Study On Hepatotoxicity And Toxicologica Mechanism Of Uyghur Traditional Herbal Colchicum Autumnale L.in Vitro

The perennial herbs of Colchicum autumnale L also called Crocus sativus or Saffron that belonged to the Lily family (Liliaceae) were Native to Europe, North Africa and Asia. The chief medical parts of Colchicum autumnale L is the corm and the colchicin is the main pharmaceutical components in it. The colchicin has been used in the treatment of gout, cancer and hepatitis in clinic; while, it also has a certain side effects during dosing, such as kidney or liver failure, nausea, intestinal paralysis, Gastric bleeding; and overdosage of it could damage the viscera and even cause death in animals, thus limiting its clinical application. To date, little known about whether the colchicin is the key toxicity components in the herb and it's mechanism of apoptosis.In this paper, we use a HPLC method to compute the content of colchicin in the crude extract and total alkaloids of Colchicum autumnale L. by standard curve which colchicin as standard. Then experiment was carried out using the different hepatocytes (rat hepatocyte line BRL 3A, human liver tumor cell line HepG2 and human normal liver cell line L-02) in vitro to detect cell morphological changes. MTT assay, cellular morphology, lactate dehydrogenase (LDH assay) membrane leakage were adopted to evaluate the hepatocytes toxicity when cells were treated with colchicin and compared their differences under different samples treatment. Finally, we use the L-02 cells to investigate cytotoxicity that induced by different Colchicum autumnale L samples and to explore whether the colchicin is the main toxic components. Besides, the enzymatic activities of ALT and AST were measured by ELISA kits to evaluate liver cell functions and AnnexinV/PI double staining method was employed to detect whether the cell cycle was blocked. The mechanism of apoptosis at molecular level was detected by Immunoblot technique to exam apoptosis proteins Bcl-2 and Bax expression. The activation of Caspase family were detected and the change of mitochondrial membrane potential and were observed by using flow cytometry and fluoroimaging methods respectively. The results showed that all the samples could induce cytotoxicity to different cells and effects are equivalent. Then, we found that the colchicin maybe the key toxicity components in the herb and different samples have same effect on L-02 when comparisons made between samples. The result of the influence to the functions of hepatocytes found that all samples could increase activities of AST and ALT, but non-significant to control, this may suggest that AST and ALT are not sensitive indicators to hepatotoxicity detection in vitro. Finally, the potential mechanism of hepatotoxicity that induced by colchicin indicated that Colchicum autumnale L. samples could induce cell apoptosis and loss of mitochondrial membrane potential and block cells in G2/M stage but non-significant between samples. In addition, they could increase the expression of Caspase 9 and 3, and down regulate anti-apoptotic protein Bcl-2 expression and up regulate pro-apoptotic protein Bax. This may suggest that colchicin could induce apoptosis in L-02 cells through endogenous apoptosis pathway that mediated by mitochondria.