CN111840334A

CN111840334A - Application of Prevotella in treating or preventing osteoporosis

Info

Publication number: CN111840334A
Application number: CN202010707796.XA
Authority: CN
Inventors: 陈雷; 金可可; 王中祥
Original assignee: First Affiliated Hospital of Wenzhou Medical University
Current assignee: First Affiliated Hospital of Wenzhou Medical University
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2020-10-30

Abstract

The invention relates to an application of Prevotella in treating or preventing osteoporosis, wherein P.histicola is used as symbiotic bacteria in normal human intestinal tracts and has excellent safety, animal experiments of estrogen-deficient mice show that the P.histicola can prevent bone loss of the mice, and the P.histicola can inhibit the expression of inflammatory factors and reduce the increase of intestinal permeability caused by estrogen deficiency by regulating the imbalance of intestinal flora caused by estrogen deficiency, thereby reducing the generation of inflammatory factors in intestinal tract walls, further inhibiting the differentiation, maturation and activation of osteoclasts guided by RANKL, reducing bone absorption and inhibiting bone loss, and has the function of resisting estrogen-deficient osteoporosis.

Description

Application of Prevotella in treating or preventing osteoporosis

Technical Field

The invention relates to the technical field of microorganism application, in particular to application of Prevotella in treating or preventing osteoporosis.

Background

Osteoporosis (osteoporotis OP) is a common senile disease characterized by a systemic bone disease characterized by a decrease in bone mass, a destruction of the microstructure of the bone tissue, an increase in bone fragility, and a tendency to fracture. The osteoporosis fracture has great harm and is one of the main causes of disability and death of old patients. Within 1 year after hip fracture, 20% of patients die from various complications, about 50% of patients cause disability, and the quality of life is obviously reduced. And medical treatment and nursing of osteoporosis and fracture need to invest a large amount of manpower, material resources and financial resources, which causes heavy burden to families and society. It is predicted that the medical costs for major osteoporotic fractures (wrist, vertebral body and hip) will be as high as 720 billion yuan, 1320 billion yuan and 1630 billion yuan in 2015, 2035 and 2050 years, respectively, in our country. At present, the anti-osteoporosis medicines mainly comprise a bone resorption inhibitor and a bone formation promoter, and the rest of the medicines comprise active vitamin D, vitamin K2, traditional Chinese medicines and the like for treatment. Although various anti-osteoporosis drugs are available, long-term administration of these drugs has side effects such as digestive tract reaction, nephrotoxicity, atypical femoral fracture, mandibular necrosis, estrogen-dependent malignancies, and the like, and is expensive. Osteoporosis is a disease requiring long-term continuous treatment, except for bisphosphonate, the curative effect of the currently used medicines is rapidly reduced after the rest medicines are taken out of medicine, but the side effect caused by long-term administration of bisphosphonate is not negligible, so the current guidelines suggest that the oral treatment course should not exceed 5 years, but the requirement of long-term maintenance for treating postmenopausal osteoporosis cannot be met.

The intestinal flora plays an important role in osteoporosis caused by estrogen deficiency. Studies have shown that disturbances in the intestinal flora are involved in the development of various metabolic diseases, such as: obesity, diabetes, osteoporosis, etc. Animal experiments showed that no estrogen-deficient bone mass reduction occurred after castration of the drug in sterile mice, but bone mass reduction occurred after transplantation of feces from mice grown in normal environment. Indicating that the intestinal flora is a necessary condition for the estrogen deficiency to cause the osteoporosis. The intestinal pathogenic bacteria ratio is increased, the intestinal wall permeability is increased and pathogenic bacteria invasion in the intestinal tract are caused by estrogen deficiency, so that local inflammatory response of the intestinal tract is increased, and related inflammatory factors are released, wherein TNF alpha, IL-1 beta and IL-6 are used as osteoclast related inflammatory factors, and the increase of the inflammatory factors often causes differentiation and maturation of osteoclasts, so that bone resorption is increased, and bone mass is lost.

