Long-term hypoxia-induced physiological response in turbot Scophthalmus maximus L.

Hypoxia affects fish's survival, growth, and physiological metabolism processes. In this study, turbot plasma glucose and cortisol contents, hepatic glycolysis (hexokinase [HK], phosphofructokinase [PFK], pyruvate kinase [PK]) and lipolysis (fatty acid synthetase [FAS], lipoprotein lipase [LPL]) enzyme activities, anti-oxidant enzyme (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH-Px]) activities, malondialdehyde (MDA), lactate and glycogen contents, gill histological parameters (lamellar length [SLL], width [SLW], interlamellar distance [ID]), respiratory frequency (RF), the proportion of the secondary lamellae available for gas exchange (PAGE), and hifs (hif-1α, hif-2α, hif-3α) expression were determined during long-term hypoxia and reoxygenation. Results showed that long-term hypoxia (3.34 ± 0.17 mg L-1) significantly elevated plasma cortisol and glucose contents; increased hepatic HK, PK, PFK, FAS, and LPL activity; decreased hepatic glycogen, lactate contents, and lipid drop numbers; and caused changes of hepatocyte (vacuolation, pyknotic, and lytic nucleus) after treatment for 4 weeks. Hepatic SOD, CAT, GSH-Px activity, and MDA contents; lamellar perimeter, SLL, ID, RF, and PAGE; and hepatic hif-1α, hif-2α, and hif-3α manifested similar results. Meanwhile, hif-1α is significantly higher than hif-2α, and hif-3α. Interestingly, females and males demonstrated no sex dimorphism significantly different from the above parameters (except hepatic FAS, LPL activity, and lipid drop number) under hypoxia. The above parameters recovered to normal levels after reoxygenation treatment for 4 weeks. Thus, long-term hypoxia promotes turbot hepatic glycogenolysis and lipolysis, induces oxidative damage and stimulates hepatic antioxidant capacity, and alters gill morphology to satisfy insufficient energy demand and alleviate potential damage, while hif-1α plays critical roles in the above physiological process.

Functionalized chitosan hydrogel promotes osseointegration at the interface of3D printed titanium alloy scaffolds.

Autogenous bone transplantation is a prevalent clinical method for addressing bone defects. However, the limited availability of donor bone and the morbidity associated with bone harvesting have propelled the search for suitable bone substitutes. Bio-inspired scaffolds, particularly those fabricated using electron beam melting (EBM) deposition technology, have emerged as a significant advancement in this field. These 3D-printed titanium alloy scaffolds are celebrated for their outstanding biocompatibility and favorable elastic modulus. Thermosensitive chitosan hydrogel, which transitions from liquid to solid at body temperature, serves as a popular carrier in bone tissue engineering. Icariin (ICA), known for its efficacy in promoting osteoblast differentiation from bone marrow mesenchymal stem cells (BMSCs), plays a crucial role in this context. We developed a system combining a 3D-printed titanium alloy with a thermosensitive chitosan hydrogel, capable of local bone regeneration and integration through ICA delivery. Our in vitro findings reveal that this system can gradually release ICA, demonstrating excellent biocompatibility while fostering BMSC proliferation and osteogenic differentiation. Immunohistochemistry and Micro-CT analyses further confirm the effectiveness of the system in accelerating in vivo bone regeneration and enhancing osseointegration. This composite system lays a significant theoretical foundation for advancing local bone regeneration and integration.

Melatonin Improves Turbot Oocyte Meiotic Maturation and Antioxidant Capacity, Inhibits Apoptosis-Related Genes mRNAs In Vitro.

