Application of lactobacillus jensenii-sourced nano-vesicles in preparation of medicine for preventing and / or treating preeclampsia

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of lactobacillus jensenii-sourced nano-vesicles in preparation of a medicine for preventing and / or treating preeclampsia. The lactobacillus jensenii-sourced nano-vesicles are obtained by separating culture supernate of strains, the components are natural, toxic and side effects are avoided, good biocompatibility and safety are achieved, in-vivo and in-vitro experiments prove that the lactobacillus jensenii-sourced nano-vesicles can effectively promote proliferation, clone formation and migration of HTR-8 / SVneo cells, and the application of the lactobacillus jensenii-sourced nano-vesicles to preparation of the nano-vesicles is promoted. The negative effects of proliferation inhibition, clone formation reduction, migration weakening, cell cycle arrest, scratch self-healing ability reduction and the like of HTR-8 / SVneo cells caused by LPS stimulation can be reversed, and the placenta injury caused by a PE animal model formed by LPS modeling can be protected, so that the application can be used for preventing and / or treating preeclampsia; a new theoretical basis is provided for application and prevention and treatment of preeclampsia of the probiotics, so that the bacillus subtilis has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of Lactobacillus jensenii-derived nanovesicles in the preparation of drugs for preventing and / or treating preeclampsia. Background Art

[0002] Preeclampsia (PE) is one of the common fetal complications during pregnancy in obstetrics. Its pathogenesis is complex, difficult to cure, and can have a huge adverse impact on the short-term and long-term prognosis of perinatal infants. However, the treatment options are limited and the curative effects are poor. Domestic and foreign studies have shown that due to reasons such as infertility treatment, multiple pregnancies, advanced maternal age, increased social pressure, nicotine use, or unbalanced nutrition, the incidence of PE has a further increasing trend. Currently, there is no complete mechanism to explain the occurrence and development of PE.

[0003] The existing treatment options for preeclampsia mainly include intervention in bad lifestyle and hormone intervention, but the curative effects are unstable and not good, and there is no drug with stable curative effect for clinical treatment. With the recent in-depth study of the gut microbiota, the relationship between the gut microbiota and preeclampsia has gradually been recognized. When preeclampsia occurs, the beneficial bacteria decrease and the harmful bacteria increase. At the same time, the increase in harmful bacteria will inversely regulate the immune aggression of the immune system on reproductive organs such as the ovaries and uterus, exacerbating the development of preeclampsia. Currently, studies have confirmed that the imbalance of the vaginal microbiota can lead to disorders of ovarian function and restricted uterine development, etc.; the vaginal microbiota not only changes during the female menstrual cycle, but also changes during the reproductive process. Lactobacilli that produce H 2 O 2 and lactic acid are the dominant bacteria in the vagina of healthy women and are important factors for protecting the female vagina from pathogen infection. In addition, the acids and some antimicrobial factors produced by the metabolism of lactobacilli can also effectively inhibit the growth and reproduction of other bacteria. However, currently, there is only one vaginal probiotic agent in China, and its main component is live Lactobacillus delbrueckii, which is not the dominant vaginal flora of Chinese women, has poor colonization ability, and cannot maintain a stable viable bacteria content, unable to meet the needs of gynecological clinics. Lactobacillus jensenii, belonging to the genus Lactobacillus, is one of the normal human flora, widely distributed in the human intestine and also distributed in the female vagina. There is no prior art research on the effect of Lactobacillus jensenii on preeclampsia.

[0004] Therefore, exploring the related action mechanism between the vaginal microbiota and PE and developing new preparations containing effective probiotic components will provide a new theoretical basis and research idea for the drug research and treatment of PE. Summary of the Invention

[0005] To overcome the above-mentioned disadvantages and deficiencies of the prior art, the primary object of the present invention is to provide the application of external vesicles of Lactobacillus Jenni origin (LJ-EVs) in the preparation of drugs for preventing and / or treating preeclampsia.

