Study on the mechanism of miRNAs on liver injury in the condition of Protoscocephalus alveolarus transhepatic portal vein infection.

OBJECTIVE: To explore the role of miRNA in liver damage caused by Echinococcus multilocularis infection. METHODS: Six female C57BL mice were randomly divided into two groups, the control group and the infection group. Mice in the control group were injected with 100 µL PBS through the hepatic portal vein, and mice in the infection group were infected with E. multilocularis via the hepatic portal vein to establish a mouse model of infection. Small RNA sequencing was performed for detecting the expression of miRNAs in the liver of mice infected with 2000 E. multilocularis after 3 months of infection, screen out miRNAs related to liver damage, and verify by RT-PCR. RESULTS: Seventy-one differentially expressed miRNAs were found in the liver in comparison with control, and a total of 36 mouse miRNAs with |FC| >0.585 were screened out, respectively. In addition, Targetscan (V5.0) and miRanda (v3.3a) software were used to predict differential miRNAs target genes and functional enrichment of target genes. Functional annotation showed that "cytokine-cytokine interaction," "positive regulation of cytokine production," "inflammatory response," and "leukocyte activation" were enriched in the liver of E. multilocularis-infected mice. Moreover, the pathways "human cytomegalovirus infection," "cysteine and methionine metabolism," "Notch signaling pathway," and "ferroptosis" were involved in liver disease. Furthermore, four miRNAs (mmu-miR-30e-3p, mmu-miR-203-3p, mmu-miR-125b-5p, and mmu-miR-30c-2-3p) related to liver injury were screened and verified. CONCLUSION: This study revealed that the expression profiling of miRNAs in the livers was changed after E. multilocularis infection, and improved our understanding of the transcriptomic landscape of hepatic echinococcosis in mice.

Development of an earthworm-based soft robot for colon sampling.

Colorectal cancer as a major disease that poses a serious threat to human health continues to rise in incidence. And the timely colon examinations are crucial for the prevention, diagnosis, and treatment of this disease. Clinically, gastroscopy is used as a universal means of examination, prevention and diagnosis of this disease, but this detection method is not patient-friendly and can easily cause damage to the intestinal mucosa. Soft robots as an emerging technology offer a promising approach to examining, diagnosing, and treating intestinal diseases due to their high flexibility and patient-friendly interaction. However, existing research on intestinal soft robots mainly focuses on controlled movement and observation within the colon or colon-like environments, lacking additional functionalities such as sample collection from the intestine. Here, we designed and developed an earthworm-like soft robot specifically for colon sampling. It consists of a robot body with an earthworm-like structure for movement in the narrow and soft pipe-environments, and a sampling part with a flexible arm structure resembling an elephant trunk for bidirectional bending sampling. This soft robot is capable of flexible movement and sample collection within an colon-like environment. By successfully demonstrating the feasibility of utilizing soft robots for colon sampling, this work introduces a novel method for non-destructive inspection and sampling in the colon. It represents a significant advancement in the field of medical robotics, offering a potential solution for more efficient and accurate examination and diagnosis of intestinal diseases, specifically for colorectal cancer.

Omics-based investigation of pathological liver injury induced by Echinococcus multilocularis infection in mice.

BACKGROUND: Alveolar echinococcosis (AE) can cause severe liver injury and be fatal if left untreated. Currently, there are no effective therapeutic options for AE-induced liver injury. Therefore, by exploring the changes of gene proteins in mice with damaged liver, we attempted to identify the key molecules of liver damage, and provide data that will enable the development of drugs targeting hepatic AE. METHODS: BALB/c mice were inoculated with protoscoleces via the hepatic portal vein. Three months later, B-ultrasound examination and Hematoxylin-eosin (H&E) staining were used to confirm liver damage in mice. RNA sequencing and Liquid chromatography-mass spectrometry (LC-MS) were used to screen differentially expressed molecules associated with liver damage through bioinformatics, and Quantitative Real-Time PCR (qRT-PCR) was used to verify their expression. RESULTS: B-ultrasound examination showed liver lesions in the infected group, and H&E staining showed liver inflammation, fibrosis and liver necrosis. RNA sequencing and LC-MS results showed changes in the levels of more than 1000 genes and proteins, with upregulation of immune and inflammation pathways. By contrast, the downregulated genes and proteins were mostly involved in various metabolic reactions. Correlation analysis was conducted between the transcriptome data and proteome data. The results revealed 240 differentially expressed genes, of which 192 were upregulated, and 48 were downregulated. Many of these genes were involved in metabolic reactions, such as Catalase (Cat), fatty acid synthase (Fasn), and IL-16 genes, which may have relevance to liver injury. The results of qRT-PCR were consistent with those of bioinformatics analysis. CONCLUSIONS: The mechanisms of liver injury in mice infected with Echinococcus multilocularis are complex, involving abnormal metabolism, oxidative stress, inflammatory response, and many other factors. This study provides the data for preliminary exploration for the development of targeted therapies against AE.

