Preparation method of anthricin nanoparticles and application of anthricin nanoparticles in preparation of medicine for treating pulmonary arterial hypertension

Abstract

The invention relates to a preparation method of anthriscus sylvestris lactone nanoparticles, which comprises the following steps: chopping dry roots of anthriscus sylvestris, extracting at room temperature to obtain an extract, suspending, and distributing by using petroleum ether, ethyl acetate and n-butyl alcohol respectively to obtain corresponding components; separating ethyl acetate components by silica gel column chromatography (CC), carrying out gradient elution, detecting and combining the components to respectively obtain subfractions, purifying the subfractions, eluting, and collecting fractions with retention time to obtain anthricin; preparing anthricin into a solution, and diluting; and dispersing, carrying out ultrasonic emulsification, stirring, centrifuging, removing unencapsulated medicines and impurities, and drying to obtain the anthricin nanoparticles. The method is novel and unique, anthriscus sylvestris lactone is effectively prepared from anthriscus sylvestris, the attenuated anthriscus sylvestris lactone nanoparticles are prepared through modification, and the attenuated anthriscus sylvestris lactone nanoparticles have an intervention effect on pulmonary arterial hypertension and can be used for preparing the medicine for treating pulmonary arterial hypertension, so that the medicinal value of anthriscus sylvestris and a new way for preparing the medicine for treating pulmonary arterial hypertension are developed, and the application prospect is wide. The medicine is an innovation of medicines for treating pulmonary arterial hypertension.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to a method for preparing nanoparticles of ginseng lactone by extracting ginseng lactone from ginseng and the use of the nanoparticles in preparing ginseng lactone nanoparticles and a method for preparing a drug for treating pulmonary hypertension. Background Art

[0002] Anthriscus sylvestris is the dried root of Anthriscus sylvestris (L.) Hoffm. of the Apiaceae family. Anthriscus lactone is a lignan monomer compound isolated from Anthriscus sylvestris. At present, the main pharmacological research of Anthriscus lactone focuses on anti-tumor, and there are relatively few studies on other pharmacological aspects. In addition, Anthriscus lactone has high toxicity and low water solubility, which greatly limits the medicinal value of Anthriscus lactone.

[0003] Pulmonary hypertension (PH) refers to a clinical and pathophysiological syndrome caused by changes in pulmonary vascular structure or function due to a variety of heterogeneous diseases and different pathogenic mechanisms, resulting in increased pulmonary vascular resistance and pulmonary artery pressure. With the continuous increase in pulmonary vascular resistance, the right heart load increases, and the patient develops right heart failure or even death. The hemodynamic diagnostic criteria for PH are: at sea level, in a resting state, the mean pulmonary artery pressure (mPAP) measured by the right cardiac catheter is >25mmHg, and the clinical manifestations are mainly dizziness, fatigue, dyspnea, chest tightness, chest pain, lower limb edema, etc. Surveys show that the incidence and mortality of pulmonary hypertension are high, and the survival time of patients with pulmonary hypertension is much lower than that of most current malignant tumor patients. Therefore, pulmonary hypertension is also known as the "cancer among cardiovascular diseases."

[0004] The pathogenesis of PH is complex and is the result of the combined action of multiple factors. Current studies believe that the malignant proliferation of pulmonary artery smooth muscle cells is the core event in the entire PH development process. Metabolic reprogramming has been widely studied in the field of tumors. Because pulmonary artery smooth muscle cells have a malignant proliferation phenotype similar to that of tumor cells, metabolic reprogramming has also been partially studied in pulmonary hypertension in recent years. It is believed that excessive enhancement of glycolysis levels exacerbates the abnormal proliferation of pulmonary artery smooth muscle cells and further worsens the disease. Overexpression of hypoxia-inducible factor (HIF-1α) has been shown to regulate cellular glycolysis levels and enhance cell proliferation levels.

[0005] Traditional Chinese medicine has complex ingredients, multiple targets, and unique effects in treating various diseases. Nanoparticles are more effective in drug modification, eliminating toxicity, and ensuring efficacy and safe use of drugs. So, can the dioscorea lactone extracted from Dioscorea odorata be modified to make dioscorea lactone nanoparticles (DPT-NPs) to reduce toxicity, study the intervention effect and potential mechanism of DPT-NPs on PH, and thus develop drugs for the treatment of pulmonary arterial hypertension, improve the medicinal value of Dioscorea odorata and dioscorea lactone, and provide a new technical approach for the development of new drugs for PH? However, there have been no public reports so far. Summary of the invention

[0006] In view of the above situation, in order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a method for preparing radix spatholoside nanoparticles and its application in the preparation of drugs for the treatment of pulmonary arterial hypertension, effectively solve the preparation of radix spatholoside nanoparticles, and realize the application in the preparation of drugs for the treatment of pulmonary arterial hypertension.

[0007] The technical solution provided by the present invention is: a method for preparing eugenolide nanoparticles, wherein the preparation method is:

[0008] (1) Preparation of dimethoate lactone (DPT):

[0009] Chop the dried root of the Chinese ginseng into small pieces, and extract at room temperature to obtain an extract;

[0010] The extract is suspended, and then partitioned with petroleum ether, ethyl acetate and n-butanol respectively to obtain a petroleum ether component, an ethyl acetate component and an n-butanol component;

[0011] Separate the ethyl acetate component by silica gel column chromatography (CC), gradient elution, identify by thin layer chromatography, combine the components with the same chromatographic spots, and obtain the target first sub-component, second sub-component, third sub-component and fourth sub-component respectively;

[0012] The fourth subcomponent of the target was purified by semi-preparative HPLC, with isocratic elution of the mobile phase at a flow rate of 3 mL / min, and the fraction with retention time tR=23.0 min was collected to obtain dioscorea lactone (DPT).

[0013] (2) Preparation of dichlorophenol nanoparticles (DPT-NPs):

[0014] The dithiocarbazone (DPT) was prepared into a solution, and then diluted with a polylactic-co-glycolic acid (PEG-PLGA) solution;

[0015] The drug was dispersed in a polyvinyl alcohol (PVA) aqueous solution, ultrasonically emulsified, stirred to volatilize dichloromethane, centrifuged to remove unencapsulated drugs and impurities, and dried to obtain dichlorophenol nanoparticles (DPT-NPs).

[0016] The dipterocarpone nanoparticles (DPT-NPs) prepared by the above method have the effects of reducing toxicity and intervening on pulmonary hypertension, and can be used in the preparation of drugs for treating pulmonary hypertension.

