CN111560425A - Application of sFgl2 in detection, prevention, alleviation and treatment of atherosclerotic diseases - Google Patents

Abstract

The invention discloses application of sFgl2 in detection, prevention, alleviation and treatment of atherosclerotic diseases. With ApoE^‑/‑And Fgl2^TgApoE^‑/‑The mouse is an animal experimental object, and the result shows that compared with a control group mouse, the aortic plaque area of the sFgl2 overexpression group mouse is obviously reduced, and the plaque stability is obviously improved. Furthermore, sFgl2 can be reduced by decreasing Ly6C in the innate immune response of AS^highM1 type M_OThe ratio of M phi and promotes M2 type M phi differentiation; in the adaptive immune response of AS, sFgl2 can increase the number of Treg cells and immunosuppressive function; in the metabolic reaction of AS, sFgl2 can reduce LDL-C levels to alleviate AS. Therefore, the sFgl2 has multi-dimensional effect in the generation and development of AS, and can be used for detection, prevention and alleviationAnd anti-inflammatory/metabolic targets for treating AS disease.

Description

Application of sFgl2 in detection, prevention, alleviation and treatment of atherosclerotic diseases

Technical Field

The invention relates to the technical field of medical experiments, in particular to application of sFgl2 in detection, prevention, alleviation and treatment of atherosclerotic diseases.

Background

In recent years, the prevalence of atherosclerotic diseases, namely AS disease (i.e., coronary heart disease, stroke and peripheral vascular disease), is still in a continuously rising stage in China and worldwide, with the high mortality rate of AS disease being the leading position, higher than that of tumors and other diseases, and about 2 out of 5 deaths die of AS. AS disease mortality, morbidity, and prevalence continue to increase, a heavy social and economic burden has been placed. Domestic and foreign studies indicate that AS diseases are mainly mediated by two pathological processes, namely inflammatory reaction and lipid metabolism. In AS diseases, LDL protein in blood is oxidized into ox-LDL protein and then penetrates under the intima of blood vessel wall to trigger the innate immune response mainly mediated by MO/M phi on one hand and the adaptive immune response mainly mediated by Treg/Teff on the other hand, and influence the blood fat level and function at the same time. The inflammation reaction and the blood fat metabolism act together to gradually initiate and aggravate the AS disease.

At present, common medicaments aiming at inflammatory reaction are lipid-lowering medicaments represented by statins/fibrates and anti-platelet aggregation medicaments represented by aspirin/clopidogrel, the curative effect has certain limitation in the treatment process of the AS disease, and the exploration aiming at inflammatory treatment targets such AS phosphatase A2(PLA2), Leukotrienes (LTs) and monocyte chemotactic protein 1(MCP-1) and the like fails in succession, so that the search for more effective new anti-inflammatory intervention targets in the AS disease or targets with dual effects of anti-inflammation and metabolism regulation is urgent to solve the treatment problem of the AS disease.

The application publication No. CN103463620A discloses the application of anti-inflammatory protein TIPE2 in preparing medicine for treating atherosclerosis, and the recombinant adenovirus of TIPE2 is used to realize the anti-inflammatory protein in ApoE^-/-In vivo of mouseThe TIPE2 anti-inflammatory protein is efficiently, stably and continuously expressed, and the function of inhibiting the formation of the atheromatous plaque is played. However, it lacks the study of the effective functions and mechanisms in anti-inflammatory or anti-inflammatory/metabolic dual roles in AS disease, nor can it be used AS a biological marker to predict plaque stability in AS disease in individuals.

The sFgl2 is a newly discovered effector in the downstream of Treg, and has certain anti-inflammatory effect in viral hepatitis, autoimmune nephropathy and heart transplantation. However, the effective function and mechanism of action of sFgl2 in more complex stromal environments of vascular diseases such AS anti-inflammatory effects in AS has not been studied and reported.

Disclosure of Invention

Technical problem to be solved

The invention aims to provide application of sFgl2 in detecting, preventing, relieving and treating atherosclerotic diseases, explores effective functions and mechanisms of sFgl2 in anti-inflammatory or anti-inflammatory/metabolic dual actions in vascular diseases with more complex matrix environment such AS AS, and solves the problem of anti-inflammatory metabolic targets or lack of anti-inflammatory/metabolic dual treatment targets in the current AS disease treatment.

