Cysticercus pisiformis, the larval stage of Taenia pisiformis, causes serious illness in rabbits that severely impacts the rabbit breeding industry. An inhibitive Th2 immune response can be induced by let-7-enriched exosomes derived from T. pisiformis cysticercus. However, the underlying molecular mechanisms are not completely understood. Here, we report that exosomal miR-let-7-5p released by T. pisiformis cysticercus played a critical role in the activation of M2 macrophages. We found that overexpression of let-7-5p in M1 macrophages decreased M1 phenotype expression while promoting polarization to the M2 phenotype, which is consistent with experimental data in exosome-treated macrophages alone. In contrast, knockdown of let-7-5p in exosome-like vesicles promoted M1 polarization and decreased M2 phenotype expression. Furthermore, down-regulation of transcription factor CCAAT/enhancer-binding protein (C/EBP)-δ resulted in the decrease of M1 phenotype markers and increase of M2 phenotype markers. These results suggested that let-7 enriched in exosome-like vesicles from T. pisiformis metacestodes can induce M2 macrophage polarization via targeting C/EBP δ, which may be involved in macrophage polarization induced by T. pisiformis metacestodes. The finding helps to expand our knowledge of the molecular mechanism of immunosuppression and Th2 immune response induced by metacestodes.

Metacestodiasis is an infectious disease caused by the larval stage of cestode parasites. This disease poses a serious health hazard to wildlife, livestock, and humans, and it incurs substantial economic losses by impacting the safety of the livestock industry, the quality of meat production, and public health security. Unfortunately, there is currently no available molecular diagnostic method capable of distinguishing cysticercus‐ and Echinococcus ‐derived microRNAs (miRNAs) from other helminthes and hosts in the plasma of metacestode‐infected animals. This study aims to develop a specific, sensitive, and cost‐efficient molecular diagnostic method for cysticercosis and echinococcosis, particularly for early detection. The study developed a rolling circular amplification (RCA)‐assisted CRISPR/Cas9 detection method based on parasite‐derived miRNA let‐7‐5p. Using a series of dilutions of the let‐7 standard, the limit of detection (LOD) of the qPCR, RCA, and RCA‐assisted CRISPR/Cas9 methods was compared. The specificity of qPCR and CRISPR/Cas9 was evaluated using four artificially synthesized let‐7 standards from different species. A total of 151 plasma samples were used to evaluate the diagnostic performance. Additionally, the study also assessed the correlation between plasma levels of let‐7‐5p, the number of Taenia pisiformis cysticerci, and the weight of Echinococcus multilocularis cysts. The results demonstrated that the RCA‐assisted CRISPR/Cas9 assay could significantly distinguish let‐7 from cestodes and other species, achieving a LOD of 10 aM; the diagnostic sensitivity and specificity for rabbit cysticercosis and mouse E. multilocularis were 100% and 97.67%, and 100% and 100%, respectively. Notably, let‐7‐5p gradually increased in the plasma of T. pisiformis ‐infected rabbits from 15 days post infection (dpi), peaked at 60 dpi, and persisted until 120 dpi. In E. multilocularis ‐infected mice, let‐7‐5p gradually increased from 15 dpi and persisted until 90 dpi. Furthermore, the expression of let‐7‐5p positively correlated with the number of cysticerci and cyst weight. These results indicated that the let‐7‐5p‐based RCA‐assisted CRISPR/Cas9 assay is a sensitive and specific detection method that can be used as a universal diagnostic method for metacestodiasis, particularly for early diagnosis (15 dpi).

Cysticercus pisiformis (C. pisiformis), the larval form of Taenia pisiformis, parasitize mainly the liver, omentum and mesentery of rabbits and cause huge economic losses in the rabbit breeding industry. MicroRNA (miRNA), a short non-coding RNA, is widely and stably distributed in the plasma and serum. Numerous data demonstrates that, after parasitic infection, miRNAs become the key regulatory factor for controlling host biological processes. However, the roles of serum miRNAs in C. pisiformis-infected rabbits have not been elucidated. In this study, we compared miRNA expression profiles between the C. pisiformis-infected and healthy rabbit serum using RNA-seq. A total of 192 miRNAs were differentially expressed (fold change ≥ 2 and p < 0.05), including 79 up- and 113 downregulated miRNAs. These data were verified by qRT-PCR (real time quantitative polymerase chain reaction) analysis. Additionally, GO analysis showed that the target genes of these dysregulated miRNAs were most enriched in cellular, single-organism and metabolic processes. KEGG pathway analysis showed that these miRNAs target genes were involved in PI3K-Akt, viral carcinogenesis and B cell receptor signaling pathways. Interestingly, after aligning clean reads to the T. pisiformis genome, four (miR-124-3p_3, miR-124-3p_4, miR-124a and novel-miR1) T. pisiformis-derived miRNAs were found. Of these, novel-miR1was upregulated in different periods after C. pisiformis infection, which was verified qRT-PCR, and pre- novel-miR-1 was amplified from the cysticerci by RT-PCR, implying novel-miR-1 was derived from C. pisiformis and has great potential for the diagnosis of Cysticercosis pisiformis infection. This is the first investigation of miRNA expression profile and function in the serum of rabbits infected by C. pisiformis, providing fundamental data for developing diagnostic targets for Cysticercosis pisiformis.

