Application of trimethylamine oxide in prediction of collateral circulation establishment condition

Abstract

The invention discloses an application of trimethylamine oxide (TMAO) in predicting collateral circulation establishment, if collateral circulation is established, the concentration of TMAO in plasma is reduced, and if the concentration of TMAO is increased, collateral artery formation after myocardial infarction or lower limb artery occlusion can be remarkably inhibited. The invention discloses for the first time that TMAO directly hinders formation of ischemic tissue collateral circulation by inhibiting reperfusion and remodeling of middle and small arteries; the TMAO level is reduced through diet limitation or drug inhibition, and collateral artery generation and blood flow recovery of a mouse heart and lower limb ischemia model can be remarkably promoted. The invention discloses the TMAO as a novel biomarker of collateral circulation disorder and a pathological mechanism of the TMAO, and provides a theoretical basis and a technical support for non-surgical treatment and precise intervention of ischemic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to the application of trimethylamine oxide (TMAO) in predicting the establishment of collateral circulation. Specifically, it relates to a gut flora metabolite TMAO that impairs the establishment of collateral arteries in cardiac ischemia and lower limb ischemia models, and is a biomarker for predicting collateral circulation disorders in ischemic diseases. Background Art

[0002] With the increasing aging of society, the incidence of severe ischemic diseases (such as coronary heart disease and peripheral vascular disease) caused by arterial obstruction or stenosis has been rising year by year, and their high mortality and disability rates have imposed a heavy disease burden on society. The current main treatment strategy is stent implantation, but approximately 20% of patients are unable to tolerate surgery due to advanced age or diffuse lesions, and some patients have a poor prognosis due to in-stent restenosis. Clinically, some patients with severe coronary or peripheral artery stenosis or occlusion do not have ischemic symptoms. This is due to the formation of good collateral circulation. Collateral circulation refers to the vascular bypass formed between the main arteries when the artery is severely narrowed, thereby providing blood supply to the distal vessels of the blocked artery; however, the vast majority of patients have difficulty in forming collateral circulation.

[0003] In recent years, trimethylamine N-oxide (TMAO), a metabolite of gut microbiota, has been shown to play a key role in the development and progression of cardiovascular diseases such as coronary heart disease, heart failure, hypertension, and peripheral ischemic disease (PAD). However, whether plasma TMAO levels affect collateral circulation and thus patient prognosis remains unclear, and its role warrants further investigation. Summary of the Invention

[0004] The first object of the present invention is to address the deficiencies of the prior art and provide an application of trimethylamine oxide (TMAO) in predicting the establishment of collateral circulation.

[0005] Preferably, if collateral circulation is established, the concentration of trimethylamine oxide in plasma is reduced.

[0006] Preferably, the collateral circulation is established for patients with acute myocardial infarction and lower limb ischemia.

[0007] The second object of the present invention is to provide the use of an agent for reducing the concentration of plasma trimethylamine oxide in the preparation of a preparation for promoting the generation of collateral circulation.

[0008] The third object of the present invention is to provide the use of trimethylamine oxide as a biomarker in the preparation of a kit for evaluating the status of collateral circulation.

[0009] Preferably, the detection object of the kit is plasma.

[0010] The fourth object of the present invention is to provide a collateral circulation status assessment kit comprising a reagent for detecting the concentration of plasma trimethylamine oxide.

[0011] A fifth object of the present invention is to provide a method for improving the establishment of collateral circulation, comprising reducing plasma trimethylamine oxide levels through dietary or drug intervention.

[0012] Preferably, the dietary intervention comprises limiting the intake of trimethylamine oxide precursors, wherein the precursors are selected from at least one of betaine, choline, creatinine or L-carnitine; when the organism suffers from myocardial infarction, the organism's left ventricular systolic function decreases after administration of trimethylamine oxide compared to a normal diet.

[0013] Preferably, the pharmaceutical intervention comprises administration of the trimethylamine oxide inhibitor 3,3-dimethyl-1-butanol (DMB).

