Application of Irisin autocrine probiotics in preparation of medicine for preventing / treating primary hypertension

Abstract

The invention discloses an application of an autocrine Irisin probiotic B.Subtills-WB600-Irisin in preparation of a medicine for preventing / treating primary hypertension, and belongs to the field of biological medicines. The invention further discloses a preparation method of the autocrine Irisin probiotic B.Subtills-WB600-Irisin. According to the application disclosed by the invention, it is found for the first time that the probiotic B.Subtills-WB600-Irisin capable of automatically secreting Irisin protein simultaneously has the effect of preventing / treating primary hypertension, can effectively reduce systolic pressure of a rat with primary hypertension, and has no obvious influence on diastolic pressure. According to the invention, autocrine Irisin probiotics (B.Subtills-WB600-Irisin) are activated and then prepared into an oral preparation, in an SHR model, primary hypertension is prevented and treated by oral administration of the bacterial preparation (1 * 10 < 9 > CFU / day), and continuous blood pressure reduction (systolic pressure reduction gt, 20 mmHg) can be realized. The invention not only provides the first type of movement simulation probiotics for hypertension treatment, but also opens up a new path for microbial therapy of other metabolic diseases (such as diabetes and obesity), and has important clinical transformation and application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Irisin, a self-secreting probiotic, in the preparation of drugs for the prevention / treatment of essential hypertension. Background Technology

[0002] Hypertension is one of the most common chronic cardiovascular diseases worldwide, affecting approximately 1.3 billion people. It is a major risk factor for stroke, myocardial infarction, heart failure, and chronic kidney disease. Currently, clinical antihypertensive drugs mainly include angiotensin-converting enzyme inhibitors, calcium channel blockers, diuretics, and angiotensin receptor antagonists. While these drugs are effective in lowering blood pressure, they still have significant limitations, such as potential side effects like arrhythmias and electrolyte disturbances; drug resistance in some patients, leading to refractory hypertension; and the fact that existing drugs can only slow disease progression and cannot reverse target organ damage, with long-term hypertension potentially causing irreversible damage such as vascular endothelial dysfunction, myocardial hypertrophy, and glomerulosclerosis. Furthermore, traditional antihypertensive drugs require daily oral or injectable administration, resulting in poor patient compliance, and long-term use may place a burden on liver and kidney function. Therefore, developing a novel antihypertensive strategy with simple administration, few side effects, long-term stability, and organ protection is an urgent research need.

[0003] Irisin, as an important muscle secretory factor induced by exercise, has shown unique potential in the treatment of hypertension. Multiple studies have shown that irisin exerts its antihypertensive effect through multiple mechanisms: (1) by activating the AMPK / eNOS signaling pathway to promote nitric oxide production in endothelial cells, thus improving vascular tone; (2) by inhibiting the renin-angiotensin system, reducing angiotensin II levels, and alleviating vasoconstriction and inflammatory responses; and (3) by inhibiting renal tubular epithelial sodium channels (ENaC) to promote urinary sodium excretion and reduce blood volume load. Animal experiments have shown that exogenous irisin injection can significantly reduce blood pressure levels in spontaneously hypertensive rats (SHR) and improve vascular endothelial function and myocardial fibrosis. However, due to its short in vivo half-life (<1h) requiring frequent injections, insufficient systemic targeting, and high production costs, irisin faces significant challenges in clinical translation. Therefore, there is an urgent need to develop a long-acting, targeted, and low-cost irisin delivery system to overcome the bottleneck in its pharmaceutical application for the prevention and treatment of essential hypertension. Summary of the Invention

[0004] This invention aims to at least partially solve one of the technical problems in the related art. Therefore, the main objective of this invention is to provide the application of Irisin, a self-secreting probiotic, in the preparation of a medicament for the prevention / treatment of essential hypertension.

[0005] The objective of this invention is achieved through the following technical solution: Application of Irisin, an autocrine probiotic, in the preparation of drugs for the prevention / treatment of essential hypertension.

[0006] In some specific embodiments, the Irisin-secreting probiotic is Irisin-secreting Bacillus subtilis (B. Subtilis WB600-Irisin). The team of this application also filed a patent application on the same day for the construction method of this Irisin-secreting Bacillus subtilis and the purification method of its protein.

[0007] The carrier of the self-secreting Irisin protein probiotic (B. Subtilis-WB600-Irisin) is Bacillus subtilis. As a Gram-positive probiotic, it has the characteristics of high safety, strong intestinal colonization ability, and sustainable and efficient expression and secretion of active proteins, making it an ideal Irisin delivery carrier.

