CN111621449B - Probiotic and application thereof in secondary osteoporosis - Google Patents

Probiotic and application thereof in secondary osteoporosis Download PDF

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CN111621449B
CN111621449B CN202010633953.7A CN202010633953A CN111621449B CN 111621449 B CN111621449 B CN 111621449B CN 202010633953 A CN202010633953 A CN 202010633953A CN 111621449 B CN111621449 B CN 111621449B
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CN111621449A (en
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赵欣
刘新红
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Shanenkang Biotechnology Suzhou Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum
    • C—CHEMISTRY; METALLURGY
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    • C12N1/20—Bacteria; Culture media therefor
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract

The invention discloses probiotics and application thereof in secondary osteoporosis, belonging to the technical field of microorganisms, wherein lactobacillus plantarum HFY15 belongs to Lactobacillus (LAB) with the preservation number of CGMCC No. 16648; the lactobacillus plantarum HFY15 is separated and identified from natural yak yoghourt; the invention uses retinoic acid to establish a rat secondary Osteoporosis (OP) model, and researches the prevention effect of HFY15 on OP; the results show that the supplementation of HFY15 increases the expression of an osteogenesis marker gene, inhibits the expression of an osteoclast gene, and stimulates the formation of bones; the research result provides a new effective strategy for preventing and treating OP.

Description

Probiotic and application thereof in secondary osteoporosis
Technical Field
The invention relates to the technical field of microorganisms, in particular to probiotics and application thereof in secondary osteoporosis.
Background
Secondary Osteoporosis (OP) is caused by certain diseases and treatments that interfere with bone density and lead to bone loss. Osteoporosis may be potentially at risk in up to 30% of postmenopausal women and 50% of men. Patients with early stage osteoporosis generally do not develop symptoms, and osteoporosis is often undetected for many years and cannot be diagnosed until a fracture is produced in an individual. Common fractures associated with osteoporosis include fractures of the hip, wrist, or spine. Osteoporosis occasionally causes symptoms, and the underlying pathogenesis of secondary osteoporosis is often multifactorial. Proper treatment of osteoporosis can reduce the risk of fracture and prevent unnecessary treatment and anti-resorptive medications.
Many diseases, drugs and lifestyle factors cause secondary osteoporosis, and the common medical conditions that lead to secondary osteoporosis are cancers that lead to bone loss, including bone, breast and prostate cancer, and hormonal imbalances that lead to hyperthyroidism, renal failure, rheumatoid arthritis, systemic lupus erythematosus, sjogren's syndrome, dermatomyositis, mixed connective tissue disease, and the like. Long-term glucocorticoid therapy can result in decreased intestinal calcium absorption, increased urinary calcium excretion, increased serum thyroid hormone, and bone loss, and long-term use of proton pump inhibitors, antiepileptics, diuretics, anticoagulants, and cyclosporin a can result in severe bone loss. In addition to disease and drug use, adverse living habits such as alcohol abuse, smoking, low physical activity and long-term vitamin intake deficiency can also lead to bone loss and, in turn, secondary osteoporosis.
In order to prevent and treat secondary osteoporosis, bisphosphonates, calcitonin, estrogens and estrogen receptor modulators are also used in the clinical treatment of osteoporosis and to increase bone density in patients, in addition to conventional approaches to change lifestyle habits and increase vitamin and calcium absorption. However, these drugs may have problems of safety, tolerance, etc., and impose a great economic burden on patients.
Disclosure of Invention
The invention aims to provide probiotics and application thereof in secondary osteoporosis so as to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides Lactobacillus plantarum HFY15, wherein Lactobacillus HFY15 is Lactobacillus (LAB), is preserved in the China general microbiological culture Collection Center (CCM) of China Committee for culture Collection of microorganisms, has the preservation date of 2018, 10 and 29 days, and has the preservation number of CGMCC No. 16648.
The invention also provides application of the lactobacillus plantarum HFY15 in preparation of a medicine for preventing or treating secondary osteoporosis.
