Traditional Chinese medicine monomer composition for preventing and treating glucocorticoid osteoporosis and hypertension and application thereof

Abstract

The invention provides a traditional Chinese medicine monomer composition for preventing and treating glucocorticoid osteoporosis and hypertension. The composition comprises the following active ingredients: beta ecdysterone, pinoresinol diglucoside and syringin according to a molar concentration combination ratio of 1: 1: 2. The invention also discloses application of the traditional Chinese medicine monomer composition in preparation of medicines or health-care foods for preventing and treating glucocorticoid osteoporosis and hypertension. The traditional Chinese medicine monomer composition can effectively improve the spine mineralized bone mass, the spine bone density, the alkaline phosphatase activity, the blood vessel diameter and the blood pressure resistance of a zebrafish model with the osteoporosis and hypertension co-disease induced by glucocorticoid. In addition, by inhibiting oxidative stress and cell apoptosis caused by glucocorticoid, bone formation is promoted, angiogenesis is promoted, and then the osteoporosis and angiotension state is improved. The traditional Chinese medicine monomer composition can effectively prevent and relieve osteoporosis and vascular adverse reactions caused by glucocorticoid, and has good application and development prospects.

Description

Technical Field

[0001] The present invention relates to a Chinese medicinal monomer composition for preventing and treating glucocorticoid-induced osteoporosis and hypertension, and also relates to application of the Chinese medicinal monomer composition. Background Art

[0002] Glucocorticoids (GC) are widely used to treat a variety of diseases, including rheumatology and immunology. However, long-term or excessive GC use can lead to glucocorticoid-induced osteoporosis (GIOP) and glucocorticoid-induced hypertension (GIHT), becoming common clinical problems. Numerous current drug treatments have demonstrated some effectiveness, but they also pose side effects and long-term safety concerns. For example, bisphosphonates, commonly used in the treatment of osteoporosis (OP), can affect bone structure and increase the risk of osteomyelitis with long-term use. Various medications commonly used in the treatment of hypertension (HT) can increase the risk of stroke, heart failure, and other conditions. Studies have shown a significant association between hypertension and osteoporosis, emphasizing the clinical significance of addressing the mechanisms of their comorbidity (GIOP and HT).

[0003] In recent years, the ingredients and medicinal value of natural medicinal herbs have garnered widespread attention. Traditional Chinese medicine considers GC (Gastrointestinal tract Pathogenicity) to be pungent, dry, sweet, and warm. Long-term, excessive use can deplete kidney essence and is considered a "drug evil." GIOP (Gastrointestinal Hypertension) is often caused by "drug evil" and falls under the category of "bone atrophy," with kidney deficiency and essence deficiency as its core pathogenesis. GIHT (Gastrointestinal Hypertension) falls under the category of "vertigo," with kidney deficiency as the underlying cause. Therefore, current Traditional Chinese Medicine (TCM) treatments for both primarily focus on kidney tonification. Achyranthes bidentata, Eucommia ulmoides, and Viscum album (Mistletoe) enter the liver and kidney meridians and are key components of the classic TCM formula, Duhuo Jishe Tang (Duhuo Jishe Tang). Their combination can tonify the kidneys and replenish qi, replenishing kidney essence depleted by GC. Studies have shown that the Achyranthes bidentata-Eucommia ulmoides herbal pair has significant therapeutic effects on GIOP. Academician Tong Xiaolin often uses this Achyranthes bidentata-Eucommia ulmoides-Mistletoe trio formula in clinical practice to treat hypertension, with significant therapeutic efficacy.

[0004] In view of the role of Achyranthes bidentata, Eucommia ulmoides, and Viscum album in preventing and treating GIOP and HT, the key to the present invention is to find a combination of their single active ingredients and how to use them in a reasonable combination to achieve the best biological effect. Summary of the Invention

[0005] To address the aforementioned deficiencies in the prior art, the present invention provides a Chinese herbal monomer composition for preventing and treating glucocorticoid-induced osteoporosis and hypertension. This composition contains the active ingredients of Achyranthes bidentata, Eucommia ulmoides, and Viscum album, which can synergistically prevent and treat glucocorticoid-induced osteoporosis and hypertension. Furthermore, the present invention further provides medical uses of the aforementioned Chinese herbal monomer composition.

[0006] According to the embodiments, the technical solution of the present invention proposes a traditional Chinese medicine monomer composition for preventing and treating glucocorticoid-induced osteoporosis and hypertension, the active ingredients of which are β-ecdysone (βEcd, βEcd, with a simplified structural formula as shown in Formula 1), pinoresinol diglucoside (PDG, with a simplified structural formula as shown in Formula 2), and syringin (SRG, with a simplified structural formula as shown in Formula 3), with a molar ratio of βEcd:PDG:SRG = 1:1:2. βEcd is one of the main active ingredients of Achyranthes bidentata, PDG is one of the active ingredients of Eucommia ulmoides, and SRG is one of the active ingredients of Viscum album.

[0007]

[0008] The traditional Chinese medicine monomer composition is mixed with pharmaceutically acceptable drug excipients to form tablets, pills, granules, powders, pastes, powders, aqueous solutions, injections or nano materials.

[0009] The preparation method of the traditional Chinese medicine monomer composition of the present invention comprises the following steps: uniformly mixing beta-ecdysterone, pinoresinol diglucoside and syringin in a molar concentration of 1:1:2 to prepare a concentration of 1 μM-10 μM.

