Application of Ackermania muciniphila or extracellular vesicles thereof in preparation of medicine for alleviating alcoholic ischemic necrosis of femoral head

Abstract

The invention belongs to the field of biological medicines, and discloses an application of Akkermansia muciniphila or an extracellular vesicle of the Akkermansia muciniphila in preparation of a medicine for alleviating the ischemic necrosis of the alcoholic femoral head. Based on the pathogenesis characteristics of the AIONFH, the Akk bacteria and the extracellular vesicles thereof are used for treating the AIONFH for the first time, the molecular mechanism of the Akk bacteria and the extracellular vesicles thereof for regulating and controlling the intestinal tract-skeleton axis function, inhibiting bone cell apoptosis and improving the bone trabecula microstructure is deeply researched, the anti-pressure ability of bone tissue is improved, and the research blank in the field is filled. Compared with a traditional treatment method, the osteonecrosis pathological change is improved through a single way, and a novel multi-target treatment strategy is provided from multiple aspects of regulating inflammatory response, recovering bone metabolism balance, promoting intraosseous blood supply and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and specifically relates to a kind of Akkermansia muciniphila ( Akkermansia muciniphila , hereinafter referred to as Akk The present invention relates to the application of Bacteria or their extracellular vesicles (EVs) in the preparation of drugs for slowing down alcohol-induced osteonecrosis of the femoral head (AIONFH), especially a new method for achieving treatment by slowing down osteocyte apoptosis and improving bone microstructure. Background Art

[0002] Alcoholic avascular necrosis of the femoral head (AIONFH) is a serious bone and joint disease caused by long-term heavy drinking, characterized by insufficient blood supply to the femoral head, leading to osteocyte death, trabecular collapse, and irreversible structural damage to bone tissue. According to statistics, millions of AIONFH patients around the world face the risk of walking difficulties or even disability every year. Existing treatments for AIONFH include surgical treatment (such as joint replacement), physical therapy, and drug therapy, but these treatments have significant limitations, such as high surgical risks, strong drug side effects, and difficulty in reversing the disease process. Therefore, finding a safe and effective treatment method has become an urgent need.

[0003] In recent years, studies have found that intestinal microbiota plays an important role in the occurrence and treatment of various systemic diseases. Akk Bacteria are an important probiotic in the human intestine and have been shown to have the following biological functions: reducing intestinal inflammation and improving intestinal barrier function; promoting intestinal health and improving metabolic disorders by regulating the balance of intestinal microbiota; and showing potential therapeutic effects in osteoporosis and fracture healing. Although existing research has revealed Akk The role of bacteria in the treatment of osteoporosis, inflammatory diseases and fractures, but no public literature or patents have mentioned Akk The application of bacteria in the treatment of alcoholic osteocyte apoptosis and AIONFH. Alcohol-induced osteocyte apoptosis is a key link in the pathogenesis of AIONFH and is also a key issue that is difficult to effectively intervene in with existing treatment options. The pathogenesis of alcoholic avascular necrosis of the femoral head (AIONFH) involves many aspects, including activation of inflammatory response, osteocyte apoptosis, enhanced osteoclast activity, decreased osteoblast activity, and insufficient angiogenesis in the bone. However, the current treatment methods for AIONFH mainly focus on surgical methods, and lack effective strategies to regulate inflammation, promote bone metabolism and angiogenesis at the molecular mechanism level. Summary of the invention The technical problem to be solved by the present invention is to overcome the shortcomings and defects mentioned in the above background technology and provide a Akk The use of bacteria or their extracellular vesicles in the preparation of drugs for slowing down alcohol-induced femoral head avascular necrosis, alcohol-induced osteocyte apoptosis or bone loss. It can effectively inhibit osteocyte apoptosis (reduce alcohol-induced osteocyte apoptosis through molecular mechanisms), repair trabecular microstructure (protect femoral head morphology by improving bone storage and reducing bone loss), and provide a Akk Biological treatment solutions based on bacteria and their extracellular vesicles avoid the side effects of traditional drugs and improve safety and biocompatibility.

[0004] In order to solve the above technical problems, the technical solution proposed by the present invention is: In a first aspect, the present invention provides a Akk Application of bacteria or their extracellular vesicles in the preparation of drugs for alleviating alcohol-induced avascular necrosis of the femoral head.

