The mice used in this study were 6–8 week old female C57BL/6J mice purchased from Jiangsu Jicui Yaokang Biotechnology Co (Production license number: SCXK (Su) 2023-0009). The mice were housed in an individually ventilated environment with a 12-hour light/12-hour dark cycle, and they were provided with food and water ad libitum. All experiments were conducted in accordance with the ethical standards set forth by the Ethics Committee of Ningxia Medical University (No.2024-G207).

The methodology employed for the isolation of the protoscolex was as previously described. In briefly, The cyst tissue was extracted from the laboratory germ rat and divided into smaller fragments. The cut tissue was filtered through an 80-mesh filter and washed twice with PBS. It was then allowed to settle naturally for five minutes, after which the supernatant was discarded. The protoscole precipitate was digested with 1% trypsin for 30 minutes, washed twice with PBS, and allowed to settle naturally. Finally, 1% eosin staining was used to identify the activity of the protoscole.

Following a one-week period of adaptive feeding, the protoscoleces (PSCs) were resuspended in phosphate buffered saline (PBS), and then 2000 protoscoleces were intraperitoneally injected into each C57BL/6N mouse. Three months following infection, the formation of lesions in the abdomen was identified via B-ultrasound in 20 mice. A total of 24 C57BL/6N mice were then randomly divided into four groups: an infection group, a 25 mg/kg oxymatrine group, a 50 mg/kg oxymatrine group, and a 100 mg/kg oxymatrine group. The mice in the infection group were administered 100 μL of phosphate-buffered saline (PBS, Hyclone) intraperitoneally on a daily basis. The mice in the 25 mg/kg oxymatrine group were intraperitoneally injected with oxymatrine solution (7.5 μL of a 10 mg/ml oxymatrine solution + 92.5 μL of PBS, SM4048-25mg, Beyotime, China) on a daily basis. The mice in the 50 mg/kg oxymatrine group were intraperitoneally injected with oxymatrine solution (15 μL of a 10 mg/ml oxymatrine solution + 85 μL of PBS) on a daily basis. The mice in the 100 mg/kg oxymatrine group were intraperitoneally injected with oxymatrine solution (30 μL of a 10 mg/ml oxymatrine solution + 70 μL of PBS) on a daily basis. All mice were administered the injection for a period of 30 consecutive days, after which samples were collected for analysis.

The concentration of isolated multilocular Echinococcus was adjusted to 2000 PSCs/ml, and 100 μl was added to each well of a 96-well plate. A solution of 0.25 mM, 0.35mM, 0.5mM, 0.75mM, 1.0mM and 1.25mM oxymatrine was administered for a period of 48 hours. Five replicate wells were set up for each intervention group. Following the intervention, 10 μl of CCK-8 reagent was added to each well and incubated at 37°C for one hour. The absorbance was then detected at 450 nm using a microplate reader.

LDH and ALT in the culture supernatant were measured using a lactate dehydrogenase assay kit (Beyotime Biotechnology, Shanghai, China) and an alkaline phosphatase assay kit(Solarbio, Beijing, China), strictly following the manufacturer’s instructions. Briefly, after the drug stimulation is completed, centrifuge the cell culture plates at 400 × g for 5 minutes using a multi-well plate centrifuge. Carefully aspirate the supernatant, add 150 μL of the LDH release reagent provided by the kit diluted 10 times with PBS (i.e., mix 1 volume of LDH release reagent with 10 volumes of PBS), gently shake the culture plates to ensure thorough mixing, and then place the plates back in the cell culture incubator for an additional 1-hour incubation. After the incubation, centrifuge at 400 × g for 5 minutes again. Subsequently, transfer 120 μL of the supernatant from each well to the corresponding wells of a new 96-well plate, and incubate at room temperature in the dark for 30 minutes. Measure the absorbance at 490 nm using a microplate reader. The formula for calculating cytotoxicity or cell death rate (%) is as follows: Cytotoxicity or cell death rate (%) = (Absorbance of treated sample - Absorbance of sample control well)/(Absorbance of maximum enzyme activity of cells - Absorbance of sample control well) × 100.

Six fecal samples were collected from each group of mice and subjected to a DNA extraction process using a commercial DNA extraction kit(TIANGEN BIOTECH, Beijing, China), following the manufacturer’s instructions. In brief, The following reagents are required: 30 μL SDS (10%), 3 μL proteinase K (20 g/L), and 4 μL RNASe. These should be added to the pretreated 500 μL sample and mixed. The sample should then be stored in a 37°C water bath for 1 hour. Next, 100 μL of 5 M NaCl should be added to each tube, and the tubes should be inverted to mix. Then, 80 μL of a 10% CTAB/NaCl mixture (0.7 M NaCl and 10% CTAB) should be added, and the tubes should be gently mixed. Finally, the tubes should be placed in a 65°C water bath for 10 minutes. Following this, an equal volume of a phenol/chloroform/isoamyl alcohol (25:24:1) mixture should be added, and the contents thoroughly mixed. The tubes should then be placed in a centrifuge at 12,000 r/min for 10 minutes, after which the resultant pellet should be discarded. The tubes should then be filled with 0.6 vol. of isopropanol, and the contents gently mixed. The tubes should then be placed in a centrifuge at 12,000 g/min for 10 minutes, after which the resultant pellet should be discarded. The precipitate was then washed with 1 mL of pre-cooled 75% ethanol, followed by centrifugation at 7500 r/min for 5 min. The ethanol was then discarded, the precipitate dried slightly on a clean bench, and dissolved in 30 μL of TE buffer. Ten fecal samples were taken from each group for microbiome analysis.

