Application of fusobacterium przewalskii and metabolite DAT of fusobacterium przewalskii in preparation of medicine for preventing and treating myocardial ischemia / reperfusion injury

Abstract

The invention discloses a novel application of DAT and bacteria for producing DAT. The application refers to application of DAT and bacteria generating DAT in preparation of drugs for preventing and / or treating myocardial ischemia / reperfusion injury. Prevention and / or treatment of myocardial ischemia / reperfusion injury are embodied in the following aspects: 1) heart injury after myocardial ischemia / reperfusion is relieved; 2) the apoptosis proportion of myocardial cells is reduced; 3) promotion of myocardial cell survival; 4) inhibiting cardiac fibrosis; 5) the area of cardiac infarction is reduced; and 6) inhibiting heart remodeling. Experimental results show that the pathological influence of ischemia / reperfusion injury of mice can be relieved by orally taking DAT or bacteria producing DAT, and the discovery provides a new thought and theoretical basis for development of DAT drugs and related bacteria.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to use of Fusobacterium prausnitzii and its metabolite DAT in preparing a drug for preventing and treating myocardial ischemia / reperfusion injury. Background Art

[0002] Cardiovascular disease (CVD) is a leading cause of morbidity and mortality worldwide, with ischemic heart disease (IHD) and stroke being the most common types, and myocardial infarction (MI) being its most severe and life-threatening manifestation. Currently, the main clinical treatments for MI include percutaneous coronary intervention (PCI) and thrombolytic therapy. However, while these treatments can restore coronary blood flow, the reperfusion process often triggers myocardial ischemia / reperfusion (I / R) injury, further aggravating the initial myocardial damage. The pathogenesis of I / R injury is complex, with serious consequences, and currently, there is a lack of comprehensive and effective treatments and specific drugs for this injury.

[0003] In recent years, studies have shown that gut microbes are closely related to cardiovascular health. The role of gut microbes in cardiovascular disease is multifaceted. For example, trimethylamine N-oxide (TMAO) and short-chain fatty acids (SCFA) are closely related to the pathogenesis of diseases such as hypertension and heart failure. Flavonifractor plautii (F. plautii) has garnered widespread attention for its anti-inflammatory properties, helping to prevent inflammation and being associated with a reduced risk of intestinal and metabolic disorders. However, no studies have yet reported whether F. plautii and its metabolites can alleviate myocardial ischemia / reperfusion injury. Summary of the Invention

[0004] The object of the present invention is to provide novel uses of deaminotyrosine (DAT) and bacteria producing DAT.

[0005] The novel use of the DAT and DAT-producing bacteria provided by the present invention is their use in preparing a medicament for preventing and / or treating myocardial ischemia / reperfusion (I / R) injury.

[0006] The prevention and / or treatment of myocardial ischemia / reperfusion injury described in the present invention is specifically embodied in at least one of the following aspects:

[0007] 1) Alleviate cardiac damage after myocardial ischemia / reperfusion; 2) Reduce the apoptosis rate of myocardial cells after myocardial ischemia / reperfusion; 3) Promote myocardial cell survival after myocardial ischemia / reperfusion injury; 4) Inhibit cardiac fibrosis after myocardial ischemia / reperfusion; 5) Reduce the area of myocardial infarction after myocardial ischemia / reperfusion; 6) Inhibit cardiac remodeling after myocardial ischemia / reperfusion.

[0008] The DAT-producing bacteria in the present invention refer to bacteria that can metabolize and produce DAT in the body after oral administration.

[0009] Furthermore, the bacteria producing DAT include but are not limited to Clostridium, Lactobacillus pentosus, Blautia, Providencia or Flavonifractor plautii.

[0010] Preferably, the DAT-producing bacterium is Fusobacterium prausnitzii.

[0011] Furthermore, in the above-mentioned uses of the present invention, the drug contains an effective dose of DAT and / or an effective dose of DAT-producing bacteria. The effective dose is expressed as a unit dosage form (e.g., the amount in a single tablet) or a unit dose per patient to be treated (e.g., a dose per unit body weight). In the present invention, the subject of drug treatment is mammals, including humans, mice, etc. Based on the equivalent dose conversion relationship between humans and experimental animals, the dose per unit body weight of humans can be inferred using the dose in animals.

