Application of LGG in preparation of product for preventing and / or treating weightlessness bone loss

Abstract

The invention discloses application of LGG in preparation of a product for preventing and / or treating weightlessness bone loss. The invention discloses application of LGG in preparation of a product for preventing and / or treating weightless bone loss. The weightless bone loss is bone loss caused by aerospace, weightlessness or flight conditions. The LGG can be used for preparing a product for preventing and / or treating weightlessness bone loss after being stimulated by a simulated weightlessness condition. The application mode comprises intragastric administration or oral administration. The LGG and the LGG subjected to weightlessness stimulation simulation can be used for preparing products for preventing and / or treating weightlessness bone loss, and have wide application prospects. The product can effectively relieve, prevent, treat or relieve weightlessness bone loss, and is convenient to carry. The preparation method disclosed by the invention is safe and efficient, the adhesion of the LGG can be improved by simulating weightlessness stimulation, the tolerance to gastrointestinal fluid and bile is also enhanced to different extents, and the LGG after simulating weightlessness stimulation can effectively resist weightlessness bone loss.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to the use of LGG in the preparation of products for the prevention and / or treatment of weight loss due to weight loss. Background Technology

[0002] With the rapid development of science and technology, humans have acquired the ability for long-duration spaceflight. However, preventing and controlling weightlessness-induced bone loss during these long-term spaceflights remains a significant challenge. Prolonged spaceflight leads to physiological effects such as decreased immune function, bone loss, and cardiovascular dysfunction. As the flight duration increases, the effects of weightlessness on the cardiovascular, vestibular, and metabolic systems undergo various adaptive changes, with bone loss worsening over time. Microgravity is one of the most important differences between space and Earth environments, and it is a major cause of bone loss. Weightlessness-induced bone loss is a long-term, progressive pathological phenomenon that can lead to osteoporosis, soft tissue calcification, kidney stones, and even gravity readjustment disorders, severely impacting astronauts' health and work efficiency. Research into the mechanisms of weightlessness-induced bone loss and its preventative measures is a crucial topic in aerospace medicine. Studies have shown that weightlessness-induced bone loss is a type of disuse bone loss, with load-bearing bones being more affected than non-load-bearing bones. It is characterized by large losses and difficulty in recovery. Weightlessness-induced bone loss results from the interaction between osteocytes, osteoblasts, and osteoclasts, with reduced osteoblast function playing a crucial role in weightlessness-induced bone loss. However, research on the body's regulatory mechanisms and pathways for bone loss under weightless or simulated weightless conditions is limited, making the development of protective measures essential.

[0003] Lactobacillus rhamnosus GG (LGG), a mature probiotic product, plays a role in regulating immune function and bone formation. Studies have found that LGG can regulate bone formation by modulating the composition of the gut microbiota and its metabolites (such as short-chain fatty acids). However, the regulatory effect of LGG on weightlessness-induced bone loss remains unclear. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide the use of LGG in the preparation of products for the prevention and / or treatment of weight loss, for the prevention, treatment or relief of weight loss.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of LGG in the preparation of products for the prevention and / or treatment of weightlessness-induced bone loss, which is bone loss caused by aerospace, weightlessness, or flight conditions.

[0007] Preferably, LGG can be used to prepare products for the prevention and / or treatment of weightlessness bone loss after being stimulated under simulated weightlessness conditions.

[0008] Preferably, the administration method includes gavage or oral administration.

[0009] Preferably, the product includes a food supplement or a medicine.

[0010] A product for the prevention and / or treatment of weightlessness-induced bone loss, comprising the LGG described in any of the above applications, with a viable count ≥1×10⁻⁶. 9 CFU / mL.

[0011] According to the above-mentioned product preparation method, the LGG is further cultured and passaged in a conventional gravity environment; or the LGG is stimulated under simulated weightlessness conditions for no less than 60 generations, and then transferred to a conventional gravity environment for further culture and passage.

[0012] The beneficial effects of this invention include:

[0013] Through experiments involving the gavage administration of LGG and LGG stimulated by simulated weightlessness to treat bone loss induced by tail-hanging simulated weightlessness, it was confirmed that LGG and LGG stimulated by simulated weightlessness can counteract bone loss induced by tail-hanging simulated weightlessness. Based on this, the LGG and LGG stimulated by simulated weightlessness of this invention can be used to prepare products for the prevention and / or treatment of weightlessness-induced bone loss, and have broad application prospects.

