CN116574659A - Bifidobacterium longum subspecies infantis capable of relieving rheumatoid arthritis and application thereof - Google Patents
Bifidobacterium longum subspecies infantis capable of relieving rheumatoid arthritis and application thereof Download PDFInfo
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- CN116574659A CN116574659A CN202310699297.4A CN202310699297A CN116574659A CN 116574659 A CN116574659 A CN 116574659A CN 202310699297 A CN202310699297 A CN 202310699297A CN 116574659 A CN116574659 A CN 116574659A
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Abstract
The invention discloses a bifidobacterium longum subspecies infancy capable of relieving rheumatoid arthritis and application thereof, belonging to the technical field of microorganisms. The invention provides a bifidobacterium longum subspecies infantis CCFM1269, and the bifidobacterium longum subspecies infantis CCFM1269 is used for preparing medicines for relieving rheumatoid arthritis, and the specific embodiments are as follows: significantly alleviating the extent of joint swelling in a mammal suffering from rheumatoid arthritis; significantly reducing the levels of pro-inflammatory cytokines IL-6, TNF-alpha, IL-22, and IL-23 in serum of a mammal having rheumatoid arthritis; significantly regulating the content of type II collagen specific antibodies in the serum of a mammal suffering from rheumatoid arthritis; significantly regulating serum osteoprotegerin and osteocalcin content of mammal with rheumatoid arthritis, and improving intestinal flora of mammal with rheumatoid arthritis at "genus" level of biological taxonomy.
Description
Technical Field
The invention relates to a bifidobacterium longum subspecies infancy capable of relieving rheumatoid arthritis and application thereof, belonging to the technical field of microorganisms.
Background
Rheumatoid arthritis (Rheumatoid arthritis, RA) is a chronic, long-term autoimmune disorder. The global epidemiological investigation shows that the disease incidence peak age of rheumatoid arthritis is 30-50 years old, the disease incidence rate is higher in developed areas than in underdeveloped areas, and women are higher than men, and the rheumatoid arthritis is closely related to daily diet and living environment conditions and has certain hereditary property. Although the pathogenesis of rheumatoid arthritis is currently not completely defined, the imbalance of the immune system is an important factor leading to the occurrence and progression of the disease. Early stage of the patient shows the characteristics of morning stiffness, rigidity and the like of the joint, along with the development of diseases, swelling deformity appears at the pathological change part, the joint function is lost, and the risk of disability exists, which is also called as 'dead cancer'. Current treatment for rheumatoid arthritis mainly includes nonsteroidal anti-inflammatory drugs, glucocorticoids, antirheumatic drugs and biological agents. Single drug therapy is rarely used in clinical treatment, and multiple drugs are generally combined to slow down the condition. These drugs often have adverse reactions or side effects, such as non-steroidal anti-inflammatory drugs that pose gastrointestinal and cardiovascular risks, and glucocorticoids have significant anti-inflammatory and analgesic effects but cannot be used for a long period of time due to numerous side effects, and antirheumatic drugs have slow onset of action and biological agents have good therapeutic effects but are expensive. Under the background, it is necessary to develop a means which is easy to obtain and has no adverse reaction to assist the drug treatment and enhance the treatment effect.
Rheumatoid arthritis, a systemic immune disease, is closely related to the abnormal response of the immune system. The intestinal tract is one of the important immune organs of the body, in which a large number of microorganisms grow, reproduce and produce various metabolites, which together with their metabolites establish a normal active immune system. Numerous studies have found that the intestinal flora composition of rheumatoid arthritis patients is significantly different from that of healthy humans, e.g. the Prevotella copri, haemophilus of the Propionibacterium would be significantly higher than that of normal humans, whereas bifidobacteria would become lower (see in particular, pianta, A., et al, evidence of the Immune Relevance of Prevotella copri, a Gut Microbe, in Patients With Rheumatoid archlitis. Archlitis Rheumatoid, 2017.69 (5): p.964-975.). In some clinical double-blind experiments, lactobacillus rhamnosus LGG can reduce inflammatory indexes of rheumatoid arthritis patients and has a certain effect of relieving diseases. Therefore, oral probiotics are expected to be used as an auxiliary intervention means for treating rheumatoid arthritis to improve the disease.
