Lactobacillus paracasei CCFM1396 with effects of relieving bone resorption and resisting inflammation and metaplasm of lactobacillus paracasei CCFM1396

Abstract

The invention discloses a Lactobacillus paracasei strain capable of relieving bone resorption and having an anti-inflammatory effect and a metagen thereof, and belongs to the technical field of microorganisms. The invention provides a novel application of the Lactobacillus paracasei CCFM1396 (Lactobacillus paracasei CCFM1396) in the treatment of the orthodontically induced excessive bone resorption, and particularly provides the application of the Lactobacillus paracasei CCFM1396 in the treatment of the orthodontically induced excessive bone resorption. The lactobacillus paracasei provided by the invention can regulate and control osteoblasts to secrete osteoprotegerin (OPG) and improve gene expression of bone morphogenetic protein (BMP), osteoprotegerin (OPG) and osteopontin (OPN). In a rat orthodontic model, the strain disclosed by the invention can effectively relieve bone resorption, inhibit the inflammatory factor level of gingival tissues and protect the stability of periodontal tissues.

Description

Technical Field

[0001] The invention relates to a Lactobacillus paracasei CCFM1396 strain capable of alleviating bone resorption and having anti-inflammatory effects and a postbiotic thereof, belonging to the technical field of microorganisms. Background Art

[0002] Orthodontics is an important treatment method in the field of dentistry, which improves oral function and facial aesthetics by correcting abnormal tooth and jaw alignment. During orthodontic treatment, tooth movement involves the remodeling process of the alveolar bone, which is mainly achieved through bone resorption by osteoclasts and bone formation by osteoblasts. Osteoclasts can break down bone tissue during bone resorption to create space for tooth movement. After osteoclasts absorb bone, osteoblasts calcify to form new bone tissue and stabilize the new position of teeth. Orthodontic force can stimulate the activity of osteoclasts and promote the absorption of alveolar bone. However, excessive orthodontic force and improper oral hygiene may induce periodontal problems such as gingival damage and alveolar bone damage, followed by excessive bone resorption of alveolar bone.

[0003] The OPG / RANKL / RANK pathway is a key pathway that regulates the balance between bone resorption and bone formation. OPG (osteoprotegerin) is secreted by osteoblasts and can competitively bind to RANKL (receptor activator of nuclear factor κB ligand) on the surface of osteoclasts RANK receptors (receptor activator of nuclear factor κB), thereby inhibiting the differentiation and activity of osteoclasts. In orthodontic treatment, by regulating this pathway, the metabolism of the alveolar bone can be affected, thereby affecting the speed and effect of tooth movement. Increasing the level of OPG or inhibiting the activity of RANKL can reduce bone resorption and help stabilize teeth, thereby achieving alveolar bone homeostasis in orthodontic patients.

[0004] At present, there are many patents involving the use of biological agents to treat alveolar bone resorption. These patents usually focus on the use of some Chinese herbal plant extracts and specific protease preparations to regulate gingival tissue inflammation, local metalloproteinase activity in gingival tissue, or local to promote osteoblast activity, inhibit osteoclast function, and thus reduce alveolar bone resorption. Patent CN116327762A found that coptisine hydrochloride can inhibit the pyroptosis pathway mediated by macrophage NLRP3 inflammasome, reduce the recognition of pathogen-associated molecular patterns (PAMPs), etc., and thus inhibit the occurrence of periodontal inflammation. However, this also indirectly reduces the individual's immunity and increases the risk of uncertainty in inaccurate dosage. In addition, in the application of microbial fields, some patented strains regulate femoral tissue bone metabolism by affecting the activity of osteoblasts and osteoclasts for the OPG / RANKL / RANK pathway and the Wnt signaling pathway, thereby slowing down the progression of osteoporosis. However, considering the complex environment of the oral cavity, the application of these strains in orthodontically induced bone resorption still needs further verification. Therefore, it is urgent to develop a probiotic that can effectively alleviate bone resorption, inhibit the level of inflammatory factors in gingival tissue, and protect the stability of periodontal tissue. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a strain of Lactobacillus paracasei and its postbiotics that can alleviate bone resorption and have anti-inflammatory effects, aiming to solve the technical problem that the prior art lacks a probiotic agent that can effectively alleviate bone resorption, inhibit the level of inflammatory factors in gingival tissue, and protect the stability of periodontal tissue.

[0006] The first technical solution provided by the present invention is a strain of Lactobacillus paracasei CCFM1396, the taxonomic name of the Lactobacillus paracasei CCFM1396 is Lacticaseibacillus paracasei, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with a deposit number of GDMCC No.64760, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The Lacticaseibacillus paracasei CCFM1396 is derived from oral dental plaque samples of healthy volunteers in Wuxi, Jiangsu Province. The strain is sequenced and analyzed, and its 16S rDNA sequence is shown in SEQ ID NO.1. The sequence obtained by sequencing is compared with the nucleic acid sequence in NCBI, and the result shows that the strain is Lacticaseibacillus paracasei, and is named Lacticaseibacillus paracasei CCFM1396.

