A Study On The Immunomodulatory And Anti-tumour Mechanism Of The Polysaccarides From The Loach

Loach is a kind of fish that belongs to the family of Misgurnus angillicaudatus. It is recorded in A Dictionary of Chinese Traditional Medicine that its whole body or mucus secreted from the skins can be used as Chinese traditional medicine. The main pharmaceutical functions of the loach in folk recipes are to nourish spleen, enrich vital energy, protect liver, bring down jaundice (or icterus index), relive internal heat, detoxify, eliminate carbuncles and loosen scabs. Recently, live loach and its dry powder have been used in folk therapy to treat infectious hepatitis, and have very excellent curative effect. Firstly, our group isolated and purified a novel polysaccharide from the mucus secreted from the loach skins. Recently, many polysaccharides and polysaccharide–protein complexes have have attracted more attention recently in the biochemical and medical areas due to their immunomodulatory and anti-cancer effects. In the paper, the immunomodulatory and anti-tumor actions of polysaccharide were studied by the methods of cellular biology. The main results in this work are as follow: 1) To understand the immunomodulatory activity of MAP, a natural polysaccharide isolated from the mucus of the loach, Misgurnus anguillicudatus (MAP), MAP was investigated by the methods of molecular biology and cellular biology. MAP was found to significantly increase the proliferation of total spleen lymphocytes cell populations and more strongly increases that of T cells. However, MAP had less influence on the proliferation of B cells. Time dependence of the secretion of cytokines showed that Th1 cell was the primary cellular target affected by MAP on T lymphocyte. MAP improved the viability of peritoneal macrophages, stimulated TNF-αand IL-6 production and induced the inducible enzyme nitric oxide synthase (iNOS) transcription and enhanced the level of NO in macrophages. Moreover, MAP exhibits optimal bioactivities at the dose of 30μg/ml, presenting immuno-reagent manner. In conclusion, MAP can enhance the immune system functions and the biological activity of the loach may mainly result from MAP selectively activating T cells and macrophages and stimulating secretion of some cytokines. 2) To determine the antiproliferative, apoptotic properties of MAP on the tumor cells, the effects of MAP on the human promyelocytic leukemia line (HL-60) and he human heptatocelluar carcinoma cells (SMMC-7721) were studied. The results showed that MAP inhibited the cells viability in time and concentration dependent characteristics. After the cells were treated with MAP, the cells exhibited the characteristic morphological changes of apoptosis including membrane blebbing, chromatin condensation and the formation of apoptotic bodies; the intercellular DNA reveals a progressive increase in non-random fragmentation into a ladder of 180–200bp in the agarose gel electrophoresis; there are the apoptotic fraction, visible as a hypodiploid or sub-G0/G1 peak of cells treated with MAP assayed by flow cytometry (FCM). The results suggested that antiproliferative effect of MAP was associated with apoptosis on HL-60 cells and SMMC-7721 cells. 3) To estimate the anti-tumour mechanism of MAP, 2, 7-Dichlorofluorescin-diacetate (DCFH-DA) was used to detect the generation of intracellular ROS by MAP. MAP treatment greatly increases the population of the cells with high DCF fluorescence, which indicates an increased level of intracellular ROS. The free calcium concentration ([Ca2+]i) was measured using the Fura-2/AM calcium probe. The results demonstrated that MAP promoted markedly an increase of [Ca2+]i which was ascribed to the release of intracellular Ca2+ storage induced by MAP. The change of mitochondria membrane potential (MMP) of the SMMC-7721 cells treated with MAP were assayed. The results showed MAP can induce the dissipation of MMP. The mechanism possibly is that MAP effect on mitochondria and boost ROS, ROS mediates a release of Ca2+ from the intracellular Ca2+ pool, increased [Ca2+]i triggers the cells a start-up of the apoptosis program. 4) The present studies showed that MAP could induce the cells apoptosis which was closely accompanied with up-regulate of p53 mRNA, increase expression of Bax mRNA and decrease expression of Bcl-2 mRNA. However, the results that the levels of Fas andFasl mRNAs were almost unchanged when the cells were treated with MAP These results suggested that cell apoptosis induced by MAP mainly was mediated by mitochondrial pathways, not involved death receptors (DRs') pathways. 5) MAP improved the NOS and enhanced the expression of the inducible enzyme nitric oxide synthase (iNOS), which induced production of nitric oxide (NO). The results suggested that action of the oxidative stimulation on mitochondrial is one of important approaches though which MAP induced SMMC-7721 cells apoptosis.