Therefore, a medicine or treatment mode using probiotics as a main component needs to be found, which can play a fundamental role in preventing and/or assisting in treating rheumatoid arthritis (Rheumatoid Arthritis, RA), and meanwhile, does not bring any potential safety hazard to patients with rheumatoid arthritis (Rheumatoid Arthritis, RA), and has low production cost.
Disclosure of Invention
The invention provides a bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Inffantis) CCFM1269, wherein the bifidobacterium subspecies infantis (Bifidobacterium longum subsp. Inffantis) CCFM1269 is preserved in the microorganism strain preservation center of Guangdong province at 9 months 26 of 2022, and the preservation number is GDMCC No:62839, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.
The invention also provides food, medicine or health care products containing the bifidobacterium longum subspecies infantis CCFM1269.
In one embodiment, the viable count of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Inffantis) CCFM1269 in the food, pharmaceutical or health care product is not less than 1×10 9 CFU/mL or 1X 10 9 CFU/g。
The invention also provides application of the bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 in preparing medicines for preventing and/or treating rheumatoid arthritis.
In one embodiment, the preventing and/or treating rheumatoid arthritis comprises at least one of the following effects: reducing joint thickness, reducing arthritis specific antibody content, increasing serum osteoprotegerin content, and increasing serum osteocalcin content.
In one embodiment, the medicament contains the bifidobacterium longum subspecies infantis CCFM1269, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment, the food or health product comprises a dairy product, a soy product, a meat product or a fruit and vegetable product produced using a starter culture comprising the bifidobacterium longum subspecies infantis CCFM1269.
In one embodiment, the method of preparing the starter is: inoculating the bifidobacterium longum subspecies infantis CCFM1269 into a culture medium according to the inoculation amount accounting for 2-4% of the total mass of the culture medium, and culturing for 24-48 hours at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; the thalli are resuspended by normal saline to obtain the ferment.
In one embodiment, the medium is MRS medium.
The invention also provides application of the bifidobacterium longum subspecies infantis CCFM1269 in preparation of medicines or health products for improving the content of osteoprotegerin and osteocalcin.
In one embodiment, the use is to use bifidobacterium longum subspecies infantis CCFM1269 as the major component of a osteoprotegerin, osteocalcin supplement.
The invention also provides application of the bifidobacterium longum subspecies infantis CCFM1269 in preparation of medicines or health-care products for regulating intestinal flora.
In one embodiment, the modulating the intestinal flora includes, but is not limited to, increasing the relative abundance of Lactobacillus and/or Ruminococcus 1 (Ruminococcus 1).
In one embodiment, the modulating the intestinal flora comprises down-regulating an abnormal increase in the relative abundance of fresnel bacteria (Fournierella).
The beneficial effects are that:
1. the invention screens out a bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Inffantis) CCFM1269, and the bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Inffantis) CCFM1269 has the effect of relieving rheumatoid arthritis, and is specifically expressed in that:
(1) Significantly reducing joint thickness, clinical score, and morbidity in rheumatoid arthritis rats;
(2) The content of pro-inflammatory cytokines TNF alpha, IL-6, IL-22 and IL-23 in the serum of the rat with rheumatoid arthritis is obviously reduced;
(3) The content of arthritis specific antibodies in the serum of the rheumatoid arthritis rats is obviously reduced;
(4) Regulating the content of osteoprotegerin and osteocalcin serum of rats with rheumatoid arthritis;
(5) The intestinal flora of rheumatoid arthritis rats is improved at the genus level.
Therefore, the bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 has great application prospect in preparing products (such as food, medicine and the like) for preventing and/or treating the rheumatoid arthritis.
2. Bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) is one of the probiotics and has been incorporated into the list of strains available for food under the health control at present, so that the screened bifidobacterium longum subsp infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 according to the invention does not bring any potential safety hazard to patients suffering from rheumatoid arthritis (Rheumatoid Arthritis, RA).