[0005] Further preferably, the method of slowing down alcoholic avascular necrosis of the femoral head is at least one of slowing down alcohol-induced osteocyte apoptosis, repairing trabecular microstructure, inhibiting inflammation, regulating bone metabolism balance, promoting bone tissue repair, balancing osteoblast and osteoclast function, improving intrabone blood supply and promoting angiogenesis.

[0006] Further preferably, the drug for slowing down alcoholic avascular necrosis of the femoral head is a drug that inhibits the release of proinflammatory factors IL-6 and TNF-α, reduces serum TRAP activity, increases osteocalcin content, and increases serum vascular endothelial growth factor levels.

[0007] The present invention proposes for the first time Akk The application of bacteria or their extracellular vesicles in the preparation of drugs for slowing down AIONFH. Akk Bacteria improve systemic inflammatory status by regulating the balance of intestinal flora, and significantly reduce the expression of local inflammatory factors in the femoral head and improve bone metabolism balance by regulating the function of the intestinal-bone axis. Specifically, it achieves multi-target intervention in the pathogenesis of AIONFH by inhibiting inflammatory factors (such as IL-6, TNF-α), regulating bone metabolism balance (such as reducing TRAP activity and increasing OCN levels), and promoting angiogenesis (by increasing VEGF expression), which has significant therapeutic potential.

[0008] In a second aspect, the present invention provides a Akk Application of bacteria or their extracellular vesicles in preparing drugs for slowing down osteocyte apoptosis.

[0009] Further preferably, the osteocyte apoptosis is alcohol-induced osteocyte apoptosis.

[0010] Further preferably, the drug is a drug that slows down alcohol-induced osteocyte apoptosis by inhibiting ERK / AKT and WNT-β-catenin signaling pathways.

[0011] The present invention first discovered Akk Bacterial extracellular vesicles can activate the ERK / AKT signaling pathway, enhance the expression of anti-apoptotic proteins, and activate the WNT-β-catenin signaling, thereby promoting the differentiation and functional activity of osteoblasts and significantly inhibiting alcohol-induced osteoblast apoptosis.

[0012] In a third aspect, the present invention provides a muciniphilic Akkermansia ( Akkermansia muciniphila ) or its extracellular vesicles in the preparation of drugs for repairing trabecular bone microstructure.

[0013] Further preferably, the drug is a drug that promotes bone storage, slows down bone loss, and slows down femoral head bone collapse.

[0014] The present invention finds Akk Bacteria can significantly increase the bone density (BMD) and trabecular thickness (Tb.Th) of the femoral head and inhibit the excessive activation of osteoclasts, thereby restoring the number of trabeculae and reducing bone loss.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. Based on the characteristics of AIONFH, the present invention first Akk Bacteria and their extracellular vesicles are used to treat AIONFH, and their molecular mechanisms of regulating the function of the intestinal-bone axis, inhibiting osteocyte apoptosis, and improving trabecular microstructure are studied in depth, and the compressive resistance of bone tissue is improved, filling the research gap in this field. Compared with traditional treatment methods, the present invention not only improves the pathological changes of osteonecrosis through a single pathway, but also starts from multiple aspects such as regulating inflammatory response, restoring bone metabolic balance, and promoting blood supply in bones, providing a new multi-target treatment strategy.

[0016] 2. The present invention has found Akk The bacteria not only demonstrated their effectiveness in the treatment of AIONFH, but also successfully expanded their therapeutic indications, such as slowing down osteocyte apoptosis and repairing trabecular microstructure, providing a new strategy for the prevention and treatment of AIONFH. Akk Biological agents for the treatment of bacterial infections provide a theoretical basis and have important potential for clinical transformation, which can provide patients with safer and more effective treatment options, thereby improving their quality of life and prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 AIONFH disease animal model and Akk The results of behavioral observations of the disease treated with bacteria; Figure A is the diet record of Wistar rats, monitoring the daily body weight (g) and diet intake (ml) of each experimental group; Figure B is a representative picture of the behavioral observation of Wistar rats outside the cage for 1 minute; Figure C is a weekly behavioral observation record of Wistar rats with ONFH onset; Note: Experimental grouping: 1-3 is the chronic alcohol stimulation group; 4-6 is the chronic alcohol stimulation + Akk bacteria treatment group; No. 7-9 was the control group.