To ensure the accuracy and reliability of sequencing data from the source, From DNA extraction to sequencing, strict sample testing and quality control were carried out, and each step strictly controls the sample quality to ensure the authenticity and reliability of the sequencing data. For the raw data (RawData) obtained after sequencing. First, the overlapping regions at both ends are used for splicing, and then low-quality sequences and chimeric sequences are filtered out to obtain high-quality CleanData. Next, based on CleanData. The sequences were denoised using QIIME2 D2DA2 to remove possible PCR amplification and sequencing errors in high-throughput sequencing data, thereby obtaining representative correct biological sequences ASV (amplicon sequence variants) and the abundance table of ASV. Then, further conduct subsequent α-diversity analysis, β-diversity analysis, species composition analysis and difference analysis, as well as functional composition and difference analysis.

Following the administration, the mice were weighed. Anesthesia was induced in the mice with isoflurane, and the cyst tissues in the abdominal cavity, liver tissues, and spleen tissues were then separated and weighed. Cyst inhibition rate = (wet weight of cysts in the control group - wet weight of cysts in the experimental group)/wet weight of cysts in the control group × 100%. Liver or spleen index = (The weight of liver/spleen/the body weight of mouse) × 100.

It is imperative that the instructions for the reagents be followed precisely. The livers of the mice in each group were excised, fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin, and cut into 6 μm slices to prepare tissue sections. The HE sections were prepared through a series of steps, beginning with dewaxing of the tissue samples. This was followed by dehydration with a range of alcohol gradients, staining with hematoxylin and eosin, and a subsequent transparentization process. Finally, the sections were sealed. They were then observed and photographed under a microscope.

The automatic biochemical analyzer, Chemray 240, was employed to detect alterations in serum liver function indicators, namely alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

The lymphocytes from the spleen and liver were separated using the density gradient centrifugation method. In summary, the spleen and liver tissues were cut into small pieces of 1-2mm and placed on an 80 mesh filter for grinding. The resulting filtrate was collected and added to the upper layer of Percoll buffer, after which the mixture was subjected to centrifugation at 450g for 30 minutes. The lymphocyte layer was then carefully aspirated using a pipette. Finally, the lymphocytes were washed twice with PBS buffer and the cell count was performed. For flow cytometry, 1×10⁶ lymphocytes were collected into a centrifuge tube and subjected to cell staining. Subsequently, 2 μl of PE-CF594 anti-mouse CD3, APC-Cy7 anti-mouse CD4, PB anti-mouse CD8, FITC anti-mouse IFN-γ, and PE anti-mouse IL-4 antibodies were added at 4°C for 30 minutes. Subsequently, the cells were washed with PBS buffer and analyzed by flow cytometry.

The data from this study were analyzed using the statistical software packages SPSS 22.0 and GraphPad Prism 8.0. The normality of all data distributions was assessed using the Shapiro-Wilk test. Data that passed the normality test were analyzed using parametric tests (unpaired two-tailed Student’s t-test or one-way ANOVA). For data that violated the normality assumption, non-parametric tests (Mann-Whitney U test or Kruskal-Wallis test) were employed. The independent sample t-test or chi-square test were employed for the comparison of two groups, while the one-way analysis of variance or nonparametric test was used for the comparison of three or more groups. A p-value <0.05 was considered to be statistically significant.

Protoscolex was administered with 0mM, 0.25mM, 0.5mM, 0.75mM, 1.0mM and 1.25mM oxymatrine for a period of 48h, the cell vitality was detected by CCK8 assay. Compared with control group, following the administration of 0.35 mM oxymatrine, a notable decline in the viability of the protoscolex was observed (P < 0.05). As the concentration of oxymatrine increases, there is a concomitant decrease in the vitality of the protoscolex. Albendazole is the drug of choice for the treatment of alveolar echinococcosis, and this study employed 0.5 μg/ml albendazole as a positive control. In comparison with the positive control group, the viability of protoscolex cells was found to be significantly reduced following the administration of oxymatrine at concentrations of 1.0 mM (P < 0.05)and 1.25 Mm (P < 0.01), as shown in Figure 1 .

