Application of new psoralen isoflavone separated from anthriscus sylvestris in preparation of medicine for treating pulmonary arterial hypertension

Abstract

The invention relates to new psoralen isoflavone separated from anthriscus sylvestris, and aims to restore endothelial cell functions and improve pulmonary arterial hypertension by regulating a peroxisome proliferator-activated receptor gamma, so that the application of the new psoralen isoflavone separated from the anthriscus sylvestris in preparation of drugs for treating pulmonary arterial hypertension is realized. The problem of developing the medicine for treating the pulmonary arterial hypertension is effectively solved, the application in preparing the medicine for treating the pulmonary arterial hypertension is realized, the new fructus psoraleae isoflavone is separated from anthriscus sylvestris, the raw materials are rich and easy to obtain, the intervention effect and the potential mechanism of the new fructus psoraleae isoflavone on the pulmonary arterial hypertension are clarified, and the new fructus psoraleae isoflavone can be used for preparing the medicine for treating the pulmonary arterial hypertension. Therefore, the new application of the new psoralen isoflavone extracted from the anthriscus sylvestris and a new medicine approach for treating the pulmonary arterial hypertension are developed, and the new psoralen isoflavone has remarkable economic and social benefits.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to the application of new psoralea corylifolia isoflavones separated from ginseng in the preparation of a medicine for treating pulmonary hypertension. Background Art

[0002] Pulmonary Hypertension (PH) is a serious pulmonary vascular disease characterized by a gradual increase in pulmonary vascular resistance and a progressive increase in pulmonary artery pressure. As pulmonary vascular resistance continues to increase, the right heart load increases, and patients experience right heart failure or even death. Although certain progress has been made in the diagnosis and treatment of PH in recent years, the disease develops rapidly, has a high mortality rate and a high disability rate, and has placed a huge burden on the social medical system. The pathogenesis of PH is complex. Genetics, drugs, toxins, left heart disease, lung disease, chronic thromboembolic disease or pulmonary artery obstructive lesions can all lead to the occurrence of PH. So far, there is no specific drug for the treatment of PH, and it is difficult to cure.

[0003] Modern pharmacological studies have shown that the main pathological changes of pulmonary hypertension include pulmonary vasoconstriction, pulmonary vascular remodeling, and in situ thrombosis. The pathogenesis of pulmonary hypertension is complex and is the result of the combined action of multiple factors. Pulmonary vascular endothelial cells are an important component of the pulmonary vascular intima and play an important role in maintaining vascular homeostasis. Under the pathological state of pulmonary hypertension, organic and functional lesions occur in pulmonary arterioles, the function of the pulmonary vascular intima is destroyed, the function of endothelial cells is impaired, the secretion of multiple vasoactive substances is unbalanced, inflammatory infiltration mediates excessive proliferation of smooth muscle cells in the middle layer of the pulmonary artery. Studies have shown that endothelial cell dysfunction is considered to be the initiating factor of pulmonary hypertension (PH). Peroxisome proliferator-activated receptor γ (PPAR-γ) is a member of the nuclear hormone receptor superfamily and plays an important role in regulating immune inflammatory responses, pulmonary vascular remodeling, and endothelial cell function.

[0004] Traditional Chinese medicine has complex ingredients, multiple targets, and unique therapeutic effects in treating diseases. Anthriscus sylvestris is the dried root of Anthriscus sylvestris (L.) Hoffm., a plant of the Apiaceae family. It has great medicinal value. The flavonoid component NBIF has been isolated from Anthriscus sylvestris. Studies have shown that flavonoids have anti-inflammatory, antioxidant, and protective effects on endothelial cell damage, but there is a lack of pharmacological studies on NBIF. So, can we clarify the intervention effect and potential mechanism of NBIF on PH through pharmacological studies, improve the medicinal value of Anthriscus sylvestris, and thus realize the application of NBIF isolated from Anthriscus sylvestris in the preparation of drugs for the treatment of pulmonary arterial hypertension and develop new drugs for the treatment of pulmonary arterial hypertension (PH), but there has been no public report so far. Summary of the invention

[0005] 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 new psoralen isoflavone isolated from the herb of Cuscuta chinensis, and through pharmacological research, regulate the peroxisome proliferator-activated receptor γ (PPAR-γ) to restore endothelial cell function and improve pulmonary hypertension, so as to realize the application of the new psoralen isoflavone isolated from the herb of Cuscuta chinensis in the preparation of drugs for the treatment of pulmonary hypertension, and effectively solve the problem of developing new drugs for the treatment of pulmonary hypertension (PH).

[0006] The technical solution solved by the present invention is: an application of new psoralen isoflavones isolated from ginseng in the preparation of drugs for treating pulmonary hypertension, which restores endothelial cell function by regulating peroxisome proliferator-activated receptor γ (PPAR-γ) and improves pulmonary hypertension, thereby realizing the application in the preparation of drugs for treating pulmonary hypertension. The new psoralen isoflavones are isolated from ginseng, and the raw materials are abundant and easy to obtain. The intervention effect and potential mechanism of new psoralen isoflavones (NBIF) on pulmonary hypertension (PH) are clarified, thereby developing new drug uses of new psoralen isoflavones extracted from ginseng and new drug approaches for treating pulmonary hypertension (PH), which have significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The chemical molecular structure diagram of the new psoralea corylifolia isoflavone of the present invention;

[0008] Figure 2 chromatograms of the present invention, wherein (B) is a chromatogram of NBIF standard, and (A) is a chromatogram of the total extract of Radix Glehniae;