High-quality eggs are essential for the sustainability of commercial aquaculture production. Melatonin is a potent candidate for regulating the growth and maturation of oocytes. Therefore, research on the effect of melatonin on marine fish oocytes in vitro has been conducted. The present study successfully established a culture system of turbot (Scophthalmus maximus) oocytes in vitro and investigated the effect of melatonin on oocyte meiotic maturation, antioxidant capacity, and the expression of apoptosis-related genes. The cultures showed that turbot Scophthalmus maximus late-vitellogenic denuded oocytes, with diameters of 0.5-0.7 mm, had a low spontaneous maturation rate and exhibited a sensitive response to 17α, 20ß-dihydroxyprogesterone (DHP) treatment in vitro. Melatonin increased by four times the rate of oocyte germinal vesicle breakdown (GVBD) in a concentration- and time-dependent manner. The mRNA of melatonin receptor 1 (mtnr1) was significantly upregulated in the oocyte and follicle after treatment with melatonin (4.3 × 10-9 M) for 24 h in vitro, whereas melatonin receptor 2 (mtnr2) and melatonin receptor 3 (mtnr3) remained unchanged. In addition, melatonin significantly increased the activities of catalase, glutathione peroxidase, and superoxide dismutase, as well as the levels of glutathione, while decreasing the levels of malondialdehyde and reactive oxygen species (ROS) levels in turbot oocytes and follicles cultures in vitro. p53, caspase3, and bax mRNAs were significantly downregulated in oocytes and follicles, whereas bcl2 mRNAs were significantly upregulated. In conclusion, the use of turbot late-vitellogenesis oocytes (0.5-0.7 mm) is suitable for establishing a culture system in vitro. Melatonin promotes oocyte meiotic maturation and antioxidative capacity and inhibits apoptosis via the p53-bax-bcl2 and caspase-dependent pathways, which have important potential to improve the maturation and quality of oocytes.

Hepatic transcriptomic analysis reveals that Hif1α/ldha signal is involved in the regulation of hypoxia stress in black rockfish Sebastes schlegelii.

Hypoxia has become a common problem for aquatic organisms due to the interaction of global climate change and human activity. Black rockfish inhabits rocky reefs in waters of Japan, Korea and China, whereas the limited hypoxia tolerance leads to mass mortality and great economic loss. In this study, high-throughput RNA-seq for transcriptomic analysis was used to investigate the hepatic response in black rockfish under hypoxia (critical oxygen tension, Pcrit; loss of equilibrium, LOE) and reoxygenation (recover normal dissolved oxygen 24 h, R24) to explore the mechanisms underlying hypoxia tolerance and adaptation. A total of 573,040,410 clean reads and 299 differentially expressed genes (DEGs) in total were obtained during hypoxia and reoxygenation. GO annotation and Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that the DEGs are mainly enriched in the biochemical metabolic pathways and HIF-1 signaling pathways. Transcriptomic analysis also identified 18 DEGs associated with HIF-1 signaling pathway (hif1α, tf, epo, hmox, gult1, mknk2, ldha, pfkfb3, hkdc, aldoa) and biological process (hif2α, apoeb, bcl6, mr1, errfi1, slc38a4, igfbp1a, ap4m1) as further validated by quantitative real-time PCR. Moreover, hif1α was positively or negatively correlated with glucose (ldha, pfkfb3, hkdc, aldoa) and lipid (apoeb) metabolism-related genes. The mRNA level of hif1α was significantly up-regulated under acute hypoxia stress and obtained the higher values than hif2α. Meanwhile, hif1α recognized the hypoxia response element located in the promoter of ldha and directly bound to the promoter to transactivate ldha expression. These results indicated that black rockfish may mainly utilize glycolysis to maintain homeostasis, and hif1α facilities hypoxia tolerance by modulating ldha expression.

Hypoxia stress induces hepatic antioxidant activity and apoptosis, but stimulates immune response and immune-related gene expression in black rockfish Sebastes schlegelii.

