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.

The mechanisms of EGFR in the regulation of axon regeneration.

To understand the relationship between epidermal growth factor receptor (EGFR) and axon regeneration and the mechanisms of how EGFR regulates the neuronal intrinsic regenerative ability, we evaluated the levels of mRNA and protein of EGFR、total mammalian target of rapamycin (mTOR), p-mTOR(Ser2448) , total Akt and p-Akt(Ser473) in rats of different developmental stage by using Western blot and real-time polymerase chain reaction analysis. Axon protein tau and neuron proteins ß-tubulin/neurofilament (NF) were assessed to evaluate the extent of the axon regeneration in cultured neuron cells. Expressions of EGFR、total mTOR, p-mTOR(Ser2448) , total Akt and p-Akt(Ser473) in cultured neuron cells were also detected using Western blot analysis. Our results showed that the expressions of EGFR and mTOR dropped off with the ageing of the rats, and Ser473 phosphorylation of Akt and Ser2448 phosphorylation of mTOR were highly expressed in foetal and newborn rats but decreased obviously in adult rats. tau, ß-tubulin and NF were upregulated when EGFR was overexpressed and down-regulated after EGFR was blocked. The phosphorylation of mTOR and Akt was apparently elevated when EGFR was overexpressed and decreased when EGFR was blocked, which suggested that EGFR has the potential to regulate the neuronal intrinsic regeneration and mTOR and PI3K/Akt pathway activation may have an important role in it.

[Advanced glycation end products inhibit testosterone production in rat Leydig cells].

OBJECTIVE: To study the expression of the receptor for advanced glycation end products (RAGE) and the inhibitory effect of advanced glycation end products (AGEs) on testosterone production in rat Leydig cells. METHODS: Rat Leydig cells were primarily cultured and the expression of RAGE in the Leydig cells was detected by RT-PCR and immunofluorescence staining. The Leydig cells were treated with AGEs at the concentrations of 25, 50, 100 and 200 microg/ml, respectively, and the testosterone content was determined by ELISA. RESULTS: RT-PCR and immunofluorescence staining exhibited the expression of RAGE in the rat Leydig cells. AGEs remarkably suppressed hCG-induced testosterone production in the Leydig cells in a concentration-dependent manner in the 50, 100 and 200 microg/ml groups as compared with the control (P < 0.01). CONCLUSION: RAGE exists in rat Leydig cells and AGEs can significantly inhibit the secretion of testosterone in primarily cultured rat Leydig cells.

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.

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.

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.

Expression of Frizzled10 in mouse central nervous system.

Frizzled transmembrane proteins (Fzd) are receptors of Wnts, and they play key roles during central nervous system (CNS) development in vertebrates. Here we report the expression pattern of Frizzled10 in mouse CNS from embryonic stages to adulthood. Frizzled10 is expressed strongly at embryonic days E8.5 and E9.5 in the neural tube and tail bud. At E10.5, Frizzled10 is expressed in the forebrain vesicle, the fourth ventricle and the dorsal spinal cord. From E12.5 to E16.5, Frizzled10 expression is mainly observed in the cortical hem/fimbria, the neuroepithelium of the third ventricular zone, midbrain, developing cerebellum, and dorsal spinal cord. At P0, with the exception of expression in the fimbria, Frizzled10 mRNA expression is limited to specific nuclei including the ventral posterior thalamic nucleus (VP) and the dorsal lateral geniculate nucleus (DLG) in the developing thalamus as well as in the proliferative ventricular zone of the developing cerebellum. From P20 to adult, Frizzled10 mRNA is detected only in the internal capsule (ic). Our data show that expression of Frizzled10 is very strong during embryonic development of the CNS and suggest that Frizzled10 may play an essential role in spatial and temporal regulation during neural development.

A useful transgenic mouse line for studying the development of spinal nociceptive circuits.