In order to solve these contradictions, there is a need for an economical and practical drug which has a prophylactic and therapeutic effect on postmenopausal osteoporosis, has few side effects, and can be taken for a long time.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the application of Prevotella in treating or preventing osteoporosis.

Further said Prevotella comprises P.

In order to realize the purpose, the invention provides an application of Prevotella in preparing a bone metabolism positive regulation medicament.

Further, the bone metabolism forward control drug is a drug for increasing bone mass or reducing bone resorption.

Further, the bone mass increasing drug is a drug for increasing the number and area of trabeculae and/or increasing the relative bone volume fraction and/or increasing the trabecular separation and/or increasing the trabecular thickness.

Further, the bone resorption reducing agent is an agent for inhibiting an increase in the number of osteoclasts and/or reducing the area of osteoclasts or an agent for inhibiting an increase in the number of osteoclasts and/or reducing the area of osteoclasts caused by estrogen deficiency.

Further, the drug for decreasing bone resorption is a drug for suppressing bone resorption by decreasing inflammatory reaction or a drug for suppressing bone resorption by decreasing inflammatory reaction due to estrogen deficiency.

Further, the drug for reducing bone resorption is a drug for inhibiting bone resorption by inhibiting expression of IL-1 β and/or TNF α.

Furthermore, the medicine for reducing bone resorption is a medicine which can up-regulate the expression of intestinal tight junction proteins occludin and ZO-1 to reduce intestinal permeability and further reduce the generation of inflammatory factors in the intestinal wall to inhibit bone resorption.

Further, the bone metabolism forward control medicament is a medicament for preventing or treating osteoporosis.

Further, the medicament for preventing or treating osteoporosis is a medicament for preventing or treating osteoporosis caused by estrogen deficiency.

The invention also provides a pharmaceutical composition for treating osteoporosis caused by estrogen deficiency, which takes Prevotella as an active ingredient and also comprises a medically acceptable pharmaceutical carrier.

Furthermore, the dosage form of the pharmaceutical composition is injection, tablet, oral preparation or capsule.

The invention has the following advantages: the P.histicola can prevent the bone loss of mice through animal experiments of estrogen-deficient mice, inhibit the expression of inflammatory factors and reduce the increase of intestinal permeability caused by estrogen deficiency by regulating the imbalance of the intestinal flora caused by estrogen deficiency, thereby reducing the generation of the inflammatory factors in the intestinal wall and reducing the level of the inflammatory factors from the intestinal tract in the circulation system. The changes can cause reduction of osteoclast-related inflammatory factors in bones, further inhibit differentiation, maturation and activation of osteoclasts guided by RANKL, reduce bone resorption, inhibit bone loss and have the effect of resisting estrogen-deficiency osteoporosis. The Histicola is used as a microecological preparation, can meet the requirements of long-term taking, economy, safety and no side effect required for preventing and treating osteoporosis, and can even replace the current commonly used medicine for resisting osteoporosis.

Drawings

FIG. 1A is a micro-CT image of the distal transverse and sagittal planes of a femur representative of three groups; panel b, VG staining pictures of the sagittal plane of the distal femur with representatives in three groups; panel C quantitative analysis of bone microstructure, including bone volume fraction BV/TV; graph d. quantitative analysis of bone microstructure, trabecular resolution tb.sp; fig. E is a quantitative analysis of bone microstructure, trabecular number tb.n; panel F is a quantitative analysis of bone microstructure, trabecular thickness tb.th;

FIG. 2A is a TRAP staining picture of the sagittal plane and skull of the distal femur representative of three groups; panels B-C are quantitative analyses of osteoclast number n.oc/BS and osteoclast area oc.s/BS per bone area, respectively; FIGS. D-E are the bone formation index P1NP and the bone resorption index CTX-1 in peripheral blood, respectively; fig. F-g expression levels of the bone resorption related indicators RANK, RANKL and OPG in tibia; panel H, osteoclast-associated inflammatory factor levels in peripheral blood; FIGS. I-K are the expression levels of the osteoclast-associated inflammatory factors IL-1 β, IL-6 and TNF α, respectively, in the tibia;

