Naringenin Improves Innate Immune Suppression after PRRSV Infection by Reactivating the RIG-I-MAVS Signaling Pathway, Promoting the Production of IFN-I.

Porcine reproductive and respiratory syndrome (PRRS) has been prevalent for nearly forty years since it was first reported. It has been one of the major diseases jeopardizing the healthy development of the world swine industry, as well as causing great economic losses to the industry's economic development. Furthermore, no way has been found to combat the disease due to the immunosuppressive properties of its pathogen porcine reproductive and respiratory syndrome virus (PRRSV) infection. We previously examined the mRNA expression of IFN-I in PRRSV-infected Marc-145 cells at different time periods using qRT-PCR, and found that the mRNA expression of IFN-I in the late stage of PRRSV infection showed suppression. Naringenin is a flavonoid found in citrus fruits and has a very wide range of pharmacological activities. Therefore, the aim of the present study was to investigate the modulatory effect of naringenin on the suppressed innate immune response after PRRSV infection. The expression of IFN-I, IL-10, and ISGs in the late stage of PRRSV infection was examined using qRT-PCR, and the results showed that naringenin improved the expression of antiviral cytokines suppressed by PRRSV infection. Further results showed that naringenin treatment significantly up-regulated the expression of proteins related to the RIG-I-MAV immune signaling pathway, and that naringenin could not significantly activate the RIG-I-MAVS signaling pathway after the addition of the RIG-I inhibitor Cyclo. Overall, these data demonstrated that naringenin could improve the innate immune response suppressed by PRRSV infection by modulating the RIG-I-MAVS signaling pathway. Therefore, our study will provide a theoretical basis for the development of naringenin as a drug against immunosuppressive viral infectious disease infections.

Retinoic acid ameliorates low-grade endotoxemia-induced mastitis by limiting inflammatory responses in mice.

Mastitis is a serious disease for humans and animals, which causes huge economic losses in the dairy industry and is hard to prevent due to the complex and unclear pathogenesis. Subacute ruminal acidosis (SARA) has contributed to the development of mastitis by inducing ruminal dysbiosis and subsequent low-grade endotoxemia (LGE), however, how ruminal metabolic changes regulate this progress is still unclear. Our previous study revealed that cows with SARA had increased ruminal retinoic acid (RA) levels, a metabolic intermediate of vitamin A that plays an essential role in mucosal immune responses. Hence, the aim of this study was to investigate the protective effect of RA on LGE-induced mastitis and the underlying mechanisms in mice. The results showed that RA alleviated LGE-induced mastitis, as evidenced by RA significantly reduced the increase in mammary proinflammatory cytokines and improved blood-milk barrier injury caused by LGE. In addition, RA increased the expression of tight junction proteins, including ZO-1, occludin and claudin-3. Furthermore, we found that RA limited the mammary inflammatory responses by inhibiting the activation of NF-κB and NLRP3 signaling pathways. These findings suggest that RA effectively alleviates LGE-induced mastitis and implies a potential strategy for the treatment and prevention of mastitis and other diseases.

Tangeretin attenuates bleomycin-induced pulmonary fibrosis by inhibiting epithelial-mesenchymal transition via the PI3K/Akt pathway.

Background: Pulmonary fibrosis (PF) is a terminal pathological change in a variety of lung diseases characterized by excessive deposition of extracellular matrix, for which effective treatment is lacking. Tangeretin (Tan), a flavonoid derived from citrus, has been shown to have a wide range of pharmacological effects. This study aimed to investigate the role and potential mechanisms of Tan on pulmonary fibrosis. Methods: A model of pulmonary fibrosis was established by administering bleomycin through tracheal drip, followed by administering Tan or pirfenidone through gavage. HE and Masson staining were employed to assess the extent of pulmonary fibrosis. Subsequently, Western blot, enzyme-linked immunosorbent assay (ELISA), RNA sequencing, and immunohistochemistry techniques were employed to uncover the protective mechanism of Tan in PF mice. Furthermore, A549 cells were stimulated with TGF-ß1 to induce epithelial-mesenchymal transition (EMT) and demonstrate the effectiveness of Tan in mitigating PF. Results: Tan significantly ameliorated bleomycin-induced pulmonary fibrosis, improved fibrotic pathological changes, and collagen deposition in the lungs, and reduced lung inflammation and oxidative stress. The KEGG pathway enrichment analysis revealed a higher number of enriched genes in the PI3K/Akt pathway. Additionally, Tan can inhibit the EMT process related to pulmonary fibrosis. Conclusion: Taken together, the above research results indicate that Tan suppresses inflammation, oxidative stress, and EMT in BLM-induced pulmonary fibrosis via the PI3K/Akt pathway and is a potential agent for the treatment of pulmonary fibrosis.