[0019] Figure 2 Figure 1 is the incidence of ONFH in Wistar rats; Figure A is a statistical table of the incidence scores and incidence of ONFH in Wistar rats; Figure B is a comparative statistical chart of the incidence of ONFH in each group of rats within 4 weeks.

[0020] Figure 3 AIONFH disease diagnosis and Akk Qualitative test of bacterial treatment; Figure A is the dissection of bilateral hip joints of rats, the taking of femoral head live specimens and the general observation; Figure B is the Micro-CT imaging results of the femoral heads of rats in each group (representative level of coronal photography); Figure C is the analysis of relevant physical indicators of the Micro-CT imaging results of the left femoral head, the indicators analyzed include: Tb.BV / TV (cancellous bone volume ratio), Tb.N (number of cancellous bone trabeculae); Tb.Th (cancellous bone thickness); Tb.Sp (cancellous bone separation); scale: 1mm, each group n = 3; LSD-t test and ANOVA analysis were used for all groups, **P<0.01; D) Pathological results of femoral head of rats in each group (HE staining).

[0021] Figure 4This is a Wistar rat AIONFH disease pathology and immunofluorescence detection test; among them: Figure A is an enlarged HE staining pathological result of each group; the blue arrow indicates: the "empty bone crack" phenomenon in the trabeculae; the black bold arrow indicates: the characteristic pathological change area of ​​the middle stage of necrosis; the red line indicates: the boundary between the necrotic trabeculae and the new bone tissue; Figure B is the detection of angiogenesis in the subchondral bone of the femoral head between groups by staining CD31, OCN, and TUNEL, the survival of osteoblasts at the edge of the trabeculae, and the apoptosis of osteocytes in the bone that constitutes the trabeculae; Figure C is the Hoechst fluorescence staining of osteocytes in the trabecular bone of the femoral head in each group. Note: Figure A: Control group: (A), (D); Alcohol + Akk Bacteria treatment group: (B), (E); alcohol modeling group: (C), (F); Figure C: The observation magnification of the upper picture is 30.4×; the observation magnification of the lower picture is 60.8×.

[0022] Figure 5 yes Akk -EVs against alcohol-induced osteoblast apoptosis cell model; Figure A is an in vitro model of alcohol-induced osteoblast apoptosis; Figure B is a test Akk -The effect of EVs on osteoblast apoptosis; Figure C is Akk -EVs treatment of alcohol-induced osteoblast apoptosis model, cell culture microscopy (magnification: 4×20SE) can also be seen Akk -EVs can effectively treat osteoblast apoptosis, and in the culture environment of the same alcohol concentration, it can promote osteoblast survival to a certain extent. Note: Figure A: # indicates P<0.05 for the comparison between the modeling group of each alcohol concentration and the control group; Figure B: # indicates P<0.05 for the comparison between the alcohol (concentration: 8%) modeling group and the control group, * indicates Akk -P<0.05 for the comparison between the EVs concentration intervention group and the alcohol (concentration: 8%) modeling group.

[0023] Figure 6 The figure shows the dry mass of the left femoral head in the AIONFH animal model (n=5). Akk Bacteria treatment group (EtOH+ Akk The results showed that the dry mass of the alcohol-stimulated group was significantly decreased compared with the control group, reflecting the phenomenon of alcohol-induced bone loss and trabecular fracture. Akk In the group treated with bacteria, the dry mass was significantly restored, indicating Akk The bacteria have the potential to improve femoral head bone mass. Error bars represent standard deviations, and the significance level between groups was set at P < 0.05.

[0024] Figure 7is the stress-strain curve of the left femoral head under axial pressure. The curves represent the alcohol stimulation group (01 L), Akk Mechanical properties of the bacteria treatment group (07 L) and the control group (18 L). From the curve shape, it can be seen that the control group showed a higher maximum stress value and a smaller displacement damage range, indicating that its bones were stronger. In contrast, the curve of the alcohol stimulation group decreased significantly, showing the fragility of the bones and the decrease in compressive properties. Akk The curve of the bacteria treatment group recovered, showing Akk Bacteria treatment has a significant effect on improving alcohol-induced bone mechanical properties. This figure helps to understand the effects of different treatment groups on bone mechanical properties through the comparison of stress-strain curves.

