Preparation of Novel Sea Cucumber Intestinal Peptides to Promote Tibial Fracture Healing in Mice by Inducing Differentiation of Hypertrophic Chondrocytes to the Osteoblast Lineage.

SCOPE: Hypertrophic chondrocytes have a decisive regulatory role in the process of fracture healing, and the fate of hypertrophic chondrocytes is not only apoptosis. However, the mechanism of sea cucumber (Stichopus japonicus) intestinal peptide (SCIP) on fracture promotion is still unclear. This study aims to investigate the effect of sea cucumber intestinal peptide on the differentiation fate of hypertrophic chondrocytes in a mouse tibial fracture model. METHODS AND RESULTS: Mice are subjected to open fractures of the right tibia to establish a tibial fracture model. The results exhibit that the SCIP intervention significantly promotes the mineralization of cartilage callus, decreases the expression of the hypertrophic chondrocyte marker Col X, and increases the expression of the osteoblast marker Col I. Mechanically, SCIP promotes tibial fracture healing by promoting histone acetylation and inhibiting histone methylation, thereby upregulating pluripotent transcription factors induced the differentiation of hypertrophic chondrocytes to the osteoblast lineage in a manner distinct from classical endochondral ossification. CONCLUSION: This study is the first to report that SCIP can promote tibial fracture healing in mice by inducing the differentiation of hypertrophic chondrocytes to the osteoblast lineage. SCIP may be considered raw material for developing nutraceuticals to promote fracture healing.

Isolation and Identification of a Novel Anti-Dry Eye Peptide from Tilapia Skin Peptides Based on In Silico, In Vitro, and In Vivo Approaches.

Tilapia skin is a great source of collagen. Here, we aimed to isolate and identify the peptides responsible for combating dry eye disease (DED) in tilapia skin peptides (TSP). In vitro cell DED model was used to screen anti-DED peptides from TSP via Sephadex G-25 chromatography, LC/MS/MS, and in silico methods. The anti-DED activity of the screened peptide was further verified in the mice DED model. TSP was divided into five fractions (TSP-I, TSP-II, TSP-III, TSP-IV, and TSP-V), and TSP-II exerted an effective effect for anti-DED. A total of 131 peptides were identified using LC/MS/MS in TSP-II, and NGGPSGPR (NGG) was screened as a potential anti-DED fragment in TSP-II via in silico methods. In vitro, NGG restored cell viability and inhibited the expression level of Cyclooxygenase-2 (COX-2) protein in Human corneal epithelial cells (HCECs) induced by NaCl. In vivo, NGG increased tear production, decreased tear ferning score, prevented corneal epithelial thinning, alleviated conjunctival goblet cell loss, and inhibited the apoptosis of corneal epithelial cells in DED mice. Overall, NGG, as an anti-DED peptide, was successfully identified from TSP, and it may be devoted to functional food ingredients or medicine for DED.

DHA- and EPA-Enriched Phosphatidylcholine Suppress Human Lung Carcinoma 95D Cells Metastasis via Activating the Peroxisome Proliferator-Activated Receptor γ.

The antineoplastic effects of docosahexaenoic acid-containing phosphatidylcholine (DHA-PC) and eicosapentaenoic acid-containing phosphatidylcholine (EPA-PC) were explored, and their underlying mechanisms in the human lung carcinoma 95D cells (95D cells) were investigated. After treatment of 95D cells with DHA-PC or EPA-PC, cell biological behaviors such as growth, adhesion, migration, and invasion were studied. Immunofluorescence and western blotting were carried out to assess underlying molecular mechanisms. Results showed that 95D cells proliferation and adherence in the DHA-PC or EPA-PC group were drastically inhibited than the control group. DHA-PC and EPA-PC suppressed the migration and invasion of 95D cells by disrupting intracellular F-actin, which drives cell movement. The protein expression of PPARγ was induced versus the control group. Furthermore, critical factors related to invasion, including matrix metallopeptidase 9 (MMP9), heparanase (Hpa), and vascular endothelial growth factor (VEGF), were drastically downregulated through the PPARγ/NF-κB signaling pathway. C-X-C chemokine receptor type 4 (CXCR4) and cofilin were significantly suppressed via DHA-PC and EPA-PC through the PPARγ/phosphatase and tensin homolog (PTEN)/serine-threonine protein kinase (AKT) signaling pathway. DHA-PC and EPA-PC reversed the PPARγ antagonist GW9662-induced reduction of 95D cells in migration and invasion capacity, suggesting that PPARγ was directly involved in the anti-metastasis efficacy of DHA-PC and EPA-PC. In conclusion, DHA-PC and EPA-PC have great potential for cancer therapy, and the antineoplastic effects involve the activation of PPARγ. EPA-PC showed more pronounced antineoplastic effects than DHA-PC, possibly due to the more robust activation of PPARγ by EPA-PC.