Astragaloside IV Regulates cGAS-STING Signaling Pathway to Alleviate Immunosuppression Caused by PRRSV Infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a global threat to pig health and results in significant economic losses. Impaired innate and adaptive immune responses are evident during PRRSV infection. Cyclic GMP-AMP synthase (cGAS), a classical pattern recognition receptor recognizing mainly intracytoplasmic DNA, induces type I IFN responses through the cGAS-STING signaling pathway. It has also been demonstrated that cGAS-STING is involved in PRRSV infection. This study utilized the qRT-PCR, ELISA, and WB methods to examine the effects of Astragaloside IV (AS-IV) on the regulation of innate immune function and cGAS-STING signaling pathway in porcine alveolar macrophages. The results showed that AS-IV attenuated the decreased innate immune function caused by PRRSV infection, restored the inhibited cGAS-STING signaling pathway, and increased the expression of interferon, ultimately exerting antiviral effects. Moreover, these results suggest that AS-IV may be a promising candidate for a new anti-PRRSV antiviral, and its mechanism of action may provide insights for developing novel antiviral agents.

The role of endoplasmic reticulum stress in regulation of intestinal barrier and inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease involving the digestive tract, characterized by abdominal pain, diarrhea, rectal bleeding, and so on, which can make patients physically weakened and live difficultly. Although IBD has been recognized for many years, the pathogenesis of IBD has not yet been established and damage to intestinal barrier is thought to be closely associated with IBD. Intestinal barrier is an innate barrier that maintains the homeostasis of the intestinal environment and impedes pathogenic bacteria and toxins, and the endoplasmic reticulum (ER) has recently been found to be involved in maintaining the integrity of intestinal barrier. Endoplasmic reticulum stress (ERS) is a status of endoplasmic reticulum damaged when unfolded or misfolded proteins accumulate in excess of the degradation systematic clearance limit of the misfolded proteins. The regulation of ERS on protein folding synthesis and maintenance of cellular homeostasis is an important factor in influencing the integrity of the intestinal barrier. This paper mainly discusses the relationship between ERS and the intestinal barrier, aiming to understand the regulatory role of ERS on the intestinal barrier and the mechanism and to improve new solutions and notions for the treatment or prevention of IBD.

Xiaochaihu Decoction Treatment of Chicken Colibacillosis by Improving Pulmonary Inflammation and Systemic Inflammation.

Chicken colibacillosis-the most common disease of poultry, is caused mainly by avian pathogenic Escherichia coli (APEC). It has a major impact on the poultry industry worldwide. The present study was conducted to investigate the therapeutic effects of Xiaochaihu Decoction (XCHD) supplementation on clinical manifestation, organ index, bacterial load in organ and inflammatory mediators in a chicken model challenged with APEC. The results showed that all doses of XCHD significantly elevated the survival rate of infected chickens. XCHD improved the clinical signs of infected chickens, reduced the organ index, reduced the bacterial load of organs, and inhibited the secretion of serum and pulmonary inflammatory factors IL-1ß, IL-6 and TNF- α. Taken together, this study demonstrates that XCHD had protective effects on APEC-infected chickens. Its mechanism includes anti-inflammatory and antibacterial effects. These findings may contribute to the further study of the mechanism of the formula and the prevention or treatment of colibacillosis in poultry. The significance of this study is that it provides a certain theoretical basis for the replacement of antibiotics by XCHD.

Baicalin inhibits APEC-induced lung injury by regulating gut microbiota and SCFA production.