[0017] The method of the present invention is novel and unique, and can effectively prepare ginseng lactone from ginseng, and after modification, it is made into attenuated ginseng lactone nanoparticles, which have an intervention effect on pulmonary arterial hypertension and can be used to prepare drugs for treating pulmonary arterial hypertension. It has opened up the medicinal value of ginseng and a new way to prepare drugs for treating pulmonary arterial hypertension. It is an innovation in drugs for treating pulmonary arterial hypertension and has huge economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The composition identification diagram of the present invention, wherein: (A) is DPT 1 H NMR spectrum (500 MHz, CDCl 3 ) Figure, (B) is the DPT 13 C NMR spectrum (125 MHz, CDCl 3 ) diagram, (C) is a DPT structural formula diagram, (D) is a total ion flow diagram of a DPT standard, and (E) is an ion flow diagram of an extract of the ginseng extracted by the method of the present invention;

[0019] Figure 2 Characterization diagrams of nanoparticles of the present invention, wherein: (A) is an infrared absorption peak diagram of diosgenin (DPT) (blue), nanoparticles (NPs) (yellow), and diosgenin nanoparticles (DPT-NPs) (red), (B) is a transmission electron microscope diagram of DPT-NPs, (C) is a particle size distribution diagram of DPT-NPs, and (D) is an HPLC chromatogram of DPT and DPT-NPs;

[0020] Figure 3 The figure shows the effect of DPT-NPs on the lung injury of rats with pulmonary hypertension induced by monocrotaline, wherein: (A) shows the effect of DPT-NPs on the lung pathological injury of rats with PH induced by monocrotaline (×400), (B) shows the effect of DPT-NPs on the pulmonary artery pressure of rats with PH induced by monocrotaline ( n=6) Figure, (C) is the effect of DPT-NPs on blood gas in rats with PH induced by monocrotaline ( n=6) Figure 2. (D) Effects of DPT-NPs on the lung function of rats with PH induced by monocrotaline ( n=6)Fig.

[0021] Note: Compared with the NC group, * P<0.05, ** P<0.01; compared with group M, # P<0.05, ##P<0.01; compared with the DPT-H group, & P<0.05, && P<0.01 (the same below);

[0022] Figure 4 The figure shows the effect of DPT-NPs of the present invention on myocardial tissue damage in rats with pulmonary hypertension induced by monocrotaline, wherein: (A) shows the effect of DPT-NPs on right heart pathological damage in rats with PH induced by monocrotaline (×400), (B) shows the effect of DPT-NPs on right heart function in rats with PH induced by monocrotaline ( n=6)Fig.

[0023] Figure 5 The figure is the effect of DPT-NPs of the present invention on the oxidative stress level of rats with pulmonary hypertension induced by monocrotaline, wherein: (A) is the effect of DPT-NPs on the ROS level in the lung tissue of rats with PH induced by monocrotaline ( n=3) Figure, (B) Effects of DPT-NPs on serum SOD, MDA, and GSH-PX levels in rats with PH induced by monocrotaline ( n=6) Figure 1, (C) Effects of DPT-NPs on the expression levels of Nrf2, NQO1, HO-1, and Keap1 proteins in lung tissue of rats with PH induced by monocrotaline ( n=3)Fig.

[0024] Figure 6 The effect of DPT-NPs of the present invention on the metabolic profile of lung tissue in rats with pulmonary hypertension induced by monocrotaline, wherein: (A) is the base peak chromatogram of the NC group, M group, and DPT-NPs group; (B) is the PCA score diagram of the NC group, M group, and DPT-NPs group ((ESI + , R 2 X=0.427,Q 2 =0.187; ESI - , R 2 X=0.442,Q 2 =0.215); (C) OPLS-DA, S-plot, permutation test diagram (ESI) of NC group and M group + , R 2 X = 0.645, R 2 Y=0.997,Q 2 =0.981); (D) OPLS-DA, S-plot, permutation test diagram (ESI - , R 2 X=0.602,R 2 Y=0.997,Q 2=0.984); (E) OPLS-DA, S-plot, and permutation test diagram (ESI) of the M group and the DPT-NPs group + , R 2 X=0.536,R 2 Y=0.991,Q 2 =0.915); (F) OPLS-DA, S-plot, permutation test diagram (ESI - , R 2 X=0.649,R 2 Y=1,Q 2 =0.901), (G) is a cluster heat map analysis diagram, (H) is a metabolic pathway analysis diagram, and (I) is a key differential metabolite network analysis diagram;

[0025] Figure 7 The effect of DPT-NPs of the present invention on pulmonary vascular remodeling and HIF-1α signaling pathway in rats with pulmonary hypertension induced by monocrotaline is shown in Figure 2, wherein: (A) is the effect of DPT-NPs on CD31 and α-SMA in the lungs of rats with PH induced by monocrotaline (green represents CD31, blue represents DAPI) ( n=6) Figure, (B) shows the effect of DPT-NPs on α-SMA in the lungs of rats with PH induced by monocrotaline (red represents α-SMA, blue represents DAPI) ( n=6) Figure 2, (C) shows the effect of DPT-NPs on the levels of serum AngⅡ, ET-1 and NO in rats with PH induced by monocrotaline ( n=6) Figure 1, (D) shows the effect of DPT-NPs on the expression levels of HIF-1α and glycolysis-related proteins in lung tissue of rats with PH induced by monocrotaline ( n=3)Fig.

[0026] Figure 8 Graph showing the effect of DPT-NPs of the present invention on abnormal proliferation of RPASMC cells induced by hypoxia, wherein: (A) shows the effect of different concentrations of DPT-NPs on the viability of RPASMC cells induced by hypoxia ( n=6) Figure, (B) shows the effect of DPT-NPs on the expression levels of α-SMA and PCNA proteins in RPASMC cells induced by hypoxia ( n=3) Figure, (C) Effects of DPT-NPs on hypoxia-induced RPASMC apoptosis level ( n=3) Figure, (D) is the effect of DPT-NPs on the mRNA levels of BAX, Bcl2, and Caspase3 in RPASMC cells induced by hypoxia ( n=6) Figure, (E) Effects of DPT-NPs on p27 and CyclinD3 levels in RPASMC cells induced by hypoxia ( n=6)Fig.

[0027] Fig. 9 Graphs showing the effects of DPT-NPs of the present invention on the glycolysis level of RPASMC cells induced by hypoxia, wherein: (A) is a graph showing the effects of HIF-1α overexpression plasmid on the fluorescence intensity of HIF-1α in RPASMC cells, (B) is a graph showing the effects of HIF-1α overexpression plasmid on HIF-1α mRNA in RPASMC cells, and (C) is a graph showing the effects of DPT-NPs on the glycolysis rate of RPASMC cells induced by hypoxia ( n=3) Figure, (D) shows the effect of DPT-NPs on the ATP production rate of RPASMC cells induced by hypoxia ( n=3) Figure, (E) Effects of DPT-NPs on the expression levels of HK-Ⅱ, PKM2, and LDHA proteins in RPASMC cells induced by hypoxia ( n=3)Fig.

[0028] Fig.10 Graph showing the effect of DPT-NPs of the present invention on the glycolysis level of RPASMC cells induced by hypoxia, wherein: (F) is the effect of DPT-NPs on the fluorescence intensity of GLUT1, HK-Ⅱ, PFKM, LDHA, and PDK1 in RPASMC cells induced by hypoxia ( n=6)Fig.

[0029] Fig.11 Graph showing the effect of DPT-NPs on the oxidative stress level of RPASMC cells induced by hypoxia, wherein: (A) shows the effect of DPT-NPs on the expression levels of Nrf2, NQO1, HO-1, and Keap1 proteins in RPASMC cells induced by hypoxia ( n=6) Figure, (B) Effects of DPT-NPs on ROS levels in RPASMC cells induced by hypoxia ( n=3)Fig.

[0030] Fig.12 Figure 2 is a diagram of the SPR and CETSA verification of the binding of DPT to HIF-1α of the present invention, wherein: (A) is the binding of DPT to HIF-1α determined by CETSA (( n=3)) Figure, (B) is the affinity level of DPT and HIF-1α analyzed by SPR. DETAILED DESCRIPTION

[0031] The specific implementation modes of the present invention are described in detail below in combination with embodiments and specific situations.

