The correlation between the methylation of PTEN gene and the apoptosis of osteosarcoma cells mediated by SeHA nanoparticles.

The invasive spreading of residual osteosarcoma cells becomes a serious threat to human health, urgently needing new bone regenerative biomaterials for orthopedic therapy. Thus, in this work, selenite-substituted hydroxyapatite (SeHA) nanoparticles were prepared for both inhibiting the recurrence of the tumor and accelerating the regenerative repair of bone defect. Physicochemical characterization showed these synthetic nanoparticles were spherical poly-crystals with the shape of snowflakes. Such structure benefited them to inhibit the cellular viability of osteosarcoma cells by about (58.90 ± 14.37)% during 24 h co-culturing. The expression level of cell growth-related genes such as PTEN, MMP-9, Cyclin D1, Cyclin A2, Annexin A2 and CDC2 decreased. Bisulfite Sequence PCR of PTEN gene exhibited about (22.40 ± 5.39)%, (45.91 ± 6.36)% and (25.90 ± 5.36)% promoter methylation in control, HA and SeHA group. Animal experiment also proved the similar effects. Almost no recurrence were observed in SeHA group. Oppositely, the slowly recurrent growth of the remnant tumor appeared in purely surgical group. The overall survival and toxicity analysis showed that, in the usage dose of 0-0.1 g, the SeHA-0.01 exhibited higher inhibitory recurrence and metastasis potentials, lower renal toxicity and better anti-inflammation function. Immunohistochemistry stain showed the reduced expression of PTEN, MMP-9, Ki-67 and Annexin A2, but slightly increased expression of DNMT1 and BMP-2. Compared the methylation status of PTEN gene in each group, it was confirming that SeHA nanoparticles hardly possessed the de-methylation effect, but the pure HA strikingly increased the methylation level of such gene. It seemed the dopant selenite ions possessed de-methylation effect onto PTEN gene. Therefore, from the viewpoint of inhibiting metastatic potentials, the SeHA-0.01 might be a feasible biomaterial to inhibit the relapse of the tumor post-surgery.

Dietary values of macroalgae Porphyra haitanensis in Litopenaeus vannamei under normal rearing and WSSV challenge conditions: Effect on growth, immune response and intestinal microbiota.

Two trials were conducted to determine the effects of dietary macroalgae Porphyra haitanensis on growth, immunity and intestinal microbiota of Litopenaeus vannamei. In trial 1, shrimp (mean initial wet weight about 0.64 g) were fed with seven diets (P0, P1, P2, P3, P4, P5 and P6) containing 0% (basal diet), 1%, 2%, 3%, 4%, 5% and 6% P. haitanensis in triplicate for 60 days. Growth performance (weight gain, WG; specific growth rate, SGR) of shrimp fed the P4 diet were significantly higher than that of shrimp fed P0, P5 and P6 diets (P < 0.05) but without significant differences with shrimp fed P1-P3 diets (P > 0.05). Hepatopancreas phenoloxidase (PO) activity of shrimp fed the P. haitanensis containing diets was significantly higher than that of shrimp fed the basal diet (P0) (P < 0.05). Total haemocyte count (THC) of shrimp fed basal diet (P0) was significantly lower than that of shrimp fed diets containing P. haitanensis. Our results declared that dietary P. haitanensis supplementation increases the abundance of beneficial bacterials such as Nitrosopumilus, Marinobacter or Bifidobacterium and reduces the abundance of harmful bacterias such as Vibrio, and especially pronounced in P4 diet treatment. In trial 2, a WSSV injection challenge test was conducted for 7-day after the rearing trial and shrimp survival was also compared among treatments. A sudden shrimp death was found from the 4th day, and values of survival of shrimp fed the P3-P4 diets were higher than that of shrimp fed other diets during 4-7 days challenge test. The immune response in trial 2 were characterized by higher superoxide dismutase activity (SOD) and PO activities, lower THC and higher HCT compared to levels found in trial 1. In conclusion, suitable dietary P. haitanensis could enhance the growth performance, antioxidant capacity and alter total bacterial numbers or microbial diversity of L. vannamei and furthermore reduce oxidative stress and immune depression challenged by WSSV injection stress, and the level of P. haitanensis supplemented in the diet should be between 2.51% and 3.14%.