[0010] Subsequent examples will demonstrate that the present Chinese herbal monomer composition can effectively improve spinal mineralized bone mass, spinal bone density, alkaline phosphatase activity, and dorsal aorta diameter in a zebrafish model of glucocorticoid-induced osteoporosis and hypertension. Furthermore, by inhibiting glucocorticoid-induced oxidative stress and apoptosis, it promotes bone formation and angiogenesis, thereby improving osteoporosis and vascular tone. This Chinese herbal monomer composition can effectively prevent and alleviate glucocorticoid-induced osteoporosis and adverse vascular reactions, and has promising application development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1Alizarin red staining results of zebrafish in each group at 0, 48, and 96 hours after drug administration [Note: N = 10; (a) Alizarin red staining results of Ctrl and Dex groups at 3 dpf zebrafish at 2x magnification after drug administration at 0, 48, and 96 hours; (b) Alizarin red staining results of Ctrl and Dex groups at 3 dpf zebrafish at 5x magnification after drug administration at 0, 48, and 96 hours; (c) Alizarin red staining results of Ctrl and Dex groups at 4 dpf zebrafish at 2x magnification after drug administration at 0, 48, and 96 hours; (d) Alizarin red staining results of Ctrl and Dex groups at 4 dpf zebrafish at 5x magnification after drug administration at 0, 48, and 96 hours. (e) Total area of ​​alizarin red staining in 4 dpf zebrafish at 96 hours after drug administration; (f) Total brightness of alizarin red staining in 4 dpf zebrafish at 96 hours after drug administration. Compared with the Ctrl group, **P<0.001].

[0012] Figure 2 The results of the vascular diameter images of each group of zebrafish after 0h, 48h, and 96h of drug administration [Note: N=10; (a): The vascular diameter images of each group of 3dpf zebrafish after 0h, 48h, and 96h of drug administration; (b): The vascular diameter images of each group of 4dpf zebrafish after 0h, 48h, and 96h of drug administration; (c) Compared with the Ctrl group, the 3dpf drug-treated group, *P<0.05; **P<0.001; compared with the Ctrl group, the 4dpf drug-treated group, ##P<0.001].

[0013] Figure 3 UPLC-Q-TOF / MS correlation spectra of the Achyranthes bidentata-Eucommia ulmoides-Mistletoe compound [Note: (a) UPLC-HRMS base peak ion chromatogram (BPC) of the Achyranthes bidentata-Eucommia ulmoides-Mistletoe compound sample in negative ion mode; (b) UPLC-HRMS base peak ion chromatogram (BPC) of the Achyranthes bidentata-Eucommia ulmoides-Mistletoe compound sample in positive ion mode; (c) UPLC UV chromatogram of the Achyranthes bidentata-Eucommia ulmoides-Mistletoe compound sample at UV254nm].

[0014] Figure 4 Venn diagram of GIOP and GIHT disease-related genes and potential therapeutic targets of Achyranthes bidentata, Eucommia ulmoides, and Viscum album [Note: (a) Merged Venn diagram of GIOP disease-related genes retrieved from DrugBank, GeneCards, PharmGkb, and OMIM databases; (b) Merged Venn diagram of GIHT disease-related genes retrieved from DrugBank, GeneCards, PharmGkb, and OMIM databases. (c) Merged Venn diagram of targets of the active ingredients of Achyranthes bidentata, Eucommia ulmoides, and Viscum album and GIOP disease-related genes; (d) Merged Venn diagram of targets of the active ingredients of Achyranthes bidentata, Eucommia ulmoides, and Viscum album and GIHT disease-related genes].

[0015] Figure 5The "active ingredient-potential target" network of Achyranthes bidentata-Eucommia ulmoides-Mistletoe for the treatment of GIOP and GIHT [Note: (a) The "active ingredient-potential target" network of Achyranthes bidentata-Eucommia ulmoides-Mistletoe for the treatment of GIOP; (b) The "active ingredient-potential target" network of Achyranthes bidentata-Eucommia ulmoides-Mistletoe for the treatment of GIHT; red circles: active ingredients of Eucommia ulmoides; green circles: active ingredients of Mistletoe; purple circles: active ingredients of Achyranthes bidentata; yellow squares: GIOP targets; blue squares: GIHT targets; lines: interactions between active ingredients and potential targets; the larger the node volume on the right, the larger its degree value].

[0016] Figure 6 Screening of core potential targets for Achyranthes bidentata-Eucommia ulmoides-Mistletoe in the treatment of GIOP and GIHT [Note: (a) PPI network, core target screening process and result diagram for Achyranthes bidentata-Eucommia ulmoides-Mistletoe in the treatment of GIOP; (b) PPI network, core target screening process and result diagram for Achyranthes bidentata-Eucommia ulmoides-Mistletoe in the treatment of GIHT].

[0017] Figure 7 GO functional analysis of potential targets of Achyranthes bidentata-Eucommia ulmoides-Mistletoe in the treatment of GIOP and GIHT [Note: (a, b) GO functional analysis of potential targets of Achyranthes bidentata-Eucommia ulmoides-Mistletoe in the treatment of GIOP; (c, d) GO functional analysis of potential targets of Achyranthes bidentata-Eucommia ulmoides-Mistletoe in the treatment of GIHT].

[0018] Figure 8 KEGG pathway enrichment analysis of potential targets of Achyranthes bidentata-Eucommia ulmoides-Mistletoe for the treatment of GIOP and GIHT [Note: (a, b) KEGG pathway enrichment analysis of potential targets of Achyranthes bidentata-Eucommia ulmoides-Mistletoe for the treatment of GIOP; (c, d) KEGG pathway enrichment analysis of potential targets of Achyranthes bidentata-Eucommia ulmoides-Mistletoe for the treatment of GIHT].

