Application of gastrodin in treating aortic valve calcification

Abstract

The present invention relates to the field of prevention and / or treatment of aortic valve calcification. The inventors have found through research that gastrodin can effectively downregulate the expression of osteogenic differentiation marker genes BMP2, RUNX2, COL1A2, and SPP1 in valve interstitial cells, inhibit valve interstitial cell calcification, thereby inhibiting or delaying valve calcification, and further provide the use of gastrodin in the preparation of a drug for preventing and / or treating individual aortic valve calcification. This provides a new option for treating aortic valve calcification by non-surgical methods.

Description

Technical Field

[0001] The present invention relates to the field of prevention and / or treatment of aortic valve calcification, and in particular to the use of gastrodin in the prevention and / or treatment of aortic valve calcification. Background Art

[0002] The natural product Gastrodin (GAS) is a pyranoglucoside, an organic compound extracted from the dried root of Gastrodia elata, an orchid plant. It has multiple pharmacological effects, such as antiplatelet aggregation, neuroprotection, free radical scavenging and immune enhancement, but the effect of Gastrodin on aortic valve calcification has not been reported.

[0003] Calcification refers to the process in which a certain tissue in the human body necrotizes under the influence of some factors, and then calcium salts in the body are deposited in the necrotic focus, which limits the lesion and stabilizes it. In essence, calcification is a defensive reaction of the body to the lesion, which is conducive to the removal of necrotic foci and the disappearance of inflammation. Although most calcifications are a defensive reaction, some calcifications can lead to adverse consequences. For example, prostate calcification may make prostatitis difficult to treat and prone to recurrence.

[0004] Aortic valve calcification (CAVD) is a heart valve disease characterized by fibrosis, sclerosis, and calcium salt deposition in the aortic valve and surrounding tissues. It is one of the common cardiovascular diseases. The clinical manifestations of CAVD mainly include aortic valve stenosis and aortic valve regurgitation. In severe cases, it can lead to heart failure and sudden death. Its pathogenesis has not yet been fully elucidated.

[0005] The cells in the valve tissue mainly include valvular endothelial cells (VEC), human valvular interstitial cells (VIC), valve precursor cells, etc. In recent years, studies have shown that the osteogenic differentiation of VIC is a key link in the occurrence and development of CAVD. VIC is the main cellular component of the aortic valve and plays an important role in maintaining the structure and function of the valve. After VIC differentiates into osteoblasts, it can express bone differentiation-related proteins, such as bone morphogenetic protein (BMP) and bone matrix protein (OPN), leading to valve calcification. VIC osteogenic differentiation plays a key role in the occurrence and development of CAVD and is currently a potential target for the treatment of CAVD.

[0006] At present, it is believed that CAVD has reached a "hopeless point", and the main treatment for aortic valve calcification is surgery, such as aortic valve replacement, aortic decalcification, and percutaneous balloon valvuloplasty. However, surgery will inevitably bring higher risks to patients, such as high perioperative mortality and poor long-term effects, and will also increase the economic burden on patients' families. There are still many clinical trials to verify some promising drug interventions that can stop or slow down its progression.

[0007] Therefore, there is a strong need for non-surgical methods to effectively treat aortic valve calcification. Summary of the invention

[0008] The problem to be solved by the present invention is how to effectively treat or prevent aortic valve calcification in an individual.

[0009] According to the above needs, in a first aspect, the present invention provides the use of gastrodin in the preparation of a drug for preventing and / or treating aortic valve calcification in an individual.

[0010] In the present invention, gastrodin refers to gastrodin with CAS No. 62499-27-8 and molecular formula C 13 H 18 O7 substance; it is a natural compound extracted from propolis, its chemical structure is as follows Figure 1 As shown in A.

[0011] In the present invention, "prevention and / or treatment" means preventing and / or delaying the occurrence of a disease or condition or normalizing it.

[0012] In one embodiment, the final concentration of gastrodin is 10 μM-800 μM.

[0013] Furthermore, in a preferred embodiment, the final concentration of gastrodin is 10 μM-400 μM.

[0014] In a more preferred embodiment, the final concentration of gastrodin is 50-100 μM.

[0015] In one embodiment, the subject is a mammal, including but not limited to rats, mice, non-human primates, humans, dogs, cats, horses, cows, sheep, pigs, goats.

[0016] Preferably, the individual is a human.

