Rhein kills Actinobacillus pleuropneumoniae, reduces biofilm formation, and effectively treats bacterial lung infections in mice.

Background. Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae family, is known for its highly infectious nature and is the primary causative agent of infectious pleuropneumonia in pigs. This disease poses a considerable threat to the global pig industry and leads to substantial economic losses due to reduced productivity, increased mortality rates, and the need for extensive veterinary care and treatment. Due to the emergence of multi-drug-resistant strains, Chinese herbal medicine is considered one of the best alternatives to antibiotics due to its unique mechanism of action and other properties. As a type of Chinese herbal medicine, Rhein has the advantages of a wide antibacterial spectrum and is less likely to develop drug resistance, which can perfectly solve the limitations of current antibacterial treatments.Methods. The killing effect of Rhein on A. pleuropneumoniae was detected by fluorescence quantification of differential expression changes of key genes, and scanning electron microscopy was used to observe the changes in A. pleuropneumoniae status after Rhein treatment. Establishing a mouse model to observe the treatment of Rhein after A. pleuropneumoniae infection.Results. Here, in this study, we found that Rhein had a good killing effect on A. pleuropneumoniae and that the MIC was 25 µg ml-1. After 3 h of action, Rhein (4×MIC) completely kills A. pleuropneumoniae and Rhein has good stability. In addition, the treatment with Rhein (1×MIC) significantly reduced the formation of bacterial biofilms. Therapeutic evaluation in a murine model showed that Rhein protects mice from A. pleuropneumoniae and relieves lung inflammation. Quantitative RT-PCR (Quantitative reverse transcription polymerase chain reaction is a molecular biology technique that combines both reverse transcription and polymerase chain reaction methods to quantitatively detect the amount of a specific RNA molecule) results showed that Rhein treatment significantly downregulated the expression of the IL-18 (Interleukin refers to a class of cytokines produced by white blood cells), TNF-α, p65 and p38 genes. Along with the downregulation of genes such as IL-18, it means that Rhein has an inhibitory effect on the expression of these genes, thereby reducing the activation of inflammatory cells and the production of inflammatory mediators. This helps reduce inflammation and protects tissue from further damage.Conclusions. This study reports the activity of Rhein against A. pleuropneumoniae and its mechanism, and reveals the ability of Rhein to treat A. pleuropneumoniae infection in mice, laying the foundation for the development of new drugs for bacterial infections.

Effects of Trichinella spiralis and its serine protease inhibitors on autophagy of host small intestinal cells.

In eukaryotes, autophagy is induced as an innate defense mechanism against pathogenic microorganisms by self-degradation. Although trichinellosis is a foodborne zoonotic disease, there are few reports on the interplay between Trichinella spiralissurvival strategies and autophagy-mediated host defense. Therefore, this study focused on the association between T. spiralis and autophagy of host small intestinal cells. In this study, the autophagy-related indexes of host small intestinal cells after T. spiralis infection were detected using transmission electron microscopy, hematoxylin and eosin staining, immunohistochemistry, quantitative real-time polymerase chain reaction, and Western blotting. The results showed that autophagosomes and autolysosomes were formed in small intestinal cells, intestinal villi appeared edema, epithelial compactness was decreased, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was expressed in lamina propria stromal cells of small intestine, and the expression of autophagy-related genes and proteins was changed significantly, indicating that T. spiralis induced autophagy of host small intestinal cells. Then, the effect of T. spiralis on autophagy-related pathways was explored by Western blotting. The results showed that the expression of autophagy-related pathway proteins was changed, indicating that T. spiralis regulated autophagy by affecting autophagy-related pathways. Finally, the roles of T. spiralis serine protease inhibitors (TsSPIs), such as T. spiralis Kazal-type SPI (TsKaSPI) and T. spiralis Serpin-type SPI (TsAdSPI), were further discussed in vitro and in vivo experiments. The results revealed that TsSPIs induced autophagy by influencing autophagy-related pathways, and TsAdSPI has more advantages. Overall, our results indicated that T. spiralis induced autophagy of host small intestinal cells, and its TsSPIs play an important role in enhancing autophagy flux by affecting autophagy-related pathways. These findings lay a foundation for further exploring the pathogenesis of intestinal dysfunction of host after T. spiralis infection, and also provide some experimental and theoretical basis for the prevention and treatment of trichinellosis.

