Clostridium butyricum GKB7-containing composition for improving osteoarthritis and degenerative arthritis

Abstract

To provide a Clostridium butyricum GKB7-containing composition for ameliorating osteoarthritis or degenerative arthritis.SOLUTION: The present invention provides a Clostridium butyricum GKB7-containing composition for ameliorating osteoarthritis or degenerative arthritis. This composition contains Clostridium butyricum GKB7 as an active ingredient, thereby delivering effects in alleviating arthralgia due to osteoarthritis, ameliorating hard bone injury, cartilage injury, and inflammation of cartilage tissue and / or synovial tissue, and reducing the expression level of IL-1β and / or TNF-α in the cartilage tissue and synovial tissue.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a probiotic-containing composition for improving musculoskeletal diseases, and in particular to a Clostridium butyricum GKB7-containing composition for improving osteoarthritis. [Background technology]

[0002] Osteoarthritis (OA), also known as degenerative arthritis, is a disease in which chronic degenerative inflammation occurs in the joints. Osteoarthritis often occurs in the hands, hips and knees. Symptoms such as bone damage, cartilage damage, subcartilage reconstruction, and inflammation of cartilage and synovial tissue occur in the affected area of ​​osteoarthritis, causing pain, stiffness and / or swelling in the affected area. Osteoarthritis is very common, affecting one-third of people over the age of 65 worldwide, according to literature-based statistics published in 2021. In countries with aging populations, osteoarthritis is a serious problem.

[0003] At present, osteoarthritis cannot be cured, and in severe cases, only surgery can be performed. Before surgery, conservative treatment may be adopted to relieve joint pain caused by osteoarthritis and / or improve joint mobility. Nonsteroidal anti-inflammatory drugs are commonly used drugs in conservative treatment, which can inhibit the action of cyclooxygenase (COX), reduce the production of prostaglandin E2 (PGE2), and further relieve osteoarthritis. However, nonsteroidal anti-inflammatory drugs can only relieve osteoarthritis, and not only cannot treat osteoarthritis, but also inhibit the synthesis of proteoglycan, which may accelerate the deterioration of osteoarthritis. In addition, long-term use of nonsteroidal anti-inflammatory drugs may cause side effects such as peptic ulcers, lower limb edema, and damage to kidney and heart function.

[0004] The gut microbiota ecosystem includes bacteria, fungi, viruses, phages and parasites from the oral cavity to the anus. The microbiota is involved in many important functions, including host immune function, regulation of intestinal permeability, nutrient absorption, pathogen resistance, synthesis and secretion of neurotransmitters and metabolic homeostasis. It is recognized that gut dysbiosis increases the permeability of the intestinal mucosa, interferes with metabolic balance, and leads to systemic inflammation, endochondral degradation and imbalance of the gut-brain axis, which is one of the key factors in osteoarthritis. However, at present, there are few studies on whether Clostridium butyricum can improve musculoskeletal diseases. Summary of the Invention

[0005] Therefore, one aspect of the present invention provides a Clostridium butyricum GKB7-containing composition for improving osteoarthritis, the Clostridium butyricum GKB7-containing composition for improving osteoarthritis comprising an effective amount of Clostridium butyricum GKB7 as an active ingredient.

[0006] Another aspect of the present invention provides a Clostridium butyricum GKB7-containing oral composition for improving osteoarthritis, which contains Clostridium butyricum GKB7 as an active ingredient, thereby improving bone damage, cartilage damage, and inflammation of cartilage tissue and / or synovial tissue in a subject.

[0007] According to the above aspect of the present invention, there is proposed a Clostridium butyricum GKB7-containing composition for improving osteoarthritis, which contains Clostridium butyricum GKB7 as an active ingredient, and the Clostridium butyricum GKB7 was deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI), Taiwan on April 1, 2022, with the accession number BCRC 911120.

[0008] According to some embodiments of the present invention, Clostridium butyricum GKB7 is a live bacterium obtained after a fermentation process, and the fermentation process is carried out for Clostridium butyricum GKB7 in an anaerobic environment at 25°C to 40°C using a fermentation substrate for 16 hours to 24 hours.

[0009] According to some embodiments of the present invention, Clostridium butyricum GKB7 is a killed bacterium obtained after a fermentation process and an inactivation process, and the fermentation process is carried out for Clostridium butyricum GKB7 in an anaerobic environment at 25°C to 40°C using a fermentation substrate for 16 hours to 24 hours.

[0010] According to some embodiments of the present invention, when the subject is a rat, the effective amount is 50 mg / kg body weight / day to 2000 mg / kg body weight / day.

[0011] According to some embodiments of the present invention, when the subject is a human, the effective amount is between 1 mg / kg body weight / day and 200 mg / kg body weight / day.

