Application of ginsenoside F4 in preparation of medicine for treating calcified aortic valve disease

Abstract

The ginsenoside F4 is found to be capable of effectively down-regulating the expression of osteogenic differentiation marker genes ALP and RUNX2 in valvular interstitial cells and inhibiting the osteogenic differentiation of the valvular interstitial cells, so that valve calcification is delayed or inhibited; the invention also provides a cell-tissue-individual three-dimensional culture system which can significantly improve osteogenic differentiation of valvular interstitial cells, human aortic valve tissue blocks and Apoe- / -male mouse aortic valves after being treated by ginsenoside F4, and further provides a new drug choice for prevention and treatment of calcified aortic valve diseases.

Description

Technical field:

[0001] The present invention relates to the field of biomedicine, and in particular to the application of ginsenoside F4 in the preparation of a medicine for treating calcific aortic valve disease. Background technology:

[0002] Calcific aortic valve disease is a type of disease in which the fibrosis and calcification of the aortic valve leaflets progressively worsen, and eventually the leaflets deform and open and close dysfunction cause left ventricular outflow tract stenosis and hemodynamic disorders. The disease process is long, and initially manifests as leaflet fibrosis and microcalcification, but at this time there is no obvious change in leaflet movement, and the aortic orifice has not yet shown obvious stenosis. In severe patients, the degree of valve calcification damage can be further aggravated, and calcific aortic valve stenosis may occur, causing left ventricular outflow tract obstruction. Patients may experience adverse events such as left ventricular hypertrophy, heart failure, and premature death. As the most common heart valve disease in countries with high economic levels, the prevalence of this disease continues to rise with the aging of the population, and the global burden is increasing. Calcific aortic valve disease is rapidly developing into a global public health issue that needs to be addressed urgently. At present, the pathogenesis of calcific aortic valve disease is still not fully understood. Similar to coronary atherosclerosis, age, gender, body mass index, smoking, hypertension, vascular sclerosis, serum lipoprotein a, serum low-density lipoprotein, serum cholesterol, diabetes, infection and kidney disease are all risk factors for this disease. At the same time, gene mutations are also widely involved in the occurrence and progression of calcific aortic valve disease. Existing experiments to explore the pathogenesis mainly focus on the study of aortic valve interstitial cells. Studies have shown that valve sterile inflammatory response, metabolic disorders of lipids such as lipoprotein a and low-density lipoprotein in the blood, imbalance of calcium and phosphorus metabolism in the body (possibly related to osteoporosis or chronic end-stage renal disease in patients), and the renin-angiotensin system are all involved in the occurrence and development of the disease. With the advancement of science and technology and the development of experimental technology, more and more studies have expanded their horizons to aortic valve endothelial cells, immune cells in the aortic valve, and their relationship with aortic valve interstitial cells and the balance of osteogenesis and osteoclastogenesis in the aortic valve. However, the existing etiological research is far from enough. Early intervention and conservative treatment of calcific aortic valve disease are still world problems, and the core mechanisms and key molecules in the progression of the disease still need more in-depth research. Unfortunately, there is currently no drug that can effectively control the progression of aortic valve calcification for clinical treatment. Although lipid-lowering drugs have always been highly anticipated, clinical trials in recent years have shown that statins can effectively reduce the level of serum low-density lipoprotein in patients, but they cannot effectively delay the progression of calcific aortic valve disease. At present, the effective treatment for calcific aortic valve disease is still surgical valve replacement. Surgical intervention mainly includes surgical aortic valve replacement and transcatheter aortic valve replacement. The valves replaced are mainly mechanical valves and biological valves. Patients with mechanical valves must take warfarin anticoagulation therapy for life after surgery, which greatly reduces the quality of life. There is also a higher risk of bleeding or thrombosis, and patients with biological valves also face a greater risk of reoperation.Therefore, the research and development of therapeutic drugs for calcific aortic valve disease is of far-reaching and urgent significance. It is very important to clarify the pathogenesis of calcific aortic valve disease as soon as possible and to find possible drug targets and effective therapeutic drugs to delay and prevent aortic valve calcification.

[0003] Ginseng has been used as a traditional Chinese medicine for thousands of years. Ginsenosides are the main bioactive components of ginseng, which are widely found in the roots, stems, leaves, flowers and fruits of ginseng. It has been found that ginsenosides can be divided into 31 subtypes, which can be divided into four categories according to the different internal aglycone structures: (1) ginsenosides: such as Rb1, Rc, Rd, etc.; (2) ginsenosides: such as Rg1, Rg2, Rh1, etc.; (3) oleanolic acid: such as Ro, Rh3, F4, etc.; (4) octillone. Ginsenosides have a wide range of effects. In recent years, it has been reported that they can play an important role in the prevention and treatment of cardiovascular diseases, nervous system diseases, hematopoietic system diseases, digestive system diseases and tumors by regulating oxidative stress, inflammatory response and immune function. Ginsenoside F4 involved in this study belongs to the oleanolic acid type ginsenoside subtype, which has been found to have the effects of regulating sugar metabolism, inhibiting cell proliferation and preventing cartilage decomposition. However, there are no reports on the relationship between ginsenoside F4 and calcific aortic valve disease. Based on the overlap between the mechanism of action of ginsenoside F4 and the pathogenesis of calcific aortic valve disease, its role and mechanism in calcific aortic valve disease deserve further study. Summary of the invention:

[0004] 1. Technical issues to be resolved

[0005] In view of the above background, the present invention has found through research that ginsenoside F4 can effectively down-regulate the expression of osteogenic differentiation marker genes ALP and RUNX2 in valvular interstitial cells, inhibit the osteogenic differentiation of valvular interstitial cells and the deposition of calcium salts in cells, thereby delaying or inhibiting valve calcification, and proposes the use of ginsenoside F4 in the preparation of drugs for the treatment of calcified aortic valve disease, and proposes a method for significantly improving valvular interstitial cells, human aortic valve tissue blocks and Apoe after treatment with ginsenoside F4. - / - The cell-tissue-individual three-dimensional cultivation system for osteogenic differentiation of the aortic valve in male mice provides new drug options for the prevention and treatment of calcific aortic valve disease and new ideas for exploring disease mechanisms.

