[Effect of dihydroartemisinin supplementation on inflammation and lipid metabolism induced by lipopolysaccharide in liver of weaned piglets].

To study the effect of dihydroartemisinin(DHA) on hepatic inflammation and lipid metabolism in weaned piglets, a liver injury model of weaned piglets was established by lipopolysaccharide(LPS)-induced method. In this study, 30 healthy weaned piglets were selected and randomly divided into control group(CON), model group(LPS) and treatment group(LD, LPS+DHA), with 10 in each group. The CON group and the LPS group were fed with a basal diet, and the LD group was fed with a basal diet+80 mg·kg~(-1) DHA. The test period was 21 days. The LPS group and the LD group were intraperitoneally injected with 100 µg·kg~(-1) LPS at 4 hours before slaughter, and the CON group was injected with the same dose of sterile physiological saline. The results showed that compared with the CON group, contents of TC, AST activity and AST/ALT ratio were significantly increased in the serum of LPS piglets(P<0.05), content of HDL-c was significantly decreased(P<0.05). In addition, in the liver, the levels of TG, NEFA, IL-1ß, IL-6 and TNF-α were increased significantly(P<0.05), and activities of LPL, HL and TL were decreased significantly(P<0.05). Compared with LPS group, content of TC, activities of AST and ALT and the AST/ALT ratio were decreased significantly(P<0.05), and HDL-c content increased significantly in the serum of LD piglets(P<0.05). The contents of TG, NEFA, IL-1ß, IL-6 and TNF-α and activity of FAS in the liver were decreased significantly(P<0.05), and the activities of LPL, HL and TL were increased significantly(P<0.05). Compared with the CON group, the mRNA expressions of IL-1ß, IL-6, TNF-α, ACCß and SREBP-1 c in the LPS group were significantly increased(P<0.05), the mRNA expressions of AMPKα, SIRT1, CPT-1 and SCD were decreased significantly(P<0.05). The above indicators were improved in the LD group compared with the LPS group. These results indicated that DHA had a certain effect in recovering LPS-induced liver inflammation and abnormal lipid metabolism.

Curcumin attenuates insulin resistance and hepatic lipid accumulation in a rat model of intra-uterine growth restriction through insulin signalling pathway and sterol regulatory element binding proteins.

The objective of the present study was to investigate the effect of curcumin on insulin resistance (IR) and hepatic lipid accumulation in intra-uterine growth restriction (IUGR). Rats with a normal birth weight (NBW) or IUGR were fed basic diets (NBW and IUGR groups) or basic diets supplemented with curcumin (NBW-C and IUGR-C groups) from 6 to 12 weeks. Rats in the IUGR group showed higher levels of glucose and homeostasis model assessment for insulin resistance index (HOMA-IR) (P < 0·05) than in the NBW group. The livers of IUGR rats exhibited higher (P < 0·05) concentration of TAG and lower (P < 0·05) activities of lipolysis enzymes compared with the normal rats. In response to dietary curcumin supplementation, concentrations of serum insulin, glucose and HOMA-IR, pyruvate, TAG, total cholesterol and NEFA in the liver were decreased (P < 0·05). The concentrations of glycogen and activities of lipolysis enzymes in the liver were increased (P < 0·05) in the IUGR-C group compared with the IUGR group. These results were associated with lower (P < 0·05) phosphorylated insulin receptor substrate 1, protein kinase B or Akt, glycogen synthase kinase 3ß and expressions of sterol regulatory element binding protein 1 and fatty acid synthase (FASN); decreased expressions for Cd36, sterol regulatory element binding protein 1c (Srebf1) and Fasn; increased (P < 0·05) expression of PPARα; and expressions for Ppara and hormone-sensitive lipase in the liver of IUGR-C rats than the IUGR rats. Maternal malnutrition caused IR and lipid accumulation in the liver. Curcumin supplementation prevented IR by regulating insulin signalling pathways and attenuated hepatic lipid accumulation.

Dietary curcumin supplementation attenuates inflammation, hepatic injury and oxidative damage in a rat model of intra-uterine growth retardation.