Baicalin is a plant-derived flavonoid from Scutellaria baicalensis Georgi with multiple bioactivities and has a protective effect against avian pathogenic Escherichia coli (APEC) infection. However, the underlying mechanism of baicalin against APEC infection is still unknown. Therefore, we aimed to explore whether the protective effects and mechanisms of baicalin on APEC-induced lung inflammation were related to the regulation of gut microbiota. The results showed that baicalin significantly reduced APEC colonization and pro-inflammatory cytokines production, and additionally recovered air-blood barrier integrity in the lungs after APEC challenge. However, depletion of gut microbiota significantly weakened the protective effects of baicalin against APEC infection as mentioned above. Furthermore, baicalin markedly restored the dysbiosis of gut microbiota induced by APEC as well as increased the abundance of short chain fatty acid (SCFA)-producing bacteria and the production of SCFAs including acetic acid, propionic acid and butyric acid, especially acetic acid. In addition, the concentrations of acetic acid and its receptor free fatty acid receptor 2 (FFAR2) were significantly upregulated in the lung tissues after baicalin treatment. In conclusion, gut microbiota played a key role in the pharmacological action of baicalin against APEC-induced lung inflammation. Baicalin remodeled the dysbiosis of gut microbiota caused by APEC and increased the production of SCFAs, especially acetic acid in the gut, and then the increased acetate may circulate to the lungs to activate FFAR2 to defend APEC infection. Together, our study suggested that baicalin inhibited APEC infection through remodeling the gut microbiota dysbiosis and increasing the SCFA production. Furthermore, baicalin may serve as an alternative antibiotic and a novel therapeutic drug to prevent or treat APEC infection.

Protective effects of gut microbiota and gut microbiota-derived acetate on chicken colibacillosis induced by avian pathogenic Escherichia coli.

Chicken colibacillosis is caused by avian pathogenic Escherichia coli (APEC), and results in huge economic losses to the poultry industry. With the investigation of the gut-lung axis, more studies have demonstrated the important role of gut microbiota in lung inflammation. The precise role of the gut microbiota in chickens-associated colibacillosis, however, is unknown. Thus, this study assessed the function of the gut microbiota in the chicken defense against APEC infection. Chicken gut microbiota was depleted by drinking water with a mixture of antibiotics (Abx), and subsequently, a model of colibacillosis was established by the intranasal perfusion of APEC. The results showed that gut microbiota protects the chicken challenge by APEC from aggravated lung histopathologic injury, up-regulated pro-inflammatory cytokine production, and increased bacterial load in lung tissues compared with controls. In addition, the air-blood barrier permeability was significantly increased in gut microbiota-depleted chickens compared to the control chickens after challenge with APEC. Furthermore, feeding acetate significantly inhibited the lung inflammatory response and the reduced air-blood permeability induced by APEC infection. The expression of free fatty acid receptor 2 (FFAR2), a receptor for acetate, was also increased in the lung after treatment with acetate. In conclusion, depletion of the gut microbiota resulted in increased susceptibility of chickens to APEC challenge, and gut microbiota derived acetate acted as a protective mediator during the APEC challenge. Novel therapeutic targets that focus on the gut microbiota may be effective in controlling colibacillosis in poultry.

Madecassoside Protects Against LPS-Induced Acute Lung Injury via Inhibiting TLR4/NF-κB Activation and Blood-Air Barrier Permeability.

Madecassoside (MA), a crucial ingredient of Centella asiatica, has been reported to exhibit a variety of bioactivities, including antipulmonary fibrosis, and antiinflammatory effects. Here we aimed to elucidate the protective effects and underlying mechanisms of MA on LPS-induced acute lung injury (ALI). The mice were treated with MA for one week and then received intratracheal of LPS to establish the ALI model. Then we evaluated the pathological changes by haematoxylin and eosin staining and measured the levels of proinflammatory cytokines and myeloperoxidase (MPO) by ELISA, the transcriptional level of tight junction proteins by qRT-PCR, as well as the expression of Toll-like receptor4/Nuclear factor kappa-B (TLR4/NF-κB) pathway by Western blot. The results showed that MA significantly inhibited LPS-induced pathological damages, lung edema, MPO, and proinflammatory cytokines production. Furthermore, MA obviously repaired alveolar epithelium integrity showing by reduced secretion of total proteins in the BALF and enhanced mRNA expression of tight junction as Occludin and zonula occludens-1 (ZO-1) comparing to LPS. Further research showed that LPS stimulation activated the TLR4/NF-κB signaling pathway and the activation was inhibited by MA. In conclusion, these data indicated that MA had protective effects against LPS-induced ALI. The therapeutic mechanisms may be associated with reducing the alveolar epithelium permeability and inflammatory response via repressing the activation of TLR4/NF-κB pathway.