[0006] The object of the present invention is achieved by the following solutions:

[0007] The application of external vesicles of Lactobacillus Jenni origin (LJ-EVs) in the preparation of drugs for preventing and / or treating preeclampsia.

[0008] In the present invention, in vitro and in vivo experiments prove that LJ-EVs can effectively promote the proliferation, colony formation and migration of HTR-8 / SVneo cells, and have a protective effect on the placental damage caused by the PE animal model established by LPS modeling, and can be used for preventing and / or treating preeclampsia.

[0009] The application of external vesicles of Lactobacillus Jenni origin in the preparation of drugs for promoting the proliferation of HTR-8 / SVneo cells.

[0010] The application of external vesicles of Lactobacillus Jenni origin in the preparation of drugs for promoting the colony formation of HTR-8 / SVneo cells.

[0011] The application of external vesicles of Lactobacillus Jenni origin in the preparation of drugs for promoting the migration of HTR-8 / SVneo cells.

[0012] The application of external vesicles of Lactobacillus Jenni origin in the preparation of drugs for promoting the scratch self-healing ability of HTR-8 / SVneo cells.

[0013] The application of external vesicles of Lactobacillus Jenni origin in the preparation of drugs for reversing the proliferation inhibition of HTR-8 / SVneo cells induced by LPS.

[0014] The application of external vesicles of Lactobacillus Jenni origin in the preparation of drugs for reversing the decrease in colony formation of HTR-8 / SVneo cells induced by LPS.

[0015] The application of external vesicles of Lactobacillus Jenni origin in the preparation of drugs for reversing the weakened migration of HTR-8 / SVneo cells induced by LPS.

[0016] The application of external vesicles of Lactobacillus Jenni origin in the preparation of drugs for reversing the decrease in scratch self-healing ability of HTR-8 / SVneo cells induced by LPS.

[0017] In the technical solution of the present invention, the drugs, which may be the same or different, respectively include a therapeutically effective amount of Lactobacillus jensenii-derived nanovesicles.

[0018] In the technical solution of the present invention, the drugs, which may be the same or different, respectively can be made into various pharmaceutical dosage forms by conventional methods, and these dosage forms include: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal tablets, granules, infusion granules, pills, pellets, suspensions, medicinal wines, tinctures, drops and other oral dosage forms for oral administration, as well as dosage forms for administration other than oral administration such as injections, such as injections.

[0019] In the technical solution of the present invention, the drugs, which may be the same or different, respectively may further contain one or more pharmaceutically acceptable carriers or excipients.

[0020] Furthermore, the carrier or excipient may include diluents, wetting agents, binders, surfactants, humectants, adsorbent carriers, lubricants, fillers, disintegrants, preservatives, etc.

[0021] The Lactobacillus jensenii-derived nanovesicles (LJ-EVs) of the present invention can be obtained by the methods for preparing nanovesicles known to those skilled in the art, such as separating the culture supernatant obtained after culturing Lactobacillus jensenii.

[0022] Specifically, the Lactobacillus jensenii-derived nanovesicles (LJ-EVs) are obtained by separating the culture supernatant obtained after culturing Lactobacillus jensenii;

[0023] Furthermore, the precipitate obtained by separation is the Lactobacillus jensenii-derived nanovesicles.

[0024] Furthermore, the separation method may be to perform ultracentrifugation on the culture supernatant for separation.

[0025] Furthermore, the speed of the ultracentrifugation may be 100,000 g or above.

[0026] Furthermore, the time of the ultracentrifugation may be 30 - 180 min.

[0027] Furthermore, the time of the ultracentrifugation may be 60 - 160 min.

[0028] Furthermore, the precipitate obtained by ultracentrifugation can be resuspended with a buffer such as PBS to obtain a suspension of Lactobacillus jensenii-derived nanovesicles.

[0029] Furthermore, the ultracentrifugation can be repeated once or more to obtain further purified Lactobacillus jensenii-derived nanovesicles.

