LAMTOR1/mTORC1 promotes CD276 to induce immunosuppression via PI3K/Akt/MMP signaling pathway in Clostridium perfringens-induced necrotic enteritis of laying hens.

Clostridium perfringens (C. perfringens) causes avian necrotic enteritis, leading to huge economic losses to the poultry industry. This pathogen induces host immunosuppression; however, the molecular mechanism is still unclear. Thus, we established a laying hen infection model to explore this mechanism. We randomly divided 20 one-old-day laying hens into the control and infection groups. The infection group was infected intragastrically with 1 × 109 colony-forming units of C. perfringens in 1 mL of sterile phosphate-buffered saline (PBS) once a day from d 17 to 20; the control group received the same volume of PBS without the bacterium. Twenty-four hours after the last challenge, we sacrificed the laying hens and collected the jejunum for analysis. The infection group presented alterations in blood biochemical parameters and necrotic lesion scores as well as damage to the jejunum. Proteomics revealed 427 upregulated and 291 downregulated proteins in the infection group. In the infection group, CD3, CD4, and CD8 messenger RNA expression (mRNA) expression was decreased; LAMTOR1 and mTORC1 mRNA expression was increased; CD276 protein expression was enhanced; and the PI3K/Akt/MMP pathway was activated in jejunum of laying hens. This is the first study to report CD276 expression in the jejunum related to immunosuppression in a laying hen model of necrotic enteritis. It provides some new key targets to potentially control avian necrotic enteritis.

Anemoside B4 attenuates necrotic enteritis of laying hens induced by Clostridium perfringens via inhibiting NF-κB and PI3K/Akt/mTOR signalling pathways.

Poultry necrotic enteritis is an important enteric disease which might be controlled by antibiotics. However, with the excessive use of antibiotics, the phenomenon of drug resistance of Clostridium perfringens is becoming increasingly prominent. Anemoside B4 exhibits important anti-inflammatory, antioxidant and immunomodulatory effects. This study was performed to estimate the effect of Anemoside B4 on chicken necrotic enteritis induced by C. perfringens in vivo and in vitro. In the in vivo experiment we investigated the efficacy of Anemoside B4 on the growth curve, biofilm formation, haemolytic activity, virulence-related gene expression and NF-κB and PI3K/AKT/mTOR activation in Caco-2 cells induced by C. perfringens. The results showed that 12.5-50 µg/mL Anemoside B4 had no antibacterial activity but could inhibit biofilm formation, attenuate haemolytic activity and virulence-related gene expression of C. perfringens and weaken NF-κB and PI3K/Akt/mTOR activation triggered by C. perfringens in Caco-2 cells. In the in vivo experiment, 60 17-day-old healthy White Leghorns were randomly divided into six groups. The growing laying hens of the control group were fed a basic diet, and those of the five challenged groups were fed a basic diet (infection group), added 0.43 g/kg Anemoside B4 (0.43 g/kg Ane group), 0.86 g/kg Anemoside B4 (0.86 g/kg Ane group), 1.72 g/kg Anemoside B4 (1.72 g/kg Ane group) and 40 mg/kg lincomycin (lincomycin group), respectively. All challenged laying hens were infected with 1 × 109 CFU C. perfringens from day 17-20. Blood and intestinal samples were obtained, and the data demonstrated that Anemoside B4 improved the blood biochemical parameters, attenuated jejunum tissue injury, increased the spleen, thymus, bursa of fabricius index, and decreased lesion scores of the jejunum and the ileum. In the jejunum, Anemoside B4 and lincomycin downregulated the expression of IL-1ß, IL-6, IL-10, TNF-α and IFN-γ at mRNA levels. Moreover, Anemoside B4 significantly enhanced both mRNA and protein levels of tight junctions ZO-1, Claudin-1 and MUC-2 in the jejunum. Anemoside B4 weakened p-P65, p-PI3K, p-Akt and p-mTOR protein expression in the jejunum infected by C. perfringens. Diets supplemented with Anemoside B4 alleviated C. perfringens-induced necrotic enteritis in laying hens by inhibiting NF-κB and PI3K/Akt/mTOR signalling pathways and improving intestinal barrier functions.