[0014] The product of this invention can effectively alleviate, prevent, treat, or reduce weightlessness-induced bone loss, and is easy to carry.

[0015] The preparation method of this invention is safe and efficient. The simulated weightlessness stimulation can improve the adhesion of LGG and enhance its tolerance to gastrointestinal fluid and bile to varying degrees. LGG after simulated weightlessness stimulation can effectively combat weightlessness-induced bone loss. Attached Figure Description

[0016] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the embodiments or prior art will be briefly introduced below.

[0017] Figure 1 The figure shows the results of the experiment in Example 1 on the effects of LGG gavage on bone loss caused by tail-hanging simulated weightlessness.

[0018] Figure 2 The changes in biological traits after simulated weightlessness stimulation (LGG) in Example 1 are shown.

[0019] Figure 3 The figure shows the results of the experiment in Example 1 on the effect of LGG gavage on bone loss caused by tail-hanging simulated weightlessness after simulated weightlessness. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] The application of LGG in the preparation of products for the prevention and / or treatment of weightlessness-induced bone loss, which is bone loss caused by space travel, weightlessness, or flight conditions. LGG (strain number: ATCC 53103) can be used to prepare products for the prevention and / or treatment of weightlessness-induced bone loss after being stimulated under simulated weightlessness conditions for at least 60 generations. Administration methods include gavage or oral administration. Products include food supplements or pharmaceuticals.

[0022] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels. In the quantitative detection methods described below, three replicate tests were performed, and the results were averaged.

[0023] Example 1: The antagonistic effect of LGG on bone loss caused by tail hanging

[0024] 1. Establishing a weightlessness-induced bone loss model in mice by tail suspension

[0025] C57BL / 6 mice (wild-type (WT) mice) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF grade. Eight-week-old C57BL / 6 mice were used. The mice were secured inside a cotton glove, leaving their tails exposed. The tails were bandaged and secured with adhesive tape. The tails were then hung on a thin chain and placed in a tail-hanging cage. The chain was fixed above the cage, and its length was adjusted to create an angle of approximately 30 degrees between the mouse and the horizontal plane. After four weeks of tail hanging, bone formation in the mice was assessed using microCT.

[0026] 2. LGG gavage

[0027] LGG glycerol culture was inoculated into MRS liquid medium and activated for 18 h. It was then transferred to a 1:100 inoculum and cultured overnight. When the OD value was adjusted to 1, it was further expanded into fresh MRS liquid medium at a 1:100 inoculum. After 10 h of culture, the OD value was measured to be 1. The culture was then centrifuged at 5000 rpm for 20 min at 4°C, and washed three times with sterile PBS. After resuspending in PBS, the OD value was measured to be 5 × 10⁻⁶.9 CFU / mL, 200 μl per mouse was administered by gavage. Before gavage, mice were fed a pentavalent antibiotic (ampicillin (1 g / L), neomycin sulfate (1 g / L), gentamicin (1 g / L), vancomycin (0.5 g / L), metronidazole (1 g / L)) aqueous solution for 2 weeks to clear intestinal flora. The bacterial suspension obtained as described above was administered daily by gavage to antibiotic-treated control mice (Ctrl+LGG) and tail-hanging mice (HU+LGG). Simultaneously, antibiotic-treated control mice (Ctrl+PBS) and tail-hanging mice (HU+PBS) were administered PBS by gavage for 4 consecutive weeks.

[0028] 3. MicroCT analysis of mouse bone formation

[0029] The femur of a dissected mouse was removed, and after the surrounding muscle tissue was removed, the femur was preserved in 75% ethanol. A high-resolution micro-CT scanner (Quantum GX2, PerkinElmer, USA) was used to perform detailed analysis of the fixed femoral specimen. Regions of interest (ROIs) were selected and analyzed using CTAn software. An ROI was defined as the area extending 10% of the proximal bone length from the distal end of the femoral growth plate. Eighty images were selected from this ROI for two-dimensional and three-dimensional reconstruction, allowing for visual and numerical analysis of indicators such as the bone volume to total tissue volume ratio (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sep). Experimental results are as follows: Figure 1 As shown, A is the phenotypic image of a high-resolution micro-CT scanner, B is the ratio of bone volume to total tissue volume, C is the amount of bone minerals per unit area, D is the bone model structure index, E is the trabecular thickness, F is the number of trabeculae, and G is the trabecular spacing.