[0032] Embodiment 1:

[0033] A method for preparing eugenolide nanoparticles, wherein the chemical molecular structure of eugenolide is:

[0034]

[0035] The preparation method is:

[0036] (1) Preparation of dimethoate lactone (DPT):

[0037] 48.0 kg of dried roots of E. chinensis were chopped into small pieces, soaked in a 70% volume concentration acetone aqueous solution at room temperature for 48 hours, extracted twice with a flash extractor, and concentrated under reduced pressure to obtain 15.0 kg of extract;

[0038] The extract was suspended in water, and extracted 15 times with petroleum ether, ethyl acetate and n-butanol, respectively, to obtain a petroleum ether component, an ethyl acetate component and a n-butanol component;

[0039] The ethyl acetate component (100.0 g) was separated by silica gel column chromatography (CC), and gradient elution was performed using a petroleum ether-ethyl acetate (volume ratio 100:0-0:100) system and an ethyl acetate-n-butanol (volume ratio 100:0-0:100) system. The components with the same chromatographic spots were combined by thin layer chromatography to obtain 7 first sub-components E1, E2, E3, E4...E7;

[0040] Use volume ratio of CH 2 Cl 2 -CH 3 OH = 100:1-1:1 gradient system, eluting the first subfraction E430.0g, after thin layer chromatography identification, combining the components with the same chromatographic spots to obtain 4 second subfractions E4.1...E4.4;

[0041] The second subcomponent E4.1 8.0 g was separated by Sephadex LH-20 with a mobile phase of CH 3 OH-H 2 O = 90:10, and four third subcomponents E4.1.1 ... E4.1.4 are obtained;

[0042] The third subcomponent E4.1.4 800.0 mg was separated by Toyopearl HW-40C CC, and the mobile phase was CH 3 OH-H 2 O = 80:20, after identification by thin layer chromatography, the components with the same chromatographic spots were combined to obtain 4 fourth sub-components E4.1.4.1, E4.1.4.2 ... E4.1.4.4;

[0043] The fourth subfraction E4.1.4.2 (180.5 mg) was purified by semi-preparative HPLC using a mobile phase of CH 3 OH-H 2 O=85:15, isocratic elution, flow rate 2mL / min, detection wavelengths 254nm and 210nm, collection retention time t R =23.0min fraction, 30.1 mg of dioscorea lactone (DPT) was obtained.

[0044] (2) Preparation of dichlorophenol nanoparticles (DPT-NPs):

[0045] Diluting dichloroacetic acid (DPT) with a polylactic acid-co-glycolic acid (PLGA) solution to 5 mg / ml, wherein the polylactic acid-co-glycolic acid (PLGA) solution is a polylactic acid-co-glycolic acid (PLGA) solution with a mass concentration of 50 mg / ml made by using dichloromethane as a solvent;

[0046] Then, the mixture was dispersed into a 1% polyvinyl alcohol (PVA) aqueous solution at a volume ratio of 1:5 and ultrasonically emulsified, and the emulsion was stirred for 12 hours to evaporate the dichloromethane, thereby obtaining a suspension of dichlorobutyric acid nanoparticles (DPT-NPs);

[0047] At room temperature, centrifuge at 2000g for 2 minutes to remove large particles, and then centrifuge at 6000g for 5 minutes to obtain a precipitate. The precipitate is washed twice with deionized water to remove unencapsulated drugs and impurities, and dried to obtain diopside nanoparticles (DPT-NPs). If necessary, disperse the diopside nanoparticles (DPT-NPs) in physiological saline (0.9%) and store at 4°C.

[0048] The dipterocarpone nanoparticles (DPT-NPs) prepared by the above method have the effects of reducing toxicity and intervening on pulmonary hypertension, and can be used in the preparation of drugs for treating pulmonary hypertension.

[0049] The nanoparticles given in the above examples can be used to prepare any amount of nanoparticles as needed. The given examples are only used to illustrate the specific implementation methods of the present invention, rather than to limit the protection scope of the present invention.

[0050] The preparation method of the present invention is novel and unique, easy to operate, and can be effectively used to extract ginseng lactone from ginseng and prepare ginseng lactone nanoparticles. Through the overexpression of hypoxia-inducible factor HIF-1α, it is proved that the glycolysis level of cells can be regulated, the proliferation level of cells can be enhanced, and the application in the preparation of drugs for treating pulmonary hypertension can be realized. After field tests, very good beneficial technical effects have been achieved. The relevant information is as follows:

[0051] 1. Testing and experimental methods

[0052] 1.1. Determination of DPT in total extract of Radix Glehniae by LC-MS

[0053] Preparation of test solution: Weigh appropriate amount of total extract of E. ginseng and DPT standard, add methanol and ultrasonically dissolve, centrifuge, and aspirate the supernatant into a sample vial for later use. HPLC conditions: Chromatographic column: AcclaimTM RSLC120 C 18 (2.1mm×100mm, 2.2μm); flow rate: 0.3mL / min; column temperature: 40℃; mobile phase: 0.1% formic acid aqueous solution (A), pure acetonitrile (B); elution program: gradient elution (0~2min, 5~16% B; 2~14min, 16~17% B; 14~20min, 17~30% B; 20~22min, 30~95% B; 22~28min, 95~5% B); injection volume: 2μL. Mass scanning range: m / z 50~1500; desolvation temperature: 230℃; desolvation flow rate: 8L / min; ion source energy: 3.0eV; calibration solution: sodium formate; flow rate: 50μL / h, test results are shown in Figure 1 With attached illustrations.

[0054] 1.2 Determination of drug loading and encapsulation efficiency of DPT-NPs

[0055] The drug loading and encapsulation efficiency were determined by high performance liquid chromatography. Appropriate amounts of DPT reference substance, DPT and DPT-NPs were weighed, dissolved in methanol, ultrasonically demulsified, filtered at 0.22 μm, and loaded onto the machine. High performance liquid chromatography conditions: C 18 Column; 290nm UV detector; mobile phase: acetonitrile: water = 53:47; column temperature 30°C; flow rate 1mL / min.

[0056] 1.3. DPT-NPs morphology and infrared characterization

[0057] The average particle size and particle size distribution of DPT-NPs were measured using a laser particle size analyzer. Each sample was measured three times in parallel. The morphology of DPT-NPs was observed and photographed using a transmission electron microscope. The construction of nanoparticles was determined using a Fourier transform infrared spectrometer.

[0058] 1.4. Evaluation of the detoxification effect of DPT-NPs

[0059] 48 SD rats, weighing 170g-200g, half male and half female, were divided into 8 groups. The experimental studies on the toxicity of euphorbia lactone were consulted to infer the dosage range of DPT that may cause toxic reactions. The dosage was determined as follows: four dosage groups of DPT (16mg / kg, 20mg / kg, 25mg / kg, 30mg / kg), four dosage groups of DPT-NPs (16mg / kg, 20mg / kg, 25mg / kg, 30mg / kg), single tail vein injection, and the death of animals within 24 hours was observed. According to the number of deaths in each group, the LD50 of DPT and DPT-NPs was calculated using SPSS software to evaluate the toxicity reduction effect and safety of DPT-NPs.

