[Effect and mechanism of aqueous extract of Strychni Semen on bone destruction in rats with type â ¡ collagen-induced rheumatoid arthritis].

To investigate the mechanism of action of aqueous extract of Strychni Semen(SA) on bone destruction in rats with type Ⅱ collagen-induced arthritis(CIA), the SD rats were randomly divided into normal group, model group, low, medium, and high dose(2.85, 5.70, and 11.40 mg·kg~(-1)) groups of SA, and methotrexate group. Except for the normal group, the CIA model was prepared for the other groups. After the second immunization, different doses of SA were given to the low, medium, and high dose groups of SA once a day, and the methotrexate group was given once every three days. 0.3% sodium hydroxymethylcellulose(CMC-Na) was given once a day to the normal and model groups for 28 d. The clinical score of arthritis was evaluated every three days. Micro computed tomography(Micro-CT) method was used to evaluate the degree of bone destruction. Histopathological changes in the joint tissue and the number of osteoclasts in CIA rats were evaluated by hematoxylin-eosin(HE) staining and tartrate-resistant acid phosphatase(TRAP) staining. The expression of interleukin-1ß(IL-1ß) in the joint tissue of rats was detected by immunohistochemistry. Western blot was used to detect key protein expression in mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) signaling pathways in the joint tissue of rats. The results showed that different doses of SA were able to improve the red and swollen inflammatory joint and joint deformity in CIA rats to varying degrees, reduce the clinical score, inhibit synovial inflammation, vascular opacification, cartilage erosion, and bone destruction, and reduce the number of TRAP-positive cells in bone tissue. Micro-CT results showed that the SA was able to increase bone mineral density, bone volume fraction, trabecular reduce, and trabecular number and reduce bone surface/bone volume and trabecular separation/spacing. Different doses of SA could down-regulate the protein expression of IL-1ß, p-JNK, p-ERK, p-p38, PI3K, and p-Akt to varying degrees. In conclusion, SA can improve disease severity, attenuate histopathological and imaging changes in joints, and have osteoprotective effects in CIA rats, and its mechanism of action may be related to the inhibition of the overactivation of MAPK and PI3K/Akt signaling pathways.

[Mechanism of bulleyaconitine A in inhibiting bone destruction of rheumatoid arthritis via Src/PI3K/Akt signaling pathway].

Based on the sarcoma receptor coactivator(Src)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway, the mechanism of action of bulleyaconitine A in the treatment of bone destruction of experimental rheumatoid arthritis(RA) was explored. Firstly, key targets of RA bone destruction were collected through GeneCards, PharmGKB, and OMIM databa-ses. Potential targets of bulleyaconitine A were collected using SwissTargetPrediction and PharmMapper databases. Next, intersection targets were obtained by the Venny 2.1.0 platform. Protein-protein interaction(PPI) network and topology analysis were managed by utilizing the STRING database and Cytoscape 3.8.0. Then, Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were conducted in the DAVID database. AutoDock Vina was applied to predict the molecular docking and binding ability of bulleyaconitine A with key targets. Finally, a receptor activator of nuclear factor-κB(RANKL)-induced osteoclast differentiation model was established in vitro. Quantitative real-time polymerase chain reaction(qRT-PCR) was used to detect the mRNA expression levels of related targets, and immunofluorescence and Western blot were adopted to detect the protein expression level of key targets. It displayed that there was a total of 29 drug-disease targets, and Src was the core target of bulleyaconitine A in anti-RA bone destruction. Furthermore, KEGG enrichment analysis revealed that bulleyaconitine A may exert an anti-RA bone destruction effect by regulating the Src/PI3K/Akt signaling pathway. The molecular docking results showed that bulleyaconitine A had better bin-ding ability with Src, phosphatidylinositol-4,5-diphosphate 3-kinase(PIK3CA), and Akt1. The result of the experiment indicated that bulleyaconitine A not only dose-dependently inhibited the mRNA expression levels of osteoclast differentiation-related genes cathepsin K(CTSK) and matrix metalloproteinase-9(MMP-9)(P<0.01), but also significantly reduced the expression of p-c-Src, PI3K, as well as p-Akt in vitro osteoclasts(P<0.01). In summary, bulleyaconitine A may inhibit RA bone destruction by regulating the Src/PI3K/Akt signaling pathway. This study provides experimental support for the treatment of RA bone destruction with bulleyaconitine A and lays a foundation for the clinical application of bulleyaconitine A.

[Mechanism of Yuxuebi Tablets in treating synovial inflammation in rheumatoid arthritis based on transcriptomics].

