P38-siRNA and application thereof in treatment of cystic echinococcosis and alveolar echinococcosis

Abstract

The invention discloses p38-siRNA and application thereof in treatment of cystic echinococcosis and alveolar echinococcosis, and belongs to the field of biomedicine. The technical problem to be solved by the invention is how to effectively treat alveolar echinococcosis or / and cystic echinococcosis. The p38-siRNA disclosed by the invention is a double-stranded RNA (Ribonucleic Acid) consisting of two single-stranded RNAs as shown in SEQ ID No. 1 and SEQ ID No. 2 in a sequence table. After the p38-siRNA disclosed by the invention is used for interfering with the protoscolex (PSCs), the growth and development of the echinococcus granulosus and the echinococcus multilocularis are obviously inhibited; in-vivo experiments prove that after interference of the p38-siRNA, focuses of the two types of echinococcosis are obviously reduced, fibrosis is weakened, inflammatory cells are reduced, and Treg cells are obviously increased. Therefore, the p38-siRNA provided by the invention can be used for treating the alveolar echinococcosis and the cystic echinococcosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to p38-siRNA and its application in the treatment of cystic echinococcosis and alveolar echinococcosis. Background Art

[0002] Echinococcosis is a serious zoonotic parasitic disease caused by the larvae of Echinococcus tapeworms parasitizing in humans and certain animals. More than 95% of echinococcosis cases are cystic echinococcosis (CE) caused by infection with Echinococcus granulosus ( Echinococcus granulosus , Eg ), and another seriously harmful one is alveolar echinococcosis (AE) caused by infection with Echinococcus multilocularis ( Echinococcus multilocularis , Em ), which is a fatal parasitic disease caused by its larvae parasitizing in the human body and is distributed worldwide. AE is severely harmful and almost originates in the liver, and is known as "parasitic cancer".

[0003] The mitogen-activated protein kinase (MAPK) family is an important signal transduction system in eukaryotic cells that mediates extracellular signals to intracellular responses. It conducts extracellular signals in the form of a three-tier kinase cascade, that is, extracellular signal → MAPK kinase kinase (MKKK) → MAPK kinase (MKK) → MAPK, regulating various important cellular physiological / pathological processes such as cell proliferation, differentiation, apoptosis, stress adaptation to the environment, and inflammatory response. The p38MAPK pathway is one of the important members of the MAPK family, regulating various biological processes such as cell proliferation, differentiation, apoptosis, and cytokine production. Echinococcus granulosus is a multicellular organism, and information communication between cells needs to be regulated by signal transduction pathways for its growth and development. It has been clearly found that p38 exists in Eg the protoscolex.

[0004] Small interfering RNA (siRNA) is a short double-stranded RNA molecule with a length of 21-25 nucleotides, having high specificity and gene silencing function, and can specifically degrade target mRNA, thereby inhibiting the expression of specific genes. This unique mechanism makes it show great potential in the treatment of various diseases, including infectious diseases, hematological malignancies, cardiovascular diseases, and neurodegenerative diseases, etc. However, there are currently few studies on specifically interfering with certain specific genes by siRNA and then affecting the growth and development of the worm. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to effectively treat alveolar echinococcosis and / or cystic echinococcosis.

[0006] To solve the above technical problem, the present invention first provides any of the following applications: 1. The application of siRNA in the preparation of products for treating cystic echinococcosis and alveolar echinococcosis, or the application in treating cystic echinococcosis and alveolar echinococcosis; The siRNA is a double-stranded RNA composed of two single-stranded RNAs shown in SEQ ID No.1 and SEQ ID No.2 in the sequence listing; 2. The application of the siRNA in the preparation of products for treating alveolar echinococcosis, or the application in treating alveolar echinococcosis; 3. The application of the siRNA in the preparation of products for treating cystic echinococcosis, or the application in treating cystic echinococcosis; 4. The application of the siRNA in the preparation of products for treating diseases caused by Echinococcus granulosus and Echinococcus multilocularis, or the application in treating diseases caused by Echinococcus granulosus and Echinococcus multilocularis; 5. The application of the siRNA in the preparation of products for treating diseases caused by Echinococcus multilocularis, or the application in treating diseases caused by Echinococcus multilocularis; 6. The application of the siRNA in the preparation of products for treating diseases caused by Echinococcus granulosus, or the application in treating diseases caused by Echinococcus granulosus.

