Ameliorative Effect of Macadamia Nut Protein Peptides on Acetaminophen-Induced Acute Liver Injury in Mice.

This study aims to examine the ameliorative effect of macadamia nut protein peptides (MPP) on acetaminophen (APAP)-induced liver injury (AILI) in mice, and develop a new strategy for identifying hepatoprotective functional foods. The molecular weight distribution and amino acid composition of MPP were first studied. Forty mice were then randomized into four groups: control group (CON), APAP model group, APAP+MPP low-dose group (APAP+L-MPP), and APAP+MPP high-dose group (APAP+H-MPP). The APAP+L-MPP (320 mg/kg per day) and APAP+H-MPP (640 mg/kg per day) groups received continuous MPP gavage for 2 weeks. A 12 h of APAP (200 mg/kg) gavage resulted in liver damage. Pathological alterations, antioxidant index levels, expression of toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB), and associated inflammatory factors were determined for each treatment group. The results revealed that the total amino acid content of MPP was 39.58 g/100 g, with Glu, Arg, Asp, Leu, Tyr, and Gly being the major amino acids. The molecular weight range of 0-1000 Da accounted for 73.54%, and 0-500 Da accounted for 62.84% of MPP. MPP ameliorated the pathological morphology and reduced the serum levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase of AILI in mice. MPP significantly increased the activities of superoxide dismutase and glutathione peroxidase in the liver compared with the APAP group. MPP inhibited the expression of TLR4, NF-κB, interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) genes in AILI mice. MPP also inhibited the expression levels of inflammatory factors (TNF-α and IL-6). Our study concludes that MPP alleviates AILI in mice by enhancing antioxidant capacity and inhibiting TLR4/NF-κB pathway-related gene activation.

Hydroxytyrosol mitigates Mycoplasma gallisepticum-induced pulmonary injury through downregulation of the NF-κB/NLRP3/IL-1ß signaling pathway in chicken.

In this study, the anti-inflammatory and antiapoptotic effects of hydroxytyrosol (HT) in Mycoplasma gallisepticum (MG)-infected chicken were investigated, and the underlying molecular mechanisms were explored. The results revealed severe ultrastructural pathological changes after MG infection in the lung tissue of chicken, including inflammatory cell infiltration, thickening of the lung chamber wall, visible cell swelling, mitochondrial cristae rupture, and ribosome shedding. MG possibly activated the nuclear factor κB (NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/interleukin (IL)-1ß signaling pathway in the lung. However, HT treatment significantly ameliorated MG-induced pathological damage of the lung. HT reduced the magnitude of pulmonary injury after MG infection by reducing apoptosis and releasing the proinflammatory factors. Compared with the MG-infected group, the HT-treated group exhibited significant inhibition of the expression of NF-κB/NLRP3/IL-1ß signaling-pathway-related genes; for example, the expressions of NF-κB, NLRP3, caspase-1, IL-1ß, IL-2, IL-6, IL-18, and TNF-α significantly decreased (P < 0.01 or <0.05). In conclusion, HT effectively inhibited MG-induced inflammatory response and apoptosis and protected the lung by blocking the activation of NF-κB/NLRP3/IL-1ß signaling pathway and reducing the damage caused by MG infection in chicken. This study revealed that HT may be a suitable and effective anti-inflammatory drug against MG infection in chicken.

Molecular mechanism of anti-inflammatory effects of the proteasome inhibitor MG-132 on Con A-induced acute liver injury in mice.

MG-132, an aldehyde-based peptide proteasome inhibitor (PI) that binds to the proteasome and reversibly inhibits proteasome activity, has been widely used in experimental research. However, it is not clear whether MG-132 has anti-inflammatory effects on liver injury. The molecular mechanism of the anti-inflammatory effect of the PI MG-132 on Con A-induced acute liver injury (ALI) mice was investigated by ELISA, HE, q RT-PCR, and IHC. The results showed that the serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and TNF-α and IL-6 contents of mice in the high and medium dose groups were reduced compared with those in the ALI group. The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels in liver tissues were significantly increased, and the malondialdehyde (MDA) content was decreased. The pathological sections of mice in the ALI group showed typical ALI manifestations such as significant central venous stasis of liver tissues, cell swelling, and inflammatory cell infiltration. The pathological damage of liver tissues was relieved significantly in the three dose groups, especially in the high-dose group. The transcriptional level of TLR4/NF-κB pathway key factors mRNA was significantly reduced, and the expression of TLR4 and NF-κB P65 protein in liver tissues was significantly and positively correlated with the contents of TNF-α and IL-1ß (p < 0.01). Our findings suggest that MG-132 can alleviate the inflammatory response to Con A-induced ALI and exert a hepatoprotective effect, and its anti-inflammatory effect is related to the inhibition of TLR4/NF-κB signaling pathway activation.