[0006] (II) Technical solution

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] The present invention discloses a first aspect, providing the use of ginsenoside F4 in the preparation of a drug for treating calcific aortic valve disease, wherein the ginsenoside F4 is CAS No. 181225-33-2; the molecular formula is C 42 H 70 O 12 of substance.

[0009] Furthermore, the ginsenoside F4 is used to prepare a drug for intervening in the osteoblastic differentiation of aortic valve interstitial cells.

[0010] Furthermore, the disease is aortic valve calcification disease induced by a high-cholesterol diet, and the aortic valve is the aortic valve of the heart.

[0011] On the other hand, the present invention also provides the use of ginsenoside F4 as an inhibitor of osteogenic differentiation of human valvular interstitial cells in vitro.

[0012] On the other hand, the present invention also provides the use of ginsenoside F4 as a drug for regulating the EGFR / PI3K / AKT signaling pathway.

[0013] On the other hand, the present invention also provides the use of ginsenoside F4 as an ALP and RUNX2 gene inhibitor.

[0014] Ginsenoside F4 is a potential antagonist of EGFR. In osteogenic-induced osteogenic differentiation cells, tissues and animal models, it was shown that ginsenoside F4 can inhibit the osteogenic differentiation of valvular interstitial cells, and it was proved that it exerts anti-calcification effects by binding to EGFR and inhibiting the downstream PI3K / AKT signaling pathway.

[0015] The present invention also provides a method for significantly improving the valvular interstitial cells, human aortic valve tissue blocks and Apoe after being treated with ginsenoside F4. - / - A cell-tissue-individual three-dimensional culture system for osteogenic differentiation of male mouse aortic valves. Human valvular interstitial cells were induced and cultured for 3-21 days using a calcification medium containing ginsenoside F4, and human aortic valve tissue blocks were cultured for 45-60 days. The calcification medium containing ginsenoside F4 was composed of: 3 μM ginsenoside F4, 50 mg / mL vitamin C, 5 mmol / L β-glycerophosphate, 100 nmol / L dexamethasone, 2% fetal bovine serum, and high glucose medium (DMEM); Ginsenoside F4 was used to give 8-week-old Apoe - / - Male mice were gavaged at a concentration of 20 mg / kg with normal saline as the solvent, twice a week for a total of 24 weeks.

[0016] Specifically, the induced culture of human valvular interstitial cells and human aortic valve tissue blocks was carried out in a mixed gas of 5% carbon dioxide volume concentration and 95% oxygen concentration, and the culture temperature was kept constant at 37°C; Apoe - / -Male mice were cultured in a pathogen-free, temperature-controlled environment with a 12-h light-dark cycle.

[0017] (III) Beneficial effects

[0018] The beneficial effects of the present invention are as follows: the present invention provides the use of ginsenoside F4 in the preparation of a drug for treating calcific aortic valve disease. Ginsenoside F4 can effectively downregulate the expression of osteogenic differentiation marker genes ALP and RUNX2 in valve interstitial cells, inhibit the osteogenic differentiation of valve interstitial cells and the deposition of calcium salts in cells, thereby inhibiting the osteogenic differentiation of valve interstitial cells and exerting an anti-aortic valve calcification effect. The present invention also provides a method for significantly improving the expression of valve interstitial cells, human aortic valve tissue blocks and Apoe after treatment with ginsenoside F4. - / - The cell-tissue-individual three-dimensional cultivation system for osteogenic differentiation of the aortic valve of male mice provides new drug options for calcific aortic valve disease and new ideas for exploring disease mechanisms. Description of the drawings:

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below.

[0020] Figure 1 A is the chemical structure of ginsenoside F4.

[0021] Figure 1 B is a graph showing the changes in the ALP gene level of human valvular interstitial cells treated with ginsenoside F4 at a concentration gradient of 0-3 μM under in vitro calcification-induced culture conditions: the NC group represents the control group with conventional non-calcification-induced culture medium, OM represents the calcification-induced culture medium, and OM+ginsenoside F4 represents the ginsenoside F4-treated group. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0022] Figure 1C shows the changes in RUNX2 gene levels in human valvular interstitial cells treated with ginsenoside F4 at a concentration gradient of 0-3 μM under in vitro calcification-induced culture conditions: NC group represents the control group with conventional non-calcification-induced culture medium, OM represents the calcification-induced culture medium, and OM+ginsenoside F4 represents the ginsenoside F4-treated group. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0023] Figure 1 D is a graph showing the changes in ALP and RUNX2 protein levels of human valvular interstitial cells treated with ginsenoside F4 at a concentration gradient of 0-3 μM under in vitro calcification-induced culture conditions: the NC group represents the control group with conventional non-calcification-induced culture medium conditions, OM represents the calcification-induced culture medium conditions, and OM+ginsenoside F4 represents the ginsenoside F4-treated group.

[0024] Figure 1 E is a statistical graph showing the improvement of ALP protein levels in human valvular interstitial cells treated with ginsenoside F4 at a concentration gradient of 0-3 μM under in vitro calcification-induced culture conditions: the NC group represents the control group with conventional non-calcification-induced culture medium, OM represents the calcification-induced culture medium, and OM+ginsenoside F4 represents the ginsenoside F4-treated group. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0025] Figure 1 F is the statistical diagram of the changes in RUNX2 protein levels in human valvular interstitial cells treated with ginsenoside F4 in a concentration gradient of 0-3 μM under in vitro calcification-induced culture conditions: NC group represents the control group with conventional non-calcification-induced culture medium, OM represents the calcification-induced culture medium, and OM+ginsenoside F4 represents the ginsenoside F4-treated group. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0026] Figure 2A is a graph showing the changes in the level of cellular calcium salt deposition of human valvular interstitial cells treated with ginsenoside F4 at a concentration gradient of 0-3 μM under in vitro calcification-induced culture conditions: the NC group represents the control group with conventional non-calcification-induced culture medium conditions, OM represents the calcification-induced culture medium conditions, and OM+ginsenoside F4 represents the ginsenoside F4-treated group.