[0060] 1.5. Animal model establishment, grouping and drug administration

[0061] After 1 week of adaptive feeding, male SD rats were randomly divided into normal group (NC), model group (M), positive drug sildenafil group (Y), total extract of Eleutherodendron chinense group (ES), low-dose DPT group (DPT-L), high-dose DPT group (DPT-H), and DPT nanoparticle group (DPT-NPs). Monocrotaline (60 mg / kg) was injected intraperitoneally, and the rat model of pulmonary hypertension was formed on 28 days. The treatment was given on 15-28 days. The dosage of each group was: Y group (10 mg / kg, injected every other day), ES group (3 g / kg, gavage), DPT-L group (0.5 mg / kg, injected into the tail vein every other day), DPT-H group (1 mg / kg, injected into the tail vein every other day), and DPT-NPs group (1 mg / kg, injected into the tail vein every other day) (the LD50 determined by the acute toxicity test was used as a reference, and 1 / 40 and 1 / 20 of it were used as the low and high doses of the pharmacodynamic experiment). The NC group and the M group were injected with equal volumes of distilled water.

[0062] 1.6. Testing of pulmonary artery pressure and right heart function

[0063] Small animal ultrasound diagnostic instrument was used to detect pulmonary artery pressure and right heart function indexes of rats. Rats were anesthetized with isoflurane, and a high-frequency matrix probe with a frequency of 250HZ was selected to find the parasternal right heart long axis section and pulmonary artery section. After adjusting the B-ultrasound image to a suitable interface, M-ultrasound was used to measure the values ​​on the middle section of the echocardiogram, and the statistics were collected and analyzed to obtain PAT / PET, PEP / PET, right heart free wall thickness, and right heart inner diameter.

[0064] 1.7. HE and Masson staining

[0065] After modeling and drug administration, the rat heart and lung tissues were dissected, fixed with 4% formaldehyde, embedded in paraffin, sliced, and stained with HE and Masson, and the lung pathological damage was observed under a microscope.

[0066] 1.8. Western Blot

[0067] Rat lung tissue was taken, and the total protein was extracted by adding RIPA lysis buffer containing PMSF according to the steps in the protein extraction kit instructions; the protein concentration was determined by the BCA method, and the loading amount was determined according to the protein concentration; the protein was separated by polyacrylamide gel electrophoresis, and the protein was transferred to a PVDF membrane; after blocking with 5% BSA for 1.5 hours, the primary antibody was incubated at 4°C overnight, and washed 5 times with 0.2% PBST, each time for 5 minutes; the secondary antibody was incubated for 1 hour in the dark, and washed 3 times with 0.2% PBST, and washed once with PBS, each time for 5 minutes. β-actin was used as an internal reference, and the target protein was semi-quantitatively analyzed using the Odyssey dual-color imaging system combined with Image Studio software.

[0068] 1.9. Blood gas test in rats

[0069] After the rats were anesthetized, blood was collected from the abdominal aorta, and 72uL of blood was drawn using a blood gas analyzer to measure the rats' arterial oxygen partial pressure (PaO 2 ), oxygen saturation (SaO 2 ) and arterial carbon dioxide partial pressure (PaCO 2 ).

[0070] 1.10. Pulmonary function test in rats

[0071] The pulmonary function test method (known technology, see Journal of Ethnopharmacology 2023, 313, 116503) uses a small animal pulmonary function meter WBP system to record the changes in pulmonary function indicators of rats within 5 minutes to obtain the rats' tidal volume (TVb), minute ventilation (MVb), and respiratory rate (F).

[0072] 1.11. Detection of ROS levels in rat lung tissue

[0073] After modeling and drug administration, fresh lung tissue of rats was taken, PBS buffer with a pH value of 7.4 was added, the tissue was chopped, centrifuged, and the supernatant was discarded. EDTA-free trypsin was added for digestion for 5 minutes, centrifuged, and the supernatant was discarded. Red blood cell lysis buffer was added for 5 minutes, centrifuged, and the supernatant was discarded. Cells were filtered with a 0.75μm filter. The DCFH-DA fluorescent probe was used to detect the level of ROS in tissue cells.

[0074] 1.12 Immunofluorescence

[0075] Fresh lung tissue specimens were fixed in 4% formaldehyde tissue fixative, and then paraffin-embedded and dewaxed. After antigen retrieval, the specimens were blocked with BSA blocking solution for 30 minutes, and after combining primary and secondary antibodies, the cell nuclei were counterstained with DAPI and the sections were sealed. Protein expression levels were quantitatively analyzed using Image Pro Plus 6.0 software.

[0076] 1.13 Metabolomics analysis

[0077] Weigh 50 mg of lung tissue, add 30 times the volume of ice methanol, homogenize, centrifuge, aspirate the supernatant, store at -20℃ overnight, add 10 times the volume of methanol, vortex, centrifuge, aspirate the supernatant into a sample vial for later use. Chromatographic column: AcclaimTM RSLC120C 18 (2.1mm×100mm, 2.2μm); flow rate: 0.3mL / min; column temperature: 40℃; mobile phase: 0.1% formic acid aqueous solution (A), pure acetonitrile (B); elution program: gradient elution (0~6min, 10~70% B; 6~14min, 70~80% B; 14~16min, 80~90% B); injection volume: 2μL. Mass scanning range: m / z 50~1500; desolvation temperature: 230℃; desolvation flow rate: 8L / min; capillary voltage: 3.5kV (ESI + ), 3.2kV(ESI - ); ion source energy: 3.0 eV; calibration fluid: sodium formate; flow rate: 50 μL / h. The collected raw data were filtered, normalized, and peak extracted using the mass spectrometry software ProfileAnalysis, and then imported into SIMCA-P software for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Combined with the mass spectrometry software DataAnalysis, as well as databases such as HMDB and KEGG, potential biomarkers were preliminarily screened and identified, and QuantiAnalysis and MEV were used to perform semi-quantitative analysis and cluster analysis of differential metabolites. Finally, the metabolic network diagram was constructed using online tools such as the MetaboAnalyst database and MBRole.

[0078] 1.14. HIF-1α overexpression plasmid transfection

[0079] RPASMC cells were cultured at 1×10 6 The cells were inoculated into 96-well plates at a density of 10 cells / well and grouped into CON group, CON-Vector group, and pcDNA-HIF-1α group. The operation steps were strictly carried out according to the operating instructions. After 48 hours of transfection, the fluorescence intensity of HIF-1α cells was detected using a high-content imaging system.

[0080] 1.15 RT-PCR

[0081] RPASMC cells in the logarithmic growth phase were cultured at 1×10 6The density of cells / well was inoculated into a 6-well plate. After transfection, drugs were added and hypoxia was maintained for 72 hours. RNA was extracted according to the instructions of the total RNA extraction kit, and the RNA concentration and purity were determined using an ultra-micro spectrophotometer. Reverse transcription was performed using a thermal cycler, and the reaction conditions were: 25°C, 5min; 42°C, 60min; 80°C, 10min; maintained at 4°C to obtain cDNA. Amplification was performed using a fluorescent quantitative PCR instrument, and the reaction conditions were: 95°C, 10s; 95°C, 5s; 60°C, 10s; a total of 40 cycles. Analyze the amplification curve and calculate the Ct value; use GAPDH as the internal reference gene and use 2 -△△Ct The relative expression of the target gene was calculated by the PCR method. The primers were synthesized by Suzhou Genewise Biotechnology Co., Ltd. The primer sequences are shown in Table 1.