FIG. 3 is a graph showing the results of P.histicola bacteria inhibiting the expression of IL-1 β, IL-6 and TNF α, which are local osteoclastogenesis-related inflammatory factors in the intestinal tract. FIG. A is a graph showing the results of expression of IL-1. beta., IL-6 and TNF. alpha. in duodenum; FIG. B is a graph showing the results of expression of IL-1. beta., IL-6 and TNF. alpha. in jejunum; FIG. C is a graph showing the results of expression of ileal IL-1. beta., IL-6 and TNF. alpha.; FIG. D is a graph showing the results of expression of colon IL-1. beta., IL-6 and TNF. alpha.; FIG. E is an immunofluorescence image of a representative labeled IL-1 β in the jejunum; FIG. F immunofluorescence image of a representative marker IL-1 β in the ileum; figure g immunofluorescence images of representative labeled TNF α in the ileum; panel h immunofluorescence images of a representative marker TNF α in the colon;

FIG. 4A is an immunofluorescent-labeled graph of the intestinal claudin OCCLudin representative of the three groups in the colon; panel B is a plot of immunofluorescence labeling of a representative gut claudin ZO-1 in the colon in three groups; FIG. C is a representative Western Blot band image; FIG. D is a representative Western Blot quantitative analysis; panel e. serum FITC-labeled dextran concentration;

fig. 5 is a schematic diagram of the prevention of estrogen-deficient osteoporosis by the bacterium p.

Detailed Description

The present invention will be further described in detail with reference to examples and effect examples, but the scope of the present invention is not limited thereto.

Animal culture

Female mice 10 weeks old, C57BL6, Free of Specific Pathogens (SPF), were housed in translucent plastic cages, 5 cages, and strictly performed in an environment simulating 12 hours of light in the natural environment and 12 hours of night (8 o' clock in the morning). All mice were randomly divided into 3 groups: sham-operated feeding of medium group (Sham + Vehicle, n ═ 10), post-oophorectomy feeding of medium group (OVX + Vehicle, n ═ 10), post-oophorectomy feeding of bacterial group (OVX + p. All mice had 1 week of acclimation to the new environment before the experiment was performed.

Histicola bacteria and culture media

The histicola species is from the german collection of microorganisms and cell cultures (DSMZ) and belongs to the lyophilized anaerobic strain, product number dsm 19854.

Preparation of animal models

Mouse bilateral ovariectomy model: after anesthesia is injected into an abdominal cavity by using 3.6 percent chloral hydrate (10ml/kg), the skin and muscle layer on the back are cut layer by layer, the skin and muscle layer enter the abdominal cavity, the ovaries on two sides are found out, the ovaries are ligated on the oviduct, the ovaries are completely cut off, and the layers are sutured in a layered mode. All mice were injected with penicillin 5 umillion/d for 3 days after surgery to prevent infection. After 1 week of postoperative recovery, experiments were performed in groups.

Mouse sham surgery model: after 3.6% chloral hydrate (10ml/kg) is used for intraperitoneal injection and anesthesia, the skin and muscle layer on the back are cut layer by layer and enter the abdominal cavity, the ovaries on both sides are found out, only partial intra-abdominal fat around the ovaries is cut off and then the ovaries are sutured layer by layer, and all mice are injected with penicillin 5 ten thousand U/d for 3 days after operation to prevent infection. After 1 week of postoperative recovery, experiments were performed in groups.

Preparation of sterile anaerobic liquid culture medium

A modified PYG medium was prepared as shown in Table 1, the prepared medium was filtered through a 0.22 μm syringe filter (Millipore, Massachusetts, American) and then poured into a sterile reagent bottle, and the reagent bottle was placed in a sterile closed container with its opening and pre-cultured together with an anaerobic gas-producing bag (2.5L) for 24 hours in order to eliminate dissolved oxygen in the medium. Immediately after the pre-culture, sealing the mixture with a sterile butyl rubber plug, and culturing the mixture in a water bath kettle at 37 ℃ for 24 hours to eliminate the possibility of culture medium pollution.

