Scutellarin enhances anti-tumor immune responses by reducing TNFR2-expressing CD4+Foxp3+ regulatory T cells.

One characteristic of tumor-associated CD4+Foxp3+ regulatory T cells (Tregs) is the high expression of tumor necrosis factor receptor type II (TNFR2), a receptor that mediates the decisive effect of tumor necrosis factor (TNF) in the activation and expansion of Tregs. There is increasing evidence that inhibition of TNFR2 can enhance anti-tumor immune responses. Therefore, we screened Chinese herbal extracts for their capacity to block TNF-TNFR2 interaction. The results showed that the treatment with a Chinese herb extract could inhibit TNFR2-induced biological responses in vitro, including the proliferation of TNFR2+ Tregs. Our subsequent study led to the identification of flavonoid compound scutellarin was responsible for the activity. Our results showed that scutellarin is able to disrupt the interaction of TNF-TNFR2 and inhibited the phosphorylation of p38 MAPK, a down-stream signaling component of TNFR2. Importantly, in vivo scutellarin treatment markedly enhanced the efficacy of tumor immunotherapy with CpG oligodeoxynucleotide in mouse CT26 colon cancer model. This effect of scutellarin was associated with the reduction of the number of tumor-infiltrating TNFR2-expressing Tregs and increased tumor infiltration of interferon-γ-producing CD8+ T cells. Our result also suggests that scutellarin or its analogs may be used as an adjuvant to enhance the anti-tumor effect of immunotherapeutic agent by eliminating TNFR2+ Treg activity.

Medium-Chain Triglycerides Attenuate Liver Injury in Lipopolysaccharide-Challenged Pigs by Inhibiting Necroptotic and Inflammatory Signaling Pathways.

This study was conducted to investigate whether medium-chain triglycerides (MCTs) attenuated lipopolysaccharide (LPS)-induced liver injury by down-regulating necroptotic and inflammatory signaling pathways. A total of 24 pigs were randomly allotted to four treatments in a 2 × 2 factorial design including diet (0 and 4% MCTs) and immunological challenge (saline and LPS). After three weeks of feeding with or without 4% MCTs, pigs were challenged with saline or LPS. MCTs led to a significant increase in eicosapentaenoic acid, docosahexaenoic acid and total (n-3) polyunsaturated fatty acid concentrations. MCTs attenuated LPS-induced liver injury as indicated by an improvement in liver histomorphology and ultrastructural morphology of hepatocytes, a reduction in serum alanine aminotransferase and alkaline phosphatase activities as well as an increase in claudin-1 protein expression. In addition, MCTs also reduced serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6 concentrations, liver TNF-α and IL-1ß mRNA expression and protein concentrations and enhanced liver heat shock protein 70 protein expression in LPS-challenged pigs. Moreover, MCTs decreased mRNA expression of receptor-interacting serine/threonine-protein kinase (RIP) 3, mixed-lineage kinase domain-like protein (MLKL) and phosphoglycerate mutase 5 and inhibited MLKL phosphorylation in the liver. Finally, MCTs decreased liver mRNA expression of toll-like receptor (TLR) 4, nucleotide-binding oligomerization domain protein (NOD) 1 and multiple downstream signaling molecules. MCTs also suppressed LPS-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation and increased extracellular signal-related kinase 1/2 phosphorylation in the liver. These results indicated that MCTs are capable of attenuating LPS-induced liver damage by suppressing hepatic necroptotic (RIP1/RIP3/MLKL) and inflammatory (TLR4/NOD1/p38 MAPK) signaling pathways.

Medium-chain TAG improve intestinal integrity by suppressing toll-like receptor 4, nucleotide-binding oligomerisation domain proteins and necroptosis signalling in weanling piglets challenged with lipopolysaccharide.