[0014] Preferably, the method promotes reperfusion of small arteries with a diameter of less than 72 μm in ischemic tissue by reducing the level of trimethylamine oxide.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This study reveals for the first time that high plasma TMAO concentrations are associated with impaired collateral circulation in patients with coronary and lower-extremity artery stenosis, providing a new biomarker for the clinical assessment of collateral circulation status. Experimental results show that elevated plasma TMAO levels significantly inhibit collateral artery formation after myocardial infarction or lower-extremity artery occlusion. Conversely, inhibiting TMAO production in vivo significantly improves collateral circulation. This study proposes that TMAO can serve as a novel marker for predicting collateral circulation in patients with coronary heart disease and peripheral vascular disease. Compared to traditional indicators (such as blood lipids and blood glucose), TMAO has higher specificity and can independently predict the risk of poor collateral circulation, filling a technological gap in collateral circulation assessment and intervention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 To investigate the relationship between plasma TMAO levels and Rentrop grade of coronary collateral circulation in an acute myocardial infarction cohort.

[0017] Figure 2 Figure 3 shows the relationship between plasma TMAO levels and the formation of lower limb collateral circulation in a cohort of patients with lower limb ischemia. (A) The TMAO level in patient C2 with good collateral circulation was significantly lower than that in patient C1 with poor collateral circulation. (B) The receiver operating characteristic (ROC) curve. (C) The levels of plasma TMAO precursors (betaine, choline, creatinine, and L-carnitine) were not associated with the formation of lower limb collateral circulation.

[0018] Figure 3Figure 3 shows the cardiac function of the adult mouse cardiac collateral circulation model. A shows the left ventricular ejection fraction (LvEF) and fractional shortening (LvFS) of the sham operation group before dietary intervention, 1 day before surgery, and after surgery (days 1, 3, 7, 14, and 28). B shows the left ventricular function of the coronary artery ligation group before dietary intervention, 1 day before surgery, and after surgery.

[0019] Figure 4 The effect of TMAO on collateral circulation formation was evaluated in a cardiac collateral circulation model 7 days after surgery. A is a cardiac clearing image after perfusion of MICROFIL angiographic contrast agent, and B is a Micro-CT image after cardiac angiography.

[0020] Figure 5 The effect of TMAO on collateral circulation formation was evaluated 28 days after surgery in a cardiac collateral circulation model. A is a cardiac clearing image after perfusion of MICROFIL angiographic contrast agent. B is a Micro-CT image after cardiac angiography. C is the arterial reperfusion volume in the infarcted area. D is the perfusion volume of collateral arteries in different diameter ranges.

[0021] Figure 6 Figure 3: Effect of TMAO on blood flow recovery in the lower limbs of mice. Figure A shows the plasma TMAO level of mice after one month of TMAO diet intervention. Figure B shows laser Doppler blood flow imaging of the lower limbs of mice. Figure C shows the statistical results of blood flow recovery in the lower limbs of mice.

[0022] Figure 7 To inhibit the effect of TMAO production on the recovery of blood flow in the lower limbs of mice, Figure A shows the plasma TMAO level of mice after one month of choline and DMB diet, Figure B shows laser Doppler blood flow imaging of the lower limbs of mice, and Figure C shows the statistical results of blood flow recovery in the lower limbs of mice.

[0023] Figure 8 The effect of TMAO on the formation of collateral arteries in the lower limbs of mice. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0025] The present invention discloses the effect of TMAO, a metabolite of intestinal flora, on the formation of collateral arteries after ischemia of the heart and lower limbs. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted in particular that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0026] The reagents and instruments used in the present invention can be purchased from the market.

[0027] The present invention is further described below with reference to implementation cases.

[0028] Example 1 Plasma TMAO is an independent risk factor for poor collateral circulation in patients with acute myocardial infarction Based on the acute myocardial infarction (OCTAMI) cohort, 211 AMI patients were included. The plasma TMAO concentration was detected by LC / MS, the collateral circulation of patients was graded by Rentrop classification, and the relationship between TMAO and collateral circulation was evaluated. It was found that the plasma TMAO concentration of AMI patients without collateral circulation was significantly higher than that of patients with collateral circulation (3.69 μM [3.24-5.26] vs. 3.00μM [1.47-3.91], P < 0.001). Logistic multivariate regression analysis showed that after adjusting for traditional cardiovascular risk factors such as age, gender, smoking, hypertension, diabetes, and onset time, TMAO was still an independent risk factor for poor collateral circulation in patients with acute myocardial infarction (OR = 0.62, 95% CI: 0.48-0.81; P < 0.001) ( Figure 1 ).