[0030] Furthermore, a filter can be used to filter out larger impurities before ultracentrifugation; the filter can be a 0.22 μm filter.

[0031] Furthermore, before ultracentrifugation, the culture supernatant can be pre-centrifuged to collect the supernatant and remove impurity precipitates.

[0032] Furthermore, the speed of pre-centrifugation can be 10000g or less.

[0033] Furthermore, the time of pre-centrifugation can be 5 - 60 min.

[0034] Furthermore, pre-centrifugation can be performed once or more. Through pre-centrifugation, non-target substances can be separated and removed, such as removing bacteria, reducing the influence of impurities on subsequent ultracentrifugation separation.

[0035] Furthermore, the culture supernatant is obtained by removing bacteria after culturing Lactobacillus jensenii.

[0036] Furthermore, Lactobacillus jensenii is cultured using BHI medium.

[0037] Furthermore, the culture is stopped when the OD value of the bacteria reaches 2 - 3.

[0038] Furthermore, it is preferred to strictly control the oxygen content during the culture process.

[0039] In the present invention, Lactobacillus jensenii-derived nanovesicles are obtained by separating the culture supernatant of the strain. The components are natural, non-toxic and have no side effects, with good biocompatibility and safety. In vitro and in vivo experiments prove that it can effectively promote the proliferation, colony formation and migration of HTR-8 / SVneo cells, and has a protective effect on the placental damage caused by the PE animal model established by LPS modeling, indicating that it can be used for the prevention and / or treatment of preeclampsia, providing a new theoretical basis for the application and prevention and treatment of probiotics in preeclampsia, and thus having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0041] Figure 1 It is a transmission electron microscope image of Lactobacillus jensenii-derived nanovesicles (LJ-EVs).

[0042] Figure 2 It is a particle size diagram of Lactobacillus jensenii-derived nanovesicles (LJ-EVs).

[0043] Figure 3 Uptake of LJ-EVs by human embryonic trophoblast cells (HTR-8 / SVneo).

[0044] Figure 4 Effect of LJ-EVs on the proliferation ability of HTR-8 / SVneo cells.

[0045] Figure 5 Effect of LJ-EVs on the colony formation of HTR-8 / SVneo cells.

[0046] Figure 6 Effect of LJ-EVs on the migration ability of HTR-8 / SVneo cells.

[0047] Figure 7 Effect of LJ-EVs on the cell cycle of HTR-8 / SVneo cells.

[0048] Figure 8 Effect of LJ-EVs on the scratch healing ability of HTR-8 / SVneo cells.

[0049] Figure 9 Distribution map of LJ-Evs in mouse organs.

[0050] Figure 10 Uptake of LJ-Evs by placental tissue.

[0051] Figure 11 Effect of LJ-EVs on the proliferation ability of HTR-8 / SVneo cells in an in vitro cell model of LPS-induced preeclampsia (PE).

[0052] Figure 12 Effect of LJ-EVs on the colony formation of HTR-8 / SVneo cells in an in vitro cell model of LPS-induced preeclampsia (PE).

[0053] Figure 13 Effect of LJ-EVs on the migration ability of HTR-8 / SVneo cells in an in vitro cell model of LPS-induced preeclampsia (PE).

[0054] Figure 14 Effect of LJ-EVs on the cell cycle of HTR-8 / SVneo cells in an in vitro cell model of LPS-induced preeclampsia (PE).

[0055] Figure 15 Effect of LJ-EVs on the scratch healing ability of HTR-8 / SVneo cells in an in vitro cell model of LPS-induced preeclampsia (PE).

[0056] Figure 16It is the placental changes in the PE mouse model induced by LPS.

[0057] Among them, *p < 0.05, **p < 0.01, ***p < 0.001. Specific implementation manners

[0058] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto. The materials involved in the following embodiments can be obtained from commercial channels without special instructions. The methods are conventional methods without special instructions.

[0059] The human embryonic trophoblast cells (HTR-8 / SVneo) used in the embodiments were purchased from Shanghai Fuheng Biotechnology Co., Ltd. and were correctly identified by STR.