(II) technical scheme

In order to realize the application of the sFgl2 in detecting, preventing, relieving and treating atherosclerotic diseases, explore the effective functions and action mechanisms of the sFgl2 in the anti-inflammatory action of vascular diseases with more complex matrix environment, such AS AS, and solve the problem that the anti-inflammatory target or the anti-inflammatory/metabolic dual treatment target is deficient in the treatment of the existing AS diseases, the invention provides the following technical scheme:

the application of sFgl2 in the following I and II:

i, application in preparing medical products for detecting, preventing, relieving and treating AS diseases;

and II, application of the polypeptide in preparing a medical product for predicting the stability of the AS plaque in an individual.

Preferably, the sFgl2 is specifically expressed in a population with unstable AS plaques.

Preferably, the stability of the AS plaque in the individual is predicted by measuring the expression of sFgl2 in a blood sample or a blood vessel wall tissue sample of the individual, and the stability of the AS plaque is predicted by detecting the content of secreted sFgl2, so AS to evaluate the incidence degree of the AS disease.

Preferably, the medical product for preventing, alleviating and treating AS diseases is realized by acting on sFgl2 target, specifically acting to increase secreted sFgl2 level and promote differentiation of M phi in blood to anti-inflammatory M2 phenotype, so that Foxp3⁺The number and immunosuppressive functions of tregs are increased, M1 cells are reduced, and LDL-C lipids in the blood are reduced.

Preferably, the medical product comprises, but is not limited to, the following ingredients: cells that can secrete sFgl2, such as tregs and CTL-associated cells; fgl2 gene sequence is coated by virus or bacterial vector, such as adenovirus-Fgl 2, lentivirus-Fgl 2, adeno-associated virus-Fgl 2 or Fgl2 plasmid; sFgl2 protein secreted by Escherichia coli or CHO cell of recombinant Fgl2 plasmid; or one of the other pathways that up-regulate sFgl2 gene or protein expression.

Preferably, the medical product forms include, but are not limited to, OTC tablets, injectable drugs, targeted drugs, capsules, drug delivery robots, vesicles, and rapid test cassettes.

Preferably, the relevant mechanism and action principle of the medical product for detecting, preventing, relieving and treating AS disease are obtained by the research of mouse animal contrast experiment, and the specific mouse animal contrast experiment scheme is AS follows:

by ApoE^-/-And Fgl2^TgApoE^-/-The mice are animal subjects, and the mice are transplanted with bone marrow (receptor ApoE after irradiation)^-/-Mice, each receiving donor ApoE^-/-Or Fgl2^TgApoE^-/-Bone marrow cells of mice), 12W is fed on western diet to obtain an AS model, and the result shows that compared with the control group of mice, the aortic plaque area of the mice of the sFgl2 overexpression group is obviously reduced, and the plaque stability is obviously improved; also, sFgl2 decreased Ly6C in the innate immune response of AS^highThe ratio of MO to M phi in the form of/M1, and promoting the differentiation of M2M phi; in the adaptive immune response of AS, sFgl2 can increase the number of Treg cells and immunosuppressive function; in thatIn the metabolic reaction of AS, sFgl2 can reduce LDL-C level to relieve AS; the sFgl2 has multi-dimensional function in the generation and development of AS, and is used AS an anti-inflammatory/metabolic regulation target for detecting, preventing, relieving and treating AS diseases.

Further, the application of the sFgl2 in preparing products for regulating and controlling MO/M phi cell polarity, T cell differentiation to Treg/Teff cells and blood lipid level in AS diseases is also included.

(III) advantageous effects

Compared with the prior art, the invention provides the application of sFgl2 in detecting, preventing, relieving and treating atherosclerotic diseases, and has the following beneficial effects:

(1) in the invention, in the innate immune response of AS, sFgl2 can be reduced by Ly6C^highM1 type M_OThe ratio of M phi and promotes M2 type M phi differentiation; in the adaptive immune response of AS, sFgl2 can increase the number of Treg cells and immunosuppressive function; in the metabolic reaction of AS, sFgl2 can reduce LDL-C levels to alleviate AS. Therefore, the sFgl2 has multidimensional function in the generation and development of AS, and can be used AS an anti-inflammatory/metabolic regulation target for preventing, relieving and treating AS diseases.

(2) The invention provides sFgl2 which can be used AS a biological marker for predicting the AS plaque stability in an individual, and the experiment shows that the sFgl2 over-expresses the aortic plaque area of a group of mice to be obviously reduced and the plaque stability is obviously improved. Plaque stability in AS disease is assessed by determining the expression of sFgl2 in a blood or tissue sample from an individual and predicting plaque stability in AS disease by determining whether sFgl2 expression levels are reduced compared to controls having stable atherosclerotic plaques in the individual. The development of the stabilizing effect of atherosclerotic plaques can also be followed by monitoring the expression of sFgl2 in individuals at different time points.

