| BACKGROUNDSub-health is an intermediate state between health and disease, which is mainly characterized as lower vitality, reaction and adaptability decline, and having no definite disease diagnosis. Its clinical manifestation was complicated, which involved in many symptoms of body, psychology and society, such as:languor, dizziness and cerebaria, palpitation and dyspnea, sleep disturbance, inappetence, easily coryza, drawdown of emotion, decrease of attention and memory, irascibility, tense social relations and so on. Epidemiological investigation showed that:the prevalence of sub-health was 60% to 70%. Sub-health status has seriously affected the people's work ability and life quality, and has become a major threat to people's and health life, which made it a medical problem to be solved to study the mechanism of sub-health, and effective prevention and control. Many investigations showed that fatigue is not only one of the important clinical manifestations among sub-health status, but also contributed to the development of sub-health and fatigue-type sub-health is one of the main types of sub-health. So discussion the mechanism of fatigue-type sub-health and active intervention of it should be the most important work in sub-health research and prevention. Immune system was an important defense system and played a crucial role in human health, and many of clinical diseases related with immune dysfunction, such as inflammation, infection, cancer, aging, reproduction, etc. Researches indicated related factors of sub-health, such as, over-work, psychological stress, staying up late, irregular life, can lead to immune dysfunction. Therefore it was necessary to investigate relationship between immune function and the incidence of fatigue-type sub-health.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 an ideal, a recognized animal model of fatigue-type sub-health means an important significance to explore the rule of occurrence, development of sub-health and formulation of control measures. In our past research, we used the method of 5% weight-loaded exhaustive swimming integrated 20 h of sleep deprivation to prepare fatigue-type sub-health status mice model, but there were still some gaps from creating a more ideal animal model of fatigue-type sub-health status. More improvements are needed in next study. In addition to the existence of the confused mechanism in sub-health study, more effective treatments were also lack. Characterized with the core thoughts of syndrome differentiation and concept of wholism, Traditional Chinese Medicine (TCM) has unique advantages in sub-health prevention and treatment. It has been confirmed in clinical and animal studies that of interventions of fatigue-type sub-health with weikang-granula have the exact effect, but the specific pharmacological mechanism was unclear, which obstructed the curement of sub-health and spread of weikang-granula.For these reasons, we would do researches to establish a mouse model of fatigue-type sub-health by simulating of the formating factors of fatigue-type sub-health, and discuss mainly the contact between fatigue-type sub-health and immune function, the effection of weikang-granula on the immune function of fatigue-type sub-health model mice by using of immune technique. This topic will be divided into two parts:PartⅠ:Establishment of fatigue-type sub-health mice model. In the course of experiments, mice were forced to stand in the water filled cage to cause their physical and psychological fatigue, psychological stress, circadian rhythm disorders, to imitate formating factors of fatigue-type sub-health. Then we analyse the performances of mice behaviors, degree of fatigue, pathological signs in tissue, blood biochemistry results and other indicators, to define and evaluate the optimal efficiency of this model.PartⅡ:The immune function changes of the fatigue-type sub-health mice with the interference of weikang-granula. Besides observation of curative effect, we also study the immune function changes of model mice before and after weikang-granula.AIM:Establish and evaluate of fatigue-type sub-health mice model; preliminary explore the mechanism of fatigue-type sub-health in terms of immune function, and the immune influence of weikang-granula in model mice, all of which lay the foundation for the mechanism research both in sub-health and weikang-granula.METHODS:PartⅠ:Establishment of fatigue-type sub-health mice model Experiment 1:Study of making model methods of fatigue-type sub-health in mice40 SPF male healthy KM mice were randomly divided into four groups:6h/d, 8h/d, 10h/d model group and normal control group (n=10,5 per box). Model group mice were treated by forced standing in water. Specific