[0060] The resuscitated HTR-8 / SVneo cell line was cultured in an incubator under conventional conditions. The cells have the characteristic of adherent growth. The HTR-8 / SVneo cells were cultured with 1640 medium (5% fetal bovine serum). The medium was changed according to the cell growth conditions. Generally, when the cell density reached 60%-70%, the cells could be passaged for subsequent experiments.

[0061] Example 1

[0062] The Lactobacillus jensenii-derived nanovesicles (LJ-EVs) were obtained by separating the culture supernatant obtained after culturing Lactobacillus jensenii, and were prepared by the following specific steps and methods:

[0063] (1) Continuously culture Lactobacillus jensenii (a conventionally commercially available strain, such as strain number SHBCC D24914, Chinese name Lactobacillus jensenii SHBCC D24914, Latin name Lactobacillus jensenii) using BHI medium;

[0064] (2) Stop culturing when the OD value of the bacteria is 2-3;

[0065] (3) After the culture is completed, collect the culture supernatant and perform pre-centrifugation treatment, such as centrifuging at 10000g or below for 5-60 min. The pre-centrifugation can be repeated once or more (such as centrifuging at 300g for 10 min, the supernatant is centrifuged at 1200g for 10 min, and the supernatant is centrifuged at 10000g for 30 min) to remove insoluble small particles such as bacterial fragments, collect the supernatant, and filter it using a 0.22 μm filter head to obtain the culture supernatant;

[0066] (4) Ultracentrifugation separation: The culture supernatant was ultracentrifuged at 100,000 g or above for 30 - 180 min. For example, when centrifuged at 100,000 g for 120 min, ultracentrifugation can be repeated once or more. The supernatant was discarded, and the obtained precipitate was Lactobacillus jensenii - derived nanovesicles (LJ - EVs); resuspended with PBS buffer to obtain a suspension of Lactobacillus jensenii - derived nanovesicles.

[0067] The same method was used to treat Escherichia coli (ATCC 25922) to obtain Escherichia coli nanovesicles (E.coil - OMVs) as a control.

[0068] The morphology of Lactobacillus jensenii - derived nanovesicles (LJ - EVs) was identified by transmission electron microscopy. It can be observed by transmission electron microscopy that LJ - EVs have the characteristics of the phospholipid bilayer of extracellular membrane vesicles, are spherical or elliptical in shape, with a double - membrane structure, and the diameter is distributed between 50 - 200 nm. The results are shown in Figure 1 ;

[0069] The particle size of LJ - EVs in the suspension was measured using a nanoparticle tracking analyzer. The results showed that the particle size of LJ - EVs was mainly distributed between 50 - 200 nm, and there was an obvious enrichment around 100 nm, with a nanoparticle size characteristic of exosomes. The results are shown in Figure 2 .

[0070] Example 2: Uptake identification of LJ - EVs by human embryonic trophoblast cells (HTR - 8 / SVneo)

[0071] (1) DIL - fluorescently labeled LJ - EVs: 10 mg of DIL dye (Fushen Biotech) was co - incubated with 10 10 particle numbers of LJ - EVs at 37 °C in the dark for 30 minutes to allow LJ - EVs to fully bind to DIL; the above - mentioned mixture was ultracentrifuged again at a centrifugal force of 130,000 g for 70 minutes. The supernatant was discarded, and the precipitate was taken and resuspended with 1 mL of PBS to obtain DIL - LJ - EVs;

[0072] (2) Human embryonic trophoblast cells (HTR - 8 / SVneo) were seeded in a 6 - well plate in advance. When the cell confluence reached 50 - 60%, 10 μL of DIL - LJ - EVs was added, and the cells were incubated at 37 °C in the dark for 6 hours; washed 2 - 3 times with PBS, 2 mL of basic 1640 medium was added to each well, and 10 μL of 1 mg / mL DAPI was added, and then placed in an incubator for 30 - 40 mins;

[0073] (3) Detection by machine: Using a fluorescence microscope (Shunyu), different magnifications were taken, and the incubation of PBS with DIL was used as a negative control; the results are asFigure 3 .