Baicalin-aluminum complex on the regulation of IPEC-1 infected with enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the main bacterial cause of diarrhea in weaned piglets. Baicalin-aluminum (BA) complex is the main active ingredient of Scutellaria baicalensis Georgi extracted-aluminum complex, which has been used to treat diarrhea in weaning piglets, however the underlying mechanism remains unclear. To investigate the effects of the BA complex on the regulation of porcine intestinal epithelial (IPEC-1) cells infected with ETEC, IPEC-1 cells were incubated with an ETEC bacterial strain at a multiplicity of infection of 1 for 6 h and then treated with different concentrations of the BA complex for 6 h. ETEC infection increased the levels of cAMP and cGMP, upregulated CFTR (cystic fibrosis transmembrane conductance regulator) mRNA, and downregulated NHE4 mRNA in IPEC-1 cells. Treatment with the BA complex inhibited ETEC adhesion and the production of cAMP and cGMP, reduced CFTR mRNA expression, and increased NHE4 mRNA expression. Overall, the BA complex weakened the adhesion of ETEC to IPEC-1 cells, and inhibited cAMP/cGMP-CFTR signaling in IPEC-1 cells.

Baicalin attenuates PD-1/PD-L1 axis-induced immunosuppression in piglets challenged with Glaesserella parasuis by inhibiting the PI3K/Akt/mTOR and RAS/MEK/ERK signalling pathways.

Infection of piglets with Glaesserella parasuis (G. parasuis) induces host immunosuppression. However, the mechanism underlying the immunosuppression of piglets remains unclear. Activation of the PD-1/PD-L1 axis has been shown to trigger host immunosuppression. Baicalin possesses anti-inflammatory and immunomodulatory functions. However, whether baicalin inhibits PD-1/PD-L1 activation and thus alleviates host immunosuppression has not been investigated. In this study, the effect of baicalin on the attenuation of piglet immunosuppression induced by G. parasuis was evaluated. Seventy piglets were randomly divided into the control group, infection group, levamisole group, BMS-1 group, 25 mg/kg baicalin group, 50 mg/kg baicalin group and 100 mg/kg baicalin group. Following pretreatment with levamisole, BMS-1 or baicalin, the piglets were challenged with 1 × 108 CFU of G. parasuis. Our results showed that baicalin, levamisole and BMS-1 modified routine blood indicators and biochemical parameters; downregulated IL-1ß, IL-10, IL-18, TNF-α and IFN-γ mRNA expression; and upregulated IL-2 and IL-8 mRNA expression in blood. Baicalin, levamisole and BMS-1 increased the proportions of CD3+ T cells, CD3+CD4+ T cells, CD3+CD8+ T cells and CD3-CD21+ B cells in the splenocyte population, increased the proportions of CD3+ T cells, CD3+CD4+ T cells and CD3+CD8+ T cells in the blood, and inhibited PD-1/PD-L1 and TIM-3 activation. Baicalin, levamisole and BMS-1 reduced p-PI3K, p-Akt, and p-mTOR expression, the p-MEK1/2/MEK1/2 and p-ERK1/2/ERK1/2 ratios and increased RAS expression. Baicalin, levamisole and BMS-1 provided substantial protection against G. parasuis challenge and relieved tissue histopathological damage. Our findings might provide new strategies for controlling G. parasuis infection and other immunosuppressive diseases.

Baicalin and probenecid protect against Glaesserella parasuis challenge in a piglet model.