The invention discloses the following technical effects:
the lactobacillus plantarum HFY15 is a lactic acid bacterium separated and identified from natural yak yoghourt in the invention. The invention establishes a secondary OP model of rats by using tretinoin and researches the prevention effect of HFY15 on secondary OP. The invention discovers that the supplement of HFY15 increases the expression of an osteogenesis marker gene and stimulates the formation of bones. The research result provides a new effective strategy for preventing and treating secondary OP.
The invention discusses the prevention effect of lactobacillus plantarum HFY15 on secondary OP of rats, and shows that the strain effectively improves the expression of rat serum and spinal cord osteogenesis marker genes, promotes the formation of bones in rats and lays a foundation for further research of HFY 15.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph of β -catenin, Lrp5, Lrp6, Wnt10B and DKK1 expression in rat spinal cord, wherein the different letters on the bar graphs in each graph indicate significant differences between groups (P <0.05), A is β -catenin, B is Lrp5, C is Lrp6, D is Wnt10B, and E is DKK 1;
FIG. 2 is the expression of OPG, RANKL and RANK in rat spinal cord, where A is OPG, B is RANKL and C is RANK;
FIG. 3 is the expression of Runx2, ALP, CTSK and TRACP in rat spinal cord, wherein A is Runx2, B is ALP, C is CTSK, D is TRACP;
FIG. 4 is a TRAP staining pathology of rat femur and tibia, magnification 100 ×; wherein, Normal is a Normal rat control; model is rats treated with tretinoin (80 mg/kg/d); medi cine is rats treated with tretinoin (80mg/kg/d) in a single dose of 0.5mL/100g zoledronic acid; HFY15 is a single dose of 1mL/100g 10 in rats treated with tretinoin (80mg/kg/d)10CFU/kg Lactobacillus plantarum HFY 15;
FIG. 5 is the results of rat femoral micro CT, wherein Normal is a Normal rat control; model is rat treated with tretinoin (80 mg/kg/d); medicine is rats treated with tretinoin (80mg/kg/d), and the single dose is 0.5mL/100g zoledronic acid; HFY15 is a single dose of 1mL/100g 10 in rats treated with tretinoin (80mg/kg/d)10CFU/kg Lactobacillus plantarum HFY 15.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
1. Materials and methods
1.1 laboratory Strain
Lactobacillus plantarum HFY15 is separated from Yak yogurt in Sichuan province, and identified as Lactobacillus acidbacteria, LAB by 16S rDNA sequence, and the strain is preserved in China general microbiological culture Collection center (CGMCC, address: institute of microbiology, China academy of sciences, No. 3, West Lu 1, North Cheng of the south facing-Yang district, Beijing city) in 29 months in 2018 with the preservation registration number of CGMCC No. 16648.
Separating and purifying lactobacillus plantarum HFY15 from a yak yoghourt sample, wherein gram staining is positive; the colony color is mostly white or milk white, the shape is circular, the edge is neat, the surface is moist and smooth, under 100 times of oil lens, the cell shape has long rod, short rod and spherical shape, and no budding reproduction exists.
The 16S rDNA base sequence of the strain HFY15 is shown in SEQ ID No. 1; the sequencing results were aligned to the NCBI website (http:// blast. NCBI. nlm. nih. gov/blast. cgi). Homology is 100% with Lactobacillus plantarum, MN 368282.1.
1.2 in vivo rat model of osteoporosis
Female Wistar rats of 8 weeks old, weighing 250-300 g each, were purchased at the center of experimental animals at university of Chongqing medicine and used as experimental animals. All rats were randomly placed in cages of 2 rats per cage, and the rat chambers were kept in 12 hour light and 12 hour dark cycles. The rats were not treated within one week after being transported to the rat room, and were allowed to adapt to laboratory conditions. Rats were randomly divided into a control group, a model group, a drug group and an HFY15 group (10 per group). Rats in the control group and model group were gazed with physiological saline daily at a rate of 1ml/100g for 2 weeks. The rats in the drug group and HFY15 group were gavaged 10/day at a ratio of 1mL/100g10CFU/kg of HFY15 bacterial suspension for 2 weeks. At the end of two weeks, all groups except the control group received retinoic acid (80mg/kg/d) for 4 weeks, and the control group received an equivalent amount of saline. During this four week period, except for tretinoin treatment, HFY15 group continued to receive daily gavage with HFY15 bacteria, while drug group rats were given tail vein injections of zoledronic acid at a rate of 0.5mL/100g once a week. After four weeks, all rats were fasted for 24h andcervical drainage was sacrificed and serum and spinal cord were collected and stored at-80 ℃. The tibia and femur were harvested and fixed in 10% (v/v) formaldehyde buffer for subsequent histological analysis and micro-computed tomography (micro-CT).