[0007] In the above applications, the treatment of diseases caused by Echinococcus multilocularis can be reflected in at least one of the following: X1. Inhibiting the growth of lesions caused by Echinococcus multilocularis; X2. Reducing the number and / or size of lesions caused by Echinococcus multilocularis; X3. Weakening the lesions produced by Echinococcus multilocularis; X4. Weakening the inflammatory reaction caused by Echinococcus multilocularis.

[0008] In the above applications, the treatment of diseases caused by Echinococcus granulosus can be reflected in at least one of the following: X1. Inhibiting the growth of lesions caused by Echinococcus granulosus; X2. Reducing the number and / or size of lesions caused by Echinococcus granulosus; X3. Weakening the lesions produced by Echinococcus granulosus; X4. Weakening the inflammatory reaction caused by Echinococcus granulosus.

[0009] The siRNA also belongs to the protection scope of the present invention.

[0010] The siRNA of the present invention can be transfected into Echinococcus to treat alveolar echinococcosis and cystic echinococcosis. After interfering with protoscoleces (PSCs) using the siRNA of the present invention, the growth and development of Echinococcus granulosus and Echinococcus multilocularis are significantly inhibited; in vivo experiments verify that after siRNA interference, the lesions of the two types of echinococcosis are significantly reduced, fibrosis is weakened, inflammatory cells (Eos, Neu) are reduced, and Treg cells are significantly increased. It shows that the siRNA of the present invention can be used to treat alveolar echinococcosis and cystic echinococcosis.

[0011] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 For the localization of Cy3-labeled siRNA in protoscoleces after 3 hours of electroporation observed by confocal microscopy after siRNA transfection into protoscoleces.

[0013] Figure 2 For the effects of each siRNA transfection for 3 days on the expression level of p38 in protoscoleces of Echinococcus granulosus and the viability of protoscoleces of Echinococcus granulosus. siRNA transfection for 3 days can reduce the expression of p38 mRNA, and among them, siRNA-768 has the best down-regulation effect. UT: untreated, untreated group; NC: negative control group.

[0014] Figure 3 For the silencing of p38 expression reducing the Eg ability of PSCs cultured in vitro to develop into cysts. PSCs were cultured for 14 days, and the formation of microcysts was observed after treatment with siRNA. UT: untreated group; NC: negative control group.

[0015] Figure 4 For the silencing of p38 expression reducing the Em ability of PSCs cultured in vitro to develop into cysts. PSCs were cultured for 7 days, and the formation of microcysts was observed after treatment with siRNA. UT: untreated group; NC: negative control group.

[0016] Figure 5 For the changes in mouse body weight, liver weight, ratio of liver weight to body weight (liver-body ratio), spleen weight, kidney weight and blood routine indexes after siRNA infection of mice. A, C are Eg groups of mice infected with PSCs, B, D are Em groups of mice infected with PSCs. con: mice not infected with PSCs; UT: untreated group; NC: negative control group.

[0017] Figure 6 Liver pathological changes in each group of mice after siRNA infection. A, C, and D are Eg the PSCs mouse infection model group, and B, E, and F are Em the PSCs mouse infection model group. A and B show the pathological changes of mouse liver tissue, C and E show the statistical results of the number of infected cysts / lesions, and D and F show the statistical results of the cyst diameter / lesion surface area, respectively. UT: untreated group; NC: negative control group.

[0018] Figure 7 Changes in lymphocytes in the liver of mice infected after siRNA transfection of PSCs. A - D are Eg the PSCs mouse infection model group, and E - H are Em the PSCs mouse infection model group. UT: untreated group; NC: negative control group.