Dissolved oxygen concentrations both in the open ocean and coast have been declining due to the interaction of global climate change and human activity. Fish have evolved different adaptative strategies to cope with possibly damage induced by hypoxic environments. Black rockfish as important economic fish widely reared in the offshore sea cage, whereas related physiological response subject to hypoxia stress remained unclear. In this study, hepatic anti-oxidant enzymes (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH-Px]), aminotransferase (AST) and alanine aminotransferase (ALT) activities, lipid peroxidation (LPO), malondialdehyde (MDA) and glutathione (GSH) content, immunological parameters and the expression of apoptosis (bax, bcl2, p53, caspase3, xiap) and immune-related genes (c3, il-1ß, ccl25, saa, hap, isg15) of black rockfish were determined during hypoxia and reoxygenation to illustrate the underlying defense response mechanisms. Results showed that hypoxia stress remarkably increased hepatic LPO and MDA content, AST and ALT activity and proportion of pyknotic nucleus. Hepatic SOD, CAT and GSH-Px activity manifested similar results, whereas GSH levels significantly decreased under hypoxia stress. The apoptosis rate of hepatocyte increased during hypoxia stress and reoxygenation. Meanwhile, p53, caspase3, bax and xiap mRNAs and bax/bcl2 rations were significantly up-regulated under hypoxia stress. However, bcl2 mRNA was significantly down-regulated. Interestingly, hypoxia stress significantly increased NBT-positive cell percent, phagocytic index, respiratory burst and ACH50 activity, and lysozyme activity. The mRNA levels of c3, ilß, ccl25, saa, hap and isg15 were significantly up-regulated in the liver, spleen and head-kidney under hypoxia stress. The above parameters recovered to normal status after reoxygenation for 24 h Thus, hypoxia stress impairs hepatic antioxidant capacity, induces oxidative damage and apoptosis via the xiap-p53-bax-bcl2 and the caspase-dependent pathways, but enhances host immunity by regulating nonspecific immune indices and related genes expression to maintain homeostasis in black rockfish. These findings will help fully understand the hypoxia tolerance mechanisms of black rockfish and provide more data for offshore open ocean farming.

Altered physiological response and gill histology in black rockfish, Sebastes schlegelii, during progressive hypoxia and reoxygenation.

Hypoxia has gradually become common in aquatic ecosystems and imposes a significant challenge for fish farming. The loss of equilibrium (LOE), 50% lethal time (LT50), plasma cortisol, glucose, red blood cells (RBC), hemoglobin (Hb), gill histological alteration, and related parameters (lamellar length [SLL] and width [SLW], interlamellar distance [ID], basal epithelial thickness [BET], lamellar surface area [LA], and gill surface area [GSA]); respiratory rate; the proportion of the secondary lamellae available for gas exchange (PAGE); and hypoxia-inducible factor (hif-1α, hif-2α) mRNA expression were determined during progressive hypoxia and reoxygenation (R-0, R-12, R-24 h) to illustrate the underlying physiological response mechanisms in black rockfish Sebastes schlegelii. Results showed that the DO concentration significantly decreased during progressive hypoxia, while DO at LOE and LT50 were 2.42 ± 0.10 mg L-1 and 1.67 ± 0.38 mg L-1, respectively. Cortisol and glucose were significantly increased at LOE and LT50, with the highest levels observed at LT50, and then gradually recovered to normal within reoxygenation 24 h. RBC number and Hb results were like those of glucose. Hypoxia stress resulted in lamellar clubbing, hypertrophy, and hyperplasia. Respiratory frequency significantly increased at LOE and decreased at LT50. Lamellar perimeters, SLL, ID, LA, GSA, and PAGE, significantly increased at LOE and LT50, with the highest values observed at LT50. However, SLW and BET significantly decreased at LOE, LT50, and R-0. These parameters recovered to nearly normal levels at R-24 h. hif-1α mRNAs in gill and liver were significantly upregulated at LOE and LT50, and recovery to normal after reoxygenation 24 h. hif-2α mRNAs in gill was similar to that of hif-1α, whereas hepatic hif-2α mRNAs remained unchanged during hypoxia-reoxygenation. These results indicated that progressive hypoxia stress elevated RBC number, Hb, cortisol, and glucose levels, induced the alteration of gill morphology, increased LA and GSA, stimulated respiratory frequency and PAGE, and upregulated the transcription of hif-1α and hif-2α in gill and liver. Reoxygenation treatment for 24 h alleviated the stress mentioned above effects. These findings expand current knowledge on hypoxia tolerance in black rockfish Sebastes schlegelii.

Reference gene validation for quantification of gene expression during ovarian development of turbot (Scophthalmus maximus).