3. The culture process of Bifidobacterium longum subsp.infantis (Bifidobacterium longum subsp. Infantis) only needs the control of culture medium and some culture conditions, has relatively low cost, and does not bring too much economic burden to patients with rheumatoid arthritis (Rheumatoid Arthritis, RA) compared with the biological preparation with high cost.
Preservation of biological materials
A bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Inffantis) CCFM1269, taxonomically designated Bifidobacterium longum subsp. Inffantis, was deposited at the microorganism strain collection, canon, 9 and 26, under the accession number GDMCC No:62839, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.
Drawings
Fig. 1: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on joint thickness in rheumatoid arthritis rats.
Fig. 2: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on clinical scores in rheumatoid arthritis rats.
Fig. 3: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on body weight of rheumatoid arthritis rats.
Fig. 4: influence of Bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on joint pathology in rheumatoid arthritis rats.
Fig. 5: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on IL-6 and TNF-alpha levels in serum of rheumatoid arthritis rats.
Fig. 6: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on IL-22 and IL-23 levels in serum of rheumatoid arthritis rats.
Fig. 7: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on CII-total IgG in serum of rheumatoid arthritis rats.
Fig. 8: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on CII-IgG1 in serum of rheumatoid arthritis rats.
Fig. 9: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on CII-IgG2A in serum of rheumatoid arthritis rats.
Fig. 10: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on CII-IgG2B in serum of rheumatoid arthritis rats.
Fig. 11: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on osteoprotegerin in serum of rheumatoid arthritis rats.
Fig. 12: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on osteocalcin in serum of rheumatoid arthritis rats.
Fig. 13: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on the abundance of Lactobacillus in rheumatoid arthritis rat faeces.
Fig. 14: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on the abundance of Ruminococcus 1 (Ruminococcus 1) in rheumatoid arthritis rat faeces.
Fig. 15: effect of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 on the abundance of fresnel bacteria (Fournierella) in rheumatoid arthritis rat faeces.
Detailed Description
The invention is further illustrated below in conjunction with specific embodiments and figures.
Female Wistar rats referred to in the following examples were purchased from the company vitelliwa of Zhejiang; bovine type II collagen solutions and freund's incomplete adjuvant referred to in the examples below were purchased from Chondrex corporation; ELISA kits for detection of IL-6, TNF- α, IL-22, IL-23, total type II collagen-specific antibody IgG, type II collagen-specific antibody IgG1, type II collagen-specific antibody IgG2A, II-type collagen-specific antibody IgG2B, osteocalcin, and osteoprotegerin, as described in the examples below, were purchased from Sen Bei Ga.
The following examples relate to the following media:
MRS solid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder, 2g/L, K2PO 4.3H2O 2.6g/L, mgSO 4.7H2O 0.1g/L, mnSO 4.0.05 g/L of Tween 80 1mL/L and 15g/L of agar.
MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder, 2g/L, K2PO 4.3H2O 2.6g/L, mgSO 4.7H2O 0.1g/L, mnSO 4.0.05 g/L of Tween 80.
Example 1: screening and strain identification of bifidobacterium longum subspecies infantis
1. Screening
Taking 0.5mL of sample stored in 30% (v/v) glycerol from breast milk sample of tin-free city of Jiangsu province, adding into 10mL centrifuge tube containing 4.5mL of physiological saline under aseptic environment to obtain 10 -1 And (3) diluting the solution, repeating the diluting steps to sequentially obtain 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 A dilution liquid; respectively sucking 100 mu L of gradient diluents with different gradients, coating the gradient diluents on an MRS solid culture medium, and culturing for 72 hours at 37 ℃ to obtain a diluted coating plate; picking typical colonies on a dilution coating plate, respectively scribing on an MRS solid culture medium, and culturing at 37 ℃ for 48 hours to obtain purified colonies; the purified colony is picked and inoculated into MRS liquid culture medium, and cultured for 48 hours at 37 ℃ to obtain the strain BJSWXB6MNIM1, named CCFM1269.