[0019] Figure 9 Effects of candidate active ingredients from mistletoe on the total area (Area) of bone mineralization, total brightness (SumBrightness), and vessel diameter (Vessel Diameter) in a zebrafish model [Note: (a) Histogram of total bone mineralization area (Area) after treatment with different monomer concentrations in a zebrafish model, N = 9-10; (b) Histogram of total brightness (SumBrightness) of mineralized bone staining in a zebrafish model, N = 9-10; (c) Histogram of vessel diameter (Vessel Diameter) in a zebrafish model, N = 8-10. Compared with Ctrl, **P < 0.001; compared with Dex, #P < 0.05, ##P < 0.001].

[0020] Figure 10Results of factorial analysis and histogram of the total area of ​​zebrafish bone mineralization (Area) [Note: (a, b) ANOVA results of the total area of ​​zebrafish bone mineralization (Area), Table A: SRG, Table B: RMZ; (c) Histogram of the total area of ​​zebrafish bone mineralization (Area). N = 9. Compared with Ctrl, **P < 0.001; compared with Dex, #P < 0.05; ##P < 0.001. T11: 10μM SRG+10μM RMZ; T12: 10μM SRG+2μM RMZ; T13: 10μM SRG; T21: 2μM SRG+10μM RMZ; T22: 2μM SRG+2μM RMZ; T23: 2μM SRG; T31: 10μM RMZ; T32: 2μM RMZ; T33: Except adding 10 μM Dex, 1 μM βEcd, and 1 μM PDG, no additional SRG and RMZ are added].

[0021] Figure 11 Results of factorial analysis and histograms of the total brightness (Sum Brightness) of zebrafish mineralized bone staining. [Note: (a, b) ANOVA results of zebrafish mineralized bone Sum Brightness, Table A: SRG, Table B: RMZ; (c) Histogram of zebrafish mineralized bone Sum Brightness results. N = 9. Compared with Ctrl, **P < 0.001; compared with Dex, #P < 0.05; ##P < 0.001.] T11: 10μM SRG+10μM RMZ; T12: 10μM SRG+2μM RMZ; T13: 10μM SRG; T21: 2μM SRG+10μM RMZ; T22: 2μM SRG+2μM RMZ; T23: 2μM SRG; T31: 10μM RMZ; T32: 2μM RMZ; T33: except for the addition of 10μM Dex, 1μM βEcd, and 1μM PDG, no additional SRG and RMZ were added].

[0022] Figure 12Results of factorial analysis and histograms of zebrafish alkaline phosphatase (ALP) levels. [Note: (a, b) ANOVA results of zebrafish ALP levels, Table A: SRG, Table B: RMZ; (c) Histogram of zebrafish ALP levels. N = 8. Compared with Ctrl, **P < 0.001; compared with Dex, #P < 0.05; ##P < 0.001.] T11: 10μM SRG+10μM RMZ; T12: 10μM SRG+2μM RMZ; T13: 10μM SRG; T21: 2μM SRG+10μM RMZ; T22: 2μM SRG+2μM RMZ; T23: 2μM SRG; T31: 10μM RMZ; T32: 2μM RMZ; T33: Except for adding 10 μDex, 1 μM βEcd, and 1 μM PDG, no additional SRG and RMZ are added].

[0023] Figure 13 Zebrafish Vessel Diameter Factorial Analysis Results and Histograms [Note: (a, b) Zebrafish Vessel Diameter Variance Analysis Results, Table A: SRG, Table B: RMZ; (c) Zebrafish Vessel Diameter Results Histogram. N = 9-10, compared with Ctrl, **P < 0.001; compared with Dex, ## P<0.001. 11 :10μM SRG+10μM RMZ; T 12 :10μM SRG+2μM RMZ; T 13 :10μM SRG;T 21 :2μM SRG+10μM RMZ; T 22 :2μM SRG+2μM RMZ; T 23 :2μM SRG;T 31 :10μM RMZ; T 32 :2μM RMZ; T 33 : In addition to the addition of 10 μM Dex, 1 μM βEcd, and 1 μM PDG, no SRG and RMZ were added].

[0024] Figure 14Histograms of the mRNA expression of related genes in each zebrafish group [Note: (a) Statistical graph of the relative mRNA expression of bone formation-related genes (GSK3β, Bmp2b, Runx2a); (b) Statistical graph of the relative mRNA expression of angiogenesis-related genes (NF-κB, VEGFAa, FLT1); (c) Statistical graph of the relative mRNA expression of oxidative stress-related genes (FOXO3a, FOXO3b) and apoptosis-related gene (Baxa); N = 3, *P < 0.05 compared with Ctrl; #P < 0.05 compared with Dex. βE-P: βEcd-PDG; βE-PS: βEcd-PDG-SRG].

[0025] Figure 15 Western blot analysis of the expression of proteins related to bone formation and angiogenesis in zebrafish in each group [Note: (a) Western blot bands of each protein expression; (bf) Statistical graphs showing the ratio of the grayscale value of the Akt1, GSK3β, β-Catenin, AMPK, and NF-κB protein bands to the grayscale value of the internal reference protein, with the results of the Ctrl group normalized to 1; N = 3, comparison between the two groups, *P < 0.05. βE-P: βEcd-PDG; βE-PS: βEcd-PDG-SRG].

[0026] Figure 16 Western blot analysis of oxidative stress-related protein expression in zebrafish of each group [Note: (a) Western blot band diagram of each protein expression; (b) Statistical diagram of the ratio of the grayscale value of the p-Ask1, JNK, p-JNK, and p-FOXO3a protein bands to the grayscale value of the internal reference protein, with the results of the Ctrl group normalized to 1; N = 3, comparison between the two groups, *P < 0.05. βE-P: βEcd-PDG; βE-PS: βEcd-PDG-SRG].

