Composition for preventing and treating echinococcosis and application thereof

Abstract

The invention discloses a composition for preventing and treating echinococcosis and application thereof, and relates to the technical field of biological medicine. The composition is prepared from a beta-carboline alkaloid compound and interleukin-25, the beta-carboline alkaloid compound is harmine or a harmine derivative. The formula of the beta-carboline alkaloid compound is shown in the description. According to the research, the harmine and the derivative thereof are combined with IL-25, so that not only can the growth of hydatid be effectively inhibited, but also the immune system of an application object can be stimulated, and the resistance of the application object to the hydatid can be enhanced. The harmine and the derivative thereof are combined with IL-25, so that the harmine and the derivative thereof have greater potential and advantages when being used for treating hydatid infection and related diseases. The discovery is of great significance to medical research and clinical treatment, and valuable clues are provided for further exploration and development of new treatment methods.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a composition for preventing and treating echinococcosis and application thereof. Background Art

[0002] Hydatid disease, also known as echinococcosis, is a zoonosis caused by the long-term parasitism of the middle tapeworm larvae of Echinococcus granulosus on the host. It is an ancient parasitic disease widely distributed worldwide and is highly prevalent in areas with developed animal husbandry. It is estimated that the global mortality rate of echinococcosis is 2%-4%, and the disease burden caused is about 871,000 disability-adjusted life years, with economic losses of up to US$3 billion. my country is one of the countries with the most serious echinococcosis epidemic in the world, with an estimated 380,000 patients and nearly 66 million people at risk of infection. However, due to its complex epidemic links and many influencing factors, the prevention and control of echinococcosis still faces many severe challenges. Therefore, the effective prevention and control of echinococcosis is an urgent need for economic development and social harmony and stability.

[0003] At present, the clinical treatment of echinococcosis is dominated by surgical removal, with drug therapy as an auxiliary. As an auxiliary treatment for echinococcosis, drug therapy provides an effective treatment for patients with multiple echinococcosis in the liver, combined with multiple organ echinococcosis, postoperative recurrence prevention, small echinococcosis lesions, and echinococcosis patients who cannot undergo surgery for various reasons. So far, WHO recommends few drug options for the clinical treatment of echinococcosis. Since the discovery of albendazole (ABZ), no other new drugs have been launched. However, albendazole has low bioavailability and obvious side effects. Although ABZ is prepared as liposomes, proliposomes, promicelles, and nanopowders for treatment, it is stopped due to various defects such as poor preparation stability and difficult process control. At present, drug therapy has become a powerful auxiliary means for the treatment of echinococcosis and the main direction of research on the disease. Therefore, it is urgent to develop accurate, efficient, and low-toxic therapeutic drugs and explore new ways of combined drug application.

[0004] Harmine (HM) is a β-carboline alkaloid compound extracted from the seeds of Peganum harmala Linn, a perennial herbaceous plant of the Zygophyllaceae family. Interleukin-25 (IL-25), also known as IL-17E, is a member of the IL-17 family. There is no report on the combined use of harmine and its derivatives and interleukin-25 for the prevention and treatment of echinococcosis. Summary of the invention

[0005] The purpose of the present invention is to provide a composition for preventing and treating echinococcosis and its application, so as to solve the problems existing in the above-mentioned prior art. The composition has greater potential and advantages in treating echinococcosis infection and related diseases, and can not only effectively inhibit the growth of echinococcosis, but also stimulate the immune system of the subject to be administered, and enhance its resistance to echinococcosis.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a composition for preventing and treating echinococcosis, comprising a β-carboline alkaloid compound and interleukin-25;

[0008] The β-carboline alkaloid compound is harmine or a harmine derivative.

[0009] Furthermore, the mass ratio of the β-carboline alkaloid compound to the interleukin-25 is 500:1.

[0010] Furthermore, the dehydrogenated harmine derivative is H-2-168.

[0011] The present invention also provides application of the above-mentioned composition in preparing medicine for preventing and treating echinococcosis.

[0012] The present invention also provides a medicine for preventing and treating echinococcosis, wherein the active ingredient comprises the above-mentioned combination.

[0013] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the auxiliary materials include solubilizers, binders, disintegrants, fillers, stabilizers, preservatives, coating materials and / or fragrances.