2. Authentication
The genome of CCFM1269 was extracted and 16S amplification was performed, 16S rDNA amplification conditions: 95 ℃ for 5min;35 cycles (95 ℃ 30s,55 ℃ 30s,72 ℃ 2 min); and at 72℃for 10min. Amplification primers: 27F: (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R: (5'-TACGGCTACCTTGTTACGACTT-3') purification of amplified products and sequence alignment procedure the procedure described in reference Exploring the Diversitym of the Bifidobacterial Population in the Human Intestinal Tract was followed. The 16S rDNA of CCFM1269 was amplified and sequenced (by Soviet Jin Weizhi Biotechnology Co., ltd.) and the 16S rDNA sequence of CCFM1269 obtained by sequencing analysis was aligned in GenBank, which showed that this strain was Bifidobacterium longum subsp infantis (Bifidobacterium longum subsp. Infantis) CCFM1269.
Example 2: preparation of Bifidobacterium longum subspecies infantis suspension
The bifidobacterium longum subspecies infantis CCFM1269 bacterial liquid is prepared according to the following method:
dipping bifidobacterium longum subspecies infantis CCFM1269 bacterial liquid, streaking on an MRS solid culture medium, and culturing at 37 ℃ for 48 hours to obtain a single colony;
picking single bacterial colony, inoculating into MRS liquid culture medium, culturing at 37deg.C for 24 hr to obtain activating solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 1% (v/v), and culturing at 37 ℃ for 24 hours to obtain first-stage seed solution;
inoculating the first-level seed liquid into MRS liquid culture medium according to an inoculum size of 1% (v/v), and culturing at 37 ℃ for 24 hours to obtain a second-level seed liquid;
inoculating the secondary seed solution into MRS liquid culture medium according to the inoculum size of 1% (v/v), and culturing at 37 ℃ for 24 hours to obtain bacterial solution; centrifuging 6000g of bacterial liquid for 15min, and collecting precipitate; washing the precipitate with physiological saline buffer solution twice, and centrifuging 6000g for 10min again to obtain thalli; the lactobacillus is resuspended to a cell concentration of 1×10 by using physiological saline 9 CFU/mL, the bifidobacterium longum subspecies infancy CCFM1269 bacterial liquid is obtained.
Example 3: effect of bifidobacterium longum subspecies infantis CCFM1269 on rheumatoid arthritis rat body weight, joint thickness, clinical score, and incidence
24 female Wistar rats of SPF (Specific Pathogen Free) class of 6 weeks old are randomly divided into 3 groups of 8 animals each, wherein the 3 groups are respectively: a normal group, a model group, a CCFM1269 group of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269.
Experiments were performed for four weeks: starting one week before molding and continuing to the end of the experiment, the normal group and the model group were each filled with 0.5mL of 0.9% (w/w) sterile physiological saline solution per day, and the CCFM1269 group was filled with 0.5mL of 1X 10 per day 9 CFU/mL of Bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 broth; in the second week to the third week, during the first day of molding, the bovine type II collagen solution (Chondrex, 20022) and Freund's incomplete adjuvant (Chondrex, 7002) are mixed and emulsified in equal volumes to form a complete emulsion, the rats are anesthetized by isoflurane, the rats are fixed and the whole rat tail root is sterilized by 75% alcohol, the rats are subjected to primary immunization, 0.2mL of the complete emulsion is precisely sucked up for subcutaneous injection at a position 1.5cm away from the rat tail root, and after one week, the same treatment method is adopted for boosting, namely 0.2mL of the complete emulsion is precisely sucked up for subcutaneous injection at a position 2.0cm away from the rat tail root, and the normal group rats are only injected with the same volume of sterile physiological saline by the same method.