[0019] Figure 8 For siRNA transfection Eg of PSCs and then infection of mice, changes in neutrophils (Neu) in the liver are shown. UT: untreated group; NC: negative control group.

[0020] Figure 9 For siRNA transfection Em of PSCs and then infection of mice, changes in eosinophils (Eos) in the liver are shown. UT: untreated group; NC: negative control group.

[0021] Figure 10 siRNA transfection Eg of PSCs and then infection of mice, changes in Treg cells in the liver are shown. UT: untreated group; NC: negative control group.

[0022] Figure 11 siRNA transfection Em of PSCs and then infection of mice, changes in Treg cells in the liver are shown. UT: untreated group; NC: negative control group. Specific implementation methods

[0023] The experimental methods in the following examples are all conventional methods, unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources, unless otherwise specified.

[0024] The livers of diseased sheep and gerbils infected with Echinococcus multilocularis were obtained from a slaughterhouse and gerbils conserved in our laboratory, respectively (Wu, C., et al. Genetic variation of mitochondrial genes among Echinococcus multilocularis isolates collected in western China . Parasit Vectors, 2017. 10(1): p. 265.). Among them, the livers of diseased sheep were identified as infected with Echinococcus granulosus ( Echinococcus granulosus, Eg ), and the gerbils were identified as infected with Echinococcus multilocularis ( Echinococcus multilocularis,Em ). Echinococcus granulosus and Echinococcus multilocularis were isolated respectively.

[0025] The data obtained from the experiments were processed using Excel software, and one-way ANOVA in GraphPad Prism software was used for variance analysis and multiple comparisons of the data.

[0026] Example 1 、 Effect of siRNA transfection of PSCs in vitro on gene expression levels 1. Preparation of p38 gene interfering RNA Three siRNAs were designed against the Egp38 gene of Echinococcus granulosus ( Echinococcus granulosus , Eg ), and the specific sequences are as follows: siRNA-571: Sense: 5’-CCGUGCUGAUGAGUUCAUdTdT-3’, Antisense: 5’AUGAAACUCAUCAGCACGGdTdT-3’, where dT represents thymidine deoxyribonucleic acid; siRNA-768: Sense: 5’-GGUGGUGUCAAGGUAUGAdTdT-3’ (SEQ ID No.1), Antisense: 5’-UCAUACCUUGAACACCACCdTdT-3’ (SEQ ID No.2), where dT represents thymidine deoxyribonucleic acid.

[0027] Design a Cy3-fluorescently labeled siRNA (siRNA-Cy3, as a control) to determine the transfection rate of protoscoleces (PSCs). A siRNA (SIR-RiboTM siRNA negative control, NC), which does not specifically target any human, mouse, rat, or Echinococcus granulosus gene, is used as a negative control. AgB3 is used as a control gene of Echinococcus granulosus itself, and two siRNAs targeting this gene (AgB3-336, AgB3-341) are designed to facilitate the detection of the importance of Egp38 for the growth and development of Echinococcus granulosus.

[0028] siRNA-Cy3: Sense: 5’-UUCUCCGAACGUGUCACGUTT-3’, Antisense: 5’-ACGUGACACGUUCGGAGAATT-3’.

[0029] SIR-RiboTM siRNA: Sense: 5’-UUCUCCGAACGUGUCACGUTT-3’, Antisense: 5’-ACGUGACACGUUCGGAGAATT-3’.

[0030] AgB3-336: Sense: 5’-GCAUGGCACUGAAGGAGUATT-3’, Antisense: 5’-UACUCCUUCAGUGCCAUGCTT-3’.

[0031] AgB3-341: Sense: 5’-GCACUGAAGGAGUAUGUCATT-3’, Antisense: 5’-UGACAUACUCCUUCAGUGCTT-3’.