Oleocanthal alleviated lipopolysaccharide-induced acute lung injury in chickens by inhibiting TLR4/NF-κB pathway activation.

This study aimed to investigate the ameliorative effect of oleocanthal (OC) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in chickens and its possible mechanisms. In total, 20 chickens were randomly divided into 4 groups: control (CON) group, LPS group, LPS + OC group, and OC group. LPS + OC and OC groups were intragastrically administered a 5 mg/kg·d OC dose for 7 d. On d 8, the LPS group and LPS + OC group were intratracheally administered 2 mg/kg LPS for 12 h. It was found that OC ameliorated the pathological morphology and significantly suppressed apoptosis after OC treatment in LPS-induced ALI chicken (P < 0.01). Antioxidant capacity was higher in the LPS + OC group compared with the LPS group (P < 0.01). OC downregulated the related genes and proteins expression of toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) pathway in LPS group (P < 0.01). In conclusion, OC supplementation can alleviate LPS-induced ALI in chickens by suppressing apoptosis, enhancing lung antioxidant capacities and inhibiting TLR4/NF-κB pathway activation.

Immunomodulatory and antioxidant effects of hydroxytyrosol in cyclophosphamide-induced immunosuppressed broilers.

As an important olive component, hydroxytyrosol (HT) has good medicinal and health effects. However, its importance in alleviating immune suppression in broilers has not been established. Therefore, we aimed at evaluating the immunomodulatory and antioxidant effects of HT in immune suppressed broilers. Immune suppressed broiler models were established via intraperitoneal injection of 80 mg/kg cyclophosphamide (Cy). Thirty two Cobb 500 male broilers were randomly allocated into 4 groups of 8 each. Broilers in the model (Cy) and HT treatment (Cy+HT) groups were intraperitoneally administered with Cy (80 mg/kg BW) once a day for 3 d. From the 4th d, broilers in the Cy+HT and HT groups were treated with 0.5 mL of 200 mg/L HT solution, once a day, for 7 d. The Cy and Con groups were orally administered with normal saline. On the 14th and 28th d, serum and duodenal samples were obtained for testing. It was found that HT increased villi height (VH)/crypt depth (CD) ratio in the duodenum and suppressed serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels. Moreover, it elevated the expressions of CD4+ and CD8+ T lymphocytes. HT upregulated the mRNA expression levels of interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10), enhanced the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and downregulated malondialdehyde (MDA) levels in Cy-induced immune-suppressed broilers. In conclusion, HT can alleviate immune-suppression as well as enhance immunity and antioxidant activities in the local mucosa of small intestines in broilers. Therefore, it can be used as an immune stimulant. More studies should be performed to confirm our findings and to elucidate on the mechanisms of HT.

Walnut oil alleviates LPS-induced intestinal epithelial cells injury by inhibiting TLR4/MyD88/NF-κB pathway activation.

In this study, we investigated the protective effects of walnut oil (WO) on mouse intestinal epithelial cells using used MODE-K cells as a model and explored the underlying mechanisms. Our data suggested that WO attenuated lipopolysaccharide (LPS)-induced pathological changes and inhibited the rate of LPS-induced apoptosis in MODE-K cells. Furthermore, WO down-regulated LPS-induced gene and protein expression of toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (MyD88), nuclear factor-κB (NF-κB), tumor necrosis factor-α, and interleukin-6. In conclusion, this study shows that WO exerts an anti-inflammatory effect on LPS-induced MODE-K cells injury by inhibiting the TLR4/MyD88/NF-κB pathway activation. Based on our data, a prominent functional food candidate can be provided for inflammatory bowel disease treatment. PRACTICAL APPLICATIONS: Walnut oil (WO) has excellent anti-inflammatory properties and is widely used in traditional dietary supplements. However, whether WO causes anti-lipopolysaccharide (LPS)-induced intestinal injury remains unclear. In this study, we investigated the protective effects of WO on mouse intestinal epithelial cells using MODE-K cells as a model and explored their potential mechanisms. Our data showed that WO ameliorated the pathological morphology, inhibited the apoptosis of LPS-induced MODE-K cell injury, decreased the release of pro-inflammatory cytokines, and down-regulated the related genes and proteins expression of the LPS-TLR4/MyD88/NF-κB inflammatory pathway. The results of this study would enhance the utilization of WO in the prevention of gastrointestinal diseases in animals and humans inflammatory bowel disease as well as in functional foods formulations.

