Yinsheng Qiu

Protective effects of N-acetylcysteine on intestinal functions of piglets challenged with lipopolysaccharide

The neonatal small intestine is susceptible to damage by endotoxin, but effective methods for prevention and treatment are lacking. N-acetylcysteine (NAC) is a widely used precursor of l-cysteine for animal cells and plays an important role in protecting cells against oxidative stress. This study was conducted with the lipopolysaccharide (LPS)-challenged piglet model to determine the effects of NAC on intestinal function. Eighteen piglets were randomly allocated into control, LPS and LPSxa0+xa0NAC groups. The control and LPS groups were fed a corn- and soybean meal-based diet, and the LPSxa0+xa0NAC group was fed the basal diet +500xa0mg/kg NAC. On days 10, 13 and 20 of the trial, the LPS and LPSxa0+xa0NAC groups received intraperitoneal administration of LPS (100xa0μg/kg BW), whereas the control piglets received saline. On day 20 of the trial, d-xylose (0.1xa0g/kg BW) was orally administrated to all piglets 2xa0h after LPS or saline injection, and blood samples were collected 1xa0h thereafter. One hour blood xylose test was used to measure intestinal absorption capacity and mucosal integrity, and diamine oxidase (DAO) was used as a marker of intestinal injury. On day 21 of the trial, pigs were killed to obtain the intestinal mucosa. Compared to the control, LPS challenge reduced (Pxa0<xa00.05) the concentrations of d-xylose (a marker of intestinal absorption) in plasma, activities of DAO in the jejunal mucosa, the ratio of villus height to crypt depth in the jejunal mucosa, RNA/DNA and protein/DNA in the jejunal and ileal mucosae, while increasing (Pxa0<xa00.05) DAO activity in plasma and caspase-3 expression in the intestinal mucosa. The adverse effects of LPS were partially ameliorated (Pxa0<xa00.05) by NAC supplementation. Moreover, NAC prevented the LPS-induced decrease in claudin-1 and occludin expression in the jejunal and ileal mucosae. Collectively, these results indicate that dietary NAC supplementation alleviates the mucosal damage and improves the absorptive function of the small intestine in LPS-challenged piglets.

N-acetylcysteine reduces inflammation in the small intestine by regulating redox, EGF and TLR4 signaling

This study determined whether N-acetylcysteine (NAC) could affect intestinal redox status, proinflammatory cytokines, epidermal growth factor (EGF), EGF receptor (EGFR), Toll-like receptor-4 (TLR4), and aquaporin-8 in a lipopolysaccharide (LPS)-challenged piglet model. Eighteen piglets (35-day-old) were randomly allocated into one of the three treatments (control, LPS and NAC). The control and LPS groups were fed a basal diet, and the NAC group received the basal diet +500xa0mg/kg NAC. On days 10, 13, and 20 of the trial, the LPS- and NAC-treated piglets received intraperitoneal administration of LPS (100xa0μg/kg BW), whereas the control group received the same volume of saline. On days 10 and 20, venous blood samples were obtained at 3xa0h post LPS or saline injection. On day 21 of the trial, piglets were killed to obtain the intestinal mucosa for analysis. Compared with the control group, LPS challenge reduced (Pxa0<xa00.05) the activities of superoxide dismutase, catalase, and glutathione peroxidase in jejunal mucosae, while increasing (Pxa0<xa00.05) the concentrations of malondialdehyde, H2O2, O2·− and the ratio of oxidized to reduced glutathione in jejunal mucosae, and concentrations of TNF-α, cortisol, interleukin-6, and prostaglandin E2 in both plasma and intestinal mucosae. These adverse effects of LPS were attenuated (Pxa0<xa00.05) by NAC supplementation. Moreover, NAC prevented LPS-induced increases in abundances of intestinal HSP70 and NF-κB p65 proteins and TLR4 mRNA. NAC supplementation enhanced plasma EGF concentration and intestinal EGFR mRNA levels. Collectively, these results indicate that dietary NAC supplementation alleviates LPS-induced intestinal inflammation via regulating redox, EGF, and TLR4 signaling.

