The Effect Of Amino Sugars On The Damaged Myocardial Cells And Myocardial Tissue

Heart disease can be divided into different kinds according to their formation mechanism and damage degree. Regardless of that, myocardial cell damage is the basic damage causes by all heart diseases.Chitosan is a kind of natural polymer glycosaminoglycan obtained in chitin, with good compatibility, biological functions, security, and biodegradability of microorganism. It is widely used in medicine, food, chemical industry and other fields in recent years. However, the lower solubility of chitosan restrained the further study of it. As the modification of Chitosan develops and its study in Medicine and biotechnology field goes deeper, the functional and safety evaluation of low molecular weight chitosan derivatives have become an important study content.Chondroitin sulfate is a kind of sugar glycosaminoglycans, which is easily dissolved in water and has important effect on preserving the relatively stableness and normal physiological function of cell environment. The experience of using medicine for a long time shows that the chondroitin sulfate effect of macromolecule is much slower. To functionally evaluate low molecular weight chondroitin sulfate after obtaining it from the degradation of chondroitin sulfate and to compare its efficiency and effectiveness with that of macromolecular chondroitin sulfate have become important contents of this study of materails.The basic materials of this topic are chitosan derivatives and chondroitin sulfate. By enzymatic degradation and purification, the low molecular weight chitosan derivatives and chondroitin sulfate are produced. Determination of the physical and chemical properties, cell level evaluation and evaluation of animal level are done to explore the protective effect of chitosan derivatives and chondroitin sulfate on the injured myocardial cells and tissues, which provide experimental basis for further development of chitosan derivatives and chondroitin sulfate.This experiment has got achievements and progresses in the following aspects:1. After purification, the carboxymethyl chitosan and chondroitin sulfate are degraded by chitosan and chondroitin sulfate and then the carboxymethyl shell oligosaccharides, chondroitin sulfate in the molecule and small molecule chondroitin sulfate are produced. 2. Experiments are conducted to determine the physical and chemical properties of the seven amino sugars. They are N-acetyl glucosamine, sulfonated Oligochitosan, high off deacetylation degree Oligochitosan, low degree of deacetylation of chitooligosaccharides, carboxymethyl chitosan, carboxymethyl chitosan oligosaccharide and chondroitin sulfate.3. Microscopic observation method and MTT method are adopted to figure out the effect of seven amino sugars mentioned above on the formal H9c2myocardial cells. The experiment shows that seven kinds of amino sugars were all not cytotoxic and they all promote the growth and proliferation of H9c2cell to varied degrees. The optimal effective density of N-acetyl glucosamine, sulfonated chitooligosaccharides, low degree of deacetylation of chitooligosaccharides, high degree of deacetylation of chitooligosaccharides and carboxymethyl chitooligosaccharides is50μg·mL-1, that of carboxymethyl chitosan is100μg·mL-1and that of small molecule chondroitin sulfate is200μg·mL-1, which provide theoretical support for the further experiments and applications of amino sugar.4. The mode of injured myocardial cells cultured in vitro is set up to assess the effect of the seven amino sugars on the injured myocardial cells. Through microscopic observation and MTT method, the experiment shows that the seven amino sugars have good influences on the growth of the injured myocardial cells. In particular, the effect of small molecule chondroitin sulfate and carboxymethyl chitosan oligosaccharide on the growth and reproduction of injured myocardial cells is prominent. The optimal effective density of small molecule chondroitin sulfate is200μg·mL-1while that of carboxymethyl chitosan oligosaccharide is50μg·mL-15. On the basis of dose regimen to copy the mode of myocardial infarction, the protective function of carboxymethyl chitosan oligosaccharide and small molecule chondroitin sulfate on the injured myocardial cells is assessed. The rat with injured myocardium is given a gastric lavage with carboxymethyl chitosan oligosaccharide and small molecule chondroitin sulfate of high, medium and low dose. The blood samples are taken out when at7dose and at14dose to test and compare the content of cardiac marker enzymes (lactate dehydrogenase, creatine kinase, aspartate aminotransferase) in serum. Tissue sections are made to conduct histopathology observation experiments. Through the test of serum marker enzymes the effects of both carboxymethyl chitosan oligosaccharide and small molecule chondroitin sulfate are found very similar. The difference analysis of the two kinds sugar of the same content shows an indistinctive difference(P>0.05). From the aspect of sugar content, middle dose group and high dose group of7d show initially the advantage of using sugar for cure (when comparing middle dose group and high dose group with ISO injury control group, there shows significant difference of P<0.01). The high dose group almost return to normal at14d (when comparing high dose group with the normal group, there shows an indistinctive difference of P>0.05). The result of tissue sections observation is consistent with that of serum marker enzymes performance, which proves that both kinds of sugar can help to restore injured myocardium.6. The same experiment method with that mentioned in article4is used to assess the protective functions of chondroitin sulfate of different molecular weights on injured myocardium. The result of test of serum marker enzymes’ contents shows that middle molecule chondroitin sulfate, especially the small molecule chondroitin sulfate can promote the restore of injured myocardium. Difference analysis of each sugar group and ISO injury control group indicates that small molecule chondroitin sulfate at7d shows significant difference (P<0.05) and when at14d and21d it shows extremely significant difference (P<0.01). Comparing each sugar group with normal group at21d, only the small molecule chondroitin sulfate group shows an indistinctive difference (P>0.05), which proves that the small molecule chondroitin sulfate group can almost return to normal at21d.