In vitro observation: the GFP-E. coli adhering to porcine erythrocytes can be removed by porcine alveolar macrophages

Although the activation of pathogen phagocytosis via complement system has been studied, erythrocyte-phagocyte interactions in pigs are not clearly understood. Therefore, we sought to investigate the ability of porcine erythrocytes to clear immune complexes (ICs) by using laser confocal microscopy and flow cytometry to observe the immune adhesion of porcine erythrocytes to fluorescent bacilli and the immune presentation process of transferring fluorescent bacilli to macrophages. Isolated porcine alveolar macrophages (PAMs) had uniform morphology and size, and a survival rate of 97.2%. The phagocytosis rate was 98.8%. After WT E. coli was labeled with Fluorescein Isothiocyanate (FITC), the bacteria showed a bright green fluorescence, and the labeling rate was 92.3%. When laser confocal microscopy was utilized to observe the co-incubation system of porcine erythrocytes, PAM, and fluorescent E. coli, the fluorescence intensity of bacilli decreased with increasing observation time and even disappeared. Flow Cytometry examination showed that the average fluorescence intensity of PAMs co-incubated with porcine erythrocytes adhered to WT-E. coli-FITC, was significantly higher than that of normal PAMs. Furthermore, when porcine erythrocytes adhered to WT E. coli were incubated with PAMs, the surface mean fluorescence intensity of porcine erythrocytes was significantly higher than that of the blank control group. This shows that PAMs can competitively bind to the oposinized E. coli adhered to the surface of porcine erythrocytes, and these oposinized pathogens can enter macrophages by the process of phagocytosis, which promoting the internalization of ICs or pathogens. During this process, the physical morphology of porcine erythrocytes was not damaged, but the levels of its main functional protein CR1-like were reduced

Analysis of the anti-PCV2 mechanism of Lactobacillus acidophilus based on non-target metabolomics and high-throughput molecular docking

Our previous studies have revealed that L. acidophilus possesses inhibitory effects on PCV2 proliferation in vivo, although the underlying mechanisms remain elusive. Probiotics like L. acidophilus are known to exert antiviral through their metabolites. Therefore, in this study, non-targeted metabolomics was used to detect the changes in metabolites of L. acidophilus after 24 h of proliferation. Subsequently, high-throughput molecular docking was utilized to analyze the docking scores of these metabolites with PCV2 Cap and Rep, aiming to identify compounds with potential anti-PCV2 effects. The results demonstrated that 128 compounds such as Dl-lactate were significantly increased. The results of high-throughput molecular docking indicated that compounds such as ergocristine, and telmisartan formed complexes with Cap and Rep, suggesting their potential anti-PCV2 properties. Furthermore, compounds like vitamin C, exhibit pharmacological effects consistent with L. acidophilus adding credence to the idea that L. acidophilus may exert pharmacological effects through its metabolites. These results will provide a foundation for the study of L. acidophilus

Anti-Invasion and Antimetastatic Effects of Porcine Recombinant NK-lysin on SMMC-7721 Human Hepatocellular Carcinoma Cells

The high invasion and metastasizing abilities of hepatocellular carcinoma (HCC) are the primary reasons for the high mortality rate of patients. Therefore, identification of agents to inhibit invasion and metastasis is very important for treatment of HCC. We analyzed the anti-invasion and antimetastatic effects of porcine recombinant NK-lysin, which was designed and expressed in vitro by our research group, on SMMC-7721 hepatocellular carcinoma cells via wound-healing assays, adhesion assays, invasion assays, real-time polymerase chain reaction (PCR), and Western blot analysis. MTT assay results indicated that NK-lysin inhibited the growth of SMMC-7721 cells in a dose- and time-dependent manner. NK-lysin reduced the ability of cell migration, adhesion, and invasion. Based on gene and protein expression analysis, NK-lysin decreased β-catenin and MMP-2 expression. These results suggested that NK-lysin has anti-invasion and antimetastatic effects on hepatocellular carcinoma cells in vitro by reducing the level of the β-catenin and MMP-2

Curcumol inhibits EMCV replication by activating CH25H and inhibiting the formation of ROs