[0032] 2. Transfection of PSCs with siRNA The siRNAs to be transfected are the siRNAs obtained in step 1, and the PSCs to be transfected are the PSCs of Echinococcus granulosus ( Echinococcus granulosus , Eg ) (denoted as Eg PSCs) and the PSCs of Echinococcus multilocularis ( Echinococcus multilocularis , Em ) (denoted as EmPSCs). First, rinse the PSCs three times with electroporation buffer (150 mM sucrose, 27 mM Na2HPO4, adjusted to pH 7.5), then add 2000 PSCs and siRNA to the electroporation buffer. siRNA-Cy3 is used as a control, and the final concentration of siRNA is 5 μM. Perform electroporation transfection with a square wave [(Square Wave Protocol (Gene Pulser II, Bio-Rad, USA)] at 125 V, 20 ms, and 1 pulse. After transfection, incubate at 37 °C for 10 min, add 1 mL of culture medium, transfer the transfected PSCs to a 24-well plate, and incubate in an incubator at 37 °C and 5% CO2. After culturing the siRNA-Cy3 transfection group for 3 h, observe the transfection situation under a fluorescence inverted microscope. Culture the remaining experimental groups for 3 h and 3 d after transfection to obtain PSCs of two types of worms transfected with each siRNA. Observe the morphological changes of PSCs under a light microscope. After collecting the remaining samples, rinse them three times with PBS and place them in a cryotube for storage at -80 °C. Untransfected PSCs are used as a control (UT).

[0033] The results showed that after three specific siRNAs designed against Egp38 were transfected into PSCs, the localization of siRNA-Cy3 in PSCs was observed under a fluorescence inverted microscope 3 h after transfection, and the transfection efficiency was about 70% - 80%, as shown in Figure 1. The natural mortality rate of PSCs was about 5 - 10% as observed by methylene blue staining of untransfected PSCs; electroporation had no significant effect on the survival of PSCs. Compared with the UT group, the NC group transfected with SIR-RiboTM siRNA and the groups of siRNA-571 and siRNA-768 did not have an obvious effect on the survival of PSCs.

[0034] 3. Real-time quantitative polymerase chain reaction (qRT-PCR) After step 2, extract the total RNA from the transfected siRNA PSCs and reverse transcribe it into cDNA. Dilute the cDNA 1:5 and take 2 μL for qRT-PCR to detect the expression level of the p38 gene, with eif3 as the internal reference gene. The primers used are as follows: p38 gene: 5’-TTCGAGAATGCAGGGTTCGC-3’ and 5’-GTCTCCAAGGAGGTTTGCGG-3’; eif3 gene: 5’-GTTACATCCCTCCGACCTTG-3’ and 5’-AAGCAGCCTCCTCTTGAGTG-3’.

[0035] All qRT-PCR systems involve pre-denaturation at 95°C for 30 sec in 1 cycle; PCR amplification for quantification at 95°C for 5 sec and 60°C for 30 sec in 40 cycles; melting curve analysis at 60 - 95°C for 5 sec in 1 cycle. The cycle threshold (Ct) values were normalized against eif3 and analyzed using the 2 -△△CT method to normalize each relative value to untreated PSC samples. The results showed that after 3 days of siRNA interference, the expression level of p38 mRNA was significantly decreased. The expression of p38 decreased after treatment with siRNA-571 but without statistical significance, while the expression of p38 decreased significantly after treatment with siRNA-768 (P<0.001) ( Figure 2 ).

[0036] Example 3. Detection of the effects after siRNA transfection of PSCs In this example, the effects of siRNA transfection of PSCs on the development of PSCs and its ability to inhibit their growth in mice were detected.

[0037] 1. Effects of siRNA on the growth of PSCs To determine whether the p38 gene was maximally knocked out by siRNA (siRNA-768) and effectively affected the development of PSCs. Under normal culture conditions, Eg PSCs and Em PSCs were first treated with 5 μM siRNA respectively, and then cultured normally for 2 weeks. PSCs without siRNA treatment were used as the untreated group (UT). The siRNAs used for intervention were SIR-RiboTM siRNA (NC, negative control), siRNA-571, siRNA-768, AgB3-336, and AgB3-341.

