Antiparasitic Efficacy of Herbal Extracts and Active Compound Against Gyrodactylus kobayashii in Carassius auratus.

Gyrodactylus spp. Nordmann, 1832 (Monogenea: Gyrodactylidae) are common ectoparasites of teleost fishes. Infection with these parasites can increase the mortality of fish and cause considerable economic losses in intensive aquaculture. To find an effective antiparasitic agent for the control of gyrodactylosis, antiparasitic efficacy of crude extracts of 36 herbal medicines was evaluated using a Carassius auratus (Cypriniformes, Cyprinidae)-Gyrodactylus kobayashii model. Among all tested medicines, methanol extract of Dioscorea collettii var. hypoglauca (Dioscoreales, Dioscoreaceae) was the most efficient, with an EC50 value of 4.17 mg/L. This extract showed 100% antiparasitic efficacy against G. kobayashii at 10 mg/L and had a therapeutic index (TI, LC50/EC50) of 5.26, which is higher than that of formaldehyde (TI = 4.58), a widely used parasiticide in aquaculture. Subsequently, the potential mechanism of antiparasitic activity of dioscin, an active compound isolated from D. collettii var. hypoglauca was investigated and the histopathological alterations in goldfish after exposure to dioscin were also studied. The in vivo trial indicated dioscin showed significant antiparasitic activity with a 24 h-EC50 value of 1.58 mg/L and it exhibited 100% antiparasitic efficacy at 0.6 mg/L. Also, G. kobayashii could be completely removed in vivo within 2 h at 0.6 mg/L dioscin. Whereas, mean survival time of this worm in vitro was 4.99 h, and some individuals even reached 12 h at the same concentration of dioscin. These results indicated that 0.6 mg/L of dioscin did not completely kill all worms within 2 h, but just temporarily remove the worms from goldfish. Scanning electron microscopy (SEM) analysis showed that most of the microvilli on the tegument surface of G. kobayashii dropped after exposure to dioscin. This might be one of the potential mechanisms of antiparasitic activity of dioscin against G. kobayashii. Furthermore, no severe histopathological alteration was observed after exposure to a high concentration of dioscin for a short time. Considering both effectiveness and safety, therapeutic baths with a high concentration of dioscin for a short time might be a more optimal choice for the treatment of gyrodactylosis in aquaculture.

Fucoxanthin extracted from Laminaria Japonica inhibits metastasis and enhances the sensitivity of lung cancer to Gefitinib.

ETHNOPHARMACOLOGICAL RELEVANCE: Laminaria japonica, a brown seaweed, has been used in Traditional Chinese Medicine (TCM) to treat a variety of diseases including lung cancer. AIM OF THE STUDY: To demonstrate the effects of Fucoxanthin (FX), a major active component extracted from Laminaria japonica on metastasis and Gefitinib (Gef) sensitivity in human lung cancer cells both in vitro and in vivo. MATERIALS AND METHODS: Invasion and migration of lung cancer cells were detected using the wound healing assay and transwell assay. Epithelial-to-mesenchymal transition (EMT) factors and PI3K/AKT/NF-κB pathways were analyzed by western blotting. RNA interference (RNAi) technology was used to silence TIMP-2 gene expression in A549 cells. The anti-metastatic effect of FX was evaluated in vivo in an experimental lung metastatic tumor model. On the other hand, cell counting kit-8 assay was used to study the cell viability of human lung cancer PC9 cells and Gef resistant PC9 cells (PC9/G) after Gef, FX or FX combined with Gef treatment. PC9 xenograft model was established to explore the anti-tumor effect of FX or combined with Gef. Immunohistochemistry staining assay and immunofluorescence staining assay were used to reveal the effects of FX on lung cancer cell proliferation and apoptosis. RESULTS: FX was able to significantly inhibit lung cancer cells migration and invasion in vitro. FX suppressed the expressions of Snail, Twist, Fibronectin, N-cadherin, MMP-2, PI3K, p-AKT and NF-κB, and increased the expression of TIMP-2. Furthermore, knockdown of TIMP-2 attenuated FX-mediated invasion inhibition. Additionally, we demonstrated that FX inhibited lung cancer cells metastasis in vivo. The anti-metastatic effects of FX on lung cancer cells might be attributed to inhibition of EMT and PI3K/AKT/NF-κB pathway. We further demonstrated that the anti-tumor activity of FX was not only limited to the drug sensitive cell lines, but also prominent on lung cancer cells with Gef resistant phenotype. Furthermore, in vivo xenograft assay confirmed that FX inhibited tumor growth and enhanced the sensitivity of lung cancer cells to Gef and this effect may be due to inhibition of tumor cell proliferation and activation of apoptosis. CONCLUSION: Collectively, our findings suggested that FX suppresses metastasis of lung cancer cells and overcomes EGFR TKIs resistance. Thus, FX is worthy of further investigation as a drug candidate for the treatment of lung cancer.

