A method for improving the secretion amount of akkermansia muciniphila extracellular vesicles and application thereof in improving the activity of bone loss treatment

Abstract

The application provides a method for improving the secretion amount of Akkermansia muciniphila extracellular vesicles and application thereof in improving the preparation of bone loss treatment activity, and relates to the technical field of biological medicine.The method comprises the following steps: oscillation culture of Akkermansia muciniphila in an anaerobic environment for 60-96 h, centrifugal collection of bacterial liquid after culture is completed, and separation and purification; during the culture, near-infrared light irradiation is started after culture for 3.5-4.5 h, and the near-infrared light irradiation is cyclically carried out after every interval of 21.5-22.5 h. The application intermittently irradiates near-infrared light in the culture process of Akkermansia muciniphila, can improve the secretion amount of extracellular vesicles, and makes the inside of the extracellular vesicles rich in microRNA which has a treatment effect on bone loss caused by various causes such as inflammation and age growth, enhances bone formation activity, and has a more significant improvement effect on bone formation activity weakened by factors such as inflammation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for increasing the secretion of extracellular vesicles of Akkermansia muciniphila and its application in formulations that enhance the therapeutic activity of bone loss. Background Technology

[0002] Osteoporosis is increasingly affecting younger people and is impacting a wider population. Photobiological regulation has been shown to improve bone metabolism through the gut-bone axis. Its core mechanism involves directly stimulating the gut microbiota, specifically enriching beneficial gut bacteria, and thus systematically inhibiting bone resorption and promoting bone formation. However, this method is relatively inefficient.

[0003] Akkermansia myxophilus ( Akkermansia muciniphila Akk (Akk@OMVs) is a probiotic discovered in recent years whose cells can secrete extracellular membrane vesicles (OMVs). These released OMVs are small, membrane-bound vesicles that stably carry bioactive substances that can directly act on bone tissue, significantly enhancing osteogenic activity. AKK's OMVs can regulate the function of multiple organs, including the skeleton, and inhibit the occurrence and development of related diseases. However, current technologies face significant limitations, lacking methods to specifically increase the production and therapeutic activity of extracellular vesicles. Therefore, there is an urgent need to provide a novel and highly effective treatment approach to improve the prevention and treatment of osteoporosis. Summary of the Invention

[0004] To address the aforementioned technical problems, the primary objective of this invention is to provide a method for increasing the secretion of extracellular vesicles (OMVs) by Akk. This method can increase the secretion of Akk's OMVs by 1.5-2 times. Furthermore, the OMVs secreted by Akk (NIR-Akk@OMVs) are enriched with microRNAs that have therapeutic effects on bone loss caused by various factors such as inflammation and aging, thus enhancing the bone formation activity of Akk@OMVs and significantly improving the weakened bone formation activity caused by factors such as inflammation.

[0005] A second objective of this invention is to provide the application of the above-described method in increasing the secretion of extracellular vesicles by Akkermansia myxophilus.

[0006] A third objective of this invention is to provide the application of the above-described method in increasing the types and quantities of microRNAs in the extracellular vesicles of Akkermansia myxophilus.

[0007] A fourth objective of this invention is to provide the application of the above-described method in the preparation of formulations that enhance the therapeutic activity of bone loss treatment.

[0008] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0009] The application provides a method for improving the secretion amount of Akkermansia muciniphila extracellular vesicles, comprising the following steps: oscillating culture of Akkermansia muciniphila in an anaerobic environment for 60-96 h, centrifugal collection of supernatant after completion of culture, and separation and purification.

[0010] During the culture, near-infrared light irradiation is started at 3.5-4.5 h, and the near-infrared light irradiation is cyclically performed after every interval of 21.5-22.5 h.

[0011] The wavelength of the near-infrared light irradiation is 700-900 nm, and the time of the near-infrared light irradiation is 1.5-2.5 h.

[0012] As an implementation form, the peak wavelength of the near-infrared light is 860 nm.

[0013] As an implementation form, the light irradiation intensity of the near-infrared light is 4.0-4.4 mW / cm 2 .