[0027] Figure 2 B is a statistical graph showing the improvement of calcium salt deposition levels of human valvular interstitial cells in vitro under calcification-induced culture conditions by treatment with ginsenoside F4 at a concentration gradient of 0-3 μM: the NC group represents the control group with conventional non-calcification-induced culture medium, OM represents the calcification-induced culture medium, and OM+ginsenoside F4 represents the ginsenoside F4-treated group. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0028] Figure 3 A is the effect of si-NC or si-EGFR treatment on the phosphorylation modification levels of PI3K and AKT proteins and the expression levels of ALP and RUNX2 proteins in human valvular interstitial cells with or without in vitro calcification induction culture conditions: the NC group represents conventional non-calcification induction culture medium conditions, and the OM represents calcification induction culture medium conditions.

[0029] Figure 3 B is a statistical diagram showing the changes in PI3K protein phosphorylation modification levels in human valvular interstitial cells treated with si-NC or si-EGFR in the presence or absence of in vitro calcification induction culture conditions: the NC group represents conventional non-calcification induction medium culture conditions, and the OM group represents calcification induction medium culture conditions. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0030] Figure 3C is a statistical diagram showing the changes in AKT protein phosphorylation modification levels in human valvular interstitial cells treated with si-NC or si-EGFR in the presence or absence of in vitro calcification induction culture conditions: the NC group represents conventional non-calcification induction medium culture conditions, and the OM group represents calcification induction medium culture conditions. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0031] Figure 3 D is a statistical diagram showing the changes in ALP protein expression levels of human valvular interstitial cells treated with si-NC or si-EGFR in the presence or absence of in vitro calcification induction culture conditions: the NC group represents conventional non-calcification induction medium culture conditions, and the OM group represents calcification induction medium culture conditions. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0032] Figure 3 E is a statistical diagram showing the changes in RUNX2 protein expression levels in human valvular interstitial cells treated with si-NC or si-EGFR in the presence or absence of in vitro calcification induction culture conditions: the NC group represents conventional non-calcification induction medium culture conditions, and the OM group represents calcification induction medium culture conditions. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0033] Figure 4 A is a drug affinity responsive target stability experiment to verify the ability of ginsenoside F4 to affect the tolerance of EGFR to proteinase K enzymatic reaction to test the binding ability of ginsenoside F4 to EGFR. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences are determined using a two-tailed unpaired t test.

[0034] Figure 4 B is a statistical graph of drug affinity responsiveness target stability experiment. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t test.

[0035] Figure 4C is a graph showing the changes in the phosphorylation modification levels of EGFR, PI3K and AKT proteins in human valvular interstitial cells treated with ginsenoside F4 at a concentration gradient of 0-3 μM under in vitro calcification-induced culture conditions: the NC group represents the control group with conventional non-calcification-induced culture medium conditions, OM represents the calcification-induced culture medium conditions, and OM+ginsenoside F4 represents the ginsenoside F4-treated group.

[0036] Figure 4 D is a statistical diagram showing the changes in the phosphorylation level of EGFR protein in human valvular interstitial cells treated with ginsenoside F4 at a concentration gradient of 0-3 μM under in vitro calcification-induced culture conditions: the NC group represents the control group with conventional non-calcification-induced culture medium, OM represents the calcification-induced culture medium, and OM+ginsenoside F4 represents the ginsenoside F4-treated group. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0037] Figure 4 E is a statistical graph showing the changes in the phosphorylation level of PI3K protein in human valvular interstitial cells treated with ginsenoside F4 at a concentration gradient of 0-3 μM under in vitro calcification-induced culture conditions: the NC group represents the control group with conventional non-calcification-induced culture medium, OM represents the calcification-induced culture medium, and OM+ginsenoside F4 represents the ginsenoside F4-treated group. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0038] Figure 4 F is the statistical diagram of the changes in the phosphorylation modification level of AKT protein in human valvular interstitial cells treated with ginsenoside F4 in a concentration gradient of 0-3 μM under in vitro calcification-induced culture conditions: the NC group represents the control group with conventional non-calcification-induced culture medium, OM represents the calcification-induced culture medium, and OM+ginsenoside F4 represents the ginsenoside F4-treated group. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0039] Figure 5A is a graph showing the effects of 3 μM concentration of ginsenoside F4 combined with or without EGFR agonist NSC 228155 on the phosphorylation modification levels of PI3K and AKT proteins and the expression levels of ALP and RUNX2 proteins in human valvular interstitial cells under in vitro calcification-induced culture conditions.

[0040] Figure 5 B is a statistical graph showing the changes in PI3K protein phosphorylation modification levels in human valvular interstitial cells under in vitro calcification induction culture conditions after treatment with 3 μM ginsenoside F4 combined with or without EGFR agonist NSC 228155. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0041] Figure 5 C is a statistical graph showing the changes in AKT protein phosphorylation modification levels in human valvular interstitial cells under in vitro calcification-induced culture conditions after treatment with ginsenoside F4 at a concentration of 3 μM or without EGFR agonist NSC 228155. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0042] Figure 5 D is a statistical graph showing the changes in ALP protein expression levels in human valvular interstitial cells under in vitro calcification induction culture conditions after treatment with 3 μM ginsenoside F4 combined with or without EGFR agonist NSC 228155. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0043] Figure 5 E is a statistical graph showing the changes in RUNX2 protein expression levels in human valvular interstitial cells under in vitro calcification-induced culture conditions after treatment with 3 μM ginsenoside F4 combined with or without EGFR agonist NSC 228155. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0044] Figure 6 A is a graph showing the changes in cellular calcium deposition levels of human valvular interstitial cells under in vitro calcification-inducing culture conditions after treatment with 3 μM concentration of ginsenoside F4 with or without EGFR agonist NSC 228155.

