Application of new parastemonine in the preparation of medicine for treating pulmonary hypertension

Abstract

The present application proposes the use of new stemonaline in the preparation of drugs for the treatment of pulmonary hypertension, and relates to the technical field of medical drug development. The experiment of the present application confirms that the mouse pulmonary hypertension model using new stemonaline can significantly reduce the right ventricular hypertrophy and right ventricular systolic pressure of pulmonary hypertension, relieve the thickening of the middle layer of the pulmonary artery, and reduce the inflammatory response of lung tissue, and the weight of the mice has not changed compared with the control group. While having a good healing effect, no side effects have been found, which reflects that new stemonaline can be used as a safe and effective drug for the targeted treatment of inflammation and related symptoms caused by pulmonary hypertension.

Description

Technical Field

[0001] The present application relates to the technical field of medical drug development, and in particular to the application of new scutellariae in the preparation of drugs for treating pulmonary hypertension. Background Art

[0002] Pulmonary hypertension (PH) is a disease that seriously endangers human health. It is the third most common cardiovascular disease after coronary heart disease and hypertension. Most patients will die of heart failure within 2 to 3 years. Typical symptoms are a chronic progressive disease characterized by progressive increase in pulmonary artery resistance, pulmonary vascular remodeling, increased lung infiltration, decreased vascular cross-sectional area, increased vascular resistance, right ventricular hypertrophy and failure. Cellular manifestations include endothelial function damage, irrational proliferation of smooth muscle cells, increased lung infiltration and imbalance of various vasomotor factors.

[0003] Under normal physiological conditions, the pulmonary vascular wall is composed of endothelial cells, internal elastic membrane, smooth muscle layer and external elastic membrane, and the contraction and relaxation of blood vessels are maintained in a balanced state. When the lung tissue is in a state of hypoxia for a long time, the inflammatory response in the patient's body will be initiated, which is manifested as increased permeability of lung tissue, infiltration of inflammatory cells, and significantly increased expression and secretion of different cytokines, chemokines, and adhesion factors. This leads to damage to endothelial cells in lung tissue, thickening of the vascular endothelium, unreasonable proliferation of smooth muscle cells, increased lung infiltration, and imbalance of various vasomotor factors. Long-term increase in pulmonary circulation resistance leads to continuous increase in right ventricular load and right ventricular hypertrophy, ultimately leading to right heart failure and death.

[0004] Because its pathogenesis is not fully understood and symptoms appear late in the development process, patients are not diagnosed and treated in time when their condition reaches the clinical treatment standard, resulting in no significant improvement in their condition even with treatment in the later stage. Inflammation in the patient's body is also one of the main pathological causes of pulmonary hypertension. Drug research targeting inflammation caused by pulmonary hypertension is still relatively rare. Therefore, the development of a novel, safe and effective drug targeting inflammation caused by pulmonary hypertension will play a significant role in the early treatment of PH. Summary of the invention

[0005] The purpose of the present application is to provide the use of new scutellariae in the preparation of drugs for treating pulmonary hypertension. The new scutellariae can be used as a drug targeting inflammation caused by pulmonary hypertension and is safe and efficient.

[0006] The present application solves the technical problem by adopting the following technical solution.

[0007] The embodiments of the present application provide the use of new stemonae in the preparation of a drug for treating pulmonary hypertension.

[0008] In the study of this application: In a cell experiment, the use of neotuberostemonine (NTS) under hypoxic conditions can reduce the secretion of cytokines in mouse pulmonary vascular endothelial cells and weaken the chemotaxis of macrophages, that is, the aggregation of inflammatory cells to blood vessels is reduced. In the mouse model, the use of NTS can significantly reduce the right ventricular hypertrophy and right ventricular systolic pressure of pulmonary hypertension in the mouse pulmonary hypertension model, relieve the thickening of the middle layer of the pulmonary artery, and reduce the inflammatory response of lung tissue. The weight of the mice did not change compared with the control group. While having a good healing effect, no side effects have been found. These results all reflect that neotuberostemonine can be used as a safe and effective drug for targeted treatment of inflammation and related symptoms caused by pulmonary hypertension.