[0015] The present invention discloses the following technical effects:

[0016] The present invention has found that the combination of dehydrogenated harmaline and its derivatives (H-2-168) and IL-25 can produce a significant inhibitory effect on the growth of hydatid cysts, and the synergistic inhibitory effect of dehydrogenated harmaline and its derivatives and IL-25 is more significant than the use of dehydrogenated harmaline or its derivatives alone. The results suggest that by combining dehydrogenated harmaline and its derivatives with IL-25, the number of hydatid cysts can be more effectively controlled or reduced, thereby providing a new strategy for treating hydatid cysts-related diseases. The present invention also found that the combination of dehydrogenated harmaline and its derivatives and IL-25 can not only effectively inhibit the growth of hydatid cysts, but also stimulate the immune system of the subject of administration and enhance its resistance to hydatid cysts. The combination of dehydrogenated harmaline and its derivatives and IL-25 has greater potential and advantages in the treatment of hydatid cysts and related diseases. This discovery is of great significance for medical research and clinical treatment, and provides valuable clues for further exploration and development of new treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is a visual picture of the vesicles in mice after drug treatment; AH are the model group, IL-25 group, ABZ group, HM group, H-2-168 group, ABZ+IL-25 group, H-2-168+IL-25 group, and HM+IL-25 group, respectively;

[0019] Figure 2 The following are the pathological morphological examinations of various organs of mice after drug treatment (HE, ×200); the scale bars in the figures are all 50 μm;

[0020] Figure 3 The results of the expression level detection of CD206, CD64 and iNOS in the liver of mice after drug treatment;

[0021] Figure 4 The results of flow cytometry determination of the expression of CD206 and CD86 in peritoneal cells of mice in the blank control group and model group;

[0022] Figure 5 The results of flow cytometry determination of the expression of CD206 and CD86 in the peritoneal cells of mice in the IL-25 group, ABZ group, and HM group;

[0023] Figure 6The results of flow cytometry determination of the expression of CD206 and CD86 in peritoneal cells of mice in the ABZ+IL-25 group and HM+IL-25 group;

[0024] Figure 7 The results of flow cytometry detection of spleen cell subsets in blank control group and model group mice;

[0025] Figure 8 The results of flow cytometry detection of spleen cell subsets in mice in the IL-25 group, ABZ group, and HM group;

[0026] Fig. 9 The results of flow cytometry detection of spleen cell subsets in ABZ+IL-25 and HM+IL-25 groups;

[0027] Fig.10 These are the results of flow cytometry detection of spleen cell subsets in mice in the H-2-168 group and H-2-168+IL-25 group. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Terminology Note:

[0034] In the present invention, the molecular formula of dehydrogenated echinopsine is C 13 H 12 N2O, molecular weight is 212.247, CAS number is 442-51-3, structural formula is:

[0035]

[0036] The molecular formula of H-2-168 is C 18 H 10 N3, molecular weight is 274.13, structural formula is:

[0037]

[0038] Example 1

[0039] 1. Materials and Methods

[0040] 1. Experimental Materials

[0041] 1.1 Source and culture of Echinococcus granulosus

[0042] The cysts of Echinococcus granulosus were obtained from the liver cysts of sheep infected with Echinococcus granulosus at Hualing Slaughterhouse in Urumqi, Xinjiang. The protoscoleci were removed by aseptic method and repeatedly washed with sterile saline containing double antibodies. The protoscoleci with activity ≥ 98% in the eosin rejection test can be cultured in a constant temperature incubator at 37°C and 5% CO2.

[0043] 1.2 Healthy mice

[0044] Kunming white healthy mice (female, 18-22g) were purchased from the Experimental Animal Research Center of Xinjiang Medical University, with a production license number of SCXK (new) 2018-0002 and a use license number of SYXK (new) 2018-0003. They were grown at a temperature of 20°C-22°C, a relative humidity of 50%-55%, and 12 hours of light / 12 hours of darkness. The feeding and management of experimental animals were the responsibility of professional staff of the Experimental Animal Research Center of Xinjiang Medical University, who ensured that the experimental animals had sufficient feed, water, and clean bedding.

[0045] 1.3 Preparation of hydatid animal model

[0046] Take Echinococcus granulosus with vitality ≥ 95%, sort out 1000 Echinococcus granulosus of uniform size in a sterile culture dish, gather them together, and inject them into Kunming white healthy mice through intraperitoneal injection to inoculate the model. Two months after infection, the mice with successful modeling were treated with drugs and drug combination.

