miR-223-3p Prevents Necroptotic Macrophage Death by Targeting Ripk3 in a Negative Feedback Loop and Consequently Ameliorates Advanced Atherosclerosis.

BACKGROUND: The formation of large necrotic cores results in vulnerable atherosclerotic plaques, which can lead to severe cardiovascular diseases. However, the specific regulatory mechanisms underlying the development of necrotic cores remain unclear. METHODS: To evaluate how the modes of lesional cell death are reprogrammed during the development of atherosclerosis, the expression levels of key proteins that are involved in the necroptotic, apoptotic, and pyroptotic pathways were compared between different stages of plaques in humans and mice. Luciferase assays and loss-of-function studies were performed to identify the microRNA-mediated regulatory mechanism that protects foamy macrophages from necroptotic cell death. The role of this mechanism in atherosclerosis was determined by using a knockout mouse model with perivascular drug administration and tail vein injection of microRNA inhibitors in Apoe-/- mice. RESULTS: Here, we demonstrate that the necroptotic, rather than the apoptotic or pyroptotic, pathway is more activated in advanced unstable plaques compared with stable plaques in both humans and mice, which closely correlates with necrotic core formation. The upregulated expression of Ripk3 (receptor-interacting protein kinase 3) promotes the C/EBPß (CCAAT/enhancer binding protein beta)-dependent transcription of the microRNA miR-223-3p, which conversely inhibits Ripk3 expression and forms a negative feedback loop to regulate the necroptosis of foamy macrophages. The knockout of the Mir223 gene in bone marrow cells accelerates atherosclerosis in Apoe-/- mice, but this effect can be rescued by Ripk3 deficiency or treatment with the necroptosis inhibitors necrostatin-1 and GSK-872. Like the Mir223 knockout, treating Apoe-/- mice with miR-223-3p inhibitors increases atherosclerosis. CONCLUSIONS: Our study suggests that miR-223-3p expression in macrophages protects against atherosclerotic plaque rupture by limiting the formation of necrotic cores, thus providing a potential microRNA therapeutic candidate for atherosclerosis.

Innate Immune Memory in Monocytes and Macrophages: The Potential Therapeutic Strategies for Atherosclerosis.

Atherosclerosis is a complex metabolic disease characterized by the dysfunction of lipid metabolism and chronic inflammation in the intimal space of the vessel. As the most abundant innate immune cells, monocyte-derived macrophages play a pivotal role in the inflammatory response, cholesterol metabolism, and foam cell formation. In recent decades, it has been demonstrated that monocytes and macrophages can establish innate immune memory (also termed trained immunity) via endogenous and exogenous atherogenic stimuli and exhibit a long-lasting proinflammatory phenotype. The important cellular metabolism processes, including glycolysis, oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, fatty acid synthesis, and cholesterol synthesis, are reprogrammed. Trained monocytes/macrophages with innate immune memory can be persistently hyperactivated and can undergo extensive epigenetic rewiring, which contributes to the pathophysiological development of atherosclerosis via increased proinflammatory cytokine production and lipid accumulation. Here, we provide an overview of the regulation of cellular metabolic processes and epigenetic modifications of innate immune memory in monocytes/macrophages as well as the potential endogenous and exogenous stimulations involved in the progression of atherosclerosis that have been reported recently. These elucidations might be beneficial for further understanding innate immune memory and the development of therapeutic strategies for inflammatory diseases and atherosclerosis.

Effects of Feeding 5-Aminolevulinic Acid on Iron Status in Weaned Rats from the Female Rats during Gestation and Lactation.