[0009] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0010] In the prior art, clinical treatments for pulmonary hypertension are relatively simple and still mainly based on vasodilators. Commonly used drugs include prostacyclins (PGI2), which are physiological products of arachidonic acid metabolism and are mainly synthesized by the vascular endothelium. They dilate pulmonary blood vessels; and phosphodiesterase type 5 inhibitors (PDE5) that selectively inhibit PDE5 (expressed in large quantities in the lungs), increase the cGMP concentration in smooth muscle cells, relax vascular smooth muscle, dilate pulmonary arteries, reduce pulmonary vascular resistance, and thus reduce pulmonary artery pressure. There are also approaches that start from the nitric oxide-cyclic GMP pathway and the endothelin pathway mechanism, but the efficacy of drugs developed from these perspectives is limited, resulting in the inability to cure pulmonary hypertension. Inflammation in the patient's body is also one of the main pathological causes of pulmonary hypertension. Drug research targeting inflammation caused by pulmonary hypertension is still relatively rare. The mouse experiment in this application confirmed that the use of new stemona alkaloids has a good healing effect on inflammation caused by pulmonary hypertension, and no side effects have been found. It is also almost non-toxic to normal cells and tissues. This reflects that new stemona alkaloids can be used as a safe and effective drug for the targeted treatment of inflammation and related symptoms caused by pulmonary hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0012] Figure 1 This is a graph showing the results of detecting the expression of cytokines in mouse vascular endothelial cells by ELISA in the present application example;

[0013] Figure 2 This is a result diagram of the Transwell chamber migration assay to detect the chemotaxis function of macrophages in the embodiment of the present application;

[0014] Figure 3 This is a result graph showing that the use of new stemonae in the examples of this application has no effect on the body weight of mice;

[0015] Figure 4 This is a result diagram showing the effect of using new stemonae on the right ventricle of mice in the examples of this application;

[0016] Figure 5 This is a result diagram showing the effect of the new stemonae on the left ventricular systolic pressure of mice after using the present embodiment;

[0017] Figure 6 This is a result diagram showing the effect of the new stemonae on inflammatory factors in the lungs of mice after using the new stemonae in the examples of this application;

[0018] Figure 7 This is a graph showing the effect of the new Stemonine on the morphology of mouse lung tissue after use in the examples of this application.

[0019] Note: The asterisk (*) symbol above the data represents the comparison between the corresponding experimental group and the Nor group; the pound sign (#) symbol represents the comparison between the corresponding experimental group and the Hyp group. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0021] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0022] The embodiments of the present application provide the use of new stemonae in the preparation of a drug for treating pulmonary hypertension.

[0023] In the study of this application: In a cell experiment, the use of neostemonine (NTS) under hypoxic conditions can reduce the secretion of cytokines in mouse pulmonary vascular endothelial cells and weaken the chemotaxis of macrophages, that is, the aggregation of inflammatory cells to blood vessels is reduced. In the mouse model, the use of NTS can significantly reduce the right ventricular hypertrophy and right ventricular systolic pressure of pulmonary hypertension in the mouse pulmonary hypertension model, relieve the thickening of the middle layer of the pulmonary artery, and reduce the inflammatory response of lung tissue. The weight of the mice did not change compared with the control group. While having a good healing effect, no side effects have been found. These results all reflect that neostemonine can be used as a safe and effective drug for the targeted treatment of inflammation and related symptoms caused by pulmonary hypertension.

[0024] In an embodiment of the present application, the use of the above-mentioned new stemonaline in the preparation of a drug for treating pulmonary hypertension is characterized in that the drug contains new stemonaline as an active ingredient.

[0025] The molecular formula of the new stemonaline is C 22 H 33 NO, with a molecular weight of 375.5, was found to have almost no toxicity to normal cells and tissues during the research phase of this application. Direct oral gavage was used to treat a mouse model of pulmonary hypertension caused by hypoxia. The results showed that it could effectively inhibit the occurrence of inflammation and relieve related symptoms. Therefore, using it as an active ingredient in the preparation of drugs for the treatment of pulmonary hypertension can make the drugs have good therapeutic effects without causing harm to the body.

[0026] In the examples of the present application, the new stemonae inhibited the expression of pulmonary inflammatory factors IL-6, TNF-α, and IL-1β.