[0038] Comparing the treated group with the untreated group of PSCs showed that after p38 silencing, Eg PSCs ( Figure 3 ) and Em PSCs ( Figure 4 ) had reduced activity. siRNA-768 effectively inhibited the Eg PSCs and Em differentiation and development of PSCs towards the cystic direction.

[0039] 2. siRNA-768 can effectively reduce the infection foci of Echinococcus granulosus ( E. granulosus ) and Echinococcus multilocularis( E. mulilocularis ) A total of 48 SPF-grade C57 / 6J mice, 6 - 8 weeks old (20 ± 2 g), were used. The mice were randomly divided into six groups of 8 mice each. The grouping of the mice was as follows: untreated PSCs group (UT group), negative control siRNA-treated group (NC group), Egp38-specific siRNA (siRNA-571 group, siRNA-768 group), AgB3 control group (AgB3-336 group, AgB3-341 group). The siRNAs used were SIR-RiboTM siRNA (NC, negative control), siRNA-571, siRNA-768, AgB3-336, and AgB3-341.

[0040] First, each siRNA was introduced into Eg PSCs or Em PSCs by electroporation transfection. The transfection amount of siRNA was 5 μM / 2000 PSCs. After successful transfection, the corresponding group of mice was inoculated at a dose of 2000 PSCs per mouse by injection through the hepatic portal vein. The UT group was injected with untreated PSCs, the NC group was injected with PSCs treated with SIR-RiboTM siRNA, the siRNA-571 group was injected with PSCs treated with siRNA-571, the siRNA-768 group was injected with PSCs treated with siRNA-768, the AgB3-336 group was injected with PSCs treated with AgB3-336, and the AgB3-341 group was injected with PSCs treated with AgB3-341. Mice without PSC injection were used as controls (Control, con). Three months after modeling (i.e., 3 months after PSC injection), the mice were dissected.

[0041] Mouse blood was collected into 1.5 mL EP tubes containing sodium heparin (as much as possible), inverted up and down, mixed well, and placed in a 4°C refrigerator for static settlement. 50 μL of whole blood from each mouse was aspirated respectively for measuring the blood routine of mice in different groups. The blood routine detected red blood cells (RBC), white blood cells (WBC), lymphocytes (Lym), monocytes (Mon), neutrophils (Neutrophilic granulocyte, Neu), eosinophils (Eosinophil, Eos), and basophils (Basophil, Bas) in the blood. The results showed that they were all within the normal range compared with the normal control group (con), and there was no statistical significance ( Figure 5 in C, D).

[0042] Measure the body weight and organ weights of mice. The results showed that after siRNA interfered with PSCs, there were no significant changes in the body weight and organ weights of mice in the siRNA-specific knockdown group (siRNA-768) compared with the model group ( Figure 5 A and B in the figure), and the behaviors and appearances of mice in each group were normal during the experiment.

[0043] Dissect and record the liver infection foci. Perform HE staining on the remaining tissues, that is, after the tissues are fixed with 4% paraformaldehyde, dehydrated and embedded, prepare tissue sections of 4 μm. Place the mouse liver tissue sections in an oven at 60 °C and preheat for ≥30 min. After dewaxing with environmental dewaxing solution Ⅰ / Ⅱ for 15 min each, place them in absolute ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 15 s each for hydration, and rinse with distilled water once. Stain the cell nuclei with hematoxylin for 50 s, then rinse with distilled water 3 times until the distilled water is colorless. Place the sections in hydrochloric acid ethanol for 2 s, and then place them in PBS for blueing for 5 min. Place the sections in eosin staining solution for 1-2 min to stain the cytoplasm, and then rinse with distilled water 3 times until the distilled water is colorless. After 75% ethanol, 80% ethanol, 95% ethanol, and absolute ethanol for 15 s each, and environmental dewaxing solution Ⅰ / Ⅱ for 2 min each, air-dry the sections and then drop neutral resin, and cover the tissue with a cover glass of the appropriate size.

