Application of parabacteroides gold in preparation of medicine for relieving myocardial ischemia-reperfusion injury

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of parabacteroides koreanum in preparation of a medicine for relieving myocardial ischemia-reperfusion injury. DNA sequencing and PCR are performed on excrement of MI / RI (myocardial ischemia reperfusion injury) model group mice, it is clear that P.goldsteinii is remarkably reduced in myocardial ischemia reperfusion injury, further research reveals that expression of myocardial necrosis factors CK-MB, LDH and cTnI of the MI / RI model mice is reduced under the action of P.goldsteinii, and expression of myocardial necrosis factors CK-MB, LDH and cTnI in myocardial necrosis factors CK-MB, LDH and cTnI in myocardial necrosis factors CK-MB, LDH and cTnI is reduced under the action of P.goldsteinii. After the P.Goldsteinii is subjected to intragastric administration, the left ventricular ejection fraction, the short-axis shortening fraction, the left ventricular end-systolic anterior wall thickness and the left ventricular end-systolic inner diameter of the heart are obviously increased, heart function decline caused by an MI / RI model is obviously relieved after the P.Goldsteinii is subjected to intragastric administration, and the P.Goldsteinii has a very good protection effect on myocardial ischemia reperfusion injury.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the application of *Pseudomonas aeruginosa* in the preparation of drugs to alleviate myocardial ischemia-reperfusion injury. Background Technology

[0002] According to the "Summary of China Cardiovascular Health and Disease Report 2024," cardiovascular disease remains the leading cause of death among urban and rural residents in China. Acute myocardial infarction (AMI), a critical illness, is primarily caused by a sharp reduction or interruption of coronary blood flow, leading to persistent myocardial hypoxia and subsequently myocardial cell necrosis and progressive damage. In the clinical treatment of AMI, myocardial reperfusion is usually achieved through thrombolytic therapy or percutaneous coronary intervention to promptly restore coronary blood flow.

[0003] While myocardial reperfusion can salvage endangered myocardial tissue and limit the expansion of infarct size, the reperfusion process can induce myocardial ischemia-reperfusion injury (MI / RI). MI / RI exacerbates cardiomyocyte death through a series of mechanisms, including oxidative stress, calcium overload, mitochondrial dysfunction, and severe inflammatory responses, and ultimately becomes an important basis for post-reperfusion cardiac dysfunction, heart failure, and cardiac death.

[0004] The relationship between gut microbiota and cardiovascular disease is a cutting-edge and hot topic in current cardiovascular research. The gut-heart axis refers to the bidirectional connection between the gut and the heart through interactions of microorganisms, metabolites, the immune system, and neuroendocrine pathways. Disruption of the gut-heart axis, alterations in gut microbiota composition, and impaired gut barrier integrity can lead to systemic inflammation driven by gut-derived metabolites, thereby exacerbating the progression of cardiovascular disease.

[0005] Parabacteroides are a core group of bacteria in the human gut microbiota, widely involved in host metabolic regulation, immune homeostasis, and energy conversion. They include various species such as *Parabacteroides distasonis*, *Parabacteroides merdae*, and *Parabacteroides goldsteinii*. Chinese patent application publication number CN115887502A discloses the use of *Parabacteroides merdae* in the preparation of drugs for the prevention and treatment of heart failure, and Chinese patent application publication number CN118903215A discloses the use of *Parabacteroides distasonis* in the preparation of drugs for the treatment or improvement of heart failure (HF).

