Use of sanguisorba officinalis saponin in treating hypoxic pulmonary arterial hypertension

Abstract

The application discloses a use of taibai araloside in treating hypoxic pulmonary arterial hypertension. In the use of hypoxia to induce pulmonary arterial hypertension, it is found that the taibai araloside can reduce right ventricular pressure and right heart hypertrophy index, and inhibit pulmonary arterial remodeling; the taibai araloside reduces the expression and activity of ACE in the lung in a concentration-dependent manner, and corrects the disorder of the RAS system in the lung caused by hypoxic pulmonary arterial hypertension. Moreover, a monomer component of the taibai araloside, anemarrhena asphodeloides glycoside and chizhenshan saponin IVa, has a stable combination with ACE. The results show that the taibai araloside has the potential to be developed into a drug for preventing and treating hypoxic pulmonary arterial hypertension in the clinic, and also provides a prevention and treatment strategy for interstitial lung disease, chronic obstructive pulmonary disease, sleep apnea syndrome, chronic high altitude disease and neonatal respiratory disease.

Description

Technical Field

[0001] This invention belongs to the field of medicine and relates to the application of saponins from Aralia elata in the treatment of hypoxic pulmonary hypertension and related diseases. Background Technology

[0002] Hypoxic pulmonary hypertension (HPH) is a condition caused by respiratory diseases and / or hypoxia, characterized by progressively increased pulmonary artery pressure, increased pulmonary vascular resistance, and leading to right ventricular hypertrophy and right ventricular failure. Clinically, it is commonly seen in interstitial lung disease, chronic obstructive pulmonary disease (COPD), sleep apnea syndrome, chronic mountain sickness, and some neonatal diseases. HPH progresses rapidly, and once it enters the irreversible stage of pulmonary vascular remodeling (PVR), treatment becomes difficult, with an extremely high mortality rate and a very poor prognosis (the 1-year survival rate is 91%, and the 3-year survival rate is less than 80%).

[0003] Currently, conventional clinical treatments for HPH include anticoagulants, oxygen therapy, diuretics, and medications such as digitalis, vasodilators, and calcium channel blockers. In recent years, with in-depth research into the mechanisms of HPH, many novel therapeutic drugs have emerged: phosphodiesterase type 5 inhibitors (such as sildenafil, tadalafil, or vardenafil), prostacyclin derivatives (such as iloprost or treprost), and endothelin receptor antagonists (such as bosentan, amphetamine, or macitan). These new drugs help improve patients' symptoms and quality of life. However, these new drugs are expensive, placing a significant financial burden on patients and severely limiting their widespread use. Therefore, to improve the prognosis of HPH patients and reduce mortality, there is an urgent need to develop effective and affordable drugs. Traditional Chinese herbal medicines are an important source for new drug development, but so far only studies have been found on the application and mechanism of some Chinese herbal compound prescriptions in related diseases (see "Study on the mechanism of Shenling Baizhu Powder in treating COPD" and "Study on the effect and mechanism of Shenling Baizhu Powder in treating stable chronic obstructive pulmonary disease", etc.), and the research content has not involved the related therapeutic effects on HPH.

[0004] Aralia elata, also known as Flying Centipede Seven, Wolf Tooth Club, etc., is a plant belonging to the genus Aralia of the Araliaceae family. It grows in the Qinling-Bashan Mountains, with abundant wild resources in the Qinling region of Shaanxi Province. Aralia elata is a plant used for both medicinal and edible purposes. Its newly sprouted buds and shoots in spring are edible and are considered a rare and nutritious wild vegetable. The root bark of Aralia elata is the main medicinal part, also known as Aralia elata bark, and is one of the "Seven Medicines of Taibai". Traditional medicine believes that Aralia elata bark is slightly bitter, slightly cold, sweet, pungent, and neutral in nature, and enters the lung, spleen, kidney, and stomach meridians. It has the effects of dispelling wind and dampness, strengthening the spleen and promoting diuresis, reducing swelling, and promoting blood circulation and removing blood stasis. It is mainly used for cough and asthma, chest pain, pulmonary distension, diabetes, hepatitis, nephritis, and other diseases. Chemical analysis shows that the effective components of Aralia elata mainly include saponins, volatile oils, polysaccharides, and trace elements. Among them, saponins from Aralia taibaiensis (sAT) are its main active ingredients. Modern molecular medicine has proven that sAT has a variety of pharmacological activities, including lowering blood sugar and lipids, anti-oxidation, protecting the cardiovascular system, anti-aging, anti-tumor, and anti-fibrotic effects (refer to CN103933090A, CN114796294A, "Study on the Effects and Mechanisms of Total Saponins and Monomeric Components of Aralia taibaiensis on Stroke," etc.). However, the therapeutic effects and mechanisms of sAT on HPH have not been reported. Summary of the Invention

[0005] The purpose of this invention is to provide the use of Aralia elata saponins in the treatment of hypoxic pulmonary hypertension.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Firstly, the use of Aralia elata saponins (sAT) in the preparation of medicaments for the prevention and / or treatment of hypoxic pulmonary hypertension is provided.

[0007] Preferably, the hypoxic pulmonary hypertension is caused by hypoxia, and the animal model used to assess the therapeutic effect is induced by hypoxia.

[0008] Preferably, the Aralia elata saponins are administered to mice with hypoxic pulmonary hypertension via gavage.

[0009] Preferably, the aralia elata saponins are used to treat one or more of the following symptoms of induced hypoxic pulmonary hypertension: elevated right ventricular pressure (e.g., right ventricular systolic pressure), right ventricular hypertrophy (e.g., right ventricular thickening), pulmonary artery remodeling, thickening of the pulmonary arteriole wall and narrowing of the lumen, and increased muscularization of pulmonary arterioles.

[0010] Preferably, the saponins from Aralia elata do not cause a decrease in systemic blood pressure while treating induced hypoxic pulmonary hypertension.

[0011] Preferably, the saponins of Aralia elata are obtained by sequentially extracting the root bark of Aralia elata with 65%~75% ethanol (ethanol solution) (80~90℃), extracting and defatting (petroleum ether, n-butanol), concentrating, preparing the sample (diluted with deionized water and centrifuged), and performing macroporous adsorption resin column chromatography (using 30%~40% ethanol for impurity removal and 65%~75% ethanol for elution), and then concentrating, redissolving (with anhydrous ethanol), and drying (40~45℃) the eluent from the column chromatography.

[0012] Preferably, the blood-entering monomeric components of the Taibaienoside (sAT) are all saponin compounds, including Araloside A, Araloside C, Narcissiflorine, Chikusetsu saponin Iva, Ginsenoside Ro, Deglucose-chikus etsusapon IVa, Ginsengsaponin Ib, and Taibaienoside V.

[0013] Preferably, network pharmacology analysis of the blood-entering monomeric components of the Aralia elata saponins (sAT) and the intersection with hypoxic pulmonary hypertension yielded 123 intersection targets. The top 13 core nodes (i.e., ALB, AKT1, EGFR, STAT3, MMP9, CASP3, JUN, ACE, ESR1, SRC, PPARG, REN, and HSP90AA1) were identified as the main targets of sAT in treating hypoxic pulmonary hypertension. KEGG pathway analysis showed that the main pathway involved in the blood-entering monomeric components of sAT is the renin-angiotensin system (RAS).

[0014] Preferably, the blood-entering monomeric components of the aralia elata saponin (sAT), namely anemone glycoside and ginsenoside IVa, exhibit stable binding interactions with angiotensin-converting enzyme 1 (ACE).

[0015] Secondly, the application of Aralia elata saponins (sAT) in the preparation of drugs for the prevention and / or treatment of complications of hypoxic pulmonary hypertension is provided.

[0016] Preferably, the saponins from Aralia elata reduce the expression and activity of ACE in the lungs in a concentration-dependent manner, reduce the content of renin and angiotensin II (Ang II) and the expression of angiotensin II type 1 receptor (AT1R), thereby correcting the pulmonary RAS system disorder caused by hypoxic pulmonary hypertension.

[0017] Thirdly, the use of Aralia elata saponins (sAT) in the preparation of medicines for the prevention and / or treatment of respiratory diseases associated with hypoxemia (or hypoxia).

[0018] Preferably, the respiratory diseases are selected from clinically common interstitial lung diseases, chronic obstructive pulmonary disease, sleep apnea syndrome, chronic mountain sickness, and some neonatal respiratory diseases.

[0019] In the first to third aspects above, the drug is taken orally, and the dosage follows the "body surface area ratio principle". Based on the effective dose of Aralia elata saponin in mice being 200 mg / kg, the reference dose for a 60 kg adult is approximately 16 mg / kg (reference dose range 15~18 mg / kg).