Rats with a normal birth weight (NBW) or intra-uterine growth retardation (IUGR) were fed basic diets (NBW and IUGR groups) or basic diets supplemented with curcumin (NC and IC groups) from 6 to 12 weeks. The body weight of IUGR rats was lower (P<0·05) than that of the controls. Rats with IUGR showed higher (P<0·05) concentrations of TNF-α, IL-1ß and IL-6; higher (P<0·05) activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in their serum; and increased (P<0·05) concentrations of malondialdehyde (MDA), protein carbonyl (PC) and 8-hydroxy-2'-deoxyguanosine (8-OHDG) in the liver compared with the NBW rats. The livers of IUGR rats exhibited a lower (P<0·05) superoxide dismutase activity and decreased (P<0·05) metabolic efficiency of the hepatic glutathione redox cycle compared with those of the NBW rats. In response to dietary curcumin supplementation, concentrations of inflammatory cytokines and activities of AST and ALT in the serum and MDA, PC and 8-OHDG in the liver were lower (P<0·05), and the hepatic glutathione redox cycle in the liver was improved (P<0·05) in the IC group than in the IUGR group. These results were associated with lower (P<0·05) phosphorylated levels of the NF-κB pathway and Janus kinase 2 (JAK2) and higher (P<0·05) mRNA expression of genes involved in the nuclear factor, erythroid 2-like 2 (Nfe2l2)/antioxidant response element (ARE) pathway in the liver of the IC rats than that of the IUGR rats. Maternal undernutrition decreased birth weight and led to inflammation, oxidative damage and injury in rats. Curcumin appeared to be beneficial in preventing IUGR-induced inflammation, oxidative damage and injury by activating the expression of the NF-κB, JAK/STAT and Nfe2l2/ARE pathways in the liver.

Intrauterine growth retardation promotes fetal intestinal autophagy in rats via the mechanistic target of rapamycin pathway.

Intrauterine growth retardation (IUGR) impairs fetal intestinal development, and is associated with high perinatal morbidity and mortality. However, the mechanism underlying this intestinal injury is largely unknown. We aimed to investigate this mechanism through analysis of intestinal autophagy and related signaling pathways in a rat model of IUGR. Normal weight (NW) and IUGR fetuses were obtained from primiparous rats via ad libitum food intake and 50% food restriction, respectively. Maternal serum parameters, fetal body weight, organ weights, and fetal blood glucose were determined. Intestinal apoptosis, autophagy, and the mechanistic target of rapamycin (mTOR) signaling pathway were analyzed. The results indicated that maternal 50% food restriction reduced maternal serum glucose, bilirubin, and total cholesterol and produced IUGR fetuses, which had decreased body weight; blood glucose; and weights of the small intestine, stomach, spleen, pancreas, and kidney. Decreased Bcl-2 and increased Casp9 mRNA expression was observed in IUGR fetal intestines. Analysis of intestinal autophagy showed that the mRNA expression of WIPI1, MAP1LC3B, Atg5, and Atg14 was also increased, while the protein levels of p62 were decreased in IUGR fetuses. Compared to NW fetuses, IUGR fetuses showed decreased mTOR protein levels and enhanced mRNA expression of ULK1 and Beclin1 in the small intestine. In summary, the results indicated that maternal 50% food restriction on gestational days 10-21 reduced maternal serum glucose, bilirubin, and total cholesterol contents, and produced IUGR fetuses that had low blood glucose and reduced small intestine weight. Intestinal injury of IUGR fetuses caused by maternal food restriction might be due to enhanced apoptosis and autophagy via the mTOR signaling pathway.

Dietary enzymatically treated Artemisia annua L. supplementation alleviates liver oxidative injury of broilers reared under high ambient temperature.

Heat stress induced by high ambient temperature is a major concern in commercial broiler production. To evaluate the effects of dietary enzymatically treated Artemisia annua L. (EA) supplementation on growth performance and liver oxidative injury of broilers reared under heat stress, a total of 320 22-day-old male broilers were randomly allotted into five groups with eight replicates of eight birds each. Broilers in the control group were housed at 22 ± 1 °C and fed the basal diet. Broilers in the HS, HS-EA1, HS-EA2, and HS-EA3 groups were fed basal diet supplemented with 0, 0.75, 1.00, and 1.25 g/kg EA, respectively, and reared under cyclic high temperature (34 ± 1 °C for 8 h/day and 22 ± 1 °C for 16 h/day). Broilers fed EA diets had higher final body weight, average daily body weight gain, and average daily feed intake, as well as liver concentration of reduced glutathione, activities of antioxidant enzymes, abilities to inhibit hydroxyl radical and superoxide radical (HS-EA2 and HS-EA3), and lower liver concentrations of reactive oxygen metabolites, malondialdehyde, and protein carbonyl (HS-EA1, HS-EA2, and HS-EA3) than HS group (P < 0.05). EA treatment downregulated the mRNA levels of heat shock proteins 70 and 90, upregulated the mRNA levels of nuclear factor erythroid 2-related factor 2 (HS-EA1, HS-EA2, and HS-EA3) and heme oxygenase 1 (HS-EA2 and HS-EA3) in liver of heat-treated broilers (P < 0.05). In conclusion, EA alleviated heat stress-induced growth depression and liver oxidative injury in broilers, possibly through improving the antioxidant capacity and regulating the pertinent mRNA expression. The appropriate inclusion level of EA in broiler diet is 1.00-1.25 g/kg.