Drawings

FIG. 1 is a graph of the extent of atherosclerosis in mice of the sFgl2 reduced experimental group:

FIG. 1A shows the reception of ApoE^-/-Or Fgl2^TgApoE^-/-AS model ApoE of control and experimental groups of bone marrow^-/-Blood of mouseThe amount of sFgl2 in the slurry;

FIG. 1B shows ApoE in control and experimental groups^-/-The aortic arch lesion area of the mouse is 2mm in scale;

fig. 1C is an oil red O stain of full-length, longitudinal-cut lesion areas of aorta of two groups of mice, at a scale bar of 5 mm;

fig. 1D is a statistic of aortic oil red O staining area, N-10,^**P<0.01。

FIG. 2 is a graph of atherosclerotic plaques in the sFgl2 stabilization experimental group:

FIG. 2A is a section of HE, oil red O, MAC-3, α -SMA and sirius red stain at the aortic annulus of two groups of mice, scale bar 200 μm;

quantitative statistics of staining for he (B), oil red o (c), Mac-3(D α -SM), a (e), and sirius red (F), N ═ 10,^*P<0.05。

FIG. 3 shows that sFgl2 reduces Ly6C in the experimental group^highType Mo ratio, promotion

Differentiation pattern to M2 type:

FIG. 3A shows CD45 in peripheral blood of two groups of mice⁺Ly-6G^-CD11b⁺Ly-6C^highAnd CD45⁺Ly-6G^-CD11b⁺Ly-6C^lowRepresentative flow cytometric plots of monocyte subpopulations, (numbers represent subpopulation as a percentage of total monocytes);

FIG. 3B is CD45 in peripheral blood⁺Ly-6G^-CD11b⁺Ly-6C^highThe quantification of the percentage of monocytes is 9-10,^*P<0.05；

FIGS. 3C-D, representative flow cytometric analysis of two monocytes in spleen (3C) and Ly-6C^highQuantitative percentage of monocyte subpopulation (3D), N9-10,^*P<0.05；

3E-F, M1 in aortic annular plaque section (MAC 3)⁺iNOS⁺) Model (III)

(3E) And M2(MAC 3)⁺ARG1⁺) Model (III)

(3F) Immunofluorescent staining of (1), scale bar 100 μm;

FIGS. 3G-H, M1(3G) and M2(3H)

The percentage of the subpopulation relative to total macrophages in the plaque, N-10,^*P<0.05；

FIGS. 3I-J, mRNA expression of pro-inflammatory factors TNF- α and IFN-gamma (3I) downstream of M1 and anti-inflammatory factors ARG-1 and IL-10(3J) downstream of M2, N is 3-6,^*P<0.05, data are expressed as mean ± sem.

Fig. 4 is a graph of sFgl2 increasing the number of Treg cells and immunosuppressive function in the experimental group:

FIG. 4A is a representative graph of flow cytometric analysis of Treg cells in control and experimental groups in lymph nodes, with the number being CD4⁺Percentage in T cells;

FIG. 4B is CD4 in lymph node⁺Quantification of the percentage of Treg cells in T cells, N-9-10, data expressed as mean ± sem,^*P<0.05；

FIGS. 4C-D, MAC3 of plaque sections at aortic annulus in two groups of mice⁺FoxP3⁺Immunofluorescent staining of cells, triangles indicate positively stained cells, scale bar 100 μm (4C) and quantitative analysis result of the above immunofluorescent staining, N6,^*P<0.05(4D)；

fig. 4E-F, representative flow chart of Tconv proliferation after co-culture of two groups of mouse Treg cells (Tconv/Treg 1/1), values representing the percentage of non-proliferating cells to total cells (4E) and the percentage of non-proliferating Treg cells at different Tconv/Treg ratios, data expressed as mean ± sem,^*P<0.05(4F)。

FIG. 5 is a graphical representation of the effect of sFgl2 on lowering LDL cholesterol levels in experimental mice:

FIG. 5A is a graph of total cholesterol levels in blood of control and experimental mice, data are expressed as mean. + -. standard error;

FIG. 5B is the triglyceride levels in blood of control and experimental mice, data are presented as mean. + -. standard error;

FIG. 5C is the high density lipoprotein level in blood of control and experimental mice, data are expressed as mean. + -. standard error;