Using female Sprague−Dawley (SD) rats as a model, the current study aimed to investigate whether feeding 5-aminolevulinic acid (5-ALA) to female SD rats during gestation and lactation can affect the iron status of weaned rats and provide new ideas for the iron supplementation of piglets. A total of 27 pregnant SD rats were randomly assigned to three treatments in nine replicates, with one rat per litter. Dietary treatments were basal diet (CON), CON + 50 mg/kg 5-ALA (5-ALA50), and CON + 100 mg/kg 5-ALA (5-ALA100). After parturition, ten pups in each litter (a total of 270) were selected for continued feeding by their corresponding mother, and the pregnant rats were fed diets containing 5-ALA (0, 50 and 100 mg/kg diet) until the newborn pups were weaned at 21 days. The results showed that the number of red blood cells (RBCs) in weaned rats in the 5-ALA100 group was significantly higher (p < 0.05) than that in the CON or 5-ALA50 group. The diet with 5-ALA significantly increased (p < 0.05) the hemoglobin (HGB) concentration, hematocrit (HCT) level, serum iron (SI) content, and transferrin saturation (TSAT) level in the blood of weaned rats, as well as the concentration of Hepcidin in the liver and serum of weaned rats and the expression of Hepcidin mRNA in the liver of weaned rats, with the 5-ALA100 group having the highest (p < 0.05) HGB concentration in the weaned rats, and the 5-ALA50 group having the highest (p < 0.05) Hepcidin concentration in serum and in the expression of Hepcidin mRNA in the liver of weaned rats. The other indicators between the 5-ALA groups had no effects. However, the level of total iron binding capacity (TIBC) was significantly decreased (p < 0.05) in the 5-ALA50 group. Moreover, the iron content in the liver of weaned rats fed with 5-ALA showed an upward trend (p = 0.085). In addition, feeding a 5-ALA-supplemented diet could also significantly reduce (p < 0.05) the expression of TfR1 mRNA in the liver of weaning rats (p < 0.05), and the expression of Tfr1 was not affected between 5-ALA groups. In conclusion, dietary supplementation with 5-ALA could improve the blood parameters, increase the concentration of Hepcidin in the liver and serum, and affect the expression of iron-related genes in the liver of weaned rats. Moreover, it is appropriate to add 50 mg/kg 5-ALA to the diet under this condition.

Identification and functional prediction of long noncoding RNAs related to intramuscular fat content in Laiwu pigs.

OBJECTIVE: Intramuscular fat (IMF) is a critical economic indicator of pork quality. Studies on IMF among different pig breeds have been performed via high-throughput sequencing, but comparisons within the same pig breed remain unreported. METHODS: This study was performed to explore the gene profile and identify candidate long noncoding RNA (lncRNAs) and mRNAs associated with IMF deposition among Laiwu pigs with different IMF contents. Based on the longissimus dorsi muscle IMF content, eight pigs from the same breed and management were selected and divided into two groups: a high IMF (>12%, H) and low IMF group (<5%, L). Whole-transcriptome sequencing was performed to explore the differentially expressed (DE) genes between these two groups. RESULTS: The IMF content varied greatly among Laiwu pig individuals (2.17% to 13.93%). Seventeen DE lncRNAs (11 upregulated and 6 downregulated) and 180 mRNAs (112 upregulated and 68 downregulated) were found. Gene Ontology analysis indicated that the following biological processes played an important role in IMF deposition: fatty acid and lipid biosynthetic processes; the extracellular signal-regulated kinase cascade; and white fat cell differentiation. In addition, the peroxisome proliferator-activated receptor, phosphatidylinositol-3-kinase-protein kinase B, and mammalian target of rapamycin pathways were enriched in the pathway analysis. Intersection analysis of the target genes of DE lncRNAs and mRNAs revealed seven candidate genes associated with IMF accumulation. Five DE lncRNAs and 20 DE mRNAs based on the pig quantitative trait locus database were identified and shown to be related to fat deposition. The expression of five DE lncRNAs and mRNAs was verified by quantitative real time polymerase chain reaction (qRT-PCR). The results of qRT-PCR and RNA-sequencing were consistent. CONCLUSION: These results demonstrated that the different IMF contents among pig individuals may be due to the DE lncRNAs and mRNAs associated with lipid droplets and fat deposition.

miR-34a Regulates Lipid Droplet Deposition in 3T3-L1 and C2C12 Cells by Targeting LEF1.