This study investigated the mechanism of Yuxuebi Tablets(YXB) in the treatment of synovial inflammation in rheumatoid arthritis(RA) based on transcriptomic analysis. Transcriptome sequencing technology was employed to analyze the gene expression profiles of joint tissues from normal rats, collagen-induced arthritis(CIA) rats(an RA model), and YXB-treated rats. Common diffe-rentially expressed genes(DEGs) were subjected to Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses. RA synovial inflammation-related target genes were retrieved from the OMIM and GeneCards databases. Venny 2.1 software was used to identify the intersection of YXB target genes and RA synovial inflammation-related target genes, and GO and KEGG enrichment analyses were performed on the intersecting target genes. Immunohistochemistry was used to assess the protein expression levels of the inflammatory factors interleukin-1ß(IL-1ß) and tumor necrosis factor-α(TNF-α) in rat joint tissues. Western blot analysis was employed to measure the expression levels of key proteins in the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway. A total of 2 058 DEGs were identified by intersecting the genes from the normal group vs model group and the model group vs YXB treatment group. A search in OMIM and GeneCards databases yielded 1 102 RA synovial inflammation-related target genes. After intersecting with the DEGs in the YXB treatment group, 204 intersecting target genes were identified, primarily involving biological processes such as immune response, signal transduction, and inflammatory response; cellular components including plasma membrane, extracellular space, and extracellular region; molecular functions like protein binding, identical protein binding, and receptor binding. These target genes were mainly enriched in signaling pathways such as PI3K/Akt, cytokine-cytokine receptor interaction, and Janus kinase/signal transducer and activator of transcription(JAK/STAT). Western blot results showed that YXB at low, medium, and high doses could significantly inhibit the expression levels of key proteins in the PI3K/Akt signaling pathway in rat joint tissues in a dose-dependent manner. Immunohistochemistry further confirmed these findings, showing that YXB not only suppressed the protein expression levels of the inflammatory factors IL-1ß and TNF-α in the joint synovial tissues of CIA rats, but also inhibited p-Akt protein expression. In conclusion, this study used transcriptomic analysis to uncover the key mechanisms of YXB in inhibiting synovial inflammation and alleviating the progression of RA, with a focus on its role in suppressing the PI3K/Akt signaling pathway.

[Mechanism of aqueous extract of Strychni Semen in improving pain in rats with rheumatoid arthritis based on TLR4/TNF-α/MMP-9 signaling pathway].

This study aims to explore the mechanism of aqueous extract of Strychni Semen(SA) in relieving pain in the rat model of rheumatoid arthritis(RA) via Toll-like receptor 4(TLR4)/tumor necrosis factor-α(TNF-α)/matrix metalloproteinase-9(MMP-9) signaling pathway. Firstly, the main chemical components of Strychni Semen were searched against TCMSP, TCMID, ETCM, and related literature, and the main targets of the chemical components were retrieved from TargetNet and SwissTargetPrediction. The main targets of RA and pain were searched against GeneCards, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD). Venny 2.1.0 was used to obtain the common targets shared by Strychni Semen, RA, and pain, and STRING and Cytoscape 3.6.1 were used to build the protein-protein interaction network. Then, molecular docking was carried out in AutoDock Vina. Finally, the rat model of type Ⅱ collagen-induced arthritis(CIA) was established. The up-down method and acetone method were employed to examine the mechanical pain threshold and cold pain threshold of rats, and the pain-relieving effect of SA on CIA rats was evaluated comprehensively. Hematoxylin-eosin(HE) staining was employed to evaluate the histopathological changes of joints in CIA rats. The expression levels of key target proteins was determined by immunohistochemistry and Western blot, and the mRNA levels of key targets were determined by real-time fluorescence quantitative polymerase chain reaction(real-time PCR). The results of network prediction showed that Strychni Semen may act on the TLR4/TNF-α/MMP-9 signaling pathway to exert the pain-relieving effect. The results of molecular docking showed that brucine, the main active component of SA, had strong binding ability to TLR4, TNF-α, and MMP-9. The results of animal experiments showed that SA improved the mechanical and cold pain sensitivity(P<0.05, P<0.01) and reduced the joint histopathological score of CIA rats(P<0.01). In addition, medium and high doses of SA down-regulated the protein and mRNA levels of TNF-α, TLR4, and MMP-9(P<0.05,P<0.01). In conclusion, SA alleviated the mechanical pain sensitivity, cold pain sensitivity, and joint histopathological changes in CIA rats by inhibiting the over activation of TLR4/TNF-α/MMP-9 signaling pathway.

A novel trypsin of Trichinella spiralis mediates larval invasion of gut epithelium via binding to PAR2 and activating ERK1/2 pathway.