FIG. 5D is the content of low density lipoprotein in blood in control and experimental mice, data are expressed as mean. + -. standard error,^*P<0.05。

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention discloses application of sFgl2(soluble fibrin-like protein 2, sFgl2) in detecting, preventing, relieving and treating Atherosclerosis (AS) diseases. The overall experimental scheme is as follows:

in the present invention, ApoE^-/-Mouse and Fgl2^TgApoE^-/-Mice were housed in an SPF animal house, college of Tongji medical university in Huazhong, maintained at 22-25 ℃ and illuminated under cyclic control. All feeding and treatment of animals meet the standard of 'guide for feeding and using laboratory animals' issued by the national academy of sciences of the United states. Selecting a batch of ApoE at 8W^-/-Mice were gamma irradiated to inactivate their bone marrow cells, and then mice were randomly divided into two groups, one group receiving ApoE^-/-Bone marrow of mice (i.e., control); the other group receives Fgl2^TgApoE^-/-Mouse bone marrow (i.e., experimental group).

After 12W feeding, mice were sacrificed for subsequent testing. Firstly, collecting blood plasma of each group of mice, measuring the content of sFgl2 by ELISA, and measuring the blood lipid level by a blood lipid measuring kit; taking mouse blood cells, lymph nodes and spleenFlow assay (Ly 6C)^high/Ly6C^lowMo and Treg cells); taking mouse aorta, and performing oil red O staining analysis, real-time quantitative PCR (RT-PCR) and immunoblotting analysis (Western Blot); mouse hearts were harvested, plaques were excised from aortic valves for plaque component staining, and staining for cell surface markers of type M1 and M2.

The statistical methods involved in the present invention:

statistical data analysis was performed using SPSS17.0 and graphpad6.0 and the experimental results values are expressed as "mean ± standard error". The difference between the two groups is analyzed by t test statistics; the comparison between the groups was performed using one-way ANOVA and Bonferroni's test, with P.ltoreq.0.05 indicating that the differences were statistically significant.

The mouse bone marrow transplantation control test group is set as follows:

selecting 8W ApoE of SPF grade^-/-Male mice served as recipient mice were given acidified autoclaved water containing roxithromycin and gentamicin 2 weeks before gamma irradiation. After the rats were transported to the irradiation center, whole body irradiation was performed twice with 4.5Gy of radiation at half an hour intervals. The irradiated recipients were then randomized into two groups, i.e., tail vein injection of ApoE^-/-Mouse bone marrow group and Fgl2^TgApoE^-/-Groups of mice.

On the day of irradiation, 8W of ApoE was removed^-/-Mouse and Fgl2^TgApoE^-/-After the mice are anesthetized and disinfected, the thighbones and the shinbones on the two sides of the mice are separated aseptically and completely, and the marrow cavities and the metaphysis are washed by using the syringes until the bones are whitish. Grinding the above bone marrow cell suspension on 200 mesh cell filter screen, centrifuging, precipitating, re-suspending, and adjusting cell concentration to 8-10 × 10⁷Perml, transferred to autoclaved 1.5mL EP tubes, 250. mu.L per tube, labeled and placed temporarily in ice boxes.

Injecting ApoE from the above step intravenously into each irradiated mouse within 3 hours^-/-Mouse or Fgl2^TgApoE^-/-One-tube bone marrow cells of mice (about 2 x 10)⁷Individual bone marrow cells).

The bone marrow transplanted mice were housed in IVC (IVC) sterilized cages in SPF animal houses and fed with sterilized conventional feed and drinking water containing antibiotics (roxithromycin and gentamicin) for 4 weeks. Thereafter, western diet and drinking water without antibiotics were fed for 3 months.

The area of the aortic AS plaque in the mice was measured AS follows:

after 3 months after high fat feeding of the mice after bone marrow transplantation, the mice were anesthetized with ether and the aorta was washed until it became white. Tissues such as periaortic fat, fascia and the like are carefully separated by using micro forceps and micro scissors, the aorta is upwards divided into a left subclavian artery, a left common carotid artery and a right subclavian artery and a right common carotid artery branch of a brachiocephalic artery, the aortic arch is photographed, and the plaque areas of a control group and an experimental group are observed.