Dietary values of Forsythia suspensa extract in Penaeus monodon under normal rearing and Vibrio parahaemolyticus 3HP (VP3HP) challenge conditions: Effect on growth, intestinal barrier function, immune response and immune related gene expression.

Two trials were conducted to determine the effects of dietary Forsythia suspensa extract (FSE) on shrimp, Penaeus monodon, first on growth performance, second on the immune response and immune related gene expression of shrimp. In trial 1, shrimp (mean initial wet weight about 3.02 g) were fed with five diets containing 0% (basal diet), 0.01%, 0.02%, 0.04% and 0.06% FSE in triplicate for 60 days. Growth performance (final body wet weight, FBW; weight gain, WG; biomass gain, BG) of shrimp fed FSE diets were higher (P < 0.05) than that of shrimp fed the basal diet. The survival among all the diets treatments were above 90% and no significant difference was revealed among them (P > 0.05). The antioxidant capacity (total antioxidant status, TAS; glutathione peroxidase, GSH-Px) appears in the trend of firstly increasing then decreasing with the increasing of dietary FSE levels. The highest value of TAS and GSH-Px were found in shrimp fed 0.02% FSE diet and were significantly higher than that of shrimp fed the basal and 0.06% FSE diets (P < 0.05). Hepatopancreas malondialdehyde (MDA) of shrimp fed FSE diets were lower (P < 0.05) than that of shrimp fed the basal diet. Total haemocyte count of shrimp fed the basal diet was lower (P < 0.05) than that of shrimp fed FSE diets. Haemolymph clotting time of shrimp had the opposite trend with the total haemocyte count of shrimp. No significant differences were found in haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) and in molecular gene expression profiles of heat shock protein 70 (Hsp 70) mRNA and hypoxia inducible factor-1α (HIF-1α) mRNA in haemolymph of shrimp among all diet treatments (P > 0.05). In trial 2, a pathogenic strain of Vibrio parahaemolyticus 3HP (VP3HP) injection challenge test was conducted for 6-day after the rearing trial and shrimp survival were also compared among treatments. Survival of shrimp fed diets supplemented with 0.01%-0.02% FSE were higher than that of shrimp fed the basal and 0.06% FSE diets (P < 0.05). Dietary FSE supplementation produced stronger hepatopancreas antioxidant capacity (TAS, GSH-Px) (P < 0.05) and higher glutathione (GSH) level (P < 0.05), lower superoxide dismutase activity (SOD) (P < 0.05), higher total haemocyte count (P < 0.05), lower haemolymph clotting time (P < 0.05), lower MDA and carbonyl protein concentration (P < 0.05), lower haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) (P < 0.05), generated lower molecular gene expression profiles of HSP 70 mRNA and higher HIF-1α mRNA (P < 0.05) than the basal diet. The immune response were characterized by lower TAS and higher antioxidant enzyme activities (SOD, GSH-Px) and higher oxidative stress level (MDA and carbonyl protein) and higher haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) compared to levels found in trail 1. However, the total haemocyte counts and haemolymph clotting times were not changed in 0.01%-0.02% FSE diets treatments between trial 1 and trial 2 (P > 0.05). The molecular gene expression profile of Hsp 70 mRNA was increased while HIF-1α mRNA was decreased when compared to trial 1. In conclusion, results suggested that dietary intake containing FSE could enhance the growth performance and antioxidant capacity of P. monodon and furthermore reduce oxidative stress and immune depression challenged by a pathogenic strain of Vibrio parahaemolyticus stress. Considering the effect of FSE on both growth performance and immune response of P. monodon, the level of FSE supplemented in the diet should be between 0.01% and 0.02%.