Immunoprotective effect and mechanism of rEg.P29 against CD4 + T cell-deficient mice with Echinococcus multilocularis infection.

Alveolar echinococcosis (AE) is a zoonotic parasitic disease caused by infection with the larval stage of Echinococcus multilocularis and a major challenge to human public health. Vaccines are the most effective way to prevent and control infectious diseases. We previously revealed that the Echinocuccus granulosus recombinant protein P29 is a good vaccine candidate against E. granulosus. However, the protective and immunological mechanism of rEg.P29 against E. multilocularis remain unclear. In this study, CD4 + T cell-deficient mice are transferred with spleen CD4 + T cells isolated from wild-type mice and subjected to rEg.P29 immunization, and then these immunized mice are infected with E. multilocularis. The cyst inhibition rate is calculated by weighing the body and cyst weights. The level of antibody is detected by ELISA. Flow cytometry is used to detect the level of IFN-γ production by CD4 + T and CD8 + T cells. The cytokines in culture supernatant are detected by ELISA. The expressions of CD44 and CD62L on memory T cells are determined by flow cytometry. The results show the cyst inhibition rate is 41.52% after adoptive transfer of CD4 + T cells. Furthermore, the levels of IgG, IgM, IgA and IgE in serum are significantly increased compared with those in the PBS group. The IFN-γ-secretion by CD8 + T cells and the level of IFN-γ in culture supernatant are obviously increased; and the number of CD4 + T cells is increased, but the number of IFN-γ producing CD4 + T cells has no significant difference compared with PBS group. In addition, the number of CD44 +CD62L ‒CD8 + memory T cells in the spleen is significantly increased, while the number of CD44 ‒CD62L + CD8 + memory T cells is not significantly altered. Collectively, rEg.P29 can alleviate E. multilocularis infection by inducing humoral immune responses and CD8 + T cell responses.

Changes in intestinal flora of mice induced by rEg.P29 epitope peptide vaccines.

OBJECTIVE: Cystic echinococcosis (CE), a zoonotic parasitic disease caused by Echinococcus granulosus, remains a public health and socioeconomic issue worldwide, making its prevention and treatment of vital importance. The aim of this study was to investigate changes in the intestinal microbiota of mice immunized with three peptide vaccines based on the recombinant antigen of E. granulosus, P29 (rEg.P29), with the hope of providing more valuable information for the development of vaccines against CE. METHODS: Three peptide vaccines, rEg.P29T , rEg.P29B , and rEg.P29T + B , were prepared based on rEg.P29, and a subcutaneous immunization model was established. The intestinal floras of mice in the different immunization groups were analyzed by 16 S rRNA gene sequencing. RESULTS: The intestinal microbiota analysis at both immunization time points revealed that Firmicutes, Bacteroidota, and Verrucomicrobiota were the predominant flora at the phylum level, while at the genus level, Akkermansia, unclassified_Muribaculaceae, Lachnospiraceae_NK4A136_group, and uncultured_rumen bacterium were the dominant genera. Some probiotics in the intestines of mice were significantly increased after immunization with the peptide vaccines, such as Lactobacillus_taiwanensis, Lactobacillus_reuteri, Lachnospiraceae_NK4A136_group, Bacteroides_acidifaciens, and so forth. Meanwhile, some harmful or conditionally pathogenic bacteria were decreased, such as Turicibacter sanguinis, Desulfovibrio_fairfieldensis, Clostridium_sp, and so forth, most of which are associated with inflammatory or infectious diseases. Kyoto Encyclopaedia of Genes and Genomes enrichment analysis revealed that the differential flora were enriched in multiple metabolic pathways, primarily biological systems, human diseases, metabolism, cellular processes, and environmental information processing. CONCLUSION: In this study, we comprehensively analyzed and compared changes in the intestinal microbiota of mice immunized with three peptide vaccines as well as their related metabolic pathways, providing a theoretical background for the development of novel vaccines against E. granulosus.

Recombinant antigen P29 of Echinococcus granulosus induces Th1, Tc1, and Th17 cell immune responses in sheep.