[0082] Table 1 PCR primer sequences

[0083]

[0084] 1.16. Detection of RPASMC apoptosis and ROS levels

[0085] RPASMC cells in the logarithmic growth phase were cultured at 1×10 6 After transfection, the cells were inoculated into 6-well plates at a density of 10 cells / well. After transfection, the drugs were added and hypoxia was continued for 72 hours. The cells were digested and collected with EDTA-free trypsin, and the cells were stained according to the instructions of the cell apoptosis and ROS detection kits. Cell apoptosis and ROS levels were detected and analyzed by flow cytometry.

[0086] 1.17. Real-time ATP generation rate and glycolysis rate detection of RPASMC cells

[0087] RPASMC cells in the logarithmic growth phase were cultured at 1×10 8 The density of cells / well was inoculated into Seahorse's dedicated 24-well plate. After transfection, drugs were added and hypoxia was maintained for 72 hours. The culture medium was replaced with the detection solution (basal culture medium containing 1mM pyruvate, 2mM glutamine and 10mM glucose). At the same time, the probe plate was hydrated 24 hours in advance. The corresponding reagents (glycolysis rate: rotenone / antimycin A, 2-DG, rotenone / antimycin A; ATP generation rate: oligomycin, rotenone / antimycin A) were added to the corresponding wells of the experimental group. The probe plate was calibrated on the machine. After calibration, the cell culture plate was tested on the machine. SeahorseWave software was used to analyze cell glycolysis and ATP generation parameters.

[0088] 1.18 High-content imaging analysis

[0089] RPASMC cells in the logarithmic growth phase were cultured at 1×10 4Cells were inoculated into 96-well plates at a density of 10 cells / well. After adding drugs and hypoxia for 72 hours, the culture medium was discarded, 150 μL of 4% paraformaldehyde solution was added to each well and fixed at room temperature for 20 minutes, and then 150 μL of 0.1% Triton solution was added for permeabilization for 20 minutes. Blocked with 1% BSA solution for 30 minutes, primary antibody was added, and incubated overnight at 4°C. Washed 3 times with 0.1% PBST solution, added secondary antibody and incubated at room temperature in the dark for 1 hour and washed 3 times, stained with DAPI solution at room temperature in the dark for 5 minutes and washed with 0.1% PBST solution. Scanning, photography and analysis were performed using the Opera PhenixTM high-content imaging system.

[0090] 1.19. Cellular Thermal Shift Assay (CETSA)

[0091] CETSA was used to explore the binding effect of DPT and HIF-1α protein. As described by previous methods, PRASMC cell lysates were collected from the DPT group (DPT) and the normal control group (DMSO), then heated at 30-70°C for 5 min, centrifuged at 12000g for 5 min, the precipitated protein was removed, and the supernatant protein concentration was detected by BCA kit. Protein immunoblotting was used to detect the expression level of HIF-1α protein.

[0092] 1.20. Surface Plasmon Resonance Analysis (SPR)

[0093] The HIF-1α protein was prepared into a 1 mg / ml solution with PBS, and then the protein solution was diluted to 50 μg / ml with sodium acetate buffer of different pH. After the sodium acetate buffer solution of appropriate pH was determined by pre-enrichment mode, the HIF-1α protein was coupled to the CM5 chip, and then the binding of DPT with different gradient concentrations in the running buffer was tested with HIF-1α. The data were fitted using BircoreT200 Evalution software to calculate K D .

[0094] 2. Test and Experimental Results

[0095] The test and experimental results were statistically processed, and the differences among the groups were compared using one-way analysis of variance using SPSS 22.0 software. The difference was statistically significant when P<0.05, and extremely significant when P<0.01.

[0096] 2.1. Component Identification

[0097] The structure of the extract of the ginseng extracted by the method of the present invention is identified as ginseng lactone DPT ( Figure 1AC). LC-MS analysis showed that the DPT standard responded at a retention time of 25.5 min, and its quasi-molecular ion peak was m / z 399.1438 [M+H] ( Figure 1 D), the corresponding quasi-molecular ion peak response at the corresponding retention time in the extract of the ginseng extracted by the method of the present invention ( Figure 1 E), which further indicates that the total extract of Radix Glehniae contains DPT.

[0098] 2.2. Characterization of DPT-NPs and determination of drug loading and encapsulation efficiency

[0099] The infrared spectrum results show that in the DPT spectrum, 2937cm -1 、2896cm -1 、2845cm -1 Corresponding to CH stretching vibration, 1764cm -1 Corresponding to C=O stretching vibration, 1587cm -1 、1504cm -1 、1482cm -1 Corresponding to C=C stretching vibration, 1421cm -1 、1330cm -1 Corresponding to CH bending vibration, 1223cm -1 Corresponding to CO stretching vibration, 1156cm -1 、1119cm -1 Corresponds to COC stretching vibration. In empty carrier nanoparticles (NPs), 3235 cm -1 Corresponding to OH stretching vibration, 2944 cm -1 Corresponding to CH stretching vibration, 1751cm -1 Corresponding to C=O stretching vibration, 1449cm -1 、1423cm -1 Corresponding to CH bending vibration, 1273 cm -1 Corresponding to CO stretching vibration, 1180cm -1 、1086cm -1 Corresponding to COC stretching vibration, 846cm -1 、693cm -1 Corresponding to CH bending vibration. In DPT-NPs, 3320cm -1 Corresponding to OH stretching vibration, 2945 cm -1 Corresponding to CH stretching vibration, 1751cm -1 Corresponding to C=O stretching vibration, 1449 cm -1 、1423cm -1 Corresponding to CH bending vibration, 1271cm -1 Corresponding to CO stretching vibration, 1178cm -1、1087cm -1 Corresponding to COC stretching vibration, 849cm -1 、704cm -1 These absorption peaks correspond to the CH bending vibrations, and they correspond to the nanoparticle carrier material PLGA; 1587 cm -1 、1504cm -1 、1482cm -1 Corresponding to C=C stretching vibration, 1421cm -1 、1330cm -1 Corresponding to CH bending vibration, 1223cm -1 Corresponding to CO stretching vibration, 1156cm -1 、1119cm -1 Corresponding to COC stretching vibration, these absorption peaks correspond to DPT. DPT-NPs have absorption peaks of both empty carrier nanoparticles and DPT, and the peak at 1754 cm in the DPT-NPs spectrum -1 The peak position shifted, suggesting that the C=O in DPT formed a hydrogen bond with the -OH in the PLGA carrier material, indicating that the two were combined. This indicates that DPT-NPs nanoparticles were successfully constructed. Figure 2 (A).

[0100] The particle size of DPT-NPs was measured by zetasize to be 152.8nm±5.77nm, and the particle size was normally distributed. Transmission electron microscopy results showed that the nanoparticles were complete spherical particles with a relatively uniform particle size distribution. Figure 2 (B)(C). The experiment used the standard substance of ophiolide as a reference substance to calculate the drug loading capacity and encapsulation efficiency of DPT-NPs. The experimental results showed that the corresponding chromatographic peak of the ophiolide standard substance appeared at around 8 minutes, and the chromatographic peak of ophiolide also appeared in the chromatogram of DPT-NPs, and the retention time was consistent, indicating that the method has good specificity. Linear regression was performed with concentration (ug / ml) as the horizontal axis and peak area as the vertical axis to construct the linear regression equation of ophiolide (y=0.00000005375804163x+0.00259923170 (r=0.9992). The drug loading capacity of DPT-NPs measured by this method was 23.74%±0.072%, and the encapsulation efficiency was 63.88%±0.19%. The results are shown in Table 2. Figure 2 (D).