The construction of an infected mouse model was achieved by intraperitoneal injection of Echinococcus multilocularis. Intervention with oxymatrine was then conducted, after which samples were collected for testing. The specific timeline is shown in Figure 2A . The results showed that compared with the control group, after intervention with oxymatrine, the weight of cysts in mice was significantly reduced, while there were no significant changes in liver weight, spleen weight, liver index, and spleen index ( Figures 2B–D ). Albendazole is the preferred drug for the treatment of patients with alveolar echinococcosis in clinical practice. In this study, albendazole served as the positive control. Following oxymatrine intervention, no significant differences were observed in cyst weight, liver weight, and spleen weight within the abdominal cavity compared to the albendazole group ( Figures 2B–D ). These in vivo and in vitro results indicate that oxymatrine can inhibit the growth of multilocular echinococcosis.

Furthermore, we investigated the impact of oxymatrine on mouse liver tissue pathology. The control group mice exhibited a significant presence of inflammatory cells near liver lesions, along with notable tissue fibrosis. Upon oxymatrine treatment, a decrease in inflammatory cell infiltration around liver tissue lesions and alleviation of liver tissue fibrosis were observed( Figure 3 ).

To investigate the immunological mechanism by which oxymatrine mitigates Echinococcus multilocularis infection, liver mononuclear cells were isolated and analyzed using flow cytometry to monitor changes in CD4⁺ T cells, CD8⁺ T cells, and B cells. The findings revealed that compared to the control group, treatment with varying doses of oxymatrine (25mg/kg, 50mg/kg, and 100mg/kg) and albendazole did not result in significant alterations in CD4⁺ T cell ( Figures 4A, C ) and B cell populations within the liver of mice ( Figures 4B, E ). However, a notable increase was observed in the number of CD8⁺ T cells ( Figures 4A, D ). These results indicate that oxymatrine may combat multilocular echinococcosis infection by enhancing CD8⁺ T cell abundance in the liver.

The Venn diagram in Figure 5A illustrates the distribution of species across different treatment groups: 760 species in the Control group, 801 species in the 25mg/kg OMT group, 829 species in the 50mg/kg OMT group, 1037 species in the 100mg/kg OMT group, and 874 species in the ABZ group were identified. Alpha diversity indices, such as Ace, Chao index, Good’s coverage, Shannon index, Simpson index, and Pielou’s evenness, are commonly utilized to assess community richness and diversity ( Figures 5B–H ). In this investigation, Ace, Chao, Shannon, and Pielou’s evenness values were significantly higher in the 25mg/kg OMT, 50mg/kg OMT, and 100mg/kg OMT groups compared to the Control group (P.05). Moreover, beta-diversity within the OMT group showed enhancements through Bray-Curtis analysis and Principal Coordinates Analysis (PCoA) utilizing the 16S data. The findings revealed substantial shifts in the gut microbiota of mice post-OMT intervention compared to the control group ( Figures 5I–K ). Additionally, UPGMA (Unweighted Pair Group Method with Arithmetic Mean) hierarchical clustering analysis indicated a high level of similarity in microbial community composition among sample groups ( Figure 5L ). Furthermore, Analysis of Similarities (Adonis) was employed to assess the similarity of each sample component, revealing significant distinctions between sample groups ( Figures 5M–O ).

To investigate variations in gut microbiota among distinct mouse groups, the top 30 abundant microorganisms at the phylum, species, and genus levels were identified and annotated. Analysis at the phylum level revealed a significant decrease in Firmicutes microbiota following OMT intervention, accompanied by significant increases in Bacteroidota and Proteobacteria microbiota compared to the control group ( Figures 6A, D ). Similarly, at the genus level, A significant increase in Muribaculaceae and Bacteroides microbiota and a decrease in Lachnospiraceae microbiota were observed after OMT intervention compared to the control group. ( Figure 6B ). Additionally, species-level analysis indicated a significant decrease in Muribaculaceae post-OMT intervention compared to the control group ( Figure 6C ).

To determine differences in gut microbiota among groups of mice after OMT intervention. The differences in the intestinal microbiota among groups were examined in detail, and the significance threshold for different species was set at P < 0.05. In total, 50 unique groups were identified at the genus level ( Supplementary Materials S1 ). After conducting a differential analysis, thirty species showing significant differences were identified, and their abundances in different groups were presented in a histogram. Following treatment with 25mg/kg OMT, the groups g:Bacteroides, g:Muribaculum, g:Parabacteroides, and g:Paramibaculum exhibited significant up-regulation compared to the control group. Conversely, the groups g:Lachnospiraceae_NK4A136_group, g:Helicobacter, g:Desulfovibrio, g:Anaerotruncus, and g:Mucispirillum showed significant downregulation ( Figure 7A ). Following treatment with 50mg/kg OMT, the g_Ligilobacillus, g_Muribaculum, g_Parabacteroides, and g_Lactococcus groups exhibited significant upregulation compared to the control group. Conversely, the g:Lachnospiraceae_NK4A136_group, g:Helicobacter, and g:Desulfovibrio groups showed significant downregulation ( Figure 7B ). Following treatment with 100mg/kg OMT, the populations of g_Ligilobacillus, g_Muribaculum, g_Dubosiella, and g_Duncaniella in the mouse intestine exhibited significant upregulation compared to the control group. Conversely, the g:Lachnospiraceae_NK4A136_group, g:Helicobacter, and g:Anaerotruncus groups showed significant downregulation ( Figure 7C ).