[0044] The in vivo intervention effect of siRNA and the histopathological results are as Figure 6 shown. After 3 months of infection, Eg In the PSCs-infected (UT) group and the irrelevant sequence interference (NC) group of mice, the liver lesions gradually increased. Each liver lesion had a varying number of cysts, and the formed cyst in the lesion was filled with cyst fluid. The lesions were also distributed in different liver lobes. The number of cysts in the UT group, NC group, siRNA-571 group, siRNA-768 group, AgB3-336 group, and AgB3-341 group were 3.67±1.58, 2.14±1.46, 1.00±0.63, 0.50±0.54, 2.0±1.41, and 2.40±2.07 respectively, and the major diameters of the measured cysts were 2.08±0.51, 2.2±0.44, 1.17±0.41, 0.6±0.55, 1.89±0.60, and 1.64±0.50 mm respectively. Whether it was the number of cysts or the cyst diameter (i.e., the major diameter), the siRNA-571 group and the siRNA-768 group were significantly lower than the untreated group and the negative control group.

[0045] EmIn the PSCs-infected (UT) group and the non-related sequence interference (NC) group, the liver lesions in mice gradually enlarged, showing honeycomb-like lesions with diameters ranging from approximately 1 to 7 mm. Each liver contained multiple lesions, and the lesions could be seen distributed in different liver lobes. The numbers of lesions in the UT group, NC group, siRNA-571 group, siRNA-768 group, AgB3-336 group, and AgB3-341 group were 6.7 ± 3.59, 3.08 ± 2.32, 0.58 ± 0.53, 1.43 ± 0.53, 2.67 ± 1.36, and 2.56 ± 1.33 respectively, and the surface areas of the lesions were 15.72 ± 14.66, 11.84 ± 8.69, 3.33 ± 1.15, 3.9 ± 3.45, 7.43 ± 2.97, and 7.50 ± 3.16 mm respectively. Whether it was the number of lesions or the surface area of the lesions, both the siRNA-571 group and the siRNA-768 group were significantly lower than the untreated group and the negative control group.

[0046] It was shown that after treatment with siRNA-768, Eg and Em both the lesions were significantly reduced. Compared with the infected (UT) group and the non-related sequence interference (NC) group, siRNA-768 could significantly weaken the growth and development of the lesions.

[0047] 3. Detection of the expression of host lymphocyte surface molecules by flow cytometry The liver tissue was placed in a culture dish with a 200-mesh nylon mesh pre-placed, ground thoroughly, and filtered through the sieve into a 15 mL centrifuge tube. After centrifuging the liver tissue homogenate suspension at 2000 rpm for 5 min, the cell pellet was collected. The liver cell pellet was subjected to 40% Percoll gradient centrifugation to remove adipose tissue, and then 1× red blood cell lysate was added to separate lymphocytes. Take 1×10 6 single nuclear cell suspension, incubate and block it with anti-CD16 / CD32 in PBS buffer (containing 0.2% bovine serum albumin, PBSA) at 4°C for 20 min. Then, antibodies against surface markers of T / B cells (CD3, CD4, CD8, CD19, CD69), EOS cells (CD45, CD11b, Ly6g, Siglec-F, CD193, Ly6C), and Treg cells (CD3, CD4, NK1.1, CD25) were added and incubated in the dark at 4°C for 30 min. After washing with PBSA, the Treg cells were added with a permeabilization solution to make holes, and then Foxp3 antibody was added for intracellular labeling. After washing with PBSA, it was transferred to a flow tube and detected and analyzed by a flow cytometer. The reagents used in this part are as follows: FITC anti-mouse CD69: Biolegend, catalog number 104505; PE anti-mouse CD19: Biolegend, catalog number 152408; PE / Cy7 anti-mouse CD8a: Biolegend, catalog number 100722; APC anti-mouse CD4: Biolegend, catalog number 100412; APC / Cy7 anti-mouse CD3: Biolegend, catalog number 100222; APC / Cy7 anti-mouse CD45: Biolegend, catalog number 103116; PE / Cy7 anti-mouse Ly6g: Biolegend, catalog number 127618; Brilliant Violet 421 anti-mouse CD170 (Siglec-F): Biolegend, catalog number 155509; PE anti-mouse CD193: Biolegend, catalog number 144506; Brilliant Violet anti-mouse Ly-6C: Biolegend, catalog number 128033; FITC anti-mouse Percp cy5.5 NK1.1: Biolegend, catalog number 108706; FITC anti-mouse CD19: Biolegend, catalog number 15240.