[0027] In the present study, it was found that after oral gavage treatment with the pulmonary hypertension mouse model group, the expression of inflammatory factors TNFα, IL6, and IL1β in the mouse lung tissue was detected by ELISA, and these inflammatory factors all showed a significant decrease, indicating that new phyllotoxin can inhibit the expression of lung inflammatory factors IL-6, TNF-α, and IL-1β.

[0028] In the embodiments of the present application, the above-mentioned medicine is in the form of tablets, granules, sprays, oral preparations or injections.

[0029] Drugs are prepared in a variety of forms to meet the needs of different patients.

[0030] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0031] Example 1

[0032] This example verifies the effect of new stemonae on mouse pulmonary vascular endothelial cells at the cellular level. (Significance marks: **, P<0.01; ##, P<0.01, the same below.)

[0033] 1. ELISA method to detect cytokine expression in mouse vascular endothelial cells

[0034] Mouse pulmonary artery endothelial cells (PAEC) were divided into normoxic group, hypoxic group, and hypoxic plus drug group. The NTS concentration in the drug-added group was 10 μmol / L. The cells were cultured in a normoxic environment containing 5% CO2 and a hypoxic environment (5% CO2, 5% O2, 90% N2) for 24 hours. The expression of cytokines IL-6, IL-1β, and CXCL-10 in the PAEC supernatant was detected by ELISA.

[0035] 2. Transwell chamber migration assay to detect macrophage chemotaxis

[0036] Macrophages RAW 264.7 and mouse PAEC were divided into normoxic group, hypoxic group, and hypoxic drug group. The cells were placed in a normoxic environment containing 5% CO2 and a hypoxic environment (5% CO2, 5% O2, 90% N2) and cultured for 24 hours. The NTS concentration of the drug-dosed group was 10μmol / L. PAEC supernatant was used as the lower layer of the transwell chamber, and RAW 264.7 cells were used as the upper layer of the transwell chamber. After 24 hours, the transwell chamber was taken out, washed twice with PBS, and fixed with anhydrous ethanol. The fixed cells were stained with toluidine blue, observed under an inverted microscope, and the cells were counted. Five fields of view were taken and imaged under an inverted microscope. The number of RAW 264.7 cells that migrated was counted.

[0037] The final ELISA results were as follows Figure 1 As shown in the figure, compared with the control group, the secretion of IL-6, IL-1β and chemokine 10 (CXCL10) in mouse pulmonary vascular endothelial cells under hypoxia increased, indicating that the secretion of inflammatory cell-related factors increased, which manifested as an increased inflammatory response in vivo; and the use of neostemonine (NTS) under hypoxia can reduce the secretion of the above cytokines in mouse pulmonary vascular endothelial cells. The results of the Transwell chamber migration assay are shown in the figure. Figure 2 As shown, under hypoxic conditions, the chemotaxis of macrophages is enhanced, while after the use of NTS under hypoxic conditions, the chemotaxis of macrophages is weakened, that is, the aggregation of inflammatory cells to blood vessels is reduced, which slows down the occurrence of inflammation.

[0038] Example 2

[0039] In this example, a hypoxic mouse PH model was constructed, and the effect of stemonae on the PH of mice was experimentally analyzed.

[0040] 1. Construction of hypoxic mouse PH model

[0041] Using the chronic hypoxia method, male C57BL / 6 mice (each weighing about 25 g) aged about six weeks were randomly divided into the following three groups:

[0042] Normoxia group (Nor): Randomly selected mice were placed in a clean environment with an ambient temperature of 22-24°C, a relative humidity of 50%, an oxygen partial pressure of 21%, a light-dark cycle of 12 hours, and free access to food and water for 28 days.

[0043] Hypoxia group (Hyp): Randomly selected mice were placed in an anaerobic box with an ambient temperature of 22-24°C, which could automatically and continuously introduce nitrogen to adjust and monitor oxygen concentration, so that the oxygen partial pressure in the anaerobic box was maintained at 9-10%, the light-dark cycle was 12 hours, and the mice were raised in a controlled environment with free access to food and water for 28 days. At the same time, appropriate amounts of anhydrous calcium chloride, calcium oxide and boric acid were placed in the anaerobic box to absorb water vapor, carbon dioxide and ammonia discharged by the mice in the anaerobic box to maintain a stable environment in the anaerobic box. After being raised in a controlled environment with free access to food and water for 3 weeks, the experiment was conducted after continuing to be raised in a normal environment for 2 weeks.