1.3 measurement of serum Biochemical indicators and cytokines
Rat blood was collected, centrifuged at 4000rpm for 10min, and the supernatant (serum) was collected. The determination of serum calcium and phosphorus levels followed the instructions of the kit (Nanjing Jiancheng bioengineering institute, Nanjing, Jiangsu, China). Cytokine levels were determined using BAP (ml037086), BGP (ml002883), IGF-1R (ml059459), TRACP-5b (ml003177) and GABA (ml064273) cytokine assay kit (Shanghai enzyme-linked Biotechnology, Inc., Shanghai, China).
1.4RNA extraction, reverse transcription and real-time qPCR
Total RNA was extracted from spinal cord using TRIzol reagent and an ultrapure RNA kit (Invitrogen, Carlsbad, Calif., USA). RNA concentration was determined using a Nanodrop1000 (Thermoscience, Wilmington, DE, USA). For real-time quantitative reverse transcription (Q RT-PCR), first strand cDNA was synthesized using the reverse Aid first strand cDNA Synthesis kit (Thermoscience). The system was reacted at 95 ℃ for 60s, then at 95 ℃ for 40 cycles for 30s, and annealed at 72 ℃ for 30 s. Finally, DNA30s was detected at 95 ℃ and DNA35s was detected at 5 ℃-ΔΔCTThe method determines relative gene expression levels. The sequences of the PCR primers used are listed in Table 1.
TABLE 1 PCR primer sequences
Figure BDA0002567099050000061
Figure BDA0002567099050000071
1.5 histological observations
Immediately after sacrifice, the tibia and femur were removed and fixed in 10% (v/v) formaldehyde buffer. Proximal tibia to femur was routinely processed, paraffin embedded, sectioned, stained with tartrate-resistant phosphatase (T RAP), and visualized under BX43 microscope (olympus, tokyo, japan). Osteoclasts with three or more nuclei were counted in 6 randomly selected fields, and the average value was calculated to represent the number of osteoclasts per histological section.
1.6 bone imaging micro-CT
After fixation of the femur, scans were performed using a Bruker micro CT sky scan1272 system (Kontich, belgium) obtained from 720 views at a voxel resolution of 20 μm, with incremental beam angles of 0.5 °, beam strengths set at 80 peak kV and 450 uA. Each run consisted of bones from the control, model, drug and HFY15 groups, and the calibration phantom was used to normalize the gray values and maintain consistency. A fixed threshold (760) is used to separate bone from bone marrow based on self-threshold and iso-depth analysis of multiple bone samples. Tissue volume, bone volume, trabecular number, trabecular thickness, trabecular separation and bone densitometry volumetric imaging data were visually analyzed using the bruker microctskyscan1272 system software application.
1.7 statistical analysis
Data are presented as mean ± standard deviation. SPSS software was used to analyze the variance and perform a new Duncan multiple range test. Differences were considered statistically significant at P < 0.05. All numbers were plotted using origine 8.0 software.
2. Results
2.1 serum Biochemical indicators and cytokine levels
As shown in table 2, the level of the model group was the lowest, and the drug group and HFY15 group were significantly higher than the control group and the model group. Serum cytokine detection showed that model rats had the lowest levels of BAP, BGP, IG F-1R and GABA in serum, and the drug and HFY15 groups were significantly higher than the control and model groups. The serum level of TRP-5b in the model group is the highest, and the serum level in the drug group and the HFY15 group is obviously lower than that in the control group and the model group.