[0008] The colonies of the Lactobacillus paracasei CCFM1396 on the MRS culture medium are small semi-transparent white circles.

[0009] The second technical solution provided by the present invention is a microbial preparation containing the Lactobacillus paracasei CCFM1396 described in the first technical solution.

[0010] In certain embodiments, in the microbial preparation, the bacterial concentration of Lactobacillus paracasei CCFM1396 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0011] Furthermore, in the microbial preparation, the bacterial concentration of the Lactobacillus paracasei CCFM1396 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0012] The third technical solution provided by the present invention is a postbiotic, which includes one or more of the fermentation supernatant, bacterial lysate and fermentation broth of Lactobacillus paracasei 1396 described in the first technical solution.

[0013] In certain embodiments, the postbiotic is a fermentation supernatant obtained by centrifuging the Lactobacillus paracasei CCFM1396 after inoculating it into an MRS medium for culturing.

[0014] In certain embodiments, the postbiotic is obtained by high-pressure homogenization and membrane filtration of the fermentation broth after inoculating the Lactobacillus paracasei CCFM1396 in MRS medium to obtain a bacterial lysate.

[0015] In certain embodiments, the postbiotics can be dried into powder or used directly by various drying methods such as vacuum drying, spray drying, vacuum freeze drying, fluidized bed drying, etc.

[0016] The fourth technical solution provided by the present invention is a product containing the live strain and / or inactivated strain of Lactobacillus paracasei CCFM1396 described in the first technical solution, or containing the postbiotics described in the third technical solution.

[0017] In certain embodiments, the bacterial concentration of Lactobacillus paracasei CCFM1396 in the product is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0018] Furthermore, the bacterial concentration of Lactobacillus paracasei CCFM1396 in the product is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0019] In certain embodiments, the product is a medicine or a daily chemical product.

[0020] In certain embodiments, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0021] In certain embodiments, the drug carrier comprises microcapsules, microspheres, nanoparticles and liposomes.

[0022] In certain embodiments, the pharmaceutical excipient comprises an excipient and an additive.

[0023] In certain embodiments, the pharmaceutical excipients include one or more of a stabilizer, a flavoring agent, a coloring agent, a filler, a binder, a wetting agent, a disintegrant, and a lubricant.

[0024] In certain embodiments, the dosage form of the drug comprises granules, capsules, tablets, pills or oral liquids.

[0025] In certain embodiments, the daily chemical product is a mouthwash or toothpaste containing the Lactobacillus paracasei CCFM1396.

[0026] The present invention also provides the use of the Lactobacillus paracasei CCFM1396 described in the first technical solution, or the microbial preparation described in the second technical solution, or the postbiotics described in the third technical solution in preparing a product for preventing and / or treating bone resorption.

[0027] The fifth technical solution provided by the present invention is the use of Lactobacillus paracasei CCFM1396 described in the first technical solution, or the microbial preparation described in the second technical solution, or the postbiotic described in the third technical solution in the preparation of a product for preventing and / or treating orthodontic-induced bone resorption.

[0028] In certain embodiments, in the product, the Lactobacillus paracasei CCFM1396 is a live strain and / or an inactivated strain.

[0029] In certain embodiments, in the product, the amount of Lactobacillus paracasei CCFM1396 added is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0030] Furthermore, in the product, the amount of Lactobacillus paracasei CCFM1396 added is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0031] In certain embodiments, the product is a medicine or a daily chemical product.

[0032] In certain embodiments, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0033] In certain embodiments, the drug carrier comprises microcapsules, microspheres, nanoparticles and liposomes.

[0034] In certain embodiments, the pharmaceutical excipient comprises an excipient and an additive.

[0035] In certain embodiments, the pharmaceutical excipients include one or more of a stabilizer, a flavoring agent, a coloring agent, a filler, a binder, a wetting agent, a disintegrant, and a lubricant.

[0036] In certain embodiments, the daily chemical product is a mouthwash or toothpaste containing the above-mentioned Lactobacillus paracasei CCFM1396.

[0037] In some embodiments, the application has at least one of the following effects:

[0038] (1) Significantly reduce the level of alveolar bone resorption in orthodontic individuals;

[0039] (2) reduce the gingival index and probing depth of orthodontic individuals;

[0040] (3) reduce the number of osteoclasts at the apical bifurcation of the alveolar bone of an individual;

[0041] (4) Reduce the expression of RANKL in individual serum.

[0042] (5) Reduce the expression levels of IL-1β, IL-6, TNF-α, and MPO in individual gingival tissues;

[0043] (6) Improve the pathological condition of individual alveolar bone tissue.

[0044] The present invention also provides application of the Lactobacillus paracasei CCFM1396 in preparing a medicine for regulating gingival tissue inflammation function and relieving bone absorption.

[0045] The present invention also provides the use of Lactobacillus paracasei CCFM1396 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of fermented food.