Continuously separating the full length of the aorta, longitudinally cutting, flatly paving and fixing the aorta on a black candle stand, incubating the aorta for 10 seconds by using 75% alcohol, pouring oil red O dye solution into the candle stand, and incubating the aorta for 30 min; and after recovering the oil red O dye solution, washing the dye solution and a candle holder around the blood vessel by PBS. Aorta was incubated with 75% alcohol for several seconds to remove background color of normal tissue of the hyperpigmented aorta. PBS was added to the candle stick, the mouse code was marked and the picture was taken with a digital camera.

As shown in fig. 1A: accept Fgl2^TgApoE^-/-Recipient mice in the bone marrow of the mice, the amount of sFgl2 in the plasma (142.3 + -14.3 ng/mL) was significantly higher than that in the control group (41.9 + -7.4 ng/mL).

As shown in fig. 1B: the results of photographing the aortic arch and the bifurcation point suggest that the plaque area of the experimental group is obviously reduced compared with the control group.

As shown in fig. 1C-D: the aorta was stained with gross oil red O and the results showed Fgl2^TgApoE^-/-The plaque area in the chimera (7.2 ± 0.76%) was smaller than that of the control group (11.6 ± 0.92%).

The components and stability of the mouse aortic AS plaque were tested AS follows:

the aortic valve rings of two groups of mice were sectioned and the thickness of the section was adjusted to 6 μm, and the sections were continuously collected. Selecting the frozen sections, and carrying out HE staining on part of the frozen sections to determine the plaque area of each group of mice; one part is stained with oil red O to clarify the lipid core area; one part is dyed with sirius red to determine the area of the type I collagen fiber; one part was immunofluorescent stained with macrophages and smooth muscle cells to clarify the proportion of the two cells in the plaque.

Wherein, the immunofluorescence staining steps of the macrophage and the smooth muscle cell are as follows:

rewarming aortic annulus slice at normal temperature for 10min, then placing into PBS (PBST) solution containing 0.5 per mill Tween-20, slowly washing on a shaking table for 10min, and repeating once; after the membrane breaking agent Triton X-100 is used for membrane breaking once, the section is soaked into PBST solution for washing, and then the tissue is sealed by donkey plasma for 2 h.

The sections were slightly soaked in PBST solution, and after removal the tissue was blotted dry with filter paper for primary antibody incubation. The method comprises the following specific steps: to clarify the infiltration of macrophages in atherosclerotic plaques, Mac-3 antibody (Abcam, USA), dilution 1: 100, respectively; to clarify the infiltration of smooth muscle cells in atherosclerotic plaques, α -SMA antibody (Abcam, USA), dilution 1: 100, respectively; adding the antibody, putting the mixture into a light-proof wet box, and incubating the mixture overnight in a refrigerator at 4 ℃; after 12h, the sections were removed and washed in PBST solution for 10 min. The above steps were repeated twice.

Incubating the secondary antibody of the species matched with the primary antibody, wherein Mac-3 corresponds to a secondary red light antibody (with a wavelength of 594) and alpha-SMA corresponds to a secondary green light antibody (with a wavelength of 488), and the incubation time is 2 h; the secondary antibody was washed away from light for 10min three times. Adding DAPI dye solution dropwise in dark, incubating for 5min, and washing for 10min in dark for three times.

After the filter paper has dried the tissue surrounding water, the encapsulated pieces are encapsulated using an anti-fluorescence quenching and photographed on a TCS SP5 multiphoton laser scanning confocal microscope (Nikon, Japan). Mean values were calculated from five randomly selected microscopic fields in the plaques and positive cells were analyzed by ImageJ (NIH, Bethesda, MD, USA).

As shown in fig. 2A-D: the results of staining and pathological analysis of aortic annular plaque sections show that,sFgl2 indicates the area of the plaque (H)&E) Necrotic core area (oil red O) and macrophage (MAC-3) decrease by 0.33 + -0.03 to 0.21 + -0.03 mm²0.08 + -0.01 to 0.04 + -0.00 mm²And 50. + -. 4.8 to 36. + -. 2.6%.

As shown in fig. 2A, E, F: immunofluorescent staining for alpha-SMA and sirius red stains showed that sFgl2 increased the percentage of smooth muscle cells and had no effect on the Collagen i region compared to the control.

The effect of sFgl2 on mouse monocyte polarity was as follows:

blood cells and spleen cells from both groups of mice were made into cell suspensions, which were transferred to 1.5ml lep tubes containing 100 μ L of cell suspension per tube.