[0074] As can be seen from the figure, DIL-LJ-EVs can be taken up by HTR-8 / SVneo cells and emit fluorescence of DIL dye intracellularly, while no fluorescence was observed in the PBS control group. This result confirms that DIL-LJ-EVs can be taken up by HTR-8 / SVneo cells and enter the cells.

[0075] Example 3: Effect of LJ-Evs on the proliferation ability of HTR-8 / SVneo cells

[0076] (1) The protein concentration of exosomes was measured by BCA. Complete cell culture media containing exosomes (LJ-Evs, E.coil-OMVs) were prepared in advance at a final concentration of 10 μg / mL respectively.

[0077] (2) HTR-8 / SVneo cells in good growth state were plated in advance at a density of 10 3 cells / well, using 96-well plates for plating. According to not less than 5 replicates per group for each time period, a total of 4 - 5 time periods were set. After continuous culture for 12 h, when the cells were completely adherent, the culture medium was replaced with the medium containing the supernatant of bacterial exosomes, and the negative group (NC) was replaced with normal complete medium; E.coil-OMVs was used as the control group.

[0078] (3) At the designed time, when the stimulation time reached, the cell plates of that time period were taken. CCK8 solution was added at 10 μL per well and incubated in the dark for 1 h. The absorbance of each well at a wavelength of 450 was measured using an enzyme-linked immunosorbent assay (ELISA) reader; recorded and analyzed to plot the cell proliferation curve; the results are shown in Figure 4 .

[0079] As can be seen from the figure, LJ-Evs significantly promoted the proliferation of HTR-8 / SVneo cells, and there were differences compared with E.coil-OMVs. Excluding non-specific results, it was clear that LJ-EVs had the effect of promoting the proliferation of HTR-8 / SVneo cells.

[0080] Example 4: Effect of LJ-Evs on the colony formation of HTR-8 / SVneo cells

[0081] (1) Take out the cells of each group that meet the experimental conditions after culturing, digest with trypsin, centrifuge, and resuspend with the medium to obtain a cell suspension, and adjust the density to 10 3 cells / mL. 400 cells were seeded in each well of a 6-well culture plate, and finally each well was filled with conventional medium to a final volume of 2 mL; the exosome group was replaced with complete cell culture medium containing exosomes prepared in advance at a final concentration of 10 μg / mL, the negative group (NC) was replaced with normal complete medium; complete cell culture medium containing the same concentration of E.coil-OMVs was used as the control group;

[0082] (2) Incubate for 2 - 3 weeks, observe the cell growth and proliferation under an inverted optical microscope every day, and change the medium every two days; when obvious clones are visible to the naked eye, fix with a fixing solution and perform staining and photography; the results are shown in Figure 5 .

[0083] As can be seen from the figure, LJ-Evs significantly promoted the colony formation of HTR-8 / SVneo cells, and there were differences compared with E.coil-OMVs. After excluding non-specific results, it was clear that LJ-EVs had the effect of promoting the colony formation of HTR-8 / SVneo cells.

[0084] Example 5: Effect of LJ-Evs on the migration ability of HTR-8 / SVneo cells

[0085] (1) Take out the cells of each group that meet the experimental conditions, digest with trypsin, centrifuge, and resuspend with the medium to obtain a cell suspension, and adjust the density to 10 5 cells / mL. Take out the transwell plate, add 100 μL of the cell suspension to the upper chamber of the transwell insert, and make up to 200 μL with serum-free medium; for the exosome group, add exosomes (LJ-Evs, E.coil-OMVs) respectively to make the final concentration of exosomes in the medium in the insert reach 10 μg / mL;

[0086] (2) Add 500 μL of complete medium to the lower chamber; transfer the transwell plate to the incubator, and after continuous culture for 24 h, take it out, discard the medium in the upper and lower chambers, add a fixing solution to fix the insert, gently wipe off the cells in the upper chamber with a cotton swab, and wash twice with PBS;

[0087] (3) After fixing for 30 min, discard the fixing solution, add 1% crystal violet solution for staining, after 30 min, discard the staining solution, and wash the insert 3 times with PBS; take pictures with a microscope and count; the results are shown in Figure 6 .