Exosome-delivered miR-153 from Trichinella spiralis promotes apoptosis of intestinal epithelial cells by downregulating Bcl2.

Trichinellosis, a helminthic zoonosis, exhibits a cosmopolitan distribution and is a public health concern. In previous studies, it was reported that the exosomes secreted by Trichinella spiralis larvae (TsExos) largely affected cell biological activities. miRNAs, as exosome-delivered cargoes, affect the biological activities of the host by targeting genes. The present study aimed to elucidate the mechanisms by which miRNAs interact with intestinal epithelial cells. First, a miRNA library of TsExos was constructed; then, based on high-throughput miRNA sequencing results, miR-153 and its predicted target genes, namely, Agap2, Bcl2 and Pten, were selected for follow-up studies. The dual-luciferase reporter assays revealed that miR-153 directly targeted Bcl2 and Pten. Furthermore, real-time qPCR and Western blotting revealed that only Bcl2 was downregulated by TsExo-delivered miR-153 in porcine intestinal epithelial cells (IPEC-J2). Bcl2, an important antiapoptotic protein, plays an essential role in cell apoptosis as a common intersecting molecule of various signal transduction pathways. Therefore, we hypothesized that miR-153 derived from TsExos causes cell apoptosis by targeting Bcl2. The results suggested that miR-153 could induce apoptosis, reduce mitochondrial membrane potential, affect cell proliferation, and cause damage and substantial oxidative stress. Furthermore, miR-153 coincubated with IPEC-J2 cells stimulated the accumulation of the proapoptotic proteins Bax and Bad, which belong to the Bcl2 family of proteins, and the apoptosis-implementing proteins Caspase 9 and Caspase 3. Moreover, studies have suggested that miR-153 can promote apoptosis by regulating the MAPK and p53 signalling pathways involved in apoptosis. Thus, exosome-mediated miR-153 delivery secreted by T. spiralis could induce apoptosis and affect the MAPK and p53 signalling pathways by downregulating Bcl2 in IPEC-J2 cells. The study highlights the mechanisms underlying the invasion of T. spiralis larva.

Intervention effects of Trichinella spiralis excretory-secretory antigens on allergic asthma in mice.

Allergic asthma is a chronic, heterogeneous and inflammatory respiratory disease, and there are few medicines at present. An increasing number of studies indicate that Trichinella spiralis (T. spiralis) and its excretory-secretory (ES) antigens are inflammatory modulator. Therefore, this study focused on the effects of T. spiralis ES antigens on allergic asthma. Asthma model was established by sensitizing mice with ovalbumin antigen (OVA) and aluminum hydroxide (Al[OH]3), the asthmatic mice were interfered using T. spiralis 43 kDa protein (Ts43), T. spiralis 49 kDa protein (Ts49), and T. spiralis 53 kDa protein (Ts53), the important components of ES antigens, to establish ES antigens intervention models. Then, asthma symptom changes, weight changes, and lung inflammation of mice were evaluated. The results showed that ES antigens could relieve symptoms, weight loss, and lung inflammation caused by asthma in the mice, and the effect of combined intervention of Ts43, Ts49, and Ts53 was better. Finally, the effects of ES antigens on type 1 helper T (Th1) and type 2 helper T (Th2) immune responses, and the differentiation direction of T lymphocytes in mice were discussed by detecting Th1 and Th2 cell-related factors and the ratio of CD4+/CD8+ T cells. The results suggested that the ratio of CD4+/CD8+ T cells decreased and the ratio of Th1/Th2 cells increased. In conclusion, this study indicated that T. spiralis ES antigens could mitigate allergic asthma in the mice by changing the differentiation direction of CD4+ and CD8+ T cells and regulating the imbalance of Th1/Th2 cells ratio.

Effects of exosomes derived from Trichinella spiralis infective larvae on intestinal epithelial barrier function.