[0047] 2. Experimental methods and results

[0048] Study on the anti-cystic echinococcosis effect of dehydrogenated echinococcus lysine and its derivatives (H-2-168) combined with IL-25 in vivo:

[0049] 2.1 Experimental Grouping

[0050] Six healthy Kunming mice were randomly selected as the blank control group; 48 mice with successful secondary modeling of Echinococcus granulosus infection and equivalent infection degree were selected and randomly divided into model group, albendazole (ABZ) group, harmine (HM) group, harmine derivative (H-2-168) group, interleukin-25 (IL-25) group, HM+IL-25 group, ABZ+IL-25 group, H-2-168+IL-25 group, 6 mice in each group. The dosage and method of each experimental group are shown in Table 1. Each experimental group was continuously administered for 14 days, and the body weight was measured regularly.

[0051] Table 1 Dosage and method of administration for each experimental group

[0052]

[0053] 2.2 Index detection

[0054] Efficacy index: The mice were modeled for 2 months. One week after the 14-day treatment, the animals were anesthetized with 1% sodium pentobarbital, dissected, and the vesicles in the mouse abdominal cavity were peeled off. The size and morphology of the vesicles were visually examined, and the wet weight of the vesicles was calculated. Other organ tissue samples were collected and weighed, and the index of each organ was calculated.

[0055] Vesicle wet weight inhibition rate = (CT) / C × 100%;

[0056] Among them, C: mean wet weight of vesicles in the model group; T: mean wet weight of vesicles in the medication group.

[0057] The formula for calculating each organ index is: organ index (mg / g) = organ weight (mg) / mouse body weight (g).

[0058] Experimental results:

[0059] After drug treatment, mice were dissected and vesicles were taken out. The wet weight of vesicles in the model group was significantly higher than that in other drug-treated groups, reaching 15.46±1.89g. The wet weight of vesicles in the HM+IL-25 group was 4.97±0.96g. Compared with the model group, ABZ group, HM group, H-2-168 group, IL-25 group, and ABZ+IL-25 group, the wet weight of vesicles in the HM+IL-25 group and H-2-168+IL-25 group was significantly reduced (all P<0.001). The inhibition rate was calculated according to the calculation formula: vesicle wet weight inhibition rate = (CT) / C×100%, and the results are shown in Table 2. The vesicles in each group were observed: the surface of the vesicles in the model group was smooth, round and transparent; the vesicles in the HM group and HM+IL-25 group collapsed, shrunk and significantly reduced in size; the number of vesicles in the HM+IL-25 group and H-2-168+IL-25 group was significantly reduced compared with the HM group and H-2-168 group (see Figure 1 ).

[0060] Table 2 Wet weight of vesicle tissue and inhibition rate of vesicles in each group after drug intervention

[0061]

[0062]

[0063] Note: Compared with the model group, *** P<0.001; compared with HM group, ### P<0.001.

[0064] 2.3 Effects of drug treatment on pathological changes of various organs in mice

[0065] After sampling, clean the organ tissues of mice and place them in 4% paraformaldehyde solution for 48 hours; trim the fixed tissues, place them in tissue embedding boxes, rinse them with running water for 1 hour, and then immerse them in 75% ethanol overnight. On the second day, perform the process of ethanol dehydration, transparency, wax immersion, paraffin embedding, and sectioning. The sections should be placed in a 60°C oven in time, baked overnight, taken out for hematoxylin-eosin (HE) staining, sliced, and observed for tissue pathology under a microscope.