[0008] In some specific embodiments, the Irisin self-secreting probiotic is one or more of the following: bacterial cells, bacterial cell lysates, and culture supernatant.

[0009] In some specific embodiments, the Irisin self-secreting probiotic and its products can reduce high systolic blood pressure.

[0010] In certain specific embodiments, regarding the description of the drug or pharmaceutical composition, when the self-secreting Irisin probiotic B. Subtilis-WB600-Irisin of the present invention is used as a drug, it can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1% to 99%, preferably 0.5% to 90% of the compound of the present invention, with the remainder being pharmaceutically acceptable, non-toxic, and inert pharmaceutically acceptable carriers or excipients for humans and animals.

[0011] In some specific embodiments, the pharmaceutically acceptable carrier is one or more selected from solid, semi-solid, and liquid diluents, superfillers, and pharmaceutical excipients. The effective extract or effective fraction is used in a dose per unit body weight.

[0012] In some specific embodiments, the oral administration may be in solid or liquid formulations, such as powders, tablets, sugar-coated tablets, capsules, syrups, pellets, etc.

[0013] Compared with the prior art, the present invention has at least the following advantages: 1) The application of the Irisin-secreting probiotic in the present invention in the preparation of drugs for the prevention / treatment of essential hypertension can achieve (1) long-term sustained release, the Irisin-secreting probiotic (B. Subtilis-WB600-Irisin) can continuously colonize the intestine and secrete Irisin, avoiding the problem of frequent injection; (2) targeted regulation, the Irisin absorbed by the intestine can preferentially act on the liver and kidney through the portal vein system, enhancing the protection of the target organs of hypertension; (3) multi-effect synergy, the engineered probiotics that secrete Irisin protein can regulate the intestinal flora (such as increasing short-chain fatty acid producing bacteria), improve hypertension-related flora imbalance (such as the increase of Firmicutes / Bacteroidetes ratio), and Irisin can further inhibit intestinal inflammation, forming a comprehensive regulatory network of "flora-immunity-metabolism".

[0014] 2) This invention activates the self-secreting Irisin probiotic (B. Subtilis-WB600-Irisin) and formulates it into an oral preparation; in the SHR model, the probiotic preparation (1×10⁻⁶) is administered orally. ^9 This product, containing CFU / day, is used for the prevention and treatment of essential hypertension, achieving sustained blood pressure reduction (systolic blood pressure reduction >20 mmHg). It not only provides the first "exercise-mimicking probiotic" for hypertension treatment but also opens new pathways for microbial therapy of other metabolic diseases (such as diabetes and obesity), demonstrating significant clinical translational applications. Attached Figure Description

[0015] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0016] Figure 1 To verify the long-term preventive effect of autocrine Irisin probiotic (B. Subtilis-WB600-Irisin) on essential hypertension, 4-week-old SHR rats were administered autocrine Irisin probiotic (B. Subtilis-WB600-Irisin) by gavage for 8 weeks. Blood pressure and related indicators were then measured using the tail cuff method. Figure 1 A represents the change in systolic blood pressure. Figure 1 B represents the changes in mean arterial pressure. Figure 1 C represents the change in diastolic blood pressure. Figure 1 D represents the changes in heart rate; Figure 2To verify the long-term therapeutic effect of autocrine Irisin probiotic (B. Subtilis-WB600-Irisin) on essential hypertension, blood pressure and related indicators were measured by tail cuff measurement after 8-week-old SHR rats were administered autocrine Irisin probiotic (B. Subtilis-WB600-Irisin) by gavage. Figure 2 A represents the change in systolic blood pressure. Figure 2 B represents the changes in mean arterial pressure. Figure 2 C represents the change in diastolic blood pressure. Figure 2 D represents the changes in heart rate; Figure 3 To verify the effect of self-secreted Irisin probiotic (B. Subtilis-WB600-Irisin) on essential hypertension using 24-hour ambulatory blood pressure monitoring, among which... Figure 3 A represents the change in systolic blood pressure over 24 hours. Figure 3 B represents the change in mean arterial pressure over 24 hours. Figure 3 C represents the change in diastolic blood pressure over 24 hours. Figure 3 D represents the changes in heart rate over 24 hours. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the scope of protection of this invention. Any formal equivalent modifications made based on the concept of this invention should be considered within the scope of this invention.

[0018] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0019] It should be understood that the examples described in this specification are merely for explaining the invention and are not intended to limit the invention in any way. Any modifications or improvements made based on the teachings of this invention fall within the protection scope of this invention.

