| Stress is the sum of the body's non-specific response to various stimuli outside the body or inside the body.Moderate stress can improve the body's ability to adapt and maintain internal environment stability;excessive stress can cause harm to the body,leading to neuroendocrine and psychological changes in the body.With the accelerated pace of life in modern society,the burden of work and life has increased,and the work pressures and psychological pressures that people face are growing.There is increasing evidence that chronic stress is an important cause of many major human diseases,which can cause a variety of major diseases such as cancer,cardiovascular disease,anxiety,depression,Alzheimer's disease(AD)and other physical and mental illnesses.As a special group,soldiers face more complicated and intense stress loads due to military factors such as extreme living environment,mental overload and high-intensity military operations.Strong military stress factors will not only directly harm military health,but also cause serious physical,mental and psychological damage,which is an important factor in reducing military operations.Therefore,the study of military stress damage prevention measures is the focus of research in various countries'military in recent years.At present,military stress is mainly controlled by psychological intervention and supplementation of amino acids and vitamins to prevent stress damage,but the onset time is slow and the effect is limited.Studies have shown that the pathophysiological changes of stress caused by any cause are the imbalance of the neuroendocrine immune network(NIM)network.The modern stress theory takes the NIM network as the core,and believes that the NIM network is the fundamental basis for maintaining the balance and stability of the environment and physiological functions in the body.The balance of NIM network in modern medical perspective is highly consistent with the overall view of "Yin and Yang,qi and blood,visceral function" balance in traditional Chinese medicine.Therefore,based on the overall view of Chinese medicine,dialectical view,balance view and modern stress theory,it is a promising research direction to explore ways and means of using Chinese medicine to intervene in stress,and to find or create drugs for preventing and treating stress damage from traditional Chinese medicine.Liuwei Dihuang active fraction combination(LW-AFC)is the first five new drugs of traditional Chinese medicine,which derived from Liuwei Dihuang Decoction and composed of active ingredients.Previous studies have shown that LW-AFC has a significant regulatory effect on NIM disorders caused by multiple causes(psychological stress,physical stress and general stress),and has improved cognitive impairment due to acute stress and the ability of learning and memory in AD model mice.However,it is unclear whether LW-AFC can improve emotional and cognitive impairment caused by chronic stress.Therefore,based on the diversity of military stressors and the complexity of stress response,this topic addresses the most common problems encountered by military stress:sleep deprivation,small fighting space,lack of drinking water,and unpredictable nervousness in the future.First,we establish single factor stress(empty bottle stimulation model,sleep deprivation stress animal model,restraint stress animal model)and chronic multiple stress animal model to simulate these stress states,and then observe different military stress patterns and different military stress effect on emotional and cognitive functions,and the effects of LW-AFC on anxiety,depression-like behaviors and learning and memory impairment induced by chronic stress,and finally explored the mechanism of LW-AFC improve chronic stress-induced mood and cognitive impairment.Chapter 1 Effects of LW-AFC on the mood of single-factor stress miceUncertain empty bottle drinking water stimulation method,sleep deprivation stress method and restraint stress method were used to simulate the common phenomenon of lack of drinking water,sleep loss and narrow space in military stress,and observe the influence of single factor stress on mouse emotion and the improvement of LW-AFC.1.Effect of empty bottle stimulation on anxiety-like behavior in miceThe mice were given once empty bottle stimulations per day for 10 min/time for 14 d to establish an uncertain empty bottle drinking stimulation model.The results showed that compared with the blank control group,the body weight of the empty bottle stimulation group and the physiological stress group decreased significantly on the 7th and 14th day of stimulation;the results of the open field test showed that the spontaneous activity of the empty bottle stimulated the mice increased significantly.The residence time in the central area has a decreasing trend;the results of the elevated plus maze test show that the regular drinking water group has a significantly reduced residence time in the open arm and a decrease in the number of access to the open arm.Compared with the regular drinking water