The Study On Developmental Toxicity And Anti-inflammation Activity Of Xiaoaiping In Zebrafish

Objective : 1.To compare the embryo developmental toxicity of Xiaoaiping(XAP),Tenacissoside G,Tenacissoside H and Tenacissoside I;To illuminate the mechanism of developmental toxicity that XAP induced.2.To assess the antitumor activity of XAP and to compare the anti-inflammation activity of Xiaoaiping(XAP),Tenacissoside G,Tenacissoside H and Tenacissoside I.3.The correlation analysis of the toxicity/efficacy and antiinflammation mechanism of tenacissoside H.Methods:1.The study of developmental toxicity.(1)Dose-toxicity relation of embryo development.The wild type AB zebrafish embryo,which will be developed to 6 hours after fertilization(6 hours post fertilization,hpf),is exposed to different concentrations according to the preliminary test,and lasts for 120 hours,respectively after exposure 24,48,72,96,120 hours(hour post exposure,hpe)were observed and recorded mortality and hatching rates.XAP,Tenacissoside G,Tenacissoside H and Tenacissoside I were treated in the same way as described above.(2)Study on the mechanism of XAP developmental toxicity.6 hpf embryos sustained in different concentration XAP for 120 hours,then,morphology,malformation rate,body length and behavioristics were recorded,the changes of the heart and liver were observed in the pathological slices and the change of the organelles in the myocardial fibers and cardiomyocytes was detected using transmission scanning electron microscope by the same time sampling.Real-time quantitative polymerase chain reaction(RT-PCR)was applied to detect oxidative stress,endoplasmic reticulum stress,apoptosis and wnt/?-cetanin pathway-related gene expression.2.Anti-tumor effect of XAP.The tumor model was established by injecting the MDA-MB-231 breast cancer cells into the yolk sac of the young zebrafish,and then the zebrafish after the injection were immersed in the different concentrations XAP(1.2,1.6,2.0 mg/m L),lasting 48 hours,the tumor cell migration of zebrafish was observed and recorded.3.Anti-inflammatory activity evaluation.Using healthy development of the 72 hpf green fluorescent macrophages(Tg: zlyz-EGFP)transgenic zebrafish as experimental animals,we set the different concentration of the Tenacissoside H according to the preliminary experiment,then,respectively,observed and recorded mortality,malfamation rate and body length at 24 hpe,48 hpe and 72 hpe.The zebrafish of 72 hpf treated with drug for 2 h and then added 20 ?M copper sulfate to incubate 1 h,and we took a microscope photo and counted the number of macrophages around the nerve mound,which is used to screen the anti-inflammatory activity of XAP,Tenacissoside G,Tenacissoside H and Tenacissoside I.0.025 mg/m L LPS treated zebrafish for 30 min,and then added the different concentration Tenacissoside H,respectively,recorded mortality,malformation rate and the number of macrophages in the tail at 24 hpe,48 hpe and 72 hpe.The expression of inflammation-related genes were detected by RT-PCR.Results:1.0.4 mg/m L XAP had a significant inhibitory effect on the hatching of zebrafish at 48 hpe,and when the concentration was higher than that of 0.8 mg/m L,there was a significant inhibition of zebrafish hatching at 72 hpe,but Tenacissoside G and Tenacissoside H had no significant inhibitory effect on zebrafish hatching,0.5 and 0.75 mg/m L Tenacissoside I had a significant inhibitory effect on the hatching of zebrafish at 72 hpe,meanwhile,there were a significant time and dose dependence on the lethal effect of Tenacissoside G,Tenacissoside H and Tenacissoside I on zebrafish embryos.After 120 hours of continuous exposure to XAP,the developmental score of 1.2 and 1.6 mg/mL exposed group was significantly lower,the malformation rate was significantly increased,the total distance of movement and the average velocity of movement decreased significantly.After 120 hours of continuous exposure to XAP,the heart rate of zebrafish decreased and the distance of SV-BA increased,and the pathological slices showed that cardiac cyclization,myocardial cell decrease,ventricular wall thinning.The cardiac electron microscopic scan showed a disorder of myocardial fibers.The kits test results showed that with the increase of the exposure dose,CAT activity increased,T-SOD activity decreased,MDA content increased,and GSH content decreased.The results of RT-PCR showed that the expression level of the oxidative stress related gene(cat,sod1 and gstp2)increased,then decreased with the increase of exposure dose;The expression of endoplasmic reticulum stress-related gene(chop,hspa5,hsp90b1 and perk)increased with the increase of exposure dose;Apoptosisrelated genes(caspase-3,p53 and bax)increased with exposure doses,while bcl-2 decreased with exposure doses;The expression of Wnt pathway-related gene(?-catenin,wnt3 a and wnt8a)was first increased and then decreased as the exposure dose increased,however,the expression of wnt11 increased significantly with the increase of XAP exposed concentration.2.The 1.2 and 1.6 mg/mL can inhibit the migration of MDA-MB-231 cells in zebrafish.Using copper sulfate model,0.1 mg/m L Tenacissoside G has anti-inflammatory activity,0.5 and 0.6 mg/m L Tenacissoside I have anti-inflammatory activity,XAP(1.2,1.6 and 2.0 mg/m L)and Tenacissoside H(0.15,0.2 and 0.25 mg/mL)also have anti-inflammatory activity.3.The normal physiological state of the zebrafish,Exposure concentration of Tenacissoside H is higher than 0.35 mg/m L induced toxicity,while the state zebrafish of LPS-induced inflammation,exposure concentration of Tenacissoside H is higher than 0.2 mg/m L can induce toxicity.Tenacissoside H concentrations below 0.05 mg/mL are no anti-inflammatory activity,higher than 0.05 mg/m L can alleviate the inflammation induced by LPS in zebrafish,reducing the size of the tail macrophages of zebra fish distribution.When the concentration was higher than that of 0.2 mg/m L,the number of macrophages in the tail of zebrafish increased and the malformation of body bending and abdominal edema increased.The results of RT-PCR showed that the inflammatory factors(tnf-?,il-1b,il-8,ptges,inos2 and cox-2)in LPS group were significantly up-regulated compared with control groups,and the inhibitory factor il-10 was down-regulated,myd88,nf-?b2,i?b?a and p38 expression was raised.Compared with the LPS model group,the expression of these genes in Tenacissoside H treatment group showed the opposite trend.Conclusion:1.The XAP can lead to the development toxicity of zebrafish embryo,including inhibiting hatching,cardiac edema and exercise capacity reduction,oxidative stress,endoplasmic reticulum stress,apoptosis and Wnt pathway to participate in these toxicity regulation mechanism.Embryo Developmental toxicity size comparison: XAP> Tenacissoside G> Tenacissoside H> TenacissosideI.2.XAP can inhibit the migration of MDA-MB-231 cells in zebrafish to exert an anti-tumor activity;Anti-inflammatory activity size comparison: Tenacissoside H> XAP > Tenacissoside G > Tenacissoside I.3.The toxicity of Tenacissoside H in the state of inflammatory is stronger than that of the normal physiological state of zebrafish.The low dosage of Tenacissoside H has anti-inflammatory effect,high dosage has developmental toxicity;Tenacissoside H can alleviate the inflammation induced by LPS,and the mechanism is related to the down-regulation of nf-?b and p38.