[0020] Example 1 This embodiment provides a process for the activation, cultivation, and oral formulation preparation of self-secreting Irisin probiotic B. Subtilis-WB600-Irisin, the specific implementation steps of which are as follows: Strain activation: A certain amount of autosecretory Irisin probiotic B. Subtilis-WB600-Irisin glycerol bacteria was inoculated onto sterilized LB liquid medium and then placed in a constant temperature incubator at 37 ℃ for 24 h at a rotation speed of 200 rpm to obtain the seed fermentation broth of the strain. The LB liquid medium contained 10 g trypsin, 5 g yeast extract, and 10 g NaCl per liter, with the remainder being deionized water.

[0021] Strains culture: A certain amount of activated autosecretory Irisin probiotic B. Subtilis-WB600-Irisin bacterial suspension was inoculated onto sterilized LB liquid medium at a ratio of 1:100, and then placed in a constant temperature incubator at 37 ℃ for about 24 h.

[0022] Cryopreservation of the oral administration strain: Take 1 mL of the cultured bacterial solution for colony counting, and put the remaining bacterial solution into a sterile container. Centrifuge at 5000 g, 4℃ for 5 minutes and discard the supernatant. Add a certain amount of sterile pre-cooled PBS to wash, and centrifuge at 5000 g, 4℃ for 5 minutes. Resuspend the bacterial cells in pre-cooled 30% sterile skim milk powder solution, dispense 1 mL into each 1.5 mL sterile centrifuge tube, and then transfer to a programmed cooling box and store at -80℃ for later use.

[0023] Activation of the strain by gavage: Take out one tube of the self-secreting Irisin probiotic B. Subtilis-WB600-Irisin prepared in step 3 from the -80℃ freezer and immediately place it in 37℃ water and gently shake to thaw.

[0024] Oral formulation preparation: Transfer the bacterial culture to a 15 mL sterile centrifuge tube, and add a certain amount of sterile water according to the counting results to make a concentration of 1×10⁻⁶. ^9 A CFU / mL solution of skim milk powder containing bacteria yields a gavage solution of self-secreting Irisin probiotic B. Subtilis-WB600-Irisin.

[0025] Colony count: The bacterial count was performed at a ratio of 1×10⁻⁶. 1 ~1×10 10 Dilute the bacterial culture using the specified dilution factor, 200 μL per tube; Then, 100 μL of bacterial suspension was taken onto the surface of LB solid medium and spread using a sterile spreader. The culture dishes were placed in a constant temperature incubator and incubated at 37°C for 24 hours. After the incubation was completed, the culture dishes of each concentration were observed and counted, and it was found that the colonies grew well.

[0026] Example 2 This embodiment provides a verification of the preventive effect of self-secreted Irisin probiotic B. Subtilis-WB600-Irisin on essential hypertension. The specific implementation steps are as follows: Experimental methods: Twelve male 4-week-old SPF-grade SHR rats were randomly divided into two groups: the SHR group and the SHR+B. Subtilis-Irisin group.

[0027] SHR group: Rats were fed a standard growth and maintenance diet, with free access to food and water, and were given 1.0 mL of a 2% skim milk powder aqueous solution by gavage; SHR+B.Subtilis-Irisin group: Rats were fed a standard growth and maintenance diet, with free access to food and water, and were given 1.0 mL of bacterial solution containing 2% skim milk powder by gavage (containing 1×10⁻⁶ mmol / L). ^9 CFU B.Subtilis-WB600-Irisin cells); Systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate of rats were measured weekly using the tail cuff method for 8 consecutive weeks. SPF-grade SHR rats (purchased from Beijing Vital River Biotechnology Co., Ltd., and certified as qualified by the Institute of Medical Animals, Army Characteristic Medical Center) were used. All SPF-grade SHR rats were housed in the Experimental Animal Management Center of the Army Characteristic Medical Center under the following environmental conditions: temperature 20-24℃, humidity 50%, and isochronous diurnal rhythm.

[0028] Experimental results are as follows Figure 1 As shown, the blood pressure of rats in the SHR group gradually increased during the first 5 weeks, reaching a plateau at week 4 with a systolic blood pressure (SBP) of approximately 200 mmHg. In contrast, the SHR+B. Subtilis-Irisin group reached a plateau at weeks 5-6 with an SBP of approximately 180 mmHg. The SBP in the SHR+B. Subtilis-Irisin group after reaching the plateau was significantly lower than that in the SHR group, with a difference of approximately 20 mmHg. These results indicate that the self-secreting Irisin probiotic B. Subtilis-WB600-Irisin can, to some extent, prevent the increase in systolic blood pressure in rats with essential hypertension.