[0009] Figure 3 The figures are the effects of NBIF of the present invention on lung tissue damage in rats with pulmonary hypertension, wherein: (A) is the effect of NBIF on lung pathological damage in rats with pulmonary hypertension; (B) is the pulmonary artery blood flow spectrum of rats with pulmonary hypertension and the effect of NBIF on PEP / PAT and PAT / PET in rats with pulmonary hypertension ( n=6); (C), (D), and (E) are the effects of NBIF on TVB, MVB, and F in rats with pulmonary hypertension ( n=6); (F), (G), (H) are the effects of NBIF on PaO2, PaCO2 and SaO2 in rats with pulmonary hypertension ( n=6);

[0010] Figure 4The figures are the effects of NBIF of the present invention on myocardial tissue damage in rats with pulmonary hypertension, wherein: (A) is the effects of NBIF on myocardial tissue pathological damage in rats with pulmonary hypertension; (B) is the echocardiogram of rats with pulmonary hypertension and the effects of NBIF on RVHI, RVFW and RVID of rats with pulmonary hypertension ( n=6); (C) Effects of NBIF on ROS levels in myocardial tissue of rats with pulmonary hypertension ( n=3)Fig.

[0011] Figure 5 The figure is a graph showing the effect of NBIF of the present invention on T lymphocyte subsets and inflammatory cells in the peripheral blood of rats with pulmonary hypertension, wherein: (A) and (B) are graphs showing the effect of NBIF on CD4+T and CD8+T cells in the peripheral blood of rats with pulmonary hypertension ( n=3); (C) and (D) are the effects of NBIF on Th2 cells in the peripheral blood of rats with pulmonary hypertension ( n=3); (E) and (F) are the effects of NBIF on Treg and Th17 cells in the peripheral blood of rats with pulmonary hypertension ( n=3); (G) Effects of NBIF on inflammatory cells in peripheral blood of rats with pulmonary hypertension ( n=3)Fig.

[0012] Figure 6 Graphs showing the effects of NBIF of the present invention on the PPAR-γ / AMPK / NF-κB signaling pathway in lung tissue of rats with pulmonary hypertension, wherein: (A) is a graph showing the effects of NBIF on the protein expression levels of the PPAR-γ / AMPK / NF-κB signaling pathway in lung tissue of rats with pulmonary hypertension ( n=3); (B) is the effect of NBIF on the expression levels of IL-1β, IL-6 and TNF-α mRNA in lung tissue of rats with pulmonary hypertension ( n=6);

[0013] Figure 7 The effect of NBIF of the present invention on the coagulation function of rats with pulmonary hypertension (A) The effect of NBIF on the platelet activation level of rats with pulmonary hypertension, wherein: (A) is the effect of NBIF on the platelet activation level of rats with pulmonary hypertension ( n=3); (B) is the effect of NBIF on FIB, APTT, PT and TT in rats with pulmonary hypertension ( n=6); (C) is the effect of NBIF on the expression levels of tPA and PAI proteins in lung tissue of rats with pulmonary hypertension ( n=3); (D) is the effect of NBIF on the expression levels of VEGF and TGF-β proteins in lung tissue of rats with pulmonary hypertension ( n=3); (E) is the effect of NBIF on the expression levels of ZO-1, VE-cadherin and occludin proteins in lung tissues of rats with pulmonary hypertension ( n=3); (F) is the effect of NBIF on the levels of ET-1, NO and AngⅡ in the serum of rats with pulmonary hypertension ( n=6);

[0014] Figure 8 The affinity level diagram of NBIF and PPAR-γ detected by SPR of the present invention, wherein: (A) is the response diagram of PPAR-γ protein coupling with CM5 chip, (B) is the binding sensor diagram of NBIF solution with different concentration gradients and PPAR-γ protein, and (C) is the affinity analysis diagram of NBIF and PPAR-γ protein;

[0015] Fig. 9 Graph showing the effect of NBIF of the present invention on the PPAR-γ / AMPK / NF-κB signaling pathway of HUVEC cells induced by hypoxia, wherein: (A) is a graph showing the effect of different modeling times on HUVEC cell viability; ( n=6); (B) is the screening diagram of Si-PPAR-γ silencing sequence ( n=6); (C) is the effect of NBIF on the PPAR-γ / AMPK / NF-κB signaling pathway in HUVEC cells induced by hypoxia detected by WB ( n=3); (D) is the effect of NBIF on the PPAR-γ / AMPK / NF-κB signaling pathway in HUVEC cells induced by hypoxia detected by immunofluorescence ( n=6);

[0016] Fig.10 Figure 2 is the effect of NBIF on mitochondrial function of HUVEC cells induced by hypoxia, wherein: (A) is the effect of NBIF on mitochondrial function of HUVEC cells JC-1 and Ca 2+ Horizontal influence diagram ( n=3); (B) is the effect of NBIF on the expression levels of MFN2 and DRP1 proteins in HUVEC cells induced by hypoxia ( n=6);

[0017] Fig.11 Graph showing the effect of NBIF of the present invention on the adhesion of HUVEC cells induced by hypoxia, wherein: (A) shows the effect of NBIF on the expression levels of ICAM-1 and VCAM-1 in HUVEC cells induced by hypoxia; n=6) Figure; (B) Effects of NBIF on the adhesion of HUVEC cells and THP-1 cells induced by hypoxia ( n=6)Fig.

[0018] Fig.12 Graphs showing the effects of NBIF on HUVEC cell barrier-related proteins and cell permeability induced by hypoxia, wherein: (A) is a graph showing the effects of NBIF on the expression levels of HUVEC cell barrier-related proteins ZO-1 and occludin induced by hypoxia ( n=6); (B) is the effect of NBIF on the transmembrane resistance of HUVEC cells induced by hypoxia ( n=6); (C) is the effect of NBIF on the permeability of sodium fluorescein in HUVEC cells induced by hypoxia ( n=6).