Walnut oil alleviates DSS-induced colitis in mice by inhibiting NLRP3 inflammasome activation and regulating gut microbiota.

Ulcerative colitis (UC) has become a global disease and closely related to changes in intestinal oxidative stress, inflammatory factors and gut microbiota. Furthermore, the NLRP3 inflammasome activation is a key cause in the pathogenesis of dextran sulfate sodium (DSS)-induced colitis. Recent data showed the potential antioxidative and anti-inflammatory advantage of walnut oil, which widely used in traditional medicine and has become a dietary supplement for some patients. Therefore, we investigated whether walnut oil exerts an anti-inflammatory effect on DSS-induced colitis mice by targeting NLRP3 inflammasome and gut microbiota. Our data showed that walnut oil ameliorated the pathological morphology, decreased the reactive oxygen species (ROS) production and pro-inflammatory cytokines release, down-regulated the related gene proteins expression of NLRP3/ASC/Caspase-1 inflammatory pathway, inhibited apoptosis, shifted from more pathogens towards probiotics, and increased the levels of short-chain fatty acids (SCFAs) in DSS-induced damaging process. Collectively, our study concludes that walnut oil exerts anti-inflammatory effect on DSS-induced colitis in mice by inhibiting the NLRP3 inflammasome activation and modulating gut microbiota, and may be a prominent functional food candidate for UC treatment.

Induction of macrophage pyroptosis-related factors by pathogenic E. coli high pathogenicity island (HPI) in Yunnan Saba pigs.

BACKGROUND: Pyroptosis plays a pivotal role in the pathogenesis of many inflammatory diseases. The molecular mechanism by which pyroptosis is induced in macrophages following infection with pathogenic E. coli high pathogenicity island (HPI) will be evaluated in our study. RESULTS: After infection with the HPI+/HPI- strains and LPS, decreased macrophage cell membrane permeability and integrity were demonstrated with propidium iodide (PI) staining and the lactate dehydrogenase (LDH) assay. HPI+/HPI--infection was accompanied by upregulated expression levels of NLRP3, ASC, caspase-1, IL-1ß, IL-18 and GSDMD, with significantly higher levels detected in the HPI+ group compared to those in the HPI- group (P < 0.01 or P < 0.05). HPI+ strain is more pathogenic than HPI- strain. CONCLUSION: Our findings indicate that pathogenic E. coli HPI infection of Saba pigs causes pyroptosis of macrophages characterized by upregulated expression of pyroptosis key factors in the NLRP3/ASC/caspase-1 signaling pathway, direct cell membrane pore formation, and secretion of the inflammatory factor IL-1ß and IL-18 downstream of NLRP3 and caspase-1 activation to enhance the inflammatory response.

The protective effect of walnut oil on lipopolysaccharide-induced acute intestinal injury in mice.

Walnut oil (WO) is widely used in traditional medicine, and it has become a dietary supplement in many countries. We isolated walnut oil from Juglans sigillata and evaluated its protective effects on acute intestinal injury, and Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway in lipopolysaccharide (LPS)-induced mice was studied. The results showed that the LPS + WO group significantly decreased serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß levels and increased the jejunum superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels compared with the LPS group. Walnut oil ameliorated the pathological morphology of the LPS-induced acute jejunum injury and decreased jejunum cells apoptosis rate and TLR4/NF-κB protein expression. Furthermore, the expression of the TLR4/NF-κB pathway key gene mRNA significantly reduced after treatment with walnut oil. This study concludes that walnut oil can exert the protective effect on LPS-induced acute intestinal injury in mice by inhibiting the TLR4/NF-κB signaling pathway.

High pathogenicity island is associated with enhanced autophagy in pathogenic Escherichia coli HPI - infected macrophages.

