Application of probiotics and their metabolite LPY in the preparation of drugs for the prevention and treatment of hypertension

Abstract

This invention relates to the application of probiotics and their metabolite LPY in the preparation of drugs for the prevention and treatment of hypertension, belonging to the field of biomedical technology. To address the problem that existing ACE inhibitors are ineffective in preventing and treating cold-induced hypertension, this invention provides the application of the intestinal symbiotic probiotic *Anaerostipes hadrus* and its metabolite LPY in the preparation of drugs for the prevention and treatment of hypertension caused by cold. This invention demonstrates that *Anaerostipes hadrus* and its metabolite LPY have significant ACE inhibitory activity, significantly reducing systolic and diastolic blood pressure in cold-induced hypertensive rats, while also lowering plasma Ang II levels, exhibiting a clear antihypertensive effect. LPY, as a natural bacterial bioactive peptide, has good biocompatibility, high safety, low side effects, and is easily absorbed by the intestines, possessing good drug-like properties and suitable for development as a specialized therapeutic drug for hypertension in cold regions.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of probiotics and their metabolite LPY in the preparation of drugs for the prevention and treatment of hypertension. Background Technology

[0002] Hypertension is the leading risk factor for cardiovascular and cerebrovascular diseases, and cold weather is a significant environmental trigger for elevated blood pressure. Epidemiological data in my country show that systolic blood pressure increases by an average of 9 mmHg in winter compared to summer; a 10°C drop in ambient temperature can lead to a 6.2 mmHg increase in systolic blood pressure. This phenomenon is particularly pronounced in the colder northern regions, where approximately 40% of my country's population is chronically exposed to cold environments, making the prevention and control of cold-induced hypertension a major public health issue. Current research suggests that cold weather raises blood pressure through mechanisms such as activating the sympathetic nervous system, the renin-angiotensin-aldosterone system (RAAS), and disrupting endothelial function (e.g., increased endothelin-1 and decreased nitric oxide). However, the specific molecular pathways are not yet fully elucidated, and targeted prevention and treatment methods are lacking.

[0003] Angiotensin-converting enzyme (ACE) inhibitors are commonly used first-line antihypertensive drugs in clinical practice, including chemically synthesized captopril, enalapril, and benazepril. These drugs exert their antihypertensive effect by inhibiting ACE activity and reducing the production of angiotensin II. However, existing ACE inhibitors have significant limitations in cold environments: First, they can only block the conversion of angiotensin I to angiotensin II and cannot inhibit excessive renin secretion caused by cold stimulation, nor do they inhibit the overactivation or alternative pathway activation of the RAAS system; second, cold can inhibit endothelial nitric oxide synthase activity, reducing nitric oxide production, and the vasodilatory effect of ACE inhibitors is partially dependent on NO, so their antihypertensive effect will be significantly weakened when endothelial function is impaired. In addition, chemically synthesized ACE inhibitors often cause adverse reactions such as dry cough and angioedema, and long-term use may lead to drug resistance, further affecting the antihypertensive efficacy.

[0004] Given the fundamental differences in the pathogenesis of cold-induced hypertension compared to primary and secondary hypertension, existing ACE inhibitors are insufficient to effectively prevent and treat cold-induced hypertension. Therefore, developing novel ACE inhibitory peptides with stronger targeting, higher safety, and fewer side effects has significant clinical value for the prevention and control of cold-induced hypertension. Summary of the Invention

[0005] To address the problem that existing ACE inhibitors are ineffective in preventing and treating cold-induced hypertension, this invention provides the application of the probiotic Corynebacterium hardrus and its metabolite LPY in the preparation of drugs for the prevention and treatment of hypertension.

[0006] The technical solution of the present invention:

[0007] Corynebacterium hardrus ( Anaerostipes hadrus abbreviation A. hadrus Its application in the preparation of drugs for the prevention and treatment of hypertension.

[0008] Furthermore, the hypertension mentioned is hypertension caused by cold.

[0009] Furthermore, the hypertension described is cold-induced hypertension complicated by endothelial dysfunction.

[0010] The application of LPY, a metabolite of Corynebacterium harlequinae, in the preparation of drugs for the prevention and treatment of hypertension. LPY is a tripeptide composed of L-leucine, L-proline, and L-tyrosine linked by peptide bonds in that order, with the molecular formula C. 19 H 25 N3O5 has a molecular weight of 365.43 g / mol.

[0011] Furthermore, the hypertension mentioned is hypertension caused by cold.

[0012] Furthermore, the culture medium preparation method for Corynebacterium harlequinae is as follows: weigh 57.5g of minced meat carbohydrate broth culture medium and 1L of distilled or deionized water, heat to boiling and continue boiling for no less than 1min, dispense into test tubes, add minced beef to the test tubes to make it occupy 1 / 3 of the liquid height, sterilize at 121℃ for 30min, cool to below 50℃, aseptically add 0.5mg of heme chloride and 5mg of microbial K1 per 100mL, place the prepared culture medium in an anaerobic chamber for deoxygenation for 24 hours before use.