[0019] Application of a new psoralea corylifolia isoflavone isolated from ginseng in the preparation of a drug for treating pulmonary hypertension DETAILED DESCRIPTION

[0020] The specific implementation modes of the present invention are described in detail below in combination with specific situations.

[0021] The present invention discloses an application of new psoralea corylifolia isoflavones isolated from Codonopsis pilosula in preparing a drug for treating pulmonary hypertension. The molecular structural formula of the new psoralea corylifolia isoflavones is:

[0022]

[0023] It is isolated from the Chinese ginseng. The new psoralen isoflavone regulates the peroxisome proliferator-activated receptor γ (PPAR-γ) to restore endothelial cell function, improve pulmonary hypertension, and achieve application in the preparation of drugs for the treatment of pulmonary hypertension. The test results have achieved very good beneficial technical effects. The relevant information is as follows:

[0024] 1. Reagents and Instruments

[0025] Monocrotaline (MCE Company, HY-15025A); AngⅡ kit, ET-1 kit (Jiangsu Enzyme Immunity Industry Co., Ltd., batch number MM-0211R1, MM-0560R1); NO one-step kit (Nanjing Jiancheng Bioengineering Institute, batch number 20220823); BCA protein concentration determination kit, sodium fluorescein, D-Hanks buffer (Beijing Solebold Company, batch number PC0020, F8140, H1045); β-actin antibody, IL-6 antibody, IL-1β, t-PA antibody, TGF-β antibody, VEGF antibody, ZO-1 antibody (Wuhan Proteintech Co., Ltd., batch number 20220823); Company, batch number 66009-1-Ig, 66146-1-Ig, 16806-1-AP, 16806-1-AP, 21898-1-AP, 19003-1-AP, 21773-1-AP); PAI-1 antibody (Beijing Bioson Biotechnology Co., Ltd., batch number bs-1704R); p-NF-KB antibody, NF-KB antibody, TLR4 antibody, occludin antibody, PPAR-γ antibody, ICAM-1 antibody, VCAM-1 antibody (batch number AF2006, AF5006, AF7017, DF7504, AF6284, AF6088, DF6082

[0026] ); CD3-PerCP-eFluor710 antibody, CD8a-PE-Cy7 antibody, CD4-FITC antibody, IFN-γ-efluor660 antibody, IL-17A-PE-Cy7 antibody, CD25-APC antibody, foxP3-PE antibody (Thermo Company, USA, batch number: 46-0030-82, 25-0084-82, 11-0040-82, 50-7310-80, 25-7177-82, 17-0390-82, 12-4774-42); IL-4PE antibody (BD Company, USA, batch number: BD-555082); total RNA extraction kit (Beijing Solebold Company, batch number PC0020, R1200); PMSF, BeyoRT TM IIIcDNA first-strand synthesis premix (5×), calcein AM (Beijing Beyotime Company, batch number ST506, D7182L, C2012).

[0027] Mini-Protean Tetra vertical electrophoresis kit (Bio-Rad, USA); Vevo 2100 small animal ultrasound system (VisualSonics, Canada); R500 general-purpose small animal anesthesia machine (Shenzhen Ruiwode Life Science Co., Ltd.); RAC-030 fully automatic coagulation analyzer (Shenzhen Raydu Life Science Co., Ltd.); Bio Tek microplate reader (Berteng Instrument Co., Ltd., USA); BC-2800vet Mindray veterinary fully automatic blood cell analyzer (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.); 040-049ABL9 blood gas analyzer (Radiometer, Denmark); Odyssey two-color infrared fluorescence imager (LI-COR, USA); BD FACS Aria III flow cytometer (BD Biosciences).

[0028] The dried root of Anthriscus sylvestris (L.) Hoffm. of the genus Anthriscus in the family Apiaceae was purchased from Leshan, Sichuan. Sildenafil (MCE Company, HY-15025A).

[0029] New psoralen isoflavone NBIF was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (B21389).

[0030] 2. Experimental Animals

[0031] 6-week-old SPF grade SD male rats, weighing 180-200 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd., animal license number: SCXK (Beijing) 2019-0008. The experimental animals were raised in a clean animal laboratory at a temperature of 18-22°C, with free access to food and water. The animal experiment was approved by the Experimental Animal Ethics Committee of Henan University of Traditional Chinese Medicine, with the ethics number DWLL201908112.

[0032] 3. Test methods

[0033] 3.1. Establishment of PH rat model and drug administration

[0034] After one week of adaptive feeding, male SD rats were randomly divided into a normal group (NC), a model group (M), a positive drug sildenafil group (Y), an extract of Eleutherodendron chinense group (ES), a low-dose NBIF-L group, and a high-dose NBIF-H group. -1 ), pulmonary hypertension rat model was formed on 28 days, and drug treatment was given on 15-28 days. -1 , injected every other day), ES group (1.5 g·kg -1 , oral administration), NBIF-L group (5 mg·kg-1 , injected every other day), NBIF-H group (10 mg·kg -1 NC group and M group were injected with equal volume of distilled water.

[0035] 3.2. Pulmonary artery pressure and right heart function test in rats

[0036] Small animal ultrasound diagnostic instrument was used to detect the pulmonary artery pressure and right heart function indexes of rats, namely PAT / PET, PEP / PET, right heart free wall thickness, and right heart inner diameter. After the rats were anesthetized with isoflurane, they were placed on the test board in a supine position, a layer of coupling agent was applied to the corresponding area of ​​the heart, and the limbs were fixed on the metal strip of the test board and the coupling agent was applied so that the body information of the rats could be input into the instrument through the metal strip. A high-frequency matrix probe with a frequency of 250HZ was selected, and the ultrasound probe was closely attached to the place where the coupling agent was applied in the precordial area. The appropriate section (parasternal right heart long axis section and pulmonary artery section) was found by twisting the probe, and after adjusting the B-ultrasound image to the appropriate interface, M-ultrasound was selected to measure the values ​​on the middle section of the echocardiogram, and the statistics were counted and the results were analyzed.

