Structure and immunostimulatory activity studies on two novel Flammulina velutipes polysaccharides: revealing potential impacts of →6)-α-D-Glcp(1→ on the TLR-4/MyD88/NF-κB pathway.

Two novel Flammulina velutipes (F. velutipes) polysaccharides, FVPH1 and FVPH2, were isolated and purified after hot water extraction. The structural characterization revealed that the backbone of FVPH1 consisted mainly of →6)-α-D-Glcp(1→, →3,4)-α-D-Galp(1→, →4)-α-L-Fucp(1→, and →4)-ß-D-Manp(1→, while the backbone of FVPH2 consisted of →3)-α-D-Galp(1→, →3,4)-α-D-Manp(1→,→6)-α-D-Glcp(1→. The branches of FVPH1 contained →6)-α-D-Glcp(1→ and α-D-Glcp(1→ and the branches of FVPH2 consisted of →3)-α-D-Galp(1→, →6)-α-D-Glcp(1→, and ß-L-Fucp(1→. FVPH2 exhibited significantly better immunostimulatory activity than FVPH1 (P < 0.05), as evidenced by the increased expression of NO, IL-1ß, IL-6, and TNF-α and pinocytic activity of RAW264.7 cells. As the most abundant structure in the polysaccharides of F. velutipes, the content of →6)-α-D-Glcp(1→ might play a crucial role in influencing the immunostimulatory activity of F. velutipes polysaccharides. The F. velutipes polysaccharide with a lower content of →6)-α-D-Glcp(1→ and a higher branching degree could significantly enhance the immunostimulatory activity of F. velutipes polysaccharides via activating the TLR-4/MyD88/NF-κB pathway more effectively.

Improved bioavailability and antioxidation of ß-carotene-loaded biopolymeric nanoparticles stabilized by glycosylated oat protein isolate.

The limited bioavailability of ß-carotene hinders its potential application in functional foods, despite its excellent antioxidant properties. Protein-based nanoparticles have been widely used for the delivery of ß-carotene to overcome this limitation. However, these nanoparticles are susceptible to environmental stress. In this study, we utilized glycosylated oat protein isolate to prepare nanoparticles loaded with ß-carotene through the emulsification-evaporation method, aiming to address this challenge. The results showed that ß-carotene was embedded into the spherical nanoparticles, exhibiting relatively high encapsulation efficiency (86.21 %) and loading capacity (5.43 %). The stability of the nanoparticles loaded with ß-carotene was enhanced in acidic environments and under high ionic strength. The nanoparticles offered protection to ß-carotene against gastric digestion and facilitated its controlled release (95.76 % within 6 h) in the small intestine, thereby leading to an improved in vitro bioavailability (65.06 %) of ß-carotene. This improvement conferred the benefits on ß-carotene nanoparticles to alleviate tert-butyl hydroperoxide-induced oxidative stress through the upregulation of heme oxygenase-1 and NAD(P)H quinone dehydrogenase 1 expression, as well as the promotion of nuclear translocation of nuclear factor-erythroid 2-related factor 2. Our study suggests the potential for the industry application of nanoparticles based on glycosylated proteins to effectively deliver hydrophobic nutrients and enhance their application.

Study on the physicochemical properties and antioxidant activities of Flammulina velutipes polysaccharide under controllable ultrasonic degradation based on artificial neural network.

The polysaccharide fraction (FVP2) with molecular weight of 1525.09 kDa and intrinsic viscosity of 3.43 dL/g was isolated and purified from Flammulina velutipes (F. velutipes), and the ultrasonic degradation model of FVP2 was established to predict the molecular weight and intrinsic viscosity at the same time based on artificial neural network. FVP2U1 (1149.11 kDa, 1.78 dL/g), FVP2U2 (618.91 kDa, 1.19 dL/g) and FVP2U3 (597.35 kDa, 0.48 dL/g) with different molecular weights or viscosity were produced by this model to explore the effect of ultrasound on the physicochemical properties and antioxidant activity of FVP2. The results showed that ultrasonic treatment did not change the types of characteristic functional groups, monosaccharide composition and glycosidic bond of FVP2, but changed the chemical composition ratio and the degree of polymerization. Under ultrasonic treatment, the intrinsic viscosity of FVP2 still decreased significantly when the molecular weight did not decrease. Compared to other components subjected to ultrasonic degradation, FVP2U1 demonstrated higher molecular weight and viscoelasticity, while exhibiting lower antioxidant activity. In the case of no significant difference in molecular weight and monosaccharide composition, FVP2U3 with lower intrinsic viscosity has stronger hydration ability, higher crystallization index, lower viscoelasticity and stronger antioxidant capacity than FVP2U2.