[0045] Figure 6 B is a statistical graph showing the effect of 3 μM ginsenoside F4 combined with or without EGFR agonist NSC 228155 on the level of calcium salt deposition in human valvular interstitial cells under in vitro calcification induction culture conditions. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0046] Figure 6 C is a graph showing the changes in calcium salt deposition levels in human aortic valve tissue explants under in vitro calcification induction culture conditions after treatment with 3 μM concentration of ginsenoside F4 with or without EGFR agonist NSC 228155.

[0047] Figure 6 D is a statistical graph showing the effects of 3 μM ginsenoside F4 combined with or without EGFR agonist NSC 228155 on alizarin red staining of human aortic valve tissue explants under in vitro calcification induction culture conditions. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0048] Figure 6 E is a statistical graph showing the effect of 3 μM ginsenoside F4 combined with or without EGFR agonist NSC 228155 on silver nitrate staining of human aortic valve tissue explants under in vitro calcification induction culture conditions. n = 3 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0049] Figure 7 A: Apoe fed with high cholesterol diet combined with ginsenoside F4 or normal saline - / - Echocardiographic results of male mice in the two groups after 24 weeks.

[0050] Figure 7B: Apoe fed with high cholesterol diet combined with ginsenoside F4 or normal saline - / - Statistical graph of changes in aortic valve peak flow velocity in male mice in two groups after 24 weeks. n = 10 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences are determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001.

[0051] Figure 7 C: Apoe fed with high cholesterol diet combined with ginsenoside F4 or normal saline - / - Statistical diagram of the changes in the mean transvalvular pressure gradient of the aortic valve in the two groups of male mice after 24 weeks. n = 10 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences are determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001.

[0052] Figure 7 D: Apoe fed with high cholesterol diet combined with ginsenoside F4 or normal saline - / - Changes in aortic valve calcium deposition in male mice in two groups after 24 weeks.

[0053] Figure 7 E: Apoe fed with high cholesterol diet combined with ginsenoside F4 or normal saline - / - Statistical diagram of aortic valve Alizarin red staining of male mice in two groups after 24 weeks. n = 10 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001.

[0054] Figure 7 F is the high cholesterol diet combined with ginsenoside F4 or normal saline feeding Apoe - / - Statistical graph of silver nitrate staining of aortic valves in two groups of male mice after 24 weeks. n = 10 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001.

[0055] Figure 8 A: Apoe fed with high cholesterol diet combined with ginsenoside F4 or normal saline - / -Figure 2. Changes in the protein phosphorylation levels of EGFR and PI3K on the aortic valve of male mice in the two groups after 24 weeks of treatment. n = 10 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences were determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001.

[0056] Figure 8 B: Apoe fed with high cholesterol diet combined with ginsenoside F4 or normal saline - / - Statistical diagram of changes in protein phosphorylation levels of EGFR on the aortic valve of male mice in the two groups after 24 weeks of treatment. n = 10 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences are determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001.

[0057] Figure 8 C: Apoe fed with high cholesterol diet combined with ginsenoside F4 or normal saline - / - Statistical diagram of changes in the protein phosphorylation level of PI3K on the aortic valve of male mice in the two groups after 24 weeks. n = 10 independent experiments, statistical values ​​are expressed as mean ± standard deviation, and all statistical differences are determined using a two-tailed unpaired t-test. ns represents no statistical difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001. Specific implementation method:

[0058] First, the experimental methods involved in the embodiments of the present invention are described in detail.

[0059] 1. Collection of Human Aortic Valve Tissue Samples

[0060] The non-calcified human aortic valves used in this experiment were obtained from patients who underwent heart transplantation for dilated cardiomyopathy at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Patients with rheumatic diseases, infective endocarditis, aortic valve or other valvular diseases were excluded. This study adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology and Wuhan Union Hospital. All participants were informed and consented. The collection of human aortic valve tissue strictly adhered to the principle of sterility. During the operation, three aortic valves were cut off and immediately rinsed with sterile cold saline, and refrigerated and stored in sterile DMEM high-glucose medium for subsequent experiments.

[0061] 2. Isolation and Culture of Human Aortic Valve Interstitial Cells

[0062] (1) Place the valve retrieved from the operating room in an ultra-clean workbench (pay attention to aseptic operation) and remove the DMEM high-glucose medium in the centrifuge tube.

[0063] (2) Transfer the valve to a six-well cell culture plate, pour sufficient sterile PBS containing double-antibody into each well, repeatedly blow up and down with a pipette or Pasteur pipette, gently rinse for 1 minute, transfer the valve to another well and continue washing, repeat 5 times.

[0064] (3) Transfer the washed valve to a sterile 15 mL centrifuge tube, add 10 mL of type I collagenase, and place in a 37°C incubator for digestion overnight.

[0065] (4) Take out the 15 mL centrifuge tube containing the digested cell turbid solution and centrifuge it at 1000 rpm at room temperature for 5 minutes.

[0066] (5) After centrifugation, discard the supernatant, then add 3 mL of high-glucose DMEM medium containing 10% fetal bovine serum (FBS), resuspend the cells, transfer to a sterile T25 cell culture flask, and place it flat in a 37°C incubator.

[0067] (6) When the cells have grown to cover more than 70% of the area, perform cell subculture. Aspirate the culture medium, add 1-2 mL of sterile PBS, and gently shake to wash 3 times. Aspirate the PBS, add 1 mL of 0.25% trypsin solution containing EDTA, digest for 1 minute, then aspirate the trypsin solution, add 6 mL of high-glucose DMEM culture medium containing 10% FBS and blow to completely blow off the cells. After observing under the microscope that no cells are attached to the wall, divide the cell solution equally into new sterile T25 or six-well plates as needed.

[0068] (7) According to experimental requirements, P2-P4 cells are usually selected for intervention and experimental operations.