[0014] As an implementation form, the culture medium of the culture is brain heart infusion.

[0015] As an implementation form, the brain heart infusion is Oxoid brain heart infusion broth CM1135 or brain heart infusion broth HB8297.

[0016] As an implementation form, the centrifugal rotation speed is 8000-12000 g, and the centrifugal time is 10-20 min.

[0017] As an implementation form, the separation and purification method is a magnetic bead method.

[0018] The application further provides application of the above method in improvement of the secretion amount of Akkermansia muciniphila extracellular vesicles.

[0019] The application further provides application of the above method in improvement of the microRNA types and quantities in Akkermansia muciniphila extracellular vesicles.

[0020] The application further provides application of the above method in preparation of a preparation with improved bone loss treatment activity.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] This invention, based on the principle of photobiological regulation, designs and implements a method for producing microRNAs with therapeutic effects on bone loss by simultaneously increasing the secretion of extracellular vesicles (OMVs) in Akkermansia muciniphila (Akk) through near-infrared irradiation. During Akk culture, intermittent, cyclic irradiation with near-infrared (NIR) light using LEDs increases the secretion of OMVs by 1.5-2 times. Furthermore, the OMVs secreted by Akk after NIR irradiation (NIR-Akk@OMVs) are enriched with microRNAs that have therapeutic effects on bone loss caused by various factors such as inflammation and aging. Experimental verification shows that NIR-Akk@OMVs not only enhances the bone-forming activity of Akk@OMVs but also significantly improves bone-forming activity weakened by factors such as inflammation. Attached Figure Description

[0023] Figure 1 The relative spectral composition of infrared LEDs;

[0024] Figure 2 The Nanosight detection results show the particle size distribution and concentration of OMV in each group. NIR-Akk@OMVs are extracellular vesicles in Example 1, and Akk@OMVs are extracellular vesicles in Comparative Example 1.

[0025] Figure 3 Transmission electron microscopy characterization of OMVs: left image shows NIR-Akk@OMVs, right image shows Akk@OMVs;

[0026] Figure 4 The left figure shows the microRNA sequencing analysis of OMV extracted from Akk bacterial culture. The right figure shows the bubble diagram of microRNA enrichment.

[0027] Figure 5 This is a control experiment for osteogenic differentiation in a TNFα-BMSC inflammation model induced by extracellular vesicles after near-infrared light irradiation. The left image is a photograph of Alizarin Red S staining; the right image is a box plot of the area stained with Alizarin Red S.

[0028] Figure 6 This is a control experiment for osteogenic differentiation of an IL-6-BMSC inflammation model induced by extracellular vesicles after near-infrared light irradiation. The left image is a photograph of Alizarin Red S staining; the right image is a box plot of the area stained with Alizarin Red S. Detailed Implementation

[0029] The application provides a method for increasing the secretion amount of Akkermansia muciniphila extracellular vesicles, comprising the following steps: oscillating culture of Akkermansia muciniphila in an anaerobic environment for 60-96 h, centrifugal collection of supernatant after completion of culture, and separation and purification.

[0030] In the application, after activation of Akkermansia muciniphila, the bacterial liquid is inoculated into a culture medium for culture, the inoculation amount of the inoculation is 0.1%-0.2%, the culture medium is preferably brain heart infusion, and the brain heart infusion is Oxoid brain heart infusion broth CM1135 or brain heart infusion broth HB8297, wherein the formula of the Oxoid brain heart infusion broth CM1135 is brain infusion solid 12.5 g / L, bovine heart infusion solid 5 g / L, proteose peptone 10.0 g / L, glucose 2.0 g / L, sodium chloride 2.5 g / L, and disodium phosphate 2.5 g / L, and the pH value of the Oxoid brain heart infusion broth CM1135 is 7.2-7.6; the formula of the brain heart infusion broth HB8297 is proteose peptone 10.0 g / L, dehydrated calf brain infusion powder 12.5 g / L, dehydrated bovine heart infusion powder 5.0 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, and disodium hydrogen phosphate 2.5 g / L, and the pH value of the brain heart infusion broth HB8297 is 7.2-7.6. In the application, the culture temperature is 36-38 DEG C, preferably 36.5 DEG C, 37 DEG C or 37.5 DEG C, and the culture time is 60-96 h, preferably 65 h, 72 h, 80 h or 90 h, so that the Akkermansia muciniphila can grow into a stable state and reach the plateau phase, and the culture is stopped.

