Preliminary Study On The Changes Of Sub-health Fatigue Rat's Hypothalamic Proteinogram Caused By Weikang Granula

BACKGROUND:The mid-1980s, the former Soviet Union professor Buheman found that, in addition to health and disease states, there is a middle state of human-health and illness, known as the Sub-health status. The investigation indicates that there are only 5% of all people in the real health status, 20% in disease and other 75% in the sub-health status. The sub-health status is popular in the developed and competition intense country and region, and more and more people suffer the sub-health status. So sun-health status has become the hot spot in the region of internationals medical science. The sub-health clinical manifestations is varied, and according to large-scale sub-health epidemiological studies suggest that fatigue is not only an important clinical manifestation of the sub-health state, but also a critical reason resulted in the development of sub-health. Thus, clarifying the mechanism of fatigue subhealth, not only to an in-depth study of the etiology of sub-health, but also further taking positive steps to prevent the development of fatigue will have a positive impact on subhealth, and make a beneficial circle to the direction of healthy.So to establish a kind of sub-health model is the key point to make the researches deepened at present study. However there are not such models for the pathogen and mechanism of sub-health haven't been found out yet. As animal experiment can be controlled,operated and repeated so easily and conveniently that it is one of the most important methods in modern biomedical research. Hence it is much necessary to establish the sub-health animal model for investigating the rule of occurrence, development and the methods to prevent and cure sub-health. Formerly study indicate that hypothalamus is important to the fatigue formation, two-dimensional electrophoresis in protome is utility technique for fatigue mechanism. Therefore,adopting the sub-health fatigue rat model and using the proteomics experimental technology to comprehensive observe hypothalamus proteinogram changes in the development of subhealth fatigue, and then applying bioinformatics methods to analyse the link between proteins and phenotype of fatigue in subhealth, identifying the biomarkers of subhealth fatigue and investigating the mechanisms in the development of subhealth fatigue, as well as the mechanism of Weikang-granula. prevention subhealth fatigue, provide a theoretical basis for the effective prevention the occurrence and development of sub-health.AIM:To establish sub-heath fatigue rat model, study the mechanism of the sub-health fatigue by two-dimensional electrophoresis of proteomic and study the mechanism of action of relieving the fatigue of sun-health status by weikang granula.METHODS:1. Combine sleep deprivation with exhaustive weight loading swimming exercising to establish rat fatigue model of sub-health status. Rats in the model group were used to establish the model of chronic fatigue by being kept in a cage with water to a height of 1.5 cm and being deprived of sleep, afterwards, rats were required to conduct exhaustive swimming exercise in a glass pool with loading 5% of rat body weight. Exhaustive swimming standard was 10 seconds after rats sank into water without outcrop. Rats in the normal control group were not given any stimulation and were fed routinely. Determine the degree of sleep deprivation and total experiment time by taking two experiments. Evaluate the model by exhaustive swimming time and blood biochemical indexes.Exp1: Determine the degree of sleep deprivation. Male healthy Sprague-Dawley 32 rats in the SPF level was adopted in this study and randomly divided into 20h/d, 16h/d, 12h/d model group and normal control group. Observe the change of exhaustive swimming time in the period of 13 days After the 13^th-day, blood sample was taken from the abdominal aorta in each group. And serum was prepared by centrifugation and was determined for the blood biochemical index of ALT, AST, TP, Alb, TBIL, BUN, Scr, UA, LDH, CK, GLU, TG, Tch and CRP. Analysis the changes of the index between the model groups and normal group and determine the degree of sleep deprivation according to the time and blood biochemical index.Exp2: Determine the days of making model. According to the changes of the exhaustive swimming time and blood biochemical index, 32 rats were divided into 1d, 4d, 5d model group and normal control group, Blood sample was taken from the abdominal aorta in each group and serum was prepared by centrifugation and was determined for the blood biochemical index of ALT, AST, TP, Alb, TBIL, BUN, Scr, UA, LDH, CK, GLU, TG, Tch and CRP. Analysis the changes of the index between the model groups and normal group and determine the days of making model to the change of blood biochemical index.2. Give the model group rat take weikang granula through oral administration. 24 rats were divided into treated group, model group and normal control group. Compare the exhaustive swimming time between the treated group and model group, and obverse the effectiveness of relieving the fatigue of sun-health status. The standard of weighting fatigue is the exhaustive swimming time.3. Statistics analyze by SPSS13.0 for windows, compare different time spots and compare more model groups and control group by One-Way ANOVA, Repeated measures data is analyzed by Repeated Measures ANOVA,α=0.05.4. Make use of two-dimensional electrophoresis of proteomic, segregate the proteinum spots of hypothalamus in the normal group, model group and the group of taking weikang granula. According to the proteinum spot analysis software----PDQuest, find and identify the difference proteinum spots. Find out the correlative proteinum about the mechanism of the fatigue in sun-health status and the reliving fatigue by the weikang granula.RESULTS:1. Exp1. The exhaustive swimming times of 20h model group changed conspicuously and became shorten gradually. Compared the o-day, the time of the 13^th-day was significantly shortened (P<0.01) in the 20h model group, and the level of AST, UA, LDH, GLU, Tch changed obviously (P<0.05 or 0.01). The exhaustive swimming time of 16h and 12h model groups was prolonged, the blood biochemical indexes of the two groups didn't change obviously comparing the normal group(P>0.05).2. Exp2. Comparing the normal group, the level of AST, LT, BUN, Scr, HBDH, CK, TG, Tch changed obviously and ware significant different (P<0.05 or 0.01) in the 5d model group; And the biochemical indexes of the 1d and 4d model group did not change obviously(P>0.05).3. The exhaustive swimming time were longer obviously in the treated group than in the model group in the 14^th-day in the experiment (P<0.05).4. According to the analyses result by two-dimensional electrophoresis of proteomic, they changed mostly in volume and rarely in isoelectric point. Compared the control group, there are 71 difference spots in model group and 65 difference spots in treated group.find that there are 18 proteinum spots that express twice more, and there are 15 proteinum spots that express twice lower in model group and there are 12 proteinum spots that express twice more, and there are 9 proteinum spots that express twice lower in treated group. 32 difference spots recover obviously or close to normal by weikang granula intervention in the 71 difference spots. All of those proteinum spots might be interrelated to the cause of how to fatigue of sub-heath status happens.CONCLUSION:1. Be successful to establish the rat fatigue model of sub-health status by sleep deprivation combined with the exhaustive swimming for 4 days.2. The weikang granula have the function of prolonging the exhaustive swimming time and relieving the sub-health fatigue.3. Find the associated proteinum about how to fatigue of sub-heath status happen and how to relieving the sub-health fatigue by weikang granula.