[0025] Figure 8 yes Akk muc group was the alcohol group, and the EtOH+A. muc group was the alcohol+ Akk The bacteria treatment group and the Vehicle group served as the control group. DETAILED DESCRIPTION

[0026] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0027] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0028] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0029] Example: 1. Materials and Methods 1. Establishment of AIONFH rat model Eight-week-old male Wistar rats (weight 200-220 g) were randomly divided into a control group and a model group. The AION FH model was established in the model group rats using the method of Okazaki et al.: liber-decarli liquid diet (Lieber et al1989), TP 4030A (ORIENTAL Yeast Co., Ltd., Tokyo, Japan), TP 4030A: 36% alcohol calories (alcohol content: 5%, wt / V, g / 100 ml), 35% fat, 18% protein, 11% carbohydrates, 36% alcohol. TP 4030A is prepared to prepare 1000 ml, which requires 132.2 grams, plus 67.3 ml of 95% alcohol (self-prepared), and the calories are 1000 kcal. The daily requirement of TP 4030A per animal was estimated when ordering: 80 ml / day for rats. (Reference: Int J Exp Pathol. 2013; 94 (5): 312-319.), and continued for 8 weeks. The control group was given an equal amount of alcohol-free liber-decarli liquid diet. All animal experiments followed the guidelines of the institutional animal ethics committee.

[0030] In order to improve the accuracy of the experiment, the success rate of the rat AIONFH model was verified in the present invention. The specific results showed that after the model was established according to the above method, the incidence of femoral head avascular necrosis in the model group was as high as 100%, and there was a significant difference compared with the control group (P<0.01).

[0031] 2. Akk Bacteria intervention program The model group rats were randomly divided into AIONFH group and Akk Bacteria intervention group. Akk The bacteria intervention group was given 2-3 ml of 1×10 9 CFU Akkermansia muciniphila The AIONFH group was given 2-3 ml of sterile PBS solution by gavage 3 times a week for 8 weeks.

[0032] 3. Behavioral Observation and Morbidity Statistical Methods Rats were observed and scored every week, including weight measurement, food consumption, and activity assessment on the last day of each week (this assessment includes: 1-minute behavioral performance of rats in a resting state in the cage and 1-minute video recording of rats placed in a fixed area outside the cage and allowed to move freely). The behavioral scoring standard for ONFH in rats is set as follows: According to the severity of ONFH in rats, the corresponding scores for their behavioral performance are set as follows: in situ (4 points), unfavorable activity (3 points), huddled (2 points), active (1 point), no special (0 points). It is stipulated that the sum of the ONFH scores must be ≥3 points. Combined with all statistical data, the ONFH incidence rate of each group was calculated.

[0033] 4. Imaging examination (Micro-CT) and analysis methods The femoral heads of rats were scanned using a Skyscan 1176 micro-CT system (Bruker, Belgium) with the following scanning parameters: voltage 60 kV, current 500 μA, pixel size 9 μm. CTAn software was used to analyze parameters such as bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular spacing (Tb.Sp).

[0034] 5. Histopathological examination methods Femoral head tissue was obtained, fixed with 4% paraformaldehyde for 24 hours, decalcified with 10% EDTA for 4 weeks, and routinely embedded in paraffin and sectioned (5 μm thickness). HE staining, OCN staining, and Tunel staining were performed. Olympus BX53 microscope was used for observation and photography.

[0035] 6. Immunohistochemistry and Immunofluorescence Staining Methods After dewaxing and hydration, the sections were subjected to antigen retrieval using citrate buffer (pH 6.0). 2 O 2 Endogenous peroxidase activity was blocked and nonspecific binding was blocked with 5% BSA. Primary antibody was incubated overnight at 4°C and secondary antibody was incubated for 1 hour at room temperature. DAB was used for color development and hematoxylin was used for counterstaining. Fluorescently labeled secondary antibodies were used for immunofluorescence staining, and DAPI was used for nuclear staining.

[0036] 7. In vitro osteoblast culture and alcohol-induced apoptosis model 1) Cell culture Cell source: Primary osteoblasts isolated from young Wistar rats (1-2 weeks old) or MLO-Y4 osteoblasts were used. Culture medium: Cells were cultured in α-MEM medium containing 10% FBS and maintained at 37°C and 5% CO 2 Passaging: Cells were used in experiments after 2-3 passages. 2) Alcohol induces osteocyte apoptosis: Alcohol treatment: osteocytes were treated with 95% ethanol solution for 24 hours to induce cell apoptosis. Concentration gradient: Alcohol treatment concentrations include: 0.5%, 1.0%, 2.0%, 4.0%, 8.0% (gradient concentration), select according to needs. 3) Akk Bacterial extracellular vesicles (EVs) Akk -EVs) Akk -EVs Source: from Akk Extracellular vesicles were extracted from bacterial culture medium and obtained by ultracentrifugation. Treatment conditions: 3 hours after alcohol treatment, different concentrations of Akk -EVs (e.g., 0.5%, 1.0%, 2.0%, 4.0%, 8.0%) were used to treat osteocytes.