Oat protein isolate-Pleurotus ostreatus ß-glucan conjugate nanoparticles bound to ß-carotene effectively alleviate immunosuppression by regulating gut microbiota.

Individuals with immune disorders cannot establish an adequate defense to pathogens, leading to gut microbiota dysbiosis. ß-Carotene can regulate immune response, but its bioavailability in vivo is very low. Herein, we developed a glycosylated oat protein-based nanoparticle to improve the application of ß-carotene for mitigating cyclophosphamide-induced immunosuppression and gut microbiota imbalance in mice. The results showed that the nanoparticles facilitated a conversion of ß-carotene to retinol or retinyl palmitate into the systemic circulation, leading to an increased bioavailability of ß-carotene. The encapsulated ß-carotene bolstered humoral immunity by elevating immunoglobulin levels, augmenting splenic T lymphocyte subpopulations, and increasing splenic cytokine concentrations in immunosuppressed mice. This effect was accompanied by the alleviation of pathological features observed in the spleen. In addition, the encapsulated ß-carotene restored the abnormal gut microbiota associated with immunosuppression, including Erysipelotrichaceae, Akkermansia, Bifidobacterium and Roseburia. This study suggested that nanoparticles loaded with ß-carotene have great potential for therapeutic intervention in human immune disorders by specifically targeting the gut microbiota.

Application of definitive screening design to optimization of the protein extraction and functional properties of proteins in Auricularia auricula.

BACKGROUND: Auricularia auricula (A. auricula) is one of the most abundant sources of plant protein in edible fungi. Problems of low protein yield exist in traditional methods of protein extraction such as alkali extraction and ultrasonic-assisted alkali after pretreatment with enzymes. Thus, the protein extraction process was investigated and optimized using a definitive screening design from A. auricula to improve the protein yield under practical operating conditions of temperature, the concentration of NaCl, meal/water ratio, extraction time and pH. RESULTS: The yield of protein isolates of the isoelectric-ammonium sulfate precipitation (9.34% w/w) was obtained almost three times and the protein content (55.23% w/w) was approximately 1.6 times that of the traditional extraction method of isoelectric precipitation. Next, the optimized method was successfully applied to the analysis of the functional properties of the protein. A. auricula protein isolate (AAPI) had better solubility, emulsification and foaming capacity than soy protein isolate (SPI) and pea protein isolate (PPI), and the oil holding capacity of AAPI exhibited extremely well, which was approximately five times that of SPI and six times that of PPI. The texture properties of AAPI gel were similar to those of PPI gels. CONCLUSION: AAPI extracted by the optimized method had a satisfactory yield and had the potential to substitute plant-originated proteins in food processing. © 2022 Society of Chemical Industry.

Effects of selenylation modification on the antioxidative and immunoregulatory activities of polysaccharides from the pulp of Rose laevigata Michx fruit.

