In Vivo and in Vitro Anti-tumour Effects of Selenium-protein Polysaccharide Extracted from Rich Selenium Agaricus Blazei
|Keywords||Rich selenium Agaricus blazei Selenium-protein polysaccharide anti-tumor BALB/c mice Immunity Serum pharmacology K562 cells Caspase-3 gene|
Agaricus blazei Murill, a native species to Brazil, has been widely planted in Japan and Indonesia since 1988. In 1995, Professor Liang Zheng in the Institutes for Botany, Chinese Academy of Sciences, successfully cultivated it with the wild strain from virgin forest in Brazil, and so it was named as Liang jing Mushroom. Main components of A. blazei, including steroid, polysaccharide, glycoprotein and nucleic acid, have been separated and purified. Among them, polysaccharide executes the activities of anti-tumour, immunity improving, anti-mutagenic and bacteria killing. In the year of 2000, this fungus has been successfully cultivated in Ziyang Country of Shan’xi Province, which is the second richest selenium area in China. Thus, it was named rich selenium A. blazei. Previous research shown that the medicinal properties of A. blazei might be influenced by different strains or growth conditions. However, the anti-tumour effect of the rich selenium A. blazei cultivated in China is still unclear.In this study, selenium-protein polysaccharide （SPP） have been isolated from the rich selenium A. blazei. The content of protein, selenium and polysaccharide were detected with Lowry-hydroxybenzene, phenol- vitriol and 2.3-two amido naphthaline fluorescence colorimetry respectively. The toxic level of selenium-protein polysaccharide was also evaluated by half of lethal dose (LD50).In most researchs of applying serum pharmacological method, the serum-treated with herbs was primarily prepared from normal status animal, and partial from pathological status animal. Although both of two serum-treated with herbs in many researches seems to have good anti-tumour effects, the difference of anti-tumour activity between them is still unknown.Thus, the present study explores anti-tumor effects of SPP in vivo and in vitro and compares the difference of anti-tumour activity of serum treated with SPP (SerumSPP) between non-tumor bearing mice and tumor bearing mice. The content as follows:1. In vivo experiment, the model of bearing tumor mice was induced. Anti-tumor effects and affects of immune system of SPP on bearing-tumor mice then were observed.2. In vitro experiment, serum pharmacology method was applied to treat the bearing and non-bearing tumour mice with SPP. The serum treated was then prepared. Growth inhibition rate of K562 cells were detected by MTT method. Apoptotic phenomenon was observed under a fluorescent microscope and through DNA electrophoresis, respectively. Caspase-3 enzymatic activity was measured by colorimetry.The results of this study shown:1. The content of protein, selenium and polysaccharide, which are active ingredient of rich A blazei, were 16.32%, 81.20% and 7.01mg/g respectively. LD50 was more than 3.0g/kg. This means that selenium-protein poly-saccharide is a low toxicological substance.2. The results of in vivo experiments show that selenium-protein polysaccharide at doses of 50.0 and 100.0 mg/kg inhibits proliferation of implanted Sarcoma 180 by 22.00 and 37.69% respectively, and significantly promotes lymphocyte transformation and natural killer （NK） cells activity of tumor bearing mice, which indicated that anti-tumour effect of SPP involved in enforcement of host immunity.3. The results of in vitro experiments show that SerumSPP could significantly inhibit proliferation of K562 cells in vitro and cause morphological typical changes of apoptosis, such as the formation of nuclear fragmentation and apoptotic bodies by acridine orange staining. Apoptosis induced by SerumSPP was also confirmed by formation of internucleosomal DNA fragmentation which is a biochemical indicator of apoptosis.4. Serum could significantly increase caspase-3 activity of K562 cells in vitro, which is indicated that apoptosis of K562 cells induced by SerumSPP may be related to up-regulation of caspase-3 gene expression.5. The difference in anti-tumour activity of SerumSPP between tumor and non-tumor bearing mice is significantly different （P<0.01）. The result indicated that, in order to represent better realic environment in vivo, researchers using serum pharmacology method should consider establishing pathological animal model.In conclusion, the present study investigated anti-tumor effects and mechanism of rich selenium A. blazei cultivated in China and elucidated the result that SPP could inhibit growth of tumor in vivo due to enhance host immunity. SerumSPP could induce apoptosis of tumor in vitro which may be related to up-regulation of caspase-3 gene expression. These results not only provide potent theoretical basis on pharmaceutical value of anti-tumor of rich selenium A. blazei but also provide useful reference and experience for anti-tumor research about applying serum pharmacological method.