[0031] In the application, near-infrared light irradiation is started after 3.5-4.5 h of culture, and the near-infrared light irradiation is cyclically performed after every interval of 21.5-22.5 h; the wavelength of the near-infrared light irradiation is 700-900 nm, the peak wavelength of the near-infrared light is 860 nm, the light intensity of the near-infrared light is 4.0-4.4 mW / cm 2 , preferably 4.1 mW / cm 2 , 4.2 mW / cm 2 or 4.3 mW / cm 2 , and the time of the near-infrared light irradiation is 1.5-2.5 h, preferably 2 h. In the application, the composition and temperature of the culture environment still need to be controlled during irradiation, and the secretion amount of Akkermansia muciniphila extracellular vesicles can be increased by cyclically irradiating near-infrared light in the process of oscillating culture.

[0032] After the culture cycle of the present application is completed, the supernatant is collected by centrifugation, the speed of the centrifugation is 8000-12000 g, preferably 9000 g, 10000 g or 11000 g; the time of the centrifugation is 5-15 min, preferably 8 min, 10 min, 12 min or 14 min, under the centrifugation condition of the present application, most of the bacteria in the bacterial suspension can be effectively removed. After centrifugation, the supernatant is filtered with a water-based filter membrane, the pore size of the filter membrane is 0.2-0.3 μm, preferably 0.22 μm, 0.25 μm or 0.27 μm, after filter membrane filtration, impurities in the supernatant can be effectively removed. Then, the filtered supernatant is separated and purified, the method of separation and purification is magnetic bead method. As an optional embodiment, the present application uses OMVs rapid purification kit for separation, after separation, the mucinophilic Akkermansia muciniphila extracellular vesicle product is obtained, the biological activity of OMVs can be maximally ensured by magnetic bead separation and purification in the present application.

[0033] Based on the present application, intermittent irradiation of near-infrared light with LED can improve the secretion amount of mucinophilic Akkermansia muciniphila extracellular vesicles, and the present application also provides the use of the above-mentioned method in improving the secretion amount of mucinophilic Akkermansia muciniphila extracellular vesicles. The secretion amount of mucinophilic Akkermansia muciniphila extracellular vesicles can be increased by 1.5-2 times by using the method of the present application.

[0034] Based on the present application, intermittent irradiation of near-infrared light with LED can enrich microRNA in mucinophilic Akkermansia muciniphila extracellular vesicles, and the present application also provides the use of the above-mentioned method in improving the type and quantity of microRNA in mucinophilic Akkermansia muciniphila extracellular vesicles. The method of the present application can enrich microRNA in mucinophilic Akkermansia muciniphila extracellular vesicles, including miR-181b-5p, miR-10a-5p, miR-125a-5p, miR-10b-5p.

[0035] The present application also provides the use of the above-mentioned method in preparing a preparation for improving bone loss treatment activity. The method of the present application can significantly improve the decrease in osteogenic differentiation ability induced by inflammation, even exceeding the osteogenic differentiation level of BMSCs without inflammation modeling.

[0036] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be described in detail below combined with examples, but they should not be understood as limiting the scope of protection of the present application.

[0037] In the following examples, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels, such as the reagents, consumables, etc. involved in the present application. If no specific use conditions are indicated, they are usually carried out according to the conventional conditions or according to the conditions recommended by the company.