[0020] Fourthly, the application of the monomeric components of Aralia elata saponins (sAT), namely anemone glycoside or ginsenoside IVa, in the preparation of drugs for the prevention and / or treatment of hypoxic pulmonary hypertension is provided.

[0021] Fifthly, the application of the monomeric components of Aralia elata saponins (sAT), namely anemone glycoside or ginsenoside IVa, in the preparation of drugs for the prevention and / or treatment of complications of hypoxic pulmonary hypertension.

[0022] Sixthly, the use of the monomeric components of Aralia elata saponin (sAT), namely anemone glycoside or ginsenoside IVa, in the preparation of medicaments for the prevention and / or treatment of the above-mentioned respiratory diseases related to hypoxemia (or hypoxia).

[0023] In aspects four to six above, the reference dose of the drug (converted from the effective dose of anemone in mice of 40 mg / kg to the reference dose for a 60 kg adult) is 3.33 mg / kg (reference dose range 3~5 mg / kg), or the reference dose of the drug (converted from the effective dose of ginsenoside IVa in mice of 40 mg / kg to the reference dose for a 60 kg adult) is 3.33 mg / kg (reference dose range 3~5 mg / kg).

[0024] The beneficial effects of this invention are reflected in: This invention establishes a hypoxic pulmonary hypertension model, administers Aralia elata saponins (sAT) to the model, and observes the therapeutic effect. Results show that SAT effectively reduces right ventricular pressure and right ventricular hypertrophy index, and inhibits pulmonary artery remodeling, achieving a therapeutic effect on hypoxic pulmonary hypertension. Furthermore, it was found that SAT does not cause changes in hemodynamics or pulmonary arteriolar morphology in normal controls, nor does it produce the side effect of decreasing systemic blood pressure while improving pulmonary artery remodeling and treating hypoxic pulmonary hypertension. This invention not only provides a new strategy for the pharmacological prevention and treatment of hypoxic pulmonary hypertension, but is also applicable to the prevention and treatment of interstitial lung disease, chronic obstructive pulmonary disease, sleep apnea syndrome, chronic mountain sickness, and some neonatal respiratory diseases related to hypoxemia, showing promising application prospects.

[0025] This invention clarifies the key mechanism of Aralia elata saponins in treating hypoxic pulmonary hypertension: the monomeric components of Aralia elata saponins (sAT), namely anemone glycoside and ginsenoside IVa, can specifically bind to angiotensin-converting enzyme 1 (ACE), inhibiting the activity and expression of ACE in the lungs, reducing the content of renin and angiotensin II (Ang II) and the expression of angiotensin II type 1 receptor (AT1R), correcting the pulmonary RAS system disorder caused by hypoxic pulmonary hypertension, and playing a therapeutic role in inhibiting pulmonary artery remodeling and improving right ventricular dysfunction. Attached Figure Description

[0026] Figure 1 Experimental results on the improvement of right ventricular function in a mouse model of hypoxic pulmonary hypertension using sAT; A: Procedure of the hypoxia-induced pulmonary hypertension and sAT intervention experiments in mice; B: Changes in body weight of mice under different treatments over 6 weeks (two asterisks) P <0.01 indicates a difference compared to the normoxia group. # P <0.05 and ## P <0.01 indicates a difference compared to the hypoxia group (n=10); C: Effect of sAT administration on pulmonary artery acceleration time (PAAT) in mice (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates comparison with the hypoxia group, n=10); D: Echocardiography of the right heart in mice under different treatments; E: Effect of SAT administration on right ventricular diameter (RVID) in mice (one asterisk). P <0.05 indicates a difference compared to the normoxic group (two asterisks). P <0.01 indicates a difference compared to the normoxia group. # P <0.05 and## P <0.01 indicates comparison with the hypoxia group, n=10); F: Effect of sAT administration on right ventricular free wall thickness (PVWT) in mice (two asterisks) P <0.01 indicates a difference compared to the normoxia group. # P <0.05 and ## P <0.01 indicates a difference compared to the hypoxia group (n=10); G: Effect of sAT administration on the systolic displacement of the tricuspid annulus (TAPSE) in mice (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates a difference compared to the hypoxia group (n=10); H: Effect of SAT administration on right ventricular output (CO) in mice (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 means compared with the hypoxia group (n=10).

[0027] Figure 2 Experimental results on the effectiveness of sAT in treating hypoxic pulmonary hypertension in mice; A: Effect of sAT administration on peak right ventricular systolic pressure (RVSP) in mice (two asterisks) P <0.01 indicates a difference compared to the normoxia group. # P <0.05 and ## P <0.01 indicates comparison with the hypoxia group, n=10); B: Effect of sAT administration on mean carotid artery pressure (mCAP) in mice; C: Effect of sAT administration on right ventricular hypertrophy index (RV / (LV+S)%) in mice (two asterisks) P <0.01 indicates a difference compared to the normoxia group. # P <0.05 and ## P <0.01 indicates a difference compared to the hypoxia group (n=10); D: Effect of sAT administration on the pulmonary arteriolar diameter ratio (WT%) in mice (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates the difference between the hypoxia group and the control group (n=30 pulmonary arterioles with an outer diameter of 50~200 µm / group, 3 pulmonary arterioles / slice, 1 slice / mouse, 10 mice / group); E: Effect of sAT administration on the pulmonary arteriole area ratio (WA%) in mice (two asterisks). P <0.01 indicates a difference compared to the normoxia group. ##P <0.01 indicates a difference compared to the hypoxia group, with n=30 pulmonary arterioles (outer diameter 50–200 µm / group), 3 pulmonary arterioles / slice, 1 slice / mouse, and 10 mice / group); F: Quantitative analysis results of α-SMA staining in pulmonary arterioles (two asterisks). P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 represents the difference between the hypoxia group and the control group (n=12 pulmonary arterioles with outer diameter of 50~200 µm / group, 3 pulmonary arterioles / slice, 1 slice / mouse, 4 mice / group); G: Effect of sAT administration on lung tissue morphology in mice (HE staining); H: Effect of sAT administration on pulmonary arteriole myoplasia (α-SMA staining); I: Effect of sAT administration on the co-expression of OPN and Ki67 (OPN and Ki67 double staining), scale bar is 50 μm.

[0028] Figure 3 A: Results of sAT inhibiting hypoxic proliferation of PASMCs; A: Results of detecting hypoxic proliferation of mouse PASMCs treated with different oxygen concentrations (21%, 10%, 5%, 3%, 1%) (two asterisks) P <0.01 indicates a comparison with the normoxic untreated control group (n=4 independent replicates); B: Hypoxic proliferation of mouse PASMCs pretreated with different concentrations of sAT (15, 30, 60 μg / mL) after 48 hours of incubation in 21% oxygen (noroxic) or 5% oxygen (hypoxic) environments (two asterisks). P <0.01 represents the difference between the control group and the normoxic untreated group. # P <0.05 and ## P <0.01 indicates comparison with the hypoxia-untreated control group (n=4 independent replicates); C: Quantitative analysis results of Edu staining after PASMCs were treated according to the conditions in B (two asterisks) P <0.01 represents the difference between the control group and the normoxic untreated group. ## P <0.01 indicates a comparison with the untreated hypoxic control group (n=4 independent replicates); D: Hypoxic proliferation of mouse PASMCs pretreated with 60 μg / mL sAT after 24 and 48 hours of incubation in 21% oxygen (normative) or 5% oxygen (hypoxic) environments (two asterisks). P <0.01 represents the difference between the control group and the normoxic untreated group. ## P <0.01 indicates a difference compared to the hypoxia + sAT 0 μg / mL 0-hour group. $$P <0.01 represents the result compared to the hypoxia + sAT 60 μg / mL 0-hour group (n=4 independent replicates); E: Representative fluorescence image of Edu staining (green) of PASMCs, scale bar 50 μm.

[0029] Figure 4-1 Results of intracellular component analysis of sAT in the blood (3 hours): Serum ion chromatogram of control mice detected in positive ion mode.

[0030] Figure 4-2 Results of serum monomer component analysis of sAT (3 hours): Serum ion chromatogram of mice in the drug-treated group detected in positive ion mode.

[0031] Figure 4-3 Results of serum monomer component analysis of sAT (3 hours): Chromatogram of serum ions detected in control mice under negative ion mode.

[0032] Figure 4-4 Results of serum monomer component analysis of sAT (3 hours): Chromatogram of serum ions in mice in the drug-treated group detected in negative ion mode.

[0033] Figure 5 The following are the network pharmacology results for sAT: A: Venn diagram of the intersection of sAT intracellular components and HPH disease targets (resulting in "potential therapeutic targets"); B: Protein-protein interaction (PPI) network obtained by importing the above "potential therapeutic targets" into the STRING database; C: "Core therapeutic target" network obtained by using the open-source cross-platform software Cytoscape and importing the above "potential therapeutic targets" PPI data; D, E, F: Gene Ontology (GO) enrichment analysis histograms; G: KEGG enrichment analysis histogram.