Protective effects of leucine on redox status and mitochondrial-related gene abundance in the jejunum of intrauterine growth-retarded piglets during early weaning period.

The aim of this study was to investigate the effects of dietary supplementation with 0.35% l-leucine on redox status and gene abundance relating to mitochondrial biogenesis and function in the jejunum of intrauterine growth-retarded (IUGR) piglets during early weaning period. According to a 2 × 2 factorial arrangement, 16 IUGR and 16 normal body weight (NBW) piglets were fed a basal diet without l-leucine supplementation or a basal diet plus 0.35% l-leucine supplementation from the age of 14 to 35 d. The results showed that compared with NBW piglets, IUGR piglets had a lower (p < 0.05) jejunal DNA concentration, a reduced (p < 0.05) manganese superoxide dismutase (MnSOD) and total antioxidant capability (T-AOC) activities and mitochondrial DNA content in the jejunum. Leucine supplementation increased (p < 0.05) MnSOD and T-AOC activities and decreased (p < 0.05) the malondialdehyde content in the jejunum of IUGR piglets. The mRNA gene abundance of nuclear respiratory factor-1 (NRF1), mitochondrial transcription factor A (TFAM), ATP synthase (ATPs), cytochrome c oxidase V (CcOX V), cytochrome c and glucokinase in the jejunum of IUGR piglets was reduced (p < 0.05) compared with NBW piglets. However, NRF1, peroxisome proliferation-activated receptor gamma coactivator-1 alpha, TFAM, ATPs and CcOX I mRNA gene abundance in the jejunum of IUGR piglets were increased (p < 0.05) by diets supplemented with leucine. These data indicate that leucine supplementation has therapeutic potential for attenuating intestinal oxidative stress and mitochondrial dysfunction in IUGR piglets during the early period of life via increasing enzyme activities and up-regulating mRNA gene abundance.

Formulation and evaluation of poly(lactic-co-glycolic acid) microspheres loaded with an altered collagen type II peptide for the treatment of rheumatoid arthritis.

The aim of this research was to evaluate the potential of water-in-oil-in-water (w/o/w) and solid-in-oil-in-water (s/o/w) emulsification techniques to prepare the altered collagen type II peptide AP268-270 (ACTP)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to make ACTP more convenient as an rheumatoid arthritis treatment. Microspheres produced by the s/o/w method had higher drug encapsulation efficiency (69.7-79.8%) than those prepared by the w/o/w method (21.8-39.3%). In vitro drug release was influenced by the microencapsulation technique, molecular weight, and composition of the polymer. After intramuscular injection of the optimal formulation to Lewis rats, the concentration of ACTP peptide in serum reached its maximum level on day 3 and then remained nearly stable for approximately 4 weeks. In a collagen-induced arthritis rat model, a single intramuscular injection of ACTP-loaded PLGA microspheres had comparable efficacy to the intravenous injection of ACTP peptide solution once every other day.

Effect of site-specific PEGylation on the fibrinolytic activity, immunogenicity, and pharmacokinetics of staphylokinase.

The bacterial plasminogen-activator staphylokinase (Sak) is a promising thrombolytic agent for treating acute myocardial infarction. To effectively reduce the immunogenicity of Sak while maintaining its fibrinolytic activity, site-specific PEGylation was performed in the present study. The chemoselective cysteine PEGylation site was selected within an immunodominant region (amino acid residues 71-87) using an in silico approach. The PEGylated Sak variants prepared in this study showed a purity of >97.0%. PEGylation at Position 80 resulted in a Sak variant Sak(E80C-PEG) which has similar fibrinolytic activity and thermostability compared with the native recombinant staphylokinase (r-Sak). The immunogenicity of Sak(E80C-PEG) in guinea pigs was greatly reduced compared with the native r-Sak. Furthermore, preliminary pharmacokinetic results suggested that the plasma clearance of Sak(E80C-PEG) from the blood stream of rabbit was significantly decreased compared with that of r-Sak, resulting in a 2.8-fold increase of initial half-life and a 3.8-fold increase of systemic availability. In summary, these results demonstrated that site-specific PEGylation yielded a novel Sak variant Sak(E80C-PEG) with remarkable advantages over the unmodified Sak.