[0037] 8. Akk -EVs preparation and processing methods By ultracentrifugation from Akk Bacterial extracellular vesicles (EVs) were isolated from bacterial culture supernatants. The culture supernatant was treated as follows: centrifuged at 300×g for 10 minutes, 2000×g for 20 minutes, and 10,000×g for 30 minutes to remove cell debris. After that, the treated supernatant was ultracentrifuged at 100,000×g for 70 minutes to collect EVs. The collected EVs were resuspended in sterile PBS and ultracentrifuged again to purify EVs to ensure purity and stability. Referring to the method of Chen et al. (2022), this extraction step ensures the structural integrity and functional activity of EVs.

[0038] 9. Femoral head dry weight (dry mass) experiment The left femoral head of Wistar rats was washed with sterile saline to remove soft tissue residues. Vacuum freeze drying (-80°C) was then performed until the femoral head was completely dehydrated. The dry mass of the femoral head (unit: gram) was measured using a high-precision electronic balance. There were 5 rat samples in each group, and the results were expressed as the average dry mass. A chart was drawn to compare the data between groups.

[0039] 10. Femoral head compression stress-strain curve experiment The left femoral head of one rat in each group was removed, the soft tissue was removed, and the femoral head was cryopreserved. The femoral head was mechanically tested using an electronic material testing machine, and the loading speed was set to 0.05 mm / s. Test range: The stress-strain curve of the femoral head under axial pressure was recorded until the femoral head structure was destroyed. The data recorded the relationship between force (unit: kN) and displacement (unit: mm). The stress-strain curve was plotted using analysis software, and the maximum stress value within the strain range (0–1.75 mm) of each group was compared.

[0040] 11. Serum sample collection: Serum was collected from the alcohol-induced AIONFH rat model, and the groups included alcohol group (EtOH), alcohol + Akk The experimental group was treated with bacteria (EtOH+A.muc) and the control group (Vehicle). Inflammatory factor detection: ELISA was used to determine the levels of IL-6, TNF-α and IL-1β in serum. Bone metabolism related index detection: AKP, OCN and TRAP levels were detected. Angiogenic factor detection: ELISA was used to detect VEGF levels. Akk VEGF in the bacterial group was significantly increased (P<0.05).

[0041] 12. Statistical analysis methods All data are expressed as mean ± SD, and statistical analysis was performed using GraphPad Prism 8.0 software. The differences between the two groups were compared using the independent sample t test; the differences between multiple groups were analyzed using one-way ANOVA, supplemented by Tukey's multiple comparison method to clarify the differences between the groups. For all statistical tests, a P value less than 0.05 was considered statistically significant.

[0042] 2. Results 1. Akk Bacteria slow down the progression of AIONFH rat model Behavioral observations and morbidity statistics show that Akk Bacterial intervention can effectively slow down the pathogenesis of chronic alcohol-induced AIONFH. Akk The incidence of AIONFH in rats treated with bacteria was significantly reduced (P<0.01), and behavioral indicators (such as gait stability and activity, Figure 1 B, C) and weight fluctuations ( Figure 1 A) significantly improved, and it is worth noting that the Akk bacteria treatment group had a slight increase in alcohol intake of 1.42% compared with the alcohol group ( Figure 2C), but the stability of its basal weight was significantly better than that of the alcohol group (P<0.05). These results indicate that Akk bacteria have a significant protective effect on alcohol-related osteonecrosis, and its potential mechanism may be closely related to regulating alcohol metabolism or inhibiting inflammatory response ( Figure 2 C).