Tilapia Skin Peptides Ameliorate Cyclophosphamide-Induced Anxiety- and Depression-Like Behavior via Improving Oxidative Stress, Neuroinflammation, Neuron Apoptosis, and Neurogenesis in Mice.

Anxiety- and depression-like behavior following chemotherapy treatment occurs in cancer patients with high probability and no specific therapeutics are available for treatment and prevention of this complication. Here, tilapia skin peptides (TSP), a novel enzymatically hydrolyzed bioactive peptide mixture, obtained from tilapia (Oreochromis mossambicus) scraps, were studied on cyclophosphamide (CP)-induced anxiety- and depression-like behavior in mice. Mice were received intraperitoneal injection of CP for 2 weeks, while TSP was administered for 4 weeks. After the end of the animal experiment, behavioral, biochemical, and molecular tests were carried out. The mice decreased preference for sugar water, increased immobility time in the forced swimming and tail suspension test, and decreased travel distance in the open field test in the Model group, compared with the Control group. Abnormal changes in behavioral tests were significantly improved after the TSP treatment. Additionally, abnormalities on superoxide dismutase, malondialdehyde, glutathione peroxidase were rescued by administration of 1000 mg/kg/d TSP in mice than that of the Model group. TSP has normalized the expression of Iba-1 and the levels of TNF-α and IL-1ß in the hippocampus of mice, which indicated that TSP could observably ameliorate neuroinflammatory response in the hippocampus of mice. TSP ameliorated the apoptosis of hippocampal neurons of CA1 and CA3 regions in the TSP group vs. the Model group. The number of doublecortin positive cells was drastically increased by administering 1000 mg/kg/d TSP in mice vs. the Model group. Furthermore, TSP reversed the Nrf2/HO-1 signaling pathway, BDNF/TrkB/CREB signaling pathway, and reduced the Bcl-2/Bax/caspase-3 apoptosis pathway. In conclusion, TSP could restore CP-induced anxiety- and depression-like behavior via improving oxidative stress, neuroinflammation, neuron apoptosis, and neurogenesis in mice hippocampus.

Tilapia skin peptides restore cyclophosphamide-induced premature ovarian failure via inhibiting oxidative stress and apoptosis in mice.

Tilapia (Oreochromis mossambicus) skin high value-added compounds have not been fully utilized in tilapia processing. Here, the protective effects of tilapia skin peptides (TSP) on primary ovarian failure (POF) and their underlying mechanisms in mice were investigated. Cyclophosphamide (CP) was injected intraperitoneally (ip) for 14 days (10 mg kg-1 d-1) to establish a mouse model of POF. At the same time, the mice were given intragastrically (ig) TSP for 30 days (250 mg kg-1 d-1, 500 mg kg-1 d-1, and 1000 mg kg-1 d-1, respectively). The ovarian index, estrous cycle, hormone level, changes in the number of follicles at various levels, and biochemical tests were carried out at the end of the experiment. The body weight and ovarian index of mice in the POF group were markedly lower than that of the control group. Treatment with TSP reversed these changes significantly. TSP administration significantly restored the estrous cycle disorder of the mice versus that of the POF group. The level changes of progesterone (P), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) induced by CP were significantly reversed by TSP treatment. TSP inhibited oxidative stress in CP-induced mice by enhancing the total superoxide dismutase (T-SOD) activity and reducing malondialdehyde (MDA) levels in the ovaries. TSP improved the apoptosis of ovarian granulosa cells in CP-induced mice compared with the POF group. Furthermore, TSP regulated the Bcl-2/Bax/caspase-3 apoptosis pathway and enhanced the Nrf2/HO-1 signaling pathway. In conclusion, TSP could improve CP-induced POF via alleviating ovarian oxidative stress and granulosa cell apoptosis.