[0012] The present invention also protects a drug for preventing and / or treating myocardial ischemia / reperfusion injury.

[0013] Furthermore, the active ingredient of the drug is DAT and / or bacteria that produce DAT.

[0014] Furthermore, in the drug, DAT and / or bacteria producing DAT can be used as one of the active ingredients or as the only active ingredient.

[0015] When preparing the drug, a carrier material may be added.

[0016] Furthermore, the carrier includes but is not limited to: a diluent, a suspending agent, a buffer, a granule, an emulsion, an excipient, an encapsulant, a spray, an adhesive, a filler, a disintegrant, a wetting agent, a transdermal absorbent, an absorption enhancer, a surfactant, a flavoring agent, a colorant or an adsorption carrier.

[0017] Furthermore, the above-mentioned drugs can be prepared into dosage forms such as tablets, capsules, solutions or injections according to conventional methods known to those skilled in the art; preferably, they can be prepared into oral preparations.

[0018] Gut microbes play a crucial role in maintaining health and preventing cardiovascular disease. However, it remains unclear which gut microbes can protect the heart from ischemia / reperfusion injury. The inventors discovered that DAT, a metabolite derived from Fusobacterium prausnitzii, can directly mitigate cardiac damage after myocardial ischemia / reperfusion and inhibit cardiac remodeling.

[0019] This study confirmed the protective effects of F. prausnitzii and its metabolite, DAT, by oral administration or administration of F. prausnitzii prior to myocardial ischemia / reperfusion in mice. Cardiac damage was assessed post-ischemia / reperfusion through tissue staining and echocardiography. These data demonstrate that oral administration of DAT or DAT-producing F. prausnitzii can prevent and / or treat myocardial ischemia / reperfusion injury in mice, providing new directions and theoretical foundations for the development of related bacteria and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the animal experiment of pretreatment with F. prausnitzii;

[0021] Figure 2 Figure 3 shows the clearance of intestinal microorganisms in mice (left) and the colonization of Fasciobacterium prausnitzii (right).

[0022] Figure 3 Effects of F. prausnitzii on cardiac injury after I / R, specifically the heart weight / body weight ratio (left) and heart weight / tibia length ratio (right);

[0023] Figure 4 Echocardiography (left) and quantification results (right) of the Fusobacterium prausnitzii treatment experiment;

[0024] Figure 5 The Sirius red staining results (left) and quantification results (right) of the Fusobacterium prausnitzii treatment experiment;

[0025] Figure 6 DAT levels in mouse serum (left) and tissue (right);

[0026] Figure 7 Schematic diagram of the DAT treatment animal experiment;

[0027] Figure 8 Effects of DAT on cardiac injury after I / R, specifically the heart weight / body weight ratio (left) and heart weight / tibia length ratio (right);

[0028] Figure 9 Echocardiography (left) and quantification results (right) of the DAT treatment experiment;

[0029] Figure 10 Sirius red staining results (left) and quantification results (right) of DAT treatment experiments;

[0030] Figure 11 Immunohistochemical staining results (left) and quantification results (right) of DAT treatment experiments;

[0031] Figure 12 TTC staining results (left) and quantification results (right) of DAT treatment experiments;

[0032] Figure 13 TUNEL staining results (left) and quantification results (right) of DAT treatment experiments. DETAILED DESCRIPTION

[0033] The present invention is further described below with reference to specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from public commercial channels unless otherwise specified.

[0034] Example 1: Fusobacterium prausnitzii / DAT alleviates cardiac injury after ischemia / reperfusion

[0035] 1. Grouping and Dosing of Animals Treated with F. prausnitzii / DAT

[0036] 1.1 Antibiotic treatment

[0037] Eight-week-old male C57BL / 6 mice were used for the experiment. A quadruple antibiotic, vancomycin (0.125 mg / ml / day), neomycin (0.25 mg / ml / day), metronidazole (0.25 mg / ml / day), ampicillin (0.25 mg / ml / day), and aspartame (0.1 mg / ml / day), was added to the mice's drinking water daily to eliminate intestinal microorganisms before immunization. The antibiotics were continued in the drinking water for 7 days.