group,the body weight of the empty bottle stimulation group and the physiological stress group decreased significantly on the 7th day of empty bottle stimulation;the results of the open field test showed that there was no significant change in the spontaneous activity of the empty bottle stimulated in the central area.The time has a downward trend;the results of the elevated plus maze test show that there is no significant change in the number of times the empty bottle stimulates mice to enter the open arm and the residence time in the open arm.The above results suggest that there is no anxiety in the mice stimulated by empty bottles for 14 d.Both simple water shortage and single cage feeding have significant effects on the behavior of mice.2.The effect of LW-AFC on the anxiety-like behavior of sleep deprived miceModified platform method was used to establish a sleep deprivation model in mice after sleep deprivation 96 h.The results showed that compared with the normal control group,the weight of mice in the large platform control group decreased significantly,suggesting that the effect of water environment on the weight of mice was not excluded in the experiment;compared with the large platform control group,the weight of mice in the sleep deprivation group decreased significantly,while LW-AFC did not significantly improve the weight of mice.The results of open-field experiment showed that compared with the normal control group,the total distance of movement in open-field increased significantly in the large-platform control group,and the residence time in the central area of open-field did not change significantly.Compared with the large-platform control group,the total distance of movement in open-field decreased significantly and the residence time in the central area of open-field decreased significantly in the sleep-deprived mice.Compared with the normal control group,there was no significant change in the time spent in the open arm and the number of times entering the open arm in the large platform control group;compared with the large platform control group,the time spent in the open arm and the number of times entering the open arm in the sleep deprived mice were significantly reduced;LW-AFC had the tendency to increase the autonomous activity of mice,the time spent in the central area of the open field and the number of times entering the open arm.These results suggest that sleep deprivation mice exhibit obvious anxiety-like behavior,and LW-AFC has a tendency to improve anxiety-like behavior in sleep deprivation mice.3.The effect of LW-AFC on the anxiety-like behavior of mice with restraint stressA restraint stress model was established by binding mice for 2 h every day for 14 d.The results showed that compared with the normal control group,the body weight of the mice on day 7 and day 14 of the restraint stress decreased,and LW and LW-AFC did not significantly improve the body weight of the mice.The experimental results of open field and elevated plus maze test showed that there was no significant change in the autonomic activity of the mice in restraint stress 7 d,and the residence time in the central area of the open field decreased.The number of times of entering the open arm and the staying time in the open arm were significantly reduced.LW and LW-AFC have a tendency to increase the residence time of mice in the central region of the open field,the number of times they enter the open arms,and the residence time in the open arms.There was an increasing trend of self-activity in mice with restraint stress 14 d.There was no significant change in the residence time in the central area of the open field;there was no significant change in the number of open arms and the residence time in the open arms.These results suggest that restraint stress 7 d can induce anxiety-like behavior in mice,while anxiety stress disappears in mice after 14 d of restraint stress.LW and LW-AFC have a tendency to improve anxiety-like behavior in mice with restraint stress for 7 d.Chapter ? Effects of LW-AFC on emotional and cognitive impairment in mice with chronic multiple stress modelsA random combination of 14 stress conditions was used to establish a chronic unpredictable mild stress(CUMS)model to simulate the stimulation of multiple complex unknown factors in military stress.Fluoxetine(FLU)was selected as a positive drug to observe the emotional and cognitive impairment of CUMS mice at 14 and 28 d in CUMS.Low dose(0.8 g/kg)and medium dose(1.6 g/kg)of LW-AFC were evaluated.And the high dose(3.2g/kg)to improve its effect,from the aging and dose effects of the two aspects of the efficacy of LW-AFC.Behavioral experiments were performed after modeling to evaluate the effects of LW-AFC on anxiety,depression-like behavior and learning and memory impairment in CUMS model mice.1.Effect of LW-AFC on body weight changes in CUMS model miceCompared with the normal group of mice,the body weight of the mice was significantly decreased at 7,14,21,and 28 d after stress,and FLU,LW,and LW-AFC did not significantly