[0013] Furthermore, the formulation of the minced meat carbohydrate broth culture medium is as follows: 30.0g casein peptone, 10.0g beef extract powder, 5.0g yeast extract powder, 5.0g dipotassium hydrogen phosphate, 0.5g L-cysteine ​​hydrochloride, 0.0005g resazurin, 4.0g glucose, 1.0g cellobiose, 1.0g maltose, 1.0g starch, pH 7.0±0.2.

[0014] Furthermore, the method for culturing Corynebacterium hadrus is to place the deoxygenated culture medium inoculated with Corynebacterium hadrus under anaerobic culture conditions and culture it at 37°C for 48 hours.

[0015] The beneficial effects of this invention are:

[0016] This invention is the first to demonstrate the presence of the gut symbiotic probiotic Corynebacterium hardrus ( Anaerostipes hadrusCorynebacterium harlequinae and its metabolite LPY exhibit significant ACE inhibitory activity and a clear antihypertensive effect. Animal experiments have confirmed that oral administration of Corynebacterium harlequinae significantly reduces systolic and diastolic blood pressure in cold-induced hypertensive rats, while also lowering plasma Ang II levels. Mechanistic studies show that Corynebacterium harlequinae not only exerts its effects by inhibiting the production of the vasoconstrictor Ang II, but also partially improves vascular function through a NO-independent pathway. This indicates that Corynebacterium harlequinae achieves antihypertensive effects through a multi-target regulatory mechanism, making it particularly suitable for hypertension induced by cold environments.

[0017] LPY, a key active metabolite of Corynebacterium harlequinae, exhibits potent and specific ACE inhibitory activity. Molecular docking experiments confirmed that LPY specifically binds to the ACE active site. In vitro experiments showed that LPY inhibits ACE activity in a concentration-dependent manner and significantly reduces Ang II production, thereby exerting an antihypertensive effect. Clinical sample analysis revealed significantly reduced plasma LPY levels in patients with cold-induced hypertension and cold-exposed rats. LPY supplementation significantly reduced systolic blood pressure, diastolic blood pressure, and circulating Ang II levels in cold-exposed rats, demonstrating clear therapeutic efficacy and making it suitable for development as a specialized treatment for hypertension in cold regions.

[0018] LPY, as a natural bacterial bioactive peptide, possesses excellent biocompatibility, high safety, and low side effects, avoiding the immunogenicity issues commonly associated with synthetic drugs. Furthermore, LPY's simple structure and small molecular weight facilitate intestinal absorption and enhance its drug-like properties, providing an ideal candidate molecule for the development of novel oral antihypertensive drugs. Supplementation with Corynebacterium harlequinae can further optimize the host metabolic environment and enhance the durability of the antihypertensive effect. This "bacterium-peptide synergy" strategy opens new avenues for personalized hypertension treatment, particularly suitable for hypertensive patients in cold regions or those with metabolic abnormalities, and holds broad clinical application prospects. Attached Figure Description

[0019] Figure 1 This is a comparison of metagenomic sequencing results of population samples from healthy volunteers and hypertensive patients in winter in Example 1;

[0020] Figure 2 This is a comparison of metagenomic sequencing results of population samples from healthy volunteers and hypertensive patients in summer, as shown in Example 1.

[0021] Figure 3 This is a linear discriminant analysis plot of the abundance of different gut microbiota species in healthy volunteers and hypertensive patients during winter in Example 1. In the plot, #1 represents... g_Lachnospira #2 is g_Butyricimonas #3 is g_Victivallis #4 is g_ Blautia 5# is g_Neobittarella#6 is g_Candidatus Cibionibacter #7 is g_ Bifidobacterium #8 is g_Anaerostipes #9 is g_Dorea #10 is g_Romboutsia 11# is g_ Muribaculaceae_unclassified, 12# is g_Intestinibacter WH represents the winter hypertension patient group, and WN represents the winter healthy volunteer group.

[0022] Figure 4 This is a microbial co-occurrence network diagram of the gut microbiota of healthy volunteers and patients with hypertension in winter in Example 1. A represents the healthy volunteer group in winter, and B represents the patient group with hypertension in winter.

[0023] Figure 5 This is a correlation analysis diagram of the differential gut microbiota between healthy volunteers and hypertensive patients in winter and their clinical blood pressure values ​​in Example 1. In the diagram, #1 represents... s_Alistiper onderdonkii #2 is s_Lachnospira eligens #3 is s_Megasphaera elsdenii #4 is s_Victicallis lenta, #5 is s_Prevotella copri #6 is s_Anaerostipes hadrus #7 is s_Bacteroides thetaiotaomicron #8 is s_ Bifidobacterium longum #9 is s_Bifidobacterium pseudocatenulatum #10 is s_ Candidatus Cibionibacter quicibialis 11# is s_Intestinibacter bartlettii #12 is s_Muribaculaceae bacterium #13 is s_Phocaeicola coprocola ;

[0024] Figure 6 This is a comparison of the relative abundance of A. hadrus in fecal samples from healthy volunteers SN (summer), hypertensive patients SH (summer), healthy volunteers WN (winter), and hypertensive patients WH (winter) in Example 1.