Adding corresponding 2.5 μ L CD45, 2 μ L Ly-6G, 2 μ L Ly-6C and 3 μ L CD11b antibody into the cell suspension, mixing with vortex oscillator, and incubating at 4 deg.C in dark for 30 min; then, 1mL of PBS buffer was added to each of the above EP tubes, and the mixture was centrifuged at 2000r/min for 10min, and the supernatant was discarded to leave a cell pellet, and the centrifugation and washing were repeated once. The resulting cell pellet was resuspended in 200. mu.L of cell fixative, transferred to a flow tube, and then assayed by flow cytometry.

As shown in fig. 3A-D: monocyte subpopulations (CD 45) in peripheral blood and spleen were analyzed using Flow Cytometry (FCM)⁺Ly6G^-CD11b⁺Ly6C^highAnd CD45⁺Ly6G^-CD11b⁺Ly6C^low). The results show that sFgl2 can promote peripheral blood and spleen Ly6C^highThe percentage of monocytes decreased from 60.5 + -3.4 to 41.7 + -3.6%, and 53.0 + -1.6 to 44.8 + -3.0%, respectively.

The effect of sFgl2 on mouse macrophage polarity was as follows:

after rewarming, washing, rupture of the membrane, sealing the aortic annulus section specimens of both groups of mice, incubation was performed with the following antibodies: mac-3 antibody (Abcam, USA), dilution 1: 100, respectively; inducible Nitric Oxide Synthase (iNOS) antibody (Abcam, USA), dilution 1: 100, respectively; arginase 1(Arg-1) antibody (Abcam, USA), dilution 1: 100. after addition of the antibody, the cells were placed in a light-shielding wet box and incubated overnight in a refrigerator at 4 ℃.

And (3) incubating the secondary antibodies of the species matched with the primary antibody, wherein the Mac-3 corresponds to a secondary red light antibody (with the wavelength of 594), and the iNOS and Arg-1 correspond to a secondary green light antibody (with the wavelength of 488), and the incubation time is 2 h. The secondary antibody was washed away from light for 10min three times. Then, DAPI dye solution is dripped in the dark, and after incubation for 5min, washing is carried out for three times in the dark, and each time lasts for 10 min.

After the filter paper has dried the tissue surrounding water, the encapsulated pieces are encapsulated using an anti-fluorescence quenching and photographed on a TCS SP5 multiphoton laser scanning confocal microscope (Nikon, Japan). Mean values were calculated from five randomly selected microscopic fields in the plaques and positive cells were analyzed by two blinded investigators using ImageJ (NIH, Bethesda, MD, USA).

As shown in fig. 3E-H: to investigate the effect of sFgl2 on macrophages in innate immune cells, we performed macrophage phenotypes in sections of aortic annular plaques by immunofluorescence staining (M1, Mac-3)⁺INOS⁺；M2，Mac3⁺Arg⁺) And (6) dyeing. The results show that sFgl2 decreased the percentage of pro-inflammatory M1 macrophages (from 48.4 ± 3.2 to 38.9 ± 2.4%) and increased the percentage of anti-inflammatory M2 macrophages (from 41.1 ± 4.0 to 50.6 ± 1.8%) compared to the control group.

As shown in fig. 3I-J: accordingly, at Fgl2, compared to the control group^TgApoE^-/-In chimeric mice, mRNA expression levels of the M2 type macrophages, the markers Arg1 (from 1.12 + -0.13 to 4.68 + -0.57) and IL-10 (from 1.10 + -0.11 to 4.79 + -1.50) increased, while expression levels of the M1 markers TNF- α and IFN- γ decreased.

The influence of sFgl2 on the number and function of mouse Treg cells is as follows:

detecting the number of Treg cells:

grinding mouse lymph nodes to prepare single cell suspension, transferring to a 1.5mL EP tube, adding 2.5 mu LCD45, 3 mu L CD3, 4 mu L CD4 and 5 mu L CD25 antibodies into the corresponding cell suspension, mixing uniformly by a vortex oscillator, and incubating for 30min at 4 ℃ in a dark place; after centrifugation and washing, 1mL of special fixing/penetrating liquid for Treg cells is added into the cell sediment, and incubation is carried out for 60min at 4 ℃ in a dark place; then 5 mu L of FoxP3 antibody is added, and after being uniformly mixed by a vortex oscillator, the mixture is incubated for 30min at 4 ℃ in a dark place; after centrifugation and washing, 200 mu L of flow cell fixing solution is used for resuspending the cells, the cells are transferred to a flow tube marked correspondingly, the flow tube is placed in a refrigerator at 4 ℃, and then the number of the Treg cells of two groups of mice is detected and analyzed by a flow cytometer.