[0037] 3.3. Pulmonary function test in rats

[0038] After the last administration of each group of rats, the pulmonary function test methods in the references were used to measure the basic pulmonary function indicators of the rats: tidal volume (TVb), minute ventilation (MVb), respiratory rate (F). The rats were placed in the cavity in a conscious state, and the test was started after the rats adapted and their breathing was stable, and the changes in the pulmonary function indicators of the rats within 5 minutes were recorded.

[0039] 3.4. Detection of coagulation function and blood inflammatory cells

[0040] Coagulation function test: After modeling and drug administration, 1 mL of venous blood was collected from the orbital venous plexus of the rats, added to a 3.8% sodium citrate tube, mixed thoroughly, and centrifuged at 3000 r·min. -1 , 15 minutes, take the upper plasma, and use the automatic coagulation analyzer to detect FIB, APTT, PT, and TT.

[0041] Blood inflammatory cell detection: After modeling and drug administration, blood was collected by sampling from the abdominal aorta and placed in a blood collection tube containing EDTA. The blood was dropped into a blood routine tester using a pipette to detect the number of inflammatory cells in the blood of the rats.

[0042] 3.5. Detection of T lymphocyte subsets in peripheral blood

[0043] Flow cytometry was used to detect T lymphocyte subsets in rat peripheral blood. After the rats were anesthetized, blood was collected from the abdominal aorta, and the whole blood sample was diluted with PBS 1:1. The diluted sample was gently spread on the lymphocyte separation solution, centrifuged at 400g for 20min (the centrifuge brake was canceled). After centrifugation, the middle white cloudy lymphocyte layer was taken, mixed with PBS, and centrifuged for 10min at 1500rpm to obtain PBMCs cells. 1ml of RPMI1640 culture medium containing stimulants was added to the PBMCs cells and placed in an incubator for 6h. Then, add extracellular staining antibodies (CD3 2.5μL, CD4 0.5μL, CD8 1.25μL), then fix and permeabilize the membrane, add extracellular staining antibodies (IL-4 2.5μL, IFN-γ2.5μL, IL-17 2.5μL, FOXP3 5μL, CD250.625μL), and set up single staining tubes for various stains. Stain at room temperature for 30 minutes. After staining, wash twice with PBS and analyze on the machine.

[0044] 3.6 Platelet activation detection

[0045] Blood was collected from the abdominal aorta of rats and collected in a blood collection tube containing 3.8% sodium citrate anticoagulant. The blood was centrifuged at 160g for 10 min, and the supernatant was taken. The blood was centrifuged at 1500g for 5 min, and the supernatant was discarded to precipitate platelets. The platelets were resuspended in PBS and dyed with 2μL CD61 and 2.5μL CD62P. The blood was stained for 20 min, centrifuged, and tested on a blood analyzer.

[0046] 3.7 Western Blotting

[0047] Western Blot was used to detect the expression of related proteins. The lung tissue frozen at -80℃ was quickly rinsed in physiological saline, homogenized in a lysis buffer containing PMSF using a homogenizer, centrifuged at 12000g for 10min, and the supernatant was taken for protein quantification by BCA method. The total protein (50μg) was separated according to the relative molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane by semi-dry method. After the membrane loaded with protein was immersed in 5% skim milk powder to block the blank site, the primary antibody that specifically binds to the antigen to be tested was added and incubated overnight, the unbound primary antibody was washed to remove the unbound primary antibody, and the secondary antibody linked to a fluorescent group was added, and the unbound secondary antibody was washed to remove the unbound secondary antibody. The Odyssey two-color infrared fluorescence imaging system was used to scan the image and quantify the data.

[0048] 3.8 RT-PCR

[0049] Rat lung tissue was obtained, and RNA was extracted according to the instructions of the total RNA extraction kit. The RNA concentration and purity were determined by 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; 4°C to obtain cDNA. Fluorescence quantitative PCR was used for amplification, and the reaction conditions were: 95°C, 10s; 95°C, 5s; 60°C, 10s; a total of 40 cycles. The amplification curve was analyzed and the Ct value was calculated; GAPDH was used as the internal reference gene, and 2 -△△Ct The relative expression of the target gene was calculated by the method. The primers were synthesized by Suzhou Genewise Biotechnology Co., Ltd., and the primer sequences are shown in Table 1.

[0050] Table 1 Primer Sequences

[0051]

[0052] 3.9. HE and Masson staining

[0053] After the last administration, the rat heart and lung tissues were dissected, fixed with 4% formaldehyde, embedded in paraffin, sliced, and stained with HE, and stained with potassium dichromate, iron hematoxylin, ponceau acid fuchsin, phosphomolybdic acid, and aniline blue. Lung pathological damage was observed under a microscope.

[0054] 3..10. Detection of affinity between NBIF and PPAR-γ

[0055] The affinity of NBIF and PPAR-γ was detected using the Biacore T200 instrument. PPAR-γ was first coupled to a CM5 chip (sodium acetate buffer pH 4.0), and then NBIF solutions with different concentration gradients were passed over the chip surface. The affinity K of NBIF and PPAR-γ protein was obtained by model fitting using the Biacore T200 Evalution software. D .

[0056] 3.11 Cell Culture

[0057] HUVEC cells were purchased from Creative Bioarray. The cells were cultured in high glucose medium (10% fetal bovine serum, 1% penicillin-streptomycin) at 37°C and 5% CO2.