[0025] Figure 7 This is a comparison chart of systolic and diastolic blood pressure in two groups of rats in Example 2;

[0026] Figure 8 This is a comparison of the plasma levels of adrenocorticotropic hormone (ACTH), adrenaline (EPI), and angiotensin II (Ang II) in two groups of rats in Example 2.

[0027] Figure 9 This is a comparison chart of the Phe concentration-effect curves obtained in the vascular ring experiment of Example 2;

[0028] Figure 10This is a comparison chart of the concentration-effect curves of Phe+L-NAME obtained in the vascular ring experiment of Example 2;

[0029] Figure 11 This is a comparison chart of the Ach concentration-effect curves obtained in the vascular ring experiment of Example 2;

[0030] Figure 12 This is a comparison chart of the SNP+L-NAME concentration-effect curves obtained in the vascular ring experiment of Example 2;

[0031] Figure 13 Example 3 A. hadrus Comparison of metabolite levels in fermentation supernatant and blank culture medium;

[0032] Figure 14 Example 3 A. hadrus Volcano plots of differential metabolites in fermentation supernatant and blank culture medium;

[0033] Figure 15 Example 3 A. hadrus Cluster analysis of the top 25 differentially expressed metabolites in fermentation supernatant and blank culture medium;

[0034] Figure 16 Example 3 A. hadrus Bar graph showing the enrichment of KEGG signaling pathway in differential metabolites between fermentation supernatant and blank culture medium;

[0035] Figure 17 Example 3 A. hadrus Differential signaling pathways and metabolite enrichment bar graphs between fermentation supernatant and blank culture medium;

[0036] Figure 18 Example 3 A. hadrus Comparative graph of the predicted antihypertensive activity of the three metabolites NFEIY, VLDE and LPY with the most significant differences in fermentation supernatant and blank culture medium.

[0037] Figure 19 This is a schematic diagram of the LPY and ACE amino acid binding sites predicted by molecular docking in Example 3;

[0038] Figure 20 This is a schematic diagram of the LPY-ACE binding pocket obtained from the molecular docking prediction in Example 3;

[0039] Figure 21 Example 4: Comparison of plasma LPY levels in summer healthy volunteers (SN), summer hypertensive patients (SH), winter healthy volunteers (WN), and winter hypertensive patients;

[0040] Figure 22 This is a comparison of LPY plasma levels in room temperature rats and cold-exposed rats, as shown in Example 4.

[0041] Figure 23 This is a comparison of systolic blood pressure in two groups of cold-exposed rats treated with LPY or control PBS, as shown in Example 4.

[0042] Figure 24 This is a comparison of diastolic blood pressure in two groups of cold-exposed rats treated with LPY or control PBS, as shown in Example 4.

[0043] Figure 25 This is a comparison of plasma Ang II concentrations in two groups of cold-exposed rats treated with LPY or control PBS, as shown in Example 4.

[0044] Figure 26 This is a comparison of the inhibitory effects of different concentrations of LPY on the ACE enzyme activity of rat pulmonary vascular endothelial cells in Example 5.

[0045] Figure 27 This is a comparison of Ang II levels in the cell supernatants obtained after treating rat lung vascular endothelial cells with LPY and control PBS, respectively, in Example 5.

[0046] Figure 28 The diagram shows the binding of LPY obtained from molecular docking in Example 5 and the known ACE inhibitor captopril to ACE. A represents the binding of captopril to ACE, and B represents the binding of LPY to ACE. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0048] The statistical methods used to present the experimental results in the following examples are as follows:

[0049] Data analysis was performed using GraphPad Prism 11.0 statistical software. Data are expressed as mean ± standard deviation or median (interquartiles). T-tests were used for comparisons between two groups; one-way ANOVA combined with Bonferroni-t tests were used for comparisons among multiple groups. Linear regression and logistic regression were used to assess the correlation between indicators. *P < 0.05, **P < 0.01, ***P < 0.001, and P < 0.05 were considered statistically significant.

[0050] Example 1

[0051] To investigate the role of gut microbiota in cold-induced hypertension, this study compared the gut microbiota of hypertensive patients and healthy volunteers in winter and summer, and found significant differences in the microbiota profiles.

[0052] (a) Collecting population samples and clinical information

[0053] Sixty healthy volunteers and 60 newly diagnosed hypertension patients were selected during the winter (December-January, the coldest months), and 60 healthy volunteers and 60 newly diagnosed hypertension patients were selected during the summer.