methods:every 9am, mice were kept in a plastic cage(29cm×18cm×15cm) filled with water to a height of 0.8 cm,5 per cage,6 cages in all, and were returned to home cage at 3,5,7pm respectively for 6h,8h, 10h groups. The normal control group was free of any stimulates. Weight-loaded forced swimming test was used to evaluate the extent of fatigue every three days in the 21 days of study. (PS:Weight-loaded forced swimming test was used to evaluate the extent of fatigue. Method:The mice were forced to swim with a load of lead rings attached to their tails that weighed approximately 7% of their body weight at a temperature of 25℃. Exhaustive swimming standard was 10 seconds after mice sank into water without outcrop). Observe behaviors changes of these mice, such as mental state, weight, diet, activities, et al, during the course of experiment.Experiment 2:Study of making model time of fatigue-type sub-health in mice150 SPF male healthy KM mice were randomly divided into 15 groups:10 groups for exhaustive swimming test:model and normal groups, each for Od,5d,9d, 12d,16d; 5 groups for blood biochemistry test:model 5d,9d,12d,16d and normal groups. The hour of model mice were kept in water filled cages was determined by the result of experiment 1. At the end of each time point in the study, exhaustive swimming time was tested only for swimming groups, and blood biochemistry groups were only taken for blood biochemistry and pathological examination for each organ tissue. Observe behaviors changes of these mice, such as mental state, weight, diet, activities, gait, et al, during the course of experiment.Experiment 3:the evaluation of fatigue-type sub-health mice model40 SPF male healthy KM mice were randomly divided into four groups:normal control group and the fatigue-type of sub-health model group, natural recovery for 7 days (rest 14 days after modeling), natural recovery for 14 days (rest 14 days after modeling). The scheme of model was determined by the results of the former experiments.7% weight-loaded forced swimming time was tested at first 2nd,8th, 15th day after modeling, for normal control group and the fatigue of sub-health model group natural recovery for 7 days group, natural recovery for 14 days group separately. Observe behaviors changes of these mice, such as mental state, weight, diet, activities, gait, et al, during the course of experiment.Part II:The immune function changes of the fatigue-type sub-health mice with the interference of weikang-granula120 SPF male healthy KM mice were randomly divided into six groups: weikang-granula high dose group, weikang-granula middle dose group, weikang-granula low dose group, fatigue-type sub-health model group, model of natural recovery group and normal control group. Each group has two subgroups for the examination of exhaustive swimming time and immune function. The former five groups were firstly prepared for the fatigue-type sub-health by the model method according to the first part of experiments, then, weikang-granula high, middle, low dose groups, and model of natural recovery group were administered by gavage for 7 days with lml weikang-granula solution of high, middle, low dose and normal saline individually. The normal control mice were free of any treatment in normal home cages. The exhaustive swimming time and spleen, thymus index and T\B lymphocyte proliferative responses were detected at the end of experiment. Observe behaviors changes of these mice, such as mental state, weight, diet, activities, gait, et al, during the course of experiment.RESULTS:Part I:Establishment of fatigue-type sub-health mice modelExperiment 1:Compared with the normal group, the mice activity and responsibility of 8h group were decreased with the extend of study, and it was significant in 10h group, but not distinctive in 6h group. The exhaustive swimming time of model mice showed marked reduction, and it was time dependence. Especially for 10h group, whose swimming time of mice dropped rapidly, with significant difference in first 3rd day to 0 day (P<0.05). The downward trend of swimming time for 6h group was gentle, and it was until the first 12th day its swimming time decrease distinctively than that of before modeling (P<0.05). Reduction rate of swimming time of 8h group was between 6h and 10h groups, with significant decrease from first 3rd day later (P<0.05).The swimming time among normal mice was not obvious and there was distinctive mutual effect between model strengthen and time (F=43.021, P=0.000)Experiment 2:Compared with normal control group, the mice in 9d group changed a lot, with their fur wet, less glossy and water stains, and their escape response also decreased, but had no significant weight loss. In comparison