Application of clostridium praeparatum in treating aortic valve calcification disease

Abstract

The invention relates to the field of prevention and / or treatment of aortic valve calcification, and the inventor finds that clostridium pralaense can effectively down-regulate expression of osteogenic differentiation marker genes BMP2 and Runx2 in valvular interstitial cells and inhibit calcification of the valvular interstitial cells through research, so that valve calcification is inhibited or delayed, and the effect of preventing and / or treating the aortic valve calcification is achieved. The invention further provides application of the clostridium praeparatum and the metabolite of the clostridium praeparatum in preparation of drugs for preventing and / or treating individual aortic valve calcification. And a new choice is provided for treating aortic valve calcification by adopting a non-surgical method.

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 Faecalibacterium prausnitzii and / or Faecalibacterium prausnitzii metabolites in the prevention and / or treatment of aortic valve calcification. Background Art

[0002] Faecalibacterium prausnitzii (F.prausnitzii), also known as Faecalibacterium prausnitzii, is one of the most important bacteria in the human intestinal flora, accounting for 5-15% of the total bacteria detected in healthy human fecal samples. It is one of the important producers of butyrate and has anti-inflammatory effects.

[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 Faecalibacterium prausnitzii and / or a metabolite of Faecalibacterium prausnitzii in the preparation of a medicament for preventing and / or treating aortic valve calcification in an individual.

[0010] In the present invention, "prevention and / or treatment" means preventing and / or delaying the occurrence of a disease or condition or normalizing it.

[0011] In one embodiment, the final concentration of Faecalibacterium prausnitzii is 1×10 9 cfu / 100μL~1×10 10 cfu / 100μL.

[0012] Further, in a preferred embodiment, the final concentration of Faecalibacterium prausnitzii is 1×10 9 cfu / 100μL~5×10 9 cfu / 100μL.

[0013] In a more preferred embodiment, the final concentration of Faecalibacterium prausnitzii is 2.5×10 9 cfu / 100μL.

[0014] 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.

[0015] Preferably, the individual is a human.

[0016] In this regard, the beneficial effect of the present invention is that Faecalibacterium prausnitzii can effectively downregulate the expression of osteogenic differentiation marker genes BMP2 and Runx2 in valve interstitial cells, inhibit valve interstitial cell calcification, and thus inhibit or delay valve calcification. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 ApoE for high fat model - / - Results of 16S sequencing of mouse feces; b. The relationship between Burkholderiales bacteriumg Parasutterella and calcification (Von Kossa staining, valve thickness, transvalvular pressure gradient and flow velocity); c. The relationship between Faecalibacterium prausnitzii Faecalibacterium and calcification (Von Kossa staining, valve thickness, transvalvular pressure gradient and flow velocity).

[0018] Figure 2 Faecalibacterium prausnitzii transplantation (FPT) can significantly inhibit ApoE in high-fat diet - / - Valvular calcification in mice.

[0019] Figure 3 This is the result of Alizarin Red test.

[0020] Figure 4 This is the result of BMP2 and Runx2 In-cell WB detection.

[0021] Figure 5 This is the result of Von Kossa staining. 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] The term "Faecalibacterium prausnitzii" belongs to the phylum Firmicutes, class Clostridia, orders Clostridiales, family Ruminococcaceae, genus Faecalibacterium prausnitzii.

[0024] Example 1: Relationship between Faecalibacterium prausnitzii and calcification in mice

[0025] ApoE in high-fat model with difficult-to-calcify and easy-to-calcify - / - Mouse feces were sequenced by 16S sequencing, and the top 10 bacterial species with significant differences were analyzed through data analysis. The results showed that only Burkholderiales bacterium gParasutterella and Faecalibacterium prausnitzii Faecalibacterium were detected in all samples ( Figure 1 a).

[0026] Among them, the abundance of Burkholderiales bacterium g Parasutterella increased significantly in the feces of mice prone to calcification, and its abundance was positively correlated with the degree of calcification, which has a calcification-promoting effect ( Figure 1 b). The abundance of Faecalibacterium prausnitzii g Faecalibacterium was significantly reduced in the feces of mice prone to calcification. At the same time, the abundance of Faecalibacterium prausnitzii g was negatively correlated with the degree of calcification, including Von Kossa staining, valve thickness (Thickness), transvalvular pressure difference (Pressure) and flow velocity (Vel) (p<0.05) ( Figure 1 c). Therefore, Faecalibacterium prausnitzii has the potential to inhibit calcification and is a probiotic that may inhibit valve calcification.

[0027] Example 2: Transplantation of Faecalibacterium prausnitzii inhibits valve calcification

[0028] Faecalibacterium prausnitzii was purchased from BNCC, with the biological collection number: ATCC 27768. Faecalibacterium prausnitzii was cultured in an anaerobic environment at 37°C using a culture medium. After obtaining a large number of bacteria, the bacterial concentration was adjusted to 2.5×10 9 cfu / 100μL, and transfer the prepared bacterial solution to the anaerobic tube for later use. In the sterile breeding environment of IVC, ApoE was fed with antibiotics - / - The mice achieved intestinal sterility on day 10, and were given high-fat ApoE daily starting on day 11. - / - The mice were fed with 100 μL of the above concentration of bacterial solution for 21 days. After the bacterial transplantation, the mice continued to be fed with high-fat diet for 6 months, during which time the bacterial transplantation was performed 3 times a week for supplementary inoculation. Experimental group: F. prausnitzii transplantation + high-fat diet (FPT); control group: normal high-fat diet (Ctrl).

[0029] The experimental results are as follows Figure 2 As shown, Von Kossa staining showed that Faecalibacterium prausnitzii transplantation (FPT) significantly reduced calcium deposition and the expression of BMP2 in valve tissue.

[0030] Example 3: Metabolites of Faecalibacterium prausnitzii inhibit valve calcification

[0031] The bacterial concentration was 2.5 × 10 9cfu / 100μL of Faecalibacterium prausnitzii supernatant was collected, and the bacteria were removed by centrifugation. The sterile supernatant was filtered through a microporous membrane and added to the cell culture medium (FPM) at a ratio of 1:10 to intervene in the osteogenic differentiation of OM-induced valvular interstitial cells (VIC). Alizarin red staining showed that the FPM treatment group could significantly inhibit the calcium deposition of VIC cells (****p<0.0001)( Figure 3 ), and significantly inhibited the expression of BMP2 and Runx2 in VIC cells ( Figure 4 ). FPM was used to intervene in ex vivo cultured valve tissue pieces, and the results showed that the sterile supernatant of Faecalibacterium prausnitzii inhibited the calcium deposition in the ex vivo cultured aortic valve tissue ( Figure 5 ).

Claims

1. Use of Faecalibacterium prausnitzii and / or a metabolite of Faecalibacterium prausnitzii in the preparation of a medicament for preventing and / or treating aortic valve calcification in an individual.

2. The use according to claim 1, in, The final concentration of Faecalibacterium prausnitzii was 1×10 9 cfu / 100μL~1×10 10 cfu / 100μL.

3. The use according to claim 1, in, The final concentration of Faecalibacterium prausnitzii was 1×10 9 cfu / 100μL~5×10 9 cfu / 100μL.

4. The use according to claim 1, in, The final concentration of Faecalibacterium prausnitzii was 2.5×10 9 cfu / 100μL.

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

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

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