TABLE 1

Setting of the Experimental groups

1) OVX + p. histicola group: the lyophilized strain was inoculated into Hungate-type tube (Hungate-type tube) containing modified PYG medium and anaerobically cultured at 37 ℃ for 20-24 hours until the concentration of the bacterial liquid reached 1X 10¹⁰cfu/ml, the mice bilateral oophorectomy model is subjected to intragastric administration, and the single intragastric administration is 1 multiplied by 10⁹CFU, the volume of the liquid or medium for intragastric administration is 0.1ml, the frequency is 1 intragastric administration every other day, and the time is maintained for 12 weeks. After the completion of the bacterial lavage, performing FITC-labeled dextran lavage the next day, wherein the dosage is 0.6mg/g, performing 3 hours later, performing intraperitoneal injection anesthesia by chloral hydrate, collecting blood under anesthesia, killing the mouse by cervical dislocation, and collecting femur, tibia, skull and intestinal tract specimens of the mouse.

2) OVX + Vehicle group: the modified PYG medium (0.1 ml) is taken to perform gavage on the bilateral ovariectomy model of the mouse, the gavage frequency is 1 time every other day, and the time is maintained for 12 weeks. After the completion of the bacterial lavage, performing FITC-labeled dextran lavage the next day, wherein the dosage is 0.6mg/g, performing 3 hours later, performing intraperitoneal injection anesthesia by chloral hydrate, collecting blood under anesthesia, killing the mouse by cervical dislocation, and collecting femur, tibia, skull and intestinal tract specimens of the mouse.

3) Sham + Vehicle group: and (3) taking 0.1ml of the improved PYG culture medium, and performing gavage on the mouse pseudo-surgical model at the frequency of gavage 1 time every other day for 12 weeks. After the completion of the bacterial lavage, performing FITC-labeled dextran lavage the next day, wherein the dosage is 0.6mg/g, performing 3 hours later, performing intraperitoneal injection anesthesia by chloral hydrate, collecting blood under anesthesia, killing the mouse by cervical dislocation, and collecting femur, tibia, skull and intestinal tract specimens of the mouse.

1. Example 1: the bacterium histicola ameliorates osteoporosis caused by estrogen deficiency in mice.

Micro-CT scanning and analysis

After the mice were sacrificed, the femurs of the mice were dissected away, the soft tissue attached to the bone was removed, and then placed in 4% tissue fixative for fixation for 24 hours. After fixation the right femur was scanned using a Skyscan 1176Micro-CT instrument (Bruker micct, Kontich, Belgium) with the following parameters: source voltage, 50 kV; source current, 500 μ A; AI 0.5mm filter; pixel size 9 μm; rotation step, 0.4 degree. The scanned pictures were reconstructed using NReconru software (Bruker micct, Kontich, Belgium) with the following parameters: ring artifact correction, 7; smoothening, 2; beam hardening correction, 40%. A more accurate analysis area is defined as 0.5mm above the growth plate, the analysis thickness is 1mm, the area of interest (ROI) of the trabecular bone within the area is determined manually, the bone properties in the ROI are determined by post-binarization of the image, the parameters of the analysis include: connection Density (Conn.D), Structural Model Index (SMI), Bone Volume fraction (Bone Volume/Tissue Volume, BV/TV), Trabecular Number (Tb.N), Trabecular Thickness (Tb.Th), Trabecular Separation (Tb.Sp). The analytical procedure used for the analysis of the parameters was CTAn (BrukermicrocoT, Kontich, Belgium).

Results of distal femur Micro-CT demonstrated that p. Results as shown in figure 1, quantitative measurements of bone microstructure related parameters demonstrated that BV/TV, tb.sp and tb.n were significantly inferior to Sham (Sham + Vehicle) in the OVX medium group, whereas treatment with p. Th did not differ significantly between the three groups. Van Gieseon (VG) staining results gave similar conclusions as Micro-CT, and the results are shown in FIG. 1B.