[0054] (1) Inclusion criteria for patients with hypertension:

[0055] Patients aged ≥18 years who are newly diagnosed with hypertension through outpatient / inpatient care;

[0056] (2) Exclusion criteria for patients with hypertension:

[0057] Those who have taken antibiotics within the past two months; patients with secondary hypertension; patients with severe liver or kidney dysfunction, infectious diseases, or malignant tumors; pregnant or breastfeeding women.

[0058] (3) Inclusion criteria for healthy patients:

[0059] The study recruited healthy control subjects without significant systemic diseases (ischemic heart disease, malignant tumors, lung diseases, and infectious diseases) through the Physical Examination Center of the First Affiliated Hospital of Harbin Medical University.

[0060] (II) Metagenomic sequencing of gut microbiota

[0061] Fecal samples were collected from the population, and DNA was extracted for library construction and sequencing to obtain raw data. First, file integrity was assessed; clean reads were quality checked using FastQC software; metagenomic assembly was performed using SPADES software; and the assembly results were statistically analyzed and evaluated using Quast software. ORF sequences were predicted using Prodigal software, and genome binning was performed using Maxbin software. The predicted ORF sequences were clustered using CD-HIT software to construct non-redundant gene sequence combinations. The clean reads were compared to the non-redundant gene set using SOAPA Lingner software, and the abundance of each gene in each sample was calculated. Gene sequences were compared with databases such as NR, CAZy, KEGG, and GOCOG using DIAMON17 software, and gene function was predicted based on sequence similarity. Finally, Metaphlan software was used to obtain five-level clustering heatmap visualization analysis results.

[0062] (III) Analysis Results

[0063] like Figure 1 and Figure 2 As shown, there are significant differences in the gut microbiota profiles of hypertensive patients and healthy volunteers regardless of whether it is winter or summer, indicating that the gut microbiota composition of hypertensive patients is significantly different from that of healthy individuals.

[0064] This embodiment performs linear discriminant analysis of differential bacterial abundance and microbial co-occurrence network analysis on the gut microbiota of healthy volunteers and hypertensive patients in winter. The results are as follows: Figure 3 and Figure 4 As shown, compared with healthy volunteers in winter, the relative abundance of 3 bacterial species was significantly increased and the relative abundance of 9 bacterial species was significantly decreased in hypertensive patients in winter. The co-occurrence network pattern of hypertensive patients in winter differed from that of healthy individuals, revealing the abundance changes of specific bacterial species in the gut microbiota of hypertensive patients.

[0065] This embodiment further performs correlation analysis on the clinic blood pressure values ​​of subjects with differentially expressed bacteria, and the results are as follows: Figure 5 As shown, the abundance of certain differentially expressed bacteria is significantly correlated with blood pressure levels.

[0066] This embodiment compares fecal samples from different subjects. A. hadrus The relative abundance, the results are as follows Figure 6 As shown, winter subjects compared to summer subjects A. hadrus The relative abundance decreased significantly in patients with hypertension during winter compared to healthy volunteers during winter. A. hadrus The relative abundance decreased more significantly and was negatively correlated with the subjects' office blood pressure levels, suggesting... A. hadrus It has become a new target for the prevention and treatment of hypertension, and it is hoped that blood pressure levels can be controlled by regulating its abundance.

[0067] Example 2

[0068] This embodiment investigated the effect of gavage supplementation using rat experiments. A. hadrus Effects on blood pressure and vascular function in cold-exposed rats.

[0069] (I) Methods for establishing a rat model of hypertension induced by cold

[0070] Male SD rats raised in SPF grade were fed standard rat diet with free access to food and water. They were then kept in a 4°C constant temperature enclosure for 4 weeks to establish a cold-induced hypertension rat model.

[0071] (two) A. hadrus Methods for bacterial culture and preparation of oral bacterial solution

[0072] A. hadrus Preparation method of bacterial culture medium:

[0073] Weigh 5.75g of minced meat carbohydrate broth culture medium into 100mL of ddH2O, heat to boiling and continue boiling for at least 1 minute, then dispense into test tubes. Add an appropriate amount of minced beef to each test tube until it reaches 1 / 3 of the liquid height. Autoclave at 121℃ for 30 minutes, then cool to below 50℃. Aseptically add 0.5mg of heme chloride and 5mg of vitamin K1 per 100mL. Place the prepared culture medium in an anaerobic chamber for deoxygenation for 24 hours before use.

[0074] The formula for the minced meat carbohydrate broth culture medium is as follows: 30.0g casein peptone, 10.0g beef extract, 5.0g yeast extract, 5.0g dipotassium hydrogen phosphate, 0.5g L-cysteine ​​hydrochloride, 0.0005g resazurin, 4.0g glucose, 1.0g cellobiose, 1.0g maltose, and 1.0g starch, with a pH of 7.0±0.2.