Intramuscular fat (IMF) content plays a key role in improving the flavor and palatability of pork. The IMF content varies between species, breeds, and individuals of the same breed. Hence, it is necessary to elucidate the mechanisms of IMF deposition to improve pork quality. Herein, the IMF content in the longissimus dorsi muscles of 29 Laiwu pigs was detected and divided into two groups, the H group (IMF > 12%) and the L group (IMF < 5%). RNA sequencing analysis showed 24 differentially expressed (DE) miRNA, and GO and KEGG analysis demonstrated that the DE miRNAs were significantly enriched in lipid metabolic process, lipid storage, Wnt, mTOR, and PPAR signaling pathways. miR-34a was found to be increased in the H group and 3T3-L1-derived adipocytes, while Lef1 was decreased. Luciferase reporter assays demonstrated that Lef1 was a potential target of miR-34a. Mechanism analysis revealed that miR-34a could increase lipid droplet deposition in 3T3-L1 and C2C12 cells by dampening the suppressive function of Lef1 on the transcription of adipogenic markers (i.e., Pparg, Cebpa, Fabp4, and Plin1). Moreover, overexpression of miR-34a could enhance the lipid deposition in the co-culture system of 3T3-L1 and C2C12 cells as well as in C2C12 cells cultured with conditioned medium from the progress of adipocyte differentiation. Taken together, our study indicated that miR-34a was an important positive modulator in the regulation of fatty metabolism and fat deposition by inhibiting the suppressive function of Lef1. These results might provide insight for the exploration of potential strategies to promote intramuscular fat deposition in livestock.

A Novel Regulatory Player in the Innate Immune System: Long Non-Coding RNAs.

Long non-coding RNAs (lncRNAs) represent crucial transcriptional and post-transcriptional gene regulators during antimicrobial responses in the host innate immune system. Studies have shown that lncRNAs are expressed in a highly tissue- and cell-specific- manner and are involved in the differentiation and function of innate immune cells, as well as inflammatory and antiviral processes, through versatile molecular mechanisms. These lncRNAs function via the interactions with DNA, RNA, or protein in either cis or trans pattern, relying on their specific sequences or their transcriptions and processing. The dysregulation of lncRNA function is associated with various human non-infectious diseases, such as inflammatory bowel disease, cardiovascular diseases, and diabetes mellitus. Here, we provide an overview of the regulation and mechanisms of lncRNA function in the development and differentiation of innate immune cells, and during the activation or repression of innate immune responses. These elucidations might be beneficial for the development of therapeutic strategies targeting inflammatory and innate immune-mediated diseases.

The role of long noncoding RNAs in livestock adipose tissue deposition - A review.

With the development of sequencing technology, numerous , long noncoding RNAs (lncRNAs) have been discovered and annotated. Increasing evidence has shown that lncRNAs play an essential role in regulating many biological and pathological processes, especially in cancer. However, there have been few studies on the roles of lncRNAs in livestock production. In animal products, meat quality and lean percentage are vital economic traits closely related to adipose tissue deposition. However, adipose tissue accumulation is also a pivotal contributor to obesity, diabetes, atherosclerosis, and many other diseases, as demonstrated by human studies. In livestock production, the mechanism by which lncRNAs regulate adipose tissue deposition is still unclear. In addition, the phenomenon that different animal species have different adipose tissue accumulation abilities is not well understood. In this review, we summarize the characteristics of lncRNAs and their four functional archetypes and review the current knowledge about lncRNA functions in adipose tissue deposition in livestock species. This review could provide theoretical significance to explore the functional mechanisms of lncRNAs in adipose tissue accumulation in animals.

Changes of gut microbiota in pregnant sows induced by 5-Aminolevulinic acid.