2. Example 2: histicola bacteria can inhibit increase of osteoclast activity caused by estrogen deficiency and improve systemic inflammatory response and bone resorption level

Decalcification, Paraffin embedding and sectioning

After Micro-CT analysis is complete, the femur will be decalcified by immersion in a solution of 14% EDTA (Sigma-Aldrich, Sydney, NSW, Australia) at 37 ℃ for 7 days, with fluid changes every two days. Paraffin embedding was performed after decalcification was completed, and the embedding scheme is shown in the following table 2:

scheme for embedding paraffin wax into femur

TABLE 2

After the embedding is finished, the specimen is placed into a square mold, liquid paraffin is poured, and after cold cutting, slicing is carried out on a paraffin slicer, wherein the slicing thickness is 6 mu m.

Dewaxing and dyeing

The cut paraffin sections were dewaxed according to the following table 3:

slice dewaxing protocol

Step (ii) of	Soaking component	Time (minutes)
			1	Xylene	2
2	Xylene	2
			3	Xylene	2
4	100% alcohol	1
			5	100% alcohol	1
6	100% alcohol	1
			7	95% alcohol	1
8	70% alcohol	1
			9	Tap water	Rinsing

TABLE 3

Sections were dewaxed and stained with hematoxylin eosin (H & E) and tartrate-resistant acid phosphatase (TRAP) strictly following the instructions, and after staining was completed, they were observed and photographed using an Olympus BX51 microscope (Olympus Corporation, Takachiho, Japan), and Image Analysis of bone histology was performed using BIOQUANT OSTEO software (Bioquant Image Analysis Corporation, Nashville, TN, USA). The mouse skull is dissected and separated, soft tissues attached to the surface of the mouse skull are removed, the mouse skull is soaked and cleaned by Phosphate Buffered Saline (PBS) for 5 minutes each time and 3 times in total, and then the mouse skull is fixed in 4% tissue fixing solution for 24 hours and cleaned by PBS for 3 times and 5 minutes each time. After the washing, the skull was immersed in the TRAP dye solution, incubated and dyed in a 37 ℃ water bath for 30 minutes, washed with running water, observed and photographed with Sony rx100m3(Sony, Tokyo, Japan).

TRAP staining showed an increase in the number of TRAP-positive osteoclasts per unit area in OVX + Vehicle group mice. The number of osteoclasts can be reduced by the Histicola strain. Meanwhile, quantitative analysis on the TRAP positive area finds that the proportion of the TRAP positive area in the unit bone surface base is obviously reduced after the P.histicola bacterium is treated, which indicates that the P.histicola bacterium can reduce the number of osteoclasts and inhibit the activity of the osteoclasts.

Serum TNF alpha, IL-1 beta and CTX-1 are obviously increased after the ovariectomy of the mice, but the IL-6 and P1NP levels are not statistically different, which indicates that the osteoporosis of the mice caused by the estrogen deficiency is mainly caused by the increase of bone absorption. The bacterium histicola can prevent the two inflammatory factors and the increase of bone absorption index caused by the lack of estrogen in the blood serum of the mice. Similarly, treatment with the bacterium p.histicola was effective in reducing TNF α expression in the tibia of estrogen deficient mice. As shown in fig. 2A-C, the p.histicola can protect the trabecular area reduction caused by estrogen reduction, as shown in fig. 2E-G, the expression levels of RANKL and OPG are greatly reduced on average in the experimental group compared to the OVX + Vehicle group peripheral blood bone resorption index CTX-1 tibial bone resorption related index RANK, indicating that the p.histicola can inhibit bone resorption, and as shown in fig. 2H-K, the experimental group is significantly reduced compared to the OVX + Vehicle group peripheral blood and tibial bone osteoclast-related inflammatory factors IL-1 β and TNF α, indicating that the p.histicola can inhibit inflammatory factor expression, thereby inhibiting inflammatory reaction to inhibit bone resorption.