[0066] HE staining results showed that the liver tissue structure of mice in the blank control group was normal, the liver lobule structure was intact, the liver cell boundaries were clear and neatly arranged, the liver cells were radially arranged around the central vein, and there was no inflammatory cell infiltration. Compared with the blank control group, the liver structure of mice in the model group was abnormal, most of the liver cell nuclei were condensed, and "empty halos" were formed around the nucleus, there was a large number of inflammatory cells infiltrating the liver portal area, and congestion appeared in the small veins in the portal area. Compared with the model group, ABZ group, HM group, H-2-168 group, IL-25 group, and ABZ+IL-25 group, the degree of inflammatory response of liver tissue in the HM+IL-25 group and H-2-168+IL-25 group showed a significant downward trend; compared with the blank control group, the model group showed a decrease in local lymphocytes, an increase in the distance between spleen cells, accompanied by a large number of red blood cell infiltration, and an increase in neutrophils. In each drug treatment group, the distance between cells was obviously reduced, and the cytoplasm tended to be uniform; compared with the blank control group, the model group had congestion of alveolar septa, thickening of alveolar walls, and a large number of neutrophils infiltration in the alveolar cavity. Compared with the model group, the inflammatory cell infiltration in the alveolar cavity of the ABZ group, HM group, H-2-168 group, IL-25 group, ABZ+IL-25 group, H-2-168+IL-25 group, and HM+IL-25 group decreased; compared with the blank control group, there were no obvious pathological changes in the kidney, heart, and brain of the model group, ABZ group, HM group, H-2-168 group, IL-25 group, ABZ+IL-25 group, H-2-168+IL-25 group, and HM+IL-25 group (see Figure 2 ).

[0067] 2.4 Immunohistochemistry for liver tissue detection

[0068] The paraffin sections were baked in a 65°C oven, dewaxed and rehydrated, and washed with 3% hydrogen peroxide for 15 minutes and PBS three times, each time for 5 minutes; citric acid repair solution was added, poured into a washing box, and placed in a microwave oven at medium heat for 13 minutes, cooled to room temperature, washed three times with PBS, circled a water blocking circle, and blocked with 5% BSA in a wet box at 37 degrees for 30 minutes; primary antibodies (CD206, FCER, INOS) were added and the wet box was kept at 4°C overnight, then washed three times with PBS at room temperature, each time for 5 minutes; secondary antibodies were added, incubated at 37°C for 30 minutes, and washed three times with PBS, each time for 5 minutes; freshly prepared DAB color developing solution was used for color development, the sections were sealed, and observed under a microscope.

[0069] Increased expression of CD206 can weaken the body's antigen presentation function, reduce adaptive immune responses, inhibit Th1 immune responses, promote damaged tissue repair and reconstruction, and vascular growth, and participate in pathological and physiological processes such as inhibiting inflammatory responses, immune tolerance, and damage repair. Studies have shown that the expression level of CD206 in patients infected with Echinococcus granulosus increases significantly in the later stage, and the polarization of macrophages in patients tends to be M2 type. The imbalance of the M1 / M2 ratio and changes in related cytokines may be one of the reasons why Echinococcus granulosus escapes the body's immune response. iNOS is mainly expressed in KCs. When the body is damaged, its expression increases in an inducible manner, catalyzing the continuous production of a large amount of NO and mediating a wide range of toxic effects. Studies have shown that the expression level of iNOS in the liver tissue of patients with echinococcosis is significantly increased, indicating that iNOS is involved in the process of liver damage and liver fibrosis caused by echinococcosis.

[0070] The immunohistochemical test results of the present invention ( Figure 3 ) showed that CD206 was expressed in small amounts in the liver tissues of mice in the blank control group that were not infected with Echinococcus granulosus, and the positive sites were mainly located in liver macrophages and liver sinusoidal endothelial cells, and the expression was in the form of yellow grids or brown-yellow reticular. The expression of CD206 in the liver tissues of mice in the model group increased, and the relative expression level was significantly different from that in the blank control group. The expression of CD206 in the liver tissues of mice in the IL-25 group showed an increasing trend, mainly expressed in kupffer cells and liver endothelial cells, and the difference was statistically significant compared with the model group. Compared with the model group, ABZ group, HM group, H-2-168 group, IL-25 group and ABZ+IL-25 group, the expression of CD206 in the HM+IL-25 group and H-2-168+IL-25 group was significantly reduced.

[0071] result( Figure 3 ) showed that CD64 protein was almost not expressed in the liver tissue of mice in the blank control group. The expression of CD64 in the liver tissue of mice in the model group infected with Echinococcus granulosus showed an increasing trend, and a large number of brown-yellow granular positive expressions were observed in the liver cell membrane and kupffer cells, which was statistically significant compared with the blank control group. The expression level of CD64 protein in the cell membrane and kupffer cells of the liver tissue of mice in the IL-25 group and HM group showed a downward trend, which was statistically significant compared with the model group. Compared with the model group, ABZ group, HM group, H-2-168 group, IL-25 group and ABZ+IL-25 group, the expression of CD64 protein in the HM+IL-25 group and H-2-168+IL-25 group was significantly reduced, and the difference was statistically significant.

