The Tilapia collagen peptide mixture TY001 protects against LPS-induced inflammation, disruption of glucose metabolism, and aberrant expression of circadian clock genes in mice.

The Tilapia collagen peptide mixture TY001 has been shown to accelerate wound healing in streptozotocin-induced diabetic mice and to protect against streptozotocin-induced inflammation and elevation in blood glucose. The goals of the present study are to further study TY001 effects on lipopolysaccharide (LPS)-induced inflammation and metabolic syndrome. LPS is known to disrupt circadian clock to produce toxic effects, the effects of TY001 on rhythmic alterations of serum cytokines and hepatic clock gene expressions were examined. Mice were given TY001 (30 g/L, ≈ 40 g/kg) through the drinking water for 30 days, and on the 21st day of TY001 supplementation, LPS (0.25 mg/kg, ip, daily) was given for 9 days to establish the inflammation model. Repeated LPS injections produced inflammation, impaired glucose metabolism, and suppressed the expression of circadian clock core genes Bmal1 and Clock; clock feedback gene Cry1, Cry2, Per1, and Per2; clock target gene Rev-erbα and RORα. TY001 prevented LPS-induced elevations of TNFα, IL-1ß, IL-6, and IL-10 in the liver, along with improved histopathology. TY001 reduced LPS-elevated fasting blood glucose and increased LPS-reduced serum insulin levels, probably via increased glucose transporter GLUT2, enhanced insulin signaling p-Akt and p-IRS-1Try612. Importantly, LPS-induced circadian elevations of serum TNFα and IL-1ß and aberrant expression of circadian clock genes in the liver were ameliorated by TY001. Immunohistochemistry revealed that the LPS decreased Bmal1 and Clock protein in the liver, which was recovered by TY001. Taken together, TY001 is effective against LPS-induced inflammation, disruption of glucose metabolism and disruption of circadian clock gene expressions. Abbreviations: TY001: Tilapia collagen peptide mixture; LPS: Lipopolysaccharide; TNFα: Tumor necrosis factor-α; IL-1ß: Interleukin-1ß; GLUT2: Glucose transporter 2.

Sensitization and synergistic anti-cancer effects of Furanodiene identified in zebrafish models.

Furanodiene is a natural terpenoid isolated from Rhizoma Curcumae, a well-known Chinese medicinal herb that presents anticancer effects in various types of cancer cell lines. In this study, we have successfully established zebrafish xenografts with 5 various human cancer cell lines; and validated these models with anti-cancer drugs used clinically for treating human cancer patients. We found that Furanodiene was therapeutically effective for human JF 305 pancreatic cancer cells and MCF-7 breast cancer cells xenotranplanted into zebrafish. Furanodiene showed a markedly synergistic anti-cancer effect when used in combination with 5-FU (5-Fluorouracil) for both human breast cancer MDA-MB-231 cells and human liver cancer BEL-7402 cells xenotransplanted into zebrafish. Unexpectedly, Furanodiene reversed multiple drug resistance in the zebrafish xenotransplanted with cis-Platinum-resistant human non-small cell lung cancer cells and Adriamycin-resistant human breast cancer cells. Furanodiene played its anti-cancer effects through anti-angiogenesis and inducing ROS production, DNA strand breaks and apoptosis. Furanodiene suppresseed efflux transporter Pgp (P-glycoprotein) function and reduced Pgp protein level, but no effect on Pgp related gene (MDR1) expression. These results suggest sensitizition and synergistic anti-cancer effects of Furanodiene that is worthy of a further investigation.

[Effect of Huangdi Anxiao Capsules on zebrafish vascular lesions induced by high glucose and high fat].

Zebrafish of different strains with 5 dpf (5 days post-fertilization) were selected and fed with 0.2% high-fat diet for 8 h and 3% glucose solution for 16 halternatively during the day and night for 4 consecutive days. The zebrafish model was established and randomly divided into model group, Huangdi Anxiao Capsules (260 mg·L⁻¹) group and pioglitazone (32 mg·L⁻¹) group. The drug treatment groups were given the water-soluble drugs, with a volume of 25 mL, and incubated in a 28 °C incubator for 4 days. To detect the exposure to the corresponding drugs, the normal control group was set up. Thirty zebrafish were included in each group. The effect of Huangdi Anxiao Capsules on vascular wall thickness, fluorescence intensity of islet beta cells, fluorescence intensity of macrophages, and blood flow velocity of zebrafish were detected. The expressions of vascular endothelial growth factor (vegfaa) and angiotensin converting enzyme (ACE) were detected by RT-PCR. The results showed that compared with the model group, Huangdi Anxiao Capsules can significantly reduce the thickness of the blood vessel wall, increase the fluorescence intensity of islet ß cells and macrophages, increase the blood flow velocity in vivo, and decrease the ACE and vegfaa expressions in zebrafish. It is suggested that Huangdi Anxiao Capsules may alleviate zebrafish vascular lesions by regulating the expressions of ACE and vegfaa.