In Vitro and in Vivo Mechanism of Bone Tumor Inhibition by Selenium-Doped Bone Mineral Nanoparticles.

Biocompatible tissue-borne crystalline nanoparticles releasing anticancer therapeutic inorganic elements are intriguing therapeutics holding the promise for both tissue repair and cancer therapy. However, how the therapeutic inorganic elements released from the lattice of such nanoparticles induce tumor inhibition remains unclear. Here we use selenium-doped hydroxyapatite nanoparticles (Se-HANs), which could potentially fill the bone defect generated from bone tumor removal while killing residual tumor cells, as an example to study the mechanism by which selenium released from the lattice of Se-HANs induces apoptosis of bone cancer cells in vitro and inhibits the growth of bone tumors in vivo. We found that Se-HANs induced apoptosis of tumor cells by an inherent caspase-dependent apoptosis pathway synergistically orchestrated with the generation of reactive oxygen species. Such mechanism was further validated by in vivo animal evaluation in which Se-HANs tremendously induced tumor apoptosis to inhibit tumor growth while reducing systemic toxicity. Our work proposes a feasible paradigm toward the design of tissue-repairing inorganic nanoparticles that bear therapeutic ions in the lattice and can release them in vivo for inhibiting tumor formation.

Lysozyme loading and release from Se doped hydroxyapatite nanoparticles.

Element-substituted hydroxyapatite (HA) based nanocomposites have become a promising therapeutic material for improving bone defect repair. Selenium substituted HA nanoparticles can both induce apoptosis of bone tumor cells and enhance osteointegration. However, the effect of selenite ions on the proteins in combination with the HA nanoparticles remains to be elucidated. Here, we investigated the influence of selenium doping concentration on the loading and release of lysozyme (LSM) as a model protein drug. The selenium substituted HA-LSM composites with different doping concentrations were synthesized and characterized. The subsequent delivery of lysozyme was studied in a phosphate buffer solution (PBS). We found that selenium substituted HA-LSM composites with Se:P=10% showed the highest amount of lysozyme loading (41.7%), whereas the amount of lysozyme loaded in undoped HA nanoparticles was the lowest (34.1%). The doped selenium interacts with lysozyme molecules, which leads to the increase of ß-sheet and unordered, and the decrease of self-association, α-helix and ß-turns in protein structures. Moreover, selenium addition significantly slows the protein release from HA-LSM composites. The composites with Se:P=10% release lysozyme at the slightly slower rate among the samples with different Se doping concentrations. It also shows that the released lysozyme retains most of its enzymatic activity.

Selenium-substituted hydroxyapatite nanoparticles and their in vivo antitumor effect on hepatocellular carcinoma.

Absence of curative treatment creates urgent need for new strategies for unresectable hepatoma. Novel selenium-substituted hydroxyapatite nanoparticles (SeHAN) were designed to serve as anticancer agent. The authors examined the nanoparticles by physicochemical techniques. The in vivo efficacy and toxicity of these nanoparticles were also investigated on a nude mice model of human hepatocellular carcinoma. The results showed that the selenite ions can be incorporated into the hydroxyapatite lattice facilely. They exhibited bundles of needles shape with a size of 160-200 nm. In the in vivo study, they showed better survival advantage. The overall survival rate of nude mice in the control, pure hydroxyapatite and SeHAN group were 50.00%, 76.92%, and 100.00% respectively. Blood biochemical studies showed that SeHAN group had significantly lower toxicities on the liver and kidney functions. Histopathological studies confirmed that massive tumor necrosis and calcium deposition were evident after SeHAN treatment. Moreover, immunohistochemistry and Western blot assay showed significantly reduced expression of the Ki-67, VEGF and MMP-9 protein in the SeHAN group. Taken together, these results suggest that the selenium-substituted hydroxyapatite nanoparticles could be a new type of promising anticancer agent to provide both survival advantage and lower toxicity.