[0101] 2.3. Evaluation of the attenuation effect of DPT-NPs

[0102] In order to verify the safety and toxicity reduction effect of DPT-NPs, an acute toxicity test was conducted. 48 SD rats, half male and half female, were divided into 8 groups, 4 dose groups of DPT and DPT-NPs, and the death of rats within 48 hours was observed after a single tail vein injection of the drug. The number of dead animals in each group is shown in Table 2 and Table 3. SPSS statistical software was used to calculate the LD50 of DPT: 18.822 mg / kg, and its 95% confidence interval: 12.630 mg / kg-22.385 mg / kg; the LD50 of DPT-NPs: 21.536 mg / kg, and its 95% confidence interval: 18.184 mg / kg-24.837 mg / kg. It suggests that DPT-NPs can increase drug safety and reduce toxicity to a certain extent.

[0103] Table 2 Statistics of rat deaths in DPT acute toxicity test

[0104]

[0105] Table 3 Statistics of rat deaths in DPT-NPs acute toxicity test

[0106]

[0107] 2.4 Effects of DPT-NPs on lung injury in rats with pulmonary hypertension induced by monocrotaline

[0108] HE sections of lung tissue showed that the walls of pulmonary vessels in the M group were significantly thicker than those in the NC group, with narrowed lumina and infiltration of inflammatory cells; after drug administration, the thickening of the pulmonary vessel walls was alleviated to varying degrees; Masson staining of lung tissue showed that collagen fibers gathered around the pulmonary vessels in the M group; after drug administration, collagen fiber deposition decreased. This suggests that DPT-NPs can improve the pathological damage of lung tissue in PH rats to varying degrees. The results are shown in Figure 3 (A). Compared with the NC group, the PAT / PET ( Figure 3 (B)), tidal volume, lung ventilation level ( Figure 3 (C) ), PO in arterial blood 2 , SaO 2 Significantly reduced ( Figure 3 (D) (P<0.01), PEP / PAT, respiratory rate, PCO 2After administration, the above indicators were improved (P<0.01 or P<0.05), and the effect of DPT-NPs was better than that of DPT (P<0.01 or P<0.05). PAT / PET and PEP / PAT are important indicators of pulmonary artery pressure. The higher the PEP / PAT ratio and the lower the PAT / PET, the higher the pulmonary artery pressure. This shows that DPT-NPs can reduce pulmonary artery pressure in rats, improve rat lung function, regulate rat blood gas levels, and improve the hypoxic state of rat lung tissue.

[0109] 2.5 Effects of DPT-NPs on myocardial tissue damage in rats with pulmonary hypertension induced by monocrotaline

[0110] HE sections of cardiac tissue showed that compared with the NC group, the myocardial tissue in the M group was disordered, disordered, and broken, with obvious necrosis and inflammatory cell infiltration between cells; after drug administration, the myocardial tissue arrangement tended to be orderly and neat. Masson staining of cardiac tissue showed that there was obvious collagen fiber deposition in the M group; after drug administration, the collagen fiber deposition between myocardial tissues decreased. This suggests that DPT-NPs can reduce myocardial tissue pathological damage in rats with pulmonary hypertension. The results are shown in Figure 4 (A). The RVHI, RVFW, and RVID of rats in group M increased significantly (P<0.01). After administration, the RVHI, RVFW, and RVID of rats decreased significantly (P<0.05 or P<0.01), and the effect of DPT-NPs was better than that of DPT (P<0.01 or P<0.05). This indicates that the increase in pulmonary artery hypertension leads to the decrease of right heart function in rats and the occurrence of right heart hypertrophy and thickening, while DPT-NPs can improve right heart hypertrophy in rats with pulmonary hypertension and regulate the right heart function of rats. The results are shown in Figure 4 (B).

[0111] 2.6 Effects of DPT-NPs on oxidative stress in rats with pulmonary hypertension induced by monocrotaline

[0112] The results showed that compared with the NC group, the ROS level in the lung tissue of rats in the M group ( Figure 5 (A) ), serum MDA level ( Figure 5 (B)), Keap1 protein expression level ( Figure 5 (C)) was significantly increased (P<0.01), and the levels of SOD and GSH-PX in serum and the expression levels of Nrf2, NQO1, and HO-1 proteins in lung tissue were significantly decreased (P<0.01); after administration, the above indicators were improved (P<0.05 or P<0.01), and the effect of DPT-NPs was better than that of DPT (P<0.01 or P<0.05). This shows that there is excessive production of ROS and imbalance of antioxidant enzymes in PH rats, and DPT-NPs can reduce the level of oxidative stress in PH rats.

[0113] 2.7 Effects of DPT-NPs on the metabolic profile of lung tissue in rats with monocrotaline-induced pulmonary hypertension

[0114] In order to explore the effect of DPT-NPs on metabolic pathways, metabolomics study of lung tissue of PH rats was conducted. The ion currents of each group are shown in Figure 6 (A) As shown. In the PCA score diagram ( Figure 6 (B)), NC group and M group separated, and the drug-treated group moved closer to the NC group. OPLS-DA analysis ( Figure 6 (C)- Figure 6 (F)), 33 common biomarkers were screened out, as shown in Table 4, 14 of which were positive source patterns and 19 were negative source patterns. The cluster heat map analysis of these differential metabolites is shown in Figure 6 As shown in (G), the metabolic pathways that they significantly affect mainly include gluconeogenesis, glycolysis, pyruvate metabolism, Warburg effect, etc. ( Figure 6 (H)). These metabolic pathways are closely related to glucose metabolism, indicating that DPT-NPs may improve rat pulmonary hypertension by affecting glucose metabolism.

[0115] 2.8 Effects of DPT-NPs on pulmonary vascular remodeling and HIF-1α signaling pathway in rats with monocrotaline-induced pulmonary hypertension

[0116] The effects of DPT-NPs on pulmonary vascular remodeling-related indicators in PH rats were detected. The results showed that the expression levels of α-SMA, a marker of smooth muscle cells in pulmonary vessels and lung tissues, and the levels of serum AngⅡ and ET-1 in rats in group M were significantly increased (P<0.01), and the expression levels of endothelial cell markers CD31 and NO were significantly decreased (P<0.01). DPT-NPs could reduce the expression level of α-SMA in rats (P<0.01), increase the expression level of CD31 (P<0.01), and improve the level of pulmonary vasoconstriction and relaxation factors in rats (P<0.01). The effect of DPT-NPs was better than that of DPT (P<0.05). Figure 7 (A)(B)(C). The expression levels of HIF-1α and glycolysis-related proteins in rat lung tissue were detected. The results showed that the expression levels of HIF-1α, GLUT1, HK-Ⅱ, PKM2, LDHA, and PDK1 proteins in the M group were significantly increased (P<0.01); DPT-NPs could improve the expression levels of the above proteins, and the effect of DPT-NPs was better than that of DPT. Figure 7 (D) The above results indicate that DPT-NPs can improve pulmonary vascular remodeling in PH rats by reducing the level of glycolysis via HIF-1α.