Echinococcosis is a common human and animal parasitic disease that seriously endangers human health and animal husbandry. Although studies have been conducted on vaccines for echinococcosis, to date, there is no human vaccine available for use. One of the main reasons for this is the lack of in-depth research on basic immunization with vaccines. Our previous results confirmed that recombinant antigen P29 (rEg.P29) induced more than 90% immune protection in both mice and sheep, but data on its induction of sheep-associated cellular immune responses are lacking. In this study, we investigated the changes in CD4+ T cells, CD8+ T cells, and antigen-specific cytokines IFN-γ, IL-4, and IL-17A after rEg.P29 immunization using enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and flow cytometry to investigate the cellular immune response induced by rEg.P29 in sheep. It was found that rEg.P29 immunization did not affect the percentage of CD4+ and CD8+ T cells in peripheral blood mononuclear cells (PBMCs), and was able to stimulate the proliferation of CD4+ and CD8+ T cells after immunization in vitro. Importantly, the results of both ELISPOT and ELISA showed that rEg.P29 can induce the production of the specific cytokines IFN-γ and IL-17A, and flow cytometry verified that rEg.P29 can induce the expression of IFN-γ in CD4+ and CD8+ T cells and IL-17A in CD4+ T cells; however, no IL-4 expression was observed. These results indicate that rEg.P29 can induce Th1, Th17, and Tc1 cellular immune responses in sheep against echinococcosis infection, providing theoretical support for the translation of rEg.P29 vaccine applications.

Alterations in Gut Microbiota Profiles of Mice Infected with Echinococcus granulosus sensu lato Microbiota Profiles of Mice Infected with E. granulosus s.l.

OBJECTIVE: Cystic echinococcosis is a kind of parasitic disease that seriously endangers human and animal health. At present, its prevention and treatment still do not achieve the desired results. The aims of this study were to explore the effect of CE on intestinal microflora in mice. METHODS: In this study, 16S rRNA metagenome sequencing and bioinformatics were used to analyze the intestinal flora of mice infected with E. granulosus s.l. Changes in intestinal microbial community abundance were investigated and the differences in microbial populations of mice infected with E. granulosus s.l. were screened. RESULTS: Our results show that at the phylum level, nine abundant taxa were identified, the relative abundance of Firmicutes and Proteobacteria were enriched in infected mice, whereas Bacteroidetes and Patescibacteria were enriched in control mice (P < 0.01). At the class level, 13 abundant taxa were identified, the relative abundance of Bacilli was enriched in control mice, but decreased in infected mice (P < 0.01). At the order level, 15 abundant taxa were identified, the relative abundance of Lactobacillales was enriched in control mice, but decreased in infected mice (P < 0.01). At the family level, 28 abundant taxa were identified, enriched bacteria in the infected mice was Streptococcaceae, while the enriched bacteria in the control group was Lactobacillaceae (P < 0.01). At the genus level, 79 abundant taxa were identified, enriched bacteria in the infected mice was Streptococcus, while the enriched bacteria in the control group was uncultured_bacterium_f_Eggerthellaceae (P < 0.01). At the species level, 80 abundant taxa were identified, enriched bacteria in the infected mice was uncultured_bacterium_g_Streptococcus, while the enriched bacteria in the control group was uncultured_bacterium_f_Eggerthellaceae (P < 0.01). 39 KEGG pathways were identified that were differentially enriched between the infected and control mice. CONCLUSION: This study comprehensively demonstrates the differential intestinal microbiota of infected mice and analyzes the metabolic pathways related to the specific microbiota. This could provide new targets and research direction for the treatment and prevention of diseases caused by E. granulosus s.l.

Identification of a dominant murine T-cell epitope in recombinant protein P29 from Echinococcus granulosus

Echinococcus granulosus causes echinococcosis, an important zoonotic disease worldwide and a major public health issue. Vaccination is an economical and practical approach for controlling E. granulosus. We have previously revealed that a recombinant protein P29 (rEg.P29) is a good vaccine candidate against E. granulosus. However, T cell immunogenic epitopes have not been identified. In the present study, we use rEg.P29-immunized mice as models to screen immunogenic epitopes for the construction of a novel multi-epitope vaccine. We search for immunodominant epitopes from an overlapping peptide library to screen the peptides of rEg.P29. Our results confirm that rEg.P29 immunization in mice elicits the activation of T cells and induces cellular immune responses. Further analyses show that a T cell epitope within amino acids 86­100 of rEg.P29 elicits significant antigen-specific IFN-γ production in CD4+ and CD8+ T cells and promotes specific T-cell activation and proliferation. Collectively, these results provide a reference for the construction of a novel vaccine against broad E. granulosus genotypes based on epitopes of rEg.P29.