[0075] A. hadrus Method for preparing bacterial culture solution for gavage:

[0076] After the culture medium is deoxygenated for 24 hours, A. hadrus The commercial bacteria were revived into deoxygenated medium, and the revival process was observed. A. hadrus The culture medium has been fully grown, and it is ready for subculturing. A. hadrus 1 mL of bacterial culture was inoculated into the deoxygenated culture medium, and then the culture medium was placed in an anaerobic chamber and incubated at 37°C for 48 h. After that, it was removed from the anaerobic chamber, placed in a centrifuge, centrifuged at 4500 g for 15 min, and the supernatant was removed.

[0077] Resuspend the bacteria in PBS in a test tube, and take 3 mL of the bacterial suspension. Analyze the turbidity using a McFarland tube, and adjust the bacterial concentration to 1 × 10⁻⁶ using PBS buffer. 9 CFU / mL. The bacterial culture sample was sealed in a 15mL sterile centrifuge tube, frozen at -80℃, and administered by gavage after thawing.

[0078] This embodiment uses A. hadrus Purchased from Gray Algae Biotechnology Co., Ltd., product number HZB576107, specifically Corynebacterium hardrus DSM 3319.

[0079] (III) Methods and results of gavage administration of A. hadrus to cold-induced hypertensive rats

[0080] A rat model of hypertension induced by cold was randomly divided into two groups, which were administered the model by gavage. A. hadrus The bacterial culture or an equal volume of PBS solution was administered by gavage at a dose of 1 mL once daily for 4 weeks. After 4 weeks of gavage, the blood pressure changes, plasma levels of adrenocorticotropic hormone (ACTH), adrenaline (EPI), and angiotensin II (Ang II) were monitored in both groups of rats.

[0081] The results are as follows Figure 7 and Figure 8 As shown, compared with the control group, A. hadrus In the gavage group, both systolic and diastolic blood pressure were significantly reduced in rats with cold-induced hypertension. Comparison of plasma ACTH, PEI, and Ang II levels showed that, compared with the control group, A. hadrus In the gavage group, the levels of PEI and Ang II in cold-induced hypertensive rats both decreased, with a significant decrease in Ang II levels. This indicates that... A. hadrus Gavage administration may help alleviate hypertension symptoms by inhibiting the RAAS system and reducing PEI and Ang II levels in hypertensive rats, especially significantly reducing Ang II levels.

[0082] (iv) Experimental methods and results of isolated vascular ring tension test

[0083] Nitric oxide (NO) is a key vasodilator released by vascular endothelial cells. Abnormalities in the NO pathway are a significant mechanism of hypertension, and enhancing NO bioavailability has become an important direction in the development of antihypertensive drugs. This example focuses on... A. hadrus Four weeks after gavage administration, two groups of rats underwent an isolated thoracic aortic vascular ring tension test to investigate the effects of gavage administration. A. hadrus The effect on vasomotor function, the specific experimental method of the vascular ring test is as follows:

[0084] (1) Solution preparation

[0085] Physiological saline solution (PSS): Prepare 500 mL of the following composition (mmol / L): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 11.1, pH 7.4 (adjusted by passing a mixture of 95% O2 and 5% CO2 gas).

[0086] High-potassium saline solution (KPSS): Prepare 10 mL of PSS by increasing the KCl concentration to 60 mmol / L (equal molar replacement of NaCl), with the remaining components the same as PSS.

[0087] Drug stock solution:

[0088] Phenylephrine (Phe): Prepared with double-distilled water at a concentration of 10... -2 The mol / L stock solution was aliquoted and stored at -20°C.

[0089] Acetylcholine (ACh): Prepared with double-distilled water at a concentration of 10... -1 The mol / L stock solution was aliquoted and stored at -20°C.

[0090] Sodium nitroprusside (SNP): Prepare 10g of solution with double-distilled water. -1mol / L stock solution, stored away from light.

[0091] Nω-nitro-L-arginine methyl ester (L-NAME): Prepared with PSS 10 -1 mol / L stock solution, prepare fresh before use.

[0092] (2) Preparation of vascular rings

[0093] The rats were anesthetized, and after the neck was severed, the thoracic cavity was quickly opened, the thoracic aorta was freed, and placed in a pre-cooled PSS solution at 4°C. The surface tissue of the blood vessel was removed under a microscope, and the blood vessel was cut into 4 ring-shaped samples, each about 2 mm long. One blood vessel ring sample was used for each group of experiments.

[0094] (3) vascular ring mounting and balance

[0095] Add 5 mL of preheated PSS solution (37°C) to the organ chamber and continuously purge with a mixture of 95% O2 and 5% CO2. Secure the vascular ring to two stainless steel triangular hooks at the lower end and connect a force sensor to the upper end. Adjust the initial length to a baseline tension of 2.0 g and record this as point "0". Raise the temperature to 37°C, replacing the preheated PSS solution every 20 minutes, and continue equilibration for 60 minutes until the tension stabilizes before commencing the experiment.