[0006] However, the current technology does not clarify the relationship between gut microbiota (especially Parabacterium kiuri) and myocardial ischemia-reperfusion injury, and there is a lack of related mechanistic studies. Summary of the Invention

[0007] This application addresses the aforementioned problems by developing an application of *P. goldsteinii* in the preparation of drugs to alleviate myocardial ischemia-reperfusion injury. Through 16S rDNA sequencing and PCR of feces from mice in a myocardial ischemia-reperfusion injury (MI / RI) model group, it was determined that *P. goldsteinii* exhibited significant differences in myocardial ischemia-reperfusion injury (MI / RI). Further research revealed a close association between *P. goldsteinii* and myocardial injury, cardiac tissue apoptosis, cardiac dysfunction, and intestinal mucosal damage caused by myocardial ischemia-reperfusion, demonstrating a good protective effect against myocardial ischemia-reperfusion injury.

[0008] To achieve the above objectives, this application first provides the use of *Pseudomonas kiuri* in the preparation of drugs to alleviate myocardial ischemia-reperfusion injury.

[0009] Preferably, the myocardial ischemia-reperfusion injury includes one or more of the following: myocardial injury, cardiac tissue apoptosis, cardiac dysfunction, and intestinal mucosal injury.

[0010] Preferably, the product number of the *Pseudomonas jini* is ATCC BAA-1180.

[0011] Preferably, the dosage form of the drug is selected from any one of oral liquid, capsule, tablet and dry powder preparation.

[0012] Preferably, the components of the drug include at least one pharmaceutically, foodologically, or health-product-acceptable carrier.

[0013] Preferably, the carrier includes one or more of excipients, additives, dispersants, and binders.

[0014] The advantages of this application compared to the prior art are mainly reflected in:

[0015] This application, through 16S rDNA sequencing and PCR of feces from mice in a myocardial ischemia-reperfusion injury model group, clearly demonstrated that *P. goldsteinii* significantly downregulated myocardial ischemia-reperfusion injury (MI / RI) compared to other *P. goldsteinii* species. In MI / RI model mice, the expression of myocardial necrosis factors CK-MB, LDH, and cTnI decreased under the influence of *P. goldsteinii*, while left ventricular ejection fraction, fractional shortening, left ventricular end-systolic anterior wall thickness, and left ventricular end-systolic diameter significantly increased. The decline in cardiac function caused by the MI / RI model was significantly alleviated after *P. goldsteinii* gavage. Simultaneously, *P. goldsteinii* intervention also significantly reduced the pathological changes in the myocardium in MI / RI, improved the degree of myocardial injury in MI / RI model mice, and alleviated colonic structural damage induced by the MI / RI model.

[0016] This application is the first to discover and reveal the relationship between *Parabacterium kiuri* and myocardial ischemia-reperfusion injury. It reveals that *Parabacterium kiuri* is closely related to myocardial injury, cardiac tissue apoptosis, cardiac dysfunction, and intestinal mucosal injury caused by myocardial ischemia-reperfusion, and has a good protective effect against myocardial ischemia-reperfusion injury, providing a new and effective solution for the treatment and drug selection of myocardial ischemia-reperfusion injury. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This image shows the 16S rDNA sequencing and PCR results of feces from mice in the Sham group and MI / RI model group, as presented in this application. Figure 1 A represents the 16S rDNA sequencing result; Figure 1 B shows the PCR results for different Parabacterium species.

[0019] Figure 2 The image shows the results of *Pseudomonas jini* improving cardiac function in MI / RI mice. Figure 2 A is a representative image of mouse cardiac ultrasound (n=6); Figure 2 B is a statistical graph of the left ventricular ejection fraction (ES). Figure 2 C represents the fractional shortening (FS) of the minor axis. Figure 2 D is a statistical chart of left ventricular end-systolic anterior wall thickness (LVAWS). Figure 2E is a statistical graph of left ventricular end-systolic diameter (LVIDS). Figure 2 F~ Figure 2 H represents the results of ELISA detection of serum myocardial necrosis factor cTnI, CK-MB and LDH levels in mice (n=6, *P<0.05 vs sham, **P<0.01 vs sham, ***P<0.001 vs sham);