improve the body weight of the mice.2.Effect of LW-AFC on anxiety-like behavior in CUMS model miceThe anxiety-like behavior of mice was tested by open field and elevated plus maze experiments.After 14 d of CUMS,the results of the open field experiment showed that compared with the normal group,the total distance of movement in the open field of the model group was significantly increased,and the residence time in the central area of the open field was significantly reduced.The results of the elevated plus maze test showed that the number of times the mice in the model group entered the open arms and the time spent in the open arms were significantly reduced.After administration of FLU,the number of times the model mice entered the open arms was significantly increased.LW was administered to increase the tendency of the model mice to stay in the central region of the open field,the number of times to enter the open arms,and the residence time in the open arms,and to give LW-AFC(3.2 g/kg)significantly increased the number of times the mouse stayed in the open arms and entered the open arms.After 28 d of CUMS,the total distance of movement of the model group mice in the open field was significantly increased,and the residence time in the central area of the open field was significantly reduced.The number of open arms and the residence time of the elevated cross maze were significantly reduced.LW-AFC(1.6 g/kg)significantly increased the residence time of the mice in the open arms,and FLU and LW had a tendency to increase the residence time of the mice in the central region of the open field,the number of access to the open arms,and the residence time in the open arms.These results suggest that CUMS can induce significant anxiety-like behavior in mice at 14 and 28 d,FLU,LW and LW-AFC can improve anxiety-like behavior in CUMS 14d mice,LW-AFC have an improvement of anxiety-like behavior in CUMS 14 and 28 d mice3.Effect of LW-AFC on depression-like behavior in CUMS model miceThe sucrose preference experiment was used to detect depression-like behavior in mice.The results showed that compared with the normal group,the saccharide water consumption rate of the mice in each group did not change significantly after 14 d of CUMS,suggesting that CUMS 14 d didn't induce to develop depression-like behavior in mice.After 28 d of CUMS,the sucrose consumption rate of the model group was significantly decreased,FLU and LW-AFC(3.2g/kg)could significantly increase the sucrose consumption rate of mice.The above results suggest that CUMS 28 d can induce significant depression-like behavior in mice.FLU and LW-AFC all have the effect of improving,and are equivalent to the effect of positive drug FLU.LW has a trend to improve depression-like behavior in mice.4.Effect of LW-AFC on recognition and memory of mouse objects in CUMS modelThe new object recognition experiment was used to detect the recognition and memory ability of mouse objects.The results showed that after 14 d of CUMS,the recognition index of new objects in the model group did not change significantly 1 h after the training period,and the recognition index of new objects decreased at 24 h after the training period,suggesting that CUMS had no short-term memory for 14 d.It has a tendency to damage long-term memory in mice.FLU,LW and LW-AFC all have a tendency to improve long-term memory in mice.After 28 d of CUMS,the recognition index of new objects in the model group did not change significantly 1 h after the training period,and the recognition index of new objects decreased at 24 h after the training period,suggesting that CUMS had no short-term memory for 28 d.The effect is that the long-term memory of the CUMS group has a tendency to damage.Both LW and LW-AFC have a tendency to improve long-term memory in mice,-while FLU has a tendency to damage long-term memory in mice.5.Effect of LW-AFC on spatial learning and memory in CUMS model miceThe water maze test was used to detect the spatial learning and memory ability of mice.The results showed that compared with the normal group of mice,after 28 d of CUMS,the escape latency of the model group mice prolonged the learning period,LW-AFC(3.2g/kg)had the tendency to shorten the escape latency of mice.During the test period,the escape latency increased significantly in the model group mice.LW-AFC(3.2g/kg)significantly reduced the escape latency of mice;the number of piercing cycles in the model group decreased,and FLU.LW and LW-AFC increased the wear of the mice.The trend of the number of times;the model group mice significantly decreased in the target quadrant residence time,FLU,LW-AFC(0.8,3.2g/kg)can significantly increase the mouse in the target quadrant residence time.The above results suggest that CUMS can damage the spatial learning and memory ability of mice in 28 d;FLU and LW-AFC can improve the spatial learning and memory impairment of CUMS model mice,and LW has an improved trend in spatial learning and memory.Chapter III Preliminary study on the mechanism of LW-AFC improving the emotional and