[0046] The sixth technical solution also provided by the present invention is the use of Lactobacillus paracasei CCFM1396 described in the first technical solution, or the microbial preparation described in the second technical solution, or the postbiotic described in the third technical solution in the preparation of a product for improving the expression of osteopontin (OPN) and / or bone morphogenetic protein (BMP) in osteoblasts.

[0047] The seventh technical solution provided by the present invention is a method for increasing the expression of osteopontin and / or bone morphogenetic protein in osteoblast precursor cells in vitro, wherein the method comprises co-culturing the Lactobacillus paracasei CCFM1396 described in the first technical solution, or the microbial preparation described in the second technical solution, or the postbiotics described in the third technical solution with osteoblast precursor cells.

[0048] The beneficial effects of the present invention are as follows:

[0049] 1. The present invention provides a strain of Lactobacillus paracasei CCFM1396, which is isolated from dental plaque of healthy adolescents. The strain has the effects of alleviating orthodontic-induced bone resorption and inhibiting periodontal inflammation, which is specifically embodied in the following aspects: (1) promoting osteoblast activity and increasing osteoblast OPG expression, preventing the binding of RANKL and RANK in alveolar bone tissue, and inhibiting the activity and differentiation of osteoclasts. (2) increasing the gene expression of osteopontin (OPN), bone morphogenetic protein (BMP), and osteoprotegerin (OPG) in MC3T3-E1 cells. (3) significantly reducing the level of alveolar bone resorption in rats induced by orthodontic models. (4) reducing the gingival index and probing depth of rats induced by orthodontic models. (5) reducing the number of osteoclasts at the apical bifurcation of alveolar bones. (6) reducing the expression of RANKL in rat serum. (7) reducing the expression levels of IL-1β, IL-6, TNF-α, and MPO in gingival tissue. (8) improving the pathological condition of alveolar bone tissue. Therefore, this Lactobacillus paracasei CCFM1396 has great application prospects in the preparation of products (such as medicines or daily chemical products, etc.) for preventing and / or treating bone resorption.

[0050] 2. Lactobacillus paracasei is a kind of probiotics and has been included in the "List of Bacteria that Can Be Used in Food" issued by the Ministry of Health. Therefore, the Lactobacillus paracasei CCFM1396 screened by the present invention will not cause any side effects to the human body, and when used in products (such as medicines or daily chemical products, etc.) for preventing and / or treating orthodontically induced bone resorption, it has higher safety.

[0051] Biomaterial Deposit

[0052] A strain of Lacticaseibacillus paracasei CCFM1396, taxonomically named Lacticaseibacillus paracasei, was deposited in the Guangdong Provincial Microbiological Culture Collection on June 14, 2024, with the deposit number GDMCC No.64760, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 :The effect of different bacterial fermentation supernatants and lysates on osteoprotegerin (OPG) in MC3T3-E1 cells.

[0054] Figure 2 :The effect of Lactobacillus paracasei on the gene expression of bone morphogenetic protein (BMP), osteoprotegerin (OPG) and osteopontin (OPN) in osteoblasts.

[0055] Figure 3 :Effects of Lactobacillus paracasei CCFM1396 on body weight and OTM in rats with orthodontic-induced bone resorption.

[0056] Figure 4 :Effects of Lactobacillus paracasei CCFM1396 on gingival index and probing depth in rats with orthodontic-induced bone resorption.

[0057] Figure 5 :The effect of Lactobacillus paracasei CCFM1396 on the expression of inflammatory factors in gingival tissue of rats with orthodontic-induced bone resorption.

[0058] Figure 6 :The effect of Lactobacillus paracasei CCFM1396 on the expression of OPG and RANKL in the serum of rats with orthodontic-induced bone resorption.

[0059] Figure 7 :Effects of Lactobacillus paracasei CCFM1396 on bone microstructure in rats with orthodontic-induced bone resorption.

[0060] Figure 8 :HE staining of Lactobacillus paracasei CCFM1396 on orthodontic-induced bone resorption in rats.

[0061] Fig. 9 :Trap staining of Lactobacillus paracasei CCFM1396 on orthodontic-induced bone resorption in rats.

[0062] In the figure, * indicates significant difference, where *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. DETAILED DESCRIPTION

[0063] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0064] The culture medium involved in the following examples is as follows:

[0065] MRS medium in Examples 1, 2 and 3: 5.0 g / L yeast powder, 10.0 g / L beef extract, 10.0 g / L peptone, 20.0 g / L glucose, 2.0 g / L anhydrous sodium acetate, 2.0 g / L diammonium hydrogen citrate, 2.6 g / L dipotassium hydrogen phosphate, 0.25 g / L manganese sulfate monohydrate, 0.5 g / L magnesium sulfate heptahydrate, 1 mL / L Tween-80, pH 6.2-6.4.

[0066] The complete cell culture medium in Examples 1 and 2 is: 92% (v / v) α-MEM culture medium + 7% (v / v) fetal bovine serum + 1% (v / v) 100× mixed solution of penicillin and streptomycin (the content of penicillin in the mixed solution is 10000 U / mL, and the concentration of streptomycin is 10 mg / mL).