[0058] 3.12 Endothelial cell transmembrane resistance measurement

[0059] HUVEC cells in the logarithmic growth phase were cultured at 1×10 5The density of cells / well was inoculated into the upper chamber of the 12-well transwell plate, and the culture medium was added to the lower chamber. After the cells stably adhered to the wall and grew, the drug NBIF was added and hypoxic stimulation was performed for 48 hours. After the hypoxic stimulation was completed, the cell transmembrane resistance was detected using an ERS-2 resistance meter. The short end of the test electrode was immersed in the inner side of the co-culture upper chamber, and the long end was immersed in the co-culture lower chamber, and the resistance value was read.

[0060] 3.13 Immunofluorescence

[0061] HUVEC cells in the logarithmic growth phase were cultured at 2×10 4 The cells were seeded into 96-well plates at a density of 10 cells / well. After the cells were stably attached to the wall and grown, the drug NBIF was added and hypoxic stimulation was performed for 48 hours. After the hypoxic stimulation was completed, the cells were washed twice with PBS solution, 150 μL of 4% paraformaldehyde solution was added to each well, fixed for 20 minutes, and then 150 μL of 0.1% Triton solution was added to each well for permeabilization at room temperature for 20 minutes. The cells were blocked with 1% BSA solution for 30 minutes, and different primary antibodies were added and incubated at 4°C overnight. Subsequently, the cells were washed three times with 0.1% PBST solution, the secondary antibody was added and incubated at room temperature in the dark for 1 hour, washed three times with 0.1% PBST solution in the dark, stained with DAPI solution at room temperature in the dark for 5 minutes, and finally washed away the excess DAPI with 0.1% PBST solution in the dark, and scanned and photographed using the Opera PhenixTM high-content imaging system.

[0062] 3.14. Fluorescein sodium permeation test

[0063] HUVEC cells in the logarithmic growth phase were cultured at 1×10 5 The cells were seeded at a density of 10 cells / well into the upper chamber of a 12-well transwell plate, and culture medium was added to the lower chamber. After the cells stably adhered to the wall and grew, the drug NBIF was added and hypoxic stimulation was performed for 48 hours. After the hypoxic stimulation was completed, the culture medium in the upper and lower chambers was discarded, and the cells were washed with PBS. D-Hanks buffer was added to the lower chamber, and 10 μg / ml sodium fluorescein was added to the upper chamber. After 1 hour, the liquid in the lower chamber was collected and placed in a 96-well opaque plate, and the absorbance was detected at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0064] 3.15. THP-1 cell adhesion assay

[0065] HUVEC cells in the logarithmic growth phase were cultured at 1×10 5 The cells were seeded into 6-well plates at a density of 10 cells / well. After the cells stably adhered to the plate and grew, NBIF was added and hypoxia was stimulated for 48 hours. 5cells / mL) were labeled with 10 μM calcein AM. After labeling, the cells were washed twice with PBS solution, and RPMI 1640 medium preheated at 37°C was slowly added and incubated in a 37°C incubator for 30 minutes. The labeled THP-1 cells were co-cultured with HUVEC cells for 1 hour, washed twice with PBS solution, and non-adherent THP-1 cells were removed. Opera PhenixTM high-content imaging system was then used to take pictures and observe the adhesion of THP-1 cells and HUVEC cells.

[0066] 4. Statistical Processing

[0067] The experimental data were processed and statistically analyzed using SPSS 20.0 statistical software, and the results were expressed as mean ± standard deviation (i.e. ). P<0.05 indicated statistical significance, P<0.05 indicated significant difference, and P<0.01 indicated extremely significant difference.

[0068] 4.1. Identification of NBIF in Radix Glehniae by HPLC

[0069] like Figure 2 As shown in the figure, the NBIF standard product has a chromatographic peak at a retention time of about 16 minutes, and the total extract of E. ginseng has a corresponding chromatographic peak at the corresponding retention time. This indicates that the total extract of E. ginseng contains NBIF.

[0070] 4.2 Effects of NBIF on lung injury in rats with pulmonary hypertension

[0071] Compared with the NC group, the M group had severe pulmonary blood vessel thickening, vascular lumen stenosis, and inflammatory cells accumulation around the pulmonary blood vessels. After administration of NBIF, the pulmonary blood vessel thickening and inflammatory cell infiltration were alleviated ( Figure 3 A); Compared with the NC group, the PEP / PAT in the M group was significantly increased, and the PAT / PET was significantly decreased (P<0.01). After the administration of NBIF, the above indicators improved ( Figure 3 B); at the same time, NBIF can improve the level of lung hypoxia in rats, significantly reduce PaCO2, increase PaO2 and SaO2 (P<0.01 or P<0.05), improve lung function in rats, and increase TVB, MVB and F (P<0.01 or P<0.05) ( Figure 3 CH).

[0072] 4.3 Effects of NBIF on right heart damage in rats with pulmonary hypertension

[0073] HE sections and Masson staining of cardiac tissue showed that the myocardial tissue of the NC group was arranged neatly and orderly, while the myocardial tissue of the M group was arranged disorderly, without hair, broken, with obvious inflammatory cell infiltration and collagen fiber deposition. After administration of NBIF, the myocardial tissue was arranged neatly and the collagen fiber deposition decreased ( Figure 4 A); at the same time, NBIF can reduce the right heart hypertrophy index, right ventricular free wall thickness and right heart inner diameter of rats, and reduce the level of ROS in rat myocardial tissue ( Figure 4 B, C) (P<0.01 or P<0.05).

[0074] 4.4 Effects of NBIF on T lymphocyte subsets in peripheral blood of rats with pulmonary hypertension

[0075] The results showed that the proportions of CD4+T cells, Th2 cells, Th17 cells and the number of inflammatory cells in the peripheral blood of rats in the model group increased significantly, and the proportions of CD8+T cells and Treg cells decreased significantly (P<0.01), indicating that there was an imbalance in the T lymphocyte subsets in the peripheral blood of rats with pulmonary hypertension. After administration of NBIF, the proportions of CD4+T cells, Th2 cells, Th17 cells and the number of inflammatory cells in rats decreased significantly, and the proportions of CD8+T cells and Treg cells increased significantly (P<0.01 or P<0.05) ( Figure 5 ).

