| Dactylogyrus is the most common parasitic disease in fish industry, which can cause greateconomic losses in aquaculture. Nowadays, with the spending of farming, the disease becomemore and more serious. To kill the parasite, many veterinary medicine and pesticide have beenused, such as mebendazole and trichlorfon. However, long-term using of these drugs hasbrought many serious problems, for example, drug residues, drug resistance, andenvironmental pollution and so on. To solve these problems, our laboratory focuses on thescreening of plant medicine. However conventional large-scale drug screening methodrequires a lot of time and energy, while new methods rely on the drug targets. Our perivousstudy found that sanguinarine has the anti-Dactylogyrus ability, sanguinarine is a kind ofalkaloids, have application prospects. However, no studies had reported the targets aboutDactylogyrus. To understand the targets of sanguinarine acts on Dactylogyrus, this study usedscanning electron microscopy, transmission electron microscopy to observe the tugment ofDactylogyrus intermedius which were immersed with sanguinarine in different time. And theactivity of antioxidant enzymes was checked whether sanguinarine can damage D.intermedius’s antioxidant system. At the same time, to evaluate the applicability ofsanguinarne, real time-PCR was used to check the expression of immune-related geneexpression in different issues of goldfish after immersed with sanguinarine and the ability ofgoldfish to infect bacteria was also examined. This paper also examined the cell viabilityinduced by sanguinarne. Results are listed as follows:1. Scanning electron microscopy observed that the damage of the tugment of D.intermedius was more and more serious with the increasing of concentration and time. Thesedamages may due to the deathe of D. intermedius.2. Transmission electron microscopy observed that the normal structual of tugment wasdamaged, and the syncytial, secretory granules, mitochondria were reduced. These damagesmay due to the deathe of D. intermedius.3. The intracellular oxidation plays a very important role in animals. In present study,total antioxidative capacity (T-AOC) and a representative antioxidant enzyme (SOD) were both significantly decreased by sanguinarine. Moreover, an increase in the MDA content wasalso observed.4. The expression of CCL-1was significantly increased in kidney and spleen, while itsignificantly decreased in gill. The expression of CXCL-8was all increased in the three testedtissues. Similarly, sanguinarine also induced significantly higher expression of IL-1β-1andIL-1β-2, in gill, kidney and spleen. A significant increase in expression of TNFα-2was alsoobserved in gill, kidney and spleen post treatment with sanguinarine. The expression ofTGF-β was significantly up-regulated in gill at day6and in kidney at day3to day6, while itsexpression was higher but not significant in spleen.5. The challenge test of Aeromonas hydrophila was conducted on day1and day6aftertreatment and four tissues of gill, kidney, spleen and liver were detected for the infectionamount. All tissues from goldfish exposed to sanguinarine showed lower bacterial loads thancontrol group. On day1, bacterial loads of treated fish were significantly lower in all thetested tissues than that of non-treated fish, On day6, bacterial loads in gill, kidney and spleenof treated group were70.5%,36.6%and17.5%of the control group (P <0.05). The amountof A. hydrophila in liver was also lower than control group, although not significant.6. MTT assay was employed to examined whether sanguinarine are toxic to the cells.The results showed that sanguinarine is safe to the CIK cells. Moreover, sanguinarine couldincrease proliferation of the CIK cells. |
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