood vessel wall, the narrow and small lumen and other lesions after being intervened in the HPH rat model, promote the obvious thinning of the blood vessel wall and the enlargement of the lumen, and show that the aucubin can obviously improve the pulmonary vascular remodeling of the HPH rat model.

Drawings

FIG. 1 is a diagram showing pathological conditions of pulmonary arterioles of rats observed under a microscope after HE staining;

FIG. 2 is a graph showing the results of testing the proliferation and migration of aucubin on rat Pulmonary Artery Smooth Muscle Cells (PASMCs) by using a scratch test;

FIG. 3 shows the result of testing the apoptosis of aucubin on rat Pulmonary Artery Smooth Muscle Cells (PASMCs) by flow-type apoptosis;

FIG. 4 shows the results of determining the effect of aucubin on the activity of rat Pulmonary Artery Smooth Muscle Cells (PASMCs) using CCK8 kit;

FIG. 5 shows the results of detecting the antioxidase content and oxidative stress index in alveolar lavage fluid using SOD, CAT, LDH and MDA kits.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The aucubin related to the invention is purchased from Shanghai source leaf biotechnology limited company, and is an injection taking the aucubin as a main active component: dissolving 20mg of aucubin in 20mL of sterile water for injection to prepare a solution with the concentration of 1mg/mL for later use; adult SD male rats were purchased from geneva biotechnology limited.

The first embodiment is as follows: the application of the injection taking aucubin as a main active ingredient in the treatment process and effect verification of pulmonary artery high-pressure vascular remodeling of hypoxic rats comprises the following steps:

s101, placing 18 SD adult male rats in an atmospheric hypoxia chamber for feeding and molding, controlling the oxygen concentration to be 10% +/-0.5%, controlling the temperature in the chamber to be 22-24 ℃, and carrying out hypoxia for 12 h/day for continuous hypoxia feeding for 4 weeks.

S102, after 4 weeks, detecting pulmonary artery pressure (mPAP) and Right Ventricular Systolic Pressure (RVSP) by using a Medlab-U biological signal acquisition and processing system through a right jugular vein catheter; the pulmonary function indexes are measured by using a small animal pulmonary function detection system, index data of the hypoxic pulmonary hypertension rat model are obtained to evaluate whether the hypoxic pulmonary hypertension rat model reaches the standard or not, and if mPAP of the rat is more than or equal to 25mmHg, RVSP of the rat is more than or equal to 28mmHg, and the pulmonary function indexes are obviously reduced, the hypoxic pulmonary hypertension rat model can be met.

S103, dividing the HPH rats reaching the standard by 2 groups, wherein each group comprises 6 HPH rats, namely a model group (HPH group) and a treatment group (HPH + AU), and then selecting 6 normal-pressure breeding SD adult male rats as a control group (CONT group); the treatment group was administered by intraperitoneal injection at a dose of 10mg/kg (injection solution concentration of 1 mg/mL) based on the actual weight of the rat, and the model group and the control group were administered twice a day with continuous administration for 2 weeks by injecting only the same amount of physiological saline.

And S104, measuring relevant indexes 2 weeks after administration, and evaluating the treatment effect.

(1) Effect of aucubin on Right Ventricular Hypertrophy Index (RVHI), right Ventricular Systolic Pressure (RVSP) and pulmonary artery pressure (mPAP) in hypoxic pulmonary hypertension rats

Rats in a control group (CONT group), a model group (HPH group) and a treatment group (HPH + AU) are respectively killed, hearts are extracted to determine the right ventricular hypertrophy index [ RV/(LV + S) ], compared with the control group, the walls of the Right Ventricles (RV) of the rats in the model group are obviously thickened, the quality of the Left Ventricles (LV) and the quality of the ventricular septum (S) are not obviously changed, and the right ventricular hypertrophy index is obviously increased, so that the pathological changes of the obvious hypertrophy of the right ventricles and ventricular remodeling of the hearts of the rats can be caused by feeding the rats under the normal pressure and low oxygen conditions. Compared with a model group, the administration treatment of aucubin has the advantages that the RVHI is obviously reduced, the RVSP and the mPAP are obviously reduced, and the aucubin has good treatment effect on hypoxic pulmonary hypertension rats.

