The Toxic Effect Of Leukamenin E On Primary Cultured Wistar Rat Cerebral Astrocyte

Toxicology is a area to study the toxic reaction, degree of damage, frequency and the mechanism of toxicity of chemical materials, in organism, and to evaluate the qualitative and quantitative toxic action as well. Because of the high-flux, high-precision, high-speed and parallelism, the experiment in vitro has becoming an advanced and pragmatic technology in biomedicine study. The toxicology will be studied in cell and molecular level in the future. Because of the complexity of Chinese herb (especially compound preparation) , it is difficult to evaluate the genetoxic by using the traditional method. However, we can easily study the target and mechanism of the genetoxic of Chinese crude drug by using the technology of cytology, which is a precise, fast, and efficient biotechnology.Astroglia (AS), taking parts in nutrition, support and protection of neuron in nervous system, is sensitive and easily damaged by the toxic substance. Many studies showed that the astroglia plays an important role in neuro-degenerative disease. Hitherto, however, there has been no any report about the cytotoxicity research in astroglia.Isodon plants, a traditional Chinese herbal with various categories (approximately 30) and far-ranging distribution in China, have been reported to be used as folk medicine of treatment to various diseases, such as heat-clearing and detoxicating, anti-bacterial and eliminate inflammation, anti-tumor and various hepatitis treatment, and so on. It reported diterpenoid was the most representative component in genus Isodon, and it was known to be the main active component in medicinal Isodon. Thereinto, C-20-un-oxidative ent-kaurane diterpenoid has the strongest bioactivity in various structure diterpenoids, and Leukamenin E is a representative one.In this study, the toxic effects of Leukamenin E on proliferation in purified and cultivated cells of astroglia of Wistar rat were studied, and some possible mechanisms were proposed. The study was mainly conducted with SRB assay, Trypan blue exclude staining, comet assay and AO/EB double fluorescent dye staining, observation of morphology were also used. Following conclusions were demonstrated from this paper:(1) Highly puritied and good state of AS were successively obtained by the methods of tissue digestion, restricted cultivation and rocking bed shaking. Two or three days after inoculation, the primary cultivated AS could completely adherence, and 9 or 12 days later, the cells can be confluent in the bottom of culture flask. Some microglia, fibroblast, neuron were contained in the primary culture. The growth speed of fibroblast could be reduced by restricted cultivation, and highly purified AS can be obtained by the next purification. In addition, restricted cultivation could gradually result in degeneration and death of neuron.(2) The results of SRB assay and Trypan blue staining showed that Leukamenin E had a strong cytotoxicity, it can significantly inhibit AS growth at low concentrations (≤1.25μM), and high concentrations (≥5μM) treatment can make cells death. The growth inhibition effect and lethal effect were in time and dose-dependent manners.(3) Leukamenin E could markedly change the morphology of AS, observed by the inverted microscope and Giemsa staining, and also in time and dose-dependent manner. In the experiment of Giemsa staining, cells showed unsharpness and eventually the disappearance of microvillus and the chromatic agglutination after treatment by Leukamenin E (1.25μM or 2.5μM). At the high concentration (5μM), nuclear fragmentation were also observed.(4) The DNA damage could be induced by Leukamenin E. In comet assay, comet rate,the rate of damaged cell was in time and dose-dependent manners in AS. And after the further statistical analysis, it exhibited that the damage cells, injury rate, OTM, TL and % tail DNA were all significantly increased in time and dose-dependent manner.(5) The results of fluorescent staining of Hoechst 33258 and double sequential AO/EB staining showed that the representative characters of apoptosis could be induced significantly by Leukamenin E in time and dose-dependent manner. That means the induction of apoptosis in AS with Leukamenin E may be one of the major mechanisms for cell death.