Dietary supplementation with dihydroartemisinin improves intestinal barrier function in weaned piglets with intrauterine growth retardation by modulating the gut microbiota.

The aim of this study was to investigate whether dietary dihydroartemisinin (DHA) supplementation could improve intestinal barrier function and microbiota composition in intrauterine growth restriction (IUGR) weaned piglets. Twelve normal birth weight (NBW) piglets and 24 IUGR piglets at 21 d of age were divided into three groups, which were fed a basal diet (NBW-CON and IUCR-CON groups) and an 80 mg/kg DHA diet (IUGR-DHA group). At 49 d of age, eight piglets of each group with similar body weights within groups were slaughtered, and serum and small intestine samples were collected. The results showed that IUGR piglets reduced growth performance, impaired the markers of intestinal permeability, induced intestinal inflammation, decreased intestinal immunity, and disturbed the intestinal microflora. Dietary DHA supplementation increased average daily gain, average daily feed intake, and body weight at 49 d of age in IUGR-weaned piglets (P < 0.05). DHA treatment decreased serum diamine oxidase activity and increased the numbers of intestinal goblet cells and intraepithelial lymphocytes, concentrations of jejunal mucin-2 and ileal trefoil factor 3, and intestinal secretory immunoglobin A and immunoglobin G (IgG) concentrations of IUGR piglets (P < 0.05). Diet supplemented with DHA also upregulated mRNA abundances of jejunal IgG, the cluster of differentiation 8 (CD8), major histocompatibility complex-I (MHC-I), and interleukin 6 (IL-6) and ileal IgG, Fc receptor for IgG (FcRn), cluster of differentiation 8 (CD4), CD8, MHC-I, IL-6 and tumor necrosis factor α (TNF-α), and enhanced mRNA abundance and protein expression of intestinal occludin and ileal claudin-1 in IUGR piglets (P < 0.05). In addition, DHA supplementation in the diet improved the microbial diversity of the small intestine of IUGR piglets and significantly increased the relative abundance of Actinobacteriota, Streptococcus, Blautia and Streptococcus in the jejunum, and Clostridium sensu_ stricto_in the ileum (P < 0.05). The intestinal microbiota was correlated with the mRNA abundance of tight junction proteins and inflammatory response-related genes. These data suggested that DHA could improve the markers of intestinal barrier function in IUGR-weaned piglets by modulating gut microbiota. DHA may be a novel nutritional candidate for preventing intestinal dysfunction in IUGR pigs.

Intrauterine growth retardation (IUGR) is defined as the restricted development of the mammalian fetus or its organs during pregnancy, which has high morbidity and mortality during the perinatal period and improves the risk of metabolic diseases in the long term. Dihydroartemisinin (DHA) is a derivative of artemisinin that possesses anti-inflammatory and immunoregulatory effects. Therefore, this experiment was conducted to investigate whether dietary DHA supplementation could improve the intestinal barrier function and microbiota composition in IUGR-weaned piglets. The result showed that IUGR could lead to intestinal barrier dysfunction. Dietary supplementation with DHA improved growth performance and attenuated intestinal barrier dysfunction by decreasing the markers of intestinal permeability, increasing the mucus layer barrier, enhancing immunity, and reducing the inflammatory response in IUGR piglets, which may be attributed to the improvement of the intestinal microbiota. Moreover, the study indicated that the gut microflora was correlated with the gene expression of tight junction proteins and immune function. This study may provide a new nutritional strategy for the maintenance of intestinal health in IUGR pigs.

Pharmacokinetic and pharmacodynamic profiles of recombinant human erythropoietin-loaded poly(lactic-co-glycolic acid) microspheres in rats.