[0096] (4) High potassium stimulation to verify vasoactivity

[0097] Add 100 μL of 60 mmol / L KPSS solution (final concentration 12 mmol / L) to induce vasoconstriction to the plateau phase (approximately 3–5 min), and record the maximum amplitude of vasoconstriction. Rinse three times with 5 mL of PSS solution preheated to 37°C each time to restore baseline tension, and allow the blood vessels to rest for 10 min.

[0098] 5. Group Experiment

[0099] (1) Phe group:

[0100] Add Phe stock solution to bring the final Phe concentration in the bath to 10. -8.5 10 -8 10 -7.5 10 -7 10 -6.5 10 -6 10 -5.5 10 -5 For concentrations of mol / L, the concentration or volume of the stock solution needs to be adjusted to ensure an accurate final concentration.

[0101] Example of final concentration calculation: 10 μL of stock solution added each time. -2After adding mol / L Phe to 5 mL of bath, the final concentration = 2 × 10⁻⁶ -5 mol / L.

[0102] Each concentration was applied for 5 minutes until the plateau phase, and the contraction amplitude was recorded. After rinsing, the baseline was restored, and the next set of experiments was conducted.

[0103] (2) Phe+L-NAME group:

[0104] First add L-NAME (10 -4 M) Incubate for 10 minutes, then add Phe stock solution to bring the final Phe concentration in the bath to 10. -8.5 10 -8 10 -7.5 10 -7 10 -6.5 10 -6 10 -5.5 10 -5 mol / L, each concentration was applied for 5 min until the plateau phase, the shrinkage amplitude was recorded, and after rinsing, the baseline was restored before proceeding to the next set of experiments.

[0105] (3) Ach group:

[0106] Add Ach stock solution to bring the final Ach concentration in the bath to 10. -8.5 10 -8 10 -7.5 10 -7 10 -6.5 10 -6 10 -5.5 10 -5 mol / L, each concentration was applied for 5 min until the plateau phase, the amplitude of diastole was recorded, and after rinsing, the baseline was restored before proceeding to the next set of experiments.

[0107] (4) SNP+L-NAME group:

[0108] First add L-NAME (10 -4 M) Incubate for 10 minutes, then add Ach stock solution to bring the final Ach concentration in the bath to 10. -8.5 10 -8 10 -7.5 10 -7 10 -6.5 10 -6 10 -5.5 10 -5 mol / L, each concentration was applied for 5 min until the plateau phase, the contraction amplitude was recorded, and the baseline was restored after rinsing.

[0109] The Phe concentration-effect curve obtained from the Phe group experiment is as follows: Figure 9 As shown, A. hadrus The vasoconstriction amplitude in the gavage group and the control group was less than 10 μmol / L. -6 No significant difference was observed at mol / L; however, when the Phe concentration increased to 10 mol / L... -6 10 -5.5 10 -5 At mol / L, the contraction amplitude in the gavage group was significantly lower than that in the control group. This indicates that the gavage... A. hadrus It has an inhibitory effect on vasoconstriction in cold-induced hypertensive rats.

[0110] The concentration-effect curve of Phe+L-NAME obtained in the Phe+L-NAME group experiment is, for example, Figure 10 As shown, A. hadrus The vasoconstriction amplitude in the gavage group and the control group was less than 10 μmol / L. -7 No significant difference was observed at mol / L; at high concentrations of Phe (10 mol / L); -7 10 -6.5 10 -6 10 -5.5 10 -5 At mol / L, the contraction amplitude in the gavage group was lower than that in the control group, but the reduction was less than that in the Phe group. L-NAME inhibits endothelial nitric oxide synthase, thereby blocking NO synthesis and eliminating the inhibitory effect of endothelial-dependent vasoconstriction on vasoconstriction. In the Phe+L-NAME group, vasoconstriction mainly reflected the direct responsiveness of smooth muscle to Phe, excluding the vasodilatory regulation by NO. The Phe group received gavage. A. hadrus The reduced contraction after gavage was more pronounced, indicating that the NO pathway is one of the main mechanisms by which blood pressure is lowered in the gavage group. However, the gavage group still showed reduced contraction under Phe+L-NAME conditions, suggesting... A. hadrus The mechanism by which cold-induced hypertension rats lower blood pressure does not depend entirely on the NO pathway; it may exert a compensatory effect through other diastolic pathways under high concentrations of Phe.

[0111] The Ach concentration-effect curves obtained from the experiments in the Ach group and the SNP+L-NAME group, for example... Figure 11 and Figure 12 As shown, A. hadrus There were no significant differences in the amplitude of vasodilation between the gavage group and the control group under different concentrations of Ach or SNP, indicating that... A. hadrus Gavage did not significantly alter the vasodilatory response of blood vessels to Ach or SNP. This further suggests that... A. hadrus The antihypertensive effect is not primarily achieved through enhancing endothelium-dependent relaxation or directly acting on SNP-sensitive pathways in vascular smooth muscle, but rather through other mechanisms that are not yet fully understood.