[0034] Figure 6 Results of SAT correction of pulmonary RAS system disorder; A: Results of pulmonary renin (two asterisks) levels. P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates a difference compared to the hypoxia group (n=10); B: Detection results of pulmonary angiotensin-converting enzyme 1 (ACE) activity (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates comparison with the hypoxia group (n=10); C: Detection results of pulmonary angiotensin II (Ang II) content (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P<0.01 indicates a difference compared to the hypoxia group (n=10); D: Changes in the expression of ACE and AT1R proteins in the lung tissue of mice under different treatments (Western Blot analysis); E: Quantitative results of ACE protein expression (two asterisks). P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates a comparison with the hypoxia group (n=3 independent replicates, where 3 independent replicates mean three replicates for each group of three different mouse samples); F: Quantification of AT1R protein expression (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates a difference compared to the hypoxia group (n=3 independent replicates).

[0035] Figure 7 The following are the molecular docking and molecular dynamics simulation results of anemone, ginsenoside IVa, and ACE in sAT: A: Surface image of the anemone-ACE complex crystal structure at 100 ns; B: Three-dimensional crystal structure of the anemone-ACE complex (ΔG of anemone-ACE = -16.61 kcal / mol); C: RMSD diagram of the anemone-ACE complex; D: RMSF diagram of ACE without anemone binding; E: Surface image of the ginsenoside IVa-ACE complex crystal structure at 100 ns; F: Three-dimensional crystal structure of the ginsenoside IVa-ACE complex (ΔG of ginsenoside IVa-ACE = -14.17 kcal / mol); G: RMSD diagram of the ginsenoside IVa-ACE complex; H: RMSF diagram of ACE without ginsenoside IVa binding.

[0036] Figure 8 Results of efficacy evaluation of narcissiflorine in treating a mouse model of hypoxic pulmonary hypertension; A: Molecular structure of narcissiflorine monomer; B: Effect of narcissiflorine on peak right ventricular systolic pressure (RVSP) in mice (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates comparison with the hypoxia group, n=10); C: Effect of narcissiflorine administration on mean carotid artery pressure (mCAP) in mice; D: Effect of narcissiflorine administration on lung tissue morphology in mice (HE staining, scale bar 50 μm); E: Effect of narcissiflorine administration on right ventricular hypertrophy index (RV / (LV+S)%) in mice (two asterisks). P <0.01 indicates a difference compared to the normoxia group.## P <0.01 indicates comparison with the hypoxia group, n=10); F: Effect of administration of narcissiflorine on the pulmonary arteriolar area ratio (WA%) in mice (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 represents the difference between the hypoxia group and the control group (n=10 pulmonary arterioles with an outer diameter of 50~200 µm / group, 1 pulmonary arteriole / slice, 1 slice / mouse, 10 mice / group); G: Effect of administration of narcissiflorine on the pulmonary arteriole diameter-to-weight ratio (WT%) in mice (two asterisks). P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates the difference between the hypoxia group and the control group (n=10 pulmonary arterioles with an outer diameter of 50~200 µm / group, 1 pulmonary arteriole / slice, 1 slice / mouse, 10 mice / group); H: Effect of administration of narcissiflorine on ACE activity in mouse lungs (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates a difference compared to the hypoxia group (n=10); I: Effect of narcissiflorine administration on ACE expression levels in mouse lungs (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates a difference compared to the hypoxia group (n=3 independent replicates).

[0037] Figure 9 Results of efficacy evaluation of chikusetsu saponin IVa in treating a mouse model of hypoxic pulmonary hypertension; A: Molecular structure of chikusetsu saponin IVa (CHS) monomer; B: Effect of CHS administration on peak right ventricular systolic pressure (RVSP) in mice (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates comparison with the hypoxia group, n=10); C: Effect of CHS administration on mean carotid artery pressure (mCAP) in mice; D: Effect of CHS administration on lung tissue morphology in mice (HE staining, scale bar 50 μm); E: Effect of CHS administration on right ventricular hypertrophy index (RV / (LV+S)%) in mice (one asterisk). P <0.05 indicates a difference compared to the normoxic group (two asterisks).P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates comparison with the hypoxia group, n=10); F: Effect of CHS administration on the pulmonary arteriole area ratio (WA%) in mice (two asterisks) P <0.01 indicates a difference compared to the normoxia group. # P <0.05 and ## P <0.01 indicates the difference between the hypoxia group and the control group (n=10 pulmonary arterioles with an outer diameter of 50~200 µm / group, 1 pulmonary arteriole / slice, 1 slice / mouse, 10 mice / group); G: Effect of CHS administration on the pulmonary arteriole diameter ratio (WT%) in mice (two asterisks). P <0.01 indicates a difference compared to the normoxia group. # P <0.05 and ## P <0.01 indicates the difference between the hypoxia group and the control group (n=10 pulmonary arterioles with an outer diameter of 50~200 µm / group, 1 pulmonary arteriole / slice, 1 slice / mouse, 10 mice / group); H: Effect of CHS administration on ACE activity in mouse lungs (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates a difference compared to the hypoxia group (n=10); I: Effect of CHS administration on ACE expression levels in mouse lungs (two asterisks) P <0.01 indicates a difference compared to the normoxia group. ## P <0.01 indicates a difference compared to the hypoxia group (n=3 independent replicates). Detailed Implementation

[0038] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0039] This invention evaluates the efficacy of Aralia elata saponins in treating hypoxic pulmonary hypertension (HPH) and its complications (specifically, pulmonary respiratory system (RAS) disorders caused by hypoxic pulmonary hypertension). It also provides in-depth and pioneering research on the pharmacological effects and mechanisms of Aralia elata saponins by analyzing the interaction between the blood-entering monomeric components and the RAS system. This invention also provides experimental evidence for the prevention and treatment of respiratory diseases such as interstitial lung disease, chronic obstructive pulmonary disease, sleep apnea syndrome, chronic mountain sickness, and some neonatal respiratory diseases.

[0040] 1. Pharmacological experiment on the treatment of hypoxic pulmonary hypertension and pulmonary RAS system disorder by saponins from Aralia elata. 1.1 Extraction and purification of Aralia elata saponins (sAT) Take 1.2 kg of commercially available dried root bark of Aralia elata (product number: 59252, Baoji Chenguang Biotechnology Co., Ltd.), pulverize it with a cell wall blender, and extract it three times with 1 L of 70% ethanol aqueous solution (each extraction was carried out at 90℃ under reflux for 1 h, the residue was filtered off and the filtrate was collected, and the residue was extracted twice more). Combine the collected filtrates. The combined filtrate was concentrated (water bath temperature: 45℃, vacuum degree: -0.08 ~ -0.09 MPa) to obtain 340 g of extract. The extract was dispersed in 0.4 L of water and then extracted and defatted three times with 0.4 L of petroleum ether (boiling range 60~90℃) (discarding the petroleum ether layer and retaining the aqueous phase). Then it was extracted four times with 0.4 L of water-saturated n-butanol (collecting the n-butanol layer). After concentration (water bath temperature: 55℃; vacuum degree: -0.08~-0.09 MPa), about 110 g of pale yellow extract was obtained, which is the crude SAT extract.

[0041] Take 100 mL of the above crude SAT extract and place it in a 250 mL separatory funnel. Add an equal volume (100 mL) of petroleum ether (60~90℃), shake vigorously for 5 min, and allow to stand at room temperature (25±2℃) for 30 min to separate into layers. After separation, discard the upper petroleum ether phase (containing fat-soluble impurities) and retain the lower water-ethanol phase. Repeat the petroleum ether defatting operation twice to ensure thorough removal of fat-soluble impurities. Place the defatted crude SAT extract in a rotary evaporator, set the temperature to 50℃ and the vacuum degree to 0.06~0.08 MPa, and concentrate until there is no alcohol odor (volume approximately 20 mL) to obtain a concentrated solution. Add 80 mL of deionized water to the concentrated solution to obtain 100 mL of diluent. Centrifuge the diluent at 5000 r / min for 10 min, discard the precipitate, and collect the supernatant, which is the loading solution.