[0069] 3. Induction of Osteogenic Differentiation in Vitro

[0070] In vitro osteogenic differentiation of human valvular interstitial cells and aortic valve tissue blocks was induced by osteogenic medium (OM medium), which contained 50 mg / mL vitamin C, 5 mmol / L β-glycerophosphate, 100 nmol / L dexamethasone, 2% fetal bovine serum, and high glucose medium (DMEM). Unless otherwise specified, human valvular interstitial cells were treated with OM for 3 days for protein and mRNA determination and for Alizarin red staining after 21 days; human aortic valve tissue blocks were treated with OM for 45-60 days for in vitro valvular osteogenic differentiation model. OM medium was changed every 3 days.

[0071] 4. Animal Model Establishment

[0072] Twenty 8-week-old Apoe - / -Male mice were divided into two groups (10 mice in each group): (1) saline group Apoe - / - Mice; (2) Ginsenoside F4 group Apoe - / - Mice (20 mg / kg of ginsenoside F4 was administered orally with saline as solvent, twice a week for 24 weeks). They were first fed with a normal diet. After the mice adapted to the environment, both groups were switched to a high-fat / high-cholesterol Western diet. After 24 weeks of continuous feeding, echocardiography was performed to evaluate the corresponding cardiac structure and function indicators. All operations strictly followed the operating guidelines formulated by the Animal Experiment Center of Huazhong University of Science and Technology and obtained animal ethics approval.

[0073] The chemical structure of ginsenoside F4 used in this example is as follows Figure 1 As shown in A.

[0074] 5. Western blot (WB)

[0075] (I) Extraction of total protein from human aortic valvular interstitial cells

[0076] (1) Discard the culture medium in the culture plate or dish, add pre-cooled PBS along the side wall with a pipette, shake gently to wash, repeat 3 times and then discard the PBS.

[0077] (2) Prepare RIPA mixed solution (RIPA: protein phosphatase inhibitor = 100:1), add 120 μL per well of the precooled six-well plate containing the cell sample, shake gently to mix, and place on ice for 30 minutes to allow for complete lysis.

[0078] (3) After the washed cells are scraped and dried and pre-cooled, the cells in the well plate are scraped off and the cell suspension is transferred into a pre-cooled sterile 1.5 mL EP tube.

[0079] (4) Place the EP tube in an ice bath and use an ultrasonic cell disruptor at 6-8% power, 2 seconds interval, 1 second working time, and sonicate 8-10 times.

[0080] (5) The protein lysate after sonication was balanced and placed in a pre-cooled high-speed centrifuge and centrifuged at 4°C, 12,000 rpm for 15 min.

[0081] (6) After centrifugation, use a pipette to carefully transfer the supernatant in the EP tube to a new pre-cooled 1.5 ml EP tube to obtain the total protein sample of the cells. Record the total volume of the protein solution. The sample can be used for subsequent protein experiments or stored in a -80°C refrigerator for long-term storage.

[0082] (ii) SDS-PAGE electrophoresis

[0083] (1) Use Bio-rad vertical electrophoresis apparatus and set up the electrophoresis gel according to the instructions.

[0084] (2) Design the protein loading sequence according to experimental requirements and add 3 μL of pre-stained protein standard marker to each loading well on both sides of the protein sample.

[0085] (3) After the sample is spotted, add electrophoresis solution to the outer tank of the electrophoresis apparatus until the liquid level exceeds the outer tank indicator line, cover the electrophoresis apparatus, set the voltage to 140-160V, and perform electrophoresis for 40-60 minutes.

[0086] (4) This experiment mainly uses precast gels with concentrations of 8%, 10%, or 4-20% according to the instructions of the precast gels of different concentrations.

[0087] (III) SDS-PAGE transfer

[0088] (1) This experiment used a 0.2 μm pore size PVDF membrane from Millipore. The PVDF membrane was cut into appropriate sizes according to experimental requirements, immersed in methanol for 30 seconds for activation, and then taken out and immersed in the equilibrium solution for 30 seconds.

[0089] (2) Take the required number of sponge gaskets, open the transfer splint, and place the sponge gaskets on the transfer splint in appropriate positions.

[0090] (3) Place the soaked PVDF membrane on the sponge gasket.

[0091] (4) After the electrophoresis is completed, rinse the gel plate with pure water (rinse the electrophoresis solution around the gel clean), pry it open, take out the gel and cover it on the surface of the PVDF membrane, and place the gel on the side with the larger molecular weight of the protein as close to the bottom of the transfer clip as possible.

[0092] (5) Use the exhaust roller moistened with pure water to gently expel the bubbles between the PVDF membrane and the gel, then place another sponge pad on the gel, close the transfer clip, and place it in the rapid transfer instrument. The positive to negative pole of the transfer system is: positive pole, sponge, PVDF membrane, gel, sponge, negative pole. After the correct installation is completed, start the program to start the transfer.

[0093] (6) According to the molecular weight of the target protein, set the transfer program with different time (the transfer time of proteins with a molecular weight below 20 kd is set to 10 minutes; the transfer time of proteins with a molecular weight between 20-180 kd is set to 16 minutes and 30 seconds; the transfer time of proteins with a molecular weight above 180 kd is set to 21 minutes).

[0094] (7) After the transfer is completed, remove the transfer clip and quickly place the PVDF membrane into the rapid blocking solution for blocking. Slowly shake the membrane on a shaker at room temperature for 10-15 minutes.

[0095] (IV) Antibody incubation

[0096] (1) After blocking, remove the PVDF membrane and wash it three times with the prepared TBST solution by rapid shaking on a shaker for 10 minutes each time.

[0097] (2) After washing, the PDVF membrane was cut into several strips of appropriate width according to the position of the protein marker and the molecular weight of the target protein, placed in a centrifuge tube containing the primary antibody, and placed in a 4°C refrigerator with slow shaking overnight.

[0098] (3) After overnight incubation with the primary antibody, remove the strips, place them in TBST solution, shake rapidly on a shaker at room temperature, and repeat the washing three times, each time for 10 minutes.