[0067] The osteogenic induction medium in Examples 1 and 2 includes complete cell culture medium + sodium β-glycerophosphate (10 mM / L), ascorbic acid (50 μg / mL), and dexamethasone (100 nM / L).

[0068] The BHI blood plate culture medium in Example 3: Qingdao Haibo BHI commercial culture medium 37.5 g / L, agar powder 20 g / L, sterile defibrinated sheep blood 100 mL / L, 10% hemin chloride-vitamin K 1 mL / L.

[0069] The strains, cells and animals involved in the following examples are as follows:

[0070] In Examples 2 and 3, Lactobacillus paracasei ATCC334 and Prevotella intermedia ATCC25611 were purchased from the American Type Culture Collection.

[0071] The rat osteoblast precursor cells MC3T3-E1 in Examples 1 and 2 were deposited in the Biotechnology Center of the School of Food Science and Engineering, Jiangnan University.

[0072] In Example 3, 24 SPF-grade WISTAR male rats, 7 weeks old, weighing 235-255 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001).

[0073] The preparation of the experimental samples involved in the following examples is as follows:

[0074] In Examples 1 and 2, the lactobacillus fermentation broth was prepared by inoculating Lactobacillus paracasei CCFM1396 and ATCC334 in MRS liquid medium at a 2% (v / v) inoculum amount, culturing at 37°C for 24 h, and adjusting the bacterial concentration to 1×10 9 CFU / mL.

[0075] Preparation of supernatant: The concentration of 1×10 9 The CFU / mL Lactobacillus live bacteria fermentation liquid was centrifuged at 12000 rpm for 10 min, and the supernatant was collected, filtered through a 0.22 μm filter membrane, and stored at -20°C.

[0076] Lysis buffer preparation: the concentration is 1×10 9 The Lactobacillus fermentation liquid with CFU / mL was homogenized in a high-pressure homogenizer (800-1200 MPa) for 10 times, filtered with a 0.22 μm filter membrane, and the filtrate, i.e., the bacterial lysate, was collected and stored at -20°C.

[0077] In Examples 2, 3, and 4, the live bacterial suspension of Lactobacillus paracasei was prepared by inoculating Lactobacillus paracasei CCFM1396 and ATCC334 in MRS liquid medium at a 2% (v / v) inoculum size, culturing at 37°C for 24 h, collecting the cells by centrifugation at 8500 g for 20 min, washing the cells twice with sterile physiological saline, resuspending the cells with 10% skim milk solution, and lyophilizing after sufficient mixing. Before the animal experiment, the freeze-dried Lactobacillus powder was counted and redissolved with sterile PBS to a bacterial suspension concentration of 1×10 9 CFU / mL.

[0078] Preparation of dead Lactobacillus paracasei suspension: The preparation method and concentration of the dead bacteria group suspension were the same as those of the live bacteria, except that it was inactivated at 105°C for 10 minutes, and the inactivation effect was checked by plate coating. After heat treatment, the bacterial suspension was centrifuged at 8500g for 20 minutes, the bacteria were washed twice with sterile saline, and then resuspended with 10% skim milk solution, mixed thoroughly, and freeze-dried. Before intervention, it was resuspended with PBS to a concentration of 1×10 9 CFU / mL.

[0079] Preparation of live Prevotella intermedia ATCC 25611 suspension: Prevotella intermedia ATCC 25611 was cultured in BHI medium at 37°C for 24 h, and the cells were collected by centrifugation and resuspended in PBS to a concentration of 1 × 10 9 CFU / mL, which was used for subsequent experiments.

[0080] Example 1 Isolation and identification of Lactobacillus paracasei CCFM1396

[0081] Collect oral swab samples from healthy adolescents and place them in EP tubes containing 1 mL of sterile saline. Pipette 0.2 mL into 1.8 mL of sterile saline to obtain 10 -1 Dilution, then draw 0.5mL 10 -1 Dilute in 4.5 mL of saline to obtain 10 -2 Dilution, follow this procedure to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6 Gradient dilution. Take 10 -4 , 10 -5 , 10 -6 Place 1 mL of each dilution in a plate, pour into MRS solid culture medium, mix gently, and after the culture medium solidifies, invert and culture at 37°C for 48 h.

[0082] Select colonies of different morphologies for streaking purification on MRS plates, pick and inoculate purified single colonies into 10 mL of liquid culture medium, and culture at 37 ° C for 48 h. Take 1.5 mL of cultured bacterial solution, centrifuge at 6000r / min for 3 min, discard the supernatant, add 1.5 mL of sterile water to wash 3 times, resuspend in 1.5 mL of sterile water, and use it as a template for bacterial identification. Set up a PCR system with a volume of 20 μL, add 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), 10 μL of 2×Taq Mixture, 0.5 μL of bacterial suspension, and 8.5 μL of double distilled water. Primer information is shown in Table 1.