[0044] The third group was the hypoxia + NTS group (Hyp + NTS). Under the same modeling conditions and methods as those of the second group, we administered NTS at a dose of 20 mg / kg·d by gavage from day 1 to day 21. The mice were weighed weekly and their general conditions were recorded.

[0045] 2. Detection and analysis of right ventricular systolic pressure (RVSP) and cardiac function.

[0046] Mice were anesthetized by injection of pentobarbital, and a Millar catheter (SPR-836F) was surgically inserted into the right jugular vein. The Power Lab monitoring device was used to monitor the changes in RVSP. The value was read after the indication stabilized, and then the chest cavity of the mouse was quickly cut open with surgical scissors, the heart and lung tissue were removed, the heart was separated from the lung lobe, and the right ventricle was slowly separated along the ventricular septum with the tip of the surgical scissors. The degree of right heart hypertrophy was assessed by weighing. At the same time, the mouse lung lobe was placed in 1× PBS buffer for washing until no blood flowed out before subsequent treatment.

[0047] 3. Detection of inflammatory factors in lung tissue

[0048] Lung tissue was taken, 9 times the volume of PBS was added according to the amount of tissue, the tissue was broken up by homogenizer, and liquid nitrogen was repeatedly frozen and thawed three times. Centrifuged at 12000rpm for 10min, and the supernatant was the protein sample required for the experiment. The expression of inflammatory factors TNFα, IL-6, and IL-1β in mouse lung tissue was detected by ELISA.

[0049] 4. Pathological observation

[0050] After the whole lung was removed from the mouse under anesthesia, a 5 mm cube of the left upper lobe was cut, fixed with 4% paraformaldehyde, and embedded for lung tissue pathology observation. H&E staining was used to observe the pulmonary vascular remodeling in mice, and photos were taken under an inverted microscope. Five fields of view were randomly photographed for each slide.

[0051] The above test results show that adding NTS under hypoxic conditions does not affect the normal body weight of mice ( Figure 3 Adding NTS under hypoxic conditions can significantly alleviate hypoxia-induced right ventricular hypertrophy in mice ( Figure 4 ), adding NTS under hypoxic conditions can significantly alleviate the increase in right ventricular systolic pressure (RVSP, Figure 5 ). The results of lung tissue inflammatory factor detection showed that the inflammatory factors TNFα, IL6, and IL1β in mouse lung tissue were significantly reduced after adding NTS under hypoxic conditions ( Figure 6 ), the morphological analysis of mouse lung tissue showed that the addition of NTS under hypoxic conditions could significantly alleviate the thickening of the pulmonary artery media ( Figure 7 ).

[0052] Based on the above results, it can be found that adding NTS under hypoxic conditions can significantly reduce pulmonary hypertension, right ventricular hypertrophy and right ventricular systolic pressure, relieve pulmonary artery media thickening, and reduce inflammatory response of lung tissue. At the same time, it will not affect the normal physiological state of mice. It has a good healing effect and no side effects have been found.

[0053] In summary, the embodiments of the present application have verified through in vitro and in vivo experiments that the new scutellariae has an improvement effect on inflammation, macrophage chemotaxis, and cardiopulmonary function changes caused by pulmonary hypertension. Therefore, the new scutellariae can be used as a safe and efficient drug targeting diseases related to pulmonary hypertension, and it has great application prospects and application value.

[0054] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

Claims

1. The use of new stemonae in the preparation of a drug for treating pulmonary hypertension, characterized in that: Neostemonine inhibits the expression of pulmonary inflammatory factors IL-6, TNF-α, and IL-1β.

2. The use of the new scutellarine according to claim 1 in the preparation of a medicament for treating pulmonary hypertension, characterized in that: The medicine is in the form of tablets, granules or sprays.

3. The use of the new scutellarine according to claim 1 in the preparation of a medicament for treating pulmonary hypertension, characterized in that: The medicine is an oral preparation or an injection.

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