基于斑马鱼模型的抗结核药物肝脏毒性比较研究

目的以肝脏绿色荧光转基因斑马鱼Tg(L-FABP:EGFP)为模型,研究一线抗结核药物异烟肼、吡嗪酰胺和乙胺丁醇对斑马鱼肝脏的影响以及比较其肝毒性大小。方法用不同浓度的异烟肼、吡嗪酰胺和乙胺丁醇分别处理发育至72h的斑马鱼,于加药后24、48和72hpe(hour post-exposure)观察斑马鱼的死亡率、畸形率和肝脏形态变化情况,荧光显微镜下观察药物对斑马鱼肝脏荧光面积和荧光强度的影响。结果随着给药浓度的增加和给药时间的延长,异烟肼、吡嗪酰胺和乙胺丁醇导致斑马鱼死亡率、畸形率升高。在72hpe,与空白对照组相比,1、2.5和5mmol/L吡嗪酰胺组斑马鱼肝脏荧光面积显著下降,5mmol/L异烟肼组斑马鱼肝脏荧光面积显著下降,10、20和30mmol/L乙胺丁醇组斑马鱼肝脏荧光面积显著下降。5mmol/L浓度下,异烟肼导致斑马鱼肝脏荧光面积降低程度高于吡嗪酰胺。结论异烟肼、吡嗪酰胺和乙胺丁醇均有肝脏毒性,乙胺丁醇肝毒性最小。1和2.5mmol/L浓度下吡嗪酰胺导致斑马鱼发生肝损伤程度强于异烟肼,但5mmol/L下异烟肼肝脏损伤程度强于吡嗪酰胺,其有关机制需进一步研究。

Kunxian Capsule Extract Inhibits Angiogenesis in Zebrafish Embryos via PI3K/AKT-MAPK-VEGF Pathway

Objective: To investigate the anti-angiogenic activity of Kunxian Capsule(KX) extract and explore the underlying molecular mechanism using zebrafish. Methods: The KX extract was prepared with 5.0 g in 100 mL of 40% methanol followed by ultrasonication and freeze drying. Freeze dried KX extract of 10.00 mg was used as test stock solution. Triptolide and icariin, the key bioactive compounds of KX were analyzed using ultra-high performance liquid chromatography. The transgenic zebrafish Tg(flk1:GFP) embryos were dechorionated at 20-h post fertilization(hpf) and treated with PTK 787, and 3.5, 7, 14 and 21 μg/m L of KX extract, respectively.After 24-h post exposure(hpe), mortality and malformation(%), intersegmental vessels(ISV) formation, and m RNA expression level of angiogenic pathway genes including phosphoinositide 3-kinase(PI3K), protein kinase B(AKT),extracellular signal-regulated kinases(ERKs), mitogen-activated protein kinase(MAPK), vascular endothelial growth factor(VEGF) and fibroblast growth factor(FGF-2) were determined. Further, the embryos at 72 hpf were treated with KX extract to observe the development of sub-intestinal vein(SIV) after 24 hpe. Results: The chromatographic analysis of test stock solution of KX extract showed that triptolide and icariin was found as 0.089 mg/g and 48.74 mg/g, respectively, which met the requirements of the national drug standards. In zebrafish larvae experiment, KX extract significantly inhibited the ISV(P<0.01) and SIV formation(P<0.05). Besides, the m RNA expression analysis showed that KX extract could significantly suppress the expressions of PI3K and AKT,thereby inhibiting the m RNA levels of ERKs and MAPK. Moreover, the downstream signaling cascade affected the expression of VEGF and its receptors(VEGFR and VEGFR-2). FGF-2, a strong angiogenic factor, was also downregulated by KX treatment in zebrafish larvae. Conclusion: KX extract exhibited anti-angiogenic effects in zebrafish embryos by regulating PI3K/AKT-MAPK-VEGF pathway and showed promising potential for RA treatment.

Tripterygium wilfordii multiglycoside-induced hepatotoxicity via inflammation and apoptosis in zebrafish

Tripterygium wilfordii multiglycoside(GTW)is a commonly used compound for the treatment of rheumatoid arthritis(RA)and immune diseases in clinical practice.However,it can induce liver injury and the mechanism of hepatotoxicity is still not clear.This study was designed to investigate GTW-induced hepatotoxicity in zebrafish larvae and explore the mechanism involved.The 72 hpf(hours post fertilization)zebrafish larvae were administered with different concentrations of GTW for three days and their mortality,malformation rate,morphological changes in the liver,transaminase levels,and histopathological changes in the liver of zebrafish larvae were detected.The reverse transcription-polymerase chain reaction(RT-PCR)was used to examine the levels of microRNA-122(miR-122)and genes related to inflammation,apoptosis,cell proliferation and liver function.The results showed that GTW increased the mortality of zebrafish larvae,while significant malformations and liver damage occurred.The main manifestations were elevated levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST),significant liver atrophy,vacuoles in liver tissue,sparse cytoplasm,and unclear hepatocyte contours.RT-PCR results showed that the expression of miR-122 significantly decreased by GTW;the mRNA levels of inflammation-related genes il1β,il6,tnfα,il10,cox2 and ptges significantly increased;the mRNA level of tgfβsignificantly decreased;the mRNA levels of apoptosis-related genes,caspase-8 and caspase-9,significantly increased;the mRNA level of bcl2 significantly decreased;the mRNA levels of cell proliferation-related genes,top2αand uhrf1,significantly reduced;the mRNA levels of liver function-related genes,alr and cyp3c1,significantly increased;and the mRNA level of cyp3a65 significantly decreased.In zebrafish,GTW can cause increased inflammation,enhanced apoptosis,decreased cell proliferation,and abnormal expression of liver function-related genes,leading to abnormal liver structure and function and resulting in hepatotoxicity.