Muscle larvae of Trichinella spiralis parasitize the host intestinal epithelium. The mechanisms of exosomes participating in the invasion of T. spiralis muscle larvae are unclear. Hence, the purpose of this study was to explore the effect of exosomes derived from T. spiralis infective larvae (TsExos) on the barrier function of porcine small intestinal epithelial cells (IPEC-J2). First, TsExos were successfully obtained, and their ingestion by epithelial cells was validated. Furthermore, the optimal induction condition was determined by the CCK8 kit, and we found that exposure to 150 µg/mL TsExos for 12/24 h decreased the viability of IPEC-J2 cells by 30%. Based on this outcome, the effects of TsExos on cell biological processes and tight junctions were studied. After coincubation of TsExos and IPEC-J2 cells, the results showed a significant increase in the content of FITC-dextran and in the levels of lactate dehydrogenase (LDH) and reactive oxygen species (ROS). The rate of apoptosis increased by 12.57%, and nuclear pyknosis and nuclear rupture were observed. After the cells were induced by TsExos, the expression of IL-1 was upregulated, but the expression of IL-10, TGF-ß, TLR-5, MUC-1 and MUC-2 was downregulated. TsExo induction also led to a decrease in the levels of ZO-1, CLDN-3, and OCLN. In conclusion, TsExos are involved in several cellular biological processes, and they function by disrupting physiological and biochemical processes, hyperactivating innate immunity, and damaging tight junctions.

Regulatory effects of Trichinella spiralis and a serine protease inhibitor on the endoplasmic reticulum stress response of intestinal epithelial cells.

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause an endoplasmic reticulum stress (ERS) response. If ERS continues or cannot be alleviated, it will cause the production of proapoptotic factors and eventually lead to apoptosis. Therefore, this study mainly explored whether Trichinella spiralis Kazal-type serine protease inhibitor (TsKaSPI) contributed to the invasion of intestinal epithelial cells during the infectious stage of T. spiralis by regulating ERS. First, in the T. spiralis infection model, H&E staining was used to analyse the damage to jejunum tissue, a TUNEL assay was used to examine cell apoptosis, and the expression of ERS-related and apoptosis-related molecules was also measured. The results showed that ERS occurred during the intestinal phase of T. spiralis infection, while remission began during the cyclic phase. Then, we selected TsKaSPI, one of the important components of T. spiralis ES antigens, for in vitro experiments. The results showed that TsKaSPI could induce apoptosis in a porcine small intestinal epithelial cell line (IPEC cells) by activating ERS and promote activation of the NF-κB signalling pathway. Inhibition experiments confirmed that the occurrence of ERS was accompanied by the activation of NF-κB, and the two processes regulated each other. Finally, we conducted in vivo experiments and administered TsKaSPI to mice. The results confirmed that TsKaSPI could activate ERS and lead to apoptosis in intestinal epithelial cells. In conclusion, T. spiralis infection and TsKaSPI can promote cell apoptosis by activating the ERS response in intestinal epithelial cells and activate the NF-κB signalling pathway to promote the occurrence and development of inflammation.

Prevalence of Toxoplasma gondii Infections in Chicken Hearts from Farmers' Markets and Supermarkets in the Tai'an Region of China.

ABSTRACT: The detection of Toxoplasma gondii in quick-frozen chickens is a good indicator of the possible transmission risk that this parasite poses to human consumers. The aim of this study was to investigate the occurrence of T. gondii in quick-frozen chicken hearts using a nested PCR assay. Heart samples (n = 720) from farmers' markets and supermarkets in the Tai'an region, People's Republic of China, were collected, and the DNA extracted was analyzed for the presence of T. gondii. The overall prevalence of T. gondii in all samples was 10.7%, but the rates for samples from farmers' markets (19.2%) and supermarkets (2.2%) were significantly different (P < 0.01). Nested PCR restriction fragment length polymorphism genotyping was performed on the 77 positive samples based on the T. gondii SAG3 and GRA6 gene loci. SAG3 genotyping revealed a mixed infection rate of 89.6% for type I and type I/II strains, and GRA6 genotyping revealed an infection rate of 98.7% for type I strains. Our results revealed a high prevalence of T. gondii in chicken hearts from farmers' markets, and most strains were mainly type I strains. Further studies are needed to determine whether quick-frozen chicken hearts are involved in disease transmission to human consumers in this area.