Taenia asiatica is a zoonotic parasite found in the human intestine and pig liver that evolved various strategies to survive the host’s defenses. Exosomes are membranous vesicles released by cells and are an important vehicle in parasite-host interactions. However, no literature exists on the specific infection mechanisms of T. asiatica against the host defense response, and further research is required to understand the parasite-host interaction. In this study, we investigated the host’s differentially expressed genes (DEGs) while stimulating them with exosomes derived from the T. asiatica adult worm (Tas-exo) on LoVo by RNA-seq analysis. Our results identified 348 genes as being significantly differentially expressed for the Tas-exo group when comparing with that of the NC group. Some of these genes are related to modulation of cell proliferation and cell autophagy. Surprisingly, autophagy and cell proliferation have crucial roles in the defense against parasites; accordingly, we detected cell proliferation and autophagy in LoVo cells by CCK8, immunofluorescence, and Western blotting, demonstrating that Tas-exo could inhibit LoVo cell proliferation and autophagy via AMPK pathway. When P62 and p-mTOR/mTOR expression were significantly increased, BeclinI and pAMPK/AMPK were significantly decreased. These results expand our understanding of parasite-host interactions mediated by exosomes.

Infection of Taenia pisiformis cysticercus is very frequently found in lagomorphs and causes serious economic losses to rabbit breeding industry. T. pisiformis cysticercus has evolved numerous strategies to manipulate their hosts. The release of exosomes is of importance in the interaction between host and parasite. However, the mechanism by which T. pisiformis cysticercus evades the host immune system for long-term survival within the host remains unclear. Using small RNA sequencing and TMT labelling proteomic, we profiled the expression patterns of miRNAs and proteins in rabbit peritoneal macrophages treated with T. pisiformis cysticercus exo-somes. Seven differentially expressed (DE)-miRNAs and six DE-proteins were randomly selected to validate the accuracy of the sequencing data by qRT-PCR or western blot. Functions of DE-miRNAs and proteins were analyzed using public data bases. And DE-miRNAs-DE-proteins correlation network were established. CCK-8 assay was used to evaluate the effect of exosomes on macrophages proliferation. Cell cycle of macrophages, isolated from T. pisiformis-infected rabbits, was determined using flow cytometry. A total of 21 miRNAs were significantly differentially expressed, including three worm-derived miRNAs. The expressions of miRNAs and proteins were consistent with the sequencing results. DE-miRNAs targets were related to cell proliferation and apoptosis. Exosomes treatment resulted in a decrease of macrophages proliferation. In vivo, T. pisiformis cysti-cercus significantly induced S phase cell arrest. Moreover, DE-proteins were related to production of interferon -gamma and interleukin-12, and immunoregulation. Correlation network analysis revealed a negative correlation relationship between DE-miRNAs and DE-proteins. Among them, novel334 and tpi-let-7-5p have potential reg-ulatory effects on IL1 & beta; and NF & kappa;B2 respectively, which imply that novel334-IL1 & beta;/tpi-let-7-5p-NF & kappa;B2 axis may be an important way that T. pisiformis cysticercus modulates host immune response through exosomes. Further understanding of these potential regulatory mechanisms will contribute to clarify the mechanism of escape mediated by T. pisiformis exosomes.

Objective: To explore the potential targets and underlying mechanism of Rubi Fructus in the treatment of type 2 diabetes mellitus. Methods: Potential active components of Rubi Fructus were proposed by literature search and as well followed the Lipinski’s Rule of Five. The therapeutic targets of potential active components and T2DM-related genes were selected through Swiss target prediction and Gene Cards. The network of potential active components and T2DM-related targets were constructed by Cytoscape 3.7.2, and STRING analysis was performed to reveal the interaction among target proteins and to build the network of protein-protein interaction. Both Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of core targets were performed using David database. Results: Totally, 68 potential active components of Rubi Fructus and 196 T2DM-related targets were found. Among them, 39 key targets were obtained by network analysis, which were primarily enriched on PI3K-Akt, HIF-1, TNF, Toll-like receptor, and Focal adhesion signaling pathways. Conclusion: For the treatment of T2DM, the active compounts of Rubi Fructus can regulate the expression of proinflammatory and oxidative stress mediators and improving insulin resistance through multiple components, targets, and pathways, which offers a scientific support to further Rubi Fructus studies and to the research on T2DM treatment.