[0030] The results showed that in mice treated with antibiotics and administered PBS by gavage, the bone loss in tail-suspended mice was significantly higher than that in normal mice; in mice treated with antibiotics and administered LGG by gavage, the bone loss in mice administered LGG by gavage was significantly lower than that in mice administered PBS by gavage. This indicates that tail-suspending simulates weightlessness and leads to bone loss in mice, while LGG gavage can counteract the bone loss induced by tail-suspending.

[0031] Example 2: Changes in the biological properties of LGG in a simulated weightless environment

[0032] A 1:100 dilution of *Lactobacillus rhamnosus* LGG glycerol culture was inoculated into a large test tube containing 5 mL of MRS liquid medium and cultured overnight at 37°C and 200 rpm on a shaker for 18 hours. Using two 1 mL syringes, approximately 0.1 mL of the bacterial culture was injected into the rotating cell culture systems of the simulated microgravity and normal gravity groups, respectively, and mixed thoroughly to remove air bubbles. The rotating cell culture systems of the simulated microgravity and normal gravity groups were placed in a 37°C incubator, the power was turned on, and the instrument rotation speed was adjusted to 25 rpm.

[0033] After 24 hours of culture, two groups of bacteria from the rotating cell culture system were removed and their internal bacterial solutions were mixed in 5 ml test tubes. Approximately 0.1 mL of bacterial solution was taken from the sampling port of the rotating cell culture system and inoculated into another rotating cell culture system filled with MRS liquid medium using a 1 mL syringe. After mixing, air bubbles were removed. Both newly inoculated rotating cell culture systems were then cultured for another 24 hours. Following the same method, LGG was passaged for 60 days in both the simulated microgravity and normal gravity environments.

[0034] LGG bacteria cultured on MRS solid plates for 18 hours under simulated microgravity and normal gravity conditions were observed. Gram staining was performed followed by optical microscopy, and further morphological analysis was conducted using transmission electron microscopy and scanning electron microscopy. Simultaneously, changes in growth curves, adhesiveness, and tolerance to artificial gastrointestinal fluids and bile were examined. Experimental results are as follows: Figure 2 As shown, A represents the bacterial culture system simulating weightlessness; B represents the phenotypes of LGG under normal gravity and simulated weightlessness stimulation under ordinary optical and electron microscopes; C represents the growth curves of LGG under normal gravity and simulated weightlessness stimulation; D represents the changes in the adhesiveness of LGG under normal gravity and simulated weightlessness stimulation; E and G represent the changes in the gastric acid resistance (pH=2, pH=3, pH=4) of LGG under normal gravity and simulated weightlessness stimulation, respectively; and H and J represent the changes in the bile resistance (1% bovine bile, 2% bovine bile, 3% bovine bile) of LGG under normal gravity and simulated weightlessness stimulation, respectively.

[0035] The results showed that, compared with LGG (NG) cultured under normal gravity, LGG (SMG) bacteria under simulated microgravity stimulation exhibited significant morphological changes, with SMG bacteria mostly being short rod-shaped. Compared with NG, the growth curve of SMG remained largely unchanged, but bacterial adhesion was significantly enhanced. Furthermore, SMG also showed varying degrees of increased tolerance to artificial gastrointestinal fluids and bile.

[0036] Example 3: Counteracting effect of simulated weightlessness stimulation LGG on tail suspension-induced bone loss