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a powerful and sensitive method used in gene expression analysis. Suitable reference genes, which are stable under all experimental circumstances and tissues significantly improve the accuracy of qRT-PCR data. In this study, the stability of six genes, namely, 18S ribosomal RNA (18s), beta-actin (actb), elongation factor 1-alpha (ef1α), glyceraldehyde-3-phosphate-dehydrogenase (gapdh), cathepsin D (ctsd), and beta-2-microglobulin (b2m) were evaluated as potential references for qRT-PCR analysis. The genes were examined in the hypothalamus-pituitary-ovary-liver (HPOL) axis throughout turbot ovarian development via using the geNorm, NormFinder and BestKeeper algorithms. Results showed that the most stable reference genes were ef1α, actb, and ctsd in the hypothalamus, pituitary, ovary and liver, respectively. The best-suited gene combinations for normalization were 18s, ef1α, and ctsd in the hypothalamus; actb, ctsd, and 18s in the pituitary; actb, and ctsd in the ovary; gapdh and ctsd in the liver. Moreover, the expression profile of estrogen receptor α (erα) manifested no significant difference normalization to the aforementioned best-suited gene during turbot ovarian development. However, no single gene or pair of genes is suitable as an internal control and account for the amplification differences among the four tissues during ovarian development. In summary, these results provide a basic data for the optimal reference gene selection and obtain highly accurate normalization of qRT-PCR data in HPOL axis-related gene expression analysis during turbot ovarian development.

Alternations in oxidative stress, apoptosis, and innate-immune gene expression at mRNA levels in subadult tiger puffer (Takifugu rubripes) under two different rearing systems.

Tiger puffer (Takifugu rubripes) is one of the major aquaculture fish species in China due to its high economic value. In this study, the transcriptions of hepatic antioxidant enzyme, stress, apoptosis, and immune-related genes of sub-adult tiger puffers (Takifugu rubripes) were evaluated under two different rearing systems [offshore sea cage aquaculture system (OSCS) and recirculating aquaculture system (RAS)]. Results showed that the mRNA expression levels of the antioxidant enzyme (mn-sod, cu/zn-sod, gpx, and gr) and stress-related (hsp70 and hsp90) genes of male tiger puffers reared in the OSCS were significantly higher than female fish reared in the OSCS and fish reared in the RAS. The anti-apoptotic gene bcl2 exhibited the similar results. By contrast, the mRNAs of the pro-apoptotic genes (p53, caspase8, caspase9, and caspase3) of male tiger puffers reared in the OSCS were significantly lower than female fish reared in the OSCS and fish reared in the RAS. Male tiger puffers reared in the OSCS displayed significantly higher complement components (c3) and inflammatory cytokine (il-6) mRNAs, whereas B-cell activating factor (baf) and tumor necrosis factor α (tnf-α) mRNAs remained unchanged. Meanwhile, the mRNA levels of pro-apoptotic (bax, caspase8) and immunity-related (c3, il-6 and il-7) genes of female tiger puffers reared in the OSCS were significantly lower and higher than female fish reared in the RAS, respectively. In conclusion, the hepatic antioxidant, anti-apoptosis, and innate immunity of tiger puffers reared in the OSCS were better than fish in the RAS, male tiger puffer obtained the best values. These results expand the knowledge on the combined RAS and OSCS alternative aquaculture model for tiger puffers and aid in their management in captive.

Molecular cloning, characterization, and mRNA expression of gonadotropins during larval development in turbot (Scophthalmus maximus).

Gonadotropins (GtHs) play a pivotal role in regulating the reproductive axis and puberty. In this study, full-length sequences coding for common glycoprotein α subunit (CGα) and luteinizing hormone ß (LHß) were isolated from female turbot (Scophthalmus maximus) pituitary by homology cloning and a strategy based on rapid amplification of cDNA end-polymerase chain reaction. Results showed that the two cDNAs consisted of 669 and 660 nucleotides encoding 129 and 139 amino acids, respectively. CGα and LHß manifested typical characteristics of glycoprotein hormones, high homologies with the corresponding sequences of available teleosts, and high homology with that of Hippoglossus hippoglossus. CGα, FSHß, and LHß mRNAs were abundant in the pituitary, but less expressed in extra-pituitary tissues. The cgα, fshß, and lhß were detected at 1-day post-hatching (dph) and peaked simultaneously at early-metamorphosis (22 dph). cgα and fshß mRNA levels were significantly increased at pre-metamorphosis, peaked in early metamorphosis, and then gradually decreased until metamorphosis was completed. Conversely, lhß mRNA levels gradually decreased at pre-metamorphosis, dramatically peaked at early metamorphosis, and then decreased during metamorphosis. In addition, the mRNA levels of cgα were significantly higher than those of fshß and lhß during turbot larval metamorphic development, whereas no significant difference was found between fshß and lhß. These results suggested (i) an early activation of the GtHs system after hatching, which was the highest expression at early metamorphosis, and (ii) FSHß and LHß were together involved in the establishment of the reproductive axis during larval development in turbot. These findings contribute to further understanding the potential roles of GtHs during fish larval development.