Hydroxytyrosol alleviates oxidative stress and neuroinflammation and enhances hippocampal neurotrophic signaling to improve stress-induced depressive behaviors in mice.

Hydroxytyrosol (HT), the main phenolic compound in olives and olive products, has antioxidative, anti-inflammatory, neuroprotective, and other physiological functions. The effects of HT on depression are unclear. The aim of this study was to explore the effects of HT on chronic unpredictable mild stress (CUMS) induced depressive-like behaviors. Mice were exposed to CUMS for 9 weeks and then treated with HT beginning in the second week and continuing for 7 weeks. Behavioral, biochemical, and molecular tests were conducted at the end of the experiment. The sucrose preference was significantly decreased in the CUMS group versus the healthy control group. Also, immobility times in forced swimming and tail suspension tests were increased in CUMS-induced mice, but treatment with HT significantly reversed this change. HT ameliorated oxidative stress in CUMS-exposed mice by enhancing superoxide dismutase activity and reducing reactive oxygen species and malondialdehyde levels in the hippocampus. HT administration significantly suppressed microglia activation and inhibited the expression of tumor necrosis factor alpha and interleukin 1 beta in the hippocampus versus the untreated group. The expression level of glial fibrillary acidic protein (GFAP) and the number of GFAP-immunoreactive astrocytes in the hippocampus were significantly augmented by HT. Furthermore, HT treatment increased the expression of hippocampal brain-derived neurotrophic factor (BDNF), phosphorylated tropomyosin receptor kinase B (p-TrkB), and phosphorylated c-AMP response element binding protein (p-CREB) compared with the untreated CUMS group. Overall, HT improved CUMS-induced depressive-like behaviors in mice by alleviating oxidative stress and neuroinflammation and by enhancing the BDNF/TrkB/CREB signaling pathway.

Polyphenols from Broussonetia papyrifera Induce Apoptosis of HepG2 Cells via Inactivation of ERK and AKT Signaling Pathways.

The extract of Broussonetia papyrifera has been proved to have antitumor activity. However, the underlying mechanism remains unclear. This study aimed to elucidate the mechanism of apoptosis of HepG2 cells induced by polyphenols from Broussonetia papyrifera (PBPs). The results revealed that PBPs inhibited the proliferation of HepG2 cells in a dose-dependent and time-dependent manner. Flow cytometry analysis showed that PBPs increased the apoptosis ratio of HepG2 cells significantly. PBPs increased intracellular reactive oxygen species (ROS) production and decreased intracellular superoxide dismutase (SOD) level of HepG2 cells. PBPs induced cell cycle arrest at G1 phase. Western blotting showed that PBPs upregulated the ratio of Bax/Bcl-2 and the expression level of Caspase-3, and activated p53 in HepG2 cells. The inhibition of proliferative relative signals (protein kinase B, PKB/AKT) and survival relative signals (extracellular signal-regulated kinase, ERK) were also observed in PBP-treated HepG2 cells. Our findings suggest that apoptosis of HepG2 cells induced by PBPs is mitochondria-mediated via inactivation of ERK and AKT signaling pathways.

Goose toll-like receptor 3 (TLR3) mediated IFN-γ and IL-6 in anti-H5N1 avian influenza virus response.

Induction of the innate immune pathways is critical for early anti-viral defense. How geese recognize viral molecules and activate these pathways is not well understood. In mammals, Toll-like receptor 3 (TLR3) recognizes double-stranded RNA. Activation of TLR3 induces the activation of NF-кB and the production of type-I interferon. In this study, the goose TLR3 gene was cloned using rapid amplification of cDNA ends. Goose TLR3 encoded an 896-amino-acid protein, containing a signal secretion peptide, 14 extracellular leucine-rich repeat domains, a transmembrane domain, a Toll/interleukin-1 receptor signaling domain, and shared 46.7-84.4% homology with other species. Tissue expression of goose TLR3 varied markedly and was highest in the pancreas and lowest in the skin. Human embryonic kidney 293 cells transfected with goose TLR3 and NF-κB-luciferase-containing plasmids responded significantly to poly i:c. The expression of TLR3, IL-6 and IFN-γ mRNA, but not IL-1 mRNA, was significantly upregulated after poly i:c or high pathogenic avian influenza virus (H5N1) stimulation in goose peripheral blood mononuclear cells cultured in vitro. Furthermore, geese infected with H5N1 showed significant upregulation of TLR3, especially in the lung and brain. We conclude that goose TLR3 is a functional TLR3 homologue of the protein in other species and plays an important role in virus recognition.