[0038] The formula of Oxoid brain heart infusion broth CM1135: brain infusion solid 12.5 g / L, beef heart infusion solid 5 g / L, proteose peptone 10.0 g / L, glucose 2.0 g / L, sodium chloride 2.5 g / L, disodium phosphate 2.5 g / L, the pH value of the Oxoid brain heart infusion broth CM1135 is 7.2-7.6. (Oxoid brain heart infusion broth CM1135 is purchased from Beijing Soler Biochemical Technology Co., Ltd.)

[0039] The formula of brain heart infusion broth HB8297: proteose peptone 10.0 g / L, dehydrated calf brain infusion powder 12.5 g / L, dehydrated beef heart infusion powder 5.0 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L, the pH value of the brain heart infusion broth HB8297 is 7.2-7.6. (Brain heart infusion broth HB8297 is purchased from Qingdao Haibo Biotechnology Co., Ltd.)

[0040] Akman's muciniphila strain BNCC323275 is purchased from North China Biotech Co., Ltd.

[0041] Example 1

[0042] A method for improving the secretion amount of Akman's muciniphila extracellular vesicles:

[0043] Akman's muciniphila strain BNCC323275 bacterial liquid is inoculated into Oxoid brain heart infusion broth CM1135B at a ratio of 1:1000 (V / V), and is placed in a strict anaerobic environment at 37±0.5℃, humidity 90%-95% and 5% CO2 for shaking culture for 72 hours. Starting from 4 hours after inoculation, NIR irradiation for 2 hours is carried out at an illumination intensity of 4.2±0.2 mW / cm 2 , the wavelength range is 700-900 nm, and the peak wavelength is 860 nm (see Figure 1 ), and NIR irradiation is repeated after 22 hours. After the culture cycle is completed, centrifugation is carried out at a speed of 10000 g for 10 minutes to remove bacterial body precipitate, and the supernatant is taken and filtered through a 0.22 µm water filter membrane, and then separated and purified immediately or stored at -80℃. The filtered supernatant is collected and separated and purified using a commercially available OMVs rapid purification kit (magnetic bead type) according to the instructions to obtain extracellular vesicles.

[0044] Example 2

[0045] A method for improving the secretion amount of Akman's muciniphila extracellular vesicles:

[0046] A suspension of *Ackermania pseudomallei* strain BNCC323275 was inoculated into brain and heart extract broth HB8297 at a ratio of 1:1000 (V / V) and incubated with shaking for 72 hours in a strictly anaerobic environment at 37±0.5℃, 90%-95% humidity, and 5% CO2. Starting 4 hours after inoculation, the culture was incubated at 4.2±0.2 mW / cm². 2 Irradiation was performed for 2 hours using near-infrared light with a wavelength range of 700-900 nm and a peak wavelength of 860 nm. This near-infrared irradiation was repeated after a 22-hour interval. After the culture cycle was completed, the cells were centrifuged at 10000 g for 10 minutes to remove the cell pellet. The supernatant was then filtered through a 0.22 µm aqueous filter and immediately purified or stored at -80°C. The filtered supernatant was collected and purified using a commercially available OMVs rapid purification kit (magnetic bead type) according to the manufacturer's instructions to obtain extracellular vesicles.

[0047] Example 3

[0048] A method to increase the secretion of extracellular vesicles by Akkermansia muciniphila:

[0049] Akkermansia myxophilus strain BNCC323275 was inoculated into Oxoid brain and heart extract broth CM1135B at a ratio of 1:1000 (V / V) and cultured with shaking in a strictly anaerobic environment of 37±0.5℃, 90%-95% humidity, and 5% CO2 for 96 hours. Starting 4 hours after inoculation, the culture was incubated at 4.2±0.2 mW / cm². 2 Irradiation was performed for 2.5 hours using near-infrared light with a wavelength range of 700-900 nm and a peak wavelength of 860 nm. This near-infrared irradiation was repeated after a 22.5-hour interval. After the culture cycle was completed, the cells were centrifuged at 10000 g for 10 minutes to remove the cell pellet. The supernatant was then filtered through a 0.22 µm aqueous filter and immediately purified or stored at -80°C. The filtered supernatant was collected and purified using a commercially available OMVs rapid purification kit (magnetic bead type) according to the manufacturer's instructions to obtain extracellular vesicles.