Thymol Protects Channel Catfish from Aeromonas hydrophila Infection by Inhibiting Aerolysin Expression and Biofilm Formation.

Aeromonas hydrophila is an opportunistic pathogen responsible for a number of diseases in freshwater farming. Moreover, the bacterium has been identified as a zoonotic pathogen that threatens human health. Antibiotics are widely used for treatments of infectious diseases in aquaculture. However, the abuse of antibiotics has led to the emergence of antimicrobial resistant strains. Thus, novel strategies are required against resistant A. hydrophila strains. The quorum sensing (QS) system, involved in virulence factor production and biofilm formation, is a promising target in identifying novel drugs against A. hydrophila infections. In this study, we found that thymol, at sub-inhibitory concentrations, could significantly reduce the production of aerolysin and biofilm formation by inhibiting the transcription of genes aerA, ahyI, and ahyR. These results indicate that thymol inhibits the quorum sensing system. The protective effects of thymol against A. hydrophila mediated cell injury were determined by live/dead assay and lactate dehydrogenase (LDH) release assay. Moreover, the in vivo study showed that thymol could significantly decrease the mortality of channel catfish infected with A. hydrophila. Taken together, these findings demonstrate that thymol could be chosen as a phytotherapeutic candidate for inhibiting quorum sensing system-mediated aerolysin production and biofilm formation in A. hydrophila.

Effects of dietary supplementation of four strains of lactic acid bacteria on growth, immune-related response and genes expression of the juvenile sea cucumber Apostichopus japonicus Selenka.

A feeding experiment was conducted to evaluate the effects of four strains of lactic acid bacteria (LAB) [i.e. Lactobacillus plantarum LL11 (LP), Weissella confuse LS13 (WC), Lactococcus lactis LH8 (LL) and Enterococcus faecalis LC3 (ES)] isolated from marine fish on growth, immune response and expression levels of immune-related gens in body wall of juvenile sea cucumber Apostichopus japonicus. As a result, sea cucumber had better growth performance fed supplementation of LP and ES than the control group (P < .05). Survival rate in each LAB supplementation group was significantly higher than that in control group after Vibrio splendidus challenge (P < .05). In regards to the enzyme activities, LP supplementation significantly imporved the enzyme activities of alkaline phosphatase (AKP) (P < .05), acid phosphatase (ACP) and superoxide dismutase (SOD), ES supplementation significantly imporved AKP activity (P < .05), and WC supplementation significantly imporved ACP activity (P < .05). However, lysozyme (LSZ) activity was not significantly changed in the four LAB supplementation treatments (P > .05). For the gene expression levels, different expression patterns were observed among four groups, heat shock proteins (HSP60, HSP70 and HSP90) and caspase-2 showed dramatic up-regulation at 30 d while NF-kappa-B transcription factor p65 was down-regulated at 15 d and up-regulated at 30 d, and nitric oxide synthase was down-regulated at both timepoints in almost all the four groups. In conclusion, the four LAB strains screened from marine fish supplemented in diets indicated positive effects on immune response for A. japonicus, especially, the L. plantarum LL11 and E. faecalis LC3 indicated better growth performance.

Protosappanin a inhibits osteoclastogenesis via reducing oxidative stress in RAW264.7 cells.

Protosappanin A (PrA), obtained from the traditional Chinese herbal medicine, Caesalpinia sappan L. (Lignum Sappan), possesses a lot of pharmaceutical activities. Typically, it is a potent antioxidant. This study makes an effort to test its protective effects against osteoporosis by partially reducing oxidative stress in RAW264.7 cells and a mouse ovariectomized (OVX) osteoporosis model. The influence that PrA affected on osteoclastic proliferation and differentiation under oxidative status was investigated. Our results revealed that PrA significantly inhibited the proliferation of RAW264.7 cells in oxidative stress conditions. Moreover, it suppressed some osteoclastic markers by TRAP staining, bone section assay and quantitative real-time PCR. PrA decreased reactive oxygen species (ROS) generation in RAW264.7 cells. In vivo, our results demonstrated that PrA supplementation improved some serum oxidative markers, including malondialdehyde (MDA) and reduced glutathione (GSH), and inhibited some osteoclastic markers, such as CTX-1 and TRAP. Importantly, it ameliorated the micro-architecture of trabecular bones by micro-CT assay. In summary, these findings showed that protection by PrA against osteoporosis is associated with a reduction in oxidative stress, suggesting that PrA may be useful in bone resorption related diseases, especially osteoporosis.

A C-type lectin that inhibits bacterial infection and facilitates viral invasion in black rockfish, Sebastes schlegelii.