[0088] As can be seen from the figure, LJ-Evs significantly promoted the migration of HTR-8 / SVneo cells, and there were differences compared with E.coil-OMVs. After excluding non-specific results, it was clear that LJ-EVs had the effect of promoting the migration of HTR-8 / SVneo cells.

[0089] Example 6: Effect of LJ-Evs on the cell cycle of HTR-8 / SVneo cells

[0090] (1) Measure the protein concentration of exosomes by BCA, and prepare complete cell culture media containing exosomes (LJ-Evs, E.coil-OMVs) in advance at a final concentration of 10 μg / mL respectively;

[0091] (2) Plate the well - growing HTR - 8 / SVneo cells in advance at a density of 10 5 cells per well. Use 6 - well plates for plating. After continuous culture for 12 h, wait until the cells are completely adherent, then replace the culture medium with the medium containing exosomes respectively. The negative control group (NC) replaces the normal complete medium; continuously culture for 48 h, digest the cells in each well of the 6 - well plate with trypsin, and resuspend them with the complete medium;

[0092] (3) Take 500 μL of the cell suspension and place it in a 1.5 - mL EP tube, one tube for each group of cells. Add frozen methanol solution to each tube for fixation overnight;

[0093] (4) After fixation overnight, centrifuge to remove the methanol solution, resuspend the cells with 1x binding buffer, add PI dye, and perform light - avoiding staining for 30 min; detect by flow cytometry, collect the cell cycle data of each group for statistical analysis, and the results are shown in Figure 7 .

[0094] As can be seen from the figure, LJ - Evs significantly promotes the accumulation of HTR - 8 / SVneo cells in the G1 phase, while reducing the arrest in the S phase and increasing the enrichment in the G2 phase; taking E. coil - OMVs as a control to remove the non - specific results of vesicles from bacteria, the effect of LJ - Evs on the cell cycle is clarified.

[0095] Example 7: Effect of LJ - Evs on the scratch - healing ability of HTR - 8 / SVneo cells

[0096] (1) Plate the well - growing HTR - 8 / SVneo cells in advance at a density of 10 5 cells per well. Use 24 - well plates for plating and continuously culture until the cell confluence reaches more than 95%;

[0097] (2) Replace the culture medium with serum - free basal medium, use a pipette tip or a scriber to make scratches, wash twice with PBS after scratching. The control group (NC) replaces the serum - free basal medium, and the exosome groups (LJ - Evs, E. coil - OMVs) are respectively added with the medium prepared by using serum - free basal medium and exosomes at a final concentration of 10 μg / mL;

[0098] (3) Take pictures at different time intervals and statistically analyze the scratch - healing situation; the results are shown in Figure 8 .

[0099] As can be seen from the figure, LJ-Evs significantly promoted the scratch self-healing of HTR-8 / SVneo cells; using E.coil-OMVs as a control to remove the non-specific results of vesicles from bacteria, the role of LJ-Evs in promoting the scratch self-healing of HTR-8 / SVneo cells was clarified.

[0100] Example 8: Tracing the organ distribution of LJ-EVs in animals after intraperitoneal injection by in vivo imaging technology

[0101] (1) The preparation of DIL-LJ-EVs was the same as step (1) of Example 2; the protein concentration was measured by BCA, and a PBS solution containing DIL-LJ-EVs was prepared at a final concentration of 10 μg / mL.

[0102] (2) The animals were C57 mice, which were intraperitoneally injected at a dose of 1 mL / Kg. After 2 h, the animals were anesthetized with 10% chloral hydrate at a dose of 100 μL / Kg. After the animals were completely anesthetized, the experimental animals were placed in a small animal in vivo imaging instrument for on-machine detection; the results are shown in Figure 9 .