[0012] According to another aspect of the present invention, there is provided an oral composition containing Clostridium butyricum GKB7 for improving osteoarthritis, the composition comprising Clostridium butyricum GKB7 having the accession number BCRC 911120 as an active ingredient, thereby improving bone damage, cartilage damage, and inflammation of cartilage tissue and / or synovial tissue in a subject.

[0013] According to some embodiments of the present invention, the expression levels of IL-1β and / or TNF-α are reduced in cartilage and / or synovial tissue of subjects administered the oral composition compared to control subjects not administered the composition.

[0014] According to some embodiments of the present invention, joint pain is reduced in subjects administered the oral composition compared to control subjects not administered the composition.

[0015] According to some embodiments of the invention, the composition is administered to the subject three to seven times per week.

[0016] According to some embodiments of the invention, the composition is administered to the subject for a period of 2 to 8 weeks. Effect of the Invention

[0017] By applying the Clostridium butyricum GKB7-containing oral composition for improving osteoarthritis and degenerative arthritis of the present invention, it is possible to improve bone damage, cartilage damage, and inflammation of cartilage tissue and / or synovial tissue of a subject, reduce the expression levels of IL-1β and / or TNF-α in cartilage tissue and / or synovial tissue, and alleviate joint pain. The above and other objects, features, advantages and embodiments of the present invention will be more clearly and easily understood from the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0018] [Figure 1]1 is a graph showing the change in weight bearing difference 6 weeks after surgery in different groups of rats according to an embodiment of the present invention. [Diagram 2] 1 is a graph showing the change in weight bearing difference 6 weeks after surgery in different groups of rats according to an embodiment of the present invention. [Diagram 3] 3A to 3G are box plots showing bone density, bone mass, bone volume fraction, bone surface area density, trabecular thickness, trabecular number and trabecular spacing for different groups according to one embodiment of the present invention. [Figure 4] 4A to 4C respectively show the International Osteoarthritis Society score, cartilage degeneration score, and synovial inflammation score for different groups according to one embodiment of the present invention. [Diagram 5] 5A and 5B are box plots showing the IHC scores of IL-1β and the IHC scores of TNF-α in cartilage tissues of different groups according to one embodiment of the present invention. [Figure 6] 6A and 6B are box plots showing the IHC scores of IL-1β and the IHC scores of TNF-α in synovial tissues of different groups according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] If a definition or use of a term in a reference contradicts or contradicts a definition of that term in this specification, the definition of that term in this specification shall control and not apply. Also, unless otherwise defined in the context, singular terms may include plurals and plural terms may include the singular.

[0020] As mentioned above, the present invention provides a Clostridium butyricum-containing composition for improving musculoskeletal disorders. The musculoskeletal disorders described herein refer to disorders in which the locomotor system (including muscles, bones, joints, and adjacent connective tissues) is damaged, causing temporary or lifelong functional limitations. Musculoskeletal disorders are usually characterized by pain, limited mobility and dexterity, and the like. Musculoskeletal disorders include joint problems such as osteoarthritis, rheumatoid arthritis, psoriatic arthritis, gout, ankylosing spondylitis, skeletal problems such as osteoporosis, osteopenia, fragility fractures, traumatic osteoporosis, muscle problems such as sarcopenia, spinal problems such as back and neck pain, and problems in many parts or systems of the body.

[0021] In some embodiments, the musculoskeletal disorder is osteoarthritis. Clinical symptoms of osteoarthritis include pain and stiffness, which worsens after activity but is relieved after rest, and stiffness that improves after activity but returns after rest. As detected by optical imaging tools, osteoarthritis often manifests as joint space narrowing, osteophyte formation, subchondral bone sclerosis or subchondral bone cysts, bone lesions, cartilage lesions, and inflammation of cartilage and / or synovial tissue.

[0022] In some embodiments, the evaluation items of osteoarthritis may include, but are not limited to, the degree of pain, the degree of bone damage, the degree of cartilage damage, and the expression levels of inflammatory cytokines in cartilage and synovial tissues. In some embodiments, the degree of pain may be evaluated by the difference in weight bearing, since osteoarthritis is generally asymmetric.

[0023] In some embodiments, the degree of bone damage may be assessed by bone mineral density (BMD), bone mineral content (BMC), bone volume fraction, bone area fraction, trabecular thickness, trabecular number, and trabecular separation. In some embodiments, the degree of cartilage damage may be scored using the rat joint histology scoring system recommended by the Osteoarthritis Research Society International (OARSI).

[0024] In some embodiments, the inflammatory cytokines may include, but are not limited to, interleukin (IL)-1β and tumor necrosis factor (TNF)-α. It should be noted that IL-1β and TNF-α promote the synthesis of proteolytic enzymes, which degrade the extracellular matrix in the joints, leading to more severe osteoarthritis.