[0029] Example 2 Plasma TMAO is an independent risk factor for poor collateral circulation in patients with lower limb ischemia Serum samples from 40 patients with lower limb arterial disease (PAD) were collected, and plasma TMAO concentrations were measured by LC / MS to grade lower limb collateral formation. The study found that PAD patients with poor collateral formation had higher plasma TMAO concentrations than those with good collateral formation (OR=1.45, 95%CI: (1.04–2.02); P=0.027), while plasma TMAO precursors were not associated with collateral formation ( Figure 2 Plasma TMAO concentration was used to predict collateral formation in PAD patients, with an area under the ROC curve of 0.73, P=0.016, and a cutoff value of 2.62 μM ( Figure 2 C in ). Logistic multivariate regression analysis showed that after adjusting for traditional cardiovascular risk factors such as age, gender, smoking, hypertension, diabetes, and hyperlipidemia, TMAO was still an independent risk factor for poor collateral circulation in PAD patients (OR=1.46, 95% CI: 0.99-2.17; P=0.05) ( Figure 2 ).

[0030] Example 3 Based on the adult mouse cardiac collateral circulation model, the effect of TMAO on cardiac function was clarified.

[0031] Dietary intervention: Six-week-old C57BL / 6J male mice weighing 21 ± 1 g were purchased from Slack and housed for one week to acclimate. At week seven, mice were fed a standard diet, a 1.2% high-choline diet (a TMAO precursor), a 0.12% TMAO diet, or a 1% v / v concentration of the TMAO inhibitor 3,3-dimethyl-1-butanol (DMB). Mouse feed was purchased from Synergy Biotechnology; the specific feed formula is shown in Table 1. DMB was purchased from Sigma-Aldrich, Cat. No. 183105. Feed and water were changed every three days. Cardiac collateral circulation modeling was performed after four weeks of feeding.

[0032] To establish a cardiac collateral circulation model in adult mice, 11-week-old male mice were anesthetized with 1.25% avertin (190 mg / kg), intubated, and connected to a ventilator. After depilation and disinfection of the precordial skin, a thoracotomy was performed between the 4th and 5th intercostal spaces, exposing the heart. A median ligature of the left anterior descending coronary artery was performed using a 10-0 suture inserted perpendicular to the long axis of the heart approximately 2.5 mm below the left atrial appendage. The ligature was then placed at a depth of 0.5 mm and a span of 1.5 mm.

[0033] Echocardiographic Assessment of Cardiac Function: Cardiac function in mice was assessed using a color Doppler ultrasound system (Shenzhen Mindray Animal Medical Technology Co., Ltd., model ZS3 SCI) before the start of the special diet, one day before surgery, and on days 1, 3, 7, 14, and 28 after surgery. Mice were anesthetized with isoflurane (Rayward, cat. no. R510-22-8), fixed, and hair removed. In B-mode ultrasound mode, the transducer was rotated 90 degrees clockwise on the left ventricular long axis, and fine-tuned to obtain a parasternal short-axis section with the two papillary muscles positioned at the 2 and 4 o'clock positions. Then, M-mode ultrasound mode was selected, with the M line positioned at the longest short-axis diameter.

[0034] Left ventricular end-diastolic dimension (LVIDd) and left ventricular end-systolic dimension (LVIDs) were obtained using M-mode echocardiography. Left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) (unit: mL) were calculated using the formula V = [7.0 / (2.4 + D)] × D³ (D is the ventricular cavity diameter at the corresponding phase). Left ventricular ejection fraction (LVEF) (%) was calculated using the following formulas: (LVEDV – LVESV) / ​​LVEDV × 100%); left ventricular fractional shortening (FS) (%) was calculated using the formulas: (LVDd - LVDs) / LVDd × 100%.

