Liver Toxicity Of Autophagy - Mediated Triptolide

Objective:Tripterygium wilfordii has a great treatment effect at rheumatoid, anti-inflammatory and anti-tumor which also cause extensive research and attention, but toxicity limits its application in clinical practice. Tripterygium chemical composition is very complex. Triptolide is the main active ingredient of Tripterygium, but it is also the main component of triptolide toxicity. So the study of toxicity of triptolide has become a major aspect of research. It has toxicity on digestive system, blood system, skin and mucous membrane system, cardiovascular system, immune system, reproductive system. The liver toxicity is one of the main aspects. Currently, there are a large number of domestic and foreign studies on the toxicity of Tripterygium extract and many different points of view, but the specific mechanism remains to be determined. This study monitors autophagy by using a variety of in vitro and in vivo experimental methods to study new mechanisms of triptolide liver toxicity.Methods:HepG2 cells were used in vitro experiment. IC50 of triptolide treated HepG2 was calculated by MTS assay; HepG2 cells were divided into five groups:triptolide low-dose group(0.3 X 10-5mg/mL), triptolide high-dose group(0.6×10-5mg/mL), rapamycin group(104nM), chloroquine group(20μmol/L), normal group(equal volume drug free medium). Autophagy of RFP-GFP-tagged LC3 adenovirus infected HepG2 cells was observed by the laser confocal fluorescence microscopy after 12 hours drug treatment; HepG2 cells were divided into six groups:triptolide low-dose group(0.3 × 10-5mg/mL), triptolide high-dose group(0.6× 10-5mg/mL), rapamycin group(104nM), chloroquine group(20μmol/L), normal group(equal volume drug free medium), triptolide-chloroquine group(triptolide:0.6×10-5mg/mL, chloroquine:104nM); HepG2 cells were detected the expression of LC3A/B-Ⅰ, LC3A/B-Ⅱ, p62 protein by Western Blot for semi-quantitative interpretation after 12 hours and 24hours drug treatment.Kunming mice were used in vivo experiment. The acute liver injury model was built by treating mice with lmg/kg triptolide in intragastric administration. Normal group was treated with equal volume distilled water. After 12h and 24h of administration, the liver was disposed with fixed liquid and frozen processing and observe by electron microscope; Mice were divided into four groups:triptolide group(IG,0.8mg/kg), triptolide-chloroquine group(triptolied:IG,0.8mg/kg; chloroquine:IP,60mg/kg), triptolide-rapamycin(triptolied:IG,0.8mg/kg; rapamycin: IP,2mg/kg), normal group(IG, equal volume distilled water). The acute toxicity was observed and the dead time was recorded; Mice were divided into five groups:triptolide group(IG,0.8mg/kg), triptolide-chloroquine group(triptolied:IG,0.8mg/kg; chloroquine:IP,60mg/kg), triptolide-rapamycin(triptolied:IG,0.8mg/kg; rapamycin: IP,2mg/kg), normal group(IG, equal volume distilled water), starved group(IG, equal volume distilled water).Autophagy was observed in RFP-GFP-tagged LC3 adenovirus infected mice by using small animal imaging.Results:IC50 of triptolide was 1.152x10-5mg/ml. A lot of autophagys and autolysosomes were caused in HepG2 cells by triptolide and the ratio of LC3A/B-Ⅱ and LC3A/B-Ⅰ, the number of p62 was decreased in HepG2 cells. Because of triptolide, mice hepatocyte nuclear morphology was changed, nuclear chromatin was marginalized obviously and a lot of organelle can be seen in cytoplasm bilayer structure which are autophagosome. The autophagys of liver and kidney can be inceresed in mice. Acute toxicity of triptolide can be improved by promoting autophagy, and thus reduce the survival rate of mice.Conclusion:Triptolide can induce autophagy in liver and accelerate the flow of autophagy and then it affect the triptolide toxicity, suggesting that triptolide may cause a chain reaction through autophagy-mediated way and produce liver toxicity.