BACKGROUND: Proteases secreted by Trichinella spiralis intestinal infective larvae (IIL) play an important role in larval invasion and pathogenesis. However, the mechanism through which proteases mediate larval invasion of intestinal epithelial cells (IECs) remains unclear. A novel T. spiralis trypsin (TsTryp) was identified in IIL excretory/secretory (ES) proteins. It was an early and highly expressed protease at IIL stage, and had the potential as an early diagnostic antigen. The aim of this study was to investigate the biological characteristics of this novel TsTryp, its role in larval invasion of gut epithelium, and the mechanisms involved. METHODOLOGY/PRINCIPAL FINDING: TsTryp with C-terminal domain was cloned and expressed in Escherichia coli BL21 (DE3), and the rTsTryp had the enzymatic activity of natural trypsin, but it could not directly degrade gut tight junctions (TJs) proteins. qPCR and western blotting showed that TsTryp was highly expressed at the invasive IIL stage. Immunofluorescence assay (IFA), ELISA and Far Western blotting revealed that rTsTryp specifically bound to IECs, and confocal microscopy showed that the binding of rTsTryp with IECs was mainly localized in the cytomembrane. Co-immunoprecipitation (Co-IP) confirmed that rTsTryp bound to protease activated receptors 2 (PAR2) in Caco-2 cells. rTsTryp binding to PAR2 resulted in decreased expression levels of ZO-1 and occludin and increased paracellular permeability in Caco-2 monolayers by activating the extracellular regulated protein kinases 1/2 (ERK1/2) pathway. rTsTryp decreased TJs expression and increased epithelial permeability, which could be abrogated by the PAR2 antagonist AZ3451 and ERK1/2 inhibitor PD98059. rTsTryp facilitated larval invasion of IECs, and anti-rTsTryp antibodies inhibited invasion. Both inhibitors impeded larval invasion and alleviated intestinal inflammation in vitro and in vivo. CONCLUSIONS: TsTryp binding to PAR2 activated the ERK1/2 pathway, decreased the expression of gut TJs proteins, disrupted epithelial integrity and barrier function, and consequently mediated larval invasion of the gut mucosa. Therefore, rTsTryp could be regarded as a potential vaccine target for blocking T. spiralis invasion and infection.

A novel Trichinella spiralis serine proteinase disrupted gut epithelial barrier and mediated larval invasion through binding to RACK1 and activating MAPK/ERK1/2 pathway.

BACKGROUND: Gut epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism by which larval penetration of gut epithelium is not completely elucidated. Previous studies showed that proteases secreted by T. spiralis intestinal infective larvae (IIL) degraded tight junctions (TJs) proteins of gut epithelium and mediated larval invasion. A new T. spiralis serine proteinase (TsSPc) was identified in the IIL surface proteins and ES proteins, rTsSPc bound to the intestinal epithelial cell (IECs) and promoted larval invasion of IECs. The aim of this study was to characterize the interacted proteins of TsSPc and IECs, and to investigate the molecular mechanisms of TsSPc mediating larval invasion of gut mucosa. METHODOLOGY/PRINCIPAL FINDING: IIFT results showed natural TsSPc was detected in infected murine intestine at 6, 12 hours post infection (hpi) and 3 dpi. The results of GST pull-down, mass spectrometry (MS) and Co-IP indicated that rTsSPc bound and interacted specifically with receptor for activated protein C kinase 1 (RACK1) in Caco-2 cells. rTsSPc did not directly hydrolyze the TJs proteins. qPCR and Western blot showed that rTsSPc up-regulated RACK1 expression, activated MAPK/ERK1/2 pathway, reduced the expression levels of gut TJs (occludin and claudin-1) and adherent protein E-cad, increased the paracellular permeability and damaged the integrity of intestinal epithelial barrier. Moreover, the RACK1 inhibitor HO and ERK1/2 pathway inhibitor PD98059 abolished the rTsSPc activating ERK1/2 pathway, they also inhibited and abrogated the rTsSPc down-regulating expression of occludin, claudin-1 and E-cad in Caco-2 monolayer and infected murine intestine, impeded larval invasion and improved intestinal epithelial integrity and barrier function, reduced intestinal worm burdens and alleviated intestinal inflammation. CONCLUSIONS: rTsSPc bound to RACK1 receptor in gut epithelium, activated MAPK/ERK1/2 pathway, decreased the expression of gut epithelial TJs proteins and disrupted the epithelial integrity, consequently mediated T. spiralis larval invasion of gut epithelium. The results are valuable to understand T. spiralis invasion mechanism, and TsSPc might be regarded as a vaccine target against T. spiralis invasion and infection.

The protective immunity induced by Trichinella spiralis galectin against larval challenge and the potential of galactomannan as a novel adjuvant.

Previous studies showed that recombinant Trichinella spiralis galectin (rTsgal) promoted larval invasion of gut epithelial cells, while anti-rTsgal antibodies inhibited the invasion. Galactomannan (GM) is a polysaccharide capable of regulating immune response. The aim of this study was to evaluate protective immunity induced by rTsgal immunization and the potential of GM as a novel adjuvant. The results showed that vaccination of mice with rTsgal+ISA201 and rTsgal+GM elicited a Th1/Th2 immune response. Mice immunized with rTsgal+ISA201 and rTsgal+GM exhibited significantly higher levels of serum anti-rTsgal antibodies, mucosal sIgA and cellular immune responses, but level of specific antibodies and cytokines of rTsgal+GM group was lower than the rTsgal+ISA201 group. Immunization of mice with rTsgal+ISA201 and rTsgal+GM showed a 50.5 and 40.16% reduction of intestinal adults, and 52.04 and 37.53% reduction of muscle larvae after challenge. Moreover, the numbers of goblet cells and expression level of mucin 2, Muc5ac and pro-inflammatory cytokines (TNF-α and IL-1ß) in gut tissues of vaccinated mice were obviously decreased, while Th2 inducing cytokine (IL-4) expression was evidently increased. Galactomannan enhanced protective immunity, alleviated intestinal and muscle inflammation of infected mice. The results indicated that rTsgal+ISA201 vaccination induced a more prominent gut local as well as systemic immune response and protection compared to rTsgal+GM vaccination. The results suggested that Tsgal could be considered as a candidate vaccine target against Trichinella infection and galactomannan might be a potential novel candidate adjuvant of anti-Trichinella vaccines.