[0042] Pretreated D101 macroporous adsorption resin was packed into a chromatography column (2.5 cm inner diameter, 50 cm length) to a height of 30 cm. The column bed was equilibrated with deionized water until the pH of the effluent reached 7.0. The above loading solution was loaded onto the resin column at a flow rate of 1 BV / h. After loading, the column bed was rinsed with deionized water at a flow rate of 2 BV / h until the absorbance of the effluent at 203 nm wavelength on a UV-Vis spectrophotometer was ≤ 0.05 (no saponins detected). The corresponding "water" eluent (containing water-soluble impurities such as sugars and amino acids) was discarded. Then, the resin column was eluted with a 30% (v / v) ethanol aqueous solution at a flow rate of 2 BV / h, with an elution volume of 2 BV (approximately 190 mL). The collected "30% ethanol" eluent (containing a small amount of polar impurities) was discarded. The resin column was then eluted with a 70% ethanol aqueous solution at a flow rate of 2 BV / h. The eluent was collected in fractions of 3 BV (approximately 285 mL) with each fraction being 25 mL. The absorbance of each fraction was measured at 203 nm using a UV-Vis spectrophotometer. Fractions with absorbance values ​​≥ 0.2 were combined to obtain a purified eluent (approximately 265 mL).

[0043] The purified eluent was placed in a rotary evaporator at 55°C and a vacuum of 0.07–0.09 MPa, and concentrated to a thick paste (5–10 mL in volume, with no obvious flowability). 5 mL of anhydrous ethanol was added to the resulting paste, dissolved, and transferred to a petri dish. The dish was then placed in a vacuum drying oven at 45°C and a vacuum of 0.08–0.10 MPa, and dried to constant weight (approximately 8–12 h, with a weight change ≤0.3%). The dried solid was finely ground using an agate mortar and pestle, passed through a 60-mesh standard sieve, and the powder passing through the sieve was collected as SAT. This SAT was stored in a sealed desiccator at 4°C.

[0044] 1.2 Determination of total saponin content in sAT The total saponins in SAT were quantified using ultraviolet-visible spectrophotometry. The specific steps are as follows: Standard solutions (0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL) were prepared using oleanolic acid (which is also an effective component of Aralia elata) standard to plot a standard curve. Six parallel 20 mg SAT samples were accurately weighed and diluted to 10 mL with methanol. 1 mL of each sample was placed in a test tube, heated in a 60°C water bath to evaporate the solvent, and 0.2 mL of freshly prepared 5% (mass fraction) vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid were added. The mixture was heated in a 60°C water bath for 15 min, then cooled to room temperature in an ice bath. 5 mL of ethyl acetate was added to each of the treated SAT solutions and standard solutions, and the absorbance was measured at 550 nm using a spectrophotometer. The total saponin content of SAT was calculated based on the standard curve.

[0045] 1.3 Construction of an animal model of hypoxic pulmonary hypertension and experimental grouping Hypoxia is one of the ways pulmonary hypertension is induced. Hypoxia causes damage to pulmonary artery endothelial cells and an imbalance of vascular factors that regulate pulmonary artery contraction / dilation, increasing the pulmonary artery systolic response and promoting arterial remodeling, ultimately leading to pulmonary hypertension. Pulmonary hypertension caused by chronic hypoxia is commonly seen in various chronic respiratory diseases, such as interstitial lung disease, chronic obstructive pulmonary disease, sleep apnea syndrome, chronic mountain sickness, and some neonatal diseases. Placing animals in a hypobaric hypoxic chamber for 2-8 weeks (maintaining an oxygen concentration of around 10% and a carbon dioxide concentration of around 5%) can replicate a hypoxic pulmonary hypertension model in animals. In this model, the animals are more likely to exhibit blood gas changes characterized by hypoxia and hypercarbon dioxide, which is more consistent with the actual situation of patients with chronic respiratory diseases in clinical practice.

[0046] C57BL / 6 mice (male, 7-8 weeks old, weighing approximately 18-22 g) were randomly divided into 6 groups of 10 mice each: (1) Normoxia group (Normoxia + sAT 0 mg / kg): Mice were placed in a normoxic environment and observed for 6 weeks (as a normal control); (2) Normoxia + sAT intervention group (Normoxia + sAT 200 mg / kg): Mice were placed in a normoxic environment and administered sAT (200 mg / kg, dissolved in 0.9% physiological saline and 0.5%~1% Tween-80 before gavage to promote sAT dissolution and avoid precipitation, and sonicated for 10~15 minutes after preparation to completely dissolve sAT and make the solution homogeneous) once a day for 2 weeks. (3) Hypoxia group (Hypoxia + sAT 0 mg / kg): Mice were placed in a low-pressure hypoxic chamber with an oxygen concentration of about 10% and a carbon dioxide concentration of about 5% for 8 hours every day for 6 consecutive weeks to replicate the mouse hypoxic pulmonary hypertension model. (4) Hypoxia + sAT intervention group (Hypoxia + sAT 50, 100, 200 mg / kg): Mice were placed in a low-pressure hypoxic chamber with an oxygen concentration of about 10% and a carbon dioxide concentration of about 5% for 8 hours every day. In the 4th week, sAT was administered by gavage in three dose groups (i.e., 50, 100, 200 mg / kg, sAT was dissolved in 0.9% physiological saline and 0.5%~1% Tween-80 was added. After preparation, the mixture was sonicated for 10~15 minutes and used immediately). The treatment was administered once a day for 2 weeks.

[0047] In the normoxic group, mice were placed in an animal room and raised under natural conditions (atmospheric pressure approximately 718 mmHg, pO2 150.6 mmHg, oxygen concentration approximately 21%). In the low-pressure hypoxic chamber (chamber pressure 380 mmHg, pO2 reduced to 79.6 mmHg, equivalent to the oxygen content at an altitude of 5540 meters, oxygen concentration approximately 10%, carbon dioxide concentration approximately 5%), soda lime and desiccants were used to deodorize and absorb CO2.

[0048] All mice were weighed weekly and treated uniformly at the end of week 6, including testing for hypoxic pulmonary hypertension-related indicators to evaluate the therapeutic effect of sAT on the hypoxic pulmonary hypertension model mice.

[0049] 1.4 Right heart echocardiography Echocardiography of mice was performed using a Vevo 2100 high-resolution imaging system (VisualSonics) equipped with an 18–38 MHz probe (MS400, mouse cardiovascular specific). The specific procedure was as follows: Mice were placed supine on a temperature-controlled plate, and their rectal temperature, heart rate, and respiratory rate were continuously recorded throughout the experiment. After anesthesia with 0.5%–1.5% isoflurane, the experimental animals were shaved and preheated ultrasound coupling agent was applied. The right ventricular diameter (RVID) and right ventricular free wall thickness (RVWT) were measured using M-mode ultrasound in the short-axis view of the right parasternal region. The tricuspid annulus systolic displacement (TAPSE) was measured by aligning the M-mode ultrasound cursor with the junction of the tricuspid valve annulus and the right ventricular free wall in the apical four-chamber view. Pulsed-wave Doppler ultrasound of the right ventricular outflow tract was also performed in the long-axis view of the left ventricle. The Doppler velocity curve measures the pulmonary artery acceleration time (PAAT) and calculates right ventricular output (CO) by combining the pulmonary artery velocity-time integral (PAVTI), pulmonary artery cross-sectional area, and heart rate.

[0050] 1.5 Hemodynamic parameter detection After anesthetizing and fixing the mice, an incision was made in the midline of the neck, and mechanical ventilation was initiated via endotracheal intubation. The ventilation parameters were set as follows: tidal volume 10 mL / kg and respiratory rate 160 breaths / min. The distal end of the left common carotid artery was separated and ligated, and the proximal end of the vessel was clamped. A small incision was made between the two ends with ophthalmic scissors, and a polyethylene catheter filled with 0.5% heparin solution was inserted into the left common carotid artery. One end of the catheter remained in the vessel and was knotted and fixed, while the other end was connected to a pressure sensor and connected in series with a pressure transducer to record the mouse's mean carotid artery pressure (mCAP). Next, the chest cavity was opened, and a heparinized 21-gauge scalp needle connected to another pressure sensor was inserted into the right ventricle to measure the right ventricular systolic pressure (RVSP). All pressure signal data were continuously recorded using a PowerLab system, and the data were analyzed using LabChart 7 software.

[0051] 1.6 Measurement of Right Ventricular Hypertrophy Index (RV / (LV+S)%) The mice were euthanized, their sternums were cut open to expose the heart, and the tissues and blood vessels around the heart, as well as the left and right atria and auricles, were removed. The pulmonary conus was located, and the right ventricle (RV) was cut open along the pulmonary conus and weighed. The remaining tissue was also weighed, which is the weight of the left ventricle and interventricular septum (LV+S). The right ventricular hypertrophy index was calculated as RV / (LV+S)×100% to reflect the degree of right ventricular hypertrophy.

