The Research Of Toxicity Of Colchicine And Its Mechanism By Using Of Zebrafish Larvae

Objective:1. To provide superior embryos, zebrafish larvae and zebrafishfor experiments we maintain the cultivation system of zebrafish be in smoothoperation.2. To evaluate the effects of colchicine on the activities of SOD,Na+-K+-ATPase and the contents of MDA, GSH in zebrafish larvae.3. Toobserve the expression of the antioxidant enzyme genes such as bcl2, gstp2,ucp2, nqo1and sod1in zebrafish larvae.4. To study the protein expression ofShh and Gli3and the change of fat content in zebrafish larvae liver.Methods:1. At the basic of cultivation system of zebrafish, we bredembryos and cultivated zebrafish larvae.2. Drug exposured experiment shouldbe carried out as follows: on the basis of preliminary experiment, we determinedthe maximum non-lethal concentration (MNLC) after72h.3dpf zebrafish larvaewere randomly divided into five groups and the drug concentrations were0.2MNLC,0.5MNLC,1.0MNLC and LC10, and Holt Buffer was selected as thecontrol group. The assays were carried out after72h as follow:(1) Measurementof the activities of antioxidant enzymes and the contents of antioxidants. Homogenated adequately to make tissue homogenate, then centrifuged at4℃,and took supernatant to determine the activities of SOD, Na+-K+-ATPase and thelevels of MDA and GSH.(2) Determination of antioxidant enzymes relatedgenes expression. By the means of RT-PCR, we detected the level of bcl2, gstp2,ucp2, nqo1and sod1expression in zebrafish larvae when drug was exposed after72h.(3) Liver pathology and the protein expression of Shh and Gli3in liverwere carried out by oil red staining, HE staining and immunohistochemicalanalysis after a series of steps: zebrafish larvaes were fixed in4%paraformaldehyde phosphate buffer for24h at4℃, then dehydrated in gradientethanol, paraffin embedded, finally sliced.Results:1. The cultivation system of zebrafish operated stably and weobtianed superior embryos, zebrafish larvaes and zebrafish for experiments.2.The6dpf MNLC for zebrafish larvae was20.6063μg mL-1, LC10was26.0615μg mL-1and LC50was34.7536μg mL-1.With drug concentrationincreasing, the activities of SOD and Na+-K+-ATPase were suppressed; while thelevel of MDA were increased when compared with control group. And thecontent of GSH had a similar trend with the activities of SOD andNa+-K+-ATPase.3. Compared with control group, the expression of bcl2, gstp2,nqo1, sod1were decreased with drug concentration increasing, while theexpression of ucp2was increased.4. Compared with control group, with thecolchicine concentration increasing, HE staining result showed that high dosegroup of zebrafish larvae liver tissue degenerated, cavity increased, liver cellswelled. Oil red staining results indicated that the zebrafish liver color deepened,which meant that the content of fat in liver was increased. The results ofimmunohistochemistry showed that the protein expression of Shh and Gli3wereincreased obviously with the increasing of drug concentration. Two kinds of protein expression increased, which meant that liver injury caused by colchicinemay be related to activation of Hedgehog signaling pathways.Conclusions:1. The cultivation of zebrafish system operated stably, andprovided embryos, zebrafish larvae and zebrafish in drug safety evaluation,which broadened the application scope of drug safety evaluation.2. We selectedzebrafish larvae as experimental subjects to study effects of colchicine onzebrafish antioxidant system.The results showed that the activities of SOD andNa+-K+-ATPase were suppressed, and the level of GSH was decreased, while thecontent of MDA was decreased, which lead to imbalance of redox state inzebrafish larvae. The toxicity of colchicine to juvenile zebrafish may beassociated with it. Zebrafish larvae hepatotoxicity mechanism may also relate tothe activation of Hedgehog signaling pathways.