[0043] 2. Akk Bacteria improve bone structure and pathological characteristics in AIONFH rats Akk The bacteria showed significant effects in improving alcohol-induced osteoporosis and AIONFH pathological features. The present invention found through Micro-CT imaging and pathological analysis that compared with the alcohol group, Akk The trabecular bone parameters of the bacterial treatment group were significantly improved ( Figure 3 The specific results are as follows: Trabecular bone quality and structure: The cancellous bone mass (BV / TV), thickness (Tb.Th) and trabecular number (Tb.N) of the Akk bacteria treatment group were significantly increased (P<0.01), while the trabecular separation (Tb.Sp) was significantly reduced, approaching the level of the control group ( Figure 3 Middle C). Pathological feature analysis showed: Akk HE staining of the femoral head in the bacteria-treated group showed that the trabeculae were more densely distributed, and the aggregation of inflammatory cells and bone necrosis were significantly reduced compared with the alcohol group. These results indicate that Akk Bacteria can effectively slow down the progression of alcohol-induced osteonecrosis by protecting trabecular bone structure and improving bone density.

[0044] 3. Akk Bacteria alleviate inflammation and osteocyte apoptosis in the femoral head of AIONFH rats and repair femoral head necrosis in chronic alcohol-induced Wistar rats The present invention further evaluates Akk The inhibitory effect of bacteria on femoral head inflammation and osteocyte apoptosis. ,Akk The bacteria significantly reduced the density of inflammatory cells in the femoral head ( Figure 4 A (B)) and "empty bone gap" formation ( Figure 4 A (E)). Specific manifestations are: Improvement of inflammation: Akk The density of inflammatory cells in the bacteria treatment group was significantly lower than that in the alcohol group (P<0.05), and was close to the level of the control group. Akk The apoptosis rate of osteoblasts in the bacterial treatment group was significantly reduced ( Figure 4 In C, osteoblast survival rate and angiogenesis indexes were significantly increased (P<0.01) ( Figure 8 ). These results indicate that AkkThe Akk bacteria played a key role in slowing down alcohol-related osteonecrosis by regulating inflammatory response and anti-apoptotic mechanism. Figure 4 A) The number and width of cracks were significantly reduced, indicating that the bone repair effect was significantly better than that of the model group. Akk The therapeutic effect of bacteria on rat AIONFH caused by chronic alcohol intake. It can be seen from the "yellow border" (the most representative area) of each group ( Figure 4 Middle A): The density of inflammatory cells in the trabecular space below the femoral head growth plate is as follows: (A) has the lowest density; (B) has a density between (A) and (C); (C) has the highest density. Akk Bacteria can counteract the severity of inflammation in the trabecular spaces within the femoral head. Zooming in on the area of ​​interest outside the "yellow border" within the femoral head of each group revealed: (D): There are fewer "empty bone cracks" in the trabeculae, and they are concentrated. (E): The "empty bone cracks" in the trabeculae increase and are scattered. One area of ​​characteristic pathological changes in the middle stage of necrosis is visible, and the degree of bone repair is relatively mild; (F): The "empty bone cracks" in the trabeculae increase more than (D), are thicker, and are scattered. Two areas of characteristic pathological changes in the middle stage of necrosis are visible, and the degree of bone repair is more severe. The scale bar is 200μm. The experimental results show that compared with rats that only drink alcohol, those that receive Akk Rats treated with the bacteria showed signs of improvement in the pathological features of the femoral head (see Figure 4 Specifically, rats in the treatment group showed increased angiogenesis in the subchondral bone of the femoral head, improved osteoblast survival at the trabecular edge, and decreased apoptotic osteocytes within the trabecular bone (see Figure 4 In addition, Hoechst fluorescence staining also revealed Akk Bacteria treatment can reduce the degree of osteocyte apoptosis in trabecular bone, indicating Akk -EVs may play a repair role by promoting osteocyte survival and resisting osteocyte apoptosis (see Figure 4 These findings suggest that Akk Bacteria may be a potential treatment for alcohol-related osteonecrosis.

[0045] Figure 4 In middle A (DF), the blue arrows indicate the "empty bone cracks" in the trabeculae. This phenomenon indicates that the bone tissue in the trabeculae is not fully grown. If the "empty bone cracks" in the trabeculae are wider and more numerous, it means that the bone loss that constitutes the trabeculae is more serious, which is consistent with the pathological signs of ONFH. The black bold arrows indicate the characteristic pathological change area in the middle stage of necrosis (the phenomenon that the necrotic trabeculae are wrapped by the new bone tissue). The red line indicates the boundary between the necrotic trabeculae and the new bone tissue.