TABLE 2 Biochemical indicators and cytokine levels in rat serum
Figure BDA0002567099050000081
2.2 expression of Wnt/beta-catenin Signal pathway Gene in spinal cord
The canonical Wnt/beta-catenin pathway plays an important role in osteoblast differentiation and proliferation. Research shows that any factor regulating the canonical Wnt/beta-catenin signaling pathway influences the differentiation and proliferation of osteoblasts. In the Wnt/beta-catenin signaling pathway, beta-catenin, Lrp5/6 and Wnt10b positively regulate bone formation, while DKK1 negatively regulates bone formation by inhibiting the pathway. The mRNA expression levels of spinal cord beta-catenin, Wnt10b, Lrp5 and Lrp6 were lowest in model rats, and those of drug spinal cord and HFY15 group were highest. The model group D KK1 expressed the highest level, and the drug group and HFY15 expressed the lowest levels, as shown in fig. 1.
2.3 expression of OPG/RANK/RANKL Signaling pathway genes in spinal cord
The Osteoprotegerin (OPG)/nuclear factor-K β Receptor Activator (RANK)/nuclear factor-K β receptor activator ligand (RANK L) signaling pathway regulates osteoclastic function during bone remodeling. Cells release RANKL, which binds RANK on the osteoclast surface, promoting osteoclast differentiation and activation through NF-K β, JNK and protein kinase B pathways. OPG can competitively inhibit the combination of RANK and R ANKL, promote the function of osteocyte, and reduce bone destruction. The expression levels of OPG and RA NKL mRNA were lowest in the model group spinal cords, highest in the drug spinal cords and HFY15 group. The model group showed the highest level of RANK expression and the drug and HFY15 groups were the lowest, as shown in fig. 2.
2.4 expression of osteogenic and osteogenic marker genes in the spinal cord
The mRNA expression level of the osteogenic marker gene Runx2 was lowest in the spinal cords of the model group and highest in the spinal cords of the drug and HFY15 groups. The model group had the lowest level of alkaline phosphatase (ALP) expression, and the drug group and HFY15 group were the highest. The osteogenic marker genes CTSK and TRACP were expressed at the highest level in the model group and at the lowest level in the drug group and HFY15 group. HFY15 strongly affected the expression of spinal cord marker genes in HFY15 group at levels close to those of drug and control groups, as shown in fig. 3.
2.5 pathological Observation of femur and tibia
As shown in fig. 4, histological micrographs of rat femur and tibia are shown. The control group had normal fracture number, morphology and fusion degree of femur and tibia. The number of osteoclasts in the model group is remarkably increased, and a plurality of fused huge multinucleated cells exist. In contrast, the osteoclast number was reduced to the level of the control group in the drug and HFY15 groups.
2.6 femoral and tibial micro CT
Fig. 5 shows the results of micro CT of rat femurs. In the control group, the percent bone volume of the femur (BV/TV), trabecular number (tb.n), trabecular thickness (tb.th), trabecular separation (tb.sp), Bone Mineral Density (BMD) were normal values. BV/TV, tb.n, tb.th, and BMD were lowest in the femoral model group, while (tb.sp) was highest in the model group. The femurs of the drug group and HFY15 group were similar to the control group; BV/TV, tb.n, tb.th, and BMD increased significantly, while tb.sp decreased significantly.
3. Conclusion
All-trans retinoic acid is an active metabolite of vitamin a in the retinoic acid family. The vitanide medicine has strong influence on cell growth, differentiation and apoptosis through a homologous nuclear receptor, and has potential application in tumor treatment and chemical prevention. Tretinoin also plays an important role in maintaining immune homeostasis and in the treatment of alzheimer's disease. Long-term use of retinoic acid can cause adverse reactions such as liver injury, bone loss, chapped skin and the like. Many studies have used retinoic acid to construct animal models of secondary osteoporosis. Therefore, we treated rats with tretinoin to establish a secondary OP model.