[0027] Figure 17 Western blot analysis of apoptosis-related protein expression in each group of zebrafish [Note: (a) Western blot bands of each protein expression; (b) Statistical graphs showing the ratios of the grayscale values ​​of Caspase3, Bak, Bad, and p-Bad protein bands to the grayscale value of the internal reference protein, with the results of the Ctrl group normalized to 1; N = 3, comparison between the two groups, *P < 0.05. βE-P: βEcd-PDG; βE-PS: βEcd-PDG-SRG]. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. These embodiments should be understood to be merely illustrative of the present invention and not intended to limit the scope of protection of the present invention. After reading the contents described herein, those skilled in the art may make various changes or modifications to the present invention, and these equivalent variations and modifications also fall within the scope defined by the claims of the present invention.

[0029] In the following examples of the present invention, experimental animals and reagents not specifically indicated were commercially available or cultured according to common methods in the art.

[0030] 1. Pharmacodynamics Experimental Plan:

[0031] The zebrafish, a small tropical freshwater fish with a genetic similarity of up to 71% with humans, is widely used in high-throughput drug screening and research on musculoskeletal and cardiovascular diseases due to its unique experimental properties of small size, low cost, genetic tractability, and optical transparency. This study used the glucocorticoid dexamethasone (Dex)-induced GIOP & HT model in zebrafish. Ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) combined with network pharmacology and factorial design experiments was used to explore the optimal combination of active ingredients from Achyranthes bidentata, Eucommia ulmoides, and Viscum album for the prevention and treatment of GIOP & HT in zebrafish, as well as their associated mechanisms of action.

[0032] 1. Construction of a dual phenotype zebrafish model of glucocorticoid-induced osteoporosis and hypertension (GIOP&HT).

[0033] Zebrafish were reared in a dedicated zebrafish culture system at a temperature of 28°C and a 14-h:10-h light / dark cycle according to the fifth edition of the zebrafish book. The juvenile zebrafish used in the experiment were spawned by mating adult male and female zebrafish (female:male = 1:1). The day of spawning was designated as 0 dpf (Days post fertilization). The collected eggs were placed in a culture dish containing embryo medium (EM, composed of 13.7 mmol / L NaCl, 0.54 mmol / L KCl, 0.025 mmol / L Na2HPO4·7H2O, 0.044 mmol / L KH2PO4, 0.10 mmol / L MgSO4·7H2O, 0.42 mmol / L NaHCO3, 0.13 mmol / L CaCl2, pH = 7.2) and cultured in a light incubator at 28.5 ± 0.5 ° C for subsequent experiments. Impurities in the dish needed to be discarded and EM was replaced every day.

[0034] Wild-type zebrafish at 3dpf or 4dpf were treated with 10μM dexamethasone (Dex) for 0h, 48h, and 96h, respectively. Alizarin red staining and vascular diameter detection were used to assess bone mineralization and blood pressure in the zebrafish to obtain the optimal dosing period for constructing the GIOP&HT zebrafish model.

[0035] 1.1 Alizarin red staining to detect bone mineralization and bone density

[0036] Alizarin red is a calcium ion dye that can be used to quantitatively analyze bone mineralization. Zebrafish were sacrificed at low temperature, fixed with 4% paraformaldehyde for 2 hours, dehydrated with 50% ethanol for 15 minutes, and incubated with 2.5 μg / mL alizarin red staining solution (dissolved and diluted in 1% KOH) for 17-18 hours. The zebrafish were washed with PBS buffer and placed under a fluorescent inverted stereomicroscope and adjusted in position. Images were acquired using NIS-Elements F4.60.00 software. The area and cumulative brightness of the alizarin red-stained region were then calculated using the professional image analysis software NIS-Elements BR Analysis 4.30.00 to reflect the amount of bone mineralization and bone density in each group of zebrafish.

[0037] 1.2 Zebrafish blood vessel diameter detection

[0038] Live zebrafish were placed on a glass slide and observed under a microscope (10×) after adjusting their body position. Blood flow in the dorsal aorta (DA) of the zebrafish was recorded using the video acquisition software View Point, and the DA blood vessel diameter was measured using the zebrafish blood flow analysis software Zebra Blood (Scale: 996.541 pixels / mm).

[0039] 2. Prediction and screening of active ingredients in Achyranthes bidentata, Eucommia ulmoides, and Viscum album

[0040] UPLC-Q-TOF-MS combined with network pharmacology was used to predict the active ingredients in Achyranthes bidentata, Eucommia ulmoides, and Viscum album for GIOP and HT. Based on this established model, a zebrafish model of GIOP was treated with various concentrations (50 μM, 10 μM, and 2 μM) of the candidate ingredients for 96 hours. Alizarin red staining and blood flow analysis were performed, and images were taken under a stereomicroscope to analyze the total area of ​​bone mineralization, cumulative brightness, and vessel diameter. The optimal concentration of each active ingredient in the intervention model was determined.

[0041] 2.1 Identification of Achyranthes bidentata-Eucommia ulmoides-Viscum album components using UPLC-Q-TOF-MS

[0042] 2.1.1 UPLC-Q-TOF-MS Test Solution Preparation

[0043] Prepare an aqueous extract of Achyranthes bidentata, Eucommia ulmoides, and Viscum album (45g each), concentrate to 150mL, and freeze at -80°C. Use a freeze dryer to produce a lyophilized powder of the traditional Chinese medicine. After dissolving, filter the powder through a 0.22μm microporous filter membrane (PES material) before use in the experiment. Accurately weigh 3g of the lyophilized powder and dissolve it in 5mL of first-grade pure water. Transfer 0.5mL to a 2mL centrifuge tube and add 1.5mL of 50% methanol. Shake well and centrifuge at 12,000 revolutions per minute (rpm) for 5 minutes. Collect the supernatant to obtain the test solution.