DHA, EPA and their combination at various ratios differently modulated Aß25-35-induced neurotoxicity in SH-SY5Y cells.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have been reported to prevent neurodegenerative diseases such as Alzheimer's disease (AD) in both experimental and clinical/epidemiological studies. However, whether DHA and EPA from natural products exert similar or different neuroprotective effects and how these n-3 PUFAs target cellular and molecular mechanisms associated with neurodegenerative disease pathogenesis are unknown. In the present study, we used amyloid-ß (Aß)25-35-treated differentiated SH-SY5Y cells as a model of AD to compare the neuroprotective effect of DHA, EPA and their combination at various ratios. Administration of 20µM Aß25-35 significantly decreased SH-SY5Y cell viability, the expression of nerve growth factor (NGF), its TrkA receptor, and the level of glutathione (GSH) and increased reactive oxygen species (ROS), nitric oxide, tumor necrosis factor (TNF)-α, brain derived neurotrophic factor (BDNF) and its TrkB receptor. Aß25-35 also increased the Bax/Bcl-2 ratio and the expression of Caspase-3 in these cells. Compared with the Aß group, pretreatment with DHA/EPA significantly reduced cell death, especially at ratio of 1:1 and 2:1 DHA/EPA or pure DHA. However, the most efficient ratio for reducing changes in ROS and GSH and for decreasing TNF-α appeared at ratio of 1:2 and 1:1, respectively. The ratio of 1:1, 2:1 and pure DHA resulted in significant increase in the level of NGF. Furthermore, pure DHA was the most efficient for reducing Bax/Bcl ratio and Caspase-3 expression. In conclusion, DHA, EPA and their combination differently modulated Aß25-35-induced neurotoxicity in SH-SY5Y cells by exerting anti-oxidative, anti-inflammatory and neurotrophic effects.

Advanced glycation end products inhibit testosterone secretion by rat Leydig cells by inducing oxidative stress and endoplasmic reticulum stress.

Diabetes severely impairs male reproduction. The present study assessed the effects and mechanisms of action of advanced glycation end products (AGEs), which play an important role in the development of diabetes complications, on testosterone secretion by rat Leydig cells. Primary rat Leydig cells were cultured and treated with AGEs (25, 50, 100 and 200 µg/ml). Testosterone production induced by human chorionic gonadotropin (hCG) was determined by ELISA. The mRNA and protein expression levels of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), which are involved in testosterone biosynthesis, were measured by reverse transcription-quantitative PCR and western blot analyssi, respectively. Reactive oxygen species (ROS) production in Leydig cells was measured using the dichlorofluorescein diacetate (DCFH-DA) probe. The expression levels of endoplasmic reticulum stress-related proteins [C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78)] in the Leydig cells were measured by western blot analysis. We found that the AGEs markedly suppressed testosterone production by rat Leydig cells which was induced by hCG in a concentration-dependent manner compared with the control (P<0.01). The mRNA and protein expression levels of StAR, 3ß-HSD and P450scc were downregulated by the AGEs in a dose-dependent manner compared with the control (P<0.01). The antioxidant agent, N-acetyl­L­cysteine (NAC), and the endoplasmic reticulum stress inhibitor, tauroursodeoxycholic acid (TUDCA), reversed the inhibitory effects of AGEs. In addition, the content of ROS in Leydig cells treated with AGEs increased significantly. The expression levels of CHOP and GRP78 were markedly upregulated by the AGEs in the Leydig cells. From these findings, it can be concluded that AGEs inhibit testosterone production by rat Leydig cells by inducing oxidative stress and endoplasmic reticulum stress.