[0035] The above results are as follows Figure 3 As shown, Figure 3A is the left ventricular ejection fraction (LvEF) and fractional shortening (FS) of the sham operation group before dietary intervention, 1 day before surgery, and after surgery (1, 3, 7, 14, and 28 days); B is the left ventricular function of the coronary artery ligation group before dietary intervention, 1 day before surgery, and after surgery. Figure 3 It can be seen that after sham surgery (non-myocardial infarction group), choline or TMAO diet had no significant effect on the left ventricular systolic function of mice; after coronary artery ligation (myocardial infarction group), the left ventricular systolic function of mice fed choline or TMAO diet was significantly decreased compared with that of mice fed normal diet, because TMAO caused collateral circulation disorders.

[0036] Example 4 Based on the adult mouse cardiac collateral circulation model, the effect of TMAO on collateral artery formation was investigated.

[0037] Cardiac vascular contrast agent perfusion: On days 7 and 28 after the establishment of the cardiac collateral circulation model, adult mouse hearts were perfused with MIRCOFIL vascular contrast agent to visualize coronary collateral vessels. Briefly, after anesthesia and euthanasia, the mice were exposed to the thoracic aorta and heart. The brachiocephalic trunk, left common carotid artery, left subclavian artery, right anterior vena cava, and left anterior vena cava were ligated in sequence. A microperfusion pump was used to retrogradely perfuse the modified MIRCOFIL contrast agent (MV-122: MV-diluent = 1:2, Flow Tech) into the cardiac arteries through the thoracic aorta. The cardiac perfusion volume was approximately 25-30 μL at a rate of 10 μL / min. Cardiac Micro-CT Scanning: Micro-CT scans of the heart following arterial angiography were performed using a μCT45 scanner (SCANCO, Switzerland). Scanning parameters were: tube current 145 μA, voltage 55 kV, full-body scan, resolution 4.0 μm, exposure time 250 ms, and scanning angle 180 degrees. A phantom (Phantom) was calibrated under the same conditions. Three-dimensional reconstruction software (SCANCO, Switzerland) was used to select and reconstruct the original images, with a threshold of 200. Evaluaticon analysis software (SCANCO, Switzerland) was used to calculate the perfusion volume below the ligation point and the diameters of different types of side branches.

[0038] Heart clearing: Place the heart in a 15 mL centrifuge tube and dehydrate with a gradient of ethanol, 10 mL of each solution, in the following order: 30% ethanol (30 min) - 50% ethanol (30 min) - 75% ethanol (12 h) - 90% ethanol (12 h) - 100% ethanol (36 h, changed every 12 h). Prepare heart clearing solution with a 1:1 ratio of benzyl benzoate (Sigma-Aldrich, Cat. No. W213802-1 KG) and benzyl alcohol (Sigma-Aldrich, Cat. No. 108006-1 L) and mix thoroughly. Add 10 mL of clearing solution to the dehydrated heart and shake on a shaker at room temperature at 100 rpm for 7 days. After clearing, photograph the cleared heart using a microscope (LEICA, Model M205 FCA) at 10× magnification.

[0039] The above results are as follows Figure 4 and Figure 5 As shown, Figure 4 To evaluate the effect of TMAO on the formation of collateral circulation in the cardiac collateral circulation model 7 days after surgery, Figure 5 The effect of TMAO on collateral artery formation was evaluated 28 days after myocardial infarction in a cardiac collateral circulation model. The results showed that elevated plasma TMAO levels significantly inhibited the formation of collateral arteries after myocardial infarction, leading to decreased left ventricular systolic function.

[0040] Example 5 Based on the adult mouse lower limb ischemia model, the effect of TMAO on lower limb blood flow recovery was investigated.

[0041] Dietary intervention: Six-week-old C57BL / 6J male mice weighing 21 ± 1 g were purchased from Slack and housed for one week to acclimate. At week seven, mice were fed a standard diet, a 0.12% TMAO diet, a 1.2% high-choline diet, or a 1.2% high-choline diet supplemented with a 1% v / v concentration of the TMAO inhibitor 3,3-dimethyl-1-butanol (DMB). DMB was purchased from Sigma-Aldrich, Cat. No. 183105. Feed and water were changed every three days. After four weeks of feeding, the left lower extremity femoral artery was ligated.