[0020] Figure 3 The results of *Pseudomonas kiuri* improving myocardial structural damage in MI / RI mice, among which Figure 3 A is a representative image of HE staining of mouse heart (n=6); Figure 3 B is a representative image of mouse heart TTC-Evens blue staining (n=6);

[0021] Figure 4 The image shows the results of *Pseudomonas jini* in improving cardiomyocyte apoptosis in MI / RI mice. Figure 4 A is a representative image of TUNEL fluorescence staining (n=6); Figure 4 B is a statistical graph of TUNEL fluorescence staining (n=6); Figure 4 C represents the JC-1 immunofluorescence staining pattern (n=6*P<0.05 vs sham,**P<0.01 vs sham,***P<0.001 vs sham);

[0022] Figure 5 The image shows the results of *Pseudomonas ginseng* alleviating intestinal mucosal damage in mice with MI / RI. Figure 5 A is a representative image of HE staining of mouse heart (n=6); Figure 5 B represents the pathological damage score of the mouse colon (n=6); Figure 5 C~ Figure 5 D represents the results of ELISA detection of LPS and DAO levels in mouse serum (n=6); Figure 5 E~ Figure 5 F represents the levels of ZO-1 and Occludin mRNA, respectively (*P<0.05 vs sham, **P<0.01 vs sham, ***P<0.001 vs sham). Detailed Implementation

[0023] The following embodiments are used to illustrate this application, but are not intended to limit the scope of this application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.

[0024] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0025] Unless otherwise specified, the percentage sign "%" in this application refers to the mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0026] The weight units mentioned in this application may be well-known weight units in the art, such as μg, mg, g, kg, etc., or multiples thereof, such as 1 / 10, 1 / 100, 10 times, 100 times, etc.

[0027] The *P. goldsteinii* product used in this application is commercially available, product number ATCC BAA-1180.

[0028] laboratory animals

[0029] Forty-eight male SPF-grade C57BL / 6J mice, aged 6-8 weeks and weighing 16-22g, were selected for the experiment and purchased from Spiford Biotechnology Co., Ltd. All operations involved in the animal husbandry process were approved by the Ethics Committee of Jiangnan University and met the requirements of animal ethics.

[0030] Example 1

[0031] The correlation between MI / RI model mice and *Pseudomonas kiuri*.

[0032] Construction and grouping of experimental animal models

[0033] Establishing an in vivo ischemia-reperfusion model: C57BL / 6 mice aged 8–10 weeks can be used to establish an in vivo mouse cardiac ischemia-reperfusion model. Mice were anesthetized with 2% isoflurane gas, intubated, and given mechanical ventilation. A small skin incision (approximately 1.2 cm) was made in the left chest, exposing the fourth intercostal space after incising the pectoralis major and minor muscles. A small hole was made in the fourth intercostal space using a needle holder to open the pleura and pericardium. With the intercostal space slightly opened by the needle holder, the heart smoothly and gently "emerged" from the hole. The left coronary artery (LCA) was located using 6-0 silk suture and ligated with a slipknot approximately 3 mm above its superior end. The ligation was considered successful when the anterior wall of the left ventricle turned pale. Immediately after ligation, the heart was returned to the thoracic cavity, leaving the longer end of the slipknot outside the chest. Manual aspiration was performed to prevent pneumothorax, and the muscles and skin were closed using a purse-string suture. A 30-minute ischemia timer was then started. Mice were then allowed to breathe room air and monitored during recovery, which typically took 3-5 minutes. The ligatures were slowly removed after 30 minutes to indicate the start of reperfusion. Reperfusion continued for 24 hours. In the sham-operated group, the thoracic cavity was opened and the sutures were immediately closed; no cardiac MI / R model was established.