Glaesserella parasuis (G. parasuis) induces vascular damage and systemic inflammation. However, the mechanism by which it causes vascular damage is currently unclear. Baicalin has important anti-inflammatory, antibacterial and immunomodulatory functions. In this study, we explored the ability of baicalin and probenecid to protect against G. parasuis challenge in a piglet model. Sixty piglets were randomly divided into a control group; an infection group; a probenecid group; and 25 mg/kg, 50 mg/kg and 100 mg/kg baicalin groups. The probenecid group and the 25 mg/kg, 50 mg/kg and 100 mg/kg baicalin groups were injected intramuscularly with 20 mg/kg body weight (BW) probenecid and 25 mg/kg BW, 50 mg/kg BW and 100 mg/kg BW baicalin, respectively. All piglets except those from the control group were injected intraperitoneally with 1 × 108 CFU of G. parasuis. The control group was injected intraperitoneally with TSB. The results showed baicalin and probenecid protected piglets against G. parasuis challenge, improved body weight and decreased temperature changes in piglets. Baicalin and probenecid attenuated IL-1ß, IL-10, IL-18, TNF-α and IFN-γ mRNA levels in the blood for 48 h, inhibited the production of the nucleosides ATP, ADP, AMP and UMP from 24 to 72 h, reduced Panx-1/P2Y6/P2X7 expression, weakened NF-kB, AP-1, NLRP3/Caspase-1 and ROCK/MLCK/MLC signalling activation, and upregulated VE-cadherin expression in the blood vessels of piglets challenged with G. parasuis. Baicalin and probenecid alleviated pathological tissue damage in piglets induced by G. parasuis. Our results might provide a promising strategy to control and treat G. parasuis infection in the clinical setting.

Quercetin Protects Blood-Brain Barrier Integrity via the PI3K/Akt/Erk Signaling Pathway in a Mouse Model of Meningitis Induced by Glaesserella parasuis.

Glaesserella parasuis (G. parasuis) causes serious inflammation and meningitis in piglets. Quercetin has anti-inflammatory and anti-bacterial activities; however, whether quercetin can alleviate brain inflammation and provide protective effects during G. parasuis infection has not been studied. Here, we established a mouse model of G. parasuis infection in vivo and in vitro to investigate transcriptome changes in the mouse cerebrum and determine the protective effects of quercetin on brain inflammation and blood-brain barrier (BBB) integrity during G. parasuis infection. The results showed that G. parasuis induced brain inflammation, destroyed BBB integrity, and suppressed PI3K/Akt/Erk signaling-pathway activation in mice. Quercetin decreased the expression of inflammatory cytokines (Il-18, Il-6, Il-8, and Tnf-α) and BBB-permeability marker genes (Mmp9, Vegf, Ang-2, and Et-1), increased the expression of angiogenetic genes (Sema4D and PlexinB1), reduced G. parasuis-induced tight junction disruption, and reactivated G. parasuis-induced suppression of the PI3K/Akt/Erk signaling pathway in vitro. Thus, we concluded that quercetin may protect BBB integrity via the PI3K/Akt/Erk signaling pathway during G. parasuis infection. This was the first attempt to explore the protective effects of quercetin on brain inflammation and BBB integrity in a G. parasuis-infected mouse model. Our findings indicated that quercetin is a promising natural agent for the prevention and treatment of G. parasuis infection.

Using network pharmacology and molecular docking to uncover the mechanism by which quercetin alleviates deoxynivalenol-induced porcine intestinal injury.

