Application of proteus mirabilis outer membrane vesicle in preparation of medicine for preventing or treating osteolytic diseases

A technology of Proteus mirabilis and outer membrane vesicles, applied in the field of application of Proteus mirabilis outer membrane vesicles in the preparation of drugs for the prevention or treatment of osteolytic diseases, to prevent or treat osteolytic diseases and improve bone metabolism imbalance Effect

Pending Publication Date: 2021-12-24
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the effect of bacterial OMVs

Method used

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  • Application of proteus mirabilis outer membrane vesicle in preparation of medicine for preventing or treating osteolytic diseases
  • Application of proteus mirabilis outer membrane vesicle in preparation of medicine for preventing or treating osteolytic diseases
  • Application of proteus mirabilis outer membrane vesicle in preparation of medicine for preventing or treating osteolytic diseases

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Example 1: Extraction and Identification of Proteus mirabilis Outer Membrane Vesicles (P.M OMVs)

[0057] 1. Bacterial culture

[0058] A single colony of Proteus mirabilis (P. mirabilis) was picked, put into BHI broth medium, and cultured at 37° C. for 16-18 hours under aerobic conditions. After the cultivation, the microplate reader detects its absorbance (OD) at a wavelength of 600nm, and when the OD value is 0.6-0.8, the bacteria are collected.

[0059] 2. Extraction, Identification and Quantification of Bacterial Outer Membrane Vesicles

[0060] After the collected bacteria were centrifuged three times at 4°C and 8000rpm, the supernatant was collected, filtered through a 0.22 μm filter, and 200 μl of the bacterial liquid was spread on an LB agar culture plate before and after filtration and placed in a constant temperature incubator at 37°C for 12 hours, the supernatant after filtration has no bacterial growth on the culture plate, and when the bacteria before fi...

Embodiment 2

[0063] Example 2: P.M OMVs inhibit RANKL-induced osteoclast differentiation, fusion and resorption activity

[0064] 1. The chromogenic endotoxin quantification (LAL) kit was used to determine the LPS content in P.M OMVs, which contained 2.64 EU endotoxin per 0.15g protein. The effects on BMMs after treatment with different concentrations of P.M OMVs and standard P.M LPS for 48, 72 and 96 hours were detected by CCK8. The specific operation is as follows:

[0065] BMMs cells were divided into 5×10 4 Seed in a 96-well plate at a density of 100 μl per well, add the prepared OMVs and LPS of various concentration gradients to the cells and incubate for 48, 72 and 96 hours, add 10 μl CCK-8 to each well, 5% CO 2 , OD values ​​were measured at 450 nm after incubation at 37°C for 3 hours. The experiment was repeated three times, and the percentage of cell viability was taken as a positive control without stimulation.

[0066] Cell viability percentage (%)=[(OD value of experimental...

Embodiment 3

[0087] Embodiment 3: P.M OMV can improve the bone loss of osteoporosis caused by OVX

[0088] C57 female mice of 8-10 weeks were intraperitoneally injected with the volume of 4% chloral hydrate (chloral hydrate volume (μl)=mouse body weight (g)×10), and after 10 minutes, the mice were in a state of deep anesthesia to start the operation. Determine the position of the mouse ovary on the back, cut the mouse skin, fat and muscle layer, find the mouse ovary, ligate and remove the ovary, suture the mouse muscle layer, fat layer and skin layer, and use alcohol cotton balls to stop bleeding, the same The other side of the ovary was removed by the same method, and the sham operation (Sham) group did not remove the ovary. After removing the ovary, the mice were kept warm under an infrared treatment lamp. After 12 hours, the mice were routinely fed. Inject PBS and OMVs once a week, and divide them into three groups: Sham+PBS group, OVX+PBS group, OVX+P.M OMVs group. After 8 weeks, mice ...

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Abstract

The invention discloses application of proteus mirabilis outer membrane vesicle in preparation of a medicine for preventing or treating osteolytic diseases. The proteus mirabilis outer membrane vesicle inhibits miR96-5p expression and promotes Abca1 expression, thereby inhibiting an MAPK/ERK pathway and causing osteoclast differentiation to be blocked; the proteus mirabilis outer membrane vesicle induces release of MPT-related cytochrome c to cause a mitochondrial structure to be damaged, increase production of active oxygen and cause increase of osteoclast apoptosis. The proteus mirabilis outer membrane vesicle not only significantly inhibits differentiation and functions of RANKL-induced osteoclasts in vitro, but also can improve bone metabolism imbalance caused by OVX and bone erosion caused by CII in vivo, and provides a new idea for preventing or treating the osteolytic diseases.

Description

technical field [0001] The invention belongs to the technical field of bone disease treatment, and in particular relates to the application of Proteus mirabilis outer membrane vesicles in the preparation of drugs for preventing or treating osteolytic diseases. Background technique [0002] The bone in the living body is undergoing the process of bone resorption and bone regeneration all the time. This process is called bone remodeling. The balance of bone remodeling plays an important role in bone homeostasis. Osteoclasts and osteoblasts are both involved in the bone remodeling process. The function of osteoclasts is to absorb old bone tissue, while the function of osteoblasts is to generate new bone. The two are in balance to maintain the balance of bone remodeling. The abnormal increase of osteoclast proliferation and activity leads to the speed of bone resorption far exceeding the speed of bone formation, breaking the balance of bone remodeling, resulting in the destructi...

Claims

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Application Information

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IPC IPC(8): A61K35/74A61P19/08
CPCA61K35/74A61P19/08
Inventor 库蒂·塞尔瓦·南达库玛王婷婷
Owner SOUTHERN MEDICAL UNIVERSITY
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