[0025] Experiments have demonstrated that Clostridium butyricum GKB7 can improve osteoarthritis. Specifically, the pain level, bone damage level, cartilage damage level, and expression levels of inflammatory cytokines in cartilage and synovial tissues of subjects administered Clostridium butyricum GKB7 are reduced compared to control subjects not administered Clostridium butyricum GKB7. The Clostridium butyricum GKB7 was deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI), Taiwan (Address: No. 331 Food Road, Hsinchu City, Postal Code: 300193) on April 1, 2022, with the accession number BCRC 911120.

[0026] Therefore, Clostridium butyricum GKB7 is applicable to the preparation of a composition for improving osteoarthritis and osteoarthritis, and the composition may contain an effective amount of Clostridium butyricum GKB7 as an active ingredient, but is not limited thereto. In some embodiments, Clostridium butyricum GKB7 is a live bacterium obtained after a fermentation process. In some embodiments, the fermentation process is carried out at 25°C to 40°C, or 35°C to 40°C, or 37°C so that Clostridium butyricum GKB7 has a preferred growth activity and / or osteoarthritis improving activity. In some embodiments, the fermentation process is carried out for 16 hours to 24 hours, or 18 hours so as to obtain a large number of live Clostridium butyricum GKB7. In some embodiments, the fermentation process is carried out in an anaerobic environment. The air composition of the anaerobic environment is not particularly limited, and the oxygen content may be 0% or close to 0%. In one embodiment, the anaerobic environment may include, but is not limited to, 75%-85% nitrogen, 5%-15% hydrogen, and 5%-15% carbon dioxide.

[0027] The fermentation substrate for the fermentation process is not particularly limited and may be, for example, a commercially available fermentation substrate. In some embodiments, the fermentation substrate may be a reinforced clostridial medium (RCM). In some embodiments, the fermentation substrate may include, but is not limited to, 1 wt% to 10 wt% sugars, 0.1 wt% to 5.0 wt% yeast extract, 0.1 wt% to 5.0 wt% peptone, 0.01 wt% to 2.00 wt% trace elements, 0.01 wt% to 0.10 wt% cysteine, 0.05 wt% to 1.00 wt% TWEEN-80, and a balanced amount of water. The sugars may include, but are not limited to, monosaccharides and / or disaccharides. In some embodiments, the sugars may be selected from the group consisting of glucose, galactose, fructose, maltose, lactose, sucrose, and combinations thereof. The trace elements may be selected from the group consisting of magnesium ions, potassium ions, sodium ions, chloride ions, and combinations thereof.

[0028] In some embodiments, Clostridium butyricum GKB7 is a killed bacterium obtained after the above fermentation and inactivation steps. The method of the inactivation step is not particularly limited, as long as it causes Clostridium butyricum GKB7 to lose proliferation activity. In some embodiments, the inactivation step may include, but is not limited to, a heat inactivation step, a radiation inactivation step, and / or a chemical inactivation step. In some specific examples, the inactivation step is a heat inactivation step at 110°C to 120°C under saturated steam pressure for 10 minutes to 20 minutes. It should be noted that although the killed bacterium of Clostridium butyricum GKB7 has lost proliferation activity, experiments have demonstrated that the killed bacterium of Clostridium butyricum GKB7 still has activity to improve osteoarthritis. Secondly, compared with live Clostridium butyricum GKB7, the storage and / or formulation of killed Clostridium butyricum GKB7 is less restricted and can be applied to many types of products.

[0029] In some embodiments, after the fermentation step or after the fermentation step and the inactivation step, Clostridium butyricum GKB7 may be selectively subjected to a solid-liquid separation step to remove the fermentation substrate. The solid-liquid separation step is not particularly limited, but it is necessary that the fermentation substrate can be removed while retaining the live or dead Clostridium butyricum GKB7. In some embodiments, the solid-liquid separation step may include, but is not limited to, a filtration step and / or a centrifugation step. It should be noted that the order of the heat inactivation step and the solid-liquid separation step is not particularly limited. In some embodiments, Clostridium butyricum GKB7 may be subjected to a solid-liquid separation step and then an inactivation step after the fermentation step.

[0030] In some embodiments, the live and / or killed Clostridium butyricum GKB7 cells may be further dried to obtain a freeze-dried powder, and the storage time may be extended. In some embodiments, the freeze-dried powder has a concentration of 5.5×10 7 CFU / g of Clostridium butyricum GKB7. The active substances of the live bacteria, killed bacteria, freeze-dried powder of live bacteria and freeze-dried powder of killed bacteria may include, but are not limited to, cell walls, extracellular matrix secreted into the cell membrane, cytoplasm and / or metabolites (e.g., lipids, sugars, proteins, glycoproteins, glycolipids and / or other biomolecules) generated after the cells have undergone a fermentation process, and it should be noted that it is not reasonably expected that the effect of improving osteoarthritis and osteoarthritis can be achieved by utilizing a single substance therein and / or excluding unknown inseparable components. Therefore, the killed bacteria of Clostridium butyricum GKB7 according to the present invention cannot be defined by components, characteristics or parameters, i.e., the present invention may not be possible or practical to define by components, characteristics or parameters.