Baicalin suppresses NLRP3 inflammasome and nuclear factor-kappa B (NF-κB) signaling during Haemophilus parasuis infection

Haemophilus parasuis (H. parasuis) is the causative agent of Glässer’s disease, a severe membrane inflammation disorder. Previously we showed that Baicalin (BA) possesses anti-inflammatory effects via the NLRP3 inflammatory pathway in an LPS-challenged piglet model. However, whether BA has anti-inflammatory effects upon H. parasuis infection is still unclear. This study investigated the anti-inflammatory effects and mechanisms of BA on H. parasuis-induced inflammatory responses via the NF-κB and NLRP3 inflammasome pathway in piglet mononuclear phagocytes (PMNP). Our data demonstrate that PMNP, when infected with H. parasuis, induced ROS (reactive oxygen species) production, promoted apoptosis, and initiated transcription expression of IL-6, IL-8, IL-10, PGE2, COX-2 and TNF-α via the NF-κB signaling pathway, and IL-1β and IL-18 via the NLRP3 inflammasome signaling pathway. Moreover, when BA was administrated, we observed a reduction in ROS production, suppression of apoptosis, and inhibition of the activation of NF-κB and NLRP3 inflammasome signaling pathway in PMNP treated with H. parasuis. To our best knowledge, this is the first example that uses piglet primary immune cells for an H. parasuis infection study. Our data strongly suggest that BA can reverse the inflammatory effect initiated by H. parasuis and possesses significant immunosuppression activity, which represents a promising therapeutic agent in the treatment of H. parasuis infection.

The anti-inflammatory effects of baicalin through suppression of NLRP3 inflammasome pathway in LPS-challenged piglet mononuclear phagocytes:

In this study, the anti-inflammatory effects and mechanisms of baicalin on LPS-induced NLRP3 inflammatory pathway were investigated in piglet mononuclear phagocytes (control, LPS stimulation, LPS stimulationu2009+u200912.5u2009µg/ml baicalin, LPS stimulationu2009+u200925u2009µg/ml baicalin, LPS stimulationu2009+u200950u2009µg/ml baicalin and LPS stimulationu2009+u2009100u2009µg/ml baicalin). The levels of reactive oxygen species (ROS), the secretion levels of IL-1β, IL-18 and TNF-α, mRNA expression levels of IL-1β, IL-18, TNF-α and NLRP3, as well as the protein levels of cleaved caspase-1 p20 were significantly increased after LPS-challenge in vitro. However, LPS stimulation did not influence apoptosis-associated speck-like protein and caspase-1 mRNA levels, which are also components of the NLRP3 inflammasome. Baicalin at 50u2009µg/ml and 100u2009µg/ml could inhibit the production of ROS, TNF-α, IL-1β and IL-18, and down-regulate mRNA expression of IL-1β, IL-18, TNF-α and NLRP3, as well as expression of cleaved caspase-1 p20. These results showed that the anti-inflammatory effects of baicalin occurred via the regulation of the release of ROS and mRNA expression of NLRP3. The anti-inflammatory activity of baicalin could be related to the suppression of NLRP3 inflammasome pathway under LPS stimulation.

Comparative transcriptional profiling of tildipirosin-resistant and sensitive Haemophilus parasuis

Numerous studies have been conducted to examine the molecular mechanism of Haemophilus parasuis resistance to antibiotic, but rarely to tildipirosin. In the current study, transcriptional profiling was applied to analyse the variation in gene expression of JS0135 and tildipirosin-resistant JS32. The growth curves showed that JS32 had a higher growth rate but fewer bacteria than JS0135. The cell membranes of JS32 and a resistant clinical isolate (HB32) were observed to be smoother than those of JS0135. From the comparative gene expression profile 349 up- and 113 downregulated genes were observed, covering 37 GO and 63 KEGG pathways which are involved in biological processes (11), cellular components (17), molecular function (9), cellular processes (1), environmental information processing (4), genetic information processing (9) and metabolism (49) affected in JS32. In addition, the relative overexpression of genes of the metabolism pathway (HAPS_RS09315, HAPS_RS09320), ribosomes (HAPS_RS07815) and ABC transporters (HAPS_RS10945) was detected, particularly the metabolism pathway, and verified with RT-qPCR. Collectively, the gene expression profile in connection with tildipirosin resistance factors revealed unique and highly resistant determinants of H. parasuis to macrolides that warrant further attention due to the significant threat of bacterial resistance.