[0046] 4. Akk -EVs protect against alcohol-induced osteocyte apoptosis in vitro In vitro construction of an alcohol-induced osteoblast apoptosis model Figure 5 Figure A shows that in vitro experiments Akk Bacterial extracellular vesicles (EVs) Akk -EVs) have a significant inhibitory effect on alcohol-induced osteoblast apoptosis. The results showed that: Anti-apoptotic effect: The apoptosis rate of osteoblasts in the alcohol-treated group was significantly increased, while the addition of Akk -EVs, the apoptosis rate was significantly reduced (P<0.01). Akk -EVs showed a dose-dependent protective effect. Improved cell survival: Microscopic observation showed that Akk -EVs-treated group had a significantly higher bone cell survival rate than the alcohol group ( Figure 5 In B and C, the cells are more complete in morphology and more numerous. These results indicate that Akk -EVs may enhance the anti-apoptosis ability of osteocytes by activating the ERK / AKT and WNT-β-catenin signaling pathways, providing important theoretical and experimental basis for the treatment of AIONFH.

[0047] 5. Akk Bacteria promote bone storage in the femoral head and slow down the collapse of the femoral head To further verify Akk The protective effect of bacteria on AIONFH was explored by measuring the dry weight of the femoral head and testing its mechanical properties. Akk The mechanism of action of bacteria on bone storage and femoral head morphology protection. First, the mass of the left femoral head of rats was measured ( Figure 6 ), found that the femoral head mass of the alcohol-induced AIONFH group (EtOH group) was significantly reduced, indicating that the destructive effect of alcohol on bone may be achieved by accelerating osteocyte apoptosis and bone loss. Akk Bacteria treatment group (EtOH+ Akk group), the femoral head shaft mass increased significantly, approaching the level of the control group, suggesting Akk This result supports the fact that bacteria can effectively slow down the bone loss caused by alcohol. Akk Bacteria may improve bone quality by promoting bone storage and protecting the structural integrity of the femur. Secondly, in the mechanical properties test ( Figure 7 ), which was further verified by stress-strain curve analysis. Akk The results showed that the peak stress of the femoral head under pressure in the alcohol group was significantly reduced, and the stress curve showed an earlier rupture point and higher brittleness, indicating that alcohol-induced mechanical properties of bone tissue were seriously reduced.Akk The stress-strain curve of the bacterial treatment group showed significant improvement, the stress peak value was significantly increased, the pressure bearing capacity of bone tissue was significantly enhanced, and the curve shape was close to the level of the control group. Akk Bacteria may slow down femoral head collapse by improving bone microstructure, reducing osteocyte apoptosis and regulating bone metabolism balance.

[0048] Through the results of femoral head dry weight measurement and mechanical properties test, the present invention first clarified Akk The bacteria promoted bone storage and protected femoral head collapse in the AIONFH model. Akk The potential therapeutic value of bacteria in protecting bone health by regulating the gut-bone axis mechanism provides a basis for the development of Akk It provides important experimental basis for the clinical biological preparations of bacteria.

[0049] 6. Akk Bacteria improve serum biochemical indexes and regulate inflammation and bone metabolism in AIONFH rats The present invention analyzes the serum biochemical indexes of rats with alcoholic avascular necrosis of the femoral head (AIONFH) and finds that Akk The bacteria can effectively improve the pathological changes induced by alcohol, such as the increase of inflammatory factors, the inhibition of angiogenesis and the disorder of bone metabolism, as shown below (the experimental results are shown in Figure 8 ): Levels of inflammatory factors: IL-6 and TNF-α levels were significantly increased in the EtOH group (alcohol group), indicating that alcohol induced a systemic inflammatory response. Akk The bacteria treatment group) significantly reduced the levels of IL-6 and TNF-α, which were close to those of the control group (Vehicle group) (IL-6: P<0.01; TNF-α: P<0.05). This result shows that Akk Bacteria can alleviate alcohol-induced systemic inflammatory response by inhibiting the release of inflammatory factors. Angiogenesis factor levels: VEGF (vascular endothelial growth factor) was significantly reduced in the EtOH group, indicating that alcohol has an inhibitory effect on angiogenesis. The VEGF level in the EtOH+A. muc group was significantly higher than that in the EtOH group (*P<0.05, P<0.01), close to the control group level, indicating that Akk Bacteria may contribute to the repair of AIONFH by promoting angiogenesis and improving intraosseous blood supply.