[0017] In this regard, the beneficial effect of the present invention is that gastrodin can effectively down-regulate the expression of osteogenic differentiation marker genes BMP2, RUNX2, COL1A2, and SPP1 in valvular interstitial cells, inhibit valvular interstitial cell calcification, and thus inhibit or delay valvular calcification. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :Effect of GAS treatment on cell viability and anti-calcification evaluation of VIC. (A) Molecular structure of GAS; (B, C) Growth status and statistics of VIC after treatment with GAS at different final concentrations; (D) IC50 detection of GAS-treated VIC; (E, F) Alizarin red staining and semi-quantitative statistical results of VIC, VIC induced by OM osteogenics, GAS-treated group, and AGP-treated group (# indicates significant difference between OMI group and Control, * indicates significant difference between GAS or AGP-treated group and OMI group, p≤0.05);

[0019] Figure 2 :GAS can inhibit the expression of OM-induced osteogenic specific markers. (A) Real-time fluorescence quantitative PCR results showed that GAS can significantly inhibit the expression of OM-induced VIC osteogenic specific markers, OMI, OM-induced VIC group; GAS 50, GAS50μM treatment group; GAS100, GAS100μM treatment group; AGP, andrographolide 10μM treatment group. (B) Immunoblotting (Western blot) results showed that GAS can significantly inhibit the expression of OM-induced VIC osteogenic specific markers; (C) Semi-quantitative statistics of the brightness of the immunoblotting results in B; (D) Intracellular immunoblotting results showed that GAS can significantly inhibit the expression of OM-induced VIC osteogenic specific markers; (E) Semi-quantitative statistics of the brightness of the intracellular immunoblotting results in D;

[0020] Figure 3 :Multi-omics analysis and validation of GAS treatment under osteogenic induction conditions (A) Volcano map of differentially expressed genes (DEGs) in the OMI group relative to the control group; (B) Volcano map of DEGs in the GAS group relative to the OMI group; (C) Wayne interaction diagram of the DEGs in the two groups, showing that 405 DEGs were common in the two groups; (D) GO annotation of the above common DEGs showed that the main DEGs gene functions were enriched in cell growth and cell connection; (E) KEGG pathway analysis of the above common DEGs showed that they were mainly enriched in pathways such as cytokine interaction and calcium signal transduction; (F, G) Metabolomics analysis of the above samples showed that the differences mainly existed in the pyruvate metabolism and glycerolipid metabolism pathways, and the differences within the groups were relatively average, and the differences between the groups were significant; (H) Quantitative analysis of markers of pyruvate metabolism and glycerolipid metabolism, # indicates significant differences relative to the Con (Control) group, * indicates significant differences relative to the OMI group, p≤0.05.

[0021] Figure 4:GAS can inhibit the valvular calcification trend of mice (A) Von Kossa staining results of heart valve sections. Compared with the Control group, the valvular calcification in the HF group increased significantly; compared with the HF group, the Von Kossa positive rate was significantly reduced after GAS treatment at two doses. # indicates a significant difference relative to the Control group, * indicates a significant difference relative to the HF group, p≤0.05; (B) Semi-quantitative statistics in A. DETAILED DESCRIPTION

[0022] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0023] Example 1: Gastrodin inhibits calcification of human valvular interstitial cells

[0024] Human valvular interstitial cells with a culture density close to 80% were starved overnight with 2% FBS+DMEM high-glucose culture medium, and then calcification was induced in the starved valvular interstitial cells with a newly configured osteoinduction medium (Cyagen, Catalog No.: HUXMA-90021). Alizarin red staining was performed 21 days after induction to examine the effects of different treatments on the calcification of valvular interstitial cells.

[0025] The cells were treated with a final concentration gradient of GAS from 25 μM to 800 μM, and the cell growth status was observed after 24 h of treatment. Figure 1 As shown in B and C, the number of viable cells was significantly reduced in the GAS treatment group at 200 μM and above, and its IC 50 Value, the result is Figure 1 As shown in D, its IC 50 The value is 293.3 μM.

[0026] Human valvular interstitial cell calcification model was established, and human valvular interstitial cells were divided into three groups, namely control group (untreated, normal culture medium), OM group (osteogenic culture medium induction), and OM+GAS group (gastrodin was added after osteogenic culture medium induction). Human valvular interstitial cells were treated with different final concentrations of gastrodin (gastrodin was purchased from Selleck (CAS: 62499-27-8), product catalog number: S2383). The results are shown in Table 1. Figure 1 As shown in E and 1F, OM can induce the calcification of VIC, and the GAS experimental group can significantly inhibit the OM-induced VIC calcification (*p<0.05), where 100 μM GAS treatment reduced the calcification degree of VIC by about 70% compared with the OM-induced group. Therefore, the results show that GAS can significantly reduce the calcification degree of VIC.