[0031] The subject may be, but is not limited to, a mammal such as a rat, a mouse, a rabbit, a cat, a dog, or a human. In some embodiments, when the subject is a rat, the effective amount of Clostridium butyricum GKB7 may be, for example, 50 mg / kg body weight / day to 2000 mg / kg body weight / day, such as 50 mg / kg to 500 mg / kg or 80 mg / kg to 100 mg / kg, so as to achieve a favorable effect with a relatively small dose. In some embodiments, when the subject is a human, the effective amount of Clostridium butyricum GKB7 may be, for example, 1 mg / kg body weight / day to 200 mg / kg body weight / day, such as 1 mg / kg body weight / day to 100 mg / kg body weight / day, or 1 mg / kg body weight / day to 50 mg / kg body weight / day. Animal experiments have shown that administering 100mg / kg to 1000mg / kg of Clostridium butyricum GKB7 can indeed improve osteoarthritis, but it should be noted that in this animal experiment, osteoarthritis was induced by anterior cruciate ligament resection, so the course of osteoarthritis is very fast. When applied clinically, the same effect should be achieved with a relatively small dose. In some embodiments, when the subject is a human, the effective amount of Clostridium butyricum GKB7 is 1mg / kg body weight / day to 10mg / kg body weight / day. In the above embodiments, the subject is either trying to prevent osteoarthritis or suffering from early osteoarthritis (at least one but less than four joints are inflamed and within two years of onset).

[0032] Clostridium butyricum GKB7 should be administered to the subject continuously for a certain period of time. In some embodiments, Clostridium butyricum GKB7 may be administered to the subject 3 to 7 times a week, for example, 5 times a week. In some embodiments, Clostridium butyricum GKB7 may be administered to the subject for 2 to 8 weeks, or longer or shorter.

[0033] When applied, the composition may be, for example, an oral composition. In some embodiments, the composition may be, for example, a pharmaceutical composition or a food composition. In one embodiment, the oral composition may optionally include a food- or medically acceptable carrier, excipient, diluent, adjuvant, preservative, filler, and / or additive.

[0034] The following describes the application of the present invention using several examples, but the examples are not intended to limit the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. EXAMPLES

[0035] 1. Preparation Example 1 1.1 Origin of the strain Strain GKB7 was isolated from the feces of a healthy human. After purifying the DNA of strain GKB7, gene sequencing was performed to obtain the nucleic acid fragment sequence of the 16s rRNA gene of strain GKB7, which is shown in sequence identification number (SEQ ID NO): 1. After sequence alignment, strain GKB7 was identified as Clostridium butyricum. The above DNA purification, gene sequencing and sequence alignment were performed by known methods and do not affect the subsequent evaluation of the improvement of osteoarthritis by strain GKB7, so they will not be described again. Clostridium butyricum strain GKB7 is Gram stain positive, strictly anaerobic, motile, and has spores and flagella. Clostridium butyricum strain GKB7 was deposited at BCRC on April 1, 2022, with the accession number BCRC 911120.

[0036] 1.2 Preparation of lyophilized powder The strain GKB7 was spread on RCM agar and cultured in an anaerobic environment (80% N2, 10% H2 and 10% CO2) at 37°C for 24 hours to obtain a single colony. The single colony was then inoculated into a culture medium, which was then subjected to a fermentation process in an anaerobic environment at 37°C for 24 hours to obtain a liquid culture. The culture medium contained 5% glucose, 1.5% yeast extract, 0.5% peptone, 0.1% MgSO4, 0.2% K2HPO4, 0.1% sodium acetate, 0.2% NaCl and a balanced amount of water. It should be noted that the type of medium does not affect the ameliorative effect of the strain GKB7 on osteoarthritis, and the culture mediums listed above are not intended to limit the present invention.

[0037] Next, the liquid culture was subjected to a centrifugation step at 1000 rpm to 15000 rpm to obtain live bacteria of the strain GKB7. Prior to the centrifugation step, the liquid culture was mixed with 15 wt% skim milk powder, which helps the bacteria to aggregate into balls, thereby increasing the recovery rate of the bacteria. The bacteria were then mixed with 6 wt% to 50 wt% skim milk powder, and then subjected to a freeze-drying step to obtain a freeze-dried powder of live bacteria of the strain GKB7. It should be noted that the freeze-drying is merely for the purpose of facilitating administration, quantification, and storage, and is not intended to limit the present invention.