TABLE 1 Effect of aucubin on Right ventricular hypertrophy index, right ventricular systolic pressure and pulmonary artery pressure in hypoxic pulmonary hypertension rats before and after administration

Group of	RVHI(％)	RVSP(mmHg)	mPAP(mmHg)
				Control group	22	19	16
Model set	31	35	27
				Treatment group	24	24	20

(2) Effect of aucubin on pulmonary artery histopathology in pulmonary hypertension rats

Respectively picking the lung tissues of rats of the control group, the model group and the treatment group, staining the lung tissues by hematoxylin-eosin (HE), and observing the histopathological changes of the pulmonary artery so as to judge the pulmonary artery remodeling condition and measure the pulmonary artery remodeling related indexes: the ratio (WT%) of the pulmonary artery tube wall thickness to the pulmonary artery outer diameter, and the ratio (WA%) of the pulmonary artery tube wall area to the pulmonary artery cross-sectional area, as shown in fig. 1 and the results of table 2, the pulmonary artery vessel wall of the rat in the model group WAs significantly thickened, which indicates muscular hypertrophy and luminal stenosis in the pulmonary artery, accompanied by fibrous thickening and abnormal cell proliferation of the pulmonary artery intima, and proved that the hypoxic pulmonary hypertension rat model WAs successfully constructed. The aucubin administration can obviously reduce the thickness of the pulmonary artery blood vessel wall, relieve the pulmonary artery myomorphism and enlarge the pulmonary artery lumen, which shows that the aucubin has obvious treatment effect on the pulmonary artery reconstruction of the pulmonary hypertension rat.

TABLE 2 Effect of aucubin on pulmonary hypertension in rats pulmonary artery histopathology results

Example two: proliferation, migration and apoptosis of rat Pulmonary Artery Smooth Muscle Cells (PASMCs) by aucubin

Culturing the PASMCs: inoculating cells into the cell suspension according to experimental requirements, dripping into the center of a glass sheet in a cell culture dish, standing for 30min, adding 2mL of F12 cell culture solution, and adding CO ₂ Culturing CONT cells (control group) in incubator at 37 deg.C for 48h, or 3% concentration of O ₂ HPH cells (model group) were cultured in a three-air incubator at 37 ℃ for 48 hours. Can be inoculated in a culture bottle for subculture and used for subsequent experiments such as cell proliferation, migration, apoptosis and the like.

(1) Test of proliferation and migration effects of aucubin on rat Pulmonary Artery Smooth Muscle Cells (PASMCs) by using scratch test

Scratch test:

(1) Uniformly marking lines which transversely penetrate through each hole by using a marker pen at the back of the 6-hole plate, wherein the number of the lines is at least 3;

(2) About 5X 10 seeds were seeded into each well, respectively ⁵ A test cell;

(3) After the cells are full, marking a trace in the hole by using a 200 mu L gun head which is vertical to a transverse line at the back;

(4) Washing the cells with PBS for 3 times, removing the scratched cells, and culturing for 48h;

(5) The scratch marks at 0 and 48 (same mark position) were taken;

(6) Calculating the area covered before and after the migration of the scratched cells, i.e.

Scratch migration area = initial scratch area-healed scratch area

The in vitro cell experiment was divided into a control group (CONT group), a model group (HPH group), and a treatment group (HPH + AU group), and aucubin was added to the HPH cell model to be evaluated to the standard at a concentration of 250. Mu.g/mL in step (2). As shown by the results of fig. 2 and table 3: compared with the model group, the migration healing area of the normal group is normalized to 1, and the healing area of the treatment group is about 0.8 times of that of the normal group, which indicates that the treatment group remarkably promotes the proliferation of cells and plays a good healing role.

(2) Method for testing apoptosis effect of aucubin on rat Pulmonary Artery Smooth Muscle Cells (PASMCs) by using flow apoptosis

In the experiment, the cell nucleus is stained red by the Annexin V which penetrates through cells at middle and late apoptosis stages and dead cells, so that the Annexin V is matched with PI for use, and the cells at different apoptosis stages can be distinguished by a flow cytometer. Collecting PASMCs in logarithmic growth phase, digesting with 0.25% pancreatin, counting, and collecting 10 × 10 ⁴ The cells were centrifuged at 1000 rpm. Times.5 min, and the supernatant was discarded. The pre-cooled PBS was washed 2 times and centrifuged at 1000 rpm. Times.5 min. Using 100. Mu.L of 1 XBinding Buffer (stock solution 10 XBinding Buffer, with ddH) ₂ O diluted to 1 × BindingBuffer) to resuspend the cells. Adding 5. Mu.L of Annexin V-FITC, mixing, adding 5. Mu.L of Propidium lodide, mixing, and reacting at room temperature for 15min in dark place. Before detection, 400. Mu.L of 1 XBinding Buffer was added, and apoptosis was detected by flow cytometry (Ex = 488nm.