Angiotensin II Receptor Blockers and Cancer Risk: A Meta-Analysis of Randomized Controlled Trials.

Angiotensin II receptor blockers (ARB) are widely used drugs that are proven to reduce cardiovascular disease events; however, several recent meta-analyses yielded conflicting conclusions regarding the relationship between ARB and cancer incidence, especially when ARB are combined with angiotensin-converting enzyme inhibitors (ACEI).We investigated the risk of cancer associated with ARB at different background ACEI levels.Search of PubMed and EMBASE (1966 to December 17, 2015) without language restriction.Randomized, controlled trials (RCTs) had at least 12 months of follow-up data and reported cancer incidence was included.Study characteristics, quality, and risk of bias were assessed by 2 reviewers independently.Nineteen RCTs including 148,334 patients were included in this study. Random-effects model meta-analyses were used to estimate the risk ratio (RR) of cancer risk. No excessive cancer risk was observed in our analyses of ARB alone versus placebo alone without background ACEI use (risk ratio [RR] 1.08, 95% confidence interval [CI] 1.00-1.18, P = 0.05); ARB alone versus ACEI alone (RR 1.03, 95%CI 0.94-1.14, P = 0.50); ARB plus partial use of ACEI versus placebo plus partial use of ACEI (RR 0.97, 95%CI 0.90-1.04, P = 0.33); and ARB plus ACEI versus ACEI (RR 0.99, 95%CI 0.79-1.24, P = 0.95).Lack of long-term data, inadequate reporting of safety data, significant heterogeneity in underlying study populations, and treatment regimens.ARB have a neutral effect on cancer incidence in randomized trials. We observed no significant differences in cancer incidence when we compared ARB alone with placebo alone, ARB alone with ACEI alone, ARB plus partial use of ACEI with placebo plus partial use of ACEI, or ARB plus ACEI combination with ACEI.

Analgesic effects of naringenin in rats with spinal nerve ligation-induced neuropathic pain.

Naringenin, a flavonoid abundant in citrus fruits, such as grapefruits, has been reported to possess anti-inflammatory properties. The present study aimed to investigate the analgesic potential of naringenin in L5 spinal nerve ligation (SNL)-induced peripheral neuropathic pain and the underlying mechanisms associated with neuroinflammation. Different doses of naringenin or saline were administered intrathecally once daily for 11 consecutive days, from 3 days prior to surgery to 7 days after surgery. Pain development was assessed 1 day prior to and 7-14 days after surgery in terms of mechanical withdrawal threshold and thermal withdrawal latency. Astrocytic and microglial activation and production of inflammatory mediators were determined on day 14 after surgery. The results demonstrated that naringenin dose-dependently attenuated the mechanical allodynia and thermal hyperalgesia induced by SNL. Furthermore, naringenin significantly inhibited SNL-induced activation of glial cells (astrocytes and microglia). Morover, the upregulated expression of inflammatory mediators in neuropathic pain was significantly inhibited by naringenin. Our findings suggested that repeated administration of naringenin may alleviate neuropathic pain, possibly through inhibiting neuroinflammation.

Fucoidan attenuates the existing allodynia and hyperalgesia in a rat model of neuropathic pain.

Fucoidan is an active constituent found in brown seaweeds, which have potential neuroprotection. The current study aimed to investigate the effects of fucoidan on the maintenance of neuropathic pain induced by L5 spinal nerve ligation (SNL) and the underlying mechanism related to the spinal neuroimmune responses. Animals were randomized into 5 groups: sham-operation with vehicle and SNL with vehicle or fucoidan (15, 50, and 100mg/kg). Different doses of fucoidan or vehicle were administered intrathecally once daily from postoperative day (POD) 11-20. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) was measured on 1 day before operation and days 10, 20, 22, 24, 26, 28, 30 after operation. Glial activation markers such as glial fibrillary acidic protein (GFAP) and macrophage antigen complex-1 (mac-1), inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6 activation, and extracellular signalregulated protein kinase (ERK) activation in the lumbar spinal cord were determined on day 30 after operation. The results showed that fucoidan caused dose-dependently attenuation of mechanical allodynia and thermal hyperalgesia. Furthermore, fucoidan could markedly inhibit neuroimmune activation characterized by glial activation, production of cytokines as well as ERK activation. The analgesic effect of intrathecal fucoidan in rats receiving SNL might partly attribute to the inhibition of neuroimmune activation associated with the maintenance of neuropathic pain.