Schizandrin attenuates inflammation induced by avian pathogenic Escherichia coli in chicken type II pneumocytes.

Avian pathogenic Escherichia coli (APEC) is a kind of highly pathogenic parenteral bacteria, which adheres to chicken type II pneumocytes through pili, causing inflammatory damage of chicken type II pneumocytes. Without affecting the growth of bacteria, anti-adhesion to achieve anti-inflammatory effect is considered to be a new method for the treatment of multi-drug-resistant bacterial infections. In this study, the anti-APEC activity of schizandrin was studied in vitro. By establishing the model of chicken type II pneumocytes infected with APEC-O78, the adhesion number, the expression of virulence genes, the release of lactate dehydrogenase (LDH), levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8 and activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were detected. The results showed that schizandrin reduced the release of LDH and the adherence of APEC on chicken type II pneumocytes. Moreover, schizandrin markedly decreased the levels of IL-1ß, IL-8, IL-6, and TNF-α, the mechanism responsible for these effects was attributed to the inhibitory effect of schizandrin on NF-κB and MAPK signaling activation. In conclusion, our findings revealed that schizandrin could reduce the inflammatory injury of chicken type II pneumocytes by reducing the adhesion of APEC-O78 to chicken type II pneumocytes. The results indicate that schizandrin can be a potential agent to treat inflammation caused by avian colibacillosis.

Schizandrin attenuates lung lesions induced by Avian pathogenic Escherichia coli in chickens.

Avian pathogenic Escherichia coli (APEC) can cause serious pathological changes and inflammation in chickens. Schizandrin has anti-inflammatory activity and can prevent damage to various tissues and organs. The purpose of this study was to investigate the protective effect of schizandrin on APEC-induced lung lesions in chickens and explore the potential mechanism of schizandrin protection. The schizandrin (50, 100, and 200 mg/kg) was intragastrically administered for 3 days. APEC was administered using intraperitoneal (i.p.) injection to induce lung lesions. Then, chickens were sacrificed by CO2 inhalation 24 h later and the lung tissues were collected for examining histopathological changes, wet/dry (W/D) ratio, myeloperoxidase (MPO) activity, malondialdehyde (MDA), levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-8 and activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Our findings showed that schizandrin markedly inhibited pathological changes, pulmonary edema, MPO activity and MDA content. Moreover, schizandrin markedly reduced the levels of TNF-α, IL-1ß, IL-6 and IL-8 in lung tissue. Importantly, the mechanism responsible for these effects was attributed to the inhibitory effect of schizandrin on NF-κB and MAPK signaling activation. In conclusion, our findings reveal that schizandrin displays anti-oxidant and anti-inflammatory activity against APEC-induced lung lesions in chickens, paving the way for rational use of schizandrin as a protective agent against lung-related inflammatory disease.

RNA-seq profiles of chicken type II pneumocyte in response to Escherichia coli infection.

Avian pathogenic Escherichia coli (APEC) causes great economic loss to the poultry industry worldwide. Chicken type II pneumocytes (CP II cells) secrete surfactants and modulate lung immunity to decrease the infection of the invading pathogen. Nevertheless, the pathogenesis of CP II cells to APEC infection remains poorly understood. Therefore, we conducted global gene expression profiling of CP II cells after APEC-O78 infection to explore the host-pathogen interaction. The differentially expressed genes of CP II cells to APEC infection were characterized by RNA-seq with EB-seq algorithm. In consequence, the mRNA of 18996 genes was identified, and CP II cells responded to APEC infection with marked changes in the expression of 1390 genes. Among them, there are 803 down-regulated mRNAs and 587 up-regulated mRNAs. The KEGG prediction and Gene Ontology terms analysis revealed that the major enriched pathways were related to NF-κB signaling pathway, apoptosis pathway, tight junction, and cytokine-cytokine receptor interaction and other pathways. We adopted qRT-PCR to verify the validity of the selected gene expression. The fold induction of qPCR was similar to the RNA-seq results. These results provide a better understanding of the pathogenesis of APEC, especially apoptosis pathway involved in APEC infection.