As the results in fig. 3 and table 3 show: compared with a model group, the aucubin can obviously inhibit the proliferation and migration of the PASMCs cells, the apoptosis is obviously increased, the wall of the pulmonary artery blood vessel is thinned, the lumen is enlarged, and the pulmonary vessel remodeling is improved.

TABLE 3 testing of Effect of aucubin on proliferation and apoptosis rates of rat Pulmonary Artery Smooth Muscle Cells (PASMCs) by flow apoptosis

Group of	Proliferation Rate (%)	Apoptosis Rate (%)
			Control group	62	8.44
Model set	91	4.64
			Treatment group	37	28.70

(3) Determination of Effect of aucubin on cell activities of rat Pulmonary Artery Smooth Muscle Cells (PASMCs) by CCK8 kit

CCK8 was used to detect STS cytotoxicity on PASMCs: the 3 rd generation PASMCs were trypsinized and centrifuged, and then 4mL of DMEM/F2 medium suspension containing 1% double antibody and 10% fetal bovine serum was added to the solution and mixed at 6X 10 ³ When the PASMCs are fused to 60%, DMEM/F2 culture medium containing 1% double antibody and 10% fetal calf serum is replaced by DMEM/F2 culture medium containing 1% double antibody and no fetal calf serum to synchronize the PASMCs for 12 hours, and DMEM/F2 culture medium containing 1% double antibody and 1% fetal calf serum containing 0 mug/mL, 5 mug/mL, 10 mug/mL, 20 mug/mL, 40 mug/mL, 60 mug/mL, 80 mug/mL aucubin and 1% fetal calf serum are respectively added. After the administration treatment, the cells were rinsed 2 times with PBS, and incubated at 37 ℃ for 30 minutes in 100. Mu.l of DMEM/F2 medium containing 10% of CCK8, and then the OD was measured by setting the absorbance at 450nm with a microplate reader. The results in FIG. 4 show that the cell viability of the aucubin dose group at 80. Mu.g/mL was maintained at 50% after 24h of drug intervention, indicating that aucubin has minimal toxicity to the PASMCs.

(4) Method for detecting antioxidant enzyme content and oxidative stress index in alveolar lavage fluid by using SOD, CAT, LDH and MDA kit

Using 3mL sterile physiological saline to repeatedly lavage the main bronchus of rats in a control group (CONT group), a model group (HPH group) and a treatment group (HPH + AU) for 3 times, collecting cleaning solution in the sterile tube, centrifuging and collecting supernatant, measuring the OD value of each sample hole according to the operation of the instructions of a Catalase (CAT), superoxide dismutase (SOD), lactate Dehydrogenase (LDH) and Malonaldehyde (MDA) detection kit, and counting the actual content of CAT, SOD, MDA and LDH in the lavage fluid of the control group and the alveolus according to the absorbance value.

As shown in table 4 and the results in fig. 5, aucubin increased the contents of antioxidases (SOD, CAT) in alveolar lavage fluid and decreased the oxidative stress indicators (LDH, MDA) as compared to the HPH group. The aucubin plays an anti-oxidation role in an anoxic pulmonary hypertension rat model, the proliferation and migration capacity of the PASMCs is obviously reduced, the apoptosis is obviously increased, and the vascular remodeling is improved, so that the aucubin further plays an important role in oxidative stress reaction in the development of the anoxic pulmonary hypertension.

TABLE 4 detection of index changes of CAT, SOD, MDA and LDH in rat alveolar lavage fluid before and after aucubin treatment by using kit

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. Application of aucubin in preparing medicament for remodeling hypoxic pulmonary hypertension blood vessel is provided.

2. The use of claim 1, wherein the aucubin is added with a pharmaceutically acceptable excipient to prepare an injection.

3. The use of claim 1, wherein the concentration of aucubin in the injection is 1mg/mL.