[0099] (4) After washing, discard the TBST solution and place the strips in freshly prepared secondary antibody solution. Incubate at room temperature with slow shaking for 1 hour. Prepare the secondary antibody solution according to the dilution ratio specified in the instructions (mix TBST solution containing 5% skim milk powder with HRP-labeled anti-mouse IgG or anti-rabbit IgG. Note that the species of the secondary antibody should be consistent with that of the primary antibody).

[0100] (5) After the secondary antibody incubation is completed, discard the liquid, place the strips in TBST solution, shake rapidly on a shaker, and repeat the washing of the membrane three times, each time for 10 minutes.

[0101] (V) Chemiluminescence

[0102] The exposure experiment was performed using Suzhou Xinsaimei Supersensitive ECL Kit. Before use, the A solution: B solution = 1:1 ratio was mixed. After the strip was spread out flat, the developer was evenly dripped on the strip and incubated for 1 minute. The incubated strip was then placed in the Shanghai Qinxiang chemiluminescence instrument to collect optical signals, collect data, and use Image J software for grayscale value analysis.

[0103] 6. Real-time quantitative PCR (RT-qPCR)

[0104] (I) Reverse transcription

[0105] The reverse transcription kit preparation system is as follows:

[0106] Reagents volume RNA 1μg / concentration 5XHiScriptⅡIQrtSuperMix 4 μL (final concentration 1x) RNase-free water Add to 20 μL Total volume 20μL

[0107] Use a gene amplification instrument to perform reverse transcription reaction at 37 degrees for 15 minutes, 85 degrees for 5 seconds, and maintain at 4 degrees. The obtained cDNA product can be used immediately for qPCR reaction or stored at -20 degrees.

[0108] (ii) Real-time PCR

[0109] Real-time PCR experiments using TB Green TM Premix Ex Taq TM Kit, prepare PCR reaction solution according to the following table (operate on ice throughout the process):

[0110] Reagents volume 2xChamQSYBRqPCRMasterMix(ROXPremix) 5μL cDNA 1μL 10uMPrimer-F 0.4μL 10uMPrimer-R 0.4μL RNAse-free water 3.2μL Total volume 10μL

[0111] Use real-time fluorescence quantitative qPCR instrument to perform PCR reaction according to the following conditions:

[0112]

[0113]

[0114] The primer sequences involved in this experiment are as follows:

[0115]

[0116] 7. Alizarin Red Staining

[0117] (I) Cells

[0118] (1) P2-P4 human aortic valve interstitial cells were digested from culture flasks and inoculated into twelve-well cell culture plates. After 24 hours of inoculation, the cells were observed under a microscope. When the cells were fused to 60-70%, intervention was performed according to experimental requirements.

[0119] (2) After cell intervention, the cell culture plate was taken out and placed in a sterile clean bench. The culture medium was aspirated and discarded. 0.5-1 ml of sterile PBS was added to each well. The plate was gently shaken and washed twice. 1 ml of osteogenic differentiation medium OM was added to each well. The plate was placed in a 37°C incubator for culture.

[0120] (3) The medium was changed every 3 days (OM, osteogenic differentiation medium, was used for medium change after intervention) for 21 days.

[0121] (4) Take out the cell culture plate, discard the culture medium, add 0.5-1 mL PBS to each well, shake gently to wash 3 times, discard the PBS, add 1 mL 4% paraformaldehyde fixative to each well, and let it stand for 15 minutes.

[0122] (5) After fixation, aspirate and discard the fixative solution, add 0.5-1 mL of double distilled water to each well, and gently shake to wash three times.

[0123] (6) Prepare 0.2% Alizarin Red dye solution.

[0124] (7) Add 500 μL of 0.2% Alizarin Red dye solution to each well of the cell culture plate and incubate at room temperature for 30 minutes.

[0125] (8) After incubation, discard the alizarin red dye solution, add 0.5-1 mL of double distilled water to each well, and gently shake and wash 5-6 times until the eluent changes from red to transparent and colorless.

[0126] (9) Observe under a microscope and take photos.

[0127] (II) Organization

[0128] (1) Paraffin-embedded sections of aortic valve were dewaxed and dehydrated, and then washed with ddH2O.

[0129] (2) Stain with 2% q Alizarin Red solution for 30-60 minutes.

[0130] (3) After staining, the samples were washed with ddH2O and then used for observation and statistics.

[0131] 8. siRNA-mediated silencing

[0132] (1) siRNA targeting EGFR (si-EGFR) and negative control siRNA without a clear target (si-NC) were designed and synthesized. The sequences are as follows (Sangon Biotechnology, Shanghai, China).

[0133]

[0134] (2) When the valvular interstitial cell confluence reached 70%, Lipofectamine 3000 (ThermoFisher Scientific, L3000075) was used to transfect si-EGFR or si-NC into the cells.

[0135] (3) The cells were cultured for 48 h before use in further experiments.

[0136] 9. Drug Affinity Responsive Target Stability (DARTS)

[0137] (1) P2-P4 valvular interstitial cells were cultured in a 10 cm cell culture dish. When the cell confluence reached 90%, proteins were extracted using BeyoLyticTM mammalian active protein extraction reagent (Beyotime Biotechnology, P0013M).

[0138] (2) The protein concentration of the lysate was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, P0011).

[0139] (3) The protein lysate was dispensed into 10 1.5 ml EP tubes, including 5 tubes for the treatment group and 5 tubes for the control group. Ginsenoside F4 or DMSO was added to the protein lysate of the two groups and incubated at 4°C for 1 h.

[0140] (4) According to the experimental design, proteinase K (Beyotime Biotechnology, ST533) was added to the protein lysate in gradients (0, 1:10000, 1:5000, 1:2000, 1:1000) and digested on ice for 5 min.

[0141] (5) Add protein loading buffer and heat in a 95°C metal bath for 10 min to terminate digestion and complete protein denaturation.

[0142] (6) The remaining target protein was then detected by western blot.

[0143] 10. Hematoxylin and Eosin staining

[0144] (1) Paraffin-embedded sections of aortic valve were dewaxed, dehydrated, and washed with ddH2O.