[0052] 1.7 Preparation of paraffin sections and HE staining of lung tissue Mice were euthanized, lung tissue was removed, and samples were taken transversely along the hilum. A tissue block of approximately 1 cm × 2 cm was cut from the upper lobe of the right lung of the mouse and placed in an embedding frame. The tissue block and the embedding frame were then fixed in 10% neutral formaldehyde buffer for 24 h. After removal, the tissue was washed, dehydrated, embedded, and prepared into paraffin blocks. The paraffin blocks were sectioned, dewaxed to water, and the paraffin sections of lung tissue from a subset of mice in each group were stained with hematoxylin and eosin (HE) to detect changes in pulmonary arterioles.

[0053] 1.8 Quantitative Analysis of Pulmonary Arterioles HE-stained sections were observed under a microscope. Pulmonary arterioles with an outer diameter between 50 and 200 μm were selected. Image analysis software was used to acquire and analyze vascular images. The inner diameter, outer diameter, wall thickness, and total vascular area of ​​the vessels were measured. Then, based on the measured values, two indicators reflecting the thickening of the vessel wall were calculated: WT% = wall thickness / outer diameter × 100% and WA% = wall area / total vascular area × 100%.

[0054] 1.9 Immunofluorescence staining Paraffin sections of lung tissue from the remaining mice in each group were dewaxed to water, infiltrated with 0.1% Triton-100, and then blocked with 1% BSA for 1 hour. After blocking, the sections were processed separately using the following two methods: (i) Add α-SMA primary antibody (catalog number: #ab5694, Abcam, dilution 1:200) and incubate overnight at 4°C. After washing three times with PBS, add fluorescent secondary antibody Alexa Fluor® 488 (catalog number: #ab150077, Abcam, dilution 1:1000) and incubate at room temperature for 1 hour. Finally, add DAPI (catalog number: #ab285390, Abcam) and incubate for 5 minutes. Observe the expression of α-SMA in mouse pulmonary arterioles using laser confocal microscopy.

[0055] (ii) Since the expression level of osteopontin (OPN) in lung tissue is one of the indicators for assessing the severity of hypoxic pulmonary hypertension, and Ki67 is an antigen related to cell proliferation, the co-expression of OPN and Ki67 by immunofluorescence double staining can show myomorphic pulmonary arterioles in a proliferating state. Therefore, after dewaxing, infiltrating, and blocking paraffin sections of lung tissue, the sections were incubated overnight at 4°C with a mixture of anti-Ki67 antibody (catalog number: #ab16667, Abcam, dilution 1:200) and anti-OPN antibody (catalog number: #ab63856, Abcam, dilution 1:200). After repeated washing with phosphate-buffered saline (PBS), for Ki67 detection, goat anti-mouse IgG secondary antibody Alexa Fluor® 594 (catalog number: #ab150120, Abcam, dilution 1:1000) was added to treat the sections; for OPN detection, goat anti-rabbit IgG secondary antibody Alexa Fluor® 488 (catalog number: #ab150077, Abcam, dilution 1:1000) was added to treat the sections. Finally, DAPI was added and incubated for 5 minutes, and the co-expression of OPN and Ki67 in mouse pulmonary arterioles was observed using a laser confocal microscope.

[0056] 1.10 Isolation and Culture of Mouse Pulmonary Artery Smooth Muscle Cells (PASMCs) The specific steps for the isolation and primary culture of PASMCs are as follows: Lung tissue from healthy mice is rapidly extracted and placed in ice-cold sterile phosphate-buffered saline (PBS); the tertiary branches of the pulmonary artery are isolated under a dissecting microscope, and the connective tissue on its surface is removed; after removing the adventitia and intima of the pulmonary arteries, the arterial tissue is cut into pieces smaller than 1 mm. 3Small pieces of tissue were added to 0.1% type II collagenase and digested at 37°C for 40-50 minutes. Immediately after digestion, an equal volume of DMEM medium (product number: 11995-065, Gibco / Thermo Fisher) containing 15% fetal bovine serum (product number: A31608-02) was added to terminate digestion. The cells were gently pipetted to mix, resulting in a digested suspension. A 70 μm cell sieve was placed on a new 15 mL centrifuge tube. The digested suspension was slowly sieved to remove undigested tissue pieces. The filtered cell suspension was centrifuged at 1000 r / min for 8 min, the supernatant was discarded, and the cell pellet at the bottom was retained. 1-2 mL of DMEM medium containing 15% fetal bovine serum was added, the cells were gently resuspended, and the cells were seeded in 25 cm⁻¹ medium. 2 Add culture medium to 5-6 mL in the culture flask, shake gently, and place in a humid incubator at 37°C with 5% carbon dioxide (CO2). Spindle cells can be seen crawling out of the tissue block after 3-7 days of culture. Change the medium normally during the period. When the cells reach 80% confluence, passage them.

[0057] The following are the steps for subculturing and identifying PASMCs before experiments: After cell fusion, cells are passaged at a ratio of 1:3 and cultured under the same conditions. Cells from passages P2 to P5 are used for experiments. Before the experiment, passaged cells are prepared into cell slides, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 (catalog number: P0096, Beyotime Biotechnology) for 10 min, blocked with 5% BSA (catalog number: P0260, Beyotime Biotechnology) at room temperature for 30 min, and then stained with α-SMA (catalog number: #ab5694, Abcam, dilution 1:200) for identification. When the proportion of α-SMA-positive cells is >90%, the cultured cells are considered to be PASMCs.

[0058] 1.11 Proliferation Experiment of PASMCs First, the proliferation of PASMCs under hypoxic conditions was detected using the MTT assay. Cells from passages P2 to P3 were used in this experiment, and the specific steps were as follows: Cells cultured to approximately 80% confluence were seeded in serum-free medium into 96-well plates (1 × 10⁶ cells per well). 4 Cells were cultured for 12 hours and then placed in environments with different oxygen concentrations (1%, 3%, 5%, 10%, 21%) for 24 or 48 hours. Then, 5 mg / mL MTT solution (dissolved in PBS, 5 μL per well) was added to each well, and incubation was continued for 4 hours. The liquid in the wells was discarded, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the generated formazan crystals. The absorbance (OD value) at 550 nm was measured by spectrophotometer.

[0059] Next, P2-P3 generation PASMCs that had undergone serum-free culture and starvation treatment were pretreated with different concentrations of sAT (15, 30, and 60 μg / mL, respectively), and an untreated control (sAT of 0 μg / mL) was set up. They were then cultured in environments with 21% oxygen (normative oxygen) and 5% oxygen (hypoxia) for 48 hours, respectively. Subsequently, the antiproliferative activity of sAT was detected by the MTT assay. Meanwhile, the anti-proliferative effect of different concentrations of SAT was verified by immunofluorescence staining using an EdU cell proliferation assay kit (catalog number: ab219801, Abcam). The specific steps were as follows: PASMCs grown on coverslips were incubated with 10 μM EdU for 2–4 h (37℃, 5% CO2); after incubation, they were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 15 min, and then blocked with 5% BSA for 30 min. The prepared Click reaction solution (provided with the kit) was added to the slides and incubated at room temperature in the dark for 30 min. After washing three times with PBS, DAPI was added for nuclear staining, and the cells were observed and imaged under a fluorescence microscope. EdU-positive cells were counted from three random fields of view to assess cell proliferation under normoxic and hypoxic conditions.

[0060] Finally, P2-P3 generation PASMCs that had undergone serum-free culture and starvation treatment were pretreated with 60 μg / mL sAT, and an untreated control (sAT of 0 μg / mL) was set up. They were then cultured in environments with 21% oxygen concentration (normative oxygen) and 5% oxygen concentration (hypoxia) for 24 or 48 hours, respectively. The antiproliferative activity of sAT was then detected by the MTT assay.

[0061] 1.12 Analysis of intracellular components of sAT entering the bloodstream Twelve healthy mice aged 6-8 weeks were selected, half male and half female. Six mice served as the normal control group, and six mice served as the treatment group. Mice in the treatment group were fasted for 12 hours and then administered sAT (200 mg / kg, dissolved in 0.9% physiological saline with 0.5%–1% Tween-80, sonicated for 10–15 minutes after preparation, and used immediately) by gavage for three consecutive days (once daily). Mice in the normal control group received the same volume of PBS solution. On the last day after administration, blood samples were collected from mice at 1 hour, 2 hours, and 3 hours (mice were anesthetized and blood was drawn from the heart before collection) to prepare serum. An equal volume of methanol (approximately 100 μL) was then added to the serum, vortexed for 3 minutes to completely dissolve the residue, and centrifuged at 4°C and 12000 r / min for 10 minutes. The supernatant was collected, and this process was repeated three times. After extraction, methanol was evaporated to dryness. The residue was reconstituted with 200 μL of methanol and then centrifuged (14000 rpm, 10 min, 4℃). The supernatant was used as the sample solution for subsequent analysis. A QExactive mass spectrometer (Thermo Fisher Scientific) coupled with a u3000 high-performance liquid chromatograph (HPLC) and a C18 column (Agilent Technologies, 2.1 × 100 mm, 1.8 μm particle size) was used for the analysis of intraserum monomeric components. The injection volume of the sample solution obtained after methanol extraction and reconstitution was set to 5 μL, and detection was performed in both positive and negative ion modes.