[0117] 2.9 Effect of DPT-NPs on hypoxia-induced abnormal proliferation of RPASMC cells

[0118] In an in vitro experiment, a hypoxia-induced abnormal proliferation model of RPASMC cells was used to explore the effect of DPT-NPs on abnormal proliferation of RPASMC cells. The results showed that concentrations of 2.5 nM and above could significantly inhibit abnormal proliferation of RPASMC cells ( Figure 8 (A)) (P<0.01), DPT-NPs can reduce the expression levels of smooth muscle cell marker α-SMA (P<0.01) and proliferating cell nuclear antigen PCNA protein in RPASMC cells (P<0.01) ( Figure 8 (B)), reduced the apoptosis resistance of cells (P<0.01) ( Figure 8 (C) Figure 8 (D)). This indicates that DPT-NPs can significantly inhibit the abnormal proliferation of RPASMC cells. The results of detecting cell cycle-related proteins showed that the fluorescence intensity of p27 in the Hypoxia group was significantly reduced (P<0.01), and the fluorescence intensity of CyclinD3 was significantly increased (P<0.01), while DPT-NPs could improve the fluorescence intensity of the above cell cycle proteins (P<0.01). Figure 8 (E) It suggests that DPT-NPs can affect the levels of cell cycle proteins and regulate cell proliferation.

[0119] 2.10 Effect of DPT-NPs on glycolysis level of RPASMC cells induced by hypoxia

[0120] To further verify whether DPT-NPs inhibit the abnormal proliferation of pulmonary artery smooth muscle cells by regulating glycolysis levels through HIF, a HIF-1α overexpression experiment was designed. The experimental results showed that the HIF-1α overexpression plasmid could significantly increase the fluorescence intensity and mRNA expression level of HIF-1α in PASMC cells. Fig. 9 (A) Fig. 9 (B) The results of the Seahorse experiment showed that the total ATP production rate and mitochondrial ATP production rate of the Hypoxia group were significantly reduced (P<0.01) ( Fig. 9 (D)), the glycolytic ATP generation rate, basal glycolytic rate and compensatory glycolytic rate increased significantly (P<0.01) ( Fig. 9 (C)); after administration of DPT-NPs, the above indicators improved (P<0.01); however, after HIF-1α overexpression, the improvement of the above indicators weakened (P<0.01). Fig. 9 (A). The results of glycolysis-related protein detection also showed that after administration of DPT-NPs, the expression level and fluorescence intensity of cell glycolysis-related proteins were significantly reduced (P<0.01); after HIF-1α was overexpressed, the improvement effect of the above indicators was weakened (P<0.01) ( Fig. 9 (E) Fig.10 (F)). This indicates that under hypoxic conditions, the energy metabolism of RPASMC cells changes from mitochondrial respiration to glycolysis, and DPT-NPs can regulate the cellular ATP production rate and energy metabolism mode through HIF-1α, while reducing the cellular glycolysis rate.

[0121] 2.11 Effects of DPT-NPs on hypoxia-induced oxidative stress in RPASMC cells

[0122] The expression levels of Nrf2, NQO1, and HO-1 proteins in the M group were significantly decreased, while the levels of ROS and Keap1 protein were significantly increased ( Fig.11 (A)(B)) (P<0.01 or P<0.05); after administration, the above indicators were improved (P<0.05 or P<0.01), and after HIF-1α overexpression, the above effects were reversed (P<0.01 or P<0.05). This suggests that there is excessive production of ROS and imbalance of the oxidative system in RPASMCs, and DPT-NPs can reduce the level of oxidative stress in RPASMCs induced by hypoxia.

[0123] 2.12. SPR and CETSA verification of the binding of DPT to HIF-1α

[0124] The results of cell thermal shift analysis showed that DPT-NP S It can enhance the thermal stability of HIF-1α protein, which proves the direct interaction between DPT and HIF-1α protein. SPR was further used to explore the affinity level of DPT and HIF-1α. The results showed that DPT and HIF-1α were bound in a dose-dependent manner. D is 5.284×10 -6 M.

[0125] Conclusion

[0126] The toxicity of eugenolide is relatively high and its water solubility is low. Making highly toxic drugs into suitable nanoformulations can reduce drug toxicity and increase drug efficacy.

[0127] PLGA is a biodegradable high molecular polymer that can be degraded into intermediate metabolites of lactic acid and glycolic acid in the human body. Due to its good biocompatibility and safety, it is widely used as a carrier for various nanoformulations, effectively delaying the release of drugs and reducing drug toxicity.

[0128] Polyethylene glycol (PEG) is the most commonly used hydrophilic compound for nano-surface modification. PEG is introduced as a hydrophilic component onto the surface of the hydrophobic PLGA polymer to form a PEG-PLGA copolymer, which can effectively improve the water solubility of the carrier and increase the stability of the drug.

[0129] Therefore, the present invention uses PEG-PLGA copolymer as a carrier to construct the eutrophil nanoparticle co-delivery system. The infrared spectrum shows that DPT and PLGA carrier have good combination, and the final prepared DPT-NPs are uniformly dispersed, with an average particle size of 152.8nm±5.77nm and an encapsulation rate of 63.88%±0.19%, which has good dispersibility and stability.

[0130] The results of acute toxicity tests showed that DPT-NPs were safe and had a toxicity-reducing effect.

[0131] Pulmonary hypertension is a refractory disease characterized by uncontrolled abnormal proliferation of pulmonary artery smooth muscle cells and pulmonary vascular remodeling, which in turn leads to right heart damage and right heart failure. In this study, a pulmonary hypertension model was induced by intraperitoneal injection of monocrotaline. The results showed that DPT-NPs could reduce pulmonary artery pressure in rats, alleviate pulmonary vascular thickening, reduce the accumulation of inflammatory cells around pulmonary vessels, improve rat lung function, and reduce rat lung tissue hypoxia and oxidative stress levels; at the same time, DPT-NPs could reduce myocardial damage in rats, reduce myocardial collagen fiber deposition, reduce right heart free wall thickness, right heart inner diameter and right heart hypertrophy index, effectively improve right heart remodeling, and the effect of DPT-NPs was better than DPT.

[0132] In order to further explore the mechanism of action of DPT-NPs in the intervention of pulmonary hypertension, the experiment used metabolomics technology to study the metabolism of lung tissue in rats with pulmonary hypertension and enrich the metabolic pathways significantly intervened by DPT-NPs. The results showed that the metabolic pathways significantly affected by DPT-NPs mainly included gluconeogenesis, glycolysis, pyruvate metabolism, Warburg effect, etc. These metabolic pathways are closely related to sugar metabolism. Therefore, DPT-NPs can affect sugar metabolism to improve pulmonary hypertension in rats.

[0133] Studies have shown that HIF-1α is a cellular transcription factor induced by hypoxia, which can participate in regulating cellular energy metabolism, cell proliferation and apoptosis. In hypoxia-induced pulmonary artery smooth muscle cells, excessive increase in reactive oxygen species drives the activation of HIF-1α. Activated HIF-1α affects multiple aspects of cell metabolism, oxidative stress levels, mitochondrial function and cell cycle. In malignantly proliferating cells, activated HIF-1α directly affects the expression levels of related proteins in glycolysis, such as GLUT1, HK-Ⅱ, PDK1, etc., enhances the level of cellular glycolysis, and aggravates abnormal cell proliferation. Combined with the previous metabolomics results, can DPT-NPs improve pulmonary vascular remodeling by regulating HIF-1α to affect glycolysis levels?