[0072] The results of the present invention ( Figure 3) showed that the liver tissue of the blank control group mice that were not infected with Echinococcus granulosus expressed a small amount of iNOS protein. Strong positive signals of iNOS protein were observed in the cytoplasm and cytoplasm of the liver cells of the model group mice, and the difference was statistically significant compared with the blank control group; compared with the model group, the iNOS protein level in the liver cells of the IL-25 group mice was significantly reduced; compared with the model group, ABZ group, HM group, H-2-168 group, IL-25 group and ABZ+IL-25 group, the expression area and degree of iNOS protein in the liver tissue of the HM+IL-25 group and H-2-168+IL-25 group mice were significantly reduced.

[0073] The results of the three indicators showed that the HM+IL-25 group and the H-2-168+IL-25 group significantly inhibited the expression of CD206, CD64, and iNOS (see Figure 3 ).

[0074] 2.5 Identification of peritoneal macrophages by flow cytometry

[0075] Anesthetize the mouse with 1% sodium pentobarbital, inject 5mL sterile serum-free RPMI1640 medium and 1mL air intraperitoneally, gently massage the mouse abdomen for 2 minutes, draw up all the cell suspension with a syringe, and collect it in a centrifuge tube (placed on ice). Rinse once with 5mL sterile serum-free RPMI1640 medium, collect and combine and transfer to a centrifuge tube. Centrifuge at 1500rpm for 5min and collect the supernatant. Wash with cold PBS, centrifuge again and discard the supernatant. Resuspend the cells in RPMI1640 complete medium containing 10% FBS. Centrifuge the extracted cells at 1500rpm for 5min. Remove the supernatant, resuspend the cells with PBS and centrifuge at 1500rpm for 5min. The supernatant was discarded, and 2 μL F4 / 80, 2 μL CD11b, 2 μL CD86 and 2 μL CD206 were added, and the mixture was incubated at room temperature in the dark for 1 h. The mixture was washed with PBS, centrifuged at 1500 rpm for 5 min, passed through a 200 μm mesh, placed in a flow tube, and the polarization of peritoneal macrophages was identified by flow cytometry.

[0076] CD86 is a characteristic molecule of M1 macrophages. Increased expression of CD86 can activate lymphocytes, participate in the metabolism of substances such as iron and fat, regulate cell proliferation and development, activate Th1 cells, and have anti-inflammatory effects. Studies have found that in the early stage of Echinococcus granulosus infection, CD86 expression increases, macrophages in mice polarize toward M1, triggering Th1-type immune response, secreting a large number of inflammatory factors, and producing immune response. The expression level of CD206 increases significantly, the polarization of macrophages in patients tends to be M2, the imbalance of the M1 / M2 ratio, and changes in related cytokines may be one of the reasons why Echinococcus granulosus escapes the body's immune response.

[0077] Flow cytometry results ( Figure 4-Figure 6and Table 3) showed that CD206 was expressed in a small amount in the peritoneal cells of the blank control group mice that were not infected with Echinococcus granulosus, and the expression of CD86 was higher than that of CD206. The expression of CD206 and CD86 in the liver tissue of the mice in the model group increased, and the expression of CD206 was slightly higher than that of CD86, which was statistically significant compared with the blank control group. CD206 in the liver tissue of the mice in the IL-25 group showed an increasing trend, and the expression of CD86 decreased, which was statistically significant compared with the model group. Compared with the model group, ABZ group, HM group, H-2-168 group, IL-25 group and ABZ+IL-25 group, the expression of CD206 in the HM+IL-25 group and H-2-168+IL-25 group was significantly reduced. Compared with the model group, the expression of CD86 in the ABZ group, HM group, H-2-168 group, IL-25 group and ABZ+IL-25 group was significantly decreased, while that in the HM+IL-25 group and H-2-168+IL-25 group was significantly increased compared with that in the ABZ group, HM group, H-2-168 group, IL-25 group and ABZ+IL-25 group.

[0078] Table 3 Expression of CD206 and CD86 in peritoneal cells of each group of mice measured by flow cytometry (mean ± S, n = 3)

[0079]

[0080] Note: Compared with the blank control group, *** P<0.001; compared with the model group, # P<0.05, ### P<0.001.

