Plasma Proteinogram Research On Prevention Rat Sub-health Fatigue With Weikang Granula

Sub-health status is a intermediate state which dynamic changes between health and disease,namely the Health-subhealth-disease.subhealth transitional changes between its upstream health and downstream disease,such change is a two-way migration and form a dynamic changes process:healthy-subhealth-disease,so subhealth is more complex than health and disease.There have no clear diseases but the lower vitality and varying degrees decreased resilience judging from the performance of the body,which is a physiological function reduced state ranging between health and disease,also known as the "third state".In this state,people feel physically and mentally maladaptation and do all kinds of medical examination and laboratory tests can not found any structural changes.Large-scale sub-health epidemiological studies suggest that fatigue is not only an important clinical manifestations of the sub-health state,but also an 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 modem 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.Therefore.adopting the subhealth fatigue rat model and using the proteomics experimental technology to comprehensive observation proteins 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.This study includes three parts.First,preparation for animal model of subhealth fatigue. In accordance with past our study as well as the relevant research data at home and abroad,we found that the occurrence of subhealth fatigue closed relate with excessive tiredness,lack of sleep.Therefore,we use the exhaustive swimming integrated 20 h of sleep deprivation method to prepare subhealth fatigue rat model, and comprehensive evaluate the model through exhaustive swimming time and blood biochemical indicators,as well as histopathological examination.Second, Pharmacodynamic Study of Weikang-granula prevention subhealth fatigue.Third, plasma proteomics research for subhealth fatigue and after the prevention of Weikang-granula. ChapterⅠPreparation of a sub-health fatigue rat modelObjective:To prepare a sub-health fatigue rat model.Method:Experiment 1:Subhealth fatigue rats modeling method research.32 rats were randomly divided into four groups:The first group（n=8,exhaustive swimming integrated sleep deprivation 20 h）,the second group（n=8,exhaustive swimming integrated sleep deprivation16h）,the third group(（n=8,exhaustive swimming integrated sleep deprivation 12 h）,the fourth group（n=8,normal control group）. Concrete method:thel,2,3 group rat loaded with 5%percent weight were exhaustive swim in swimming-pool with the depth of 50 cm and 24±2℃water.Then,get the 1,2,3 group in mice cage（wire cover,rats can not escape and free water and food）at 13:00,17:00,21:00 respectively for sleep deprivation.Record the exhaustive swimming time and rat behaviour such as mental state,weight,diet,activities,gait, et al.Experiment 2:Subhealth fatigue rats modeling time research.32 rats were randomly divided into four groups:normal control group,1 day group,4 days group,5 days group,8 rats per group.Adopting the eaxaustive swimming integrated sleep deprivation 20 h method to prepare model.Exaustive swimming standard:Rats nose into the water for 10 s.Record the exhaustive swimming time.Each group fasting one day in the end of the experiment,and take the blood biochemistry and each organ tissue pathological examination.Results:Experiment 1:1、Exaustive swimming time:there were statistical difference in the last exhaustive swimming time of the 1^st group compared with the first of the 1^st group and the last exhaustive swimming time of the 2^st,3^st group（P＜0.05）.2、General performance:Compared with Group 4,the activity and response capabilities of rats in Group 1 have a significant drop,in group 2 have not obvious decline,in Group 3 have the normal activity and response capabilities.Experiment 2:1、Exaustive swimming time:there were no statistical difference between each group in last exhaustive swimming time（P＞0.05）.Compared the last with the first exhaustive swimming time have statistical differene in each group（P＜0.05）.2、General performance:compared with normal control group,the activity and response capabilities were decreased in 4 days group and 5 days group.3、Blood biochemistry test results:the levels of biochemical indicators in the normal range in every group except 5 days group.The levels of blood HBDH,BUN in 5 days group compared with the rest group have statistical difference（P＜0.05）. The levels of blood CK,CHOL in 5 days group compared with 1 days group and normal control group have statistical difference（P＜0.05）.The levels of blood AST, ALT,CREA,TG in 5 days group compared with normal control group have statistical difference（P＜0.05）.4、Pathological examination results:the pathological examination no obvious organ abnormalities in each group.Conclusion:On the condition of indoor temperature（22～26℃）,humidity（65±5%）, light-controlled environment（light and dark,12h alternation）,the method,rats were exaustive swimming in a 120cm×60cm×60cm swimming pool filled with water （24±2℃） to a height of 50cm,and sleep deprivation 20 hours in mice cage filled with water to a height of 1.5 cm,for 4 consecutive days,might establish a rat model of sub-health. ChapterⅡThe effect of Weikang-granula on subhealth fatigue preventionObjective:To observation the effect of Weikang-granula on subhealth fatigue prevention.Method:24 rats were divided into three groups:the normal control group,the model groupand the weikang granule high dose group.The normal control group was take no intervention except intragastric administration（11:00am,Sodium Chloride 2 ml） when the model group and the weikang granule high dose group were modeling. The model group and the weikang granule high dose group were kept exaustive swimming in a 120cm×60cm×60cm swimming pool filled with water（24±2℃） to a height of 50cm,and sleep deprivation 20 hours in mice cage filled with water to a height of 1.5 cm,for 4 consecutive days.Also,The weikang granule high dose group received daily gavages for 4 days with lixivium of Weikang granule（0.28g/ml）.And the model group need to take intragastric administration（11:00am,Sodium Chloride 2 ml） also.Record the exhaustive swimming time and the general performance.All rats were executed and got blood at the 6^th day.Results:1、The exhaustive swimming time:compared with the weikang granule high dose group,the exhausted swimming time of the model group decreased significantly at the 5^th day（P＜0.05）.2、Genenral performance:the activity and response capabilities in weikang granule high dose group better than in model group.Conclusion:Weikang granule have a good effect on prevention subhealth fatigue. ChapterⅢPlasma proteinogram research on prevention subhealth fatigue with Weikang-granulaObjective:To investigate the plasm proteinogram of each group for the next step to discover the plasma differential expression protein between normal group,model group,weikang group,make a foundation for 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.Method:Make plasma concentration and desalination in each group by acetone precipitation,quantitate protein samples by Bradford method,mix with the same group samples for two-dimensional gel electrophoresis,stain gel by silver nitrate, use Power look1100 scanner to scan images,analyse the differentially expressed proteins point data by two-dimensional pattern analysis software PDQuest 7.1.0.Results:There have 120 points significant difference between the proteinogram of the model group and the normal group.Compared with other two groups,17 proteins recovered expression in the proteinogram of Weikang high-dose group.Conclusion:The occurrence of sub-health fatigue of rat may be related to 17 protein points which compared with model group recovered express in high-dose weikang group.