[0042] Femoral artery ligation: Mice were anesthetized with 1.25% avertin. The limbs were immobilized, with the soles of the hind legs facing upward and the legs in a near-straight line. 6-0 non-absorbable surgical sutures (Kangliyuan, model 20060080) were prepared and divided into three strands. The skin was cut approximately 1 cm along the course of the femoral artery. Subcutaneous tissue and muscle were bluntly dissected to free the femoral artery. The proximal and distal ends of the femoral artery were ligated, and the artery was severed between the two knots. The skin was sutured, and laser Doppler blood flow imaging of the lower limb was performed. A decrease in blood flow of more than 70% on the ligated side (left side) with the normal side (right side) as a reference was considered successful.

[0043] Laser Doppler blood flow imaging: On days 0, 3, 7, and 14 after surgery in the mouse lower limb ischemia model, a small animal laser Doppler instrument (Moor instruments, model MOORFLPI-2) was used to detect blood flow in both lower limbs of the mice, and the ratio of the left ROI / right ROI was used as the result for statistical analysis.

[0044] The results showed that the recovery of blood flow in the ischemic lower limbs of mice fed with TMAO diet was significantly slowed down ( Figure 6 ), and the TMAO inhibitor 3,3-dimethyl-1-butanol (DMB) can reverse the effects of TMAO ( Figure 7 ).

[0045] Example 6 Based on the adult mouse lower limb ischemia model, the effect of TMAO on collateral artery remodeling was investigated ( Figure 7 ).

[0046] Lower limb arterial angiography: MIRCOFIL contrast agent was perfused 21 days after femoral artery ligation. Mice were euthanized under anesthesia, and the abdominal aorta below the renal artery was isolated. Normal saline and 4% paraformaldehyde were perfused through the abdominal aorta. 1 mL of MIRCOFIL contrast agent (Flow Tech Inc., volume ratio: MV-122: MV-diluent = 4:5) was prepared and 100 μL of curing agent was added. MIRCOFIL contrast agent was pumped into the abdominal aorta at a rate of 200 μL / min, paused after 4 minutes, and the lower limb venous return was monitored. Adequate perfusion was considered complete when continuous venous return was observed. The lower limb was rinsed with PBS, allowed to rest at room temperature (25°C) for 30 minutes, and then fixed overnight in 4% paraformaldehyde at 4°C. After 48 hours, the solution was replaced with PBS.

[0047] The lower limb artery clearing procedure was the same as in Example 3. After clearing, the gracilis and adductor musculature of the lower limb were removed and the formation of collateral arteries in the lower limbs was photographed using a microscope (LEICA, model M205 FCA) at a magnification of 10×. The clearing images showed that TMAO and choline diet inhibited the diameter and remodeling of the collateral arteries in the lower limbs, thereby inhibiting their blood flow recovery ( Figure 8 ).

[0048] The above description is merely a preferred embodiment of the present invention and does not limit the embodiments of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications shall be included within the scope of protection of the claims of the present invention.

Claims

1. Application of trimethylamine oxide in predicting the establishment of collateral circulation.

2. The use according to claim 1, characterized in that If collateral circulation is established, the concentration of trimethylamine oxide in plasma will decrease.

3. The use of agents that reduce plasma trimethylamine oxide concentration in the preparation of preparations that promote the formation of collateral circulation.

4. The use according to claim 3, characterized in that The preparation for promoting collateral circulation generation is aimed at patients with acute myocardial infarction or lower limb ischemia.

5. Application of trimethylamine oxide as a biomarker in the preparation of a kit for assessing collateral circulation status.

6. A method for improving the establishment of collateral circulation, characterized in that: This includes lowering plasma TMAO levels through dietary or pharmacological interventions.

7. The method according to claim 6, characterized in that The dietary intervention comprises limiting the intake of a trimethylamine oxide precursor, wherein the precursor is selected from at least one of betaine, choline, creatinine or L-carnitine.

8. The method according to claim 7, characterized in that Compared with a normal diet, the left ventricular systolic function of the organism decreased after administration of trimethylamine oxide.

9. The method according to claim 6, characterized in that Pharmacological intervention included administration of the trimethylamine oxide inhibitor 3,3-dimethylbutan-1 (DMB).

10. The method according to any one of claims 7 to 9, characterized in that: The method promotes reperfusion of small arteries with a diameter of less than 72 μm in ischemic tissue by reducing the level of trimethylamine oxide.

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