Effect Of Moxibustion On Learning And Memorizing Capacity And Apoptosis Of Hippocampal Neurons In Sub-health Mental Fatigue Model Rats

Objective:The experiment aims to investigate the effect and the possible mechanisms of moxibustion on Yintang(GV29) and Shenque(RN08) for learning and memorizing capacity in sub-health mental fatigue model rats, providing the modern medical theoretical basis for the clinical application of moxibustion on Yintang(GV29) and Shenque(RN08) for the treatment of sub-health mental fatigue. The detailed purpose of this project includes the following four aspects:(1)To establish and appraise the sub-health mental fatigue model in rats induced by partial sleep deprivation, and to observe the effect of the moxibustion on Yintang(GV29) and Shenque(RN08) on learning and memorizing capacity in rats.(2)To investigate the effects of partial sleep deprivation on hypothalamus-pituitary-adrenocortex axis (HPA) related hormone levels in rats, and to investigate the regulatory role of the moxibustion in HPA axis related hormone levels.(3) To observe morphological changes of hippocampal neurons in sub-health mental fatigue model rats caused by partial sleep deprivation, and to investigate the effects of the moxibustion on Yintang (GV29) and Shenque (RN08) on the morphology of hippocampal neurons.(4)To detect the expressionnal changes of apoptosis relatedproteins in hippocampal neurons in model rats caused by partial sleep deprivation, and to investigate the effect of the moxibustion on Yintang(GV29) and Shenque(RN08) on the expression of apoptosis related proteins in hippocampal neurons in model rats.Methods:To apply the researching method of randomized controlled animal trials on this subject. The experiment was divided into the following four parts:(1) 30 healthy adult Sprague-Dawley (SD) rats (in half male and female) were randomly divided into 3 groups:Normal group, model group and moxibustion group (10 rats per group). Rats in model group and moxibustion group were placed in sleep deprivation tank with no more than 5-6 hours of sleep a day for partial sleep deprivation, while the rats in normal group were put into a tank of water environment under simulated conditions. The whole process lasted for 14 days. During the period of modeling, another moxibustion of Yintang (GV29) and Shenque (RN08) were given to the rats in moxibustion group,15 minutes for each point at a time per day. The general condition of the rats in 3 groups was observed everyday and the body weights were recorded weekly throughout the experiment. In the second week of the experiment, eight-arm maze was used for the the learning training in all the rats. Learning and memorizing capacity were then tested.(2) On the day when the experiment finished,all rats were put to death. At the time of sacrifice, blood samples of each group were collected. Two steps by double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of serum adrenocorticotropic hormone (ACTH) and endogenous glucocorticoid(GC) in each rat.(3) At the time of sacrifice, brain samples of each group were collected. Histological and pathological changes of hippocampal neurons in CA3 area were observed for the 3 groups of rats. TdT-mediated dUTP nick end labeling (TUNEL) assay was used to evaluate the apoptosis of hippocampal neurons.(4) The immunohistochemistry staining was used to test the expression of Bcl-2, Bax and Caspase-3 in hippocampal neurons of CA3 area.Results:After the above experiments, the following results were obtained.(1) General condition:During the initial period of.the modeling experiment, the appearance of the model group and the moxibustion group had no obvious change, but the excitement and alertness of their behaviour improved. And they responded quickly to the stimulus of the surroundings. With the extension of the modeling time, the outer coat color of the model and moxibustion group gradually turned yellow and few gloss. The rats started to get hair loss, displayed the "irritability" phenomenon and escaped to the stimulation such as capture and touch. One week after modeling, model group became emaciated obviously with unkempt coat, which was of dull brown and easy to drop. The fatigue and weakness behaviour appeared. From the beginning to the end of the experiment, the appearance of the normal group performed approximately like it did before the experiment. The appearance and behaviour of the moxibustion group was situated between the normal group and the model group since one week after modeling.