Molecular function of gonadotrophins and their receptors in the ovarian development of turbot (Scophthalmus maximus).

Gonadotropins (GtHs) and their receptors (follicle-stimulating hormone receptor, FSHR; luteinizing hormone receptor, LHR) are involved in the regulation of gametogenesis and play important roles during the reproductive cycles in vertebrate species, including fish. This minireview focuses on the molecular characterization and quantification of GtHs (common glycoprotein α subunit CGα, FSHß, and LHß) and their receptors (FSHR and LHR) throughout the reproductive cycle of female turbot Scophthalmus maximus. Information about GtHs, FSHR, LHR as well as other ligand-receptors interaction from different teleosts are also included in this review for the implications they may have on the functions of GtHs, FSHR and LHR in the reproductive development of turbot. These findings may enhance our understanding of the physiological roles of the GtHs, FSHR and LHR in controlling of flatfish ovarian development during the reproductive cycle and contributing to the improvement of management strategies for turbots in captivity.

Involvement and expression of growth hormone/insulin-like growth factor member mRNAs in the ovarian development of turbot (Scophthalmus maximus).

Accumulating evidence suggests that the growth hormone (GH)/insulin-like growth factor (IGF) system participates in fish reproduction. To understand the physiological functions of the GH/IGF system, the mRNA expression profiles of all known members of the GH/IGF system, including hepatic and ovarian gh, GH receptor (ghr), IGFs (igf-i, igf-ii), IGF-I receptor (igf-ir) and IGF binding protein (igfbp1, igfbp2), pituitary gh, and hepatic vitellogenin (vtg) were investigated during ovarian development in turbot Scophthalmus maximus. Results showed that ghr, igf-i, igf-ii, igf-ir, and igfbp2 were expressed in the liver and ovary, whereas igfbp1 and gh were undetected. The hepatosomatic index (HSI) and gonadosomatic index (GSI) gradually increased and peaked during the late vitellogenesis (Latvtg) and migratory nucleus (Mig-nucl) stages, respectively. The mRNA expression profiles of ovarian ghr, igf-ii, hepatic igf-ir, vtg, and pituitary gh were similar to the HSI; ovarian igf-i and igf-ir expression was close to the GSI. However, the hepatic mRNA levels of ghr, igf-i, and igf-ii peaked at the early vitellogenesis (Evtg) stage, and then drastically declined during ovarian development. The mRNA expression of hepatic igfbp2 decreased and reached the lowest at the atresia (Atre) stage, whereas that of ovarian igfbp2 increased and peaked at Latvtg stage. Furthermore, significant correlations between pituitary gh, ovarian ghr, igf-i, and igf-ii, and hepatic ghr, igf-i, igf-ir, and igf-ii were observed, respectively. These results suggest that GH/IGF members appear to play distinct roles in the regulation of ovarian development in turbot and will be valuable for fish reproduction and broodstock management of aqua-cultured fish species.

A new 3D printed titanium metal trabecular bone reconstruction system for early osteonecrosis of the femoral head.