AIM: To characterize the pharmacokinetic and pharmacodynamic profiles of the recombinant human erythropoietin (rhEPO)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres in rats. METHODS: The rhEPO-loaded microspheres were prepared using a solid-in-oil-in-water emulsion method. Pharmacokinetics and pharmacodynamics of the rhEPO-loaded microspheres were evaluated in male Sprague-Dawley rats. The serum rhEPO level was determined with ELISA. The level of anti-rhEPO antibody in the serum was measured to assess the immunogenicity of rhEPO released from the microspheres. RESULTS: rhEPO was almost completely released from the PLGA microspheres in vitro, following zero-order release kinetics over approximately 30 d. After intramuscular injection (10,000 or 30,000 IU rhEPO/kg) in the rats, the serum rhEPO concentration reached maximum levels on d 1, then decreased gradually and was maintained at nearly steady levels for approximately 4 weeks. Furthermore, the release of rhEPO from the PLGA microspheres was found to be controlled mainly by a dissolution/diffusion mechanism. A good linear correlation (R(2)=0.98) was obtained between the in vitro and in vivo release data. A single intramuscular injection of the rhEPO-loaded PLGA microspheres (10,000 or 30,000 IU rhEPO/kg) in the rats resulted in elevated hemoglobin and red blood cell concentrations for more than 28 d. Moreover, the immunogenicity of rhEPO released from the PLGA microspheres was comparable with that of the unencapsulated rhEPO. CONCLUSION: The results prove the feasibility of using the PLGA-based microspheres to deliver rhEPO for approximately 1 month.

Hsa_circ_0081534 facilitates malignant phenotypes by sequestering miR-874-3p and upregulating FMNL3 in nasopharyngeal carcinoma.

OBJECTIVE: Circular RNAs (circRNAs) are connected to nasopharyngeal carcinoma (NPC) development and progression. CircRNA hsa_circ_0081534 (circ_0081534) has been reported to be associated with NPC progression, but its underlying regulatory mechanisms are largely unknown. Thus, the study aims to investigate the mechanism by which circ_0081534 regulates NPC progression. METHODS: Quantitative reverse transcription polymerase chain reaction was conducted to detect circ_0081534 expression. Loss-of-function assays were conducted to evaluate the role of circ_0081534, including 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU) staining, colony formation, flow cytometry, transwell, western blotting, and xenograft assays. Targeting relationship was identified through dual-luciferase reporter assay and RNA immunoprecipitation. RESULTS: Our data exhibited that circ_0081534 was upregulated in NPC samples and cells. Knockdown of circ_0081534 repressed NPC cell proliferation, migration, invasion, EMT, and triggered NPC cell apoptosis. Also, circ_0081534 silencing decreased NPC cell growth in xenograft models. Circ_0081534 functioned as a miR-874-3p sponge, and downregulation of miR-874-3p alleviated the suppressive effects of circ_0081534 silencing on NPC cell malignant phenotypes. MiR-874-3p targeted FMNL3, and circ_0081534 regulated FMNL3 expression through serving as a miR-874-3p sponge. Upregulation of FMNL3 relieved the inhibitory effects of circ_0081534 downregulation on NPC cell malignant phenotypes. CONCLUSION: circ_0081534 interference repressed NPC progression partly by modulating the miR-874-3p/FMNL3 axis.

Sustained release of low molecular weight heparin from PLGA microspheres prepared by a solid-in-oil-in-water emulsion method.

Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres for the sustained release of low molecular weight heparin (LMWH) were prepared by a soild-in-oil-in-water (s/o/w) emulsion method. Prior to encapsulation, the LMWH micro-particles were fabricated by a modified freezing-induced phase separation method. The micro-particles were subsequently encapsulated into PLGA microspheres. Process optimization revealed that the NaCl concentration in the outer phase of s/o/w emulsion played a critical role in determining the properties of the microspheres. When the NaCl concentration increased from 0% to 5%, the encapsulation efficiency significantly increased from 51.5% to 76.8%. The initial burst release also decreased from 37.3% to 12.4%. In vitro release tests showed that LMWH released from PLGA microspheres in a sustained manner for about 14 days. Single injection of LMWH-loaded PLGA microspheres into rabbits resulted in an elevation of an anti-factor Xa activity for about 6 days. Furthermore, the integrity of the encapsulated LMWH was preserved during encapsulation process.

Simultaneous removal of T and B cell epitope in recombinant staphylokinase by structure-based mutagenesis of immuno-dominant Arg77 and Glu80 residues.