[0062] 1.13 Network Pharmacological Analysis The SMILES structures of each core active saponin component obtained through blood-entry monomer component analysis (downloaded from PubChem, e.g., Aralia elata saponin A:C) were obtained. 47 H 74 O 18 Import them one by one into the SwissTargetPrediction and PharmMapper databases, selecting "mice" as the species. Mussapiens ) and people ( Homo sapiens (), to obtain the predicted target of the corresponding component; import the PubChem_CID / InChI of the "core active saponin component" (copied from PubChem) one by one into BATMAN-TCM, and select " Mussapiens "and" Homo sapiens "For species, with parameter adjustments ≥ 20 and scores ≥ 0.5, obtain the predicted targets for the corresponding components; merge targets from multiple databases, remove duplicate targets, and establish the "sAT-Active Target Dataset".

[0063] Download disease targets by entering "Hypoxic Pulmonary Hypertension" into the OMIM and Genecards databases respectively. Then, select the top 2000 targets by median score using an algorithm, merge the targets in the database, remove duplicate targets, and create the "HPH Disease Target Dataset".

[0064] The intersection of the “sAT-Active Target Dataset” and the “HPH Disease Target Dataset” was obtained using the online website Venny2.1. A Venn diagram was then drawn, the number of intersection targets was marked, and the scope of the core research targets was clarified.

[0065] Import the aforementioned "potential therapeutic targets" into the STRING database, setting the species to "Homo sapiens and..." Mussapiens With a confidence threshold ≥ 0.4, nodes without interactions are hidden, and TSV format data (referring to the PPI network) is exported. The PPI data is imported into the open-source cross-platform software Cytoscape, and a network is constructed (nodes = target points, edges = interaction strengths). The CytoHubba plugin in Cytoscape is used to calculate topological parameters: degree, betweenness centrality, and closeness centrality. The top 30 target points in descending order of degree score are selected as "core therapeutic targets" (e.g., ALB, AKT1, EGFR, ACE, etc.). The "core active saponin components" and "core therapeutic targets" are integrated, and a saponin component-target network (referring to the "core therapeutic target" network) is constructed using Cytoscape.

[0066] The online database DAVID was used to perform GO functional enrichment and KEGG pathway enrichment analysis on "potential therapeutic targets". P<0.05 and FDR<0.05 were set, and a visualization was generated.

[0067] 1.14 Molecular docking and molecular dynamics simulation The 3D structures (SDF format) of each saponin component obtained from blood-borne monomer component analysis were downloaded from PubChem. Hydrogenation and Gasteiger charge calculation were performed using AutoDockTools 1.5.6, and the structures were saved in PDBQT format. The crystal structures of the "core therapeutic targets" (e.g., ACE: PDB ID 6H5W) were downloaded from the PDB database, water molecules and ligands were removed, Gasteiger charges were added, and active pockets were defined using AutoGridBox. Semi-flexible docking was performed using AutoDock Vina, with exhaustiveness set to 32, and the binding energy (ΔG) was output. Based on ΔG < -5.0 kcal / mol as "good binding activity," the "component-target" pairs with the optimal binding energy were screened, and the binding sites were visualized using PyMOL. Then, the optimal conformations of the "core therapeutic target" and the blood-bound monomer component with "good binding activity" were selected as the initial conformations for molecular dynamics simulation. During the simulation using the AMBER 14 force field, the saponin-target complex was dissolved in a dodecahedral box with 0.9% sodium chloride, and the box boundary was 12 Å away from the complex. Finally, by setting the starting temperature for minimizing the simulation annealing to 298 K and using a Berendsen thermostat to reduce the influence of temperature control, a molecular dynamics simulation of 100 nanoseconds was performed with a step size of 2 femtoseconds.

[0068] 1.15 Detection of ACE activity in mouse lung tissue The ACE activity of mouse lung tissue was determined using an ACE activity assay kit (catalog number: ab239703, Abcam). The specific steps were as follows: lung tissue homogenates of mice in each group were prepared, quantified, and diluted with gradient concentration buffers provided in the kit; the substrate and reagents were added to a 96-well plate and reacted at room temperature for 30 minutes; then the ACE activity was quantified at 340 nm using a spectrophotometer.

[0069] 1.16 ELISA method for detecting the levels of Renin and Ang II in mouse lung tissue Tissue samples were taken from the right lower lobe of mice in each group. 100 mg of tissue was accurately weighed and placed in an EP tube. 1 mL of physiological saline was added, and the tissue was homogenized using a hand homogenizer. The homogenate was then centrifuged at 4500 rpm for 10 min at 4°C, and the supernatant was collected. The levels of Renin and Ang II in the lung tissue were detected according to the instructions of the ELISA kits (Renin ELISA kit number: E-EL-M0701c, Ang II ELISA kit number: E-EL-M0031c, both purchased from Elabscience).

[0070] 1.17 Detection of changes in ACE and AT1R expression in mouse lung tissue The expression of ACE and AT1R was detected by Western blotting. The specific steps were as follows: 100 mg of tissue was accurately weighed from the left lower lobe of mice in each group, and RIPA lysis buffer (Catalog No.: C500005-0100, Sangon Biotech) containing protease inhibitors and phosphatase inhibitors was added. The tissue was fully lysed on ice, centrifuged, and the supernatant was collected. The protein concentration was determined by the BCA method. Then, the expression changes of ACE and AT1R were analyzed by vertical gel electrophoresis, membrane transfer, antibody binding (primary antibodies: ACE and AT1R antibodies were purchased from Abcam Antibody, catalog Nos. ab254222 and ab124734, respectively; β-actin antibody was purchased from Abcam Antibody, catalog No. ab8224; secondary antibodies: catalog Nos. 31430 and 31460, purchased from Thermo Fisher Scientific), chemiluminescence, and gel imaging.

[0071] 2. Pharmacological experiments on the therapeutic effects of monomeric components of Aralia elata saponins on hypoxic pulmonary hypertension and pulmonary RAS system disorders. The therapeutic effects of the monomeric components of sAT on a mouse model of hypoxic pulmonary hypertension were evaluated using commercially available Chikusetsu saponin IVa (CHS, catalog number: 51415-02-2, Baoji Chenguang Biotechnology Co., Ltd.) and Narcissiflorine (catalog number: 59252-95-8, Baoji Chenguang Biotechnology Co., Ltd.).

[0072] C57BL / 6 mice (male, 7-8 weeks old, weighing approximately 18-22 g) were randomly divided into 5 groups of 10 mice each: (1) Normoxia group (Normoxia+CHS / Narcissiflorine 0 mg / kg): Mice were placed in a normoxic environment and observed for 6 weeks (as a normal control); (2) Normoxia + CHS or Narcissiflorine intervention group (Normoxia + CHS / Narcissiflorine 40 mg / kg): Mice were placed in a normoxic environment and administered CHS (40 mg / kg, with sterile double-distilled water as the gavage solvent) or Narcissiflorine (40 mg / kg, Narcissiflorine was first dissolved in DMSO, then 0.5% sodium carboxymethyl cellulose aqueous solution was added, and the mixture was sonicated for 10-20 min to mix well, with the final concentration of DMSO ≤ 1%) once a day for 2 weeks. (3) Hypoxia group (Hypoxia+CHS / Narcissiflorine 0 mg / kg): Mice were placed in a low-pressure hypoxic chamber with an oxygen concentration of about 10% and a carbon dioxide concentration of about 5% for 8 hours every day for 6 consecutive weeks to replicate the mouse hypoxic pulmonary hypertension model. (4) Hypoxia + CHS or Narcissiflorine intervention group (Hypoxia + CHS / Narcissiflorine 20 mg / kg, 40 mg / kg): Mice were placed in a low-pressure hypoxic chamber with an oxygen concentration of about 10% and a carbon dioxide concentration of about 5% for 8 hours every day. In the 4th week, CHS (20 mg / kg, 40 mg / kg, with sterile double-distilled water as the gavage solvent) or Narcissiflorine (20 mg / kg, 40 mg / kg, Narcissiflorine was first dissolved in DMSO, then 0.5% sodium carboxymethyl cellulose aqueous solution was added, and the mixture was sonicated for 10~20 min to mix well. The final concentration of DMSO was ≤ 1%) was once a day for 2 weeks.