(2) The change of body weight:There was no significant difference between normal group weighed on the day 7 and before the experiment, (P>0.05); Significant reductions in body weights in model group and moxibustion group on day 7 than initial weight were observed (P<0.05); There was no significant difference in body weight changes between the model group and the moxibustion group on the 7th day of the experiment(P>0.05). The model and moxibustion groups had distinctly lower body weights than normal group on the 7th and 14th day of the experiment (P<0.05)(3) Eight-arm maze training results:There were significantly differences in foraging time among each group (P<0.05), while the model group was the longest one, followed by the moxibustion group, and the normal group was the shortest. Pairwise comparison among the 3 groups had significant difference (P<0.05). Since the 3rd day of the training test, the error number of the arms for the normal and moxibustion group was fewer compared with the model group. The comparison between the moxibustion group and the model group had no significant difference, (P>0.05). Since the 5th day of the training test, the correct number of the arms for normal and moxibustion group was more than the model group (P<0.05)(4) The concentration of ACTH and GC in serum:the ACTH concentration in serum of model and moxibustion group was significantly higher than the normal group (P<0.05). While the moxibustion group was significantly lower than model group (P<0.05). The GC concentration in serum of model group was significantly higher than the normal group (P<0.05),While the moxibustion group was significantly lower than the model group (P<0.05). The comparison between the moxibustion and normal group hasdno significant difference (P>0.05)(5) Morphological observations on hippocampal neurons sectionThere were large amount of well-defined pyramidal cells in the CA3 area of the hippocampus in normal group under microscope. The irregular pyramidal cells with decreased numbers of model group could be observed as well. And the pyramidal cells of the moxibustion group were well-defined.Apoptosis of hippocampus:Few TUNEL-positive cells in the hippocampus tissue of the normal group could be observed under microscope. And the positive cells of the model group spread widely throughout the entire hippocampal tissue. The positive cells of the moxibustion group were significantly fewer than the model group (P<0.05). The comparison between the moxibustion and normal group had no significant difference (P>0.05)(6) Results of the immunohistochemical staining in hippocampal sectionThe results of Bcl-2 expression test:Bcl-2 positive cells in model and moxibustion group were increased significantly compared to those in normal group (P<0.05); Bcl-2 positive cells in moxibustion group were also significantly increased compared to those in model group (P<0.05).The results of Bax expression test:Bax positive cells in model and moxibustion group were increased compared to those in normal group (P<0.05); Bax positive cells in moxibustion group were significantly decreased compared to those in model group (P<0.05).The results of Caspase-3 expression test:Caspase-3 positive cells in model and moxibustion group were increased compared to those in normal group (P<0.05); Caspase-3 positive cells in moxibustion group were decreased compared to those in model group, the difference was significant (P<0.05).Conclusion:(1) Sub-health mental fatigue rat model, induced by partial sleep deprivation methods with less than 5-6 hours of sleep a day for 14 days, was similar to the clinical manifestations of human sub-health mental fatigue. It is identified to be an ideal animal model for studying sub-health mental fatigue.(2) The moxibustion on Yintang(GV29) and Shenque (RN08) acupoint has the function of improving subhealth-like symptoms (such as decreased learning and memorizing capacity) in sub-health mental fatigue rat.(3) The hippocampus damage caused by HPA axis hyperactivity and high GC level is one of the mechanisms for the onset of sub-health mental fatigue. The moxibustion on Yintang(GV29) and Shenque(RN08) acupoint has the function of regulating HPA axis in sub-health mental fatigue rat. This is also one of the mechanisms for improving the subhealth-like symptoms of the mental fatigue rat by moxibustion.(4) The moxibustion on Yintang(GV29) and Shenque(RN08) acupoint can prevent the neurons loss in hippocampus pyramidal cell layer in the mental fatigue rat and reduce the damage of hippocampal neurons, which is beneficial to maintain the normal structure of hippocampal neurons; Moxibustion can also inhibit the apoptosis in hippocampal neurons and finally protect hippocampal neurons.(5) The upregulation of Bax and Casepase-3 proteins expression in hippocampal neurons of the model rat induced by sleep deprivation promote the incidence of apoptosis in the hippocampus. It is another possible mechanism of the sub-health mental fatigue. The moxibustion on Yintang(GV29) and Shenque (RN08) acupoint can reduce the expression of pro-apoptotic proteins Bax and apoptosis-related enzymes Casepase-3, increase the expression of Bcl-2 to inhibit the hippocampus neuronal apoptosis of the rats and finally protect hippocampus neurons. It is another main mechanism for improvement of learning and memorizing capacity in mental fatigue rat by moxibustion.