| BackgroundSenescence is natural process of human beings. And antiaging research is a hot theme of science field. Being estimated biologically, expectancy life span of human beings is 8-10 folds than our sexual mature period (namely 100 - 150 years old). At present, average life span is about 61 years old in the world. It is 70 years in developed country among which average life span of Japanese is 78 years old. Therefore, nowadays human life span hasn't reached the limit of the natural life span.Modern medical researches consider that the death of aging cells are cell apoptosis actually. Cell apoptosis is an active death process controlled by gene, which multi-cell organism maintain their autologous tissue and keep the balance between cell proliferation and death. In those many motivations, free radical produced in body metabolism or extrinsic factor can induce cell apoptosis.Apoptotic cells react with aging in two ways. First, they clear damaged or dysfunctional cells, and are replaced by fibrous tissue to maintain the homeostasis. Second, they clear irregenerative cells (eg. neuron, cardiomyocytes) which can not be replaced, and cause pathological change. In this way cells apoptosis cause decrease the cells number of brain or heart, then result in aging pathological change in some critical organ such as brain mantle and myocardium.It has been confirmed that inhibiting cadiomyocytes apoptosis can protect heart on certain degree. Scientists showed great interests on the effect of drugs anti-apoptosis on cardiomyocytes. The researches of some traditional drugs curing cardiovascular diseases (eg. ACEI) have showed that these drugs inhibit cardiomyocytes apoptosis obviously in animal experiments.Ever since a long time ago, China medicine play an important role in cardiovascular diseases treatment. Scientists are paying attention to the relation between China medicine and cardiomyocytes apoptosis, and have obtained some success. the researches have to be done further, especially on the effective simple component of China medicine.We have confirmed that Tremella Polysaccharides(TP) has effect on clearing oxygen free radical and preventing lipid peroxidation. So we presume that TP has the effect of anti-apoptosis on cardiomyocytes. In this study we use methods in modern pharmacology, cytobiology, histochemistry to observe the effect of TP on apoptosis induced by oxidative stress in neonatal rat cardiomyocytes in vitro culture, and study its effect on cardiomyocytes apoptosis in aging mice model in vivo. It can identify the mechanism of TP's anti-apoptosis and anti-aging effect and its value of clinical appliancation.Objective1,To study anti-apoptotic mechanism of TP at the level of cell and whole animal;2,To find the best effect dose of TP to prevent cardiomyocytes apoptosis;3,To provide experimental theoretic foundation for clinical applications of TP.Methods1,Purification and certification of TP raw powder.2,Study the effects of TP on apoptosis of cardiomyocytes induced by oxidative stress neonatal rat. Primary cultured cardiomyocytes were randomly assigned to three experimental groups: (1)control group(A); (2)apoptotic model group induced by H2O2(B); (3)TP group(C). Morphologic changes of cardiomyocytes were observed by transmission electron microscope. The cell viability was determined by trypan blue exclusion. The apoptotic cells were dyed by Annexin V-FITC/PI and the apoptotic index was evaluated by flow cytometry (FCM).3,Making aging animal model by injecting D-galactose and treated with TP.One hundred ICR mice were divided into five groups. In addition to normal control,every group had been injected with the dose of D-galactose 120mg/kg/d for 8 weeks to establish subacute aging model. At the same time,low dose(L), middle dose(M) and high dose(H) groups were treated with 100,200 and 400mg/kg TP respectively by intragastric (ig) administration. 8 weeks later,the cardiac muscles were collected, to be studied in histology: HE stain and SA-β-Gal stain. Then were examined the antioxidase GSH-Px, SOD and oxidant production LP, MDA.Results1,We get purified TP powder with 80% polysaccharides certificated by vapor phase chromatography.2,Experiment in vitro:(1)Typical morphologic changes of apoptosis were found in the model group;(2)Compared with those of control group, the absorbance of trypan blue and the apoptotic index in the model group increased significantly.(3)After treatment with TP, the ultrastructure changes induced by oxidative stress were improved; the absorbance of trypan blue and the apoptotic index decreased remarkably.3,Animal experiment:(1) Pathology result by light microscope: there are no significant differences between aging groups and normal group.(2) SA-β-Gal stain: Nucleus of negative cells are red re-stained by eosin, and kytoplasm of positive cells are indigo blue or royal blue. Use semiquantitative scoring to calculate values of each group. Values of cardiomyocytes in all aging groups are higher than normal group(P<0.05), which means numbers of positive cells are higher. The method of making aging model is efficient. In TP treatment groups, high dose group(400mg/kg) has the lowest value(P<0.05), which means positive cells are least in this group and the activity of SA-β-Gal is lowest.(3)In situ apoptotic cells detection by TUNEL: the apoptosis index (AI) in normal group is lower than any other groups(P<0.05). Comparing AI between low dose group(L) and aging control group(C), AI in two group are different but the difference is not significant(P>0.05). AI in middle dose group(M) and high dose group(H) are both lower than in aging control group(C) (P<0.05).(4)The antioxidation of TP in mice cardiac muscles: the activities of SOD and GSH-Px in the groups treated with TP were higher than aging control group(C), and the content of MDA and LP in the groups treated with TP were lower than C group(P<0.05). The activities of SOD and GSH-Px in M group and H group are higher than L group(P<0.05), and the content of MDA and LP in them are lower than L group(P<0.05). There is dose-effect relationship between the treatment doses of TP and the activities of SOD and GSH-Px and the content of MDA and LP in TP treatment groups.Conclusions1,Our laboratory has built up a method to purify TP raw powder, which can get high quality TP powder.2,TP can inhibit the apoptosis induced by oxidative stress in neonatal rat myocardial cell, which can be protection for cadiomyocytes.3,Animal aging model was obtained by injecting D-galactose.4,TP has anti-apoptotic and antioxidative effects on cardiomyocytes in aging mice model induced by D-galactose.5,The anti-apoptotic and antioxidative effects of TP has some dose-effect relationship. |
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