Roles of amino acids in preventing and treating intestinal diseases: recent studies with pig models

Animal models are needed to study and understand a human complex disease. Because of their similarities in anatomy, structure, physiology, and pathophysiology, the pig has proven its usefulness in studying human gastrointestinal diseases, such as inflammatory bowel disease, ischemia/reperfusion injury, diarrhea, and cancer. To understand the pathogenesis of these diseases, a number of experimental models generated in pigs are available, for example, through surgical manipulation, chemical induction, microbial infection, and genetic engineering. Our interests have been using amino acids as therapeutics in pig and human disease models. Amino acids not only play an important role in protein biosynthesis, but also exert significant physiological effects in regulating immunity, anti-oxidation, redox regulation, energy metabolism, signal transduction, and animal behavior. Recent studies in pigs have shown that specific dietary amino acids can improve intestinal integrity and function under normal and pathological conditions that protect the host from different diseases. In this review, we summarize several pig models in intestinal diseases and how amino acids can be used as therapeutics in treating pig and human diseases.

Autophagy and tight junction proteins in the intestine and intestinal diseases

The intestinal epithelium (IE) forms an indispensible barrier and interface between the intestinal interstitium and the luminal environment. The IE regulates water, ion and nutrient transport while providing a barrier against toxins, pathogens (bacteria, fungi and virus) and antigens. The apical intercellular tight junctions (TJ) are responsible for the paracellular barrier function and regulate trans-epithelial flux of ions and solutes between adjacent cells. Increased intestinal permeability caused by defects in the IE TJ barrier is considered an important pathogenic factor for the development of intestinal inflammation, diarrhea and malnutrition in humans and animals. In fact, defects in the IE TJ barrier allow increased antigenic penetration, resulting in an amplified inflammatory response in inflammatory bowel disease (IBD), necrotizing enterocolitis and ischemia-reperfusion injury. Conversely, the beneficial enhancement of the intestinal TJ barrier has been shown to resolve intestinal inflammation and apoptosis in both animal models of IBD and human IBD. Autophagy (self-eating mechanism) is an intracellular lysosome-dependent degradation and recycling pathway essential for cell survival and homeostasis. Dysregulated autophagy has been shown to be directly associated with many pathological processes, including IBD. Importantly, the crosstalk between IE TJ and autophagy has been revealed recently. We showed that autophagy enhanced IE TJ barrier function by increasing transepithelial resistance and reducing the paracellular permeability of small solutes and ions, which is, in part, by targeting claudin-2, a cation-selective, pore-forming, transmembrane TJ protein, for lysosome (autophagy)-mediated degradation. Interestingly, previous studies have shown that the inflamed intestinal mucosa in patients with active IBD has increased claudin-2 expression. In addition, inflammatory cytokines (for example, tumor necrosis factor-α, interleukin-6, interleukin-13, and interleukin-17) whose levels are increased in IBD patients cause an increase in claudin-2 expression and a claudin-2-dependent increase in TJ permeability. Thus, the role of claudin-2 in intestinal pathological processes has been attributed, in part, to the increase of intestinal TJ permeability. Claudin-2 represents a new therapeutic target in treating IBD, diarrhea and malnutrition in animals and humans.

PK-PD Integration Modeling and Cutoff Value of Florfenicol against Streptococcus suis in Pigs