Selenylation modification has been widely utilized to improve the activity of polysaccharides and to develop novel sources of selenium (Se) supplements. A purified pulp polysaccharide of Rose laevigata Michx fruit (PPRLMF-2) was selenized into Se-PPRLMF-2 in this study. PPRLMF-2 + Se was formulated by Na2SeO3 according to the Se content of Se-PPRLMF-2. To investigate the effects of selenylation modification on the structure and functions of PPRLMF-2, the characteristics, antioxidative and immunoregulatory activities of PPRLMF-2 before and after selenylation were compared. The results showed that compared with PPRLMF-2, Se-PPRLMF-2 became an irregular fibrous network, and its Mw decreased and C-6 substitution predominated in 13C NMR spectra. Se-PPRLMF-2 significantly increased chemical antioxidant activity and reduced the oxidative damage of erythrocytes, which was not due to Se alone. Se-PPRLMF-2 significantly increased immunomodulatory activity on macrophages, which was related to Se alone. Se-PPRLMF-2 could be a good potential source of antioxidants, immune enhancers and dietary Se supplements.

Effects of ultrasound on the degradation kinetics, physicochemical properties and prebiotic activity of Flammulina velutipes polysaccharide.

The controllable ultrasonic modification was hindered due to the uncertainty of the relationship between ultrasonic parameters and polysaccharide quality. In this study, the ultrasonic degradation process was established with kinetics. The physicochemical properties and prebiotic activity of ultrasonic degraded Flammulina velutipes polysaccharides (U-FVPs) were investigated. The results showed that the ultrasonic degradation kinetic models were fitted to 1/Mt-1/M0 = kt. When the ultrasonic intensity increased from 531 to 3185 W/cm2, the degradation proceeded faster. The decrease of polysaccharide concentration contributed to the degradation of FVP, and the fastest degradation rate was at 60 °C. Ultrasound changed the solution conformation of FVP, and partially destroyed the stability of the triple helix structure of FVP. Additionally, the viscosity and gel strength of FVP decreased, but its thermal stability was improved by ultrasound. Higher ultrasonic intensity led to larger variations in physicochemical properties. Compared with FVP, U-FVPs could be more easily utilized by gut microbiota. U-FVPs displayed better prebiotic activity by promoting the growth of Bifidobacterium and Brautella and inhibiting the growth of harmful bacteria. Ultrasound could be effectively applied to the degradation of FVP to improve its physicochemical properties and bioactivities.

Purification and comparative study of bioactivities of a natural selenized polysaccharide from Ganoderma Lucidum mycelia.

The development of selenized polysaccharides is a promising strategy for the dietary selenium supplementation. The purpose of this research is to determine the influence of selenium on the structure and bioactivity of a polysaccharide fraction (MPN) isolated from Ganoderma lucidum mycelia. After biological selenium enrichment, the selenium content in the selenized polysaccharide (SeMPN) was 18.91 ± 1.8 µg/g. SeMPN had a slightly lower molecular weight than MPN, but the carbohydrate content and monosaccharide composition remained identical. Additionally, the band at 606 cm-1 in MPN changed to 615 cm-1 in SeMPN as revealed by FT-IR spectra. No significant changes were observed in the types and ratios of glycosidic linkages, as determined by NMR spectroscopy. Extracellular and intracellular antioxidant assays demonstrated that SeMPN was more effective than MPN in scavenging free radicals, inhibiting AAPH-induced erythrocyte hemolysis, and protecting catalase (CAT) and glutathione peroxidase (GSH-Px) activity in H2O2-injured PC12 cells. Additionally, SeMPN had a higher increase effect on RAW 264.7 cells's pinocytic and phagocytic capacity, as well as their production of NO, TNF-α, and IL-6. SeMPN could be as potential functional selenium supplementation.

Purification and characterization of immunomodulatory peptides from enzymatic hydrolysates of duck egg ovalbumin.

Duck egg white (DEW) is considered as an abandoned protein resource. For a higher-value utilization, preparation of immunomodulatory peptides from the extracted ovalbumin in DEW was explored. Among the hydrolysates catalyzed by five proteases, papain hydrolysate (PH) shows the highest degree of hydrolysis and the strongest immunomodulatory activity. PH could significantly enhance the phagocytic capacity and promote the NO, TNF-α, and IL-6 secretion of RAW 264.7 cells, involving toll-like receptor 2 and 4. After purification, nine identified peptides were synthesized to confirm the immunomodulatory effect, and five of them exhibit a strong activity. The peptide, TQIDKVVHFDKLPGF, presents the highest immunomodulatory activity. Moreover, the results of molecular docking indicate that nine peptides interacted with toll-like receptor 2 and 4 and all show good affinity. Furthermore, three peptides with high affinity and strong immune activity were selected for interaction site map analysis. Three peptides could form hydrogen bonds with the receptor and bind stably, which contributes to the immunomodulatory activity of the peptide. Results suggest that DEW can be a promising source of immunomodulatory peptides.