[0083] Table 1: Primer information

[0084]

[0085] The amplified 16S rDNA sequence was sequenced as shown in SEQ ID NO.1.

[0086] The obtained 16S rDNA sequence was confirmed by NCBI's BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST). The closer the Query Cover and Identification are to 100%, the more feasible it is. If there are multiple species in the comparison results, the Complete genome species will be prioritized while taking into account the numerical value. According to the Blast results, the bacteria was identified as Lactobacillus paracasei. Then, 1.5 mL of the identified bacteria was taken into a 2 mL bacteria storage tube, centrifuged at 6000 r / min for 3 minutes, the supernatant was removed in a clean bench, 1 mL of 30% sterile glycerol was added, and after being thoroughly mixed with a vortex oscillator, it was stored in a -80°C refrigerator.

[0087] Example 2 Ability of Lactobacillus paracasei to promote osteoblast osteoprotegerin (OPG) expression in vitro

[0088] OPG is a glycoprotein secreted by osteoblasts and belongs to the tumor necrosis factor receptor superfamily. It can compete with RANKL (receptor activator of nuclear factor κB ligand) for binding to RANK (receptor activator of nuclear factor κB), thereby inhibiting the differentiation and activity of osteoclasts.

[0089] MC3T3-E1 cells were removed from liquid nitrogen and thawed quickly in a 37°C water bath. The thawed cell suspension was added to a centrifuge tube containing 5 times the volume of complete medium. After centrifugation, the supernatant was discarded and the cells were resuspended in 10 mL of complete medium. The cells were cultured at 37°C and 5% CO2, and the medium was replaced every 48 hours. After counting the cells, 1×10 4 Cells were seeded on a 6-well plate at a density of 10 cells / well. 2 mL of osteogenic induction medium containing 5% (v / v) bacterial supernatant or lysate was added to each well, and cell morphology was observed regularly. MC3T3-E1 cells that differentiated into osteoblasts usually became flatter. After 21 days of culture, the cell culture supernatant of each group was collected, and the expression of OPG was determined using the Senbega OPG Elisa Kit. The grouping information of the cell experiment is shown in Table 2.

[0090] Table 2: Group information table

[0091]

[0092] The expression of OPG in MC3T3-E1 cells after intervention with Lactobacillus paracasei Figure 1As shown. The OPG concentration in the cell culture supernatant of the control group was 117.78 ng / L, and the OPG content in the supernatant group and lysate group of Lactobacillus paracasei CCFM1396 increased significantly, reaching 322.75 ng / L and 339.29 ng / L, respectively (p < 0.0001). After the intervention of Lactobacillus paracasei ATCC334, the OPG concentration in the cell culture supernatant decreased.

[0093] In summary, Lactobacillus paracasei CCFM1396 can increase the expression of OPG in osteoblasts in vitro.

[0094] Example 3 Ability of Lactobacillus paracasei to promote the expression of bone morphogenetic protein (BMP), osteoprotegerin (OPG), and osteopontin (OPN) genes in osteoblasts in vitro

[0095] BMPs stimulate the differentiation of osteoblast precursor cells into mature osteoblasts, while inhibiting their differentiation into osteoclasts. BMPs also bind to specific receptors on the surface of bone-forming cells, initiating signaling cascades that lead to the activation of genes required for bone matrix production. BMPs regulate bone homeostasis through the Wnt / β-catenin and RANKL / OPG pathways.

[0096] The MC3T3-E1 cell culture and induction test methods were the same as in Example 2. 4 After 21 days of differentiation induction with an initial concentration of 100 μg / mL, cellular RNA was extracted according to the detailed instructions provided by the Novozyme RNA extraction kit, and the operation was performed step by step. All experimental steps were performed in an RNase-free environment to avoid RNA degradation. The extracted RNA was measured for purity (A260 / A280 ratio) and concentration using a NanoDrop spectrophotometer.

[0097] After intervention with Lactobacillus paracasei CCFM1396, the expression of BMP, OPG, and OPN genes in MC3T3-E1 cells was as follows Figure 2 After treatment with the supernatant of Lactobacillus paracasei CCFM1396, the Fold Change values ​​of BMP, OPG and OPN genes were 1.65, 1.45 and 2.08, respectively, which were significantly higher than those of the control group; while treatment with the lysate of Lactobacillus paracasei CCFM1396 significantly increased the expression of BMP and OPN genes.

[0098] The above results indicate that after treatment with Lactobacillus paracasei CCFM1396, the gene expression levels of BMP, OPG, and OPN in the induced MC3T3-E1 cells increased, thereby improving the activity of osteoblasts.

[0099] Example 4 Application of Lactobacillus paracasei CCFM1396 in alleviating orthodontic-induced bone resorption in rats

[0100] Twenty-four SPF Wistar rats, male, 7 weeks old, weighing 235-255 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001). The 24 rats were evenly divided into 4 groups according to their body weight: control group (6 rats), model group (6 rats), live Lactobacillus paracasei group (6 rats), and dead Lactobacillus paracasei group (6 rats).