[0038] 2. Preparation Example 2 The liquid culture was subjected to a heat inactivation step at saturated vapor pressure of 110°C to obtain a heat inactivated liquid culture. The heat inactivated liquid culture was then subjected to a centrifugation step to obtain killed cells of the GKB7 strain. The killed cells of the GKB7 strain were then subjected to a freeze-drying step to obtain a freeze-dried powder of killed cells of the GKB7 strain.

[0039] 3. Evaluation method and results 3.1 Animal models Eight-week-old male Sprague Dawley (SD) rats were housed in stainless steel rat cages, and the temperature and humidity of the environment were 22±2°C, 60%-80%, and the light and dark periods were 12 hours each. During the study, the rats were allowed free access to food (standard chow diet) and distilled water. The rats weighed 300g-350g and had matured skeletal development.

[0040] The rats were divided into a sham-operated control group (n=6), a surgical control group (n=6) and an experimental group 1 (n=8). The right hind limbs of the rats in the sham-operated control group, the surgical control group and the experimental group 1 were subjected to corresponding surgeries, the right hind limbs of the rats in the surgical control group and the experimental group 1 were subjected to anterior cruciate ligament transect (ACLT) including arthrotomy and anterior cruciate ligament fiber transection, and osteoarthritis was induced in the right hind limbs of the rats by ACLT, and the right hind limbs of the rats in the sham-operated control group were subjected to arthrotomy but their anterior cruciate ligament fibers were not transected.

[0041] Two days after surgery, 1 mL of distilled water was added to the diet of each group. Rats in experimental group 1 were given 100 mg / kg bw / day (5.5 × 10 7 To obtain a freeze-dried powder of live bacteria of the GKB7 strain (equivalent to CFU / g, equivalent to a human dose of 16.1 mg / kg body weight / day), the live freeze-dried powder of the GKB7 strain was added to the distilled water of experimental group 1 five days a week (Preparation Example 1).

[0042] Then, different doses of live and killed freeze-dried powders of GKB7 were tested. Eight-week-old rats were divided into six groups: sham control group 2, surgical control group 2, experimental group 2, experimental group 3, experimental group 4, and positive control group. There were eight animals per group. The right hind limbs of rats in surgical control group 2, experimental group 2, experimental group 3, experimental group 4, and positive control group were subjected to anterior cruciate ligament transection surgery.

[0043] After the rats had recovered for 1-2 days, 1 mL of distilled water was added to the feed of each group, 5 days a week, along with the corresponding doses of live GKB7 freeze-dried powder and killed GKB7 freeze-dried powder for each group. This allowed the rats in experimental group 2 to receive a high dose (1 g / kg bw / day, equivalent to a human dose of 161.3 mg / kg body weight / day) of live GKB7 freeze-dried powder (Preparation Example 1), the rats in experimental group 3 to receive a low dose (100 mg / kg bw / day, the same dose as experimental group 1) of live GKB7 freeze-dried powder (Preparation Example 1), and the rats in experimental group 4 to receive 1 g / kg bw / day of killed GKB7 freeze-dried powder (Preparation Example 2). Celecoxib (50 mg / kg) was added to the distilled water in the positive control group. Celecoxib is an over-the-counter analgesic that inhibits the activity of cyclooxygenase (COX), thereby reducing inflammatory pain by inhibiting the synthesis of prostaglandin E2 (PGE2).

[0044] 3.2 Static weight bearing test (also called incapacitance test) Before surgery, the left and right hindlimbs of rats in the sham-operated control group, the operated control group, and the experimental group 1 were placed on separate sensor plates for 10 seconds, and the difference in weight load between the left and right hindlimbs (unit: basis weight, normalized by the rat's weight) was measured when the rat stood on its hindlimbs. After surgery, the above measurements were performed weekly. Generally, normal rats distribute their body weight equally between the left and right hindlimbs, so the greater the difference in weight load between the left and right hindlimbs, the greater the degree of pain in the rat's right hindlimbs. The results of different groups were compared by paired t-test.

[0045] FIG. 1 is a graph showing the change in weight load difference 6 weeks after surgery in different groups of rats according to one embodiment of the present invention, where the horizontal axis shows time (unit: weeks) and the vertical axis shows weight load difference (unit: g), with curves 110, 120 and 130 showing the sham surgery control group, the surgery control group and experimental group 1, respectively, with the symbol "*" indicating that there is a statistically significant difference (p<0.05) between the surgery control group and the sham surgery control group, and the symbol "#" indicating that there is a statistically significant difference (p<0.05) between experimental group 1 and the surgery control group.

[0046] As shown in FIG. 1, the weight bearing difference of the operation control group (curve 120) was significantly higher than that of the sham operation control group (curve 110), and the weight bearing difference increased over time, which means that ACLT indeed induces osteoarthritis and causes joint pain. Conversely, the weight bearing difference of experimental group 1 significantly decreased 2 weeks after ACLT surgery, and continued to decrease over time, and 3 weeks after ACLT surgery, it was only one-third of the weight bearing difference of the operation control group. This result shows that the live freeze-dried powder of strain GKB7 can significantly alleviate joint pain caused by osteoarthritis after 14 days (2 weeks) of continuous administration, and can achieve more ideal effects after 21 days (3 weeks) of continuous administration.