[0076] 4.5 Effects of NBIF on PPAR-γ / AMPK / NF-κB signaling pathway in rats with pulmonary hypertension

[0077] The results showed that compared with the NC group, the levels of PPAR-γ and p-AMPK / AMPK in the lung tissues of the rats in the M group were significantly decreased (P<0.01), and the levels of p-NF-κB / NF-κB, TLR4, IL-6, and IL-1β were significantly increased (P<0.05 or P<0.01); after administration of NBIF, the expression levels of the above proteins were improved (P<0.05 or P<0.01) ( Figure 6 ). This suggests that NBIF may further reduce the inflammatory response of lung tissue and improve lung injury in PH rats by affecting the PPAR-γ / AMPK / NF-κB signaling pathway.

[0078] 4.6 Effects of NBIF on endothelial cell function in rats with pulmonary hypertension

[0079] Compared with the NC group, the activated partial thromboplastin time (APTT) and the expression level of pulmonary tissue plasminogen activator (t-PA) protein in the M group were significantly decreased (P<0.01) ( Figure 7 B, C), the platelet activation level, plasma fibrinogen level (FIB) and lung tissue plasminogen activator inhibitor (PAI) level were significantly increased (P < 0.01) ( Figure 7A, B). After administration of NBIF, the above indicators improved to varying degrees (P<0.01 or P<0.05). This indicates that the blood of rats with pulmonary hypertension in group M is in a hypercoagulable state, and NBIF can regulate platelet activation and the levels of anticoagulant and procoagulant factors, reducing blood coagulation and thrombus formation. The expression level of vascular endothelial growth factor VEGF in the lung tissue of rats in group M was significantly decreased, and the level of transforming growth factor TGF-β was significantly increased (P<0.01)( Figure 7 D); the expression levels of endothelial cell barrier-related proteins ZO-1, VE-cadherin, occludin, and Cx43 were significantly decreased (P<0.01) ( Figure 7 E); serum ET-1 and AngⅡ levels increased significantly (P<0.01), and NO level decreased significantly (P<0.01). After administration of NBIF, the above indicators improved to varying degrees (P<0.01 or P<0.05) ( Figure 7 F) These results suggest that pulmonary hypertension rats have vascular contraction and relaxation imbalance and endothelial cell dysfunction, and NBIF can regulate the secretion of endothelial cell growth factor and improve endothelial cell barrier damage.

[0080] 4.7 Analysis of NBIF and PPAR-γ affinity

[0081] PPAR-γ protein binds to the CM5 chip under the condition of sodium acetate buffer at pH 4.0, and the response signal is 10490.5RU ( Figure 8 A), indicating that the protein binds specifically to CM5. The binding signal of NBIF solution to PPAR-γ protein in the concentration range of 0.4 μM to 51.2 μM is shown in Figure 2. Figure 8 As shown in B, the affinity of NBIF to PPAR-γ protein is KD = 14.46 μΜ ( Figure 8 C), indicating that NBIF binds specifically to PPAR-γ.

[0082] 4.8 Effect of NBIF on the PPAR-γ / AMPK / NF-κB signaling pathway in HUVEC cells induced by hypoxia

[0083] The experimental results showed that with the increase of hypoxia time, the viability of HUVEC cells decreased significantly (P<0.01), among which the viability of cells was the lowest after hypoxia for 48h. Therefore, the optimal modeling conditions for HUVEC cells were determined as follows: hypoxia for 48h ( Fig. 9 B). RT-PCR was used to detect the level of PPAR-γ mRNA in HUVEC cells. The results showed that Si-PPAR-γ-2 had the highest silencing efficiency (P<0.01), so sequence 2 was selected for subsequent experiments ( Fig. 9A). WB experimental results showed that the expression levels of PPAR-γ and p-AMPK / AMPK in the Model group were significantly decreased (P<0.01); the expression levels of the above proteins in the NBIF group were significantly increased (P<0.01); after the addition of Si-PPAR-γ, the expression levels of the above proteins were significantly decreased (P<0.01) ( Fig. 9 C). High content results showed similar effect trends (P<0.05 or P<0.01) ( Fig. 9 D).

[0084] 4.9 Effects of NBIF on mitochondrial function of HUVEC cells induced by hypoxia

[0085] The experimental results showed that the mitochondrial membrane potential of cells in the Model group ( Fig.10 A) The fluorescence intensity levels of DRP1 and MFN2 were significantly decreased (P<0.01) ( Fig. 9 B), cellular Ca 2+ The level was significantly increased (P<0.01) ( Fig.10 A); NBIF group mitochondrial membrane potential, DRP1, MFN2 fluorescence intensity levels were significantly increased (P < 0.01), cell Ca 2+ After adding Si-PPAR-γ, the improvement of the above indicators was significantly reduced (P<0.05 or P<0.01) ( Fig.10 A, B). These results suggest that NBIF may improve mitochondrial dysfunction in HUVEC cells by regulating the expression of PPAR-γ.

[0086] 4.10 Effect of NBIF on Hypoxia-Induced HUVEC Cell Adhesion

[0087] The experimental results showed that the fluorescence intensity levels of cell adhesion factors ICAM-1 and VCAM-1 in the Model group were significantly increased (P<0.01) ( Fig.11 A), the number of cells adhering to THP-1 increased significantly (P<0.01) ( Fig.11 B); NBIF can reduce the expression level of adhesion factors (P<0.01) and the number of adhesion to THP-1 inflammatory cells (P<0.01); after adding Si-PPAR-γ, the improvement level of the above indicators was significantly reduced (P<0.01) ( Fig.11 A, B). These results suggest that NBIF may reduce the adhesion of THP-1 to endothelial cells through PPAR-γ, improve the adhesion level of endothelial cells, and restore endothelial cell function.

