| Genistein is a kind of soy isoflavones, and also a kind of tyrosine kinase inhibitors, which widespreads in legumes such as broom, acacia, huaijiao and other medicinal plants and whose content is rich in soybean hypocotyl. Genistein has estrogen-like effect and effect of anti-turner and is good for treatment of osteoporosis and other pharmacological effects reported by numbers papers in the word. However, as a single class of plant extracts, its effect of prevention and treatment of diabetes and its pharmacological mechanism are lack of study systemly. Diabetes is called xiaohezheng in Chinese traditional medicine, which is a common metabolic disorded disease. It is divided into two kinds of I (insulin-dependent) and II (non-insulin-dependent) at present by clinic. In recent years, with people's living standards improving, the incidence of this disease is uprising, but there are no one kind of drug could eradicate it completely. The medicine people used to treat diabetes are more chemical drugs than others for its fast effect and direct functions but there always have some side effects such as blood glucose declined too fast to lead hypoglycemia, the results are instable etc. Wnt signal pathway in human stem cells in patients with type 2 diabetes is increased. In addition, the level of CD30 and related gene expression in wnt and other signal pathway in hESCs reflects hESCs self-renewal capacity and genetic stability of the nuclear type. The increase of CD30 and related gene expression in wnt and other signal pathway in hESCs indicates the tendency of genomic instability and transformation to cancer cells.There are three experiment parts which respectively study effect of genistein on T1DM and T2DM rats as well as expression of CD30 and related gene in wnt and other signal pathway in hESCs cultured in vitro.Part 1 The effect of genistein on T1DM:In vivo, we used intraperitoneal injection of alloxan one time to induce typeâ… diabetic rat model. Then divided the diabetic rats into 5 groups:typeâ… diabetic rat model group, high-dose genistein group (30mg/kg/day), middle-dose genistein group (18mg/kg/day), low-dose genistein group (9 mg/kg/day) and the vehicle tween -80 group. And the nomal rats whose number and age matched to other groups were as control group. The control group and T1DM model group were given equal volume of saline during the experimentl, and the vehicle group were daily given the same volume distilled water containing a small amount of tween-80. The quantity of eating, drinking, urine output and body weight were recorded daily, at the same time, rats'hair color,active and growth condition were observed, the value of blood glucose was tested periodicly. After continue lavaging genistein for 4 weeks, all the animals were fasting 12 h. Blood sample was collected from rats tail and blood glucose were measured. After continuously lavaging genistein for 1 week, the rats were fasting 12 h and were anesthetized by sodium pentobarbital. Blood sample were collected from heart. Serum glucose and insulin were determined according to reagent kit request. At last, the animals were killed and liver and hind leg muscles were gained for determination of liver and muscle glycogen.In vitro, rat islet cells were isolated and primary cultured. The islet cells damaged by alloxan were treated with different concentrations of genistein for different time, then the survival rate and proliferation ability of each group of islet cells were measured by MTT.By the experiments in vivo and in vitro, we first testify that genistein can reduce high blood glucose resulting from pancreatic damaged and the decrease of the number of islet cells by alloxan. The good effect of genistein on diabetic rats (to improve islet cell viability and proliferation) may be based on high-dose genistein. Genistein could dose-dependent decrease higher blood glucose and reduce the damage of islets and the effective genstein dose is apparently higher than the dose daily food intake. Therefore, the effectively increasing and supplement intake of genistein, are considered one of the ways to improve the symptoms of diabetes by dietary modification. In addition, the results showed for the first time that high dose of genistein could decrease the reduction of the number of islet cells damaged by alloxan. And high dose of genistein can contribute to islet cell number and to the function recover in vivo and in vitro. These results indicate the potential prevention and possible cellular mechanisms of genistein on diabetes.Part 2 The effect of genistein on T2DM:We used one time intraperitoneal injection of low dose streptozotocin and fed rats with high fat diet at the same time to induce T2DM (non-insulin-dependent) rat model. The experiment animals were divided into several groups as foregoing test. Then different doses of genistein were given to rats by gavage for 6 weeks. At the end of the experiment, the parameters of blood glucose, OGTT, serum insulin, C-peptide, TC, TG, LDL-C, liver glycogen and muscle glycogen were measured according to relevant reagent kits. The effects of genistein on type II diabetes in rats were evaluated.Our results suggest that genistein could significantly reduce serum insulin levels, make the value of C-peptide tend to be normal and it also could reduce TC, TG, LDL-C, liver and muscle glycogen content, increase HDL-C and HDL-C/LDL-C ratio of T2DM, indicating that genistein can effectively correctT2DM rats'insulin resistance and disorder of lipid metabolism, improve impaired glucose tolerance and liver and muscle glucose metabolism during its transit, reduce syndrome of atherosclerosis sclerotic coronary artery disease in T2DM. The mechanism of above action may be, by increasing reverse cholesterol transport, promotion of plasma HDL-C removing cholesterolin peripheral tissue, reducing obesity and insulin resistance, making glucose metabolism into virtuous circle of development in the body.Part 3 The effect of genistein on hESCs morphology and CD30 gene related expression:To human embryonic stem cell lines CA1, H1 and H9 in vitro, we treated it with different concentrations of genistein or PP2 continuously, and the morphological changes of hESCs observed by micrograph. Combined with the results of QPCR and west blotting, we analyze the effect of genistein on expression of CD30 and gene related in wnt and other signal pathway. We selected appropriated concentration of genistein to further treat hESCs and we could gain the possible molecular mechanisms and signal path of genistein on hESCs morphology and gene expression of CD30 by comparing and analysis.These results show that genistein12.5uM is the best concentration to treat hESCs in vitro. The effects of maintenance morphological characteristics and self-renewal of hESCs as well as prevention its differentiation were not significant when the concentration below 12.5uM; Also, it shows a certain toxicity when the concentration was above it. In addition, hESCs CA1 were treated continuously with genistein 12.5uM 13 days can make the expression of CD30 decrease by 60%, to a minimum, E-cadh expression decrease by 60%, Nanog reduce by 50%, while other gene expression were almost no significant change. Further result indicate that hESCs H9 continuously treated with genistein 12.5uM 14 days, the expression of CD30 reduced by 80% to a minimum, E-cadh decreased 60%, Nanog decreased 50%, NF-kB decreased nearly 60%, and other related gene expression did not change. Therefore, 12.5uM genistein could have potential ability of prevention genetic instability and karyotype change. but it is varying duration for different hESCs cell lines to achieve the best treatment effect. From the experimental results, in order to achieve the desired inhibition effect of expression of CD30 and gene related, the time of 12.5uM genistein treating hESCs H9 is longer than CA1 treated.
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