[0073] In the normoxic group, mice were placed in an animal room and raised under natural conditions (atmospheric pressure approximately 718 mmHg, pO2 150.6 mmHg, oxygen concentration approximately 21%). In the low-pressure hypoxic chamber (chamber pressure 380 mmHg, pO2 reduced to 79.6 mmHg, equivalent to the oxygen content at an altitude of 5540 meters, oxygen concentration approximately 10%, carbon dioxide concentration approximately 5%), soda lime and desiccants were used to deodorize and absorb CO2.

[0074] At the end of week 6, all mice were uniformly treated, including the detection of indicators related to hypoxic pulmonary hypertension, such as right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RV / (LV+S)%), HE staining of pulmonary arterioles, and quantitative analysis of pulmonary arteriole wall thickness and vascular area (specific procedures are the same as 1.6, 1.7, and 1.8); the experiment also included the determination of ACE activity in mouse lung tissue using an ACE activity assay kit (specific procedures are the same as 1.15), and the detection of ACE expression changes in mouse lung tissue using Western blotting (specific procedures are the same as 1.17). In addition, mean carotid artery pressure (mCAP) was examined in the experiment. The purpose of detecting mCAP was mainly to observe whether the drug could reduce pulmonary artery pressure without reducing systemic circulatory pressure.

[0075] 3. Experimental Results (1) Evaluation of the efficacy of sAT in hypoxia-induced pulmonary hypertension A hypoxic pulmonary hypertension model mouse was established by comparing normal controls with mice that had undergone 6 weeks of replication (i.e., hypoxia-induced pulmonary hypertension mice, or simply hypoxia mice; see detailed experimental procedures below). Figure 1 A) After comparison, the results showed that during the 6-week observation period, the hypoxic mice gradually exhibited anorexia and lethargy, with little increase in body weight. Figure 1 B), the mouse heart rate was maintained at 400 beats / minute (bpm). Hypoxic mice exhibited severe right ventricular dysfunction, manifested as a shortened pulmonary artery acceleration time (PAAT). Figure 1 C Figure 1 D), right ventricular diameter (RVID) significantly increased ( Figure 1 E, Figure 1 D), right ventricular free wall thickness (PVWT) significantly increased ( Figure 1 F, Figure 1 D), Tricuspid annular systolic displacement (TAPSE) was significantly reduced ( Figure 1 G, Figure 1 D) and right ventricular output (CO, Figure 1 H) was significantly reduced. Furthermore, the peak right ventricular systolic pressure (RVSP) was significantly increased in hypoxic mice. Figure 2 A), the index reflecting right ventricular hypertrophy (i.e., RV / (LV+S)%) also increased significantly. Figure 2 C). Histological evaluation of the lungs revealed significant alveolar edema, extensive exudation and hemorrhage in hypoxic mice, and compared with normal controls, the smooth muscle layer of pulmonary arterioles was significantly thickened and the lumen was narrowed. Figure 2 G), the indicators representing pulmonary arteriolar thickening, WT% and WA% both showed a significant increase. Figure 2 D、 Figure 2 E). Furthermore, α-SMA staining of pulmonary arterioles showed an increase in myoplastic pulmonary arterioles in hypoxic mice (E). Figure 2 H, Figure 2 F), the results of immunofluorescence double staining for OPN and Ki67 co-expression showed an increase in myomorphic pulmonary arterioles in a proliferative state (F). Figure 2 I). These results indicate that 6 weeks of hypoxia can induce significant symptoms and manifestations of hypoxic pulmonary hypertension in mice.

[0076] Administration of sAT (the total saponin content of sAT was measured to be 66.62%, the same below) improved the general condition of hypoxic mice and significantly increased their body weight. Figure 1 B). Furthermore, sAT significantly improved right ventricular function in hypoxic mice in a concentration-dependent manner: sAT increased pulmonary artery acceleration time (PAAT). Figure 1 C Figure 1 D), right ventricular diameter (RVID) decreased ( Figure 1 E, Figure 1 D), right ventricular free wall thickness (PVWT) decreased ( Figure 1 F, Figure 1D), Increased systolic displacement of the tricuspid annulus (TAPSE) ( Figure 1 G, Figure 1 D) and an increase in right ventricular output (CO) Figure 1 H), the mouse heart rate was maintained at 400-500 beats / minute (bpm). Furthermore, sAT significantly inhibited the increases in RVSP, RV / (LV+S)%, WT%, and WA% induced by hypoxia in a concentration-dependent manner. Figure 2 A, Figure 2 C Figure 2 D and Figure 2 E), lung histological evaluation also showed that administration of sAT could reverse hypoxia-induced thickening of the smooth muscle layer and luminal narrowing of the pulmonary arterioles in mice. Figure 2 G). α-SMA staining of pulmonary arterioles showed that administration of sAT significantly reduced myoplastic pulmonary arterioles in hypoxic mice. Figure 2 H, Figure 2 F), sAT also inhibited the co-expression of OPN and Ki67 in a concentration-dependent manner ( Figure 2 I). These results indicate that sAT has a significant therapeutic effect on hypoxia-induced pulmonary hypertension in a concentration-dependent manner, inhibiting hypoxia-induced pulmonary artery remodeling and improving right ventricular function. Furthermore, sAT did not cause hemodynamic or pulmonary arteriolar morphological changes in normoxic mice (i.e., normal controls), and while improving hypoxia-induced pulmonary artery remodeling, pulmonary hypertension, and right ventricular dysfunction, it did not cause a decrease in systemic blood pressure. Figure 2 B).

[0077] (2) sAT effectively inhibits the hypoxic proliferation of PASMCs. PASMCs showed significant hypoxic proliferation after 48 hours of culture at different oxygen concentrations below normoxic (21%) (10%, 5%, 3%, 1%). Figure 3 A) 5% oxygen concentration resulted in the most significant hypoxic proliferation of PASMCs, and 5% oxygen concentration was subsequently used as the optimal hypoxic stimulation condition for PASMCs. sAT inhibited hypoxia-induced excessive proliferation of PASMCs in a concentration-dependent manner, but had no effect on the proliferation of cells cultured under normoxic conditions. Figure 3 B). 60 μg / mL of sAT alleviated the excessive proliferation of PASMCs induced by hypoxia culture for 24 and 48 hours, but had no effect on cells cultured under normoxic conditions. Figure 3 D). Based on representative fluorescence images of PASMCs cultured for 48 hours under normoxic and hypoxic conditions with or without 15, 30, or 60 μg / mL sAT ( Figure 3 E), hypoxia significantly increased the number of Edu-positive PASMCs, while sAT reversed this phenomenon in a concentration-dependent manner. Figure 3C). These results indicate that sAT can effectively reverse pulmonary artery remodeling by inhibiting hypoxia-induced excessive proliferation of PASMCs.

[0078] (3) Blood component analysis of SAT Eight monomeric components of sAT were identified from the blood of mice administered sAT via gavage, all of which were saponin compounds. Figure 4-1 , Figure 4-2 and Figure 4-3 , Figure 4-4 The glycosides include Araloside A, Araloside C, Narcissiflorine, Chikusetsu saponin Iva, Ginsenoside Ro, Deglucose-chikus etsusapon IVa, Ginsengsaponin Ib, and Taibaienoside V, as detailed in Table 1.

[0079] Table 1. Identification results of SAT monomeric components entering the bloodstream

[0080] Note: In Table 1, NEG represents negative ion detection mode and POS represents positive ion detection mode.

[0081] (4) Results of network pharmacology analysis By intersecting eight sAT monomeric components entering the bloodstream with the disease HPH, 123 intersection targets were obtained. Figure 5 A: 286 active targets, 1228 disease targets, and 123 intersection targets. These 123 potential targets were uploaded to the STRING 11.0 database to construct a protein-protein interaction (PPI) network. Figure 5 B), and then the network was reconstructed using Cytoscape software ( Figure 5 C). Among them, the top 13 core nodes (ALB, AKT1, EGFR, STAT3, MMP9, CASP3, JUN, ACE, ESR1, SRC, PPARG, REN, HSP90AA1) were identified as the main targets for sAT therapy of HPH. GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on the intersection targets using the DAVID platform. Figure 5 D、 Figure 5 E, Figure 5 F displays the top 30 detailed information items in biological processes (BP), molecular functions (MF), and cellular components (CC). Figure 5 KEGG pathway analysis in G showed that the eight sAT monomeric components involved in the blood mainly included cancer signaling pathways and the renin-angiotensin system (RAS).