[0048] Alexa Fluor 647 anti -mouse Foxp3: Biolegend, catalog number 126408 The results showed that Eg Three months after infection, the number of B cells in the siRNA-768 group increased, but there was no significant difference. The number of CD8+ T cells increased significantly (p<0.05) ( Figure 7 ). Three months after Em infection, the proportion of B cells in the siRNA-571 group and the siRNA-768 group was lower than that in the control group (p<0.05), and there was no significant change in CD4+ T and CD8+ T cells ( Figure 7 ). After siRNA-768 interference, for Eg Neu cells were significantly reduced after PSCs infection (p<0.05, Figure 8 ). After treatment with siRNA-768, EmPSCs-infected mice had a decrease in Eos cells, but it was not statistically significant. After treatment with siRNA-571, the Eos cells decreased significantly (p < 0.05). Figure 9 ). After interference with siRNA-768, Eg the Treg cells in PSCs-infected mice increased significantly (p < 0.001). Figure 10 ), Em the Treg cells in PSCs-infected mice also increased significantly (p < 0.05). Figure 11 ).

[0049] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements of the present invention, including those that depart from the scope disclosed in this application but are made by conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Use of siRNA in the preparation of products for treating cystic echinococcosis and alveolar echinococcosis, or use in treating cystic echinococcosis and alveolar echinococcosis; The siRNA is a double-stranded RNA composed of two single-stranded RNAs shown in SEQ ID No.1 and SEQ ID No.2 in the sequence listing.

2. Use of the siRNA described in claim 1 in the preparation of products for treating alveolar echinococcosis, or use in treating alveolar echinococcosis.

3. Use of the siRNA described in claim 1 in the preparation of products for treating cystic echinococcosis, or use in treating cystic echinococcosis.

4. Use of the siRNA described in claim 1 in the preparation of products for treating diseases caused by Echinococcus granulosus and Echinococcus multilocularis, or use in treating diseases caused by Echinococcus granulosus and Echinococcus multilocularis.

5. Use of the siRNA described in claim 1 in the preparation of products for treating diseases caused by Echinococcus multilocularis, or use in treating diseases caused by Echinococcus multilocularis.

6. Use of the siRNA described in claim 1 in the preparation of products for treating diseases caused by Echinococcus granulosus, or use in treating diseases caused by Echinococcus granulosus.

7. The application according to any one of claims 4 to 6, characterized in that: The treatment of diseases caused by Echinococcus multilocularis is manifested in at least one of the following: X1. Inhibiting the growth of lesions caused by Echinococcus multilocularis; X2. Reducing the number and / or size of lesions caused by Echinococcus multilocularis; X3. Weakening the lesions produced by Echinococcus multilocularis; X4. Weakening the inflammatory response caused by Echinococcus multilocularis.

8. The application according to any one of claims 4-7, characterized in that: The treatment of diseases caused by Echinococcus granulosus is manifested in at least one of the following: X1. Inhibiting the growth of lesions caused by Echinococcus granulosus; X2. Reducing the number and / or size of lesions caused by Echinococcus granulosus; X3. Weakening the lesions produced by Echinococcus granulosus; X4. Weakening the inflammatory response caused by Echinococcus granulosus.

9. The siRNA described in claim 1.

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