After the molding was completed, the body weight of each group of rats was measured by a weight meter, the joint thickness of each group of rats was measured by a screw micrometer, the clinical score of each group of rats was measured by observing the swelling and redness degree of ankle and finger joints, and safranine-fast green pathological sections of rat ankle joints were prepared (see reference document Integrated Serum and Fecal Metabolomics Study of Collagen-Induced Arthritis Rats and the Therapeutic Effects of the Zushima Tablet for details). The measurement results are shown in FIGS. 1 to 4, respectively.
As can be seen from fig. 1, the joint swelling started 2 weeks after the first immunization, and the joint thickness of the rats in the model group was significantly higher than that of the rats in the normal group at week 3, and the joint thickness of the rats in the CCFM1269 group was lower than that of the rats in the model group, wherein the joint thicknesses of the rats in the control group, the model group, and the CCFM1269 group were 4.3, 6.7, and 5.5mm, respectively.
As can be seen from fig. 2, the change in clinical score of each group of rats was similar to the change in joint thickness, and the clinical score began to increase at week 2 until week 3 after the first immunization, the average score of the model group was at most 7.1 points, and the CCFM1269 significantly down-regulated the joint score of the arthritic model mice to 5.6 points.
As can be seen from fig. 3, the body weight of the rats in the model group was significantly lower than that in the normal group by 242.9g at week 3 after the first immunization, the body weight of the rats in the ccfm1269 group was 256g between the model group and the control group, and the body weight of the control group was 270.9g at the highest.
As can be seen from fig. 4, the ankle joint of the rats in the model group was severely damaged, and the model group was significantly more swollen than the control group and the CCFM1269 group. Soft tissue surrounding the model group suffered significant inflammatory infiltration (arrow point); CCFM1269 has no obvious inflammatory infiltration, which indicates that CCFM1269 can obviously relieve pathological damage of ankle joints.
It can be seen that bifidobacterium longum subspecies infancy CCFM1269 can assist in the treatment and prevention of rheumatoid arthritis.
Example 4: effect of Bifidobacterium longum infant subspecies CCFM1269 on IL-6 and TNF-alpha content in serum of rheumatoid arthritis rats
24 female Wistar rats of SPF (Specific Pathogen Free) class of 6 weeks old are randomly divided into 3 groups of 8 animals each, wherein the 3 groups are respectively: a normal group, a model group, a CCFM1269 group of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269.
Experiments were performed for four weeks: starting one week before molding and continuing to the end of the experiment, the normal group and the model group were each filled with 0.5mL of 0.9% (w/w) sterile physiological saline solution per day, and the CCFM1269 group was filled with 0.5mL of 1X 10 per day 9 CFU/mL of Bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 broth; the second week to the third week are molding period, and when molding is performed for the first day, the bovine type II collagen solution (Chondrex, 20022) and Freund's incompletely mixedAdjuvant (Chondrex, 7002) is mixed and emulsified in equal volume to form a complete emulsion, isoflurane is used for anesthetizing the rat, after the rat is fixed and the whole rat tail root is disinfected by 75% alcohol, the rat is immunized for the first time, 0.2mL of complete emulsion is precisely sucked for subcutaneous injection at a position which is 1.5cm away from the rat tail root after the first time immunization, and the same treatment method is adopted for boosting immunization after one week, namely 0.2mL of complete emulsion is precisely sucked for subcutaneous injection at a position which is 2.0cm away from the rat tail root, and normal group rats are only injected with sterile physiological saline with the same volume by adopting the same method.
After the molding is finished, the rats are killed, the serum of the rats is taken, the content of IL-6, TNF-alpha, IL-22 and IL-23 in the serum of each group of rats is measured by ELISA kit, and the detection result is shown in figures 5-6.
As shown in FIG. 5, the serum concentration of IL-6 in rats in the model group was 261ng/L, which was significantly higher than that in the normal group (121 ng/L); IL-1β levels were significantly reduced to 179ng/L in the serum of the CCFM1269 group of rats compared to the model group of rats. The concentration of TNF-alpha in the serum of the rats in the model group is 368ng/L, which is obviously increased compared with that of the normal group (190 ng/L); compared to model rats, the levels of TNF- α in the serum of CCFM1269 rats were significantly reduced to 259ng/L.