[0050] Bone metabolism related indicators: OCN (osteocalcin) levels decreased in the EtOH group, but increased significantly in the EtOH+A. muc group, close to the control group, indicating Akk Bacteria can promote the functional activity of osteoblasts and enhance bone formation.

[0051] AKP (alkaline phosphatase) activity decreased in the EtOH group. Akk The activity of the bacteria-treated group was restored, indicating its promoting effect on bone formation.

[0052] TRAP (tartrate-resistant acid phosphatase) activity was significantly increased in the EtOH group, indicating that alcohol induced enhanced osteoclast activity. Akk TRAP activity in the bacterial treatment group was significantly reduced and close to that in the control group, suggesting Akk Bacteria can inhibit osteoclast activity and slow down bone resorption.

[0053] Adiponectin (ADPH) level: ADPH was significantly reduced in the EtOH group, suggesting that alcohol has an inhibitory effect on the expression of adiponectin, which may be closely related to alcohol-induced microvascular dysfunction and inflammatory response. In contrast, the ADPH level in the EtOH+A. muc group was significantly higher than that in the EtOH group (*P<0.05, P<0.01), and was close to the control group level. This result shows that Akk Bacteria may play a multi-target role in the repair of avascular necrosis of bone (AIONFH) by promoting adiponectin expression, improving microvascular function and alleviating systemic inflammation.

[0054] Summary: Experimental data show that Akk The serum VEGF level in the bacterial intervention group was increased by 63.7% and the serum OCN level was increased by 45.6% compared with the model group, and the IL-6 and TNF-α levels were significantly reduced (reduced by 52.4% and 48.2%, respectively, P<0.01). Akk The bacteria improved alcohol-induced systemic inflammatory response and bone metabolism disorders at the systemic level by regulating serum inflammatory factors (such as IL-6, TNF-α) and bone metabolism indicators (such as OCN, TRAP), and promoted angiogenesis by increasing VEGF levels. These results further verified Akk The bacteria have a significant multi-target therapeutic effect in alleviating the pathogenesis of AIONFH, providing strong data support for the technical effects of the present invention.

[0055] Combining the results of a series of in vivo and in vitro experiments, the Akk Bacteria and their extracellular vesicles ( Akk -EVs) in the treatment of AIONFH. The experiment showed that Akk Bacteria can significantly slow down the progression of AIONFH and improve bone structure and pathological characteristics. Its core mechanism may include inhibiting inflammatory response and reducing osteocyte apoptosis. Akk-EVs showed significant anti-apoptotic effects in in vitro experiments, further confirming their potential role in protecting bone health. Akk The application of bacteria and related treatment strategies in alcohol-induced avascular necrosis of the femoral head provides new theoretical support and data basis, and expands Akk The clinical translation potential of bacteria in the treatment of bone diseases.

Claims

1. A strain of Akkermansia muciniphila ( Akkermansia muciniphila ) or its extracellular vesicles in the preparation of a drug for slowing down alcoholic avascular necrosis of the femoral head.

2. The use according to claim 1, characterized in that: The method of slowing down alcoholic avascular necrosis of the femoral head is at least one of slowing down alcohol-induced osteocyte apoptosis, repairing trabecular microstructure, inhibiting inflammation, regulating bone metabolism balance, promoting bone tissue repair, balancing osteoblast and osteoclast functions, improving bone blood supply and promoting angiogenesis.

3. The use according to claim 2, characterized in that: The drug for slowing down alcoholic avascular necrosis of the femoral head is a drug that inhibits the release of proinflammatory factors IL-6 and TNF-α, reduces serum TRAP activity, increases osteocalcin content, and increases serum vascular endothelial growth factor level.

4. A strain of Akkermansia muciniphila ( Akkermansia muciniphila ) or its extracellular vesicles in the preparation of drugs for slowing down osteocyte apoptosis.

5. The use according to claim 4, characterized in that: The osteocyte apoptosis is alcohol-induced osteocyte apoptosis.

6. The use according to claim 5, characterized in that: The drug is a drug that slows down alcohol-induced osteocyte apoptosis by inhibiting ERK / AKT and WNT-β-catenin signaling pathways.

7. A muciniphilic Akkermansia Akkermansia muciniphila ) or its extracellular vesicles in the preparation of drugs for repairing trabecular bone microstructure.

8. The use according to claim 7, characterized in that: The medicine is a medicine for promoting bone storage, slowing down bone loss, and slowing down femoral head bone collapse.

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