[0027] Example 2: Gastrodin inhibits the expression of OM-induced osteogenic specific markers

[0028] The human valvular interstitial cell calcification model constructed as in Example 1 above was used, and the human valvular interstitial cells were divided into 3 groups, namely a control group (untreated, normal culture medium), an OM group (osteogenic culture medium induction) and an OM+CAPE group (gastrodin was added after osteogenic culture medium induction). Human valvular interstitial cells were treated with 50 and 100 μM final concentrations (final concentration in culture medium) of gastrodin, and osteogenic-specific genes BMP2, RUNX2, COL1A2, and SPP1 were detected.

[0029] Quantitative qRT-PCR results showed that compared with the control group, osteogenic medium significantly upregulated the expression of ALP and RUNX2 (osteoblast differentiation marker genes) (*p<0.05). However, with the addition of gastrodin to the culture medium, BMP2, RUNX2, COL1A2, and SPP1 were all downregulated ( Figure 2 A) and showed statistically significant differences (#p<0.05).

[0030] Western blot results showed that GAS could significantly inhibit the expression of VIC osteogenic markers induced by OM ( Figure 2 B), and provided its semi-quantitative statistics ( Figure 2 C).

[0031] Intracellular immunoblotting results also showed that GAS could significantly inhibit the expression of OM-induced VIC osteogenic specific markers ( Figure 2 D), and provided its semi-quantitative statistics ( Figure 2 E).

[0032] All the above results indicate that GAS can significantly inhibit the expression of OM-induced VIC osteogenic specific markers BMP2, RUNX2, COL1A2, and SPP1.

[0033] Example 3: Mechanism of Gastrodin Inhibiting Valvular Calcification

[0034] According to the method of the above example, GAS was used to intervene in VIC induced by OM for osteogenic differentiation, and multi-omics analysis including transcriptome and metabolome was performed. The results of gene expression profile analysis showed that after VIC was induced by OM for osteogenic differentiation, there were 1307 differentially expressed genes (DEGs), of which 823 were upregulated and 484 were downregulated ( Figure 3 A). After further intervention with GAS, a total of 552 DEGs were identified, of which 247 were upregulated and 305 were downregulated ( Figure 3 B). Venn diagram interaction analysis showed that 405 DEGs were common in OMI / Con and GAS / OMI groups ( Figure 3 C). GO analysis showed that these common DEGs were mainly enriched in cellular and metabolic processes ( Figure 3 D). KEGG gene function enrichment analysis showed that the above DEGs were mainly enriched in cytokine-cytokine receptor interaction, metabolic pathways and mineral absorption pathways ( Figure 3 E). The above KEGG and GO analyses showed that DEGs were highly enriched in cellular processes and metabolic pathways. Therefore, we examined the effect of GAS treatment on the metabolic regulation of VIC cells under OMI conditions. The results of metabolic pathway enrichment analysis showed that the differential metabolites were mainly enriched in glycolysis, pyruvate metabolism, and glycerolipid metabolism pathways, and Figure 3 G shows the metabolome enrichment results of the parallel group ( Figure 3 F, G). In addition, LDHA, LDHB and PKM2 are key genes regulating glycolysis. Compared with the OM group, GAS could significantly inhibit the expression of LDHA, LDHB and PKM2 genes in VIC ( Figure 3 H).

[0035] Example 4: GAS can alleviate valve calcification in mice fed a high-fat diet

[0036] A heart valve calcification model of high-fat-fed mice was used. The specific method was to divide the one-month-old mice into three groups. The control group was fed normally, and the CAVD group (heart valve calcification) and CAVD+GAS group were fed a high-fat diet for 16 consecutive weeks. Then the CAVD+GAS group was treated with GAS intervention at a dose gradient of 10 mg / kg and 30 mg / kg for 8 consecutive weeks. After 24 weeks, the heart valves of the mice were evaluated by the classic Von Kossa staining method for marking calcification points. The results showed that the high-fat diet alone could cause significant calcification of the heart valves, and both doses of GAS feeding could significantly reduce the valve calcification trend caused by high-fat feeding ( Figure 4 A and B), indicating that GAS can significantly alleviate heart valve calcification, which is helpful for the prevention and treatment of aortic valve calcification.

Claims

1. Use of gastrodin in the preparation of a medicament for preventing and / or treating individual aortic valve calcification.

2. The use according to claim 1, wherein The final concentration of the gastrodin is 10 μM-800 μM.

3. The use according to claim 1, wherein The final concentration of the gastrodin is 10 μM-400 μM.

4. The use according to claim 1, wherein The final concentration of gastrodin is 50-100 μM.

5. The use according to any one of claims 1 to 4, wherein The individual is a mammal.

6. The use according to any one of claims 1 to 4, wherein The individual is a human.

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