Protective Effects And Mechanism Of Puerarin And Flavonoids In Hickory Leaf Against Tetrabromobisphenol A-induced Developmental Toxicity In Zebrafish Embryos

Objective:We discussed the toxic effects of TBBPA on zebrafish embryos, looking for its potential target organs for toxicity and interpretation of toxicity mechanism on morphological, zymologic and molecular levels. At the same time we districted and separated the flavonoids in hickory leaf, filtered and discussed the protective effects of several chinese medicine effective constituents and the flavonoids in hickory (C.cathayensis Sarg) leaf. Furthermore, we discussed the protective mechanism, particularly the antioxidation, anti-apoptosis and cardiac protection mechanism, to provide related basis for the environmental risk assessment and the effects on human health of TBBPA, and to provide related experimental supports on further development and utilization of puerarin and flavonoids in hickory leaf.Methods (1) Wild-type (AB strain) zebrafish were maintained to get the embryo. To discuss weather TBBPA could induce apoptosis in zebrafish embryo, morphological tests and AO staining were operated after exposure to different concentration of TBBPA. Through the DN A ladder test and ROS enzyme immunoassay, we further confirmed the apoptosis and influence on ROS level induced by TBBPA. Then, we detected the expression level of apoptotic related genes by Real-time PCR to filter the functional genes and try to explain the toxic mechanism of TBBPA on zebrafish embryo.(2) We tested the influence of on the morphological toxic effects of TBBPA on zebrafish embryo and forecasted weather the protective effects is related to anti-apoptosis by AO staining and ROS enzyme immunoassay. Furthermore, we detected the expression level of cardiac developmental related genes by Real-time PCR to try to explain the protective mechanism.(3) The hickory leaves were abstracted by ultrasonic treatment, extracted in chloroform, separated by gel column, depurated by silica gel column chromatography, then according to sampling points in silica gel plate, the flavonoids in hickory leaf were collected and analysed by HPLC. Then, we discussed its protective effects by the morphological test and investigated the protective mechanism by ROS enzyme immunoassay and by detecting apoptotic genes expression with Real-time PCR.Results (1) The result of morphological indexes test showed that exposure to TBBPA caused malformation and oosperm coagulation after96h which was significantly increased across0.5and1.0mg/L groups compared to the control group. The survival rate was reduced in the1.0mg/L group. Heart development related morphological indexes such as hemodynamic disorder after24h and pericardial edema after48h were significantly increased across0.1,0.5, and1.0mg/L groups in a concentration-dependent manner. Furthermore, pericardial edema after48h and hemodynamic disorder after24h in1.0mg/L were both significantly increased (p<0.001). No significant changes were observed on the heart rate. As presented in the result of AO staining, no obvious apoptotic cells were found in the control group or0.05mg L-1TBBPA-treated group, while, considerable numbers of apoptotic cells appeared mainly in the heart area in all other TBBPA-treated groups. The result of DNA ladder text showed there occurred obvious DNA ladder in the1mg/L TBBPA concentration group which could further confirm apoptosis appears. ROS enzyme immunoassay showed that the ROS level was significantly up-regulated in0.5mg/L and1.0mg/L groups. The expression of apoptosis related genes, such as P53、Bax and Caspase9, were significantly up-regulated in all TBBPA exposed groups compared to the control group. In contrast, Bcl-1was down-regulated in the0.1,0.5and1.0mg/L concentrations. Enzyme activity level of Caspase9was significantly up-regulated in high concentrations of TBBPA, which has conformance with its mRNA expression level.(2) Puerarin showed protective effects on morphological toxicity induced by TBBPA on zebrafish embryo. However, the results of AO staining and ROS enzyme immunoassay showed that there is no significant difference between P+T and T group, which indicates that the protective effects of puerarin might not be related to its anti-apoptosis. Three heart development related genes, Tbxl, Raldh2, and Bmp2b were measured. The gene expression of all of them were significantly down-regulated induced by TBBPA compared to the control group, while they were significantly up-regulated (p<0.01) under the protection of puerarin in P+T group compared to the T group particularly the expression of Bmp2b (p<0.001). In addition, no significant changes of all gene expression were demonstrated in P group compared to the control group. Therefore, the protective mechanism of puerarin against toxicity induced by TBBPA on zebrafish embryo might be related to its influence on normal expression of heart development related genes, Tbxl, Raldh2, and Bmp2b. Nevertheless, further researches are needed to be done.(3) The flavonoids in hickory showed protective effects on morphological toxicity induced by TBBPA on zebrafish embryo, such as oosperm coagulation, malformation and hemodynamic disorder. ROS enzyme immunoassay showed that ROS level in S+T group was significantly down-regulated compared with T, and so was the expression of P53. In contrast, Bcl-2was up-regulated and so was it in S compared with the control. Therefore, the flavonoids in hickory might have anti-oxidation and anti-apoptosis effects, which may be related to the chalcone.Conclusion The toxic effects induce by TBBPA in zebrafish embryo such as oosperm coagulation, malformation, hemodynamic disorder, and low survival were observed, and the potential toxic target might be the heart. The toxic mechanism might be related to the up-regulated ROS level and the expression of apoptotic genes, as well as the down-regulated anti-apoptotic genes, which further induces apoptotic in heart. The flavonoids in hickory and puerarin showed protective effects on toxicity induced by TBBPA in zebrafish embryo, especially performance in the inhibiting effects of puerarin on abnormal expression of heart development related genes and the anti-oxidation and anti-apoptosis effects of the flavonoids in hickory.