[0029] Example 3 This embodiment provides a verification of the therapeutic effect of self-secreted Irisin probiotic B. Subtilis-WB600-Irisin on essential hypertension, and the specific implementation steps are as follows: Experimental methods: Twelve male 8-week-old SPF-grade SHR rats were randomly divided into two groups: the SHR group and the SHR+B. Subtilis-Irisin group.

[0030] SHR group: Rats were fed a standard growth and maintenance diet, with free access to food and water, and were given 1.0 mL of a 2% skim milk powder aqueous solution by gavage; SHR+B.Subtilis-Irisin group: Rats were fed a standard growth and maintenance diet, with free access to food and water, and were given 1.0 mL of bacterial solution containing 2% skim milk powder by gavage (containing 1×10⁻⁶ mmol / L). ^9 CFU B.Subtilis-WB600-Irisin cells); Systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate of rats were measured weekly using the tail cuff method for 8 consecutive weeks. SPF-grade SHR rats (purchased from Beijing Vital River Biotechnology Co., Ltd., and certified as qualified by the Institute of Medical Animals, Army Characteristic Medical Center) were used. All SPF-grade SHR rats were housed in the Experimental Animal Management Center of the Army Characteristic Medical Center under the following environmental conditions: temperature 20-24℃, humidity 50%, and isochronous diurnal rhythm.

[0031] Experimental results are as follows Figure 2 As shown, the blood pressure of 8-week-old SHR rats remained relatively stable during the 8-week observation period, with a systolic blood pressure (SBP) of 200 ± 20 mmHg. Compared to the SHR group, the SHR+B. Subtilis-Irisin group showed a significantly lower SBP of approximately 180 mmHg after 8 weeks of treatment, indicating that B. Subtilis-WB600-Irisin gavage can reduce systolic blood pressure in SHR rats to some extent. Furthermore, B. Subtilis-WB600-Irisin also reduced diastolic blood pressure in SHR rats by approximately 10 mmHg. These results suggest that the self-secreting Irisin protein probiotic B. Subtilis-WB600-Irisin can treat elevated blood pressure in essential hypertensive rats to some extent.

[0032] Example 4 This embodiment provides a 24-hour ambulatory blood pressure monitoring demonstration of the effect of self-secreted Irisin probiotic B. Subtilis-WB600-Irisin on essential hypertension. The specific implementation steps are as follows: Experimental Methods: Three rats (12 weeks old) were randomly selected from each of the SHR group and the SHR+B. Subtilis-Irisin group in Example 3. The rats were anesthetized with sodium pentobarbital (30 mg / kg), and an ambulatory blood pressure monitoring implant was inserted via iliac artery cannulation. The wound was then sutured. One week after recovery, the systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate of each group were monitored using an ambulatory blood pressure monitoring device. Environmental Characteristics: Temperature 25℃, humidity 50%, and isochronous day and night.

[0033] Experimental Groups: The experimental groups are as follows.

[0034] SHR group: Rats were fed a standard growth and maintenance diet, with free access to food and water, and were given 1.0 mL of a 2% skim milk powder aqueous solution by gavage; SHR+B.Subtilis-Irisin group: Rats were fed a standard growth and maintenance diet, with free access to food and water, and were given 1.0 mL of bacterial solution containing 2% skim milk powder by gavage (containing 1×10⁻⁶ mmol / L). ^9 CFU B.Subtilis-WB600-Irisin cells).

[0035] Experimental results are as follows Figure 3 As shown, the blood pressure of the SHR group rats was significantly higher than that of the SHR+B.Subtilis-Irisin group, indicating that the engineered probiotic B.Subtilis-WB600-Irisin, which secretes irisin, can significantly reduce the blood pressure of SHR rats by gavage.

[0036] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. Application of Irisin, an autocrine probiotic, in the preparation of drugs for the prevention / treatment of essential hypertension.

2. The application according to claim 1, characterized in that, The Irisin self-secreting probiotic is Irisin Bacillus subtilis.

3. The application according to claim 1 or 2, characterized in that, Irisin, a self-secreting probiotic, is one or more of the following: bacterial cells, bacterial lysates, and culture supernatant.

4. The application according to claim 3, characterized in that, The Irisin self-secreting probiotic can reduce high systolic blood pressure.

5. The application according to claim 1, characterized in that, The drug comprises an effective dose of Irisin self-secreting probiotic and / or a pharmaceutically acceptable carrier.

6. The application according to claim 1, characterized in that, The dosage form of the drug is one of tablets, capsules, oral liquid, or lyophilized powder.

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