Trichinella spiralis galectin binding to toll-like receptor 4 induces intestinal inflammation and mediates larval invasion of gut mucosa.

Previous studies showed that Trichinella spiralis galectin (Tsgal) facilitates larval invasion of intestinal epithelium cells (IECs). However, IEC proteins binding with Tsgal were not identified, and the mechanism by which Tsgal promotes larval invasion is not clear. Toll-like receptors (TLRs) are protein receptors responsible for recognition of pathogens. The aim of this study was to investigate whether recombinant Tsgal (rTsgal) binds to TLR-4, activates inflammatory pathway in gut epithelium and mediates T. spiralis invasion. Indirect immunofluorescence (IIF), GST pull-down and co-immunoprecipitation (Co-IP) assays confirmed specific binding between rTsgal and TLR-4 in Caco-2 cells. qPCR and Western blotting showed that binding of rTsgal with TLR-4 up-regulated the TLR-4 transcription and expression in Caco-2 cells, and activated p-NF-κB p65 and p-ERK1/2. Activation of inflammatory pathway TLR-4/MAPK-NF-κB by rTsgal up-regulated pro-inflammatory cytokines (IL-1ß and IL-6) and down-regulated anti-inflammatory cytokine TGF-ß in Caco-2 cells, and induced intestinal inflammation. TAK-242 (TLR-4 inhibitor) and PDTC (NF-κB inhibitor) significantly inhibited the activation of TLR-4 and MAPK-NF-κB pathway. Moreover, the two inhibitors also inhibited IL-1ß and IL-6 expression, and increased TGF-ß expression in Caco-2 cells. In T. spiralis infected mice, the two inhibitors also inhibited the activation of TLR-4/MAPK-NF-κB pathway, ameliorated intestinal inflammation, impeded larval invasion of gut mucosa and reduced intestinal adult burdens. The results showed that rTsgal binding to TLR-4 in gut epithelium activated MAPK-NF-κB signaling pathway, induced the expression of TLR-4 and pro-inflammatory cytokines, and mediated larval invasion. Tsgal might be regarded as a candidate molecular target of vaccine against T. spiralis enteral invasive stage.

Molecular characterization of a novel serine proteinase from Trichinella spiralis and its participation in larval invasion of gut epithelium.

BACKGROUND: A novel serine proteinase of Trichinells spiralis (TsSPc) has been identified in the excretion/secretion (ES) antigens, but its role in larval invasion is unclear. The aim of this study was to clone and express TsSPc, identify its biological and biochemical characteristics, and investigate its role on larval invasion of gut epithelium during T. spiralis infection. METHODOLOGY/PRINCIPAL FINDINGS: TsSPc has a functional domain of serine proteinase, and its tertiary structure consists of three amino acid residues (His88, Asp139 and Ser229) forming a pocket like functional domain. Recombinant TsSPc (rTsSPc) was expressed and purified. The rTsSPc has good immunogenicity. On Western blot analysis, rTsSPc was recognized by infection serum and anti-rTsSPc serum, natural TsSPc in crude and ES antigens was identified by anti-rTsSPc serum. The results of qPCR, Western blot and indirect immunofluorescence test (IIFT) showed that TsSPc was expressed at diverse stage worms, and mainly localized at cuticle, stichosome and intrauterine embryos of this nematode. The rTsSPc had enzymatic activity of native serine protease, which hydrolyzed the substrate BAEE, casein and collagen I. After site directed mutation of enzymatic active sites of TsSPc, its antigenicity did not change but the enzyme activity was fully lost. rTsSPc specifically bound to intestinal epithelium cells (IECs) and the binding sites were mainly localized in cell membrane and cytoplasm. rTsSPc accelerated larval invasion of IECs, whereas anti-rTsSPc antibodies and TsSPc-specific dsRNA obviously hindered larval invasion. CONCLUSIONS: TsSPc was a surface and secretory proteinase of the parasite, participated in larval invasion of gut epithelium, and may be considered as a candidate vaccine target molecule against Trichinella intrusion and infection.

[Intervention effect of Qufeng Gutong Cataplasm on myofascial pain syndrome in rats and its mechanism].