Canine distemper virus increased the differentiation of CD4+CD8+ T cells and mRNA expression of inflammatory cytokines in peripheral blood lymphocyte from canine.

BACKGROUND: Canine distemper virus (CDV) can cause a highly contagious disease to canid. However, how CDV affects peripheral blood lymphocyte (PBL) remains unclear. METHODS: In this study, CDV infected PBL was cultured to investigate the effect of CDV on the differentiation of lymphocytes and the mRNA expression of inflammatory cytokines in PBL. RESULTS: The results showed that CDV changed the phenotype of lymphocytes and increased the percentage of CD4+CD8+ T cells. To explore the effect of immune response of lymphocytes to CDV, the mRNA expression of pro- and anti-inflammatory cytokines was examined. Interleukin (IL-6, IL-12B), and tumor necrosis factor (TNF)-α mRNA expression was significantly increased at 12-48 h after CDV infection. IL-10 mRNA expression was dramatically enhanced at 12-36 h after CDV infection. However, IL-4 and transforming growth factor (TGF-ß) were not response to CDV infection. These results indicated that PBL differentiated intoCD4+CD8+ T cells and improved the inflammatory response to CDV infection. CONCLUSIONS: After CDV infection, PBL differentiated into CD4+CD8+ T cells and initiated inflammatory response.

Baicalin alleviated APEC-induced acute lung injury in chicken by inhibiting NF-κB pathway activation.

Bacterial pneumonia is a leading cause of death in the animal husbandry. Acute lung injury (ALI), most often seen as a part of systemic inflammatory process, characterized by progressive hypoxemia, edema, and neutrophil accumulation in the lung. Baicalin has been reported to inhibit inflammatory response, but its role in ALI remains unknown. The purpose of our study was to determine the protective effect and possible mechanism of baicalin against avian pathogenic Escherichia coli (APEC)-induced ALI in chicken. Chickens were conditioned with baicalin 1 week before intratracheally instilled with APEC. Then, chickens were sacrificed by CO2 inhalation 12 h later and the lung tissues were collected for examining histopathological changes, wet/dry (W/D) ratio, myeloperoxidase (MPO) activity, levels of pro-inflammatory cytokines and activation of NF-κB signaling pathway. The results showed that pre-treatment of chickens with baicalin significantly alleviated the death rate, histopathological changes in lung tissues. The W/D ratio, MPO activity and production of cytokines, such as IL-1ß, TNF-α, IL-6 of lung tissues were also decreased following treatment with baicalin. Furthermore, the mechanism responsible for these effects was attributed to the inhibitory effect of baicalin on nuclear factor-κB (NF-κB) signaling activation. These data thus support the application of baicalin as a potential medicine for the treatment of E. coli-induced ALI by regulating NF-κB signaling pathway.

Anti-bacterial activity of baicalin against APEC through inhibition of quorum sensing and inflammatory responses.

Avian pathogenic Escherichia coli (APEC), collectively known as causative agent of extraintestinal infections, is an important cause of morbidity and mortality in poultry. Currently, quorum sensing (QS), biofilm formation and virulence factors are considered as novel prospective targets for antimicrobial therapy to control APEC invasion. In addition, inflammatory responses are also served as the major pathological features of APEC invasion. This study was aimed to explore the effect of baicalin on APEC and APEC-induced inflammatory responses. After treatment with baicalin, we mainly examined the AI-2 secretion, biofilm formation, expression of virulence genes of APEC, and the levels of inflammatory cytokines, as well as the expression of NF-κB pathway. Our results showed that baicalin significantly inhibited the QS via decreasing the AI-2 secretion, biofilm formation, and the expression of virulence genes of APEC such as LsrB, LsrK, LuxS, pfs, H-NS, fimA, fimB, fyuA, csgA, csgB, and rpoS. Moreover, baicalin significantly attenuated the release of lactate dehydrogenase (LDH), and the adhesion of APEC to chicken type II pneumocytes to reduce cell damage. Furthermore, baicalin also inhibited the expression of pro-inflammatory cytokines and NF-κB activation. Thus, our data revealed that baicalin could interfere with the quorum sensing, biofilm formation and virulence genes expression to relieve the APEC pathogenicity. Additionally, baicalin decreased the inflammatory responses of chicken type II pneumocytes induced by APEC. Taken together, these data provide a novel potential pharmaco-therapeutic approach to chicken colibacillosis.