Noxious peripheral stimuli are detected by nociceptive DRG sensory neurons; this information is transmitted to the superficial dorsal horn of the spinal cord, relayed to the brain, then perceived as pain. Previous studies have shown that Wnt signaling plays a crucial role during the development of nociceptive circuits. We have reported a transgenic mouse line in which Tau-LacZ is driven by the promoter of one Wnt receptor, Frizzled10. Here, we further characterize this transgenic mouse line and present our findings that nociceptive circuits were specifically labeled by the Tau-LacZ transgene in developing spinal cord and DRG. Tau-LacZ specifically labeled commissural axons crossing the floor plate and transmitting nociceptive information to the brain. Thus this mouse line will provide a powerful genetic tool for studying the development of spinal nociceptive circuits and the mechanisms of commissural axon guidance.

Generation of Frizzled10-Cre transgenic mouse line: a useful tool for the study of dorsal telencephalic development.

Wnt signaling plays an important role in regulating cortical and hippocampal development, but many of the other molecular mechanisms underlying dorsal telencephalic development are largely unknown. We are taking advantage of the highly regionalized expression patterns of signaling components of the Wnt pathway to generate new mouse lines that will be useful for studying forebrain development. Here, we describe a transgenic mouse line where Cre is driven by the promoter of the Wnt receptor, Frizzled10. In these mice, Cre activity is mainly detected in the dorsal telencephalon during development and is confined to the pyramidal cell fields in the adult hippocampus. The Cre recombinase has very high efficiency when assayed by crossing the transgenic line with the ROSA26 reporter line. Thus, this Cre line will be useful for the study of dorsal telencephalic development and conditional inactivation of target genes in the cortex and hippocampus.

Antinociceptive effects of fucoidan in rat models of vincristine-induced neuropathic pain.

Chemotherapeutic drugs commonly induce peripheral neuropathic pain, which limit their clinic use. In the present study, the effect of fucoidan on the development of vincristine­induced neuropathic pain was evaluated and the underlying mechanism was examined. A neuropathy model was established in Sprague­Dawley rats by intraperitoneal injection of vincristine sulfate 50 µg/kg once a day for 10 consecutive days. Fucoidan (50, 100 or 200 mg/kg.) and pregabalin (10 mg/kg) were injected for 14 consecutive days. Behavioral assessments were then performed and the expression of GABAB receptor was determined. The results showed that a single treatment with fucoidan did not prevent the induction of vincristine­induced mechanical or cold allodynia. However, repeated fucoidan administration attenuated vincristine­induced mechanical and cold allodynia in a dose­dependent manner. Additionally, the analgesic effects of fucoidan contributed to an upregulation in the expression of GABAB receptor in the spinal cord. Furthermore, all the effects of fucoidan against vincristine­induced neuropathy were reversed by saclofen, a selective GABAB receptor antagonist. These results suggested that the antinociceptive effects of fucoidan may be through activation of GABAB receptor, and fucoidan may be a promising drug for the treatment of chemotherapeutic drug-induced neuropathic pain.

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.

Simple risk factors to predict urgent endoscopy in nonvariceal upper gastrointestinal bleeding pre-endoscopically.

The goal of this study is to evaluate how to predict high-risk nonvariceal upper gastrointestinal bleeding (NVUGIB) pre-endoscopically. A total of 569 NVUGIB patients between Match 2011 and January 2015 were retrospectively studied. The clinical characteristics and laboratory data were statistically analyzed. The severity of NVUGIB was based on high-risk NVUGIB (Forrest I-IIb), and low-risk NVUGIB (Forrest IIc and III). By logistic regression and receiver-operating characteristic curve, simple risk score systems were derived which predicted patients' risks of potentially needing endoscopic intervention to control bleeding. Risk score systems combined of patients' serum hemoglobin (Hb) ≤75 g/L, red hematemesis, red stool, shock, and blood urine nitrogen ≥8.5 mmol/L within 24 hours after admission were derived. As for each one of these clinical signs, the relatively high specificity was 97.9% for shock, 96.4% for red stool, 85.5% for red hematemesis, 76.7% for Hb ≤75 g/L, and the sensitivity was 50.8% for red hematemesis, 47.5% for Hb ≤75 g/L, 14.2% for red stool, and 10.9% for shock. When these 5 clinical signs were presented as a risk score system, the highest area of receiver-operating characteristic curve was 0.746, with sensitivity 0.675 and specificity 0.733, which discriminated well with high-risk NVUGIB. These simple risk factors identified patients with high-risk NVUGIB of needing treatment to manage their bleeding pre-endoscopically. Further validation in the clinic was required.

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.