[0101] The experimental groups are shown in Table 3.

[0102] Table 3 Animal experiment groups

[0103]

[0104] The rats were randomly divided into 4 groups according to their body weight, with 6 rats in each group. The rats in all groups were kept normally throughout the experiment.

[0105] Except for the control group, the other groups underwent orthodontic modeling on day 0 (the specific operation was: a nickel-titanium spring was used to apply a 50g traction force between the first molar and the central incisor of the rat. This step required first using a grinding tool to grind a groove perpendicular to the midline of the dental arch in the maxillary central incisor for fixing the stainless steel ligature. The maxillary central incisor and the first molar both needed to be fixed with fluid resin to tie the silk thread, and to ensure that the silk thread fracture did not damage the epithelial barrier. Except for the left first molar, the occlusal surfaces of the remaining upper molars were fixed with fluid resin to make the occlusal surfaces convex to eliminate the occlusal interference of the maxillary first molar in orthodontic movement), and were required to be infected with Prevotella intermedia for a total of 12 days (days 1-6 and 8-13) (the specific operation method of infection was: 1×10 9 1 mL of Prevotella intermedia suspension with a CFU / mL was slowly irrigated into the rat's oral cavity and placed in the cage after leaving it for 1 minute).

[0106] For the live and dead bacteria groups of Lactobacillus paracasei CCFM1396, 6 hours after infection with Prevotella intermedia, the rats’ oral cavity was irrigated with 1 mL of saliva combined with live and dead bacterial suspensions of Lactobacillus CCFM1398, respectively. The concentration of the bacterial suspension was 1 × 10 9 CFU / mL.

[0107] The gingival index and probing depth of the periodontal tissue of all rats were measured on day 0, day 7 and day 14, and the rat saliva was collected with a sterile swab and stored in a 2 mL centrifuge tube containing 1 mL of 30% glycerol. The weight of the rats, the distance from the first molar to the central incisor (OTM), the gingival index and the probing depth were also recorded.

[0108] The experimental period was 13 days (days 0 to 13). All rats were killed on the 14th day, and serum, gingival tissue and alveolar bone tissue were collected for subsequent experimental histopathology and alveolar bone microstructure analysis, and the levels of soluble interleukins (IL-6, IL-1β) and tumor necrosis factor (TNF-α) in gingival tissue, myeloperoxidase (MPO), osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) in serum were detected.

[0109] Determination method: On the 14th day, before the rats were killed, 10 mL of blood was taken from the abdominal aorta of the rats. After standing for 30 minutes, the blood was centrifuged at 3000 r / min for 15 minutes at 4°C, and the supernatant was taken as the blood sample. After the rats were killed, the lingual and buccal gingival tissues of the first molars were peeled off. 20 mg of gingival tissue was weighed and mixed with 180 μL PBS buffer, placed in a 1.5 mL centrifuge tube, 3 grinding zirconium beads were added, and ground in a high-throughput tissue grinder. The tissue homogenate was then centrifuged at 12000g at 4°C for 15 minutes, the supernatant was collected, and stored at -20°C.

[0110] Then, the levels of RANKL and OPG in serum and TNF-α, IL-6, and MPO in gingival tissue were determined according to the kit instructions.

[0111] Bone microstructure analysis: After the rats were killed, the right maxillary tissue of the rats was peeled off and fixed in 4% paraformaldehyde solution for 48 hours. The fixed maxilla of the rats was washed with PBS and dried. The maxillary samples were scanned using a high-resolution Micro-CT imaging system. The specific parameters were: voltage, 100 kV; current, 88 μA; field of view, 18 μm, imaging rotation 360°; acquisition time, 4 min. The ratio of the bone volume (BV) at the modeling site to the total alveolar bone volume (TV) (BV / TV) was measured and calculated.

[0112] Histopathological observation: Alveolar bone tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sliced ​​into 5 μm thick slices, and stained with hematoxylin and eosin (HE) and Trap. Alveolar bone tissue slice samples were observed under a pathological slice scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary) at a magnification of 20 times.

[0113] Experimental results:

[0114] (1) Changes in rat body weight and tooth movement distance (OTM)

[0115] like Figure 3As shown in A, rats showed significant weight loss after orthodontic modeling (p<0.0001). After intervention with live and dead bacteria of Lactobacillus paracasei CCFM1396 for 13 days, the weight of rats recovered compared with the model group. The recovery effect of dead bacteria of Lactobacillus paracasei CCFM1396 was more significant, and the result was statistically significant (p<0.0001). There was no significant difference in OTM between the groups ( Figure 3 B), indicating that Lactobacillus paracasei does not cause excessive tooth movement and remodeling.

[0116] (2) Changes in periodontal tissue gingival index and probing depth

[0117] Changes in the gingival index can reflect changes in the degree of gingival inflammation. A decrease in the gingival index means improvements in symptoms such as gingival redness, swelling, and bleeding. Changes in probing depth are usually associated with improvements in periodontal tissue. A shallow probing depth suggests healthy periodontal tissue and a lower risk of periodontal disease.