[0145] (2) Stain with modified Harris hematoxylin solution for 3 minutes and wash with ddH2O for 5 minutes.

[0146] (3) Counterstain with eosin alcohol solution for 1 minute.

[0147] (4) Dehydrated in ethanol and washed with xylene for 2 minutes before use for observation and statistics.

[0148] 11. Silver nitrate staining (Von Kossa staining)

[0149] (1) Dewaxing and dehydration of paraffin-embedded aortic valve sections.

[0150] (2) Incubate the sections with 5% silver nitrate solution for 30 min and then expose them to strong light for 1 h.

[0151] (3) After washing, the sections were treated with 5% sodium sulfate for 2 min and neutral red for 3 min.

[0152] (4) The deposition of calcium salts in the aortic valve leaflets can be observed under a light microscope and used for subsequent statistics.

[0153] 12. Immunofluorescence staining

[0154] (1) Paraffin-embedded sections of aortic valves were dewaxed, dehydrated, washed and sealed.

[0155] (2) The treated tissue sections were incubated with specific primary antibodies and fluorescently conjugated secondary antibodies.

[0156] (3) Use 4',6-diamidino-2-phenylindole (DAPI) to stain the nucleus.

[0157] (4) Seal the slides, observe and collect images under a fluorescence microscope in the dark for subsequent statistics.

[0158] 13. Data statistics and analysis

[0159] The experimental data were analyzed using GraphPad Prism 10 (GraphPad Software, Inc., CA, USA). The values ​​are expressed as mean ± standard deviation (SD). The normality of the data was determined by the Shapiro-Wilk normality test. If the data passed the normality test (alpha = 0.05), parametric tests such as unpaired t-test or ordinary one-way analysis of variance were used. If the data did not pass the normality test, nonparametric tests (Mann-Whitney test) were used. P < 0.05 was considered statistically significant.

[0160] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0161] Example

[0162] 1. Construction of a cell model of osteogenic differentiation of human valvular interstitial cells induced by calcification induction medium.

[0163] Human aortic valve interstitial cells with a culture density of about 70% were starved overnight with DMEM high-glucose medium containing 2% fetal bovine serum. On the second day, human valve interstitial cells were induced to differentiate into osteoblasts with a newly prepared osteogenic induction medium (50 mg / mL vitamin C, 5 mmol / L β-glycerophosphate, 100 nmol / L dexamethasone, and the solvent was high-glucose DMEM medium containing 2% fetal bovine serum). After 3 days of induction, the gene and protein expression changes of calcification marker genes RUNX2 and ALP in different treatment groups were detected. After 21 days of induction, the effects of different treatments on calcium salt deposition in valve interstitial cells were detected.

[0164] 2. Ginsenoside F4 inhibits the osteogenic differentiation phenotype of human valvular interstitial cells.

[0165] The human valvular interstitial cell osteogenic differentiation model constructed in step 1 above was used, and the human valvular interstitial cells were divided into three different treatment groups, namely the control group (normal culture medium), the OM group (osteogenic induction culture medium) and the OM + ginsenoside F4 group (osteogenic induction culture medium + ginsenoside F4). Ginsenoside F4 was purchased from MedChemExpress (https: / / www.medchemexpress.cn / ligustroflavone.html), product catalog HY-N2503, and the concentration was the final concentration in the culture medium. The chemical structure of ginsenoside F4 is as follows Figure 1 As shown in A. After 3 days of treatment, RNA and protein were extracted from each group to detect the expression changes of calcification marker genes ALP and RUNX2 gene and protein; after 21 days of treatment, each group was stained with Alizarin red to observe the changes in osteogenic differentiation phenotype.

[0166] Depend on Figure 1 BF showed that after OM treatment of human valvular interstitial cells for 3 days, the expression of ALP and RUNX2 genes and proteins was significantly upregulated compared with the control group. After adding ginsenoside F4 with a concentration gradient of 0-3μM to the OM treatment group, the upregulation of RUNX2 and ALP genes and proteins induced by OM culture medium was inhibited, and the effect was concentration-dependent.

[0167] Depend on Figure 2 AB shows that after 21 days of treatment in each group, alizarin red staining was performed on human valvular interstitial cells in each group. The results showed that after 21 days of treatment with OM, the positive staining of alizarin red in cells increased significantly compared with the control group. After adding ginsenoside F4 with a concentration gradient of 0-3 μM to the OM treatment group, the increase in the positive staining of alizarin red in cells induced by OM culture medium was inhibited, and the effect was concentration-dependent.

[0168] The above results indicate that ginsenoside F4 can inhibit the osteogenic differentiation phenotype transition of human valvular interstitial cells in vitro.

[0169] 3. Ginsenoside F4 can bind to the epidermal growth factor receptor (EGFR) and alleviate the enhanced effect of aortic valve osteogenic differentiation phenotype caused by a calcific environment through the PI3K / AKT signaling pathway.

[0170] Data query and computer prediction suggest that EGFR may play a major role in the regulation of calcific aortic valve disease by ginsenoside F4. At the same time, the PI3K / AKT signaling pathway is involved in this process. In order to explore whether the EGFR / PI3K / AKT pathway plays a role in the osteogenic differentiation of human valvular interstitial cells, si-EGFR was designed to inhibit the expression of intracellular EGFR. The results showed that (1) OM can activate the PI3K / AKT pathway; (2) Inhibition of EGFR expression can inhibit the activation of the PI3K / AKT pathway with or without OM culture medium induction; (3) Inhibition of EGFR expression can reduce the expression of OM-induced human valvular interstitial cell calcification-related proteins ALP and RUNX2, such as Figure 3 AE. This indicates that the EGFR / PI3K / AKT pathway plays a role in the osteogenic differentiation of human valvular interstitial cells induced by OM, and inhibiting the activation of this pathway can alleviate the osteogenic differentiation of human valvular interstitial cells.