[0044] 2.1.2 Chromatographic conditions

[0045] Chromatographic column: Waters ACQUITY UPLC HSS T3 (2.1×150 mm, 1.8 μm); column temperature: 30°C; flow rate: 0.3 mL / min; detection wavelength: 254 nm, 190-400 nm; recording time: 90 min; injection volume: 2 μl; mobile phase ratio: phase A: acetonitrile, phase B: 0.1% formic acid aqueous solution, gradient see Table 1.

[0046] Table 1. Mobile phase gradient

[0047]

[0048] 2.1.3 Mass spectrometry conditions

[0049] Mass spectrometry detection mode: ESI-Negative / Positive ion mode; mass spectrometry parameters are shown in Table 2.

[0050] Table 2. Mass spectrometry parameters

[0051]

[0052] 2.2 Prediction of the active ingredients and mechanisms of action of Achyranthes bidentata-Eucommia ulmoides-Mistletoe for the prevention and treatment of GIOP and GIHT based on UPLC-Q-TOF-MS results using network pharmacology analysis

[0053] 2.2.1 Main components of Achyranthes bidentata-Eucommia ulmoides-Mistletoe and their targets

[0054] The main components of Achyranthes bidentata, Eucommia ulmoides, and Viscum album identified by LC-MS and their targets were retrieved using the TCMS database, and the obtained targets were standardized using the Unipro protein database. The species was limited to "Human" and the filtering condition was "Reviewed". The active components of Achyranthes bidentata, Eucommia ulmoides, and Viscum album and the targets of the corresponding active components were obtained.

[0055] 2.2.2 GIOP and GIHT disease-related genes

[0056] DrugBank, GeneCards, OMIM, and PharmGk databases were used to search for GIOP and GIHT disease-related genes, using the search terms "glucocorticoid-induced osteoporosis" and "glucocorticoid-induced hypertension," respectively. The targets obtained from each database were merged and duplicates were removed.

[0057] 2.2.3 Construction of the “Active Ingredients-Potential Targets” Network for Achyranthes bidentata-Eucommia ulmoides-Mistletoe in the Treatment of GIOP and GIHT

[0058] Using R 4.3.1 software, the targets of the active ingredients of Achyranthes bidentata, Eucommia ulmoides, and Viscum album were intersected with GIOP and GIHT disease-related genes, identifying potential targets for the Achyranthes bidentata-Eucommia ulmoides-V. viscosa compound for the treatment of GIOP or GIHT. The active ingredient and potential target information was imported into Cytoscape 3.8.0 software to construct an "active ingredient-potential target" network for the compound in the treatment of GIOP and GIHT, respectively.

[0059] 2.2.4 Screening of core potential targets for Achyranthes bidentata-Eucommia ulmoides-Mistletoe in the treatment of GIOP and GIHT

[0060] Protein-protein interaction networks (PPIs) of potential targets were constructed using the STRING database, with the minimum interaction score set at 0.900. Core targets were screened out by calculating the node scores using Cytoscape 3.8.0 software and the CytoNCA plug-in.

[0061] 2.2.5 GO (Gene Ontology) function and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis

[0062] GO function enrichment and KEGG pathway enrichment analysis were performed on potential targets using R language and R 4.3.1 software.

[0063] 3. Screening of the optimal combination of Achyranthes bidentata, Eucommia ulmoides, and Viscum album active ingredients for the prevention and treatment of GIOP & HT in zebrafish models

[0064] Previously, a combination of β-ecdysone (βEcd), an active ingredient from Achyranthes bidentata, and pinoresinol diglucoside (PDG), an active ingredient from Eucommia ulmoides, was shown to effectively intervene in GIOP by promoting bone formation (Chinese Patents CN111973605B, ZL201910422112.9). Therefore, this study focused on identifying the optimal combination of active ingredients from mistletoe with βEcd-PDG. Bioinformatics analysis previously identified candidate active ingredients from mistletoe with preventive and therapeutic effects on GIOP and GIHT as rhamnazin (RMZ), syrigin (SRG), and 3'-methyleriodictyol (also known as homoeriodictyol, HED).

[0065] 3.1 Effectiveness testing and optimal concentration detection of candidate active ingredients from mistletoe

[0066] The effectiveness of the three active ingredients mentioned above and the optimal concentrations were explored using a zebrafish model. The dosage concentrations are shown in Table 3. After administration, the amount of bone mineralization, bone density, and blood vessel diameter of the zebrafish were measured.

[0067] Table 3. RMZ, SRG, and HED dosing groups

[0068]

[0069] 3.2 Screening the optimal combination of Achyranthes bidentata-Ulcommia ulmoides-Viscum album active ingredients for the prevention and treatment of zebrafish GIOP & HT based on factorial design

[0070] A factorial design approach was used, with βEcd-PDG as loading factors and varying concentrations of candidate active ingredients from mistletoe as influencing factors. Alizarin red staining, alkaline phosphatase (ALP) activity, and vascular diameter were used to identify the optimal combination of active ingredients from Achyranthes bidentata, Eucommia ulmoides, and Viscum album. The factors and levels of the factorial design are shown in Table 4. In addition to the addition of 10 μM Dex, 1 μM βEcd, and 1 μM PDG, the respective drug groups in the table were added to assess bone mineralization, bone density, and vascular diameter in zebrafish.

[0071] Table 4 2×3 two-factor factorial design table L4(2 3 )

[0072]

[0073] Note: Level 1: 10 μM; Level 2: 2 μM; Level 3: 0.