[0037] 1. Surgical administration of LGG to simulate weightlessness

[0038] Glyceryl glycerol strains subjected to normal gravity stimulation (NG) and simulated microgravity stimulation (SMG) were inoculated into MRS liquid medium and activated for 18 h. They were then transferred at a 1:100 inoculum and cultured overnight. Once the OD values ​​were adjusted to 1, the cultures were expanded to a 1:100 ratio in fresh MRS liquid medium. After 10 h of culture, the OD value was measured to be 1. The cultures were then centrifuged at 5000 rpm for 20 min at 4°C and washed three times with sterile PBS. After resuspending in PBS, the OD value was measured to be 5 × 10⁻⁶. 9 CFU / mL, 200 μl per mouse was administered by gavage. Before gavage, mice were fed a pentavalent antibiotic (ampicillin (1 g / L), neomycin sulfate (1 g / L), gentamicin (1 g / L), vancomycin (0.5 g / L), metronidazole (1 g / L)) aqueous solution for 2 weeks to clear intestinal flora. The bacterial suspension obtained as described above was administered daily to antibiotic-treated control mice (Ctrl+NG, Ctrl+SMG) and tail-hanging mice (HU+NG, HU+SMG) via gavage with NG and SMG respectively. Simultaneously, antibiotic-treated control mice (C+PBS) and tail-hanging mice (HU+PBS) were administered PBS via gavage. This gavage treatment was continued for 4 weeks.

[0039] 2. MicroCT analysis of mouse bone formation

[0040] The femur of the hind limb was removed from the dissected mouse, and after the surrounding muscle tissue was removed, the femur was preserved in 75% ethanol. The fixed femur specimen was analyzed in detail using a high-resolution micro-CT scanner (Quantum GX2, PerkinElmer, USA). During the scanning process, an 18mm field of view, 70kV X-ray voltage, and 114μA current were used, and images were acquired at a 9-micron voxel resolution. Three-dimensional reconstruction and analysis of the images were performed using DataViewer (version 1.5.6), CT Analyzer (version 1.16.4.1), and CTvox (version 3.3.0) software, respectively. Regions of interest (ROIs) were selected and analyzed using CTAn software. An ROI was defined as the area extending 10% of the proximal bone length from the distal end of the femoral growth plate. Eighty image layers were selected from this region for 2D and 3D reconstruction. Visual and numerical analyses were then performed on parameters such as the bone volume to total tissue volume ratio (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sep). Experimental results are as follows: Figure 3 As shown, A is the phenotypic image of a high-resolution micro-CT scanner, B is the ratio of bone volume to total tissue volume, C is the amount of bone minerals per unit area, D is the bone model structure index, E is the trabecular thickness, F is the number of trabeculae, and G is the trabecular spacing.

[0041] The results showed that in mice treated with antibiotics and administered PBS by gavage, the bone loss in tail-suspended mice was significantly higher than that in normal mice; in tail-suspended mice treated with antibiotics and administered NG and SMG by gavage, the bone loss was significantly lower than that in mice administered PBS by gavage; indicating that tail-suspending simulates weightlessness leads to bone loss in mice, while NG and SMG gavage can counteract the bone loss induced by tail-suspending in mice.

[0042] In summary, the study investigated the effects of LGG and LGG after simulated weightlessness stimulation on tail-suspended bone loss in mice. The results confirmed that tail-suspended mice experienced more severe bone loss compared to normal control mice. Gavage administration of LGG and LGG after simulated weightlessness stimulation alleviated tail-suspended bone loss, indicating that LGG and LGG after simulated weightlessness stimulation have a protective effect against tail-suspended bone loss.

[0043] This demonstrates that LGG and LGG administered via gavage after simulated weightlessness can counteract bone loss caused by simulated weightlessness.

[0044] The above description is only a preferred embodiment of the present invention. Those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of LGG in the preparation of products for the prevention and / or treatment of weight loss due to gravity, characterized in that, Weightlessness-induced bone loss refers to bone loss caused by spaceflight, weightlessness, or flight conditions.

2. The application according to claim 1, characterized in that, LGG can be used to prepare products for the prevention and / or treatment of weightlessness after being stimulated under simulated weightlessness conditions.

3. The application according to claim 1, characterized in that, Administration methods include gavage or oral administration.

4. The application according to claim 1, characterized in that, Products include food supplements or medicines.

5. A product for preventing and / or treating weightlessness-induced bone loss, characterized in that, Including the LGG used in any of the applications described in claims 1-4, with a viable count ≥ 1 × 10⁻⁶ 9 CFU / mL.

6. The method for preparing the product according to claim 5, characterized in that, This includes continuing to culture and passage the LGG in a normal gravity environment; or stimulating the LGG under simulated weightlessness conditions for at least 60 generations, and then transferring it to a normal gravity environment for continued culture and passage.

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