[0171] Drug affinity responsiveness target stability experiments showed that ginsenoside F4 can enhance EGFR's tolerance to proteinase K enzymatic reactions, such as Figure 4 AB, which is consistent with the results of molecular docking simulated by computer, indicating that ginsenoside F4 has good binding ability with EGFR. At the same time, the changes in phosphorylation modification of core molecules of EGFR / PI3K / AKT pathway before and after ginsenoside F4 intervention were detected. The results showed that ginsenoside F4 could inhibit the expression of p-EGFR, p-PI3K and p-AKT induced by OM, and the effect was concentration-dependent, such as Figure 4 CF, indicating that ginsenoside F4 could inhibit OM-induced activation of the EGFR / PI3K / AKT pathway in human valvular interstitial cells.

[0172] To further verify the relationship between ginsenoside F4, EGFR / PI3K / AKT signaling pathway and aortic valve calcification, 2 μM EGFR agonist NSC 228155 (MCE, HY-101084) was added to the OM culture medium. The experimental results showed that ginsenoside F4 could reduce the expression of p-PI3K, p-AKT, ALP and RUNX2 in human valvular interstitial cells induced by OM, but this effect could be reversed by NSC 228155. Figure 5 AE. Alizarin red staining results showed that NSC 228155 could also reverse the reducing effect of ginsenoside F4 on OM-induced calcium salt deposition in human valvular interstitial cells. Figure 6 AB. The same intervention was applied to human aortic valve tissue pieces for 45-60 days, and the obtained tissue sections were stained with alizarin red and silver nitrate, and the same results were obtained: NSC 228155 could reverse the reducing effect of ginsenoside F4 on OM-induced aortic valve calcium salt deposition, such as Figure 6 The above results once again indicate that ginsenoside F4 can inhibit OM-induced osteogenic differentiation of human valvular interstitial cells and aortic valve calcification via the EGFR / PI3K / AKT pathway in vitro.

[0173] To investigate the regulatory effect of ginsenoside F4 on aortic valve calcification in vivo, Apoe - / - Male mice were given ginsenoside F4 or saline. After 24 weeks of high cholesterol diet feeding, the peak flow velocity of the aortic valve orifice and the mean transvalvular pressure gradient of the aortic valve between the two groups of mice showed significant differences under ultrasound, such as Figure 7 AC. The aortic valves of mice were sliced ​​and stained with alizarin red and silver nitrate. The results showed that the calcium salt deposition in the aortic valve leaflets of mice in the ginsenoside F4 group was significantly less than that in the normal saline group. Figure 7 DF. At the same time, immunofluorescence staining was performed on the sections of aortic valve leaflets of mice in the two groups. The results showed that compared with the saline group, the phosphorylation modification of EGFR and PI3K proteins in the aortic valve leaflets of mice in the ginsenoside F4 group was inhibited. Figure 8 The above results indicate that ginsenoside F4 can also inhibit the expression of Apoe1 in vivo induced by high cholesterol diet via the EGFR / PI3K / AKT pathway. - / - Aortic valve calcification in mice.

[0174] In conclusion, ginsenoside F4 can alleviate OM-induced osteogenic differentiation of human valvular interstitial cells and calcification of human aortic valve leaflets, as well as Apoe1+ / 2-hydroxy-1,2-digestive dysregulation induced by high cholesterol diet through the EGFR / PI3K / AKT pathway. - / - Aortic valve calcification in mice. Ginsenoside F4 is a natural compound with potential for the treatment of calcific aortic valve disease.

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the protection scope of the present invention. In addition, after reading the technical content of the present invention, those skilled in the art may make various changes, modifications or variations to the present invention, and all of these equivalent forms also fall within the protection scope defined by the present application.

Claims

1. Use of ginsenoside F4 in the preparation of a drug for treating calcific aortic valve disease, wherein the ginsenoside F4 is a subtype of oleanolic acid-type ginsenoside, with a CAS number of 181225-33-2 and a molecular formula of C 42 H 70 O 12 .

2. The use of ginsenoside F4 according to claim 1 in the preparation of a drug for treating calcific aortic valve disease, characterized in that: The ginsenoside F4 is used for preparing a drug for intervening in the osteoblastic differentiation of aortic valve interstitial cells.

3. The use of ginsenoside F4 according to claim 1 in the preparation of a drug for treating calcific aortic valve disease, characterized in that: The disease is high cholesterol diet-induced aortic valve calcification disease.

4. Use of ginsenoside F4 as an inhibitor of osteogenic differentiation of human valvular interstitial cells in vitro.

5. Use of ginsenoside F4 as a drug for regulating the EGFR / PI3K / AKT signaling pathway.

6. Use of ginsenoside F4 as an ALP and RUNX2 gene inhibitor.

7. A method for significantly improving the expression of valvular interstitial cells, human aortic valve tissue and Apoe after treatment with ginsenoside F4 - / - A cell-tissue-individual three-dimensional culture system for osteogenic differentiation of male mouse aortic valve, characterized in that: The human valvular interstitial cells were cultured for 3-21 days using a calcification medium containing ginsenoside F4, and human aortic valve tissue blocks were cultured for 45-60 days. The calcification medium was composed of: 3 μM ginsenoside F4, 50 mg / mL vitamin C, 5 mmol / L β-glycerophosphate, 100 nmol / L dexamethasone, 2% fetal bovine serum, and high-glucose medium DMEM. Ginsenoside F4 was used to induce the culture of 8-week-old Apoe - / - Male mice were gavaged at a concentration of 20 mg / kg with normal saline as the solvent, twice a week for a total of 24 weeks.

8. The method according to claim 7, wherein the ginsenoside F4 treatment significantly improves the expression of valvular interstitial cells, human aortic valve tissue and Apoe - / - A cell-tissue-individual three-dimensional culture system for osteogenic differentiation of male mouse aortic valve, characterized in that: The induced culture of human valvular interstitial cells and human aortic valve tissue pieces was carried out in a mixed gas of 5% carbon dioxide and 95% oxygen, and the culture temperature was kept constant at 37°C; Apoe - / - Male mice were cultured in a pathogen-free, temperature-controlled environment with a 12-h light-dark cycle.

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