[0118] The changes of gingival index and periodontal probing depth are as follows Figure 4 As shown. In the dead bacteria group of Lactobacillus paracasei CCFM1396, the gingival index decreased from 3 to 1.6 on the 7th day, and the probing depth decreased from 1.33mm to 0.33mm on the 7th day, which was significantly lower than that of the model group, and remained at a low level on the 14th day. In the live bacteria group, the gingival index decreased from 3 to 1.2 on the 14th day, and the probing depth decreased from 1.67mm to 0.6mm, which was significantly lower than that of the model group. This shows that after oral irrigation with saliva combined with Lactobacillus CCFM1398, the level of gingival inflammation in rats was reduced, the periodontal bleeding problem was alleviated, and the depth of periodontal pockets also decreased significantly.

[0119] In conclusion, Lactobacillus paracasei CCFM1396 can reduce the incidence of gingival tissue bleeding and gingival inflammation, and reduce the formation of periodontal pockets in orthodontic model rats.

[0120] (3) Secretion of inflammatory factors IL-1β, IL-6, TNF-α, MMP-8, and MPO in rat gingival tissue and expression of OPG and RANKL in serum

[0121] The massive secretion of IL-1β, IL-6, TNF-α, MMP-8, and MPO causes periodontal inflammation and damages the periodontal tissue itself, accelerates the entry of pathogenic bacteria and toxins, and promotes excessive bone resorption by activating multiple downstream signaling pathways including NF-κB. OPG is a soluble protein that belongs to the tumor necrosis factor receptor superfamily. RANKL is a transmembrane protein mainly produced by osteoblasts and activated T cells. RANKL can bind to the RANK receptor on osteoclast precursor cells, promote the differentiation and maturation of osteoclasts, and increase bone resorption. OPG can bind to RANKL, preventing RANKL from activating its receptor RANK, thereby inhibiting the differentiation and activity of osteoclasts and reducing bone resorption.

[0122] The secretion of inflammatory factors IL-1β, IL-6, TNF-α, MMP-8, and MPO in rat gingival tissue Figure 5 After orthodontic and pathogenic bacteria stimulation, the levels of IL-1β, IL-6, TNF-α, MMP-8, and MPO in the model group were significantly increased compared with the control group; after intervention with live Lactobacillus paracasei CCFM1396, the levels of IL-1β, IL-6, TNF-α, and MPO were significantly decreased compared with the control group; after intervention with dead Lactobacillus paracasei CCFM1396, the levels of IL-6 and MPO were significantly decreased compared with the control group. After intervention with live Lactobacillus paracasei CCFM1396, the level of IL-1β decreased from 25.30 ng / L to 14.57 ng / L (p<0.05), the level of IL-6 decreased from 660.17 ng / L to 502.23 ng / L (p<0.001), the level of TNF-α decreased from 324.35 ng / L to 173.45 ng / L (p<0.01), and the level of MPO decreased from 128.94 ng / L to 91.42 ng / L (p<0.0001). After intervention with dead Lactobacillus paracasei CCFM1396, the level of IL-6 decreased from 660.17 ng / L to 483.90 ng / L (p<0.001), and the level of MPO decreased from 128.94 ng / L to 105.32 ng / L (p<0.001).

[0123] The expression of OPG and RANKL in rat serum Figure 6 After orthodontic and pathogenic bacteria stimulation, the serum OPG level and RANKL level in the model group increased significantly, while after intervention with dead bacteria of Lactobacillus paracasei CCFM1396, the OPG level decreased significantly from 1130.33 ng / L to 939.92 ng / L (p<0.05); after intervention with live bacteria of Lactobacillus paracasei CCFM1396, the RANKL level decreased significantly from 301.57 pmol / L to 229.55 pmol / L (p<0.05).

[0124] In summary, Lactobacillus paracasei CCFM1396 can reduce periodontal tissue inflammation and alleviate bone resorption by reducing the secretion of inflammatory factors, MMP-8 and MPO in gingival tissue. After the orthodontic model was established, the level of bone resorption in rats was significantly aggravated. The use of Lactobacillus paracasei CCFM1396 can slow down the bone remodeling process, reduce the ratio of RANKL / OPG, and alleviate orthodontic-induced bone resorption.

[0125] (4) Microstructure of rat alveolar bone tissue

[0126] MicroCT technology can be used to visually observe the root resorption of the first molar and calculate the ratio of the alveolar bone volume (BV) to the total volume (TV). This ratio is related to the risk of alveolar bone resorption. The lower the ratio, the higher the risk of bone resorption.