[0081] 2.6ELISA kit to detect the levels of cytokines IL-10 and CCL12 in the supernatant of mouse peritoneal macrophages

[0082] The peritoneal cell supernatant was collected according to the method in 2.5, the supernatant was diluted twice and then the experimental operation was carried out according to the instructions of the ELISA kit. The effect of each treatment group on the secretion level of IL-10 and CCL12 in the peritoneal supernatant of mice infected with Echinococcus granulosus was detected by microplate reader, and the standard curve was drawn to calculate the content of IL-10 and CCL12 in the supernatant of peritoneal macrophages of mice infected with Echinococcus granulosus after drug intervention. The results are shown in Table 4.

[0083] Studies have shown that in the early stage of infection with Echinococcus cysts, IL-10 is not expressed or expressed at a low level in the liver of mice. In the late stage of infection, the expression level of IL-10 in the liver of mice increased significantly, suggesting that the rapid reproduction of Echinococcus cysts in the mouse body and liver may be related to the significant increase in the expression level of IL-10. While IL-10 plays an anti-inflammatory role in the middle and late stages of infection, it also promotes the rapid growth of Echinococcus cyst tissue in the body. The proinflammatory factor CCL12 may be involved in the infiltration of monocyte-derived macrophages in the liver tissue, and it plays an important role in liver damage and fibrosis caused by hepatic echinococcosis. Macrophage-related cytokines are involved in the process of liver damage and fibrosis caused by hepatic echinococcosis, and the proportion of monocyte-derived macrophages in the liver tissue of mice infected with Echinococcus cysts is significantly increased.

[0084] The content of IL-10 and CCL12 cytokines in the serum of each group of mice was detected by double antibody sandwich enzyme-linked immunosorbent assay (ELISA). As shown in Table 4, compared with the control group, the expression levels of IL-10 and CCL12 in the model group were higher than those in the blank control group, and the difference was statistically significant; compared with the model group, the differences in the IL-25 group, ABZ group, HM group, H-2-168 group and ABZ+IL-25 group were statistically significant. Compared with the model group, ABZ group, HM group, IL-25 group and ABZ+IL-25 group, the expression levels of IL-10 and CCL12 in the HM+IL-25 group and H-2-168+IL-25 group were significantly reduced. The results showed that the combination of dehydrogenated harmine and IL-25 group significantly inhibited the expression of IL-10 and CCL12.

[0085] Table 4 ELISA determination of IL-10 and CCL12 levels in the supernatant of peritoneal macrophages of each group of mice (mean ± S, n = 3)

[0086]

[0087] Note: Compared with the blank control group, * P<0.05, *** P<0.001; compared with the model group, ### P<0.001.

[0088] 2.7 Detection of immune factors in mouse spleen cells by flow cytometry

[0089] Anesthetize the mouse with 1% sodium pentobarbital, spray 75% ethanol on the dissection site and surrounding area to sterilize, dissect the mouse, remove the spleen aseptically, place it in a sterilized culture dish containing 2mL PBS, and use ophthalmic scissors to remove other tissues around the spleen. Grind the spleen and filter it using a 100μm disposable coring cell strainer. Centrifuge at 1500 rpm for 10 minutes, discard the supernatant, add 5mL PBS buffer containing 10% FBS, and blow and mix the tissue homogenate. Pipette 2.5mL mouse lymphocyte separation solution into the bottom of a 15mL centrifuge tube, slowly add the tissue homogenate along the wall of the tube to the surface of the mouse lymphocyte separation solution, and centrifuge at 2000rpm for 20min. Pipette the second layer of white fog and place it in a 15mL centrifuge tube, wash with PBS, and centrifuge at 1500rpm for 5min. After discarding PBS, add 1mL red blood cell lysis solution to lyse the red blood cells. Incubate at 4℃ for 2min, then add 5mL PBS to rinse and terminate the reaction, centrifuge at 20℃, 2000rpm for 5min, and discard the supernatant. Place spleen lymphocytes in EP tubes, add labeled antibodies CD4, CD8, CD21, and CD28 respectively, place at 37℃ in the dark for 30min, wash with PBS, transfer to flow cytometer tubes, and analyze by flow cytometry.