OBJECTIVE: To reduce immunogenicity of recombined staphylokinase (r-Sak) , site-directed mutagenesis of Arg77 and Glu80 residue was performed to simultaneously remove T and B cell epitope in r-Sak molecule. METHODS: The solvent accessible surface areas of residues 77 and 80 in r-Sak were used to analyze rational design of Sak mutation. The Sak mutants were expressed in E coli DH5alpha. After purified by a 3-step chromatography, their fibrinolytic activities and immunological properties were analyzed. RESULTS: Immunogenicity tests suggested that Sak induced a Th2-type immune response. Substitution of Glu80 with alanine or serine successfully reduced its solvent accessible surface area while simultaneously removing part of the T and B cell epitope. Changing Arg77 to glutamine, asparagine, or lysine removed only part of the T cell epitope. Of six dually substituted variants, Sak (R77Q/E80A) and Sak(R77Q/E80S) variants effectively eliminated part of the B and T cell epitopes, which markedly reduced their immunogenicity.

Dietary dihydroartemisinin supplementation alleviates intestinal inflammatory injury through TLR4/NOD/NF-κB signaling pathway in weaned piglets with intrauterine growth retardation.

The aim of present study was to evaluate whether diets supplemented with dihydroartemisinin (DHA) could alleviate intestinal inflammatory injury in weaned piglets with intrauterine growth retardation (IUGR). Twelve normal birth weight (NBW) piglets and 12 piglets with IUGR were fed a basal diet (NBW-CON and IUCR-CON groups), and another 12 piglets with IUGR were fed the basal diet supplemented with DHA at 80 mg/kg (IUGR-DHA group) from 21 to 49 d of age. At 49 d of age, 8 piglets with similar body weight in each group were sacrificed. The jejunal and ileal samples were collected for further analysis. The results showed that IUGR impaired intestinal morphology, increased intestinal inflammatory response, raised enterocyte apoptosis and reduced enterocyte proliferation and activated transmembrane toll-like receptor 4 (TLR4)/nucleotide-binding and oligomerization domain (NOD)/nuclear factor-κB (NF-κB) signaling pathway. Dihydroartemisinin inclusion ameliorated intestinal morphology, indicated by increased villus height, villus height-to-crypt depth ratio, villus surface area and decreased villus width of piglets with IUGR (P < 0.05). Compared with NBW piglets, IUGR piglets supplemented with DHA exhibited higher apoptosis index and caspase-3 expression, and lower proliferation index and proliferating cell nuclear antigen expression in the intestine (P < 0.05). Dihydroartemisinin supplementation attenuated the intestinal inflammation of piglets with IUGR, indicated by increased concentrations of intestinal inflammatory cytokines and lipopolysaccharides (P < 0.05). In addition, DHA supplementation down-regulated the related mRNA expressions of TLR4/NOD/NF-κB signaling pathway and upregulated mRNA expressions of negative regulators of TLR4 and NOD signaling pathway in the intestine of piglets with IUGR (P < 0.05). Piglets in the IUGR-DHA group showed lower protein expressions of TLR4, phosphorylated NF-κB (pNF-κB) inhibitor α, nuclear pNF-κB, and higher protein expression of cytoplasmic pNF-κB in the intestine than those in the IUGR-CON group (P < 0.05). In conclusion, DHA supplementation could improve intestinal morphology, regulate enterocyte proliferation and apoptosis, and alleviate intestinal inflammation through TLR4/NOD/NF-κB signaling pathway in weaned piglets with IUGR.

Simultaneous elimination of T- and B-cell epitope by structure-based mutagenesis of single Glu80 residue within recombinant staphylokinase.

To reduce the immunogenicity of recombinant staphylokinase, structure-based mutagenesis of Glu80 residue in wild-type staphylokinase (wt-Sak) was rationally designed and carried out by a modified QuikChange site-directed mutagenesis. Sak mutants, including Sak(E80A) and Sak(E80S), were successfully expressed in E. coli DH5a as a soluble cytoplasmic proteins and accounted for more than 40% of the total cellular proteins. The expressed proteins were purified by a three-step chromatographic purification process. SDS-PAGE and HPLC analyses results indicated that the purified proteins were almost completely homogeneous and the purities of Sak mutants exceeded 97%. Analysis of fibrinolytic activity revealed that substitution of E80 residue with serine and alanine resulted in slightly increased specific activities of Sak mutants. Investigation of the immunogenicity of Sak mutants showed that the amount of specific anti-Sak IgG antibodies elicited by Sak(E80A) and Sak(E80S) in BALB/c mice decreased approximately 35% and 27%, respectively compared with wt-Sak. The abilities of Sak mutants to stimulate proliferation of T cells from BALB/c mice and to bind mouse anti-Sak polyclonal serum were significantly lower than those of wt-Sak. These results suggested that substitution of Glu80 residue by alanine and serine successfully eliminated part of T- and B-cell epitope of Sak molecule. Our findings suggested that simultaneous elimination of T- and B-cell epitopes was a useful method to reduce the immunogenicity of wt-Sak molecule and provided a strategy for engineering safe Sak-based fibrinolytics for the clinical treatment of acute myocardial infarction.