[0103] As can be seen from the figure, after intraperitoneal injection, LJ-EVs can be enriched in various organs, enter the blood, and exert biological functions.

[0104] Example 9: Uptake identification of LJ-EVs by placental tissue

[0105] (1) The preparation of DIL-LJ-EVs was the same as step (1) of Example 2; the protein concentration was measured by BCA, and a PBS solution containing DIL-LJ-EVs was prepared at a final concentration of 10 μg / mL.

[0106] (2) The animals were C57 pregnant mice. Pregnant mice with a gestational age of 9-12 days were selected and intraperitoneally injected at a dose of 1 mL / Kg respectively; they were continuously fed normally for 1 day, and then the experimental animals were sacrificed.

[0107] (3) The mice were quickly dissected and the target tissues such as ovaries and uterus were removed; some tissues were cut open with tissue scissors, washed with PBS, frozen at -80 °C for WB use, some were soaked in 10% neutral formalin for fixation, and some were soaked in 30% sucrose solution. There should be enough fixative, generally more than 10 times the volume of the tissue block. For the tissue fixed in the sucrose solution, it should be quickly transferred to the -80 refrigerator for freezing after 4 h of fixation.

[0108] (4) After the tissue was fixed and frozen hard, it was sectioned on a cryostat.

[0109] (5) The sections were laid flat on glass slides and observed and photographed under an upright fluorescence microscope; the results are shown in Figure 10 .

[0110] In this example, placental tissue sections were prepared by cryosectioning technology and observed in combination with an inverted fluorescence microscope to examine the uptake of LJ-EVs by placental tissue. As shown in the figure, LJ-EVs could reach the placenta of pregnant mice, and the sections showed that LJ-EVs could be significantly endocytosed by placental villous cells.

[0111] Example 10: Regulatory effect of LJ-EVs on the PE cell model induced by LPS

[0112] (1) Grouping: Control group (NC, cultured in normal complete medium); LPS group (10 μg / mL); LJ-EVs group (medium with a concentration of 10 μg / mL); LPS + LJ-EVs group (cultured in medium with a concentration of 10 μg / mL of LJ-EVs after LPS treatment);

[0113] (2) HTR-8 / SVneo cells were continuously cultured with medium containing LPS (10 μg / mL) for 24 h to induce the formation of an in vitro cell model of PE in HTR-8 / SVneo cells;

[0114] (3) Referring to Examples 3-7, the proliferation ability, colony formation, migration ability, cell cycle, scratch healing ability, etc. of the cells were observed, and the results are shown in Figures 11 - 15 .

[0115] As shown in the figure, LJ-EVs could effectively reverse the effects of LPS-induced inhibition of HTR-8 / SVneo cell proliferation, decreased colony formation, weakened migration of villous cells, cell cycle arrest, and decreased scratch healing ability, indicating that LJ-EVs had an effective protective effect on cell damage caused by LPS at the in vitro cell level.

[0116] Example 11: Regulatory effect of LJ-EVs on the PE animal model induced by LPS

[0117] (1) The animals were pregnant C57 mice, with 8 mice in each group. Pregnant mice with a gestational age of 9-12 days were selected for intervention, and the gestational age was counted from the day when obvious sperm plugs were formed in the mice as the starting day of pregnancy;

[0118] (2) Pregnant mice were intraperitoneally injected with LPS at a dose of 1 mg / Kg; fed normally continuously; drug intervention was carried out at mid-pregnancy, i.e., on days 12-15, and the drug was administered continuously for 3 days to form a placental inflammation model to achieve the effect of the PE model;

[0119] (3) After modeling by injecting LPS in step (2), pregnant mice modeled with LPS were intraperitoneally injected with LJ-EVs at a dose of 1 mg / Kg as the LJ-EVs+LPS group; pregnant mice without modeling were intraperitoneally injected with LJ-EVs at a dose of 1 mg / Kg as the LJ-EVs group; the injection was performed once every 5 days.