As shown in FIG. 6, the serum concentration of IL-22 in rats in the model group was 29.3ng/L, which was significantly higher than that in the normal group (19.8 ng/L); the serum IL-22 levels were significantly reduced to 18.4ng/L in the CCFM1269 group of rats compared to the model group of rats. The concentration of IL-22 in the serum of the rats in the model group is 55.8ng/L, which is obviously higher than that of the serum of the rats in the normal group (37.3 ng/L); the serum IL-22 levels were significantly reduced to 46.7ng/L in the CCFM1269 group of rats compared to the model group of rats.
It can be seen that Bifidobacterium longum subspecies infantis CCFM1269 reduced the levels of proinflammatory factors IL-6, TNF-alpha, IL-22 and IL-23 in serum of rheumatoid arthritis rats.
Example 5: effect of Bifidobacterium longum infant subspecies CCFM1269 on type II collagen-specific antibody IgG subtype content in serum of rheumatoid arthritis rats
24 female Wistar rats of SPF (Specific Pathogen Free) class of 6 weeks old are randomly divided into 3 groups of 8 animals each, wherein the 3 groups are respectively: a normal group, a model group, a CCFM1269 group of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269.
Experiments were performed for four weeks: starting one week before molding and continuing to the end of the experiment, the normal group and the model group were each filled with 0.5mL of 0.9% (w/w) sterile physiological saline solution per day, and the CCFM1269 group was filled with 0.5mL of 1X 10 per day 9 CFU/mL of Bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 broth; in the second week to the third week, during the first day of molding, the bovine type II collagen solution (Chondrex, 20022) and Freund's incomplete adjuvant (Chondrex, 7002) are mixed and emulsified in equal volumes to form a complete emulsion, the rats are anesthetized by isoflurane, the rats are fixed and the whole rat tail root is sterilized by 75% alcohol, the rats are subjected to primary immunization, 0.2mL of the complete emulsion is precisely sucked up for subcutaneous injection at a position 1.5cm away from the rat tail root, and after one week, the same treatment method is adopted for boosting, namely 0.2mL of the complete emulsion is precisely sucked up for subcutaneous injection at a position 2.0cm away from the rat tail root, and the normal group rats are only injected with the same volume of sterile physiological saline by the same method.
After the molding is finished, the rats are killed, the serum of the rats is taken, the total type II collagen specific antibodies IgG, type II collagen specific antibodies IgG1 and type II collagen specific antibodies IgG2A, II type collagen specific antibodies IgG2B in each group of serum of the rats are measured by ELISA kit, and the level of the type II collagen specific antibodies reflects the intensity of the arthritis symptoms of the rats. The detection results are shown in FIGS. 7-10.
As shown in FIG. 7, the concentration of the total type II collagen specific antibody IgG in the rats of the model group was 9.67. Mu.g/L, which was significantly higher than that in the normal group (7.59. Mu.g/L); compared with the model group of rats, the level of the total type II collagen specific antibody IgG in the serum of the CCFM1269 group of rats is obviously reduced to 7.56 mug/L which is close to the normal level.
As shown in FIG. 8, the concentration of the model group rat type II collagen-specific antibody IgG1 was 6.89. Mu.g/L, which was significantly higher than that of the normal group (5.19. Mu.g/L); the level of type II collagen-specific antibody IgG1 in the serum of the group of CCFM1269 rats was significantly reduced to 5.38 μg/L, approaching normal levels, compared to the model group of rats.
As shown in FIG. 9, the concentration of the model group rat type II collagen-specific antibody IgG2A was 3.04. Mu.g/L, which was significantly higher than that of the normal group (1.71. Mu.g/L); the level of type II collagen-specific antibody IgG2A in the serum of the group of CCFM1269 rats was significantly reduced to 2.39 μg/L compared to the model group of rats.
As shown in FIG. 10, the concentration of the model group rat type II collagen-specific antibody IgG2B was 1.75 μg/L, which was significantly higher than that of the normal group (1.18 μg/L); the level of type II collagen-specific antibody IgG2B in the serum of the group of CCFM1269 rats was significantly reduced to 1.34 μg/L, approaching normal levels, compared to the model group of rats.