[Effect of NaCl in outer water phase on the characteristics of BSA-loaded PLGA sustained-release microspheres fabricated by a solid-in-oil-in-water emulsion technique].

The aim of this study is to investigate the critical factor affecting the properties of PLGA microspheres fabricated by a solid-in-oil-in-water (S/O/W) emulsion technique with BSA as a model protein. Prior to encapsulation, the BSA microparticles were fabricated by a modified freezing-induced phase separation method. The microparticles were subsequently encapsulated into PLGA microspheres by S/O/W emulsion method, then Motic BA200 biological microscope, confocal laser scanning microscope, scanning electron microscope were used to observe the structure of S/O/W emulsion and PLGA microspheres. The protein content extracted or released from BSA microspheres was measured by Bradford protein assay method. It was found that NaCl added in the outer aqueous phase effectively suppressed material exchange between the inner and outer phase of S/O/W emulsion. Then, the structure and permeability of obtained microspheres were influenced. As a result, with the increase of NaCl concentration in the outer aqueous phase, the encapsulation efficiency of microspheres significantly increased from 60% to more than 85%, the burst release of microspheres reduced from 70% to 20%, and the particle size decreased from 103 microm to 62 microm. Furthermore, the rehydration of encapsulated protein was also retarded and then integrity of BSA was successfully protected during encapsulation process. In vitro release test showed that BSA released from PLGA microspheres in a sustained manner for more than 30 days.

Dietary Supplemented Curcumin Improves Meat Quality and Antioxidant Status of Intrauterine Growth Retardation Growing Pigs via Nrf2 Signal Pathway.

Intrauterine growth retardation (IUGR) exhibits programming consequences and may induce oxidative stress in growing animals and humans. This study was conducted to investigate the hypothesis that dietary curcumin may protect growing pigs from IUGR-induced oxidative stress via the Nrf2 pathway. Twelve normal birth weight (NBW) and 24 IUGR female piglets were selected and fed control diets supplemented 0 (NBW), 0 (IUGR) and 200 (IUGR + Cur) mg/kg curcumin from 26 to 115 days of age (n = 12). Growth performance, meat quality, redox status and its related Nrf2 pathway were determined. Results showed that IUGR pigs exhibited decreased body weight on 0 d, 26 d and 56 d (p < 0.01) but had no difference on 115 d among NBW, IUGR and IUGR + Cur groups (p > 0.05). Compared with NBW and IUGR groups, a significant decrease in drip loss (24 h and 48 h) was observed in the IUGR + Cur group (p < 0.01). IUGR pigs had higher concentrations of malondialdehyde (MDA) (p < 0.01) and protein carbonyl (PC) (p = 0.03) and lower activities of glutathione peroxidase (p = 0.02), catalase (p < 0.01) and peroxidase (p = 0.02) in leg muscles than NBW pigs. Dietary-added 200 mg/kg curcumin decreased concentrations of MDA and PC and improved the activities of catalase, superoxide dismutase (SOD) and peroxidase as compared to the IUGR group (p < 0.05). Additionally, dietary curcumin enhanced protein (NQO1) and mRNA expression of genes (Nrf2, NQO1, gamma-glutamyltransferase 1 (GGT1), heme oxygenase-1 (HO-1), glutathione S-transferase (GST) and catalase (CAT)) as compared to the IUGR group (p < 0.05). These results suggest that dietary curcumin could serve as a potential additive to enhance redox status and improve meat quality of IUGR growing pigs via the Nrf2 signal pathway.

Effects of dietary dihydroartemisinin supplementation on growth performance, hepatic inflammation, and lipid metabolism in weaned piglets with intrauterine growth retardation.

