Study On The Potential Toxic Effects Of Anthraquinone Metabolites And Monomers Of Rhubarb On Human Colon Cells

Rhubarb is a traditional Chinese medicine and has been widely used in clinic as a laxative.Anthraquinones,the main active components of rhubarb,could be digested in the intestinal into complex mixtures which play rapid purgation but have potential side effects.However,it is in challenge to study the mechanisms by using common methods due to the complexity of the metabolites and the limitation of in vivo experiments.In this study,we prepared metabolites of sennoside A,total anthraquinone and alcohol extract of rhubarb by using the in vitro intestinal bacterial incubation system established by our team members.The metabolites were given to human colon cells to investigate their potential toxic effects regarding to autophagy and apoptosis.Using HPLC,metabolites of the three rhubarb products metabolites were quantified with the content of bound anthraquinones and free anthraquinones.NCM460 and HT29 cells viability were observed and determined using SRB assay.Acridine orange staining and western blot were used to assess their effects on autophagy.The apoptosis was evaluated based on mitochondrial membrane potential staining and western blot.Aquaporin(AQP)alteration was investigated by using RT-q PCR and western blot.The results showed that the content of sennoside A was decreased along with the culture time,meanwhile the contents of rhein,emodin and aloe-emodin was increased.So the in vitro intestinal bacterial incubation system could mimic the interstinal metabolic process in which the bound anthraquinone was metabolized into free anthraquinone.The cell viability assay showed that sennoside A did not influence the viability of NCM460 and HT29 cells,but the metabolites,rhein,emodin and aloe-emodin significantly inhibited the cell viability in a dose-dependent manner.Under fluorescence microscopy,we could observe autophagy and apoptosis cells treated with the metabolites,rhein,emodin and aloe-emodin,as the treated NCM460 and HT29 cells showed increased acidic vesicle organelles or decreased mitochondrial membrane potential.The western blot also showed up-regulated autophagic and apoptotic related proteins,and down-regulated anti-apoptotic proteins in the treated cells.But there was no influecne on NF-κB p65 and p-Histone H2 A.X,indicating their weak actions on the DNA double strand break.In addition,RT-q PCR and western blot assays showed that the metabolites,rhein,emodin and aloe-emodin could modulate some AQPs m RNA levels and down-regulated the AQP3 levels.In summary,free anthraquinones are main components of the rhubarb metabolites.They had multiple actions such as inducing autophagy and apoptosis,and modulating multiple AQPs subtypes.The status would produce side-effect to the colons if they were not repaired under long terms.