Therefore, the bifidobacterium longum subspecies infantis CCFM1269 can reduce the content of the total type II collagen specific antibody IgG, type II collagen specific antibody IgG1 and type II collagen specific antibody IgG2A, II type collagen specific antibody IgG2B in the serum of the rat with rheumatoid arthritis, thereby relieving the symptom of the arthritis of the rat.
Example 6: effect of Bifidobacterium longum infant subspecies CCFM1269 on the content of osteoprotegerin and osteocalcin in serum of rheumatoid arthritis rats
24 female Wistar rats of SPF (Specific Pathogen Free) class of 6 weeks old are randomly divided into 3 groups of 8 animals each, wherein the 3 groups are respectively: a normal group, a model group, a CCFM1269 group of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269.
Experiments were performed for four weeks: starting one week before molding and continuing to the end of the experiment, the normal group and the model group were each filled with 0.5mL of 0.9% (w/w) sterile physiological saline solution per day, and the CCFM1269 group was filled with 0.5mL of 1X 10 per day 9 CFU/mL of Bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 broth; the second week to the third week are molding periodsOn the first day of molding, bovine type II collagen solution (Chondrex, 20022) was mixed with freon incomplete adjuvant (Chondrex, 7002) in equal volumes and emulsified to form a complete emulsion, the rats were anesthetized with isoflurane, fixed and sterilized with 75% alcohol over their entire rat tail roots, the rats were immunized initially, 0.2mL of the complete emulsion was precisely aspirated and injected subcutaneously 1.5cm from the rat tail roots, and one week later, the same treatment was used to boost the immunity, i.e., 0.2mL of the complete emulsion was precisely aspirated and injected subcutaneously 2.0cm from the rat tail roots, and normal rats were injected with the same volumes of sterile physiological saline only by the same method.
After the molding is finished, the rats are killed, the serum of the rats is taken, and the content of the osteoprotegerin and the osteocalcin in the serum of each group of rats is measured by ELISA kit, so that the osteoprotegerin plays an important role in preventing bone destruction caused by rheumatoid arthritis. Osteocalcin levels reflect the disease progression of rheumatoid arthritis. Serum osteocalcin levels were significantly reduced, indicating reduced osteoblast activity and reduced bone turnover. The detection results are shown in FIGS. 11-12.
As shown in FIG. 11, the serum osteoprotegerin concentration was 325ng/L in the model group, which was significantly lower than that in the normal group (438 ng/L) and CCFM1269 group (482 ng/L).
As shown in FIG. 12, the serum osteocalcin concentration was 2.12ng/L in the model group rats, significantly lower than that in the normal group (3.21 ng/L) and CCFM1269 group (3.38 ng/L).
It can be seen that bifidobacterium longum subspecies infantis CCFM1269 can improve the content of osteoprotegerin and osteocalcin in serum of rats with rheumatoid arthritis, and has the effect of increasing the content of the osteoprotegerin and osteocalcin in serum.
Example 7: influence of Bifidobacterium longum infant subspecies CCFM1269 on the abundance of flora in Rheumatoid arthritis rat feces
24 female Wistar rats of SPF (Specific Pathogen Free) class of 6 weeks old are randomly divided into 3 groups of 8 animals each after being fed for 1 week under the conditions of free feeding and drinking water, wherein the feeding room temperature is 22-24 ℃, the humidity is 40-60 percent, 12 hours/12 hours are alternately day and night, and the 3 groups are respectively: a normal group, a model group, a CCFM1269 group of bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269.