[0134] In order to meet the energy and material needs of rapid proliferation, cells reprogram their metabolic pathways, which are mainly manifested in abnormal glucose metabolism, amino acid metabolism and lipid metabolism, thereby promoting cell growth. This mechanism has been widely studied in tumor cells. Studies have shown that in the development of PH, pulmonary artery smooth muscle cells have a tumor-like malignant phenotype that resists apoptosis. The abnormal enhancement of glycolysis in pulmonary artery smooth muscle cells is considered to play a key role in the development of pulmonary hypertension. The enhancement of glycolysis aggravates the proliferation of pulmonary artery smooth muscle cells. In the cytoplasm, the process of converting glucose into pyruvate is called glycolysis. Under normal aerobic conditions, pyruvate enters the mitochondria and is completely oxidized to produce carbon dioxide and water, which is the aerobic oxidation of sugar. Studies have shown that the enhancement of glycolysis in pulmonary artery smooth muscle cells can quickly provide energy for the malignant proliferation of cells. In addition, its intermediates can provide raw materials for intracellular metabolic pathways. Current studies have shown that the key metabolic enzymes that lead to enhanced glycolysis in pulmonary artery smooth muscle cells are glucose transporter (GLUT1), hexokinase (HK-Ⅱ), pyruvate kinase 2 (PKM2), pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase (LDHA), etc. GLUT1 is a glucose transporter responsible for transporting glucose into cells for use. The upregulation of GLUT1 levels occurs in most malignant proliferating cells. HK is the first step in initiating the glycolysis reaction, responsible for catalyzing the phosphorylation of glucose into 6-phosphate glucose (G6P). HK has four isozymes. HK-Ⅱ is a key enzyme that regulates cell energy metabolism and cell proliferation. Studies have shown that under pathological conditions, HK-Ⅱ can not only increase the level of glycolysis and promote cell proliferation, but also inhibit the release of mitochondrial cytochrome C, thereby inhibiting cell apoptosis. PKM is a key enzyme that generates pyruvate in the last step of glycolysis. Most normal cells express PKM1, but under pathological conditions, PKM2 is highly expressed. Currently, various studies have shown that increased activity of PKM2 can increase the rate of glycolysis and promote cell proliferation through the pentose phosphate pathway. PDK1 plays a key role in the energy metabolism of sugars and is a key factor in connecting glycolysis and the tricarboxylic acid cycle. It shuts down mitochondrial metabolism by inhibiting pyruvate dehydrogenase (PDH). Studies have shown that under pathological conditions of pulmonary hypertension, PDK1 activity is significantly increased, resulting in reduced aerobic oxidation of sugars.

[0135] The results of in vivo experiments showed that DPT-NPs could reduce the expression levels of HIF-1α and glycolysis-related proteins in the lung tissue of rats with pulmonary hypertension, and at the same time regulate the expression levels of α-SMA, a marker of smooth muscle cells in rat pulmonary vessels and lung tissue, increase the expression level of endothelial cell marker CD31, regulate the level of vasoconstriction and relaxation factors in rats, and improve pulmonary vascular remodeling in rats. In order to further verify whether DPT-NPs inhibit the abnormal proliferation of pulmonary artery smooth muscle cells by regulating the glycolysis level through HIF-1α, in vitro experiments were conducted. The results showed that DPT-NPs could regulate the expression of RPASMC cell cycle proteins and oxidative stress levels, and inhibit the abnormal proliferation of RPASMC cells; at the same time, DPT-NPs could reduce the cell glycolysis ATP production rate and glycolysis rate, increase the mitochondrial ATP production rate, and regulate the expression level of glycolysis-related proteins, while this effect disappeared after overexpression of HIF-1α.

[0136] In summary, a safe, attenuated and effective nanoparticle drug delivery system was prepared in the experiment, and DPT-NPs reduced the glycolysis level through HIF-1α and inhibited the abnormal proliferation of PASMC cells, thus improving the condition of rats with pulmonary hypertension.

Claims

1. A method for preparing scutellariae lactone nanoparticles, characterized in that: The chemical molecular structure of the euphorbia lactone is:

2. The preparation method is: (1) Preparation of dimethoate lactone (DPT): 48.0 kg of dried roots of E. chinensis were chopped into small pieces, soaked in a 70% volume concentration acetone aqueous solution at room temperature for 48 h, extracted twice with a flash extractor, and concentrated under reduced pressure to obtain 15.0 kg of extract; The extract was suspended in water, and extracted 15 times with petroleum ether, ethyl acetate and n-butanol, respectively, to obtain a petroleum ether component, an ethyl acetate component and a n-butanol component; The ethyl acetate component was separated by silica gel column chromatography (CC), and gradient elution was performed using a petroleum ether-ethyl acetate (volume ratio 100:0−0:100) system and an ethyl acetate-n-butanol (volume ratio 100:0−0:100) system. The components with the same chromatographic spots were combined by thin layer chromatography to obtain seven first sub-components E1, E2, E3, E4…E7; The first subfraction E4 was eluted using a gradient system of CH2Cl2-CH3OH with a volume ratio of 100:1-1:

1. After identification by thin layer chromatography, the components with the same chromatographic spots were combined to obtain four second subfractions E4.1…E4.

4. The second subfraction E4.1 was separated by Sephadex LH-20 with the mobile phase being CH3OH-H2O in a volume ratio of 90:10 to obtain four third subfractions E4.1.1…E4.1.4; The third subcomponent E4.1.4 was separated by Toyopearl HW-40C CC, the mobile phase was CH3OH-H2O with a volume ratio of 80:20, and the components with the same chromatographic spots were combined by thin layer chromatography to obtain four fourth subcomponents E4.1.4.1, E4.1.4.2...E4.1.4.4; The fourth subcomponent E4.1.4.2 was purified by semi-preparative HPLC with a mobile phase of CH3OH-H2O in a volume ratio of 85:15, isocratic elution, a flow rate of 2 mL / min, detection wavelengths of 254 nm and 210 nm, and retention time t R = 23.0 min fraction, 30.1 mg of dimethoate (DPT) was obtained; (2) Preparation of dichlorophenol nanoparticles (DPT-NPs): Diluting dichloroethane (DPT) with a polylactic acid-co-glycolic acid (PLGA) solution to 5 mg / ml, wherein the polylactic acid-co-glycolic acid (PLGA) solution is a polylactic acid-co-glycolic acid (PLGA) solution with a mass concentration of 50 mg / ml made by using dichloromethane as a solvent; Then, it was dispersed into a 1% polyvinyl alcohol (PVA) aqueous solution at a volume ratio of 1:5 and ultrasonically emulsified. The emulsion was stirred for 12 h to evaporate the dichloromethane, and a suspension of dichlorophenol nanoparticles (DPT-NPs) was obtained. At room temperature, centrifuge at 2000 g for 2 min to remove large particles, and then centrifuge at 6000 g for 5 min to obtain a precipitate. The precipitate was washed twice with deionized water to remove unencapsulated drugs and impurities, and dried to obtain diopside nanoparticles (DPT-NPs).

3. The dioscorea lactone nanoparticles (DPT-NPs) prepared according to the method of claim 1, characterized in that: The particle size of the dioscorea lactone nanoparticles (DPT-NPs) is 152.8 nm ± 5.77 nm, the drug loading is 23.74% ± 0.072%, and the encapsulation efficiency is 63.88% ± 0.19%.

4. Use of the eucomaclide nanoparticles according to any one of claims 1 or 2 in the preparation of a drug for treating pulmonary arterial hypertension.

5. Use of the euphorbia lactone nanoparticles according to any one of claims 1 or 2 in the preparation of a drug for treating toxicity reduction and intervention on pulmonary hypertension.

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