[0074] 4. Mechanisms of the optimal combination for the prevention and treatment of GIOP and HT in zebrafish models

[0075] Based on the results of the factorial design experiment, the relative mRNA expression levels of bone formation-related genes (GSK3β, Bmp2b, Runx2a), angiogenesis-related genes (NF-κB, VEGFAa, FLT1), oxidative stress-related genes (FOXO3a, FOXO3b), and apoptosis-related gene (Baxa) were detected by RT-qPCR. The protein expression levels of bone formation-related genes (Akt1, GSK3β, β-Catenin), angiogenesis-related genes (AMPK, NF-κB), oxidative stress-related genes (p-Ask1, JNK, p-JNK, p-FOXO3a), and apoptosis-related genes (Caspase3, Bak, Bad, p-Bad) were detected by Western-blot. The optimal combination of Achyranthes bidentata-Ulcommia ulmoides-Viscum album active ingredients and its improvement effect on the abnormal changes of the above factors were observed.

[0076] 4.1 RT-qPCR detection of zebrafish-related gene mRNA expression

[0077] The zebrafish were killed at low temperature, washed with cold phosphate-buffered saline (PBS), and total RNA was extracted by Trizol method. After RNA concentration was tested, RT reverse transcription was performed to cDNA and stored at -20℃ for later use. qPCR reaction was performed using real-time fluorescence quantitative gene amplification instrument (ABI 7500, Applied Biosystems). First, the cDNA was diluted stepwise to prepare the standard curve of each primer to verify the quality of the primer. Then, the PCR of each primer was run. The computer software was set up with the following program: 95℃, 1min; 40 cycles: 95℃, 5s; 58℃, 15s; 72℃, 30s; after the program was completed, the results were exported and analyzed using 2 -△△CT Gene expression levels were calculated and statistical analysis was performed.

[0078] 4.2 Western Blot Detection of Zebrafish Related Gene Protein Expression

[0079] Zebrafish were sacrificed at low temperature, washed with cold PBS, added to the lysis buffer, and sonicated in an ice bath. The cells were then placed on ice for 30 minutes. After complete lysis, the cells were centrifuged (15,000 g, 4°C, 25 minutes). The supernatant was collected for protein analysis, and the protein was denatured at 95°C for 10 minutes. Polyacrylamide gel electrophoresis (SDS-PAGE, 10% separating gel and 4% stacking gel) was performed. After transfer and blocking, the cells were incubated with the primary antibody overnight, washed thoroughly with TBST buffer, and then incubated with the corresponding secondary antibody and washed. The cells were developed using enhanced chemiluminescence (ECL), exposed on a gel image analyzer, and photographed. Grayscale values ​​were analyzed using Image J software (IJ1.46r).

[0080] 2. Experimental results:

[0081] 1. Effective Construction of a GIOP & HT Dual Phenotype Zebrafish Model

[0082] like Figure 1 、 Figure 2 As shown, 10 μM dexamethasone (Dex) treatment of 4 dpf zebrafish for 96 hours significantly reduced the area of ​​mineralized bone in the zebrafish spine, bone density, and blood vessel diameter, with statistically significant differences (P < 0.01). This indicates that 96 hours of treatment of 4 dpf zebrafish with 10 μM Dex can effectively establish a GIOP & HT zebrafish model.

[0083] 2. Composition Analysis of Achyranthes Bidentatae-Ulcommia-Viscum Albiflorum

[0084] The Achyranthes bidentata-Eucommia-Mistletoe compound sample was analyzed using UPLC-Q-TOF / MS. The data acquisition software was Analyst TF 1.7.1, and the data processing software was Peakview 1.2. The mass spectrometry data were matched with the Natural Products HR-MS / MS Spectral Library 1.0 database. The compounds were initially screened based on the scores of each chromatographic peak. The compounds were further confirmed based on the primary and secondary information of each chromatographic peak. The relevant spectra are shown in Figure 3 Compounds not included in the database were identified based on literature reports, mass spectrometry fragmentation patterns, etc. Based on the multi-level mass spectrometry information of the samples, combined with the natural product high-resolution mass spectrometry database and related literature [1-9] , 54 compounds were identified from the Achyranthes bidentata-Eucommia ulmoides-Mistletoe compound sample, as shown in Table 5.

[0085] 3. Network pharmacology analysis of the active ingredients, possible targets and pathways of Achyranthes bidentata-Eucommia ulmoides-Mistletoe in the treatment of GIOP and GIHT

[0086] 3.1 Main components and targets of Achyranthes bidentata-Eucommia ulmoides-Mistletoe

[0087] The main components of the Achyranthes bidentata-Eucommia-Mistletoe compound identified by UPLC-Q-TOF-MS were searched using the TCMSP database to obtain their targets. The targets were normalized using the Uniprot protein database, resulting in 20, 28, and 7 active components for Achyranthes bidentata, Eucommia ulmoides, and Mistletoe, respectively, for a total of 66 targets. Among the candidate components of Mistletoe, rhamnazin (RMZ), syrigin (SRG), and homoeriodictyol (HED) were identified.

[0088] 3.2 GIOP and GIHT disease-related genes

[0089] DrugBank, GeneCards, PharmGkb, and OMIM databases were used to search for GIOP and GIHT disease-related genes, and 29, 673, 459, and 12 GIOP-related genes and 29, 732, 517, and 24 GIHT-related genes were obtained, respectively. After merging the genes in each database and removing duplicates, 1083 GIOP disease-related genes and 1460 GIHT disease-related genes were obtained ( Figure 4 ).