Presently, biomechanical support therapy for the femoral head has become an important approach in the treatment of early osteonecrosis of the femoral head (ONFH). Previous studies have reported that the titanium metal trabecular bone reconstruction systems (TMTBRS) achieved satisfactory clinical results for the treatment of early femoral head necrosis. Electron beam melting technology (EBMT) is an important branch of 3D printing technology, which enables the construction of an interface that is required for support of bone in-growth. However, the effect of TMTBRS created using EBMT for clinical applications for early ONFH is still unknown. At present, there are no reports on this topic worldwide. The purpose of this study was to assess the safety of a new 3D printed TMTBRS implant and to evaluate its clinical efficacy in early ONFH.Thirty patients who underwent surgery for ONFH were selected. The stages of ONFH were classified according to the Association Research Circulation Osseus (ARCO) classification. They were followed-up and radiological examination was performed at 6, 12, and 24 months post-surgery to assess TMTBRS stability and bone growth in the bone trabecular holder portion surface. To evaluate hip function, postoperative Harris and Visual Analogue Scale (VAS) scores were used.The postoperative Harris score increased significantly and VAS score decreased significantly at the 12-month follow-up compared to the 24-month follow-up, wherein the Harris score declined slightly and the VAS score was slightly elevated with the aggravation of ONFH. With the passage of time, postoperative improvement rates were 100% for IIA, 70% for IIB, and 0% for IIC. Hip-preserving rates were 100% for IIA, 100% for IIB, and 50% for IIC.The effect of TMTBRS treatment for early ONFH in ARCO IIA and ARCO IIB is satisfactory. However, it is not recommended for a relatively large area of necrosis such as in ARCO IIC.

Systematic Review of Three Electrical Stimulation Techniques for Rehabilitation After Total Knee Arthroplasty.

BACKGROUND: The comparative effectiveness of neuromuscular electrical stimulation (NMES), transcutaneous electrical nerve stimulation (TENS), and electroacupuncture (EA) for improving patient rehabilitation following total knee arthroplasty (TKA) is controversial. Therefore we conducted this systematic review to assess the available evidence. METHODS: The PubMed, OVID, and ScienceDirect databases were comprehensively searched and studies were selected and analyzed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations. Data were extracted and qualitatively synthesized for several outcomes. RESULTS: Data were analyzed from 17 randomized controlled trials involving 1285 procedures: 8 NMES studies (608 procedures), 7 TENS studies (560 procedures), and 2 EA studies (117 procedures). Qualitative analysis suggested that NMES was associated with higher quadriceps strength and functional recovery after TKA. Recovery benefits were maximal when the stimulation was performed once or twice a day for 4-6 weeks at an intensity of 100-120 mA and frequency of 30-100 Hz. The electrode should be sufficiently large (100-200 cm2) to reduce discomfort. TENS at an intensity of 15-40 mA and frequency of 70-150 Hz provided effective analgesia after TKA. EA at an intensity of 2 mA and frequency of 2 Hz may also provide postoperative analgesia of TKA. CONCLUSION: As adjunct modalities, NMES and TENS can effectively improve rehabilitation after TKA without triggering significant intolerance, and maximal benefits depend on optimized parameters and intervention protocols. EA may be an effective adjunct modality for analgesia after TKA.

Roles of insulin-like growth factors in metamorphic development of turbot (Scophthalmus maximus).

Larval turbot (Scophthalmus maximus) undergo metamorphosis, a late post-embryonic developmental event that precedes juvenile transition. Insulin-like growth factors (IGFs) are important endocrine/autocrine/paracrine factors that provide essential signals to control of the embryonic and postnatal development of vertebrate species, including fish. Accumulating evidence suggests that IGFs are involved in regulating the metamorphic development of flatfish. This mini review focus on the functions of all known IGFs (IGF-I and IGF-II) during the metamorphic development of turbot. Information about IGFs and insulin-like growth factors binding proteins (IGFBPs) from other teleosts is also included in this review to provide an overview of IGFs functions in the metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGFs system in controlling of flatfish metamorphosis and contributing to the improvement of broodstock management strategies for larval turbot.

Molecular characterization and quantification of estrogen receptors in turbot (Scophthalmus maximus).