[0127] The microstructure of rat alveolar bone tissue Figure 7 As shown. The BV / TV ratio at the root apex of the first molar in the control group was 43.38%, and the BV / TV ratio at the root apex of the first molar in the model group was significantly reduced to 24.81% (p < 0.0001). After intervention with Lactobacillus paracasei CCFM1396, the BV / TV ratio in the live bacteria group increased to 40.59%, close to the control group, and significantly increased compared with the model group (p < 0.001). The BV / TV ratio in the dead bacteria group was 32.43%, which was significantly increased compared with the model group (p < 0.05).

[0128] In conclusion, Lactobacillus paracasei CCFM1396 can increase the BV / TV ratio at the root apex of the first molar in the alveolar bone, thereby alleviating orthodontic-induced bone resorption.

[0129] (5) Histopathological analysis of rat alveolar bone

[0130] HE staining and Trap staining of rat alveolar bone tissue can effectively evaluate the integrity, inflammation, and number and location of osteoclasts per unit area of ​​the alveolar bone tissue in each group of rats.

[0131] HE staining can determine the acuteness of inflammation and also observe changes in tissue morphology. Figure 8As shown in the figure, the alveolar periodontal tissue structure of rats in the control group was intact, and the maxillary bone sections of rats in the blank group showed a complete periodontal tissue structure after HE staining. The gingiva was tightly attached to the tooth surface, no periodontal pocket was formed, and no inflammatory cells were observed to aggregate. The structure of the periodontal tissue and alveolar bone was uniform, and no abnormalities were observed. The maxillary bone sections of rats in the model group showed that the integrity of the periodontal tissue was impaired. Gaps appeared between the gingiva and the tooth surface, forming periodontal pockets. A large number of inflammatory cells were observed to aggregate in the apical area, the gap between the first molar and the second molar was enlarged, and the thickness of the periodontal apical tissue was uneven, indicating that the orthodontic model triggered an inflammatory response in the periodontal tissue and bone resorption.

[0132] The HE staining results of the live and dead bacteria groups of Lactobacillus paracasei CCFM1396 showed that the tissue inflammatory infiltration was alleviated after intervention, the inflammatory cells no longer gathered at the alveolar bone root apex, and the damage to the periodontal tissue was also alleviated. This indicates that Lactobacillus paracasei CCFM1396 can significantly alleviate the symptoms of alveolar bone resorption in orthodontic model rats and inhibit the occurrence of inflammation.

[0133] Trap staining can effectively evaluate the integrity of alveolar bone tissue, inflammation, and the number and location of osteoclasts per unit area in rats of each group. Fig. 9 As shown in the figure, the number of osteoclasts in the model group increased significantly compared with the control group (p < 0.01), and was mainly distributed at the edge of the alveolar root apex bifurcation. The live and dead bacteria staining results of Lactobacillus paracasei 1396 showed that Lactobacillus paracasei 1396 could alleviate orthodontic-induced excessive bone resorption, and osteoclasts no longer gathered at the alveolar root apex bifurcation. The number of osteoclasts also decreased from 10 osteoclasts to 7.2 and 7 at 20 times the field of view, respectively.

[0134] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Lactobacillus paracasei CCFM1396, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with the deposit number GDMCC No.64760.

2. the microbial preparation of Lactobacillus paracasei CCFM1396 described in claim 1.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation, the bacterial concentration of Lactobacillus paracasei CCFM1396 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A postbiotic, characterized in that: The postbiotics include one or more of the fermentation supernatant, bacterial lysate and fermentation broth of the Lactobacillus paracasei CCFM1396 according to claim 1.

5. A product, characterized in that: Contains the live strain and / or inactivated strain of Lactobacillus paracasei CCFM1396 according to claim 1, or contains the postbiotics according to claim 4.

6. The product according to claim 5, characterized in that The products are medicines or daily chemical products.

7. Use of the Lactobacillus paracasei CCFM1396 according to claim 1, or the microbial preparation according to claim 2 or 3, or the postbiotic according to claim 4 in the preparation of a medicine for preventing and / or treating orthodontic-induced bone resorption.

8. The use according to claim 7, characterized in that: The application has at least one of the following functions: (1) Significantly reduce the level of alveolar bone resorption in orthodontic individuals; (2) reduce the gingival index and probing depth of orthodontic individuals; (3) reduce the number of osteoclasts at the apical bifurcation of the alveolar bone of an individual; (4) Reduce the expression of RANKL in individual serum. (5) Reduce the expression levels of IL-1β, IL-6, TNF-α, and MPO in individual gingival tissues; (6) Improve the pathological condition of individual alveolar bone tissue.

9. Use of the Lactobacillus paracasei CCFM1396 according to claim 1, or the microbial preparation according to claim 2 or 3, or the postbiotic according to claim 4 in the preparation of a product for increasing the expression of osteopontin and / or bone morphogenetic protein in osteoblasts.

10. A method for increasing the expression of osteopontin and / or bone morphogenetic protein in osteoblast precursor cells in vitro, characterized in that: The method comprises co-culturing the Lactobacillus paracasei CCFM1396 described in claim 1, or the microbial preparation described in claim 2 or 3, or the postbiotic described in claim 4 with osteoblast precursor cells.

Images