Expression profiling of exosomal miRNAs derived from different stages of infection in mice infected with Echinococcus granulosus protoscoleces using high-throughput sequencing.

Echinococcosis is a worldwide zoonosis. The mechanism of the establishment, growth, and persistence of parasites in the host has not been fully understood. Exosomes are found to be a way of information exchange between parasites and hosts. They exist in various body fluids widely. There are few studies on host-derived exosomes and their miRNA expression profiles at different infection time points. In this study, BALB/c mice were intraperitoneally infected with protricercariae. Exosomes were extracted from plasma (0, 3, 9, and 20 weeks post infection), and the expression profiles of exosome miRNA in the peripheral blood of mice were determined using RNA-sequencing. Compared to the 0 week groups, 24, 35, and 22 differentially expressed miRNAs were detected in infected mouse at the three infection stages, respectively. The results showed that there were significant differences in the miRNAs of exosomes at different infection time points. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to annotate the different miRNAs. The results showed that the biological pathways of parasites changed significantly at different stages of infection, with many significant and abundant pathways involved in cell differentiation, inflammation, and immune response, such as MAPK signaling pathway, Th17 cell differentiation, Wnt signaling pathway, FoxO signaling pathway, Notch signaling pathway, etc. These results suggest that miRNA may be an important regulator of interactions between Echinococcus granulosus and host. The data provided here provide valuable information to increase understanding of the regulatory function of microRNAs in the host microenvironment and the mechanism of host-parasite interaction. This may help us to find targets for Echinococcus granulosus to escape host immune attack and control Echinococcus granulosus infection in the future.

Coding and Noncoding RNA Expression Profiles of Spleen CD4+ T Lymphocytes in Mice with Echinococcosis.

Cystic echinococcosis (CE) is a severe and neglected zoonotic disease that poses health and socioeconomic hazards. So far, the prevention and treatment of CE are far from meeting people's ideal expectations. Therefore, to gain insight into the prevention and diagnosis of CE, we explored the changes in RNA molecules and the biological processes and pathways involved in these RNA molecules as E. granulosus infects the host. Interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17A, and tumor necrosis factor (TNF)-α levels in peripheral blood serum of E. granulosus infected and uninfected female BALB/c mice were measured using the cytometric bead array mouse Th1/Th2/Th17 cytokine kit. mRNA, microRNA (miRNA), long noncoding RNA (lncRNA), and circular RNA (circRNA) profiles of spleen CD4+ T cells from the two groups of mice were analyzed using high-throughput sequencing and bioinformatics. The levels of IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF-α were significantly higher in the serum of the CE mice than in control mice (P < 0.01). In total, 1,758 known mRNAs, 37 miRNAs, 175 lncRNAs, and 22 circRNAs were differentially expressed between infected and uninfected mice (|fold change| ≥ 0.585, P < 0.05). These differentially expressed molecules are involved in chromosome composition, DNA/RNA metabolism, and gene expression in cell composition, biological function, and cell function. Moreover, closely related to the JAK/STAT signaling pathways, mitogen-activated protein kinase signaling pathways, P53 signaling pathways, PI3K/AKT signaling pathways, cell cycle, and metabolic pathways. E. granulosus infection significantly increased the levels of IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF-α in mouse peripheral blood of mice and significantly changed expression levels of various coding and noncoding RNAs. Further study of these trends and pathways may help clarify the pathogenesis of CE and provide new insights into the prevention and treatment of this disease.

The Recombinant Eg.P29-Mediated miR-126a-5p Promotes the Differentiation of Mouse Naive CD4+ T Cells via DLK1-Mediated Notch1 Signal Pathway.