[0090] In the immune response to parasitic infection, the host CD4 + The cells may first become activated and release cytokines such as IL-2, which in turn stimulate CD8 + T cells, CD8 + After T cells are activated, they exert their effects through direct cytotoxicity or secretion of cytokines, thereby amplifying the effects of CD4+ T cells. + T and CD8 + T cells increased significantly, but CD4 + The number of T cells increases faster than CD8 + However, during chronic infection with Echinococcus granulosus in mice, mouse spleen CD8 + T cells begin to proliferate continuously. Similarly, CD8 + T cells continue to proliferate in an active state. CD4 / CD8 + The dynamic balance of T cell ratio reflects the immune regulation state and immune level of the body. + A decrease in the T cell ratio indicates that the body's immune function is suppressed. Clinical patient in vivo test data show that with the formation of echinococcosis cysts, CD4 + The immune killing response dominated by T cells is weakened, forming a CD8 + An immunosuppressive state characterized by a gradual increase in the number of T cells.

[0091] CD21 is one of the important markers of B lymphocytes, which secrete antibodies and play a role in the humoral immune component of the adaptive immune system. Literature reports that infection with Echinococcus granulosus can lead to an increase in the number of mediating B cells in mice, thereby inhibiting anti-infective immunity and the proliferation of other pro-inflammatory lymphocytes.

[0092] CD28 can effectively enhance T cell receptors and induce T cell proliferation and differentiation. + CD28-T cells will increase, play an immunosuppressive role, and promote the development of Echinococcus granulosus.

[0093] Compared with the blank control group, the CD4 + T, CD8 + Compared with the model group, the proportion of T cells in the spleen of mice in the ABZ group, HM group, IL-25 group, ABZ+IL-25 group, and HM+IL-25 group was significantly lower than that in the blank control group. + Compared with the model group, ABZ group, HM group, H-2-168 group, IL-25 group and ABZ+IL-25 group, the CD4 / CD8 + The proportion increased significantly, and the difference was statistically significant (see Figure 7-10 , Table 5). Compared with the blank control group, the expression level of CD21 in the model group mice was higher than that in the blank control group, and the difference was statistically significant. Compared with the model group, the expression level of CD21 in the spleen of the IL-25 group mice was lower than that in the model group. Compared with the model group and IL-25 group, the expression levels of CD21 in the spleen of the ABZ group, HM group, H-2-168 group, ABZ+IL-25 group, HM+IL-25 group, and H-2-168+IL-25 group were higher than those in the model group and IL-25 group. Compared with the model group, ABZ group, HM group, IL-25 group, and ABZ+IL-25 group, the expression level of CD21 in the HM+IL-25 group was significantly increased, and the difference was statistically significant (see Figure 7-10 , Table 5). Compared with the blank control group, the expression of CD28 in the model group was higher than that in the blank control group, and the difference was statistically significant. Compared with the model group, the expression of CD28 in the spleen of mice in the ABZ group, HM group, H-2-168 group, IL-25 group, ABZ+IL-25 group, H-2-168+IL-25 group and HM+IL-25 group was lower than that in the model group. Compared with the model group, H-2-168 group and ABZ+IL-25 group, the expression of CD28 in the HM+IL-25 group and H-2-168+IL-25 group was significantly reduced, and the difference was statistically significant (see Figure 7-10 , Table 5).

[0094] Table 5 Flow cytometry detection of splenocyte subsets (mean ± S, n = 3)

[0095]

[0096] Note: Compared with the blank control group, * P<0.05, ** P<0.01, *** P<0.001; compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0097] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A composition for preventing and treating echinococcosis, characterized in that: Including β-carboline alkaloid compounds and interleukin-25; The β-carboline alkaloid compound is dehydrogenated harmine or a hydrogenated harmine derivative.

2. The composition according to claim 1, characterized in that The mass ratio of the β-carboline alkaloid compound to the interleukin-25 is 500:

1.

3. The composition according to claim 1, characterized in that The harmaline derivative is H-2-168.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a drug for preventing and treating echinococcosis.

5. A drug for preventing and treating echinococcosis, characterized in that: The active ingredient comprises the composition according to any one of claims 1 to 3.

6. The drug according to claim 5, characterized in that The drug also includes pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that The auxiliary materials include solubilizers, binders, disintegrants, fillers, stabilizers, preservatives, coating materials and / or fragrances.

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