This paper aims to investigate the intervention effect of Qufeng Gutong Cataplasm(QFGT) on myofascial pain syndrome(MPS) in rats and to preliminarily explain its mechanism from the perspective of improving muscle inflammation and pain. Male SD rats were divided into 6 groups, namely normal group, model group, positive control drug(Huoxue Zhitong Ointment, HXZT) group, and low, medium, and high-dose QFGT groups(75, 150, and 300 mg·d~(-1)). The rat model of MPS was established by striking combined with centrifugation for 8 weeks, during which QFGT and HXZT were used for corresponding intervention. Standard VonFrey fiber was used to evaluate the mechanical pain threshold, and acetone was used to detect the cold pain threshold. The electrophysiological activity of muscle at trigger point was detected, and the electromuscular analysis of trigger point was performed. CatWalk gait analyzer was used to detect pain-induced gait adaptation changes. The hematoxylin-eosin(HE) staining was used to observe the pathological changes in muscle and skin tissues at the trigger point of rats. Immunohistochemistry was used to detect the expression of capsaicin receptor transient receptor potential vanilloid 1(TRPV1) in muscle tissues and interleukin(IL)-33 in skin tissues at the trigger point. The protein expression levels of TRPV1, protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), IL-1ß, and tumor necrosis factor-α(TNF-α) in muscle tissues at the trigger point were detected by Western blot. The results showed that as compared with the model group, the mechanical pain threshold and cold pain threshold of rats in other groups were increased after treatment with QFGT. The spontaneous electromyography(EMG) activity was observed in the model group, but QFGT alleviated the EMG activity in a dose-dependent manner. Gait analysis showed that standing duration, average intensity, swing speed, maximum contact point, maximum contact area, paw print length, paw print width, and paw print area were significantly improved in all QFGT groups. Pathological results showed that the disorder of muscle arrangement at the trigger point was decreased, muscle fiber adhesion and atrophy were reduced, and inflammatory cell infiltration was alleviated after treatment with QFGT. In addition, QFGT and HXZT both inhibited the protein expression of TRPV1, PI3K, Akt, p-Akt, IL-1ß, and TNF-α in the muscle tissues of rats with MPS. However, there was no significant difference in the pathological structure and expression of IL-33 in the treated skin as compared with the normal group. The related results have proved that QFGT can inhibit the release of inflammatory factors by inhibiting the TRPV1/PI3K/Akt signaling pathway in the muscle trigger point of rats with MPS and finally attenuate the atrophy and adhesion of local muscles and inflammatory infiltration, thereby relieving the muscle pain of rats with MPS, and local administration has no skin irritation.

[Mechanism of artesunate on bone destruction in experimental rheumatoid arthritis based on transcriptomics and network pharmacology].

The present study investigated the mechanism of artesunate in the treatment of bone destruction in experimental rheumatoid arthritis(RA) based on transcriptomics and network pharmacology. The transcriptome sequencing data of artesunate in the inhibition of osteoclast differentiation were analyzed to obtain differentially expressed genes(DEGs). GraphPad Prism 8 software was used to plot volcano maps and heat maps were plotted through the website of bioinformatics. GeneCards and OMIM were used to collect information on key targets of bone destruction in RA. The DEGs of artesunate in inhibiting osteoclast differentiation and key target genes of bone destruction in RA were intersected by the Venny 2.1.0 platform, and the intersection target genes were analyzed by Gene Ontology(GO)/Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment. Finally, the receptor activator of nuclear factor-κB(RANKL)-induced osteoclast differentiation model and collagen-induced arthritis(CIA) model were established. Quantitative real time polymerase chain reaction(q-PCR), immunofluorescence, and immunohistochemistry were used to verify the pharmacological effect and molecular mechanism of artesunate in the treatment of bone destruction in RA. In this study, the RANKL-induced osteoclast differentiation model in vitro was established and intervened with artesunate, and transcriptome sequencing data were analyzed to obtain 744 DEGs of artesunate in inhibiting osteoclast differentiation. A total of 1 291 major target genes of bone destruction in RA were obtained from GeneCards and OMIM. The target genes of artesunate in inhibiting osteoclast differentiation and the target genes of bone destruction in RA were intersected to obtain 61 target genes of artesunate against bone destruction in RA. The intersected target genes were analyzed by GO/KEGG enrichment. According to the results previously reported, the cytokine-cytokine receptor interaction signaling pathway was selected for experimental verification. Artesunate intervention in the RANKL-induced osteoclast differentiation model showed that artesunate inhibited CC chemokine receptor 3(CCR3), CC chemokine receptor 1(CCR1) and leukemia inhibitory factor(LIF) mRNA expression in osteoclasts in a dose-dependent manner compared with the RANKL-induced group. Meanwhile, the results of immunofluorescence and immunohistochemistry showed that artesunate could dose-dependently reduce the expression of CCR3 in osteoclasts and joint tissues of the CIA rat model in vitro. This study indicated that artesunate regulated the CCR3 in the cytokine-cytokine receptor interaction signaling pathway in the treatment of bone destruction in RA and provided a new target gene for the treatment of bone destruction in RA.

Oral immunization of mice with recombinant Lactobacillus plantarum expressing a Trichinella spiralis galectin induces an immune protection against larval challenge.