Sow health is related to farm productivity and sustainability, but the increased resistance of bacteria to antibiotics in the pig industry has led to a decline in resistance to disease and environmental pollution. 5-Aminolevulinic acid (5-ALA) is considered a feed additive to replace antibiotics, but the effect of 5-ALA on gut microbiota has not been studied. In this study, we fed 12 healthy Landrace × Large White two-line hybrid sows with different concentrations of 5-ALA; blood and fecal samples were obtained after 110 days of pregnancy, and 16S rRNA amplicon sequencing was performed. The alpha diversity of the gut microbiota in sows was not significant among the sows fed different concentrations of 5-ALA. PCoA revealed a significant (P < 0.05) difference in the gut microbiota composition with different 5-ALA groups. LEfSe revealed that 5-ALA increased relative abundance of Streptococcus, while Myroides was enriched in CK group. Functional prediction of Tax4Fun showed that different concentrations of 5-ALA significantly (P < 0.05) increased the mean relative abundance of KEGG pathways involved in core microbiota cellular processes, environmental information processing, and genetic information processing. In summary, 5-ALA changed bacterial community composition of gut microbiota, reduced colonization of some pathogenes and increased the relative abundance of some probiotics. These results provide a theoretical basis for the healthy breeding of pigs.

A polysaccharide extracted from alfalfa activates splenic B cells by TLR4 and acts primarily via the MAPK/p38 pathway.

Alfalfa polysaccharide (APS) has been proposed to exhibit growth-promoting and immune-enhancing bodily functions in vivo. However, little is known about its downstream immunomodulatory and intrinsic molecular mechanisms. Herein, mouse splenic lymphocytes were isolated to characterize the immunomodulatory effects and molecular mechanisms of APS in vitro. The results demonstrated that APS selectively improved the cell viability and IgM production of B cells, but no effects on T cell viability or secretion of IL-2, IL-4 and IFN-γ were observed in vitro. The receptor blocking assay showed that TLR4 was the primary receptor involved in APS-mediated B cell activation, which was confirmed by the results obtained using C57BL/10ScNJ (TLR4 gene-deficient) mice. Moreover, APS activated the TLR4-MyD88 signaling pathway at the translational level by significantly increasing the protein expression of TLR4 and MyD88. Downstream pathway blocking assay demonstrated that both the MAPK and NF-κB pathways were involved in APS-induced B cell activation. Additionally, APS significantly enhanced the phosphorylation of p38, ERK, and JNK and activated the nuclear translocation of the NF-κB p65 subunit. Therefore, we concluded that APS specifically activates the immune functions of splenic B cells by TLR4, acting through the MAPK and NF-κB signaling pathways, and potently activates the p38 pathway.

Effects of Different Selenium Sources on Meat Quality and Shelf Life of Fattening Pigs.

The aim of this study was to evaluate the effects of different Se sources on the meat quality and shelf life of fattening pigs. The control diet was supplemented with 0.3 mg/kg of Se from sodium selenite (SS), and experimental diets included 0.3, 0.3 and 0.15 + 0.15 mg/kg of Se from Se-enriched yeast (SY), selenomethionine (Se-Met) and SS + Se-Met, respectively. The results showed that using organic Se or Se + Se-Met in fattening pigs' diet could increase average daily gain (ADG) (p < 0.05), decrease F/G (p < 0.05), reduce (p < 0.01) moisture, drip loss and cooking loss of longissimus thoracis, as well as increase (p < 0.05) protein and fat contents of longissimus thoracis. Diet supplementation with SY or Se + Se-Met could increase (p < 0.01) back fat thickness and skin thickness, and SY could increase (p < 0.01) belly fat rat. Adding SY or Se + Se-Met could reduce (p < 0.01) L value (45 min, 24 h). Adding Se-Met could decrease (p < 0.01) b value (45 min, 24 h), adding Se + Se-Met could reduce b value (45 min), and adding SY could reduce the b value (24 h). However, there were no (p < 0.05) significant effects on dressing percentage, carcass sloping length, eye muscle area, pH, a value (45 min) and a value (24 h) of longissimus thoracis. Moreover, the TVB-N contents of longissimus thoracis on the first and fifth days, the numbers of Lactobacillus on the third to seventh days and the numbers of E. coli in in the fifth to seventh days of longissimus thoracis were reduced (p < 0.01) by diet supplementation with organic Se. In conclusion, all the results indicate that replacing inorganic Se in diet with organic Se could improve meat quality of fattening pigs. In addition, organic Se could reduce the total volatile basic nitrogen (TVB-N) contents of longissimus thoracis and reduce the numbers of E. coli and Lactobacillus in longissimus thoracis, prolonging the shelf life of pork. These results demonstrated that organic Se supplementation was more effective than SS supplementation for meat quality and the shelf life of fattening pigs.