[0112] Based on the previous plasma hormone level test results, A. hadrus Gavage administration indirectly improves vasomotor function by reducing the levels of vasoconstrictive substances such as Ang II. This indicates that... A. hadrus It can lower blood pressure by regulating vascular function through multiple targets, and is especially suitable for cold-induced hypertension with endothelial dysfunction, providing a new microbial intervention strategy for the prevention and treatment of cold-induced hypertension.

[0113] Example 3

[0114] Gut microbiota can influence the function of distal target organs through their metabolites. To investigate... A. hadrus Mechanisms for regulating cold-induced hypertension. This example collects... A. hadrus Non-target metabolomics sequencing was performed on fermentation supernatant and blank culture medium supernatant.

[0115] According to Example 2 A. hadrus Microbial culture methods A. hadrus Fermentation culture was carried out at 37℃ for 48 h, and the fermentation broth was collected. After centrifugation at 12000g for 5 min, the supernatant was collected and extracted for pretreatment. Metabolites were separated and detected by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LS-MS), or nuclear magnetic resonance (NMR).

[0116] We used Metabo Analysis 4.0 to filter low-quality metabolite types, compress the data, and standardize it. We applied principal component analysis to assess the overall distribution of the samples, and used discriminant analysis (PLS-DA) to build a predictive model and check the prediction accuracy. We used volcano plots to visualize differential metabolites, and used databases such as HMDB, KEGG, Reactome BioCyc, and MetaCyc to annotate differential metabolites. We then performed species abundance, differential metabolite function, and pathway enrichment analysis between the two groups of samples.

[0117] Figure 13 A comparison of metabolite levels in Anaerostipes hadrus fermentation supernatant and blank culture medium; as shown in the figure. Figure 13 As shown, there were significant differences in the levels of metabolites between the two groups.

[0118] Figure 14 and Figure 15 The images show volcano plots of differentially expressed metabolites in Anaerostipes hadrus fermentation supernatant and blank culture medium, respectively, and cluster analysis of the top 25 differentially expressed metabolites. Figure 14 The results showed that, compared with the control group, A. hadrus 110 metabolites were significantly downregulated and 32 metabolites were significantly upregulated in the fermentation supernatant. Figure 15The most significant differences were observed in three small peptides: NFEIY with the amino acid sequence Asn-Phe-Glu-IIe-Tyr, VLDE with the amino acid sequence Val-Leu-Asp-Glu, and LPY with the amino acid sequence Leu-Pro-Tyr.

[0119] Figure 16 and Figure 17 In Example 3 A. hadrus Bar graphs showing the enrichment of KEGG signaling pathways and differential signaling pathways and metabolites in fermentation supernatant and blank culture medium; Figure 16 The results showed that differential signaling pathways were mainly enriched in the renin-angiotensin-aldosterone system (RAAS) and amino acid synthesis signaling pathways, among which the RAAS plays an important regulatory role in the occurrence and development of hypertension. Figure 17 The results showed that LPY was the main differential metabolite in the renin-angiotensin-aldosterone system RAAS pathway.

[0120] Deep learning was used to perform probabilistic predictive analysis of the antihypertensive activity of the three small peptides NFEIY, VLDE, and LPY, which showed the most significant differences. The results are as follows: Figure 18 As shown, LPY has the highest probability of exhibiting antihypertensive activity.

[0121] The three-dimensional structures of ACE protein and LPY were obtained from the PDB and PubChem databases, respectively. After preprocessing including structural adjustment and conversion, the amino acid binding sites and docking pocket positions of LPY and Ang II to form the key enzyme ACE were predicted, and molecular docking experiments were performed. Results are as follows: Figure 19 and Figure 20 As shown, LPY and AngII have a binding site for the key enzyme ACE, which can bind specifically.

[0122] Example 4

[0123] This embodiment uses mass spectrometry to verify the plasma LPY concentrations collected in Example 1 from healthy volunteers in summer, patients with hypertension in summer, healthy volunteers in winter, and patients with hypertension in winter. The results are as follows: Figure 21 As shown, plasma LPY concentrations were lower in subjects during winter than in summer, and the decrease in plasma LPY levels was more significant in hypertensive patients during winter than in healthy volunteers.

[0124] This embodiment uses mass spectrometry to verify the plasma LPY concentrations of rats subjected to cold exposure for 4 weeks in a 4°C constant-temperature enclosure and greenhouse rats. The results are as follows: Figure 22 As shown, the plasma LPY level in cold-exposed rats was significantly lower than that in rats at room temperature.

[0125] The above experimental results indicate that plasma LPY concentration is related to season, hypertension status, and cold exposure. The concentration decreases in winter or in cold environments, and hypertension may exacerbate this trend.