[0034]

[0035] 16S rDNA sequencing and PCR were performed on fecal samples from Sham and model mice. The 16S rDNA sequencing steps were as follows: fecal DNA was extracted for testing, followed by PCR amplification, product purification, quality control, and sequencing. The raw data (Raw Data) was first spliced ​​based on the overlapping regions at both ends, and then low-quality and chimeric sequences were filtered to obtain high-quality Clean Data. Next, based on the Clean Data, sequence noise reduction was performed using QIIME2 D2DA2 to remove potential PCR amplification and sequencing errors in the high-throughput sequencing data, obtaining representative correct biological sequences (ASVs) and an ASV abundance table. Further analysis was then conducted on alpha diversity, beta diversity, species composition, and differential composition, as well as functional composition and differential composition.

[0036] The results are as follows Figure 1 As shown, P. goldsteinii was downregulated most significantly in the model group, which may be an important reason for myocardial ischemia-reperfusion injury.

[0037] Experimental Example 1

[0038] P. goldsteinii can improve cardiac function after MI / RI.

[0039] Following the method in Example 1, a mouse model of myocardial ischemia-reperfusion was established via MI / RI surgery. Six- to eight-week-old C57BL / 6J mice were randomly divided into four groups: Sham, P. goldsteinii, MI / RI, and MI / RI+P. goldsteinii. Parabacteroides Goldsteinii was administered at a rate of 2 × 10⁻⁶. 8 [CFU] / animal / day administered orally for 2 weeks, 0.25ml each time, concentration 8×10 8 CFU / ml, with the control group receiving an equal volume of physiological saline via gavage. This established an in vivo mouse model of myocardial ischemia-reperfusion. Echocardiography was performed on mice in different groups, and the results are as follows: Figure 2 As shown. From Figure 2The results showed that, compared with MI / RI mice, P. goldsteinii mice administered via gavage had significantly higher left ventricular ejection fraction (ES), fractional shortening (FS), left ventricular anterior wall thickness at end-Systole (LVAWS), and left ventricular internal dimension at end-Systole (LVVIDS) compared with MI / RI mice. Figure 2 A~ Figure 2 E). ELISA was used to detect myocardial necrosis factors (CK-MB, LDH, and cTnI) in the serum of mice from different groups. It was observed that the expression of CK-MB, LDH, and cTnI was decreased in the model group mice under the influence of *P. goldsteinii*. Figure 2 F~ Figure 2 The decline in cardiac function caused by the MI / RI model was effectively alleviated by gavage with P. goldsteinii.

[0040] Experimental Example 2

[0041] P. goldsteinii can improve myocardial damage caused by MI / RI.

[0042] To verify the effect of *P. goldsteinii* on myocardial structural damage induced by the MI / RI model, hematoxylin-eosin (H&E) staining was performed on the hearts of four different groups of mice in Experiment 1: Mouse sections were dewaxed and rehydrated, stained with eosin for 5 minutes, rinsed with running water, stained with hematoxylin for 2 minutes, rinsed with running water for 5 minutes (blue reversal), observed under a microscope, and then mounted. Pathological changes in the mouse hearts were observed, and the results are as follows: Figure 3 As shown in Figure A, it can be seen that the MI / RI mice treated with P. Goldsteinii showed reduced myocardial structural damage and a lessened inflammatory response compared to the MI / RI mice. This demonstrates that P. Goldsteinii can improve the structural disorder of myocardial tissue caused by MI / RI model mice and reduce the infiltration of inflammatory cells.

[0043] The hearts of four different groups of mice in Experiment 1 were subjected to TTC-Evens blue staining for pathological observation, and the results are as follows: Figure 3 As shown in B: The infarct area of ​​MI / RI mice treated with P. Goldsteinii was significantly reduced compared to MI / RI mice, and a large area of ​​pale white infarct area was visible in the MI / RI group.

[0044] In conclusion, P. goldsteinii can improve myocardial injury, cardiac tissue structural disorder, and inflammatory cell infiltration in cardiac tissue caused by MI / RI surgery.

[0045] Experimental Example 3

[0046] P. goldsteinii can reduce the level of apoptosis in the heart tissue of MI / RI model mice.