The aims of this study were to investigate the effects of dietary supplementation with dihydroartemisinin (DHA) on growth performance, hepatic inflammation, and lipid metabolism in intrauterine growth retardation (IUGR)-affected weaned piglets. Eight piglets with normal birth weight (NBW) and 16 IUGR-affected piglets were selected and fed either a basal diet (NBW and IUGR groups) or the basal diet supplemented with 80 mg/kg DHA (IUGR-DHA group) from 21 to 49 day of age. Blood and liver samples were collected on day 49. DHA supplementation significantly alleviated the compromised growth performance and liver damage in IUGR-affected piglets. Additionally, DHA supplementation decreased the activities of alanine aminotransferase and aspartate aminotransferase, as well as the serum levels of non-esterified fatty acids (NEFA), very-low-density lipoprotein, and total cholesterol. In the liver, the concentrations of interleukin 1 beta, interleukin 6, tumor necrosis factor alpha, triglycerides, and NEFA were decreased. Fatty acid synthesis was decreased by DHA supplementation, whereas the activities of lipoprotein lipase, hepatic lipase, and total lipase were increased. Dietary DHA supplementation led to upregulation of the expression of AMPK/SIRT1 signaling pathway-related genes, whereas that of inflammatory factor-related genes were downregulated. In conclusion, dietary inclusion of 80 mg/kg DHA can alleviate IUGR-induced impairments in piglets.

Dietary Dihydroartemisinin Supplementation Attenuates Hepatic Oxidative Damage of Weaned Piglets with Intrauterine Growth Retardation through the Nrf2/ARE Signaling Pathway.

The object of present study was to evaluate the effects of dihydroartemisinin (DHA) supplementation on the hepatic antioxidant capacity in IUGR-affected weaned piglets. Eight piglets with normal birth weight (NBW) and sixteen IUGR-affected piglets were selected. Piglets were weaned at 21 days. NBW and IUGR groups were fed a basal diet and the ID group was fed the basal diet supplemented with 80 mg/kg DHA for 28 days. The result indicated that compared with NBW piglets, IUGR-affected piglets increased (p < 0.05) the concentration of malondialdehyde (MDA) and decreased (p < 0.05) the serum activities of total superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). In addition, IUGR-affected piglets showed increased (p < 0.05) hepatic concentrations of protein carbonyl (PC), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and oxidized glutathione (GSSG), and an increased GSSG:GSH value. IUGR-affected piglets exhibited lower (p < 0.05) activities of GSH-Px, T-SOD, total antioxidant capacity (T-AOC), and the concentration of glutathione (GSH). DHA supplementation decreased (p < 0.05) the serum concentration of MDA and increased the serum activities of T-AOC, T-SOD, GSH-Px, and CAT. The ID group showed decreased (p < 0.05) concentrations of MDA, PC, 8-OHdG, and GSSG, and a decreased GSSG:GSH value in the liver. The hepatic activity of T-SOD and the concentration of GSH were increased (p < 0.05) in the liver of ID group. IUGR-affected piglets downregulated (p < 0.05) mRNA expression of nuclear erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and CAT. DHA supplementation increased (p < 0.05) mRNA expression of Nrf2, HO-1, GPx1, and CAT in the ID group. In addition, the protein expression of Nrf2 was downregulated (p < 0.05) in the liver of IUGR-affected piglets and DHA supplementation increased (p < 0.05) the protein content of Nrf2 and HO-1. In conclusion, DHA may be beneficial in alleviating oxidative damage induced by IUGR through the Nrf2/ARE signaling pathway in the liver.

Effect of Curcumin on Growth Performance, Inflammation, Insulin level, and Lipid Metabolism in Weaned Piglets with IUGR.

The possible causes of intrauterine growth retardation (IUGR) might stem from placental insufficiency, maternal malnutrition, inflammation in utero, and other causes. IUGR has had an adverse influence on human health and animal production. Forty weaned piglets with normal birth weights (NBWs) or IUGR were randomly divided into four treatments groups: NBW, NC (NBW with curcumin supplementation), IUGR, and IC (IUGR with curcumin supplementation) from 26 to 50 d. Levels of cytokines, glucose, and lipid metabolism were evaluated. IUGR piglets showed slow growth during the experiment. Piglets with IUGR showed higher levels of serum pro-inflammatory cytokines, insulin resistance, and hepatic lipid accumulation. Curcumin supplementation reduced the production of serum pro-inflammatory cytokines, attenuated insulin resistance and hepatic triglyceride, and enhanced the hepatic glycogen concentrations and lipase activities of IUGR piglets. The hepatic mRNA expressions of the insulin-signaling pathway and lipogenic pathway were influenced by IUGR and were positively attenuated by diets supplemented with curcumin. In conclusion, IUGR caused slow growth, insulin resistance, and increased hepatic lipid levels. Diets supplemented with curcumin improved growth, attenuated insulin resistance, and reduced lipid levels in the liver by regulating the hepatic gene expressions of the related signaling pathway in IUGR piglets.