Estrogens regulate various reproductive processes via estrogen receptor (ER)-mediated signaling pathway in vertebrates. In this study, full-length sequences coding for ERα, ERß1 and ERß2 were isolated from female turbot (Scophthalmus maximus) by homology cloning and a strategy based on rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR). The nucleotide and amino acid sequences of turbot ERs showed high homologies with the corresponding sequences of other fish species and significant homology with the Japanese flounder (Paralichthys olivaceus) and the European sea bass (Dicentrarchus labrax). Turbot ERs contained six typical nuclear receptor-characteristic domains and exhibited high evolutionary conservation in the functional domains. Quantitative real-time polymerase chain reaction analysis revealed that the erα and erß (ß1, ß2) mRNAs were abundant in the liver and ovary, respectively. Furthermore, hepatic mRNA levels of erα and vitellogenin (vtg) were found increased gradually from pre-vitellogenesis to late-vitellogenesis stages, with the highest values observed at the late-vitellogenesis stage, and then decreased from migratory-nucleus to atresia stages. However, mRNA levels of erα in the ovary remained unchanged during ovarian development. Hepatosomatic index, gonadosomatic index, serum estradiol-17ß and the mRNA levels of erß1 and erß2 in the ovary manifested results similar to the expression of erα mRNAs in the liver. These findings indicated that ERα is mainly involved in hepatic vitellogenesis, and ERßs may play crucial roles to regulate ovarian development in turbot. Overall, this study improves understanding of the physiological functions of turbot ERs, which will be valuable for fish reproduction and broodstock management.

Naringin enhances osteogenic differentiation through the activation of ERK signaling in human bone marrow mesenchymal stem cells.

OBJECTIVES: Naringin has been reported to regulate bone metabolism. However, its effect on osteogenesis remains unclear. The aim was to investigate the effect of naringin on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) through the activation of the ERK signaling pathway in osteogenic differentiation. MATERIALS AND METHODS: Annexin V-FITC assay and MTT assay were used to measure the effect of naringin on cytotoxicity and proliferation of hBMSCs, respectively. Alkaline phosphatase activity analysis, Alizarin Red S staining, Western blotting, and real-time PCR assay were used to evaluate both the potential effect of naringin on osteogenic differentiation and the role of ERK signaling pathway in osteogenic differentiation. RESULTS: Our results showed that naringin had no obvious toxicity on hBMSCs, and could significantly promote the proliferation of hBMSCs. Naringin also enhanced the osteogenic differentiation of hBMSCs and increased the protein and mRNA expression levels of osteogenic markers such as Runx-2, OXS, OCN, and Col1 in a dose-dependent manner. In addition, we found that the enhancing effect of naringin on osteogenic differentiation was related to the activation of phosphor-ERK, with an increase in duration of activity from 30 min to 120 min. More importantly, both the enhancing effect of naringin on osteogenic differentiation and the activity effect of naringin on ERK signaling pathway were reversed by U0126 addition. CONCLUSION: Our findings demonstrated that naringin promoted proliferation and osteogenesis of hBMSCs by activating the ERK signaling pathway and it might be a potential therapeutic agent for treating or preventing osteoporosis.

Ameliorative effect of vitamin E on hepatic oxidative stress and hypoimmunity induced by high-fat diet in turbot (Scophthalmus maximus).

This study was conducted to examine the effects of vitamin E on growth performance, oxidative stress and non-specific immunity of turbot (Scophthalmus maximus) fed with high-fat diet. Results showed that high-fat diet significantly increased hepatosomatic index, viscerosomatic index, hepatic malondialdehyde level and decreased catalase and superoxide dismutase activities, whereas final weight, specific growth rate and survival rate remained unchanged. Meanwhile, nitro blue tetrazolium positive leucocytes of head kidney, respiratory burst activity in head-kidney macrophage, phagocytic index and serum lysozyme activity were significantly reduced after feeding with high-fat diet. Furthermore, fish fed with high-fat diet promoted higher expression of heat shock protein (hsp70, hsp90), and inhibited expression of complement component 3 (c3) in the liver and tumor necrosis factor-α (tnf-α), interleukine 1ß (il-1ß), toll like receptor 22 (tlr-22) in the spleen and head-kidney, respectively. However, simultaneous supplementation with 480 mg kg-1 vitamin E protected turbot against high-fat diet-induced hepatic oxidative stress, hypoimmunity through attenuating lipid peroxidation, renewing antioxidant enzymes activities and nonspecific immune responses, and modulating the expression of stress protein (hsp70, hsp90) and immune-related genes (c3, tnf-α, il-1ß, tlr-22). In conclusion, the obtained results indicate the vitamin E as a wildly used functional feed additive contributes potentially to alleviate high-fat diet-induced hepatic oxidative stress and hypoimmunity, maintain the health, and improve the broodstock management for turbot.