Cystic echinococcosis (CE) is a zoonotic parasitic disease spread worldwide caused by Echinococcus granulosus (Eg), which sometimes causes serious damage; however, in many cases, people are not aware that they are infected. A number of recombinant vaccines based on Eg are used to evaluate their effectiveness against the infection. Our previous report showed that recombinant Eg.P29 (rEg.P29) has a marvelous immunoprotection and can induce Th1 immune response. Furthermore, data of miRNA microarray in mice spleen CD4+ T cells showed that miR-126a-5p was significantly elevated 1 week after immunization by using rEg.P29. Therefore, in this perspective, we discussed the role of miR-126a-5p in the differentiation of naive CD4+ T cells into Th1/Th2 under rEg.P29 immunization and determined the mechanisms associated with delta-like 1 homolog (DLK1) and Notch1 signaling pathway. One week after P29 immunization of mice, we found that miR-126a-5p was significantly increased and DLK1 expression was decreased, while Notch1 pathway activation was enhanced and Th1 response was significantly stronger. The identical conclusion was obtained by overexpression of mmu-miR-126a-5p in primary naive CD4+ T cells in mice. Intriguingly, mmu-miR-126a-5p was significantly raised in serum from mice infected with protoscolex in the early stages of infection and markedly declined in the late stages of infection, while has-miR-126-5p expression was dramatically reduced in serum from CE patients. Taken together, we show that miR-126a-5p functions as a positive regulator of Notch1-mediated differentiation of CD4+ T cells into Th1 through downregulating DLK1 in vivo and in vitro. Hsa-miR-126-5p is potentially a very promising diagnostic biomarker for CE.

Metabolic profiling of liver and faeces in mice infected with echinococcosis.

BACKGROUND: Echinococcosis is a severe zoonotic parasitic disease which severely affects the health of the hosts. The diagnosis of echinococcosis depends mainly on imaging examination. However, the patient is often in the late stage of the disease when the symptoms appear, thus limiting the early diagnosis of echinococcosis. The treatment and prognosis of the patients are hampered because of long-term asymptomatic latency. Metabolomics is a new discipline developed in the late 1990s. It reflects a series of biological responses in pathophysiological processes by demonstrating the changes in metabolism under the influence of internal and external factors. When the organism is invaded by pathogens, the alteration in the characteristics of metabolites in cells becomes extremely sensitive. Here, we used a metabolomics approach involving liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to determine the molecular mechanism of cystic echinococcosis (CE) and to develop an effective method for CE diagnosis. METHODS: Twenty 8-week-old female BALB/c mice were divided into normal and Echinococcus granulosus infection groups. To develop the E. granulosus infection model, mice were infected with protoscoleces. Six weeks later, the abdomens of the mice showed significant bulging. An LC-MS/MS system-based metabolomics approach was used to analyse the liver and faeces to reveal the metabolic profiles of mice with echinococcosis. RESULTS: We found that the metabolism of nucleotides, alkaloids, amino acids, amides, and organic acids in mice is closely interrelated with E. granulosus infection. In the liver, the metabolic pathways of tyrosine and tryptophan biosynthesis; phenylalanine, valine, leucine and isoleucine biosynthesis; and phenylalanine metabolism were notably associated with the occurrence and development of hydatid disease, and in the faeces, pantothenate and CoA biosynthesis are thought to be closely associated with the development of CE. CONCLUSION: The metabolomics approach used in this study provides a reference for a highly sensitive and specific diagnostic and screening method for echinococcosis.

Screening, construction, and serological identification of the diagnostic antigen molecule EG-06283 for the diagnosis of Echinococcus granulosus.

Several strategies exist to prevent and control echinococcosis, a global parasitic disease. However, most treatments are ineffective and adverse effects are common. Therefore, we aimed to screen protoscolex antigen molecules of Echinococcus granulosus to identify a diagnostic biomarker for hydatid disease. Published E. granulosus transcriptome sequencing data were analyzed to screen for antigen molecules that are highly expressed in protoscoleces but not in oncospheres. The membrane protein EG-06283 (annotated as Frizzled-4) was selected from 16 antigens, and its gene fragment was subjected to codon optimization and synthesis. rEG-06283 expression was induced in the pET-24a/EG-06283/BL21 strain; subsequently, the protein was purified and subcutaneously injected into ICR mice at weeks 0, 2, 4, and 6. Blood sampling occurred periodically to quantify serum immunoglobulin G (IgG) levels via enzyme-linked immunosorbent assays (ELISA). Immunogenicity was determined by western blot assays using sera from normal mice and mice with secondary hydatid infections. The antigen's immune reactivity and diagnostic value were validated using sera of patients with hydatid disease. ELISA results confirmed that the antigen molecule induced specific IgG production in mice, resulting in significantly higher levels than those in the adjuvant and control groups (P < 0.05). The western blot results indicated that the protein was recognized by antibodies in the sera of mice with hydatid infection and the antisera of immunized mice. Quantification of protein levels in the sera of patients with hydatid disease significantly differed from levels in healthy participants (P < 0.05). These results indicate that rEG-06283 is a potential diagnostic antigen for E. granulosus infections.