cognitive impairment induced by chronic stress in miceLW-AFC can improve the anxiety,depression-like behavior and learning and memory impairment of chronic multi-stress mice.On the basis of this,the LW-AFC may be used to explore the efficacy of HPA axis function,neurotransmitters and related protein expression to research the possible mechanism of action.1.Effect of LW-AFC on HPA axis function of CUMS model miceThe plasma CORT,ACTH and hypothalamic CRH levels in mice were detected by radioimmunoassay and enzyme-linked immunosorbent assay.The results showed that compared with the normal group,the plasma CORT level in the CUMS 14d mice was significantly increased,the ACTH content in the plasma was significantly decreased,the CRH content in the hypothalamus was significantly decreased,and the FLU,LW and LW-AFC were significantly decreased.The plasma CORT content,LW and LW-AFC(1.6g/kg)can significantly increase the ACTH content in plasma.LW-AFC(3.2g/kg)can significantly increase the CRH content released from the hypothalamus.The plasma CORT level in CUMS 28d model mice was significantly increased,the ACTH content in plasma was significantly increased,and the CRH content in the hypothalamus was significantly increased.FLU and LW significantly reduced the plasma CORT content in mice,LW and LW-AFC can significantly reduce the ACTH content in mouse plasma.These results suggest that CUMS can cause abnormal HPA axis function in mice,and FLU,LW and LW-AFC have a regulatory effect on HPA axis dysfunction in CUMS mice.2.Effect of LW-AFC on monoamine neurotransmitters in hippocampus of CUMS miceThe content of monoamine neurotransmitters in mouse hippocampus was detected by high performance liquid chromatography.The results showed that compared with the normal group,the contents of 5-HT,5-HIAA,DOPAC and HVA in the hippocampus of the CUMS 14d model group were significantly decreased,and the DA content was decreased.FLU increased the DA in the hippocampus of mice.The trend of DOPAC and HVA content,LW can significantly increase the content of 5-HIAA and DOPAC in mouse hippocampus.LW-AFC can significantly increase 5-HT,5-HIAA,NE,DA,DOPAC and HVA in mouse hippocampus.content.The content of DA in hippocampus of CUMS 28d model group was significantly decreased,the content of 5-HT,5-HIAA and HVA decreased,and the content of NE and DOPAC did not change significantly.LW and LW-AFC had a trend to increase 5-HT and 5-HIAA content in hippocampus of mice.FLU and LW-AFC(0.8g/kg)can significantly increase the DA content in the hippocampus of mice.These results suggest that CUMS can reduce the content of monoamine neurotransmitter in mouse brain.LW-AFC regulates the content of monoamine neurotransmitters in CUMS 14d model mice by the synergistic action of 5-HT and DA system.LW-AFC primarily regulates the content of monoamine neurotransmitters through the DA system for CUMS 28d model mice.3.Effect of LW-AFC on NMDAR-BDNF signaling pathway in hippocampus of CUMS miceWestern Blot was used to detect the expression of related proteins in the NMDAR-BDNF signaling pathway in hippocampus of mice.The results showed that compared with the normal group,the expression of NR2B and BDNF protein in hippocampus of CUMS 14d model mice increased significantly,the expression levels of NR2A and NRSF proteins did not change significantly,and the expression of GR protein decreased.FLU and LW had the expression of NR2B and BDNF protein in hippocampus of mice was decreased,and LW-AFC(3.2g/kg)significantly decreased the expression of NR2B protein.The expression of NR2A protein in hippocampus of CUMS 28d model mice was significantly increased,the expression of NR2B protein was increased,the expression level of BDNF protein was significantly decreased,the expression of NRSF protein was increased,and the expression of GR protein was not changed.FLU,LW and LW-AFC had a tendency to decrease the expression of NR2A protein.LW can significantly increase the expression level of BDNF protein.LW-AFC(0.8g/kg,1.6g/kg)can significantly reduce the expression level of NRSF protein.These results suggest that CUMS can change the levels of NR2A and NR2B protein expression in mice brain.CUMS 14d model mice mainly show changes in NR2B protein expression levels,and CUMS 28d model mice mainly show changes in NR2A protein expression levels.LW-AFC may exert anti-chronic effects through the NMDAR-BDNF signaling pathway.Through the above research,this study mainly got the following conclusions:(1)LW-AFC has the tendency of improving anxiety-like behavior in restraint stress and sleep deprivation stress mice and improves the impairment of anxiety,depression and learning and memory ability in CUMS model mice.(2)LW-AFC may play an anti-chronic stress role by regulating HPA axis dysfunction,monoamine neurotransmitter content and NMDAR-BDNF signaling pathway in CUMS mice.(3)The reserach suggests that LW-AFC may be a potential preventive and therapeutic drug for chronic stress-induced emotional and cognitive impairment. |
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