The Effects of Partially or Completely Substituted Dietary Zinc Sulfate by Lower Levels of Zinc Methionine on Growth Performance, Apparent Total Tract Digestibility, Immune Function, and Visceral Indices in Weaned Piglets.

The study aimed to evaluate the effects of replacing zinc sulfate (ZnSO4) with a lower level of zinc methionine (ZnMet) on the growth performance, apparent total tract digestibility (ATTD) of nutrients, serum metabolites and immune functions of weaned piglets. Thirty-five weaned Duroc × Landrace × Large White male piglets (10.69 ± 0.26 kg) were randomly allotted to five diets. The control diet was supplemented with 100 mg/kg of Zn from ZnSO4, and experimental diets included 75 + 12.5, 50 + 25, 25 + 37.5, and 0 + 50 mg/kg of Zn from ZnSO4 and ZnMet, respectively. The results showed that no differences were observed in growth performance, ATTD of nutrients and serum metabolites among treatments, while serum white blood cell count, lymphocyte count, IgM contents and spleen index were higher (p < 0.01) in piglets fed with 50 + 25 mg/kg of Zn. Zinc digestibility (p < 0.05), IgA content (p < 0.001) and thymus index (p < 0.05) were increased when at least 50% of ZnSO4 was replaced by ZnMet. All the results indicated that using a lower level of ZnMet in weaned piglet's diet instead of ZnSO4 had no adverse impacts on ATTD of nutrients and serum metabolites; and a 50 + 25 mg/kg of Zn (from ZnSO4 and ZnMet, respectively) diet showed the best advantages for parameters relating to immune functions.

Immunomodulatory, antioxidant and intestinal morphology-regulating activities of alfalfa polysaccharides in mice.

The effects of alfalfa polysaccharides (APS) on immunomodulatory and antioxidant functions, as well as intestinal morphology were investigated in vivo in this study. Sixty-four mice were randomly divided into four groups and administered 0, 200, 400 or 800 mg/kg/d body weight APS via gavage for 28 days. The blood parameters and metabolites, viscera indices, antioxidant enzyme activities and intestinal morphology were measured. The results showed that the oral administration of APS improved the immune functions of mice, significantly enhanced the white blood cells and lymphocyte counts, and led to improvements in spleen and thymus indices. APS exhibited significant antioxidant activity by enhancing total antioxidant capacity, superoxide dismutase and glutathione peroxidase activities in heart, kidney and liver, and decreasing the malondialdehyde levels of heart and liver. Moreover, administration of APS potently enhanced the small intestinal villous height and the villus-to-crypt ratio, and decreased the crypt depth of duodenum in mice. Therefore, we can conclude that APS possesses pronounced immunomodulatory activities, and plays an important role in the prevention of oxidative stresses and in the improvement of intestinal morphology in the immunological system in vivo. APS thus shows potential for the development as an effective natural immunomodulatory and antioxidant agent.

Alfalfa polysaccharide prevents H2O2-induced oxidative damage in MEFs by activating MAPK/Nrf2 signaling pathways and suppressing NF-κB signaling pathways.