Comparative study of DHA-enriched phosphatidylcholine and EPA-enriched phosphatidylcholine on ameliorating high bone turnover via regulation of the osteogenesis-related Wnt/ß-catenin pathway in ovariectomized mice.

Here, we compared the effects of marine DHA-enriched phosphatidylcholine (DHA-PC) and EPA-enriched phosphatidylcholine (EPA-PC) on high bone turnover in a model of osteoporosis induced by bilateral ovariectomy in vivo, and further investigated the possible protective mechanisms. Meanwhile, DHA-PC and EPA-PC clearly ameliorated the microstructure of the trabecular bone and accelerated bone mineral apposition rate, additionally increasing bone mineral density and biomechanical properties of the bone. Furthermore, gene and protein expression levels suggest that DHA-PC and EPA-PC inhibited overactive osteogenesis via down-regulation of the expression of the osteogenesis-related Wnt/ß-catenin signaling pathway. In conclusion, DHA-PC and EPA-PC reduced excessive osteogenesis via normalization of Wnt/ß-catenin expression. These results may contribute to the elucidation of the anti-osteoporotic properties of DHA-PC and EPA-PC and further develop their potential application value as a functional food.

Suppression of lipopolysaccharide-induced activation of RAW 264.7 macrophages by Se-methylseleno-l-cysteine.

Se-methylseleno-l-cysteine (l-SeMC) is a natural source of organic selenium for humans. Although it has a structure similar to that of l-Cysteine (l-Cys), its anti-inflammatory properties and possible underlying mechanisms have not been explored. Here, we compared the anti-inflammatory activities of inorganic selenium (selenite), l-Cys, and l-SeMC in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophages and focused on the related molecular and biochemical events. The results showed that, anti-inflammatory activity of l-SeMC was much stronger compared to both individual l-Cys treatment and l-Cys/selenite combinations. The organic form of selenium may play a crucial role in the effects of l-SeMC. Further study confirmed that l-SeMC suppressed the RNA expression of iNOS, TNF-α, IL-1ß, IL-6, COX-2, and MMP-9, as well as the release of NO, TNF-α, IL-6, IL-12p70, COX-2, and PGE2 from LPS-activated RAW264.7 macrophages in a concentration-dependent manner. Moreover, l-SeMC prevented LPS-induced changes in cell morphology. l-SeMC concentrations between 50 and 200 µM exhibited an anti-inflammatory effect closed to that exhibited by 20 µM dexamethasone. Our results demonstrated that l-SeMC effectively inhibited the activation of RAW 264.7 macrophages induced by LPS, and suggested that l-SeMC could be a potential functional food component for the prevention or treatment of inflammatory diseases.

The opposite effects of Antarctic krill oil and arachidonic acid-rich oil on bone resorption in ovariectomized mice.