Downregulated miR-29a/b/c during Contact Inhibition Stage Promote 3T3-L1 Adipogenesis by Targeting DNMT3A.

Differentiation of 3T3-L1 cells into adipocytes involves a highly-orchestrated series of events including contact inhibition (CI), clonal expansion, growth arrest, and terminal differentiation. Recent study demonstrated that 3T3-L1 preadipocytes will not be differentiated into mature adipocytes without CI stage, which indicated that CI stage plays an important role during 3T3-L1 adipogenesis. However, the molecular mechanism is not yet fully understood. In the present study, we found that the expression level of miR-29a/b/c was decreased and the expression of DNMT3A was up-regulated during CI stage, respectively. Furthermore, overexpression of miR-29a/b/c during CI stage inhibits adipogenesis significantly but not at other stages. In addition, miR-29a/b/c repressed DNMT3A expression by directly targeting its 3' untranslated region (3' UTR). Our data reveal a novel mechanism of miR-29a/b/c in the regulation of adipogenesis.

Acute hyperthermic responses of heat shock protein and estrogen receptor mRNAs in rainbow trout hepatocytes.

Heat shock proteins (HSPs) are induced upon elevated temperature in fishes. HSPs also function as molecular chaperones for cellular proteins, including steroid hormone receptors. Estrogen receptors (ERs) are critical for the hormone signaling necessary during the liver production of the yolk precursor protein vitellogenin in oviparous vertebrates. Considering the possible regulatory role of HSPs on the ER signaling pathway, the present study characterized the mRNA expression of all known isoforms of HSP70 (hsp70a, hsp70b), HSP90 (hsp90a1a, hsp90a1b, hsp90a2a, hsp90a2b, hsp90b1, hsp90b2), and ERs (erα1, erα2, erß1, erß2) in Rainbow Trout hepatocytes following an acute heat shock (1h at 25°C) compared to a control treatment (12°C). The results showed that the mRNA levels of hsp70a, hsp70b, hsp90a1b, hsp90a2a, and hsp90b2 were significantly increased after heat shock, while erα1 mRNA levels were significantly reduced by this treatment. hsp90a1a, hsp90a2b, hsp90b1, erα2, erß1 and erß2 were unaffected by this acute hyperthermic treatment. Comparatively, the responses of the two hsp70 isoforms were much greater than the hsp90 isoforms. Acute heat shock treatment of hepatocytes followed by a 24h exposure to 17ß-estradiol (E2) exposure also resulted in decreased expression of erα1 mRNA, but not vitellogenin (vtg) mRNA. This study showed that some hsp70 and hsp90 isoforms display a robust response to an acute hyperthermic treatment in Rainbow Trout hepatocytes. Among the transcripts measured here, the erα1 isoform uniquely showed significantly decreased mRNA levels upon acute heat treatment.

Expression of insulin-like growth factors at mRNA levels during the metamorphic development of turbot (Scophthalmus maximus).

Insulin-like growth factors I and II (IGF-I and IGF-II) are important regulators of vertebrate growth and development. This study characterized the mRNA expressions of igf-i and igf-ii during turbot (Scophthalmus maximus) metamorphosis to elucidate the possible regulatory role of the IGF system in flatfish metamorphosis. Results showed that the mRNA levels of igf-i significantly increased at the early-metamorphosis stage and then gradually decreased until metamorphosis was completed. By contrast, mRNA levels of igf-ii significantly increased at the pre-metamorphosis stage and then substantially decreased during metamorphosis. Meanwhile, the whole-body thyroxine (T4) levels varied during larval metamorphosis, and the highest value was observed in the climax-metamorphosis. The mRNA levels of igf-i significantly increased and decreased by T4 and thiourea (TU, inhibitor of endogenous thyroid hormone) during metamorphosis, respectively. Conversely, the mRNA levels of igf-ii remained unchanged. Furthermore, TU significantly inhibited the T4-induced mRNA up-regulation of igf-i during metamorphosis. The whole-body thyroxine (T4) levels were significantly increased and decreased by T4 and TU during metamorphosis, respectively. These results suggested that igf-i and igf-ii may play different functional roles in larval development stages, and igf-i may have a crucial function in regulating the early metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGF system to control flatfish metamorphosis and contribute to the improvement of broodstock management for larvae.