[0120] (4) Feed continuously for 4 weeks, and end the experiment on the 2nd day after the last administration. All mice were weighed on the day of the end of the experiment. Blood was collected from the orbital cavity of mice: The mice to be experimented were anesthetized with ether. During the experiment, pay attention to the anesthesia time of the mice and observe the state of the mice after anesthesia to avoid failure of blood collection due to blood coagulation. During blood collection, a capillary tube about 1 cm long was inserted under the lower orbital cavity of the mice, and the capillary tube was quickly aligned with the EP tube mouth, and the capillary tube was gently rotated to allow the blood to flow out into the centrifuge tube (during blood collection, the heart can be gently pressed with the index finger to promote blood flow). The centrifuge tube containing blood was left standing at room temperature for half an hour until obvious stratification of the blood occurred, and then centrifuged at high speed for 30 minutes. After centrifugation, the upper serum was aspirated into a newly coded centrifuge tube and stored in a -80°C refrigerator. After blood collection, the animals were sacrificed by cervical dislocation, and bilateral ovaries, uterus, placenta, etc. were fixed in 4% paraformaldehyde solution overnight, and the other half of each organ was frozen at -80°C for subsequent experiments.

[0121] (5) HE analysis of the placental inflammation level;

[0122] The results are shown in Table 1 and Figure 16 as follows. The HE results showed that in the LPS group, the placental villi were underdeveloped, and obvious atrophy occurred in the placental villous layer. Compared with the LJ-EVs group, the villous layer in the LJ-EVs group was full and rich in blood vessels. Compared with the LPS group, the atrophy of the villous layer improved in the LPS+LJ-EVs group; in the statistical table of the pregnancy period of mice, it was found that the placental volume and weight in the LPS+LJ-EVs group showed a significant recovery compared with the LPS group, indicating that LJ-EVs can effectively protect the placenta from damage caused by LPS.

[0123] Table 1

[0124]

[0125]

[0126] Note: *The gestational age at termination refers to the time of delivery or miscarriage of pregnant mice, which is regarded as the time of pregnancy termination.

[0127] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of Lactobacillus jensenii-derived nanovesicles in the preparation of drugs for preventing and / or treating pre-eclampsia.

2. Application of Lactobacillus jensenii-derived nanovesicles in the preparation of drugs that promote the proliferation, cloning or migration of HTR-8 / SVneo cells.

3. Application of Lactobacillus jensenii-derived nanovesicles in the preparation of drugs that promote self-healing of HTR-8 / SVneo cell scratches.

4. Application of Lactobacillus jensenii-derived nanovesicles in the preparation of drugs for reversing LPS-induced proliferation inhibition, decreased colony formation or weakened migration of HTR-8 / SVneo cells.

5. Application of Lactobacillus jensenii-derived nanovesicles in the preparation of drugs for reversing LPS-induced decreased scratch self-healing ability of HTR-8 / SVneo cells.

6. The use according to any one of claims 1 to 5, characterized in that: The medicine comprises a therapeutically effective amount of nanovesicles derived from Lactobacillus jensenii.

7. The use according to any one of claims 1 to 5, characterized in that: The drug is prepared into various pharmaceutical dosage forms by conventional methods, including tablets, capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, wine preparations, tinctures, drops and other dosage forms for oral administration such as injections.

8. The use according to any one of claims 1 to 5, characterized in that: The drug is prepared into various pharmaceutical dosage forms by conventional methods, including: sugar-coated tablets, film-coated tablets, enteric-coated tablets, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, wine preparations, tinctures, oral dosage forms of drops and dosage forms other than oral administration of injections.

9. The use according to any one of claims 1 to 5, characterized in that: The medicine also contains one or more pharmaceutically acceptable carriers or excipients.

10. The use according to claim 9, characterized in that: The carrier or auxiliary material includes at least one of a diluent, a wetting agent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, a disintegrant, and a preservative.

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