[0090] 3.3 The “active ingredient-potential target” network of Achyranthes bidentata-Eucommia ulmoides-Mistletoe for the treatment of GIOP and GIHT

[0091] The targets of the active ingredients of Achyranthes bidentata, Eucommia ulmoides and Viscum album were intersected with the GIOP and GIHT disease-related genes, and 29 potential targets for the Niu Du Ji Sheng Xiao Fang in treating GIOP and 30 potential targets for treating GIHT were obtained. Figure 4 The "active ingredient-potential target" network of Achyranthes bidentata-Ulcommia ulmoides-Mistletoe for the treatment of GIOP and GIHT was constructed using Cytoscape 3.8.0 software. Figure 5 The nodes on the left side of the network represent the active ingredients of Achyranthes bidentata, Eucommia ulmoides, and Viscum album, while the nodes on the right represent potential disease targets. The lines connecting the nodes represent the interactions between the active ingredients and potential targets. The degree value indicates the number of edges connected to a node; a larger value indicates that the node plays a more important role in the network.

[0092] 3.4 Screening of core potential targets for Achyranthes bidentata-Eucommia ulmoides-Mistletoe in the treatment of GIOP and GIHT

[0093] The 29 GIOP potential targets and 30 GIHT potential targets were obtained through the STRING database and PPI networks were constructed ( Figure 6Potential targets were imported into Cytoscape 3.8.0 software, and the CytoNCA plug-in was used to calculate the node interaction scores. Core targets were screened based on betweenness centrality (BC), closeness centrality (CC), degree centrality (DC), eigenvector centrality (EC), local average connectivity (LAC), and network centrality (NC). The first screening conditions of GIOP were BC=5.298262848, CC=0.682926829, DC=15, EC=0.190305158, LAC=10.93333333, NC=12.88809524; the second screening conditions were BC=0.2, CC=1, DC=11, EC=0.29234764, LAC=9.818181818, NC=11. No genes were screened out in the second screening, so the first screening result was the final result, and 12 GIOP core targets were obtained, namely GSK3β, CASP3, IL6, ESR1, PTGS2, PPARG, RELA, HSP90AA1, BCL2, CDK2, HMOX1, and CASP9. The first screening conditions of GIHT were BC=5.856239267, CC=0.6374396135, DC=14, EC=0.1889883875, LAC=10.85128205, NC=12.44025974; the second screening conditions were BC=0.622222222, CC=1, DC=12, EC=0.286341935, LAC=10.5, NC=12. No genes were screened out in the second screening, so the first screening result was the final result, and 13 core GIHT targets were obtained, namely GSK3β, PPARG, CDK2, CASP3, ICAM1, ESR1, HMOX1, IL6, BCL2, PTGS2, NFKBIA, RELA, and HSP90AA1. The above core targets may be of great significance in the prevention and treatment of GIOP and GIHT by Achyranthes bidentata-Eucommia ulmoides-Mistletoe.

[0094] 3.5 GO functional analysis of potential targets of Achyranthes bidentata-Eucommia ulmoides-Mistletoe for the treatment of GIOP and GIHT

[0095] GO functional analysis of potential targets of GIOP enriched 1197 biological process (BP) entries, of which the top 10 were response to steroid hormone, epithelial cell apoptotic process, regulation of blood pressure, response to lipopolysaccharide, negative regulation of apoptotic signaling pathway, response to molecule of bacterial origin, response to UV, negative regulation of extrinsic apoptotic signaling pathway, response to temperature stimulus, and response to hypoxia; 13 cell component (CC) entries included organelle outer membrane, outer membrane, Bcl-2 family protein complex, and mitochondrial membrane. complex), nuclear pore complex, etc.; 106 molecular function (MF) entries, including DNA-binding transcription factor binding, RNA polymerase II-specific DNA-binding transcription factor binding, nuclear receptor activity, ligand-activated transcription factor activity, nuclear steroid receptor activity, NF-κB binding, etc. ( Figure 7 )。

[0096] GO functional analysis of potential targets of GIHT enriched 1209 biological process (BP) entries, of which the top 10 were response to steroid hormone, regulation of blood pressure, epithelial cell apoptotic process, response to lipopolysaccharide, negative regulation of apoptotic signaling pathway, response to molecule of bacterial origin, response to UV, negative regulation of extrinsic apoptotic signaling pathway, response to temperature stimulus, and intracellular receptor signaling pathway; 14 cell component (CC) entries, including caveolae, membrane rafts, organelle outer membrane, and mitochondrial membrane. membrane), outer membrane, Bcl-2 family protein complex, nuclear pore complex, etc.118 molecular function (MF) entries, including DNA-binding transcription factor binding, RNA polymerase II-specific DNA-binding transcription factor binding, nuclear receptor activity, ligand-activated transcription factor activity, nuclear steroid receptor activity, NF-κB binding, etc. (; Figure 7 ).

[0097] 3.6 KEGG enrichment analysis of potential targets of Achyranthes bidentata-Eucommia ulmoides-Mistletoe for the treatment of GIOP and GIHT

[0098] KEGG enrichment analysis of potential GIOP targets revealed 116 signaling pathways (P < 0.05). The top 30 pathways were ranked by significance. Figure 8 As shown, including apoptosis (Apoptosis-multiple species), lipid and atherosclerosis (Lipid and atherosclerosis), p53 signaling pathway (p53signaling pathway), IL-17 signaling pathway (IL-17signaling pathway), etc.

[0099] KEGG enrichment analysis of GIHT potential targets revealed 116 signaling pathways (P < 0.05). The top 30 pathways were ranked by significance. Figure 8 As shown, including lipid and atherosclerosis, IL-17 signaling pathway, apoptosis (Apoptosis-multiple species), p53 signaling pathway, etc.

[0100] In summary, after the components of the Achyranthes bidentata-Eucommia ulmoides-Viscum album aqueous extract were identified by UPLC-Q-TOF-MS, network pharmacology was used to predict the active ingredients effective for GIOP and GIHT and their potential targets and related pathways for the treatment of GIOP and GIHT. Common therapeutic targets for both diseases, such as RELA (NF-κB), CASP3 (Caspase-3), BCL2, and GSK3, as well as important GO functions or KEGG pathways, such as BCL-2 family protein complex, apoptosis-multiple species, NF-κB binding, and regulation of blood pressure, were predicted. This provides a reference for subsequent research on the mechanism of the effective active ingredients of this compound in preventing and treating GIOP and GIHT.