Evaluation of collagen mixture on promoting skin wound healing in zebrafish caused by acetic acid administration.

The aim of this study is to use zebrafish embryos as a quick platform for wound healing studies. At beginning, we optimized a protocol to induce skin lesion by acetic acid injection. The acetic acid injection induced regional inflammation wound hyperpigmentation by recruiting pigment cells to the wound area. Later, we applied established platform to evaluate the effect of tilapia's collagen peptide mixtures, including demonstration on promoting skin wound healing and eliminating inflammatory response. Results showed that after treating TY001, one of the above fish collagen peptide mixtures, not only repair and proliferation were induced, but also death and apoptosis cells were cleared within cutaneous lesion. Moreover, inflammatory response was suppressed along with collagen mixture treatment. Finally, the TY001-associated signaling was validated by real time-PCR, and numbers of gene associated with tissue repair and vessel proliferation were induced. To sum up, our findings provided a permissive model that may apply to generate a platform for further screening on repair and restoration technology. In addition, the tilapia fish collagen peptide mixture we applied on our model has great potential on developing clinical application on wound healing.

Closantel Suppresses Angiogenesis and Cancer Growth in Zebrafish Models.

Angiogenesis has emerged as an important therapeutic target in several major diseases, including cancer and age-related macular degeneration. The zebrafish offer the potential for high-throughput drug discovery in a whole vertebrate system. In this study, we have taken advantage of the transgenic Tg (fli1a:EGFP) zebrafish line to screen the U.S. Drug Collection Library and identified 11 old drugs with antiangiogenic activity, including Closantel, an FDA-approved broad-spectrum salicylanilide antiparasitic drug for a variety of types of animals. Closantel was confirmed to have antiangiogenic activity in zebrafish with a half-inhibitory concentration (IC50) at 1.69 µM on the intersegmental vessels and 1.45 µM on the subintestinal vessels. Closantel also markedly suppressed cancer growth in zebrafish xenotransplanted with human lymphoma, cervical cancer, pancreatic cancer, and liver cancer cells, generally in a dose-dependent manner. These data reveal that Closantel has antiangiogenesis and anticancer effects and could be a potential drug candidate for animal and human cancer treatments. Further study is needed to clarify the mechanisms involved in the antiangiogenesis and anticancer effects of Closantel.

Rapid analysis of hypolipidemic drugs in a live zebrafish assay.

INTRODUCTION: Hyperlipidemia is the most common form of dyslipidemia, which is the key risk factor for cardiovascular disease and stroke. The development of effective and safe drug treatments for hyperlipidemia has been proven challenging. METHODS: In this study, taking advantage of the transparency of larval zebrafish, we developed a zebrafish hyperlipidemia model for drug screening and efficacy assessment. Zebrafish at 5 d.p.f (days post fertilization) were fed with 0.1% egg yolk for 48 h (hours), followed by drug treatment for 24h or 48 h. Tested drugs were administered into the zebrafish by direct soaking. Drug effect was evaluated based on quantitative analysis of Oil Red O (ORO) in zebrafish vena caudalis. RESULTS: All 5 human hypolipidemic drugs (simvastatin, lovastatin, ezetimibe, bezafibrate and hyodesoxycholic acid) showed significant hypolipidemic effects (p<0.01) in a dose-dependent manner in the zebrafish hyperlipidemia model. 'We also found a well-known Chinese tea Pu-erh tea significantly reduced lipids in this model (p<0.001 and p<0.01). DISCUSSION: Our results demonstrate that the zebrafish hyperlipidemia model developed and validated in this study could be used for in vivo hyperlipidemia studies and drug screening and for assessing hypolipidemic drugs with different mechanisms.

Human cardiotoxic drugs delivered by soaking and microinjection induce cardiovascular toxicity in zebrafish.