[0038] 1.2 Oral administration of F. prausnitzii / DAT administration

[0039] In this study, the Fusobacterium flavonifractor plautii (F. plautii, FP) strain used was purchased from the American Type Culture Collection (ATCC) with the number ATCC 49531.

[0040] In the experiment of oral administration of F. prausnitzii, mice were treated with antibiotics to eliminate intestinal microorganisms for one week, and then randomly divided into the following three groups: ① sham group (open chest, but no cardiac I / R surgery); ② I / R+PBS group (cardiac I / R surgery, PBS was administered orally for 7 days before surgery); ③ I / R+FP group (cardiac I / R surgery, F. prausnitzii was administered orally at a dose of 5×10 8 CFU / day gavage for 7 days) Figure 1 ).

[0041] In the DAT administration experiment, mice were randomly divided into the following three groups: ① Sham group (open chest, but no cardiac I / R surgery); ② I / R group (cardiac I / R surgery); ③ I / R+DAT group (DAT was administered in drinking water at a final concentration of 20 mg / mL, and DAT was given in drinking water for 7 days before and after cardiac I / R surgery, for a total of 14 days).

[0042] 2. Colonization of Fusobacterium prausnitzii

[0043] Mice were given oral antibiotics 7 days before oral administration of F. prausnitzii to eliminate intestinal microorganisms. Then, 16s rDNA was used to verify the elimination of intestinal microorganisms 7 days after oral administration of F. prausnitzii. Figure 2 Left) and the colonization of Fusobacterium prausnitzii ( Figure 2 (Right) The results showed that the content of F. prausnitzii in the feces of mice in the FP gavage group was significantly increased, indicating that F. prausnitzii had successfully colonized the mouse intestines.

[0044] 3. Pretreatment with Fasciobacterium prausnitzii protects cardiac function after ischemia / reperfusion injury

[0045] Seven days after the inoculation, we performed I / R surgery and collected samples 2 weeks after the surgery. Compared with the sham group, the heart weight / body weight ratio and heart weight / tibia length ratio of the PBS inoculation I / R group were significantly increased, while the hypertrophic response of the FP inoculation I / R group was significantly reduced ( Figure 3 ). In addition, by cardiac ultrasound, we observed that 2 weeks after I / R, the cardiac contractility of the PBS-gavaged I / R group was significantly reduced and the left ventricular cavity was enlarged compared with the sham-operated group. In contrast, the left ventricular fractional shortening (LVFS%) and ejection fraction (LVEF%) of the FP-gavaged I / R group were significantly improved, and the left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD) were reduced compared with the PBS-gavaged I / R group ( Figure 4 ).

[0046] In addition, we performed histopathological analysis on the heart tissues of all groups. Sirius red staining showed that compared with the sham group, the area of fibrotic scar tissue in the hearts of mice in the PBS gavage I / R group was significantly larger, while the area of fibrotic scar tissue in the FP gavage I / R group was significantly smaller ( Figure 5 Taken together, these findings suggest that prophylactic oral administration of F. prausnitzii can mitigate I / R-induced cardiac injury.

[0047] 4. DAT treatment protects cardiac function after ischemia / reperfusion injury

[0048] The DAT levels of mice in the PBS and FP I / R groups were determined by HP-LC. It was found that the DAT levels in the serum and heart tissues of the mice in the FP group were significantly increased compared with those in the PBS group. Figure 6 We then examined the effects of DAT on post-I / R cardiac injury by directly supplementing DAT (administered in drinking water at a final concentration of 20 mg / mL) ( Figure 7The results showed that, consistent with the observations in the Fusobacterium prausnitzii pretreatment experiment, DAT pretreatment significantly reduced the heart weight / body weight ratio and heart weight / tibia length ratio compared with the I / R group, and DAT significantly reduced I / R-induced myocardial hypertrophy ( Figure 8 Similarly, cardiac ultrasound results also showed that DAT alleviated the cardiac dysfunction caused by I / R, as shown by significant improvement in LVFS and LVEF, and reduction in LVEDD and LVESD one week after surgery ( Figure 9 ). In conclusion, DAT reproduced the cardioprotective effects of FP against I / R-induced cardiac injury.