The aims of the present study were to establish optimal doses and provide an alternate COPD for florfenicol against Streptococcus suis based on pharmacokinetic-pharmacodynamic integration modeling. The recommended dose (30 mg/kg b.w.) were administered in healthy pigs through intramuscular and intravenous routes for pharmacokinetic studies. The main pharmacokinetic parameters of Cmax, AUC0-24h, AUC, Ke, t1/2ke, MRT, Tmax, and Clb, were estimated as 4.44 μg/ml, 88.85 μg⋅h/ml, 158.56 μg⋅h/ml, 0.048 h-1, 14.46 h, 26.11 h, 4 h and 0.185 L/h⋅kg, respectively. The bioavailability of florfenicol was calculated to be 99.14% after I.M administration. A total of 124 Streptococcus suis from most cities of China were isolated to determine the minimum inhibitory concentration (MIC) of florfenicol. The MIC50 and MIC90 were calculated as 1 and 2 μg/ml. A serotype 2 Streptococcus suis (WH-2), with MIC value similar to MIC90, was selected as a representative for an in vitro and ex vivo pharmacodynamics study. The MIC values of WH-2 in TSB and plasma were 2 μg/ml, and the MBC/MIC ratios were 2 in TSB and plasma. The MPC was detected to be 3.2 μg/ml. According to inhibitory sigmoid Emax model, plasma AUC0-24h/MIC values of florfenicol versus Streptococcus suis were 37.89, 44.02, and 46.42 h for the bactericidal, bacteriostatic, and elimination activity, respectively. Monte Carlo simulations the optimal doses for bactericidal, bacteriostatic, and elimination effects were calculated as 16.5, 19.17, and 20.14 mg/kg b.w. for 50% target attainment rates (TAR), and 21.55, 25.02, and 26.85 mg/kg b.w. for 90% TAR, respectively. The PK-PD cutoff value (COPD) analyzed from MCS for florfenicol against Streptococcus suis was 1 μg/ml which could provide a sensitivity cutoff value. These results contributed an optimized alternative to clinical veterinary medicine and showed that the dose of 25.02 mg/kg florfenicol for 24 h could have a bactericidal action against Streptococcus suis after I.M administration. However, it should be validated in clinical practice in the future investigations.

Baicalin modulates NF-κB and NLRP3 inflammasome signaling in porcine aortic vascular endothelial cells Infected by Haemophilus parasuis Causing Glässer’s disease

Haemophilus parasuis (H. parasuis) can cause vascular inflammatory injury, but the molecular basis of this effect remains unclear. In this study,we investigated the effect of the anti-inflammatory, anti-microbial and anti-oxidant agent, baicalin, on the nuclear factor (NF)-κB and NLRP3 inflammasome signaling pathway in pig primary aortic vascular endothelial cells. Activation of the NF-κB and NLRP3 inflammasome signaling pathway was induced in H. parasuis-infected cells. However, baicalin reduced the production of reactive oxygen species, apoptosis, and activation of the NF-κB and NLRP3 inflammasome signaling pathway in infected cells. These results revealed that baicalin can inhibit H. parasuis-induced inflammatory responses in porcine aortic vascular endothelial cells, and may thus offer a novel strategy for controlling and treating H. parasuis infection. Furthermore, the results suggest that piglet primary aortic vascular endothelial cells may provide an experimental model for future studies of H. parasuis infection.

Evaluation of recombinant protein superoxide dismutase of Haemophilus parasuis strain SH0165 as vaccine candidate in a mouse model

Haemophilus parasuis can cause a severe membrane inflammation disorder. It has been documented that superoxide dismutase (SOD) is a potential target to treat systemic inflammatory diseases. Therefore, we constructed an experimental H. parasuis subunit vaccine SOD and determined the protective efficacy of SOD using a lethal dose challenge against H. parasuis serovar 4 strain MD0322 and serovar 5 strain SH0165 in a mouse model. The results demonstrated that SOD could induce a strong humoral immune response in mice and provide significant immunoprotection efficacy against a lethal dose of H. parasuis serovar 4 strain MD0322 or serovar 5 strain SH0165 challenge. IgG subtype analysis indicated SOD protein could trigger a bias toward a Th1-type immune response and induce the proliferation of splenocytes and secretion of IL-2 and IFN-γ of splenocytes. In addition, serum in mice from the SOD-immunized group could inhibit the growth of strain MD0322 and strain SH0165 in the whole-blood killing bacteria assay. This is the first report that immunization of mice with SOD protein could provide protective effect against a lethal dose of H. parasuis serovar 4 and serovar 5 challenge in mice, which may provide a novel approach against heterogeneous serovar infection of H. parasuis in future.