[0050] Example 4

[0051] A method to increase the secretion of extracellular vesicles by Akkermansia muciniphila:

[0052] Akkermansia myxophilus strain BNCC323275 was inoculated into Oxoid brain and heart extract broth CM1135B at a ratio of 1:1000 (V / V) and cultured with shaking for 84 hours in a strictly anaerobic environment at 37±0.5℃, 90%-95% humidity, and 5% CO2. Starting 4 hours after inoculation, the culture was incubated at 4.2±0.2 mW / cm².2 The light intensity of the light source was 700-900 nm, the peak wavelength of the near-infrared light was 860 nm, the NIR irradiation was performed for 1.5 hours, and the near-infrared light irradiation was repeated after an interval of 21.5 hours. After the completion of the culture cycle, centrifugation was performed at 10,000 g for 10 minutes to remove the bacterial precipitate, and the supernatant was collected and filtered through a 0.22 μm water filter membrane. The filtered supernatant was immediately separated and purified or stored at -80°C. The filtered supernatant was collected and subjected to rapid purification using a commercially available OMVs rapid purification kit (magnetic bead type) according to the manufacturer's instructions to obtain extracellular vesicles.

[0053] Comparative Example 1

[0054] In comparison with Example 1, no near-infrared light irradiation was performed, and the remaining steps were the same as in Example 1.

[0055] Test Example 1

[0056] The extracellular vesicles collected in Example 1 and Comparative Example 1 were dissolved in 1x PBS solution to obtain OMVs suspensions, and each group was repeated 4 times.

[0057] 1. The particle size and concentration of the extracellular vesicles were detected using a Nanosight NS300 according to the manufacturer's instructions, Figure 2 The results of the Nanosight NS300 detection are shown in Table 1. As can be seen from the results, the OMVs concentration in Example 1 was significantly higher than that in Comparative Example 1. The average OMV concentration of each group in Example 1 and Comparative Example 1 is shown in Table 1. It can be seen that the concentration of extracellular vesicles in Example 1 was increased by 1.5-2.5 times compared with Comparative Example 1, indicating that the secretion of extracellular vesicles was increased by an average of 1.78 times after intermittent near-infrared light irradiation.

[0058] Table 1: Nanosight detection results of the average OMV concentration of each sample

[0059]

[0060] In combination with transmission electron microscopy analysis, the particle size and morphology of the extracted extracellular vesicles were consistent with the conventional understanding of extracellular vesicles, and the transmission electron microscopy image is shown in Figure 3 .

[0061] 2. There are various substances that have a regulatory effect on life activities, such as microRNAs, in the interior of the extracellular vesicles. After near-infrared light irradiation, several microRNAs were enriched in the extracellular vesicles produced by the strain, Figure 4 is a microRNA sequencing analysis chart of the OMVs extracted from the Akk bacterial solution, Figure 4The left-middle figure shows the microRNA enrichment volcano plots for Example 1 and Comparative Example 1. Red represents the microRNA enriched in Example 1, and blue represents the microRNA enriched in Comparative Example 1. The screening criterion was |Fold Change| > 1.5. p <0.05. Figure 4 The right-middle figure is a bubble chart of GO pathway enrichment analysis. The size of the bubble represents the number of target genes enriched in that pathway, and the bubble color represents the significance of enrichment, i.e., after adjustment by the BH method. p The value is represented by FoldEnrichment on the x-axis, indicating the degree of enrichment of target genes. These microRNAs enriched in this invention can indirectly or directly enhance bone formation by silencing inflammatory pathways such as TNF, NF-κB, and MAPK, as well as calcium metabolism pathways, thereby treating and preventing osteoporosis.