[0088] 4.11 Effects of NBIF on barrier-related proteins and cell permeability of HUVEC cells induced by hypoxia

[0089] The experimental results showed that the fluorescence intensity of ZO-1 and occludin in the Model group cells ( Fig.12 A) The cell transmembrane electrical resistance (TEER) was significantly reduced (P<0.01) ( Fig.12 B), the permeability of sodium fluorescein was significantly increased (P<0.01) ( Fig.12 C), NBIF can improve the above indicators (P<0.01); after adding Si-PPAR-γ, the improvement of the above indicators was weakened (P<0.01). This suggests that NBIF may alleviate hypoxia-induced endothelial cell barrier damage by regulating PPAR-γ.

[0090] V. Conclusion

[0091] Pulmonary hypertension 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 pathogenesis, resulting in increased pulmonary vascular resistance and pulmonary artery pressure. With the long-term increase in pulmonary artery pressure, right heart failure will eventually occur, and severe patients will die. The main pathological changes of pulmonary hypertension include pulmonary vascular remodeling, in situ thrombosis, right heart remodeling, etc. The incidence of pulmonary hypertension is as high as 1%, but there is currently no effective treatment. NBIF is a flavonoid compound isolated from ginseng. Studies have shown that NBIF has good antioxidant, anti-tumor and anti-inflammatory activities. The occurrence and development of pulmonary hypertension is closely related to endothelial cell dysfunction and immune inflammation. Therefore, the present invention studies whether NBIF has an anti-pulmonary hypertension effect. Experiments show that the pulmonary hypertension model is induced by intraperitoneal injection of monocrotaline. In clinical practice, the diagnosis of pulmonary hypertension is often assisted by ultrasound imaging. Among them, PAT / PET and PEP / PAT are important indicators of pulmonary artery pressure. The higher the PEP / PAT ratio, the lower the PAT / PET, indicating that the pulmonary artery pressure is higher. The results showed that for lung tissue, NBIF can reduce pulmonary artery pressure in rats, alleviate pulmonary vascular thickening, reduce the accumulation of inflammatory cells around pulmonary vessels, and improve rat lung function; for myocardial tissue, NBIF can alleviate and improve myocardial damage in rats, reduce myocardial collagen fiber deposition, reduce right heart free wall thickness, right heart inner diameter and right heart hypertrophy index, and effectively improve right heart remodeling; hypoxia and decreased arterial oxygen partial pressure are the pathological basis of pulmonary hypertension, and decreased arterial oxygen partial pressure is closely related to increased pulmonary artery pressure. Further experiments found that NBIF can reduce arterial carbon dioxide partial pressure (PaCO2) in rats, and increase arterial oxygen partial pressure (PaO2) and blood oxygen saturation (SaO2) in rats.

[0092] Experiments have shown that various immune cells are involved in the occurrence and development of pulmonary hypertension. In the pathological state of pulmonary hypertension, it is often accompanied by an imbalance of immune inflammatory cells such as macrophages, dendritic cells, neutrophils, and T lymphocytes. The imbalance of T lymphocyte subsets is closely related to the occurrence of pulmonary hypertension. The increase in CD4+T cells exacerbates inflammation around pulmonary vessels. As the disease further deteriorates, CD8+T cells are constantly exhausted, further indicating that the reduction of CD8+T cells may be related to the apoptosis-anti-apoptosis phenotype conversion of pulmonary arterial endothelial cells. The immune response of Th2 cells is involved in pulmonary vascular remodeling and the formation of muscularization of pulmonary arterioles. The imbalance of the Th17 / Treg cell ratio also promotes the further deterioration of pulmonary vascular inflammation. Th17 cells have a proinflammatory effect, and Treg cells can play an effective immunoregulatory role to reduce pulmonary vascular inflammatory damage. It further shows that the reduction in the number and activity of Treg cells leads to further spread of endothelial cell inflammation, proliferation of RPASMC cells, and imbalance between endothelial cell contraction and relaxation. Therefore, the number of white blood cells, lymphocytes, and neutrophils in the plasma of rats with pulmonary hypertension was detected, and the proportion of T lymphocyte subsets (CD4+T, CD8+T, Th2, Th17, Treg) was detected by flow cytometry to clarify the regulatory effect of NBIF. The results showed that NBIF can reduce the number of inflammatory cells in peripheral blood, regulate the balance of T lymphocyte subsets, and improve the disorder of pulmonary vascular immune regulation, proving that NBIF has an anti-pulmonary hypertension effect. But what is the potential mechanism of NBIF?

[0093] Peroxisome proliferator-activated receptor γ (PPAR-γ) is an important component of nuclear hormone receptors. PPAR-γ plays an important role in regulating cell growth, maintaining vascular homeostasis, improving endothelial cell dysfunction, and alleviating immune inflammatory responses. Adenosine monophosphate-activated protein kinase (AMPK) is an important serine / threonine kinase involved in cell proliferation and apoptosis, cell energy metabolism and other processes. Experiments have shown that activated PPAR-γ can promote AMPK phosphorylation to inhibit NF-κB activation, thereby inhibiting the activation of downstream inflammatory signaling pathways and the expression of inflammatory factors such as IL-6 and IL-1β. Therefore, can NBIF regulate PPAR-γ to play a protective role in rats with pulmonary hypertension? The experimental results showed that NBIF can regulate the level of PPAR-γ / AMPK / NF-KB signaling pathway, reduce the expression of IL-6 and IL-1β inflammatory factors, and thus reduce pulmonary vascular inflammation in rats with pulmonary hypertension.