Cardiovascular toxicity is a major challenge for the pharmaceutical industry and predictive screening models to identify and eliminate pharmaceuticals with the potential to cause cardiovascular toxicity in humans are urgently needed. In this study, taking advantage of the transparency of larval zebrafish, Danio rerio, we assessed cardiovascular toxicity of seven known human cardiotoxic drugs (aspirin, clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride) and two non-cardiovascular toxicity drugs (gentamicin sulphate and tetracycline hydrochloride) in zebrafish using six specific phenotypic endpoints: heart rate, heart rhythm, pericardial edema, circulation, hemorrhage and thrombosis. All the tested drugs were delivered into zebrafish by direct soaking and yolk sac microinjection, respectively, and cardiovascular toxicity was quantitatively or qualitatively assessed at 4 and 24 h post drug treatment. The results showed that aspirin accelerated the zebrafish heart rate (tachycardia), whereas clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride induced bradycardia. Quinidine and terfenadine also caused atrioventricular (AV) block. Nimodipine treatment resulted in atrial arrest with much slower but regular ventricular heart beating. All the tested human cardiotoxic drugs also induced pericardial edema and circulatory disturbance in zebrafish. There was no sign of cardiovascular toxicity in zebrafish treated with non-cardiotoxic drugs gentamicin sulphate and tetracycline hydrochloride. The overall prediction success rate for cardiotoxic drugs and non-cardiotoxic drugs in zebrafish were 100% (9/9) as compared with human results, suggesting that zebrafish is an excellent animal model for rapid in vivo cardiovascular toxicity screening. The procedures we developed in this report for assessing cardiovascular toxicity in zebrafish were suitable for drugs delivered by either soaking or microinjection.

Diurnal variation of hepatic antioxidant gene expression in mice.

BACKGROUND: This study was aimed to examine circadian variations of hepatic antioxidant components, including the Nrf2- pathway, the glutathione (GSH) system, antioxidant enzymes and metallothionein in mouse liver. METHODS AND RESULTS: Adult mice were housed in light- and temperature-controlled facilities for 2 weeks, and livers were collected every 4 h during the 24 h period. Total RNA was isolated, purified, and subjected to real-time RT-PCR analysis. Hepatic mRNA levels of Nrf2, Keap1, Nqo1 and Gclc were higher in the light-phase than the dark-phase, and were female-predominant. Hepatic GSH presented marked circadian fluctuations, along with glutathione S-transferases (GST-α1, GST-µ, GST-π) and glutathione peroxidase (GPx1). The expressions of GPx1, GST-µ and GST-π mRNA were also higher in females. Antioxidant enzymes Cu/Zn superoxide dismutase (Sod1), catalase (CAT), cyclooxygenase-2 (Cox-2) and heme oxygenase-1 (Ho-1) showed circadian rhythms, with higher expressions of Cox-2 and CAT in females. Metallothionein, a small non-enzymatic antioxidant protein, showed dramatic circadian variation in males, but higher expression in females. The circadian variations of the clock gene Brain and Muscle Arnt-like Protein-1(Bmal1), albumin site D-binding protein (Dbp), nuclear receptor Rev-Erbα (Nr1d1), period protein (Per1 and Per2) and cryptochrome 1(Cry1) were in agreement with the literature. Furthermore, acetaminophen hepatotoxicity is more severe when administered in the afternoon when hepatic GSH was lowest. CONCLUSIONS: Circadian variations and gender differences in transcript levels of antioxidant genes exist in mouse liver, which could affect body responses to oxidative stress at different times of the day.

Diurnal-and sex-related difference of metallothionein expression in mice.

BACKGROUND: Metallothionein (MT) is a small, cysteine-rich, metal-binding protein that plays an important role in protecting against toxicity of heavy metal and chemicals. This study was aimed to define diurnal and sex variation of MT in mice. METHODS: Adult mice were maintained in light- and temperature-controlled facilities for 2 weeks with light on at 8:00 and light off at 20:00. The blood, liver, and kidneys were collected every 4 h during the 24 h period. Total RNA was isolated, purified, and subjected to real-time RT-PCR analysis and MT protein was determined by western blot and the Cd/hemoglobin assay. RESULTS: The diurnal variations in mRNA levels of MT-1 and MT-2in liver were dramatic, up to a 40-foldpeak/trough ratio. MT mRNA levels in kidneys and blood also showed diurnal variation, up to 5-fold peak/trough ratio. The diurnal variation of MT mRNAs resembled the clock gene albumin site D-binding protein (Dbp), and was anti-phase to the clock gene Brain and Muscle ARNT-like Protein 1 (Bmal1) in liver and kidneys. The peaks of MT mRNA levels were higher in females than in males. Hepatic MT protein followed a similar pattern, with about a 3-fold difference. CONCLUSION: MT mRNA levels and protein showed diurnal- and sex-variation in liver, kidney, and blood of mice, which could impact the body defense against toxic stimuli.