Detecting the function of the Treg cells:

CD4 was first sorted from lymph node single cell suspensions using flow cytometry (BD Biosciences)⁺CD25^-And CD4⁺CD25⁺A cell. Second sorting of the resulting CD4⁺CD25^-The cells were transferred to a 1.5mL EP tube at high pressure and incubated with CFSE (eBioscience, USA) working solution for 30min at low temperature; after centrifugation and washing of the cells, 96-well plates pre-coated with anti-CD3/CD28 were removed according to C57-CD4⁺CD25^-：ApoE^-/--CD4⁺CD25⁺1: 0,1: 1 and 1: 2, and C57-CD4⁺CD25^-：Fgl2^TgApoE^-/--CD4⁺CD25⁺1: 0,1: 1 and 1: 2, adding RPMI-1640 medium containing 10% fetal bovine plasma, and mixing and culturing for 72 hours. Finally, detecting C57-CD4 in each hole by using a flow type computer⁺CD25^-Cell proliferation, statistical mapping.

As shown in fig. 4A-B: we continued to detect sFgl2 for Tregs in spleen and lymph nodes using flow cytometry (CD 45)⁺CD3⁺CD4⁺CD25⁺) The proportion of cells in CD4+ T cells varied. Flow results showed that sFgl2 increased the proportion of Treg cells in lymph nodes to CD4+ T cells from 5.37 ± 0.94 to 9.33 ± 1.30% compared to control.

As shown in fig. 4C-D: we detected Tregs in the aortic sinus of mice (FoxP 3)⁺) Cells, sFgl2 was found to also increase the number of Treg cells in AS plaques (from 60.0 ± 3.5 to 85.4 ± 9.2 cells/mm)²)。

As shown in fig. 4E-F: fgl2 compared with control group^TgApoE^-/-Treg cells in chimeras can enhance Tc at different Tconv/Treg ratios (1: 0, 1: 1 and 1: 2)onv ability to inhibit cell proliferation.

The effect of sFgl2 on mouse lipids was as follows:

after the model building of the mouse is finished, blood is taken from the inner canthus vein, the mouse is kept still for 1 hour at 37 ℃, then the blood is centrifuged for 10 minutes at 2000r/min, the upper layer of blood plasma is taken and subpackaged into small EP tubes of 200 mu L, and the mouse is stored in a refrigerator at-80 ℃ after the information such as the mouse number, the blood sampling date and the like are marked on the tube wall. When blood lipid is measured, the sample is taken out from a refrigerator and rewarmed, and a blood lipid detection kit (Nanjing institute of bioengineering) is used for detecting the contents of total cholesterol, triglyceride, high density lipoprotein and low density lipoprotein cholesterol.

As shown in fig. 5A-D: sFgl2 decreased LDL (from 3.34 ± 0.90 to 0.79 ± 0.29, P ═ 0.027) levels in experimental mice compared to controls, while there was no significant effect on TC (from 5.15 ± 0.78 to 3.47 ± 0.88, P ═ 0.175), TG (from 2.08 ± 0.58 to 0.91 ± 0.19, P ═ 0.099), and HDL (from 3.01 ± 0.98 to 2.30 ± 0.46, P ═ 0.565).

The invention uses ApoE^-/-And Fgl2^TgApoE^-/-The mice are animal subjects, and the mice are transplanted with bone marrow (receptor ApoE after irradiation)^-/-Mice, each receiving donor ApoE^-/-Or Fgl2^TgApoE^-/-Bone marrow cells of mice) and 12W are fed on western diet to obtain an AS model, and the result shows that compared with the control group of mice, the aortic plaque area of the mice of the sFgl2 overexpression group is obviously reduced, and the plaque stability is obviously improved. Furthermore, sFgl2 can be reduced by decreasing Ly6C in the innate immune response of AS^highM1 type M_OThe ratio of M phi and promotes M2 type M phi differentiation; in the adaptive immune response of AS, sFgl2 can increase the number of Treg cells and immunosuppressive function; in the metabolic reaction of AS, sFgl2 can reduce LDL-C levels to alleviate AS. Therefore, the sFgl2 has multidimensional function in the generation and development of AS, and can be used AS an anti-inflammatory/metabolic regulation target for preventing, relieving and treating AS diseases.