[0047] Static weight-bearing tests were also performed on sham control group 2, surgical control group 2, experimental group 2, experimental group 3, experimental group 4 and the positive control group, the methods of which have been described above and will not be repeated here.

[0048] FIG. 2 is a graph showing the change in weight load difference 6 weeks after surgery for rats in different groups according to one embodiment of the present invention, where the horizontal axis shows time (unit: weeks) and the vertical axis shows weight load difference (unit: g), with curves 210, 220, 230, 240, 250 and 260 showing sham surgery control group 2, surgery control group 2, experimental group 2, experimental group 3, experimental group 4 and positive control group, respectively, with the symbol "*" indicating that there is a statistically significant difference (p<0.05) between surgery control group 2 and sham surgery control group 2, and the symbol "#" indicating that there is a statistically significant difference (p<0.05) between experimental group 2, experimental group 3, experimental group 4 and positive control group and surgery control group 2.

[0049] As shown in FIG. 2, compared to the surgical control group 2 (curve 220), the difference in weight bearing was small among experimental group 2 (curve 230), experimental group 3 (curve 240), experimental group 4 (curve 250) and the positive control group (curve 260), indicating that the live freeze-dried powder of GKB7 strain and the killed freeze-dried powder of GKB7 strain can indeed relieve joint pain caused by osteoarthritis. Curve 230 representing experimental group 2 and curve 250 representing experimental group 4 almost overlap, indicating that the same dosage of the live freeze-dried powder of GKB7 strain and the killed freeze-dried powder of GKB7 strain have the same improving effect on osteoarthritis, explaining that the improving effects on osteoarthritis and osteoarthritis are the same. Although the dosages of the rats in experimental group 2 and experimental group 4 are different from those of experimental group 3, both have the effect of relieving joint pain. Therefore, the following experiment is the effect of administering a low dose of live freeze-dried powder of GKB7 strain on the rats in experimental group 1, but those skilled in the art will understand that the administration of a high dose of live freeze-dried powder and killed freeze-dried powder of GKB7 strain can both achieve the same effect.

[0050] 3.3 Bone image analysis Six weeks after the ACLT operation, the rats in the sham operation control group, operation control group and experimental group 1 were sacrificed, and the bone parameters of the knee joint of the right hind leg of the rats were analyzed by micro-computerized tomography (Micro-CT, μ-CT), including bone mineral density, bone mass, bone volume fraction, bone surface area density, trabecular thickness, trabecular number and trabecular space. The micro-computerized tomography showed that the rats in the operation control group indeed had bone damage, including bone loss and bone destruction (not shown), but the degree of bone damage in the rats in experimental group 1 was lower than that in the operation control group (not shown).

[0051] 3A to 3G are box plots showing bone mineral density (FIG. 3A), bone mass (FIG. 3B), bone volume fraction (FIG. 3C), bone surface area density (FIG. 3D), trabecular thickness (FIG. 3E), trabecular number (FIG. 3F), and trabecular space (FIG. 3G) in different groups according to one embodiment of the present invention. The horizontal axis indicates the group, which is, from left to right, the sham-operated control group, the operated control group, and the experimental group 1, respectively. The vertical axes of FIGS. 3A to 3G are bone mineral density (unit: g / cm3), bone mass (unit: mg), and bone volume fraction (i.e., the ratio of bone volume to total volume, BV / TV), respectively. and unit: %), bone surface area density (i.e., the ratio of bone surface area to total volume, abbreviated as BS / TV, unit: 1 / mm), trabecular thickness (abbreviated as Tb.Th, unit: mm), trabecular number (abbreviated as Tb.N, unit: 1 / mm), and trabecular space (abbreviated as Tb.Sp, unit: mm). The symbol "*" indicates that there is a statistically significant difference (p<0.05) between the surgical control group and the sham surgical control group, and the symbol "#" indicates that there is a statistically significant difference (p<0.05) between experimental group 1 and the surgical control group.

[0052] As shown in Figures 3A-3G, compared with the sham-operated control group, the operation control group had significantly decreased bone mineral density, bone mass, bone volume fraction, bone surface area density, trabecular thickness and trabecular number in the knee joint of the right hind leg of rats, but significantly increased trabecular space, indicating that the induction of osteoarthritis by ACLT can indeed cause bone damage such as bone loss and bone destruction. Conversely, the above bone parameters of the right hind leg knee joint of rats in experimental group 1 were significantly improved, indicating that the live freeze-dried powder of the GKB7 strain can indeed ameliorate bone damage caused by osteoarthritis.