[0062] 3. Using mouse bone marrow mesenchymal stem cells (Huatuo Biotechnology Co., Ltd., HTX2114) as a model, inflammation was induced for 24 hours in 24-well plates by adding TNFα and IL-6 (PEPROTECH; 315-01A, 216-16) at a concentration of 25 ng / mL to complete HDMEM medium (90% Gibco HDMEM, 10% Vicente FBS, 1% Vicente PS double antibody). Then, osteogenic differentiation induction medium (Pronosai, PD-003) was added to cells with a final concentration of 10... 9 OMV obtained in Example 1 and Comparative Example 1 at cell / mL (orders per second) was used to induce osteogenic differentiation of cells for 10 days according to the manufacturer's instructions. Following this, ARS staining was performed, and the proportion of stained area in each well was calculated using Fiji software to directly characterize the osteogenic differentiation level. All modeling and induction processes were conducted at 37 °C, 90%-95% humidity, and 5% CO2. Experimental results are as follows: Figure 5 , Figure 6 And as shown in Table 2. Among them, Figure 5 The results of the control experiment for osteogenic differentiation of the TNFα-BMSC inflammation model induced by extracellular vesicles after near-infrared light irradiation are shown. The NIR-Akk@OMV group represents the osteogenic induction differentiation medium supplemented with OMV produced in Example 1; the Akk@OMV group represents the osteogenic induction differentiation medium supplemented with OMV produced in Comparative Example 1; the TNFα group represents the TNFα inflammation model induced after induction; and the Ctrl group represents the control group that was induced without inflammation modeling. Figure 6The results of the control experiment for osteogenic differentiation of the IL-6-BMSC inflammation model induced by extracellular vesicles after near-infrared light irradiation, wherein NIR-Akk@OMV group represents the addition of OMV produced in Example 1 to the osteogenic induction differentiation medium; Akk@OMV group represents the addition of OMV produced in Comparative Example 1 to the osteogenic induction differentiation medium; IL-6 group represents induction after IL-6 inflammation modeling; Ctrl group represents the control group without inflammation modeling for induction. Table 2 is the average area ratio of each group in the BMSC inflammation model osteogenic differentiation control experiment, and the results show that Example 1 can significantly improve the decrease in osteogenic differentiation ability caused by inflammation induction, even exceeding the osteogenic differentiation level of BMSC without inflammation modeling.

[0063] Table 2 Average area ratio of each group in the BMSC inflammation model osteogenic differentiation control experiment

[0064]

[0065] The above description is only an embodiment of the present application, and does not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation using the content of the present application specification, or direct or indirect application in other related technical fields, is also included in the patent protection scope of the present application.

Claims

1. A method for increasing the secretion of extracellular vesicles by Akkermansia muciniphila, characterized in that, Includes the following steps: Akkermansia myxophilus strain BNCC323275 was cultured in an anaerobic environment with shaking for 72–96 h. After the culture was completed, the supernatant was collected by centrifugation and then purified. During the culture period, near-infrared light irradiation begins after 3.5 to 4.5 hours of culture, and the near-infrared light irradiation is repeated every 21.5 to 22.5 hours. The wavelength of the near-infrared light irradiation is 860 nm, and the light intensity of the near-infrared light is 4.0~4.4 mW / cm². 2 The near-infrared light irradiation time is 1.5~2.5 h.

2. The method according to claim 1, characterized in that, The culture medium used for the culture was brain and heart extract.

3. The method according to claim 2, characterized in that, The brain and heart extract is Oxoid brain and heart extract broth CM1135 or brain and heart extract broth HB8297.

4. The method according to claim 1, characterized in that, The centrifugation speed is 8000~12000 g, and the centrifugation time is 10~20 min.

5. The method according to claim 1, characterized in that, The separation and purification method is the magnetic bead method.

6. The application of the method according to any one of claims 1 to 5 in increasing the secretion of extracellular vesicles by Akkermansia muciniphila.

7. The application of the method according to any one of claims 1 to 5 in increasing the types and quantities of microRNAs in extracellular vesicles of Akkermansia muciniphila.

8. The use of the method according to any one of claims 1 to 5 in the preparation of an agent that enhances the therapeutic activity of bone loss.

Images