Like other body tissues, bone constantly undergoes cellular or bone metabolism, which is divided into two phases: formation and remodeling. Bone formation plays a major role in the growth and development of individuals, while bone remodeling continues throughout the life cycle. Differentiation of osteoblasts into osteocytes is a complex process involving multiple signaling pathways, of which the Wnt/β -catenin and OPG/RANK/RANKL signaling pathways have been the most studied. Research shows that genes involved in Wnt/beta-catenin and OPG/RANK/RANKL signal pathways have important influence on osteogenesis. After modeling, the invention directly detects related gene expression, and discusses the influence of HFY15 on bone mass, QPCR analysis shows that the expression levels of model groups of beta-catenin, Wnt10b, Lrp5, Lrp6, OPG, RANKL and Runx2 are lowest, and the expression levels of negative regulatory genes of DKK1, ALP, CTSK and TRACP are increased, which shows that the construction of the model of the secondary osteoporosis is successful. Also, analysis of the qPCR results indicated that HFY15 has a positive regulatory effect on rat bone formation, similar to that of zoledronic acid. Many genes are involved in both pathways, and the present invention does not further verify changes in gene expression levels by western blot. However, the results of enzyme-linked immunosorbent assay (ELISA) showed changes in IG F-1R content and BAP, BGP and TRACP expression levels, confirming the QPCR results.
A small amount of tretinoin can promote bone formation, but a large amount of tretinoin can damage the ovary of a rat, cause the reduction of estrogen secretion, weaken the inhibition effect on osteoclast and increase the activity and the quantity of the osteoclast. During bone remodeling, osteoclasts from mononuclear macrophages fuse into multinucleated osteoclasts, resorb bone, and reduce bone mass. Osteoclasts with three or more nuclei are counted, which accurately reflects the occurrence of bone remodeling. The present invention stains bone tissue sections with TRAP to make osteoclasts have a strong cytoplasmic brunness and a bluish color of the nucleus. After the bone tissue is sectioned and stained, the present invention counts multinucleated osteoclasts of 40 rats. The model group had the most multinucleated osteoclasts, while the treated and HFY15 groups had fewer osteoclasts. This indicates that HFY15 is effective in preventing retinoic acid from promoting bone resorption, and it is difficult to count the total number of multinucleated osteoclasts due to the large bone tissue of rats, and error may be induced. Thus, the present invention randomly selects three fields of view from each slice and counts the number of multinucleated osteoclasts in each slice, averaged from each view.
Quantitative PCR, ELISA and histological micrographs reflect only the localization of osteogenic and bone resorption and do not directly or effectively represent the actual change in bone mass. Therefore, it is necessary to accurately detect the bone mass using a technique capable of directly responding to the change in the bone mass. micro-CT is a non-invasive technique that combines imaging and high resolution histological examination. The difference in X-ray attenuation coefficients between bone and other body tissues allows bone imaging using micro-CT to directly indicate bone mass. The present invention randomly extracts 6 femurs and 6 tibias from four groups for CT scanning to reflect the real condition of the bone block. The laboratory isolated and purified HFY15 Lactobacillus was shown to have a prophylactic effect on secondary OPs, similar to zoledronic acid.
Probiotics are a group of active microorganisms that benefit the host by colonizing the body and altering the composition of part of the host's flora. Regulating the functions of mucosa and immune system of a host or regulating the balance of intestinal flora, promoting nutrient absorption and maintaining the health of intestinal tracts, thereby generating single or mixed microorganisms, having definite function and being beneficial to the health of the host. Before studying the effect of probiotics on life activities, it must be ensured that the probiotics are able to colonize the intestine and resist damage by gastric acid and bile salts. The invention carries out relevant experiments on the separated and purified strain HFY 15. The survival rate of HFY15 after artificial gastric acid and bile salt treatment was 86.26% ± 10.78% and 53.45% ± 2.74%, respectively. Prior to the start of the experiment, the present invention injected rats with HFY15 for two weeks to ensure colonization of HFY15 in the rats. The results show that HFY15 has good prevention and treatment effects on retinoic acid-induced osteoporosis.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> Chongqing second college of education
School planning, construction and development center of the Ministry of Education
<120> probiotics and application thereof in secondary osteoporosis
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Claims (1)

1. A Lactobacillus plantarum (A)Lactobacillus plantarum) The application of HFY15 in preparing the medicine for preventing or treating secondary osteoporosis is characterized in that the HFY15 has been preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation date of 2018, 10 months and 29 days and the preservation number of CGMCC No. 16648.
CN202010633953.7A 2020-07-02 2020-07-02 Probiotic and application thereof in secondary osteoporosis Active CN111621449B (en)

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