[0053] 3.4 Histological analysis After analyzing the bone images, the knee joint tissues of the right hind limbs of the rats were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 37°C for 24 hours, and then decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 2 weeks. After dehydration with increasing concentrations of ethanol, the knee joint tissues were embedded in paraffin blocks and cut into tissue sections with a thickness of 5 μm. The tissue sections were then stained with hematoxylin and eosin (H&E) and Safranin-O / Fast Green, and the histopathological changes of the knee joint tissues were observed under an optical microscope. As can be seen from the tissue sections, the knee joint tissue of the rats in the surgical control group indeed showed pathological changes in the cartilage tissue and cartilage damage such as hyperplasia of the synovial tissue (not shown), but the degree of cartilage damage in the rats in experimental group 1 was lower than that of the rats in the surgical control group (not shown).

[0054] The rat joint histological scoring system (including the International Osteoarthritis Society score, cartilage degeneration score and synovial inflammation score) recommended by the International Osteoarthritis Society was used to quantify the degree of cartilage damage in the knee joint tissue. The International Osteoarthritis Society score, cartilage degeneration score and synovial inflammation score are well known to those skilled in the art, and will not be described again here.

[0055] Figures 4A to 4C each show the International Osteoarthritis Society score, cartilage degeneration score, and synovial inflammation score for different groups according to one embodiment of the present invention, where the horizontal axis shows the group, which from left to right are the sham surgery control group, the surgery control group, and the experimental group 1, respectively, and the vertical axis shows the International Osteoarthritis Society score, cartilage degeneration score, and synovial inflammation score (unit: points), respectively, with the symbol "*" indicating that there is a statistically significant difference (p<0.05) between the surgery control group and the sham surgery control group, and the symbol "#" indicating that there is a statistically significant difference (p<0.05) between the experimental group 1 and the surgery control group.

[0056] As shown in Figures 4A-4C, the International Osteoarthritis Society score, cartilage degeneration score, and synovial inflammation score of the surgical control group were significantly improved compared to the sham-operated control group, and the symptoms of osteoarthritis induced by ACLT certainly include cartilage damage. However, compared to the surgical control group, the International Osteoarthritis Society score, cartilage degeneration score, and synovial inflammation score of experimental group 1 were significantly decreased, indicating that the live freeze-dried powder of GKB7 strain can indeed improve cartilage damage caused by osteoarthritis.

[0057] 3.5 Immunohistochemistry (IHC) The tissue sections were immersed in a 3% hydrogen peroxide solution to suppress endogenous catalase activity, and then in a PBS solution containing 3% bovine serum albumin to reduce the occurrence of nonspecific background. After primary antibody incubation with anti-IL-1β or anti-TNF-α antibodies at 4°C, the tissue sections were subjected to secondary antibody incubation at room temperature for 1 hour, and then stained with diaminobenzidine (DAB). The tissue sections were observed under an optical microscope. The IHC scores indicate the immunoreactive signals of IL-1β and TNF-α, and the higher the IHC score, the stronger the immunoreactive signal, i.e., the higher the expression level of IL-1β or TNF-α.

[0058] 5A-5B are box plots showing the IHC scores of IL-1β (FIG. 5A) and TNF-α (FIG. 5B) in cartilage tissues of different groups according to one embodiment of the present invention, with the horizontal axis showing the groups, from left to right being the sham operation control group, the operation control group, and experimental group 1, respectively, and the vertical axis showing the IHC scores of IL-1β and TNF-α (unit: points). As shown in FIG. 5A and FIG. 5B, the IHC scores of IL-1β and TNF-α in the operation control group were significantly increased compared to the sham operation control group, indicating high expression of inflammatory cytokines such as IL-1β and TNF-α in cartilage tissues of osteoarthritis induced by ACLT. Conversely, compared with the surgical control group, the IHC scores of IL-1β and TNF-α in experimental group 1 were significantly decreased, indicating that the live lyophilized powder of GKB7 strain could indeed reduce the expression levels of inflammatory cytokines in osteoarthritis cartilage tissue.

[0059] 6A-6B are box plots showing the IHC scores of IL-1β (FIG. 6A) and TNF-α (FIG. 6B) in synovial tissues of different groups according to an embodiment of the present invention, with the horizontal axis showing the groups, from left to right, the sham-operated control group, the surgical control group, and the experimental group 1, respectively, and the vertical axis showing the IHC scores of IL-1β and TNF-α (unit: points). As shown in FIG. 6A and FIG. 6B, compared with the sham-operated control group, the surgical control group had significantly increased IHC scores of IL-1β and TNF-α, indicating that the expression of inflammatory cytokines in synovial tissues of osteoarthritis induced by ACLT was indeed high. Conversely, compared with the surgical control group, the experimental group 1 had significantly decreased IHC scores of IL-1β and TNF-α, indicating that the live freeze-dried powder of GKB7 strain can indeed reduce the expression of inflammatory cytokines in synovial tissues of osteoarthritis.