Example 3: the Histicola can regulate local inflammatory reaction of intestinal tract

Intestinal specimens of the OVX + P. histicola group, the OVX + Vehicle group and the Sham + Vehicle group were taken, and evaluation of local inflammatory response of intestinal tracts was determined by mRNA expression of inflammatory factors associated with different intestinal tracts. As shown in fig. 3, no significant difference was shown between the three groups of mice in the duodenum. In the jejunum, the expression of IL-1. beta. was significantly increased in mice of the OVX + Vehicle group relative to those of the Sham + Vehicle group. In the ileum, the expression of IL-1. beta. and TNF. alpha. was significantly increased in mice of the OVX + Vehicle group relative to those of the Sham + Vehicle group. In the colon, the expression of TNF α was significantly increased in OVX + Vehicle group mice relative to Sham + Vehicle group mice. Treatment with the bacterium histicola is effective in ameliorating the over-expression of these inflammatory factors.

Example 4: histicola can reduce intestinal permeability of OVX mice

By examining the dextran concentration of intestinal specimens of OVX + p. histicola, OVX + Vehicle and Sham + Vehicle groups, it was found that the FITC-labeled dextran concentration in serum of mice of the OVX + Vehicle group was significantly increased relative to the Sham + Vehicle group as shown in fig. 4. The dextran concentration of OVX + P.histicola group is obviously reduced compared with that of OVX + Vehicle group, and the result shows that the dextran concentration can effectively reduce the increase of intestinal permeability caused by estrogen deficiency. Quantitative and positioning research on colon tight junction proteins ZO-1 and Occludin shows that estrogen deficiency can cause the level of the colon tight junction proteins to be reduced, so that the distribution of the colon tight junction proteins on the intestinal wall is widened, and P.histicola can keep the level of the colon tight junction proteins close to a normal level after the estrogen deficiency, so that the intestinal permeability is reduced, the invasion of pathogenic bacteria in the intestinal tract is inhibited, and the local inflammatory reaction of the intestinal tract is inhibited to inhibit bone absorption.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims

1. Application of Prevotella in preparing medicines for regulating bone metabolism.

2. The use of Prevotella according to claim 1 in the preparation of a medicament for the positive regulation of bone metabolism, wherein: the bone metabolism forward control drug is a drug for increasing bone mass or reducing bone resorption.

3. Use of prevotella for the preparation of a medicament for the positive regulation of bone metabolism according to claim 2, wherein said medicament for increasing bone mass is a medicament for increasing trabecular bone number and trabecular bone area and/or increasing relative bone volume fraction and/or increasing trabecular bone resolution and/or increasing trabecular bone thickness.

4. Use of Prevotella according to claim 2 in the preparation of a medicament for the positive regulation of bone metabolism, wherein the medicament for reducing bone resorption is a medicament for inhibiting increase in osteoclast number and/or reducing osteoclast area or a medicament for inhibiting increase in osteoclast number and/or reducing osteoclast area caused by estrogen deficiency.

5. Use of Prevotella for the preparation of a medicament for the positive control of bone metabolism according to claim 2, wherein the medicament for reducing bone resorption is a medicament for inhibiting bone resorption by reducing inflammatory response or a medicament for inhibiting bone resorption by reducing inflammatory response due to estrogen deficiency.

6. The use of Prevotella for the preparation of a medicament for the positive control of bone metabolism according to claim 5, wherein the medicament for reducing bone resorption is a medicament for inhibiting bone resorption by inhibiting the expression of IL-1 β and/or TNF α.

7. The use of Prevotella in the preparation of a medicament for the positive control of bone metabolism according to claim 5, wherein the medicament for decreasing bone resorption is a medicament for decreasing intestinal permeability by upregulating the expression of claudin and ZO-1, thereby decreasing the production of inflammatory factors in the intestinal wall to inhibit bone resorption.

8. The use of Prevotella according to claim 1 in the preparation of a medicament for the positive regulation of bone metabolism, wherein: the bone metabolism forward control medicament is a medicament for preventing or treating osteoporosis.

9. The use of Prevotella according to claim 8 in the preparation of a medicament for the positive regulation of bone metabolism, wherein: the medicament for preventing or treating osteoporosis is a medicament for preventing or treating osteoporosis caused by estrogen deficiency.

10. A pharmaceutical composition for treating osteoporosis caused by estrogen deficiency comprises Prevotella as active ingredient, and pharmaceutically acceptable carrier.