This study was conducted to evaluate whether medium-chain TAG (MCT) could alleviate Escherichia coli lipopolysaccharide (LPS)-induced intestinal injury by regulating intestinal epithelial inflammatory response, as well as necroptosis. A total of twenty-four weanling piglets were randomly allotted to one of four treatments in a 2×2 factorial arrangement including diet type (5 % maize oil v. 4 % MCT+1 % maize oil) and immune stress (saline v. E. coli LPS). The piglets were fed diets containing maize oil or MCT for 21 d. On 21 d, piglets were injected intraperitoneally with saline or LPS. The blood and intestinal samples were collected at 4 h post injection. Supplementation with MCT improved intestinal morphology, digestive and barrier function, indicated by increased jejunal villus height, increased jejunal and ileal disaccharidases (sucrase and maltase) activities, as well as enhanced protein expression of claudin-1. Furthermore, the protein expression of heat-shock protein 70 in jejunum and the concentration of TNF-α in plasma were reduced in the piglets fed diets supplemented with MCT. In addition, MCT down-regulated the mRNA expression of toll-like receptor 4 (TLR4) and nucleotide-binding oligomerisation domain proteins (NOD) signalling-related genes in jejunum and ileum. Finally, MCT inhibited jejunal and ileal enterocyte necroptosis indicated by suppressed mRNA expression of the receptor-interacting protein 3 and mixed-lineage kinase domain-like protein. These results indicate that MCT supplementation may be closely related to inhibition of TLR4, NOD and necroptosis signalling pathways and concomitant improvement of intestinal integrity under an inflammatory condition.

Glycine enhances muscle protein mass associated with maintaining Akt-mTOR-FOXO1 signaling and suppressing TLR4 and NOD2 signaling in piglets challenged with LPS.

Pro-inflammatory cytokines play a critical role in the pathophysiology of muscle atrophy. We hypothesized that glycine exerted an anti-inflammatory effect and alleviated lipopolysaccharide (LPS)-induced muscle atrophy in piglets. Pigs were assigned to four treatments including the following: 1) nonchallenged control, 2) LPS-challenged control, 3) LPS+1.0% glycine, and 4) LPS+2.0% glycine. After receiving the control, 1.0 or 2.0% glycine-supplemented diets, piglets were treated with either saline or LPS. At 4 h after treatment with saline or LPS, blood and muscle samples were harvested. We found that 1.0 or 2.0% glycine increased protein/DNA ratio, protein content, and RNA/DNA ratio in gastrocnemius or longissimus dorsi (LD) muscles. Glycine also resulted in decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) in gastrocnemius muscle. In addition, glycine restored the phosphorylation of Akt, mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and Forkhead Box O 1 (FOXO1) in gastrocnemius or LD muscles. Furthermore, glycine resulted in decreased plasma tumor necrosis factor-α (TNF-α) concentration and muscle TNF-α mRNA abundance. Moreover, glycine resulted in decreased mRNA expresson of Toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain protein 2 (NOD2), and their respective downstream molecules in gastrocnemius or LD muscles. These results indicate glycine enhances muscle protein mass under an inflammatory condition. The beneficial roles of glycine on the muscle are closely associated with maintaining Akt-mTOR-FOXO1 signaling and suppressing the activation of TLR4 and/or NOD2 signaling pathways.

Fish oil enhances intestinal barrier function and inhibits corticotropin-releasing hormone/corticotropin-releasing hormone receptor 1 signalling pathway in weaned pigs after lipopolysaccharide challenge.

Stress induces injury in intestinal barrier function in piglets. Long-chain n-3 PUFA have been shown to exhibit potential immunomodulatory and barrier protective effects in animal models and clinical trials. In addition, corticotropin-releasing hormone (CRH)/CRH receptor (CRHR) signalling pathways play an important role in stress-induced alterations of intestinal barrier function. We hypothesised that fish oil could affect intestinal barrier function and CRH/CRHR signalling pathways. In total, thirty-two weaned pigs were allocated to one of four treatments. The experiment consisted of a 2×2 factorial design, and the main factors included immunological challenge (saline or lipopolysaccharide (LPS)) and diet (5 % maize oil or 5 % fish oil). On d 19 of the trial, piglets were treated with saline or LPS. At 4 h after injection, all pigs were killed, and the mesenteric lymph nodes (MLN), liver, spleen and intestinal samples were collected. Fish oil decreased bacterial translocation incidence and the number of translocated micro-organisms in the MLN. Fish oil increased intestinal claudin-1 protein relative concentration and villus height, as well as improved the intestinal morphology. In addition, fish oil supplementation increased intestinal intraepithelial lymphocyte number and prevented elevations in intestinal mast cell and neutrophil numbers induced by LPS challenge. Moreover, fish oil tended to decrease the mRNA expression of intestinal CRHR1, CRH and glucocorticoid receptors. These results suggest that fish oil supplementation improves intestinal barrier function and inhibits CRH/CRHR1 signalling pathway and mast cell tissue density.