[0094] Pulmonary vascular endothelial cells are an important component of the pulmonary vascular endothelium, and vascular endothelial cells play an important role in maintaining vascular function. Under physiological conditions, endothelial cells maintain vascular homeostasis, regulate vascular permeability, control vasoconstriction and vasodilation, and inhibit excessive platelet aggregation. Under the pathological state of pulmonary hypertension, endothelial cells are stimulated by inflammatory reactions and hypoxia factors, and endothelial homeostasis is unbalanced, which in turn causes endothelial cell dysfunction. Experiments have shown that endothelial cell dysfunction is considered to be the initiating factor of PAH. These intracellular dysfunctions include endothelial cell barrier damage, increased permeability, imbalanced cytokine secretion, abnormal coagulation function, imbalanced vasoconstriction and vasodilation factors, and endothelial-mesenchymal transformation. The PPAR-γ / AMPK / NF-KB signaling pathway can reduce endothelial cell immune inflammatory response and endothelial cell dysfunction. Therefore, the experiment detected endothelial cell function-related indicators to explore whether NBIF plays a protective role on endothelial cells through the PPAR-γ / AMPK / NF-KB signaling pathway.

[0095] The experimental results show that in terms of vasoconstriction and relaxation, NO is the most important endothelial cell vasodilator factor produced by endothelial cells, and endothelin-1 (ET-1) is an effective vasoconstrictor. In terms of coagulation function, FIB and APTT are important indicators of coagulation function. In the acute exacerbation period of patients with pulmonary hypertension, the FIB level increases, the APTT decreases, and the blood is in a hypercoagulable state; excessive platelet activation will further aggravate the occlusion of the pulmonary vascular cavity and increase the risk of thrombosis; plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) are the main components of the human fibrinolytic system. t-PA can activate plasminogen, promote the degradation of fibrin, and inhibit thrombosis; PAI is an inhibitor of t-PA and can promote the formation of thrombus. NBIF can improve the coagulation function of rats with pulmonary hypertension, inhibit the level of platelet activation in rats, and reduce thrombosis. In terms of abnormal secretion of endothelial cell factors, the increase of VEGF will aggravate the effect of vascular permeability and the inflammatory response process. TGF-β1 is a strong cell proliferation regulator. Under the condition of pulmonary hypertension, TGF-β1 promotes the phenotypic transformation of pulmonary artery endothelial cells and the abnormal proliferation of pulmonary artery smooth muscle cells. Experimental results show that NBIF can regulate the abnormal secretion of rat cell growth factors. In terms of endothelial cell barriers, the pulmonary vascular endothelium is a permeable barrier for the exchange of substances between blood vessels and blood. When the pulmonary vascular endothelial cells are damaged and endothelial dysfunction occurs, the endothelial permeability will increase, the leakage of vascular inflammatory factors will increase, and the pulmonary vascular lesions will be aggravated. Tight junction-related transmembrane protein ZO-1, vascular endothelial cadherin VE-cadherin, and transmembrane protein occludin are the main factors of tight junctions between endothelial cells. The decrease in their expression levels will increase vascular permeability. NBIF can regulate the expression levels of cell barrier-related proteins in rats with pulmonary hypertension and improve cell permeability. NBIF can restore endothelial cell function through the PPAR-γ / AMPK / NF-KB signaling pathway, thereby improving rat pulmonary hypertension.

[0096] In order to further verify whether NBIF plays a protective role on endothelial cells through the PPAR-γ / AMPK / NF-κB signaling pathway, an in vitro experiment was conducted. Endothelial cell damage was induced by hypoxia, and the PPAR-γ silencing sequence was added to detect cell mitochondrial function, cell adhesion ability, and cell barrier-related indicators. The fission and fusion of mitochondria are mainly regulated by mitochondrial motor protein Drp1 and mitochondrial fusion-related protein MFN2. MFN2 is located in the outer membrane of mitochondria and can promote the fusion of two adjacent mitochondria and repair mitochondrial damage. Drp1 can promote mitochondrial fission and is beneficial to cell proliferation. Abnormal mitochondrial fusion and fission lead to pulmonary artery endothelial cell dysfunction, excessive endothelial cell migration and endothelial-mesenchymal transition. The enhancement of endothelial cell inflammatory response is closely related to the occurrence of endothelial cell dysfunction. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are important adhesion molecules of endothelial cells. When stimulated by inflammatory response, their expression levels increase, mediating the adhesion of immune inflammatory cells to endothelial cells. In addition, enhanced endothelial cell permeability and endothelial barrier damage lead to increased leakage of inflammatory factors, further aggravating pulmonary vascular inflammation. The experimental results showed that after silencing PPAR-γ, the effect of NBIF on PPAR-γ protein and this pathway was weakened; at the same time, NBIF could improve the mitochondrial function of HUVEC cells, restore cell barrier damage, and reduce the adhesion level of THP-1 cells and endothelial cells.

[0097] In summary, NBIF improves pulmonary hypertension by restoring endothelial cell function through the PPAR-γ / AMPK / NF-κB signaling pathway, and can become a potential drug for the treatment of PH. It can also exert anti-PH effects by regulating PPAR-γ, and can be used in the preparation of drugs for the treatment of pulmonary hypertension. It has opened up new ways for the medicinal value of E. ginseng and the treatment of pulmonary hypertension, with significant social and economic benefits.

Claims

1. Use of new psoralea corylifolia isoflavones isolated from Radix Glehniae in the preparation of drugs for treating pulmonary arterial hypertension.

2. The application of a new psoralea corylifolia isoflavone isolated from E. corylifolia in the preparation of a drug for treating pulmonary arterial hypertension by regulating peroxisome proliferator-activated receptor γ to restore endothelial cell function and improve pulmonary arterial hypertension.

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