Wherein sFgl2 includes: cells that can secrete sFgl2 such as tregs and CTLs; the Fgl2 gene sequence is enveloped by virus or bacterial vector, such as adenovirus-Fgl 2, lentivirus-Fgl 2, adeno-associated virus-Fgl 2 or Fgl2 plasmid, etc.; recombinant Fgl2 plasmid, Escherichia coli or CHO cell, etc. secreted sFgl2 protein; or one of the other pathways that up-regulate sFgl2 gene or protein expression.

The stability of plaques in AS disease is determined by determining the expression of sFgl2 in a blood or tissue sample of an individual and predicting the stability of plaques in AS disease by determining whether the expression level of sFgl2 is reduced compared to a control. The development of the stabilizing effect of atherosclerotic plaques can also be followed by monitoring the expression of sFgl2 in individuals at different time points.

In conclusion, the invention can provide that the expression level of sFgl2 is inversely related to the disease degree of AS diseases, improve the level of secreted sFgl2 by acting on an sFgl2 target spot, promote the differentiation of M phi in blood to an anti-inflammatory M2 phenotype, increase the number of Foxp3+ Tregs and the immune suppression function, reduce Treg cells in blood, reduce lipids such AS LDL-C in blood and the like, can well detect, prevent, alleviate and treat AS diseases, and has a solid basis for the application of the invention.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (8)

1. The use of sFgl2 in the following I and II:

   i, application in preparing medical products for preventing, relieving and treating AS diseases;

   and II, application in preparing a medical product for detecting AS plaque stability in individuals.
2. 2. The use according to claim 1, wherein the medical product for the prevention, alleviation and treatment of AS disease is achieved by acting on sFgl2 targets, specifically by increasing the secreted sFgl2 level, promoting the phenotypic differentiation of M Φ in the blood towards the anti-inflammatory M2, resulting in Foxp3⁺Increase of Treg quantity and immunosuppressive function, and decrease of Ly6C^highM1 type M_Othe/M phi ratio and lowering LDL-C lipids in the blood.
3. 3. The use of claim 1 or 2, wherein the medical product comprises, but is not limited to, the following ingredients: cells that can secrete sFgl2, such as tregs and CTL-associated cells; a viral or bacterial vector coating Fgl2 gene sequence, such as adenovirus-Fgl 2, lentivirus-Fgl 2, adeno-associated virus-Fgl 2 or Fgl2 plasmid; recombinant Fgl2 plasmid of Escherichia coli or CHO cells secreted sFgl2 protein; or one of the other pathways that up-regulate sFgl2 gene or protein expression.
4. 4. The use of any of claims 1-3, wherein the medical product form includes, but is not limited to, OTC tablets, injectable drugs, targeted drugs, capsules, drug delivery robots, vesicles, and rapid test cassettes.
5. 5. The use of claim 1, wherein sFgl2 is specifically expressed in a population with unstable AS plaques.
6. 6. The use of claim 1, wherein the stability of AS plaque in an individual is determined by measuring the expression of sFgl2 in a blood sample or a blood vessel wall tissue sample of the individual, and the degree of AS disease incidence is assessed by predicting the stability of AS disease plaque by detecting the level of secreted sFgl 2.
7. 7. The use according to any one of claims 1 to 6, wherein the therapeutic products for detecting, preventing, alleviating and treating AS diseases have the relevant mechanisms and action principles thereof obtained by the study of mouse animal control experiments, and the specific experimental scheme of the mouse animal control experiments is AS follows:

   by ApoE^-/-And Fgl2^TgApoE^-/-The mice are animal subjects, and the mice are transplanted with bone marrow (receptor ApoE after irradiation)^-/-Mice, each receiving donor ApoE^-/-Or Fgl2^TgApoE^-/-Bone marrow cells of mice), 12W is fed on western diet to obtain an AS model, and the result shows that compared with the control group of mice, the aortic plaque area of the mice of the sFgl2 overexpression group is obviously reduced, and the plaque stability is obviously improved; also, sFgl2 decreased Ly6C in the innate immune response of AS^highThe ratio of MO to M phi in the form of/M1, and promoting the differentiation of M2M phi; in the adaptive immune response of AS, sFgl2 can increase the number of Treg cells and immunosuppressive function; in the metabolic reaction of AS, sFgl2 can reduce LDL-C levels to alleviate AS; the sFgl2 has multi-dimensional effect in the generation and development of AS, and can be used AS an anti-inflammatory/metabolic regulation target for detecting, preventing, relieving and treating AS diseases.
8. Application of sFgl2 in preparing products for regulating MO/M phi cell polarity, T cell differentiation to Treg/Teff cell and blood lipid level in AS diseases.

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