[0047] TUNEL staining: Paraffin sections of mouse hearts were dewaxed and rehydrated.

[0048] Add 50 μl of TUNEL detection solution to the tissue, incubate at 37°C in the dark, mount with anti-fluorescence quenching mounting solution, and observe under a fluorescence microscope.

[0049] The number of apoptotic cells in the MI / RI model group was significantly reduced under the treatment of P. goldsteinii.

[0050] JC-1: After dewaxing and rehydrating paraffin sections of mouse hearts, JC-1 working solution was prepared according to the specified ratio and dropped onto the tissue. The tissue was incubated at 37°C for 20 minutes. After incubation, the tissue was washed three times with staining buffer and observed under a fluorescence microscope.

[0051] JC-1 is an ideal fluorescent probe widely used for detecting mitochondrial membrane potential (MMT). It can detect the MMT of cells, tissues, or purified mitochondria. When the MMT is high, JC-1 aggregates in the mitochondrial matrix, forming J-aggregates that produce red fluorescence. When the MMT is low, JC-1 cannot aggregate in the mitochondrial matrix and remains a monomer, producing green fluorescence.

[0052] The results were obtained by TUNEL staining and JC-1 immunofluorescence staining as follows: Figure 4 As shown, the mitochondrial membrane potential increased in the MI / RI model group under the treatment of P. goldsteinii; and cardiomyocyte apoptosis was reduced in the MI / RI model mice under the treatment of P. goldsteinii.

[0053] Experiment Example 4

[0054] P. goldsteinii alleviates intestinal mucosal damage in MI / RI model mice.

[0055] To investigate the effect of *P. goldsteinii* on colonic structural damage induced by the MI / RI model, HE staining was used to observe the pathological changes in the colons of four different groups of mice in Experiment 1. The severity of colonic tissue damage and glandular structure loss was assessed from 1 to 5, with the sham group rated 1 and the most severe model group rated 5. Figure 5 A~ Figure 5 B shows that the MI / RI mice treated with P. Goldsteinii had less colonic structural damage and reduced inflammatory response compared to the MI / RI mice, indicating that P. Goldsteinii can improve the destruction of colonic tissue integrity and disappearance of glandular structure caused by MI / RI model mice.

[0056] ELISA was performed on mouse serum to detect LPS and DAO levels, and a decrease in LPS and DAO expression was observed in the model group mice under the influence of P. goldsteinii. Figure 5 C~ Figure 5 D). Occludin (a closure protein) and ZO-1 (Zonula Occludens-1) are two key proteins that constitute tight junctions between cells, working together to maintain the barrier function of epithelial and endothelial tissues. qPCR detection of ZO-1 and Occludin expression in mice revealed that gavage administration of *P. goldsteinii* upregulated the expression of ZO-1 and Occludin genes in MI / RI model mice. Figure 5 E- Figure 5 F). In conclusion, P. goldsteinii can improve colonic injury caused by MI / RI surgery.

[0057] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this application.

Claims

1. Application of *Pseudomonas kiuri* in the preparation of drugs to alleviate myocardial ischemia-reperfusion injury.

2. The application according to claim 1, characterized in that, The myocardial ischemia-reperfusion injury includes one or more of the following: myocardial injury, cardiac tissue apoptosis, cardiac dysfunction, and secondary intestinal mucosal injury.

3. The application according to claim 1, characterized in that, The product number of the *Pseudomonas jini* is ATCC BAA-1180.

4. The application according to claim 1, characterized in that, The dosage form of the drug is selected from any one of oral liquid, capsule, tablet, and dry powder preparation.

5. The application according to claim 1, characterized in that, The components of the drug include at least one pharmaceutically, foodologically, or health food-grade carrier.

6. The application according to claim 5, characterized in that, The carrier includes one or more of the following: excipients, additives, dispersants, and binders.

Images