Osteoporosis, a chronic disease that affects over 200 million people worldwide, presents a substantial medical and socioeconomic burden on the modern society. However, long-term intake of diets supplemented with different polyunsaturated fatty acids (PUFAs) can affect bone metabolism; thus, this study investigated the comparative effects of Antarctic krill oil (AKO, containing n-3 PUFAs) and arachidonic acid-rich oil (AAO, containing n-6 PUFAs) on bone resorption in a mice model of postmenopausal osteoporosis. Mice were orally administered with AKO (200 mg kg-1) or AAO (220 mg kg-1) once daily for 30 days, ovariectomized, followed by the continued administration of the respective samples for 90 days. Biomechanical and histomorphometric analyses revealed that AKO increased the bone mineral density (BMD) to enhance the biomechanical properties by increasing the mineral apposition rate and repairing the microstructure of the trabecular bone, whereas AAO had the opposite effect. The fatty acid analysis of the vertebra showed that AKO increased the n-3 PUFA (especially for DHA) content, thereby decreasing the ratio of n-6/n-3 PUFAs, which was negatively correlated with the BMD. However, AAO had the opposite effect due to high amounts of arachidonic acid. To explore the underlying mechanism responsible for these observations, we compared the classical bone resorption OPG/RANKL/NF-κB pathway mediated by PGE2/EP4. The ratio of n-6/n-3 PUFAs in the bone affected the production of PGE2, a factor regulating the OPG/RANKL pathway, thereby regulating osteoclastogenesis by stimulating the NF-κB pathway. The results of ELISA, qRT-PCR, and western blot demonstrated that AKO reduced the secretion of PGE2 and the expression of EP4, upregulating the ratio of OPG/RANKL in the bone, thereby decreasing TRAF6 expression to inhibit the activation of the NF-κB signaling pathway, and finally inhibiting the expression of nuclear transcription factors (c-fos and NFATc1) to prevent excessive osteoclastogenesis (TRACP, MMP-9, and Cath-K). Arachidonic acid is a precursor of PGE2 synthesis. AAO showed the opposite trend through the same pathway. Thus, AKO could significantly improve osteoporosis via the OPG/RANKL/NF-κB pathway mediated by PGE2/EP4 to inhibit osteoclastogenesis, whereas AAO aggravated osteoporosis via the same pathway. This is the first study to systematically compare the effects and mechanism of AKO and AAO in regulating bone resorption in osteoporotic mice to support recommendations on fatty acid types in dietary oils for an osteoporotic population.

Structural characterization of polysaccharides with potential antioxidant and immunomodulatory activities from Chinese water chestnut peels.

Chinese water chestnut peels are a kind of vegetable processing waste containing many active components such as polysaccharides, the structure of which remains unknown. To elucidate the structure of polysaccharides from Chinese water chestnut peels, two polysaccharides named WVP-1 and WVP-2 were isolated. WVP-1 (3.16 kDa) consisted of mannose (1.75 %), glucose (84.69 %), galactose (6.32 %), and arabinose (7.24 %), while WVP-2 (56.97 kDa) was composed of mannose (3.18 %), rhamnose (1.52 %), glucuronic acid (1.42 %), galacturonic acid (4.83 %), glucose (11.51 %), galactose (36.02 %), and arabinose (41.53 %). Linkage and NMR data indicated that WVP-1 was composed mainly of →4)-α-d-Glcp(1→ and a certain proportion of →3)-ß-d-Glcp-(1→, including linear and branched polysaccharides simultaneously. WVP-2 was a pectin-like polysaccharide with →4)-α-d-GalpA6Me-(1→ units and the branch points of →3,4)-α-l-Arap-(1→, →3,6)-ß-d-Galp-(1→. WVP-2 exhibited stronger potential antioxidant and immunomodulatory activities than WVP-1 in vitro. These results provide a foundation for the further study of polysaccharides from Chinese water chestnut peels.

Structural characterization and immunomodulatory activity of a novel polysaccharide from Pueraria lobata (Willd.) Ohwi root.

A novel polysaccharide with a molecular weight of 12.3 kDa, was isolated from the root of Pueraria lobata (Willd.) Ohwi using cold water extraction method. Methylation analysis, Periodate oxidation, Smith degradation, and nuclear magnetic resonance analysis showed that 1,4-α-D-Glcp and 1,3,6-α-D-Glcp were the main linkage types of this polysaccharide. It exhibited immunomodulatory activity by enhancing the pinocytic and phagocytic capacities and promoting the secretion of nitric oxide, interleukin (IL)-6, and tumor necrosis factor alpha by RAW 264.7 cells. The beta glucan receptor, scavenger receptor I, and toll-like receptor 4 were identified as the main receptors of the polysaccharide on the membrane of RAW 264.7 cells. These results suggested that this polysaccharide is a candidate functional food supplement for hypoimmune populations.