[0101] 4. Factorial design to screen the best combination of candidate active ingredients of Achyranthes bidentata, Eucommia ulmoides, and Viscum album

[0102] like Figure 9 The candidate components of mistletoe are rhamnazin (RMZ), syrigin (SRG), and homoeriodictyol (HED). The results showed that 10μM SRG, 2μM SRG, 10μM RMZ, and 2μM RMZ significantly improved the decrease in zebrafish spinal mineralized bone area, bone density, and blood vessel diameter caused by 10μM Dex (P < 0.05).

[0103] like Figure 10 、 Figure 11 、 Figure 12 、 Figure 13 As shown in the figure, the factorial design method was used to perform variance analysis on the results of zebrafish spine mineralized bone area, bone density, ALP activity and blood vessel diameter, and it was concluded that βEcd-PDG-SRG was the best combination, with statistically significant differences (P < 0.05). The optimal combination molar concentration ratio was βEcd:PDG:SRG = 1:1:2.

[0104] 5. The optimal combination improves bone formation and angiogenesis in zebrafish models, and effectively inhibits oxidative stress and apoptosis

[0105] like Figure 14As shown in the results, compared with the 10 μM Dex group, βEcd-PDG-SRG significantly increased the mRNA expression of Bmp2b, Runx2, NF-κB, VEGFAa, and FLT1 genes in the model zebrafish (P < 0.05), and significantly reduced the mRNA expression of GSK3β, FOXO3a, FOXO3b, and Baxa genes in the model zebrafish (P < 0.05), indicating that the βEcd-PDG-SRG active ingredient combination can effectively improve the zebrafish bone formation and angiogenesis inhibition caused by Dex, and effectively inhibit the oxidative stress and apoptosis caused by high-dose Dex, and the effect is better than the βEcd-PDG active ingredient combination.

[0106] like Figure 15 、 Figure 16 、 Figure 17 As shown in the figure, Western Blot test results showed that Dex significantly reduced the expression of Akt1, β-Catenin, NF-κB, p-JNK, p-FOXO3a, and p-Bad proteins in zebrafish (P < 0.05), and significantly increased the expression of GSK3β, AMPK, p-Ask1, JNK, Caspase3, Bak, and Bad proteins in zebrafish (P < 0.05); the βEcd-PDG-SRG active ingredient combination significantly increased the expression of Akt1, β-Catenin, and NF-κB in the model zebrafish , p-JNK, p-FOXO3a, and p-Bad protein expressions (P < 0.05), and weakened the expression of GSK3β, AMPK, p-Ask1, JNK, Caspase3, Bak, and Bad proteins in the model zebrafish (P < 0.05), indicating that βEcd-PDG-SRG can improve the inhibition of bone formation and angiogenesis in the model zebrafish caused by Dex, and improve the oxidative stress and apoptosis caused by Dex, and the improvement effect of βEcd-PDG-SRG is better than that of βEcd-PDG.

[0107] 3. Experimental Conclusions

[0108] 1. Treatment of 4dpf zebrafish with 10μM dexamethasone (Dex) for 96h significantly reduced bone mineralization and density, and reduced blood vessel diameter, effectively establishing a dual phenotype zebrafish model of glucocorticoid-induced osteoporosis and hypertension (GIOP&HT).

[0109] 2. The active ingredients of mistletoe, syringoside (SRG) and rhamnosine (RMZ), can improve the reduction of spinal mineralized bone mass, decreased spinal bone density and vascular tension in the GIOP&HT zebrafish model caused by high concentration of Dex (10μM).

[0110] 3. The factorial design method screened out the combination of β-ecdysterone-pinoresinol diglucoside-syringoside (βEcd-PDG-SRG, the active ingredients of Achyranthes bidentata, Eucommia ulmoides, and Viscum album, respectively) as an effective combination for intervening in the GIOP&HT zebrafish model, and the optimal combination molar concentration ratio was βEcd:PDG:SRG = 1:1:2.

[0111] 4. The βEcd-PDG-SRG active ingredient combination exerts a preventive and therapeutic effect on zebrafish GIOP&HT by promoting bone formation and angiogenesis, and its effect is better than the βEcd-PDG active ingredient combination. Its mechanism of action may be related to its intervention in glucocorticoid-induced oxidative stress and thus inhibition of cell apoptosis.

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Claims

1. A Chinese medicine monomer composition for preventing and treating glucocorticoid-induced osteoporosis and hypertension, characterized in that: The active ingredients of the traditional Chinese medicine monomer composition and the molar ratio thereof are beta-ecdysterone: pinoresinol diglucoside: syringin=1:1:

2.

2. The Chinese medicine monomer composition according to claim 1, characterized in that The traditional Chinese medicine monomer composition is mixed with pharmaceutically acceptable drug excipients to prepare tablets, pills, granules, powders, ointments, powders, injections, aqueous solutions, injections or nano materials.

3. Use of the Chinese medicine monomer composition according to claim 1 or 2 in the preparation of drugs for preventing and treating glucocorticoid-induced osteoporosis and hypertension.

4. Use of the Chinese medicinal monomer composition according to claim 1 or 2 in the preparation of health food for preventing and treating glucocorticoid-induced osteoporosis and hypertension.

5. The method for preparing the Chinese medicine monomer composition according to claim 1, characterized in that: β-ecdysterone, pinoresinol diglucoside and syringin are mixed evenly at a molar concentration of 1:1:2 to prepare a concentration of 1 μM-10 μM.

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