[0049] Sirius red staining showed that there was a large amount of scar tissue in the hearts of mice in the I / R group, and the scar tissue in the I / R group treated with DAT was significantly smaller ( Figure 10 ). In addition, we examined the expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), which are known to be proinflammatory cytokines upregulated after I / R. Our immunohistochemical analysis showed that DAT treatment significantly reduced the increased expression of these proinflammatory cytokines after I / R ( Figure 11 ). Together, these data suggest that DAT attenuates the immune response in I / R-injured hearts and protects the myocardium.

[0050] 5. DAT treatment promotes cardiomyocyte survival after ischemia / reperfusion injury

[0051] 2,3,5-Triphenyltetrazolium chloride (TTC) staining showed that one day after surgery, the unstained area of TTC staining in the hearts of mice in the I / R group increased compared with that in the Sham group, while the area of myocardial infarction caused by I / R was reduced in the I / R group treated with DAT ( Figure 12 We also observed that the TUNEL-positive rate of mice in the I / R group increased one day after surgery compared with the sham-operated group, indicating that cardiomyocyte apoptosis occurred after I / R, and DAT treatment significantly alleviated this situation. The above studies show that DAT treatment reduces cardiomyocyte apoptosis after I / R and promotes the survival of cardiomyocytes in I / R mice ( Figure 13 ).

Claims

1. Use of deaminotyrosine (DAT) in the preparation of a medicament for preventing and / or treating myocardial ischemia / reperfusion injury.

2. The use according to claim 1, characterized in that: The prevention and / or treatment of myocardial ischemia / reperfusion injury is specifically embodied in at least one of the following aspects: 1) alleviating cardiac damage after myocardial ischemia / reperfusion; 2) reducing the apoptosis rate of myocardial cells after myocardial ischemia / reperfusion; 3) promoting myocardial cell survival after myocardial ischemia / reperfusion injury; 4) inhibiting cardiac fibrosis after myocardial ischemia / reperfusion; 5) reducing the area of myocardial infarction after myocardial ischemia / reperfusion; and 6) inhibiting cardiac remodeling after myocardial ischemia / reperfusion.

3. Use of DAT-producing bacteria in the preparation of a medicament for preventing and / or treating myocardial ischemia / reperfusion injury.

4. The use according to claim 3, characterized in that: The DAT-producing bacteria are bacteria that can metabolize and produce DAT in vivo after oral administration.

5. The use according to claim 4, characterized in that: The bacterium that can metabolize and produce DAT in the body after oral administration is Fusobacterium plautii.

6. The use according to any one of claims 3 to 5, characterized in that: The prevention and / or treatment of myocardial ischemia / reperfusion injury is specifically embodied in at least one of the following aspects: 1) alleviating cardiac damage after myocardial ischemia / reperfusion; 2) reducing the apoptosis rate of myocardial cells after myocardial ischemia / reperfusion; 3) promoting myocardial cell survival after myocardial ischemia / reperfusion injury; 4) inhibiting cardiac fibrosis after myocardial ischemia / reperfusion; 5) reducing the area of myocardial infarction after myocardial ischemia / reperfusion; and 6) inhibiting cardiac remodeling after myocardial ischemia / reperfusion.

7. The use according to any one of claims 1 to 6, characterized in that: The subject of administration of the drug is a human or a mammal.

8. A pharmaceutical composition for preventing and / or treating myocardial ischemia / reperfusion injury, wherein the active ingredient is DAT and / or DAT-producing bacteria.

9. The pharmaceutical composition according to claim 8, characterized in that: The DAT and / or the bacteria producing the DAT can be used as one of the active ingredients of the medicine or as the only active ingredient.

10. The pharmaceutical composition according to claim 8, characterized in that: The DAT-producing bacteria are bacteria that can metabolize and produce DAT in vivo after oral administration.

Images