Structural characterization and immunomodulatory activity of a novel acid polysaccharide isolated from the pulp of Rosa laevigata Michx fruit.

A novel acid polysaccharide (PPRLMF-2) with the Mw of 137,123 Da and a triple-helix conformation was first isolated from the pulp of Rosa laevigata Michx fruit. Structural characterization showed that PPRLMF-2 consisted of rhamnose (7.6%), arabinose (26.5%), xylose (3.5%), mannose (0.9%), glucose (5.7%), galactose (31.9%) and galacturonic acid (23.9%). The methylation and NMR (1D and 2D) analysis revealed that PPRLMF-2 contained 16 types of glycosidic linkages. The immunomodulatory activity assays indicated that PPRLMF-2 could significantly enhance phagocytosis, the secretion and mRNA expression of cytokines in RAW 264.7 cells. Additionally, SR, GR, TLR-2, and TLR-4 were the main pattern recognition receptors (PRRs) of PPRLMF-2 to upregulate the p-ERK, p-JNK, p-p38, and p-p65. These results suggested that PPRLMF-2 could recognize the PRRs of the macrophages to enhance the immunomodulatory activity via activation of the MAPKs and NF-κB signaling pathways. This study provides important implications of PPRLMF-2 as an attractive immunomodulatory functional food.

Structural Characterization and Immunomodulatory Activity of a Polysaccharide from Eurycoma longifolia.

A polysaccharide, Ali-1, was isolated from the roots of Eurycoma longifolia, a popular traditional medicinal herb in Malaysia. The structure of Ali-1 was characterized by monosaccharide, methylation, and NMR data analyses. The average molecular weight of Ali-1 is 14.3 ku, and it is composed of arabinose (14.31%), xylose (57.69%), galacturonic acid (13.03%), and glucuronic acid (14.86%). The main chain comprises (1→4)-linked xylose residues. It has branch points in the main chain; (1→2,4)-linked xylose residues, 1,2-linked glucuronic acid residues, and 1,2-linked arabinose residues form the branches, and the branches are terminated with T-linked galacturonic acid residues and T-linked arabinose residues. Ali-1 significantly improves the pinocytic and phagocytic abilities of RAW264.7 cells and facilitates cytokine secretion according to an immunostimulation assay. These results demonstrate that Ali-1 has potential as a functional supplement for people with compromised immune systems.

Two novel polysaccharides from the torus of Saussurea laniceps protect against AAPH-induced oxidative damage in human erythrocytes.

Two major polysaccharides (SLT-3, SLT-4) were isolated from the torus of Saussurea laniceps. Their molecular weight, monosaccharide compositions and the ability to protect human erythrocytes from oxidative damage induced by AAPH were assessed. Results showed that the Mw of SLT-3 and SLT-4 were 10,113 Da and 12,392 Da. SLT-3 was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose in a molar ratio of 0.25:0.53:0.19:15.35:0.51:1.10:0.63:1.73, whereas SLT-4 was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose in a molar ratio of 0.92:5.61:0.93:19.50:2.42:5.27:3.01. Pretreatment with SLT-3 and SLT-4 reduced MDA content, inhibited the generation of intracellular ROS and maintained the balance of GSH and GSSG in AAPH-treated erythrocytes. Furthermore, the activities of intracellular antioxidant enzymes, such as SOD, GSH-Px and CAT, were attenuated in polysaccharide treated cells. The results provide an important basis for the development of S. laniceps as a natural antioxidant.

A novel low molecular weight Enteromorpha polysaccharide-iron (III) complex and its effect on rats with iron deficiency anemia (IDA).