[0060] In summary, the present invention has been described by exemplifying the Clostridium butyricum GKB7-containing composition for improving osteoarthritis and osteoarthritis simply by a specific dosage amount, a specific dosage form, a specific detection method, or a specific evaluation method. However, for those skilled in the art, other dosage amounts, other dosage forms, other detection methods, or other evaluation methods may also be applied to the Clostridium butyricum GKB7-containing composition for improving osteoarthritis and osteoarthritis without departing from the spirit and scope of the present invention, and are not limited to the above. For example, the composition may be, for example, a pharmaceutical composition or a food composition, and may selectively contain medically and food-acceptable carriers, excipients, and / or additives, and may be in the form of a powder, tablet, granule, suppository, microcapsule, ampoule, liquid spray, or embolization agent. In other examples, the strain GKB7 may be cultured using other suitable media or other culture environments, and the obtained bacterial cells should also have the above-mentioned improving effect on osteoarthritis. In other embodiments, effects achievable with a low dose of live bacteria (experimental group 1) include improving bone damage, cartilage damage, inflammation of cartilage tissue and / or synovial tissue due to osteoarthritis, etc., which can also be achieved with a high dose of live bacteria (experimental group 2) and a high dose of killed bacteria (experimental group 4).

[0061] According to the above examples, the Clostridium butyricum GKB7-containing composition for improving osteoarthritis and osteoarthritis of the present invention can not only relieve joint pain caused by osteoarthritis by using Clostridium butyricum GKB7, but can also improve bone damage, cartilage damage, and inflammation of cartilage tissue and / or synovial tissue caused by osteoarthritis, and has the advantage that it can be used as an active ingredient in the preparation of various compositions in the future and applied to the improvement of osteoarthritis and ultimately other musculoskeletal diseases.

[0062] While the present invention has been disclosed above in terms of several specific embodiments, other embodiments are possible, and therefore it is not intended that the spirit or scope of the invention be limited to the embodiments described herein. [Explanation of symbols]

[0063] 110, 120, 130, 210, 220, 230, 240, 250, 260 curve [Deposit of biological materials] Clostridium butyricum GKB7 was deposited at the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), Taiwan on April 1, 2022, with the accession number BCRC 911120. [Reference sequence] TIFF0007607709000001.tif236168TIFF0007607709000002.tif190168

Claims

1. A Clostridium butyricum GKB7-containing composition for improving osteoarthritis, comprising 5.5×10 7 CFU / g of live or dead Clostridium butyricum GKB7 as an active ingredient, wherein the dead cells are obtained by subjecting live cells to a heat inactivation process under saturated vapor pressure at 110° C. to 120° C. for 10 to 20 minutes, and the Clostridium butyricum GKB7 was approved by the Bioresource Collection and Research Center of the Food Industry Research and Development Institute (FIRDI) of Taiwan on April 1, 2022. and a composition for improving osteoarthritis, which has been deposited at the Boston Cancer Research Center (BCRC) and has accession number BCRC 911120, the composition being administered to a subject at an effective dose of 50 mg / kg body weight / day to 2,000 mg / kg body weight / day, the subject being a rat, thereby reducing the expression level of IL-1β in cartilage tissue and / or synovial tissue of the subject.

2. The composition of claim 1, wherein the composition is administered to the subject three to seven times per week.

3. The composition of claim 1, wherein the composition is administered to the subject for between 2 and 8 weeks.

4. The composition of claim 1, further comprising a food or medically acceptable carrier, excipient, diluent, adjuvant, preservative, filler, and / or additive.

5. The composition described in claim 1, wherein the composition is a pharmaceutical composition or a food composition.

6. A Clostridium butyricum GKB7-containing composition for improving osteoarthritis, comprising 5.5 x 107 CFU / g of live or dead cells of Clostridium butyricum GKB7 having the accession number BCRC 911120 as an active ingredient, wherein the dead cells are obtained by subjecting live cells to a heat inactivation step under saturated vapor pressure at 110°C to 120°C for 10 to 20 minutes, and the composition is administered to a subject at an effective dose of 1 mg / kg body weight / day to 100 mg / kg body weight / day, the subject being a human, thereby reducing the expression level of IL-1β in the cartilage tissue and / or synovial tissue of the subject.

7. The composition according to claim 6, wherein the expression level of TNF-α in the cartilage tissue and / or the synovial tissue of a subject to which the composition is administered is reduced compared to a control subject to which the composition is not administered.

8. The composition of claim 6, wherein the subject to which the composition is administered has reduced joint pain compared to a control subject to which the composition is not administered.

9. The composition of claim 6, wherein the composition is administered to the subject three to seven times per week.

10. The composition of claim 6, wherein the composition is administered to the subject for between 2 and 8 weeks.

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