High pathogenicity island (HPI), which is widely distributed in Escherichia coli (E. coli), can enhance the pathogenicity of E. coli. Thus the HPI positive E. coli could pose a threat to human and animal health. It remains to be elucidated how HPI affects the virulence of pathogenic E. coli. Autophagy is an important mechanism to maintain cellular homeostasis and an innate immunity responses of organisms against pathogens. The interaction between pathogenic E. coli possessing HPI (E. coli HPI) and host autophagy system has not been reported. In this study, it was demonstrated that pathogenic E. coli induced autophagy in 3D4/21 macrophages and HPI was associated with enhanced autophagy through transmission electron microscopy, immunofluorescence and real-time PCR. The PI3K/Akt/mTOR pathway is an important negative regulatory pathway for autophagy. Through detecting the expression of key genes of PI3K/Akt/mTOR pathway, it was speculated that HPI enhanced the inhibition of the signaling pathway stimulated by pathogenic E. coli. Furthermore, HPI inhibited the secretion of IFN-γ, while the presence of HPI did not significantly affect the secretion of IL-1ß. This work is the first attempt to explore the interplay between HPI carried by pathogenic E. coli and host cell autophagy. The findings might enable better understanding of the contribution of HPI to pathogenicity.

Effect of walnut (Juglans sigillata) oil on intestinal antioxidant, anti-inflammatory, immunity, and gut microbiota modulation in mice.

The study investigated the anti-oxidant, anti-inflammatory, immunity, and gut microbiota modulation in mice (n = 60; 15 mice/group) after intragastric administration of walnut oil (WO; three groups (low (LD), medium (MD), and high doses (HD): 2.5, 5, and 10 ml/kg, respectively) and normal control (NC, saline). WO significantly increased the median villous height/crypt depth (VH/CD) ratio, the activities of superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in intestinal mucosa. WO exerted the anti-inflammatory effects by decreasing the expression of tumor necrosis factor-α (TNF-α) in the duodenal mucosa. All groups shared 157 operational taxonomic units (OTUs; 97% similarity) representing nine phyla. The relative abundance in gut microbiota shifted from more pathogenic bacteria-Helicobacter (NC: 22% versus MD: 3%) toward probiotic-Lactobacillus (NC: 19% versus MD: 40%). The immune organ index (spleen) and contents of secretory immunoglobulin A (S-IgA) were increased from small intestine. In conclusion, WO decreased the oxidative stress, inflammation, and improved the immunity and beneficial gut microbiota in the mice. PRACTICAL APPLICATIONS: Walnut oil (WO) is widely used in traditional medicine around the world and is prescribed as beneficial food oil in agro-industry. However, the intestinal benefits of WO have not been explored extensively, and even its therapeutic mechanism still remains unknown in modern medicine. In this study, WO from Juglans sigillata was investigated for its preventive and protective effects on the intestinal mucosa in mice including anti-oxidant, anti-inflammatory, immunity, and gut microbiota modulation. WO decreased the oxidative stress, inflammation, and improved immunity and beneficial gut microbiota in the mice. WO has shown strong probiotic effect on the gut, and thus, can be considered as a potential candidate in food. The study outcome would enhance utilization of WO for the prevention of gastrointestinal diseases (e.g., Helicobacter, etc.) both in animals and human (inflammatory bowel diseases, IBD) and the formulation of functional foods.

Pathogenic E. coli HPI upregulate the expression of inflammatory factors in porcine small intestinal epithelial cells by ubiquitin proteasome pathway.

To investigate the effects of pathogenic Escherichia coli high pathogenicity island (HPI) on the expression of inflammatory factors via ubiquitin proteasome pathway. Firstly, the UBC-sus-263 shRNA plasmid was successfully established and transfected into porcine small intestine epithelial cells (IPEC-J2) by liposome to silence the ubiquitinntion gene. Then the IPEC-J2 was infected with E. coli HPI+ and HPI- strains, respectively. Finally, the mRNA of intracellular NF-κB and IκB-α,and the protein levels of NF-κB, IκB-α, TNF-α and IL-1 in IPEC-J2 cell line transfected with UBC-sus-263 shRNA (Ub-shRNA) were detected. The results showed that the Ub-shRNA was effectively inhibited ubiquitination pathway in the IPEC-J2 cell. After infected with HPI+, the mRNA and protein levels of NF-κB and IκB-α were dramatically decreased in Ub-hsRNA transfected IPEC-J2 cells compared to the control and HPI--infected groups. Consistently, the production of downstream cytokines such as TNF-α and IL-1 were highly expressed after HPI+-infection than that of HPI--infected groups. However, whether the HPI+ or HPI-, both could induce increasingly expression of NF-κB and IκB-α and its downstream cytokines in normal IPEC-J2 cells. Thus, the E. coli HPI can upregulate the expression of IκB-α to promote the releasing of TNF-α and IL-1 via the ubiquitination pathway.