Iron deficiency anemia (IDA) is a common nutritional disease that affects normal erythropoiesis. Traditional iron supplements usually cause gastrointestinal irritation. In this study, a novel low-molecular-weight polysaccharide from Enteromorpha prolifera (LPE) was prepared by oxidation degradation, and LPE-iron (III) complex was synthesized and characterized. The molecular weight of LPE-iron (III) complex was 21.25kDa, and iron content was 25%. The therapeutic effects of LPE-iron (III) complex on IDA were investigated in rats. The hematological indices and organ coefficients of the rats were analyzed. Results showed a dose-dependent relationship, and a prior intragastric administration of LPE-iron (III) complex (2mg Fe/kg body weight) exhibited considerable effect when compared with the positive control. Therefore, LPE-iron (III) complex could be exploited as a new iron fortifier.

Sialoglycoprotein isolated from the eggs of Gadus morhua enhances fracture healing in osteoporotic mice.

Osteoporosis is a common disease in the elderly, which is related to fracture healing delay. In this study, the effects of treatment with sialoglycoprotein isolated from the eggs of Gadus morhua (Gm-SGP) on tibial fracture healing in ovariectomized (OVX) osteoporotic female C57BL/6J mice for 56 days post-fracture were investigated. The result showed that Gm-SGP treatment significantly increased serum angiogenic factors and bone formation markers on day 5 and 11 post-fracture when compared with the OVX group. In addition, histological results in the Gm-SGP group showed a stronger endochondral ossification, a stronger bony consolidation and a stronger bony callus remodeling capability on day 11, 24 and 35 post-fracture, respectively, in comparison with the OVX group. Meanwhile, micro-computerized tomography revealed that the Gm-SGP group had stronger bony callus remodeling capability as evidenced by higher BV/TV and Tb.N but lower Tb.Sp and shorter lengths of callus maximum cross section than the OVX group on day 24 post-fracture. Besides, the tibial callus bending stiffness was significantly enhanced in the Gm-SGP group as compared with the OVX group on day 56 post-fracture. Moreover, gene expression suggested that Gm-SGP promoted vascular invasion and endochondral ossification on day 11 post-fracture as well as bone formation on day 11 and 24 post-fracture via up-regulating the expression of angiogenesis factors (including VEGF, PDGF and Ang1), entochondrostosis factors (including Col2a1, Aggrecan, Col10a1 and MMP-13) and osteogenesis markers (including Col1a1, BMP-2 and OCN). This research suggests that Gm-SGP significantly improve fracture healing which is delayed by OVX-induced osteoporosis. The present study may contribute to providing important implications for the utilization of Gm-SGP from fish eggs as a functional food to enhance fracture healing.

Eicosapentaenoic Acid-Enriched Phosphatidylcholine Attenuated Hepatic Steatosis Through Regulation of Cholesterol Metabolism in Rats with Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world. Disturbed cholesterol metabolism plays a crucial role in the development of NAFLD. The present study was conducted to evaluate the effects of EPA-PC extracted from sea cucumber on liver steatosis and cholesterol metabolism in NAFLD. Male Wistar rats were randomly divided into seven groups (normal control group, model group, lovastatin group, low- and high-dose EPA groups, and low- and high-dose EPA-PC groups). Model rats were established by administering a diet containing 1% orotic acid. To determine the possible cholesterol metabolism promoting mechanism of EPA-PC, we analyzed the transcription of key genes and transcriptional factors involved in hepatic cholesterol metabolism. EPA-PC dramatically alleviated hepatic lipid accumulation, reduced the serum TC concentration, and elevated HDLC levels in NAFLD rats. Fecal neutral cholesterol excretion was also promoted by EPA-PC administration. Additionally, EPA-PC decreased the mRNA expression of hydroxymethyl glutaric acid acyl (HMGR) and cholesterol 7α-hydroxylase (CYP7A), and increased the transcription of sterol carrying protein 2 (SCP2). Moreover, EPA-PC stimulated the transcription of peroxisome proliferators-activated receptor α (PPARα) and adenosine monophosphate activated protein kinase (AMPK) as well as its modulators, liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent kinase kinase (CAMKK). Based on the results, the promoting effects of EPA-PC on NAFLD may be partly associated with the suppression of cholesterol synthesis via HMGR inhibition and the enhancement of fecal cholesterol excretion through increased SCP2 transcription. The underlying mechanism may involve stimulation of PPARα and AMPK.