BACKGROUND: Trichinella spiralis is an important foodborne parasite that presents a severe threat to food safety. The development of an anti-Trichinella vaccine is an important step towards controlling Trichinella infection in food animals and thus ensure meat safety. Trichinella spiralis galectin (Tsgal) is a novel protein that has been identified on the surface of this nematode. Recombinant Tsgal (rTsgal) was found to participate in larval invasion of intestinal epithelium cells (IECs), whereas anti-rTsgal antibodies impeded the invasion. METHODS: The rTsgal/pSIP409- pgsA' plasmid was constructed and transferred into Lactobacillus plantarum strain NC8, following which the in vitro biological properties of rTsgal/NC8 were determined. Five groups of mice were orally immunized three times, with a 2-week interval between immunizations, with recombinant NC8-Tsgal, recombinant NC8-Tsgal + α-lactose, empty NC8, α-lactose only or phosphate-buffered saline (PBS), respectively. The vaccinated mice were infected orally with T. spiralis larvae 2 weeks following the last vaccination. Systemic and intestinal local mucosal immune responses and protection were also assessed, as were pathological changes in murine intestine and skeletal muscle. RESULTS: rTsgal was expressed on the surface of NC8-Tsgal. Oral immunization of mice with rTsgal vaccine induced specific forms of serum immunoglobulin G (IgG), namely IgG1/IgG2a, as well as IgA and gut mucosal secretion IgA (sIgA). The levels of interferon gamma and interleukin-4 secreted by cells of the spleen, mesenteric lymph nodes, Peyer's patches and intestinal lamina propria were significantly elevated at 2-6 weeks after immunization, and continued to rise following challenge. Immunization of mice with the oral rTsgal vaccine produced a significant immune protection against T. spiralis challenge, as demonstrated by a 57.28% reduction in the intestinal adult worm burden and a 53.30% reduction in muscle larval burden, compared to the PBS control group. Immunization with oral rTsgal vaccine also ameliorated intestinal inflammation, as demonstrated by a distinct reduction in the number of gut epithelial goblet cells and mucin 2 expression level in T. spiralis-infected mice. Oral administration of lactose alone also reduced adult worm and larval burdens and relieved partially inflammation of intestine and muscles. CONCLUSIONS: Immunization with oral rTsgal vaccine triggered an obvious gut local mucosal sIgA response and specific systemic Th1/Th2 immune response, as well as an evident protective immunity against T. spiralis challenge. Oral rTsgal vaccine provided a prospective approach for control of T. spiralis infection.

Epidemiology of trichinellosis in the People's Republic of China during 2009-2020.

Trichinellosis is an important meat-borne zoonotic parasitic disease caused by ingesting raw or semi-cooked meat of pigs and other animals infected with Trichinella sp. muscle larvae. Epidemiological data on human and animal Trichinella sp. infection in the People's Republic of China (PRC) during 2009-2020 were analyzed in this review. The results showed that the endemic foci of human trichinellosis are principally localized in southwestern areas, and eight outbreaks covering 479 cases and 2 deaths were reported. Pork is still the primary source of trichinellosis outbreaks. Seven out of 8 outbreaks (87.50%) were caused by ingesting raw or semi-cooked pork. The seroprevalence of swine anti-Trichinella IgG ranged from 0 to 42.11% in 11 provinces/autonomous regions  (P/As), and swine Trichinella infection was detected in six P/A slaughterhouses. The Trichinella-infected pigs came from small backyard farms and outdoor free-ranging pigs in western and southwestern PRC. To prevent trichinellosis, the traditional pig-rearing mode should be improved, more industrialized pig farms should be developed, all pigs should be raised in piggeries under controlled management conditions, and mandatory inspection of Trichinella sp. in slaughtered pigs should be implemented in rural areas of western and southwestern PRC. A One Health approach with participation from governments, public health officials, and medical and veterinary practitioners is vital for controlling zoonotic foodborne trichinellosis.

Proteases secreted by Trichinella spiralis intestinal infective larvae damage the junctions of the intestinal epithelial cell monolayer and mediate larval invasion.

The intestinal epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism of larval invasion of the gut epithelium is not fully elucidated. The aim of this study was to investigate whether the excretory/secretory proteins (ESPs) of T. spiralis intestinal infective larvae (IIL) degrade tight junction (TJ) proteins, to assess the main ESP proteases hydrolysing TJ proteins using various enzyme inhibitors and to define the key invasive factors in IIL invasion of the gut epithelium. The results of immunofluorescence, Western blot and Transwell assays showed that serine proteases and cysteine proteases in the ESPs played main roles in hydrolysing occludin, claudin-1 and E-cad and upregulating claudin-2 expression. Challenge infection results showed that IIL expulsion from the gut at 12 hpi was significantly higher in mice which were infected with muscle larvae (ML) treated with a single inhibitor (PMSF, E-64, 1,10-Phe or pepstatin) or various mixtures containing PMSF and E-64 than in mice in the PBS group or the groups treated with an inhibitor mixture not containing PMSF and E-64 (P < 0.0001). At 6 days post-infection, mice which were infected with ML treated with PMSF, E-64, 1,10-Phe or pepstatin exhibited 56.30, 64.91, 26.42 and 31.85% reductions in intestinal adult worms compared to mice in the PBS group (P < 0.0001). The results indicate that serine proteases and cysteine proteases play key roles in T. spiralis IIL invasion, growth and survival in the host and that they may be main candidate target molecules for vaccines against larval invasion and development.

Oral immunization with attenuated Salmonella encoding an elastase elicits protective immunity against Trichinella spiralis infection.