Experiments were performed for four weeks: starting one week before molding and continuing to the end of the experiment, the normal group and the model group were each filled with 0.5mL of 0.9% (w/w) sterile physiological saline solution per day, and the CCFM1269 group was filled with 0.5mL of 1X 10 per day 9 CFU/mL of Bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis) CCFM1269 broth; in the second week to the third week, during the first day of molding, the bovine type II collagen solution (Chondrex, 20022) and Freund's incomplete adjuvant (Chondrex, 7002) are mixed and emulsified in equal volumes to form a complete emulsion, the rats are anesthetized by isoflurane, the rats are fixed and the whole rat tail root is sterilized by 75% alcohol, the rats are subjected to primary immunization, 0.2mL of the complete emulsion is precisely sucked up for subcutaneous injection at a position 1.5cm away from the rat tail root, and after one week, the same treatment method is adopted for boosting, namely 0.2mL of the complete emulsion is precisely sucked up for subcutaneous injection at a position 2.0cm away from the rat tail root, and the normal group rats are only injected with the same volume of sterile physiological saline by the same method.
After the molding is finished, collecting the feces of the rat, extracting genome DNA in the feces, carrying out specific PCR amplification on the V3-V4 region, sequencing the 16S rDNA, and analyzing the change of the feces flora, wherein the analysis results are shown in figures 13-15.
As shown in fig. 13-15, the microbiota in the fecal sample of the model group of rats was significantly altered compared to the fecal sample of the normal group of rats, wherein the relative abundance of Lactobacillus in the fecal sample of the model group was significantly reduced compared to the fecal sample of rats of the normal group, and bifidobacterium longum subspecies infantis CCFM1269 up-regulated the relative abundance of Lactobacillus in the fecal sample of rats to the level of the normal group. Also, bifidobacterium longum subspecies infantis CCFM1269 can significantly down-regulate the relative abundance of fresnel genus (Fournierella) compared to the model group; and bifidobacterium longum subspecies infantis CCFM1269 may significantly up-regulate the relative abundance of normal group Ruminococcus 1 (Ruminococcus 1) compared to normal group. The Bifidobacterium longum subspecies infantis can normalize abnormal flora of rats with rheumatoid arthritis and maintain normal intestinal canal steady state.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp.inffantis) CCFM1269, deposited at the Cantonese microorganism strain collection at 9 and 26 of 2022 under the accession number GDMCC No:62839, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.
2. Use of bifidobacterium longum subspecies infantis CCFM1269 in accordance with claim 1 for the manufacture of a medicament for the prevention and/or treatment of rheumatoid arthritis.
3. The use according to claim 2, wherein the prevention and/or treatment of rheumatoid arthritis comprises at least one of the following actions: reducing joint thickness, reducing arthritis specific antibody content, increasing serum osteoprotegerin content, and increasing serum osteocalcin content.
4. The use according to claim 2 or 3, wherein the viable count of bifidobacterium longum subspecies infantis CCFM1269 in the food, pharmaceutical or health-care product is not less than 1 x 10 9 CFU/mL or 1X 10 9 CFU/g。
5. Use of bifidobacterium longum subspecies infantis CCFM1269 in the manufacture of a medicament or health product for increasing the content of osteoprotegerin, osteocalcin according to claim 1.
6. The use according to claim 5, wherein the health product comprises a dairy, soy, meat or fruit and vegetable product produced using a starter culture comprising the bifidobacterium longum subspecies infancy CCFM1269.
7. The use according to claim 6, wherein the preparation method of the starter is: inoculating the bifidobacterium longum subspecies infantis CCFM1269 into a culture medium according to the inoculation amount accounting for 2-4% of the total mass of the culture medium, and culturing for 24-48 hours at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; the thalli are resuspended by normal saline to obtain the ferment.
8. Use of the bifidobacterium longum subspecies infantis CCFM1269 of claim 1 in the manufacture of a medicament or health care product useful in modulating intestinal flora.
9. The use according to claim 8, wherein the modulation of intestinal flora includes, but is not limited to, increasing the relative abundance of Lactobacillus and/or Ruminococcus 1 (Ruminococcus 1).
10. A medicament for preventing or treating rheumatoid arthritis, comprising the bifidobacterium longum subspecies infantis CCFM1269 of claim 1, and a pharmaceutical carrier and/or pharmaceutical adjuvant.
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