with each time point normal control group, the exhaustive swimming time was notably down among model of 5d,9d,12d,16d groups (P=0.000). In terms of blood biochemistry results, the levels of BUN, ALT, AST, TG were different among model groups and its ascending or descending extend related with model time. The order of variation scope from large to small is model 16d group>12d group>9d group>5d group. In model 16d group, the levels of BUN, ALT and AST significantly increased, and TG level obviously decreased, in comparison with normal control group (P<0.05); the fluctuations in blood biochemical changes of model 12d,9d,5d groups were in the normal range, with no significant difference between the normal group (P>0.05); With changes just after 16d group, the model 12d group had latent difference tendency in contrast with normal group; model 5d group had lest obvious changes among model groups and had the similar level with the normal group; the index fluctuation of model 9d group was between 12d and 5d groups, and the levels of BUN, ALT and AST raised or lowered mildly within normal range. Each group showed no abnormal changes in routine blood test and pathological lesions. Experiment 3:According to the former experiments results, we adapted the modeling method that forced mice to stand in water 8 hours per day for 9 days to cause experimental mice fatigue-type sub-health status and we found the swimming time of model mice decrease significantly, indicating an obvious fatigue of model mice. Although the fatigue degree abated after a rest of 7days free of modeling factors, swimming time was still less than normal level (P<0.05); and the swimming time became no significant difference compared with that of normal control mice with a recovery of 14 days (P>0.05).PartⅡ:The immune function changes of the fatigue-type sub-health mice with the interference of weikang-granulaCompared with model group, exhaustive swimming time of weikang-granula intervention groups and natural recovery group, was significantly increased (P<0.05), and the swimming time of weikang-granula intervention groups was marked higher than that of natural recovery group (P<0.05), in which the swimming time of weikang-granula high and middle dose groups restore the highest and had no significant difference compared with the normal group (P>0.05). Thymus index and spleen index of model mice were significantly reduced compared with normal group (P<0.05). With intervention by weikang-granula or natural recovery, the thymus and spleen index appeared a recovery trend, of which weikang-granula high and middle dose groups returned the highest levels had no significant difference compared with the normal group (P>0.05). Results of T\B lymphocyte proliferative responses showed that T\B lymphocyte stimulation index stimulated by ConA/LPS among model mice highly dropped compared with normal mice (P<0.05); With intervention by weikang-granula or natural recovery, T\B lymphocyte stimulation index and OD value appeared a recovery trend, and weikang-granula high dose group showed more greater effect than other groups, the T\B lymphocyte stimulation index and OD value of which had no Statistics difference in comparison with normal group (P>0.05).CONCLUSION:1. In this study, we finally determined the way of forcing mice to stand in water 8 hours per day for 9 days to imitate formatting factors of fatigue-type sub-health status, such as physical and psychological fatigue, psychological stress, circadian rhythm disorders, and successfully prepared this mouse model of fatigue-type sub-health; evaluation of this model showed it well stability and reproducibility. This provides a technology platform on research of the pathological mechanism on fatigue-type sub-health and its drug treatment.2. Mice of fatigue-type sub-health status exists immune dysfunction, and the occurrence of fatigue-type sub-health may have some connection with immune dysfunction.3. After the stop of modeling factors and a period of 14days for rest, fatigue-type sub-health mice could recovery to the normal state, suggesting that fatigue-type sub-health status can be transformed to the normal state, and natural recovery can promote the transformation process.4. The effect of Weikang-granula intervention on fatigue-type sub-health mice is significantly higher than natural recovery group. Weikang-granula can significantly alleviate fatigue in mice models, extend the swimming time and make model mice return to normal state. In addition, it can also adjust the immune function of fatigue-type sub-health mice and promote them to the normal level, and this may be the one of mechanisms of Weikang-granula effect in fatigue-type sub-health treatment. |
PDF Full Text Request