Protective Effect of Piceatannol Against Acute Lung Injury Through Protecting the Integrity of Air-Blood Barrier and Modulating the TLR4/NF-κB Signaling Pathway Activation.

Acute lung injury (ALI) is a common and complex inflammatory lung syndrome with higher morbidity and mortality rate. Piceatannol (PIC) has anti-inflammation and anti-oxidant properties. The study was designed to explore the effect and the action mechanisms of PIC on lipopolysaccharide (LPS)-induced ALI. Twenty-four hours after LPS challenge, mice from different treatment groups were euthanized, and the bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected. Then the degree of pulmonary edema, lung pathological changes, myeloperoxidase (MPO) activity, and the production of pro-inflammatory cytokines were detected. Additionally, the messenger RNA (mRNA) expressions associated with cell adhesion molecules and tight junction were analyzed through quantitative real-time (qRT)-PCR, and the TLR4/NF-κB activation was examined by western blot. The results showed that PIC significantly inhibited LPS-induced lung edema, histopathological damage, MPO activity, cell infiltration, and pro-inflammatory cytokines production. Moreover, PIC notably suppressed mRNA expressions associated with inflammation and cell adhesion molecules. Furthermore, PIC also alleviated LPS-induced damage of air-blood barrier through reducing the levels of total proteins in BALF and recovering the expression of occludin and ZO-1 in the lung tissues. We also found that PIC remarkably restrained the LPS-induced TLR4/NF-κB pathway activation in lung tissues. In conclusion, PIC may be potential to treat LPS-induced acute lung injury (ALI) via regulating air-blood barrier and TLR4/NF-κB signaling pathway activation.

Rutin inhibits quorum sensing, biofilm formation and virulence genes in avian pathogenic Escherichia coli.

The study aimed to investigate whether rutin affects the quorum sensing (QS) of avian pathogenic Escherichia coli (APEC). In this study, APEC-O78 was selected as the test strain. We mainly examined the effects of rutin on the AI-2 secretion by bioluminescence assay, biofilm formation through a crystal violet staining method, and expression of virulence genes of APEC by qRT-PCR. We found that rutin can significantly interfering with QS through reducing the secretion of AI-2, inhibited the biofilm formation, and reduced the expression of virulence genes of APEC. Moreover, rutin markedly decreased adhesion and damage of APEC to chicken type II pneumocytes. These results suggested rutin reduces cell damage of APEC-infected chicken type II pneumocytes through interfering with QS via decreasing AI-2 production, biofilm formation, and the expression of virulence genes. This paper may provide a new evidence for colibacillosis prevention in chicken.

In vitro antiviral efficacy of caffeic acid against canine distemper virus.

Canine distemper (CD) is a highly contagious disease caused by the canine distemper virus (CDV), and mortality can be as high as 100%. However, there is no specific treatment for CD. In this study, the antiviral activity of the caffeic acid against CDV was evaluated in vitro. The results showed that the IC50 of the caffeic acid against CDV at 1 and 2 h post infection (PI) is 23.3 and 32.3 µg/mL, respectively. Consistently, at 1 and 2 h PI, the caffeic acid exhibited a reduced (23.3-57.0% and 37.2-38.1%) viral inhibitory effect in vero cells. Furthermore, the caffeic acid plus Ribavirin (RBV) has greater antiviral activity against CDV than the caffeic acid or RBV individually. In addition, the caffeic acid reduced the total viral RNA synthesis by 59-86% at 24-72 h. Therefore, our data provided the experimental evidence that the caffeic acid effectively inhibited CDV infection in vero cells, which may potentially be used to treat clinical disease associated with CDV infection.