C-type lectins (CTLs) are important pattern recognition receptors (PRRs) that play vital roles in innate immunity. In teleosts, a number of CTLs have been reported, but their in vivo effects on host defense are still limited. In this study, a CTL homolog (SsLec1) was identified from black rockfish, Sebastes schlegelii, and its structure, expression and biological function was analyzed. The open reading frame of SsLec1 is 633 bp, with a 5'- untranslated region (UTR) of 36 bp and a 3'- UTR of 117 bp. The deduced amino acid sequence of SsLec1 shares the highest overall identity (73.20%) with the CTL of Oplegnathus fasciatus. SsLec1 possesses conserved CTL features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine residues, the mannose-type carbohydrate-binding motif, the conserved calcium binding sites and a putative signal peptide. The expression of SsLec1 was highest in liver and could be induced by experimental infection with Listonella anguillarum. Recombinant SsLec1 (rSsLec1) purified from E. coli was able to bind and agglutinate the Gram-negative fish pathogens Vibrio ichthyoenteri and Vibrio vulnificus. The agglutinating ability of rSsLec1 was abolished in the presence of mannose or ethylenediaminetetraacetic acid. Further analysis showed that rSsLec1 could enhance phagocytosis by macrophages. In vivo experiments indicated that rSsLec1 could inhibit bacterial infection and promote viral invasion. Taken together, these results suggest that SsLec1 is a novel CTL that possesses apparent immunoregulation property and plays a critical role in host defense against pathogens invasion.

Effects of Bu-Shen-An-Tai recipe and its two components on endometrial morphology during peri-implantation in superovulated mice.

The aim of this study was to investigate the effect of Bu-Shen-An-Tai recipe (BSATR) and its two components (Bushen recipe, and Huoxue recipe) on endometrial morphology during peri-implantation in superovulated mice. Mice were randomly divided into five groups, including the normal (N), model (M), Bushen (BS), Huoxue (HX) and Bu-Shen-An-Tai (BH) groups. The uteri were collected on day 4 of pregnancy, and the endometrium thickness, microvessel density (MVD) and number of pinopodes observed. Compared with the M group, the endometrial thickness in the BS, HX and BH groups was significantly increased and there was a significant difference in endometrial thickness between the BS and the BH groups. The mean MVD was significantly lower in the M group than in the N group, and there was a significant increase in MVD in the BS, HX and BH groups as compared with the M group. Compared with the M group, the pinopode scores in the endometrium were significantly increased in the HX and BH groups; and the BS group had significantly higher pinipode scores than the HX and BH groups. In conclusion, the results of the present study demonstrated that the recipes (Bushen, Huoxue and BSATR) could improve the endometrial environment by regulating the endometrial thickness, MVD and the number of pinopodes at the window of implantation. Moreover, the Huoxue recipe and the BSATR were more efficient than the Bushen recipe, with the BSATR tending to have the most beneficial effects.

Bromodichloromethane induces cell proliferation in different tissues of male F344 rats by suppression of E-cadherin expression via hypermethylation or transcriptional activation of c-myc and cyclin D1.

The aim of this study was to investigate the mechanism of bromodichloromethane (BDCM) - induced cell proliferation in different tissues of male F344 rats. Rats were administered at doses of 0 and 100mg/kg/day BDCM dissolved in corn oil by gavage for 5 days/week for 1, 4, 8 and 12 weeks. Then the colon, kidney and liver were collected. No histologic lesions were observed in the colon of rats exposed to BDCM, while there were mild nephrotoxicity and marginal hepatotoxicity related to BDCM treatment. Moreover, BDCM enhanced cell proliferation in the colon and kidney but not in the liver. In colons, hypermethylation in E-cadherin promoter might be associated with inhibition of mRNA and protein expression after 12 weeks of BDCM exposure. In kidneys, BDCM decreased E-cadherin mRNA expression, accompanying with transcriptional activation of c-myc and cyclin D1. However, suppression of E-cadherin mRNA and protein expression occurred in the absence of significant changes in DNA methylation. Therefore, suppression of E-cadherin expression via hypermethylation or transcriptional activation of c-myc and cyclin D1 may be involved in BDCM-induced cell proliferation in different tissues of male F344 rats.

[Effects of phenolic alkaloids of Menispermum dauricum on the hemodynamics and coronary circulation in anesthetized dog].

AIM: To observe the effect of phenolic alkaloids of Menispermum dauricum (PAMD) on the hemodynamics, coronary circulation and oxygen metabolism of the myocardium in anesthetized dogs. METHODS: In this study, the changes of LVSP, LVEDP and +/- dp/dtmax, the flow of coronary artery and myocardial energy metabolism were measured in anesthetized dog with PAMD or NS. RESULTS: In the anesthetized dogs, compared with pre-treatment status, PAMD at 3.5 and 7.0 mg.kg-1 caused decreases in the left ventricular systolic pressure(LVSP), +/- dp/dtmax, heart rate, the rate of oxygen utilization, the coronary and general peripheral resistance. It was found to increase myocardial oxygen and coronary flow. There were no significant change in the left ventricular end diastolic pressure (LVEDP). CONCLUSION: PAMD can ameliorate hemodynamics, coronary circulation and myocardial metabolism.