Allogeneic bone marrow transplantation compared to peripheral blood stem cell transplantation for the treatment of hematologic malignancies: a meta-analysis based on time-to-event data from randomized controlled trials.

Controversy remains regarding the transplant outcomes of human leukocyte antigen-identical related bone marrow transplantation (BMT) and peripheral blood stem cell transplantation (PBSCT) for the treatment of patients with hematological malignancies. To provide an estimate of the effect of BMT and PBSCT on clinical outcomes in patients with hematological malignancies, we conducted a meta-analysis based on time-to-event data from 17 randomized controlled trials. PubMed, EMBASE, and Cochrane Central Register of Controlled Trials (CENTRAL), from 1972 through July 2010, and conference proceedings through July 2009 and reference lists, without any language restriction, of randomized trials that compared the transplant outcomes after BMT and PBSCT in patients with hematological malignancies were searched for details. Two independent reviewers extracted the data. The outcomes examined were engraftment, graft-versus-host disease (GVHD), relapse, transplant-related mortality (TRM), leukemia-free-survival (LFS), and overall survival (OS). Compared to PBSCT, BMT had lower neutrophil (HR, 2.08; 95% CI, 1.80 to 2.42; p < 0.00001) and platelet (HR, 2.77; 95% CI, 1.78 to 4.30; p < 0.00001) engraftment. BMT was associated with a significant decrease in the development of grades II-IV (HR, 0.75; 95% CI, 0.63 to 0.90; p = 0.002) and III-IV (HR, 0.63; 95% CI, 0.47 to 0.84; p = 0.001) acute GVHD as well as overall (HR, 0.70; 95% CI, 0.59 to 0.83; p < 0.0001) and extensive (HR, 0.60; 95% CI, 0.39 to 0.91; p = 0.002) chronic GVHD. BMT was associated with a higher incidence of relapse (HR, 1.91; 95% CI, 1.34 to 2.74; p = 0.0004). Comparable TRM (1.08; 95% CI, 0.56 to 2.10; p = 0.81), LFS (HR, 1.04; 95% CI, 0.83 to 1.30; p = 0.73), and OS (HR, 1.06; 95% CI, 0.81 to 1.39; p = 0.65) were demonstrated for both treatments. An inverse linear relationship was observed between the acute GVHD difference (PBSCT minus BMT) and the outcome of OS (p = 0.016). Our meta-analysis suggest that BMT leads to slower hematological recovery, increasing rates of relapse, and a lower risk of GVHD, but no significant difference in LFS and OS. A lower incidence of acute GVHD is associated with a superior OS.

Innate immune responses of epididymal epithelial cells to Staphylococcus aureus infection.

The epithelium is an active participant in the host response to infection. We hypothesized that epididymal epithelia play a role in the innate immune responses by sensing the presence of pathogens, expressing and secreting inflammatory cytokines that recruit inflammatory cells in response to invading pathogens. Our results indicated that TNF-alpha and IL-1beta could be secreted by the primary cultured rat epididymal cauda epithelia infected with Staphylococcus aureus. Epididymal epithelial-induced nitric oxide synthase (iNOS) expression was up-regulated after S. aureus infection and nitric oxide (NO) was also found to be produced significantly. NF-kappaB inhibitor BAY11-7082 inhibited TNF-alpha secretion completely and p38 mitogen-activated protein kinases (MAPKs) inhibitor SB203580 decreased TNF-alpha secretion partly, indicating that NF-kappaB and p38 signal pathways were involved in this inflammation response. Toll-like receptor (TLR)-2 and -4 were shown to be expressed in primary cultured rat epididymal epithelia. After infection the level of TLR2 expression was up-regulated rather than TLR4. These results demonstrated that epididymal epithelium have an innate immune response through activation of p38 MAPK and NF-kappaB after TLR2 activation by S. aureus infection.