Elastase belongs to the serine protease family. Previous studies showed that Trichinella spiralis elastase (TsE) was highly expressed in intestinal infective larvae (IIL). Recombinant TsE (rTsE) promoted the larval intrusion of enteral epithelium cells (IECs), whereas anti-rTsE antibodies and siRNA impeded larval intrusion. Subcutaneous vaccination of mice with rTsE showed a partial protective immunity, suggesting that TsE might be a promising vaccine target against Trichinella infection. In this study, complete TsE cDNA sequence was cloned into pcDNA3.1, and the rTsE DNA was transformed into attenuated S. typhimurium strain ΔcyaSL1344. Oral vaccination of mice with TsE DNA elicited a systemic Th1/Th2/Treg mixed immune response and gut local mucosal sIgA response. Immunized mice exhibited a significant immune protection against T. spiralis larval challenge, as demonstrated by a 52.48% reduction of enteral adult worms and a 69.43% reduction of muscle larvae. The protection might be related to the TsE-induced production of intestinal mucus, specific anti-TsE sIgA and IgG, and secretion of IFN-γ, IL-2, IL-4 and IL-10, which protected gut mucosa from larval intrusion, suppressed worm development and impeded female reproduction. The results demonstrated that attenuated Salmonella-delivered TsE DNA vaccine provided a prospective strategy for the control of Trichinella infection in food animals.

Vaccination of mice with recombinant novel aminopeptidase P and cathepsin X alone or in combination induces protective immunity against Trichinella spiralis infection.

Trichinella spiralis is a major foodborne zoonotic parasitic nematode which has a serious threat to meat food safety. Development of anti-Trichinella vaccine is requisite for control and elimination of Trichinella infection in food animals to ensure meat safety. Aminopeptidase P (TsAPP) and cathepsin X (TsCX) are two novel proteins identified in T. spiralis intestinal infectious L1 larvae (IIL1). The objective of this study was to investigate the protective immunity elicited by immunization with TsAPP and TsCX alone and TsAPP-TsCX in combination in a mouse model. The results demonstrate that subcutaneous vaccination of mice with rTsAPP, rTsCX or rTsAPP + rTsCX elicited a systemic humoral response (high levels of serum IgG, IgG1/IgG2a and IgA) and significant local gut mucosal sIgA responses. The vaccination with rTsAPP, rTsCX or rTsAPP + rTsCX also induced a systemic and local mixed Th1/Th2 response, as demonstrated by clear elevation levels of IFN-γ and IL-4 in vaccinated mice. Vaccination of mice with rTsAPP+rTsCX exhibited a 63.99 % reduction of intestinal adult worms and 68.50% reduction of muscle larva burdens, alleviated inflammation of intestinal mucosal and muscle tissues, and provided a higher immune protection than that of vaccination with rTsAPP or rTsCX alone. The results demonstrated that TsAPP and TsCX might be considered novel candidate target molecules for anti-Trichinella vaccines.

Immune responses in mice vaccinated with a DNA vaccine expressing a new elastase from Trichinella spiralis.

The elastase, which belongs to the serine protease family, hydrolyses various proteins and may be involved in the parasite invasion. In this study, complete sequence of elastase-1 (TsE) the nematode Trichinella spiralis (Owen, 1835) was cloned into the plasmid pcDNA3.1 as TsE DNA vaccine. After intramuscular vaccination, serum anti-Trichinella antibodies (IgG and subclass IgG1/IgG2a, and IgA), total and specific intestinal mucosal sIgA in mice vaccinated with pcDNA3.1/TsE were measured by ELISA. The results showed that vaccination with pcDNA3.1/TsE induced a systemic humoral immune response (high levels of serum IgG and subclass IgG1/IgG2a and IgA) and local intestinal mucosal immune responses (high levels of TsE-specific sIgA). Vaccination of mice with TsE DNA vaccine also triggered a systemic and local concomitant Th1/Th2 response, as demonstrated by significant elevation of Th1 (IFN-γ and IL-2) / Th2 (IL-4 and IL-10) cytokine levels after the spleen, mesenteric lymph node and Peyer's patch cells from vaccinated mice were stimulated with recombinant TsE (rTsE). The vaccination of mice with pcDNA3.1/TsE displayed a 17% reduction of intestinal adult worms and a 39% reduction of muscle larvae. Our results indicated that TsE DNA vaccine elicited a systemic concomitant Th1/Th2 response and an enteral local sIgA response, and produced a partial protection against infection with T. spiralis. The TsE may be regarded as a potential candidate vaccine target against Trichinella infection. The oral polyvalent vaccines should be developed to improve the protective efficacy of anti-Trichinella vaccines.

Protective immunity in mice vaccinated with a novel elastase-1 significantly decreases Trichinella spiralis fecundity and infection.