Abstract Background Zedoary turmeric oil extracted from the roots of curcuma (Curcuma aeruginosa Roxb.) is used for the treatment of myocarditis in China. EMCV infection causes abortion in pregnant sows and myocarditis in piglets. Our previous studies demonstrated that curcumol significantly increased the expression of IFN-β in EMCV infected HEK-293T cells. The present results showed that curcumol inhibits EMCV replication by interfering the host cell cholesterol homeostasis and reducing ROs production through activation of the JAK/STAT signaling pathway. Method This study was designed to explore whether curcumol can inhibit the replication of encephalomyocarditis viruses (EMCV) in cell culture. The expression level of JAK1, IRF9, STAT2, P-STAT2, CH25H, PI4KA and OSBP in EMCV-infected HEK-293T cells treated with curcumol, ribavirin or hydroxypropyl-β-CD (HPCD) were determined by Western blotting (WB). The cholesterol level in EMCV infected HEK-293T cells treated with curcumol and HPCD were detected using Amplex™ Red Cholesterol Assay Kit. The antiviral effects of curcumol and HPCD on EMCV were also quantitatively detected by real-time fluorescence quantitative PCR (q-PCR). The amount and morphology of ROs were observed by transmission electron microscopy (TEM). Results The results demonstrated that curcumol significantly (P < 0.05) increased the expression of JAK1, IRF9, P-STAT2 and CH25H proteins, while that of STAT2, PI4KA and OSBP were remained unchanged. Compared with virus group (0.134 μg.μg-1 proteins), the total cholesterol level was significantly (P < 0.05) reduced by curcumol (0.108 μg.μg-1 proteins) and HPCD (0.089 μg.μg-1 proteins). Compared with virus group (88237 copies), curcumol (41802 copies) and HPCD (53 copies) significantly (P < 0.05) reduced EMCV load. Curcumol significantly reduced the production of ROs in EMCV-infected HEK-293T cells and activated CH25H through the JAK/STAT signaling pathway. Conclusion Curcumol inhibited EMCV replication by affecting the cholesterol homeostasis and the production of ROs in HEK-293T cell

Matrine combined with Osthole inhibited the PERK apoptosis of splenic lymphocytes in PCV2-infected mice model

Abstract Background Porcine circovirus type 2 (PCV2) is one of the major pathogens commonly found in pigs, which causes immunosuppression and apoptosis. Vaccination and a single drug cannot totally prevent and treat PCV2 infection. Our previous in vitro study reported that the synergistic anti-PCV2 effect of Matrine and Osthole was better than that of Matrine or Osthole alone, This study was aimed to evaluate the synergistic anti-PCV2 effect as well as the underline molecular mechanism of Matrine and Osthole in Kunming (KM) mice model infected with PCV2. KM mice were randomly divided into 8 groups namely control group, PCV2 infected, Matrine combined with Osthole high dose treatment (40 mg/kg + 12 mg/kg), medium dose treatment (20 mg/kg + 6 mg/kg), low dose treatment (10 mg/kg + 3 mg/kg), Matrine treatment (40 mg/kg), Osthole treatment (12 mg/kg) and Ribavirin positive control (40 mg/kg) groups. PCV2 was intraperitoneally (i.p.) injected in all mice except the control group. 5 days of post-infection (dpi), mice in different treatment groups were injected i.p. with various doses of Matrine, Osthole and Ribavirin once daily for the next 5 consecutive days. Results The synergistic inhibitory effect of Matrine and Osthole on PCV2 replication in mouse liver was significantly heigher than that of Matrine and Osthole alone. The expression of GRP78, p-PERK, p-eIF2α, ATF4, CHOP, cleaved caspase-3 and Bax proteins were significantly reduced, while that of Bcl-2 was significantly increased in Matrine combined with Osthole groups, which alleviated the pathological changes caused by PCV2, such as interstitial pneumonia, loss of spleen lymphocytes, infiltration of macrophages and eosinophils. Conclusions The synergistic anti-apoptotic effect of Matrine and Osthole was better than their alone effect, Both Matrine and Osthole had directly inhibited the expression of PCV2 Cap and the apoptosis of spleen cells induced by PCV2 Cap through the PERK pathway activated by endoplasmic reticulum (ER) GRP78. These results provided a new insight to control PCV2 infection and provide good component prescription candidate for the development of novel anti-PCV2 drugs

Fascin-1 Promotes Cell Metastasis through Epithelial–Mesenchymal Transition in Canine Mammary Tumor Cell Lines

Canine mammary tumors (CMTs) are the most common type of tumor in female dogs. In this study, we obtained a metastatic key protein, Fascin-1, by comparing the proteomics data of in situ tumor and metastatic cell lines from the same individual. However, the role of Fascin-1 in the CMT cell line is still unclear. Firstly, proteomics was used to analyze the differential expression of Fascin-1 between the CMT cell lines CHMm and CHMp. Then, the overexpression (CHMm-OE and CHMp-OE) and knockdown (CHMm-KD and CHMp-KD) cell lines were established by lentivirus transduction. Finally, the differentially expressed proteins (DEPs) in CHMm and CHMm-OE cells were identified through proteomics. The results showed that the CHMm cells isolated from CMT abdominal metastases exhibited minimal expression of Fascin-1. The migration, adhesion, and invasion ability of CHMm-OE and CHMp-OE cells increased, while the migration, adhesion, and invasion ability of CHMm-KD and CHMp-KD cells decreased. The overexpression of Fascin-1 can upregulate the Tetraspanin 4 (TSPAN4) protein in CHMm cells and increase the number of migrations. In conclusion, re-expressed Fascin-1 could promote cell EMT and increase lamellipodia formation, resulting in the enhancement of CHMm cell migration, adhesion, and invasion in vitro. This may be beneficial to improve female dogs’ prognosis of CMT