[0082] (5) sAT reduces the expression and content of ACE and corrects the disorder of the pulmonary RAS system. Network pharmacology studies revealed that angiotensin-converting enzyme 1 (ACE) and renin (REN) in the respiratory tract system (RAS) are the core targets of sAT in the effective treatment of HPH. Therefore, the expression of RAS-related molecules in the lungs was detected by Western blotting and ELISA experiments. The results showed that sAT reduced the expression and activity of ACE in the lungs in a concentration-dependent manner. Figure 6 D、 Figure 6 E and Figure 6 B), reduced the levels of renin (REN) and angiotensin II (Ang II). Figure 6 A and Figure 6 C) and the expression of angiotensin II type 1 receptor (AT1R) Figure 6 D、 Figure 6 F), thereby correcting the pulmonary RAS system disorder caused by HPH.

[0083] (6) The monomeric components of sAT, namely ginsenoside Iva and anemone glycoside, can specifically bind to ACE. The results of molecular docking of eight blood-entering monomeric components of sAT with the core targets of the RAS system using AutoDockTools software showed that both ginsenoside Iva and anemone glycoside in sAT could specifically bind to ACE. The docking patterns were visualized using PYMOL software. Figure 7 A, Figure 7 B. Figure 7 E and Figure 7 F), the binding energies of ginsenoside IVa and anemone glycoside to ACE were -14.17 kcal / mol and -16.61 kcal / mol, respectively. Lower binding energies indicate stronger affinity between the component and the target site (tests showed that the other six monomers could not stably bind to ACE, and the other eight monomers could bind to other molecules, but with higher binding energies and instability). Molecular dynamics simulations showed that throughout the simulation, anemone glycoside and ginsenoside Iva remained stably present at the center of the ACE binding site, with their RMSD values ​​fluctuating between 1.0 Å and 1.5 Å, respectively. Figure 7 C Figure 7 G), RMSF values ​​remained below 2 Å ( Figure 7 D、 Figure 7The results (H) indicate that the binding of anemone glycoside and ginsenoside Iva to ACE stabilizes the protein conformation of the entire complex system. These results suggest that there is a stable binding interaction between ginsenoside Iva and anemone glycoside in sAT and ACE.

[0084] (7) Evaluation of the efficacy of anemone glycoside and bamboo rhizome saponin IVa on hypoxia-induced pulmonary hypertension The above results not only revealed the efficacy and mechanism of action of sAT in treating hypoxic pulmonary hypertension, suggesting that sAT can effectively inhibit the hypoxic proliferation of PASMCs, reverse pulmonary vascular remodeling, and improve right ventricular function by inhibiting the activity and expression of ACE in the lungs and improving the disorder of the pulmonary RAS system; but also pointed out the more clearly defined monomeric components of sAT that exert its therapeutic effect (i.e., anemone glycoside and bamboo ginsenoside Iva). Therefore, further experiments (evaluating efficacy and the inhibition of ACE) verified that anemone glycoside and bamboo ginsenoside Iva can be effective components for treating hypoxia-induced pulmonary hypertension: using commercially available anemone glycoside monomer ( Figure 8 A), Bamboo rhizome saponin IVa monomer ( Figure 9 A) Treatment of hypoxic mice (mice with a 6-week-old hypoxic pulmonary hypertension model) showed that anemone significantly inhibited the increase in RVSP, RV / (LV+S)%, WA% and WT% caused by hypoxia in a concentration-dependent manner. Figure 8 B. Figure 8 E, Figure 8 F and Figure 8 G); Lung histological evaluation showed that administration of anemonesin could reverse hypoxia-induced thickening of the smooth muscle layer and luminal narrowing of the pulmonary arterioles in mice. Figure 8 D); Anemone also inhibited the content (expression) and activity of ACE in the lungs of hypoxic mice in a concentration-dependent manner. Figure 8 I and Figure 8 H). Bamboo root saponin IVa also significantly inhibited the increase in RVSP, RV / (LV+S)%, WA%, and WT% induced by hypoxia in a concentration-dependent manner. Figure 9 B. Figure 9 E, Figure 9 F and Figure 9 G), reversed the thickening of the smooth muscle layer and luminal narrowing of the pulmonary arterioles in mice caused by hypoxia. Figure 9 D), inhibiting ACE levels (expression) and activity ( Figure 9 I and Figure 9 H). Furthermore, anemone glycoside and bamboo rhizome saponin IVa did not cause hemodynamic or pulmonary arteriolar morphological changes in normoxic mice (i.e., normal controls), and while improving hypoxia-induced pulmonary arterial remodeling, pulmonary hypertension, and right ventricular dysfunction, they did not cause a decrease in systemic blood pressure. Figure 8 C Figure 9 C). These results indicate that anemone glycoside and bamboo ginsenoside Iva can effectively treat hypoxic pulmonary hypertension and improve pulmonary RAS disorder.

[0085] It should be noted that although there were still significant differences between the hypoxic intervention group and the normoxic group in terms of sAT (or anemone glycoside, bamboo rhizome saponin IVa), this does not affect the results of the demonstration of its therapeutic effect as an effective drug component. The specific reasons are as follows: The normoxic group represents a completely healthy physiological state, while the hypoxic group typically represents a severe injury / disease model with severe pathological damage and a complex disease course. It is difficult for them to fully recover to a normal healthy level within this experimental period after drug treatment, which aligns with the objective laws of disease treatment. On the other hand, drug intervention has an ameliorative effect on pathological damage: compared to the hypoxic group, the intervention group showed significant improvement in hypoxic pulmonary hypertension-related indicators, indicating that the drug can effectively reduce damage, alleviate pathological progression, and improve the physiological function and prognosis of the model animals, fully demonstrating the drug's clear therapeutic effect. In other words, the therapeutic drug does not necessarily need to achieve a "complete cure" indistinguishable from the normoxic group; a statistically significant improvement compared to the hypoxic group is sufficient to confirm the drug's therapeutic effect.

[0086] 4. Experimental Conclusions This invention experimentally demonstrates the therapeutic effect of oral sAT on hypoxic pulmonary hypertension. The efficacy is manifested in the following ways: during a 6-week observation period, it effectively improved the poor general condition and prevented weight gain in mice with hypoxia-induced pulmonary hypertension; reduced the peak right ventricular systolic pressure (RVSP) and the index reflecting right ventricular hypertrophy (RV / (LV+S)%) in mice with hypoxia-induced pulmonary hypertension; inhibited pulmonary artery remodeling; reduced the indices reflecting pulmonary arteriolar thickening (WA% and WT); improved pulmonary arteriolar luminal stenosis and wall thickening; and reduced the formation of myomorphic pulmonary arterioles. The mechanism of action of sAT may involve the inhibition of the renin-angiotensin system (RAS) and the correction of pulmonary RAS system disorders. Furthermore, the blood-entering monomeric components of sAT, namely, ginsenoside Iva and anemone glycoside, can specifically bind to ACE, inhibiting ACE activity and reducing ACE expression, and also exhibit therapeutic effects on hypoxic pulmonary hypertension.

[0087] In summary, this invention reveals the efficacy of sAT and its specific monomeric components as effective candidate drugs for treating hypoxic pulmonary hypertension. It not only provides a new strategy for the prevention and treatment of hypoxic pulmonary hypertension, but also offers ideas and strategies for the prevention and treatment of interstitial lung disease, chronic obstructive pulmonary disease, sleep apnea syndrome, chronic mountain sickness, and some neonatal respiratory diseases as well as hypoxemia.

Claims

1. Application of Aralia elata saponins in the preparation of drugs for the prevention and treatment of hypoxic pulmonary hypertension.

2. The application according to claim 1, characterized in that: The hypoxic pulmonary hypertension is caused by respiratory diseases and / or hypoxia.

3. The application according to claim 1, characterized in that: The saponins from Aralia elata are used to treat one or more of the following symptoms of hypoxic pulmonary hypertension: increased right ventricular pressure, right ventricular hypertrophy, pulmonary artery remodeling, thickening of the pulmonary arteriole wall and narrowing of the lumen, and increased muscularization of pulmonary arterioles.

4. The application according to claim 1, characterized in that: The monomeric components of the Taibai Aralia elata saponins include anemone glycoside and / or bamboo rhizome saponin IVa.

5. The application according to claim 1, characterized in that: The saponins from Aralia elata correct pulmonary RAS system disorders caused by hypoxic pulmonary hypertension.

6. Application of Aralia elata saponins in the preparation of drugs for the prevention and treatment of complications of hypoxic pulmonary hypertension.

7. Application of Aralia elata saponins in the preparation of drugs for the prevention and treatment of respiratory diseases related to hypoxemia.

8. Application of anemone glycoside or bamboo ginsenoside IVa in the preparation of drugs for the prevention and treatment of hypoxic pulmonary hypertension.

9. Application of anemone glycoside or bamboo ginsenoside IVa in the preparation of drugs for the prevention and treatment of complications of hypoxic pulmonary hypertension.

10. Application of anemone glycoside or bamboo ginsenoside IVa in the preparation of drugs for the prevention and treatment of respiratory diseases associated with hypoxemia.

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