Trichinella spiralis is an important foodborne parasitic nematode that represents an enormous threat to the food safety of pork meat. The development of a preventive vaccine is valuable for the prevention and control of Trichinella infection in domestic pigs to ensure pork safety. Elastase is a trypsin-like serine protease that hydrolyzes the host's diverse tissue components and participates in parasite penetration, and it might be a novel vaccine target molecule. The aim of this study was to assess the protective immunity produced by vaccination with a novel Trichinella spiralis elastase-1 (TsE) in a mouse model. The results demonstrate that subcutaneous vaccination of mice with rTsE elicited a systemic humoral response (high levels of serum IgG and subclass IgG1/IgG2a and IgA) and significant local enteral mucosal sIgA responses. Anti-rTsE IgG recognized the native TsE at the cuticle, stichosome of intestinal infective larvae and adult worm (AW), and intrauterine embryos of female AW. The rTsE vaccination also produced a systemic and local mixed Th1/Th2 response, as demonstrated by clear elevation levels of Th1 cytokines (IFN-γ, IL-2) and Th2 cytokines (IL-4, IL-10) after spleen, mesenteric lymph node and Peyer's patch cells from immunized mice were stimulated with rTsE. The immunized mice exhibited a 52.19% reduction in enteral AW and a 64.06% reduction in muscle larvae after challenge infection. The immune response triggered by rTsE vaccination protected enteral mucosa from larval intrusion, suppressed larval development and reduced female fecundity. The results indicate that TsE may represent a novel target molecule for anti-T. spiralis vaccines.

Molecular characterization of a Trichinella spiralis elastase-1 and its potential as a diagnostic antigen for trichinellosis.

BACKGROUND: Trichinella spiralis muscle larval (ML) excretion/secretion (ES) antigen is the most widely used diagnostic antigen of trichinellosis, but preparation of ES antigen requires collecting worms from infected animals, and detection of specific IgG against ML ES antigen may result in a false negative at the early stage of infection. The aim of the study was to characterize T. spiralis elastase-1 (TsEla) and to evaluate its potential as diagnostic antigen for trichinellosis. METHODS: The complete cDNA sequences of the TsEla gene were cloned and expressed, and recombinant (rTsEla) was purified. TsEla transcription and expression in different T. spiralis life-cycle stages was investigated by qPCR and western blotting, and its location in the nematodes was evaluated using an immunofluorescence assay (IFA). The antigenicity of rTsEla was investigated by western blotting analysis and ELISA. Anti-Trichinella IgG, IgM and IgE of experimentally infected mice and specific IgG antibodies of trichinellosis patients were assayed by rTsEla-ELISA and ES-ELISA. RESULTS: The results of the qPCR and western blotting showed that TsEla was expressed in various T. spiralis life stages. Natural TsEla was detected in the soluble proteins and ES proteins of different life stages. IFA revealed that TsEla was identified in the whole nematodes of various stages, especially in the cuticle, stichosome and genital primordium of the parasite. Serum anti-Trichinella IgM, IgG and IgE in infected mice was first detected by rTsEla-ELISA at 6, 10 and 12 days post-infection (dpi), and reached 100% at 8, 14 and 14 dpi, respectively. When rTsEla-ELISA and ES-ELISA were used to detect anti-Trichinella IgG in sera of trichinellosis patients, the sensitivity was 97.37% (37/38) and 89.74% (34/38) (P > 0.05), and the specificity was 99.10% (220/222) and 98.20% (218/222), respectively (P > 0.05). The rTsEla cross-reacted with only one serum sample out of 20 samples from paragonimiasis patients and 7 samples from clonorchiasis patients. CONCLUSIONS: rTsEla is valuable to early diagnosis of trichinellosis and could be an alternative diagnostic antigen to the ML ES antigens.

Protective immunity against Trichinella spiralis infection induced by TsNd vaccine in mice.

BACKGROUND: We have previously reported that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and vaccination of mice with recombinant TsNd protein (rTsNd) produced a partial protective immunity. The aim of this study was to investigate the immune protection induced by TsNd DNA vaccine. METHODS: The full-length cDNA sequence of TsNd gene was cloned into pcDNA3.1 and used to immunize BALB/c mice by intramuscular injection. Transcription and expression of TsNd were detected by RT-PCR and IFT. The levels of specific IgA, IgG, IgG1 and IgG2a, and cytokines were assayed by ELISA at weeks 0, 6 and 8 post-immunization. The immune protection of TsNd DNA vaccine against challenge infection was investigated. RESULTS: Immunization of mice with TsNd DNA elicited a systemic Th1/Th2 immune response and a local mucosal IgA response. The in vitro transcription and expression of TsNd gene was observed at all developmental stages of T. spiralis (ML, IIL, AW and NBL). Anti-rTsNd IgG levels were increased after immunization and levels of IgG1 were obviously higher than that of IgG2a. Intestinal specific IgA levels of immunized mice were significantly higher than those of vector and PBS control mice. Cytokine profiling also showed a significant increase in Th1 (IFN-γ, IL-2) and Th2 (IL-4, 10) responses in splenocytes of immunized mice on stimulation with rTsNd. Vaccination of mice with pcDNA3.1-TsNd displayed a 40.44% reduction in adult worms and a 53.9% reduction in larval burden. CONCLUSIONS: TsNd DNA induced a mixed Th1/Th2 immune response and partial protection against T. spiralis infection in mice.