Alfalfa polysaccharide (APS) is a bioactive component extracted from alfalfa that exhibits potent antioxidant properties. However, the cellular and molecular mechanisms underlying these properties remain unclear. To explore the molecular mechanism by which APS exerts antioxidant effects, an H2O2-induced oxidative stress mouse embryonic fibroblast (MEF) model was established. Cell proliferation, antioxidant enzyme activity, immune cytokine expression, and related protein expression were examined in APS-supplemented or non-supplemented conditions. The results suggested that APS strengthened the antioxidative capacity of MEFs, increasing cell proliferation, superoxide dismutase activity (SOD), and the total antioxidant capacity (T-AOC). In addition, APS reduced the secretion of interleukin (IL)-6 and IL-8 as well as expression of the proinflammatory gene retinoic acid-inducible gene I (RIG-I). APS was also able to activate the mitogen-activated protein kinase (MAPK) pathway, which promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus. However, expression of nuclear factor-κB (NF-κB) was decreased after APS treatment. Overall, these results suggest that APS relieves H2O2-induced oxidative stress in MEFs by activating MAPK/Nrf2 signaling and suppressing NF-κB signaling. To the best of our knowledge, this is the first study to link APS with MAPK/Nrf2, NF-κB and RIG-I, thus providing new perspectives regarding the mechanisms of the antioxidant activity of APS.

Polysaccharide from alfalfa activates RAW 264.7 macrophages through MAPK and NF-κB signaling pathways.

Alfalfa polysaccharide (APS), a bioactive compound extracted from alfalfa, has been proposed to exhibit potential growth-promoting and immune-enhancing functions. But, little is known about the cellular immunomodulatory and intrinsic molecular mechanisms. Here we extracted the APS, and performed in vitro experiments to characterize the immunomodulatory functions as well as the molecular mechanisms of APS on RAW 264.7 macrophages cells. Chemical analyses showed that APS was mainly composed of fucose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid. The results of in vitro assays demonstrated that 50 and 100 µg/mL APS increased the cell viability of RAW 264.7 cells. The secretion and gene expression of NO/iNOS, IL-6 and TNF-α in APS-induced macrophage cell were significantly enhanced. However, APS-induced TNF-α production was decreased by blocking the MAPK or NF-κB signaling pathways, especially for the blockade of p38. Moreover, APS enhanced the phosphorylation of p38, ERK, and JNK, promoted the degradation of IκBα, and increased the nuclear translocation of NF-κB p65 subunit. Therefore, we demonstrated that APS could improve the immune functions of RAW 264.7 macrophages cells by promoting the cell viability and increasing secretion and gene expressions of NO/iNOS, IL-6 and TNF-α through the MAPK and NF-κB signaling pathways.

Legume-Cereal Intercropping Improves Forage Yield, Quality and Degradability.

Intercropping legume with cereal is an extensively applied planting pattern in crop cultivation. However, forage potential and the degradability of harvested mixtures from intercropping system remain unclear. To investigate the feasibility of applying an intercropping system as a forage supply source to ruminants, two consecutive experiments (experiments 1 and 2) involving a field cultivation trial and a subsequent in vivo degradable experiment were conducted to determine the forage production performance and the ruminally degradable characteristics of a harvested mixture from an alfalfa/corn-rye intercropping system. In experiment 1, the intercropping system was established by alternating alfalfa and corn or rye with a row ratio of 5:2. Dry matter (DM) and nutrient yields were determined. In experiment 2, forages harvested from the different treatments were used as feedstuff to identify nutrient degradation kinetics and distribution of components between the rapidly degradable (a), potentially degradable (b) and the degradation rate constant (c) of 'b' fraction by in sacco method in Small-Tail Han wether Sheep. The intercropping system of alfalfa and corn-rye provided higher forage production performance with net increases of 9.52% and 34.81% in DM yield, 42.13% and 16.74% in crude protein (CP) yield, 25.94% and 69.99% in degradable DM yield, and 16.96% and 5.50% in degradable CP yield than rotation and alfalfa sole cropping systems, respectively. In addition, the harvest mixture from intercropping system also had greater 'a' fraction, 'b' fraction, 'c' values, and effective degradability (E value) of DM and CP than corn or rye hay harvested from rotation system. After 48-h exposure to rumen microbes, intercropping harvest materials were degraded to a higher extent than separately degraded crop stems from the sole system as indicated by visual microscopic examination with more tissues disappeared. Thus, the intercropping of alfalfa and corn-rye exhibited a greater forage production potential, and could be applied as forage supply source for ruminants. The improved effective degradability of harvest mixture material could be attributed to greater degradable components involving the rapidly degradable fractions (a), potentially degradable (b) fractions, and degradable rate constant