Deoxynivalenol is a widespread feed contaminant that leads to vomit, which results in serious symptom such as increased intestinal permeability and even intestinal mucosal necrosis. Recent studies have reported the role of quercetin in alleviating deoxynivalenol-induced intestinal injury; however, the mechanisms and targets remain unclear. Thus, we aimed to identify the mechanisms of action by using a combination of network pharmacology and molecular docking. We identified 151 quercetin targets, 235 deoxynivalenol targets and 47 porcine intestinal injury targets by searching compound database and PubMed database, among which there were two common targets. The PPI network showed that the key proteins involved are NQO1 and PPAR-γ. The PPI network showed that the key proteins involved were NQO1 and PPARG. GO analysis found that genes were enriched primarily in response to oxidative stress. The PPI network showed that the key proteins involved are NQO1 and PPAR-γ. The genes are enriched primarily in response to oxidative stress. KEGG analysis showed enrichment of the HIF, reactive oxygen species and other signaling pathways. The molecular docking results indicated key binding activity between NQO1-quercetin and PPAR-γ-quercetin. By using network pharmacology, we have revealed the potential molecular mechanisms by which quercetin alleviates deoxynivalenol-induced porcine intestinal injury, which lays the foundation for the development of drugs to treat deoxynivalenol-induced intestinal injury in pigs.

Baicalin Weakens the Virulence of Porcine Extraintestinal Pathogenic Escherichia coli by Inhibiting the LuxS/AI-2 Quorum-Sensing System.

Porcine extraintestinal pathogenic Escherichia coli (ExPEC) is a pathogenic bacterium that causes huge economic losses to the pig farming industry and considerably threatens human health. The quorum sensing (QS) system plays a crucial role in the survival and pathogenesis of pathogenic bacteria. Hence, it is a viable approach to prevent ExPEC infection by compromising the QS system, particularly the LuxS/AI-2 system. In this study, we investigated the effects of baicalin on the LuxS/AI-2 system of ExPEC. Baicalin at concentrations of 25, 50, and 100 µg/mL significantly diminished the survival ability of ExPEC in hostile environments and could inhibit the biofilm formation and autoagglutination ability in ExPEC. Moreover, baicalin dose-dependently decreased the production of AI-2 and down-regulated the expression level of luxS in PCN033. These results suggest that baicalin can weaken the virulence of PCN033 by inhibiting the LuxS/AI-2 system. After the gene luxS was deleted, AI-2 production in PCN033 was almost completely eliminated, similar to the effect of baicalin on the production of AI-2 in PCN033. This indicates that baicalin reduced the production of AI-2 by inhibiting the expression level of luxS in ExPEC. In addition, the animal experiment further showed the potential of baicalin as a LuxS/AI-2 system inhibitor to prevent ExPEC infection. This study highlights the potential of baicalin as a natural quorum-sensing inhibitor for therapeutic applications in preventing ExPEC infection by targeting the LuxS/AI-2 system.

PD-1/PD-L1 axis induced host immunosuppression via PI3K/Akt/mTOR signalling pathway in piglets infected by Glaesserella Parasuis.

Glaesserella parasuis, an important respiratory bacterial pathogen, causes Glässer's disease in piglets, with potential immunosuppression. We established a piglet infection model and explored the immunosuppression mechanism to improve our understanding of the host immune response to G. parasuis. Twenty piglets were randomly divided into two groups (n = 10). The infection group was intraperitoneally challenged with 2 × 108 CFU of G. parasuis in 2 mL TSB. The control group was intraperitoneally injected with equivalent TSB. After 72 h, the piglets were sacrificed, and spleen tissue was collected. PD-1/PD-L1 expression was determined. The splenocytes were isolated to detect CD3+ T, CD3+CD4+ T, CD3+CD8+ T and CD3-CD21+cell differentiation. Via data-independent acquisition (DIA), we compared the proteomics of healthy and infected spleen tissues. Glaesserella parasuis modified CD3+ T, CD3+CD4+ T, CD3+CD8+ T and CD3-CD21+ cell differentiation and PD-1/PD-L1 expression in the spleen. The infection group had 596 proteins with significant differences in expression, of which 301 were significantly upregulated and 295 downregulated. Differentially expressed proteins (DEPs) were mainly related to immune responses. This is the first study on PD-1/PD-L1 expression in the spleen associated with immunosuppression in a piglet model to explore the protein changes related to immune responses via DIA.