[0126] To clarify the role of LPY in cold-induced hypertension, SD rats were divided into two groups: an LPY group and a control group. LPY was prepared using a solid-phase synthesis method and formulated into an injectable solution with PBS buffer. SD rats in the LPY group were intraperitoneally injected with the LPY solution at a dose of 10 mg LPY / kg body weight, while the control group was injected with an equal volume of PBS. The injections were administered daily for four weeks, during which both groups were exposed to a 4°C environment. Blood pressure and plasma Ang II concentrations were monitored in both groups after four weeks.

[0127] The results are as follows Figures 23-25 As shown, LPY supplementation significantly reduced systolic blood pressure, diastolic blood pressure, and plasma Ang II levels in cold-exposed rats.

[0128] Example 5

[0129] This embodiment verifies the inhibitory effect of LPY on ACE enzyme activity through cell experiments.

[0130] The cells used in this embodiment are primary lung vascular endothelial cells, and the specific extraction method is as follows:

[0131] (1) The rat was euthanized by cervical dislocation. It was quickly fixed supine on the dissecting board. The abdominal skin was disinfected with 75% alcohol. The skin and abdomen were cut along the midline of the abdomen to expose the thoracic cavity. The abdominal wall was cut to expose the heart and lungs. The lungs were carefully cut off and placed in a culture dish containing pre-cooled sterile PBS. The lungs were gently rinsed 2-3 times to remove surface blood and impurities.

[0132] (2) Transfer the rinsed lungs to centrifuge tubes, add an appropriate amount of collagenase II solution, mix gently, seal the centrifuge tubes with sealing film, and place them in a 37°C water bath for 30-60 minutes, gently shaking every 5 minutes to ensure complete digestion. After digestion, remove the centrifuge tubes, centrifuge at 1000 r / min for 5 minutes, discard the supernatant, add erythrocyte lysis buffer to the precipitate, gently pipette to mix, let stand at room temperature for 5 minutes, centrifuge again at 1000 r / min for 5 minutes, and discard the supernatant.

[0133] (3) The precipitate was reselected with an appropriate amount of endothelial cell culture medium and then passed through 70μm and 40μm cell sieves in sequence to obtain a cell suspension. After centrifuging the suspension again, the cells were resuspended with endothelial cell culture medium and seeded into a culture flask coated with gelatin. The flask was placed in a 37℃, 5% CO2 incubator and cultured for 12h. The culture medium was then gently poured off, and the cells were gently rinsed twice with sterile PBS. Fresh endothelial cell culture medium was added and the cells were cultured for a longer period of time.

[0134] The cultured primary pulmonary vascular endothelial cells were placed in 6-well plates, with 1 × 10⁶ cells per well. 5 Cells were treated with 1 μM Ang II and cultured for 24 h. Then, 0 μM, 200 μM, 400 μM, and 800 μM LPY were added respectively. After 24 h, the cell culture supernatant was extracted, and the intracellular ACE enzyme activity was detected using an ELISA kit. Results are as follows: Figure 26 As shown, ACE activity is negatively correlated with LPY concentration, and there may be a concentration-dependent relationship.

[0135] In this embodiment, primary rat pulmonary vascular endothelial cells were pretreated with Ang I. The cultured primary pulmonary vascular endothelial cells were placed in 6-well plates, with 1 × 10⁶ cells per well. 5 Cells were treated with 1 μM Ang II and cultured for 24 h. Then, 200 μM LPY and an equal volume of PBS buffer were added. After 24 h, the cell culture supernatant was extracted, and the Ang II level in the supernatant was detected using an ELISA kit. Results are as follows: Figure 27 As shown, the Ang II level in the LPY group was significantly lower than that in the control group.

[0136] The above results suggest that the metabolite LPY may inhibit ACE enzyme activity and reduce Ang II production by specifically binding to ACE.

[0137] This embodiment further compares the ability of LPY to bind to ACE with the known ACE inhibitor captopril through molecular docking. Figure 28 As shown, captopril forms 5 hydrogen bonds with residues surrounding ACE, while the LPY peptide forms 6 hydrogen bonds with residues surrounding ACE within the catalytic pocket. This suggests that LPY has a stronger affinity for ACE than captopril, demonstrating that LPY has a stronger potential to inhibit ACE than captopril.

Claims

1. Application of Anaerostipes hadrus DSM 3319 in the preparation of drugs for the prevention and treatment of hypertension.

2. The application of Anaerostipes hadrus DSM 3319 according to claim 1 in the preparation of drugs for the prevention and treatment of hypertension, characterized in that, The hypertension mentioned is hypertension caused by cold.

3. The application of Anaerostipes hadrus DSM 3319 according to claim 2 in the preparation of drugs for the prevention and treatment of hypertension, characterized in that, The hypertension mentioned is cold-induced hypertension combined with endothelial dysfunction.

Images