The Investigation Of Single Intraperitoneal Injection Of TNF-α On Cognition And Anxiety-like Behavior In Mice And The Intervention Effects Of LW-AFC

TNF-α is a pleiotropic cytokinc,and it can activate mitogen-activated protein(MAP)kinase,nuclear factor kappa-B(NF-κB)and other signaling pathways when it is combined with its typeⅠ or type Ⅱ receptors,and it is widely involved in cell differentiation and apoptosis,promote inflammation and tumor development.As an important immune factor,TNF-α can also directly stimulate the hypothalamus-pituitary-adrenal(HPA)axis to enhance sympathetic nervous system activity,inhibit hippocampal nerve function,damage long-term potentiation(LTP),and promote the catabolism of serotonin precursor substance tryptophan to affect learning and memory function and emotional response.Clinical trials have shown that TNF-α antagonists Adalimumab,Infliximab,and Etanercept have significantly improved fatigue and depressive symptoms in patients with autoimmune diseases.Memory impairment in patients with postoperative cognitive dysfunction(POCD)is closely related to the acute increase in plasma TNF-α concentration.The acute increase of TNF-α level in the brain of patients with traumatic brain injury(TBI)can cause secondary nerve damage and neuron loss,which are closely related to the memory impairment of patients.Animal studies have found that acute stress can rapidly increase the level of TNF-α in the hippocampus of mice and cause memory impairment in mice.Peripheral administration of etanercept(TNF-α receptor antagonist)can significantly improve memory impairment in mice.The current research on the effects of TNF-α on the central learning and memory function and mood mainly adopts the model of repeated administration of TNF-α.For example,repeated administration of TNF-α to simulate chronic inflammatory response can cause anxiety and depression-like behavior in mice.It also damages the conditioned fear memory of mice,and whether single administration of TNF-α has an effect on cognition and emotion is still unclear.The traditional Chinese medicine compound Liuwei Dihuang Decoction consists of six traditional Chinese medicine(rehmannia glutinosa,dogwood,peony bark,yam,poria,Alisma).Liuwei-active fraction combination(LW-AFC)is the active ingredient group extracted from Liuwei Dihuang Decoction by the research team,mainly composed of polysaccharides,oligosaccharides and glycosides.At present,it has obtained clinical approval and is in phase Ⅱclinical trials.Previous studies have shown that LW-AFC can improve cognitive function impairment and emotional disturbance caused by stress,as well as improve the learning and memory ability of Alzheimer disease(AD)model mice,and restoring the balance of the NIM network is the modern pharmacological basis for LW-AFC to improve cognitive and emotional disorders in mice.However,in the NIM network,the immune system mainly regulates the function of the nervous system through the cytokine network,and the regulatory effect of LW-AFC on the behavioral changes of mice caused by the acute increase of TNF-α in the cytokine network is still unclear.Therefore,in this study,single intraperitoneal injection of TNF-α was used to simulate the rapid increase in TNF-α levels in mice,and to investigate its effects on spatial learning and memory and anxiety-like behaviors in mice,and from the effects of neurosynaptic plasticity and neuronal damage to explore the possible ways that TNF-α affects learning,memory and emotional behaviors of mice.On this basis,we observed the intervention effects and possible ways of LW-AFC on anxiety-like behaviors of TNF-α model mice.一、The effects of single intraperitoneal injection of TNF-α on cognition and anxiety-like behaviors in mice1.The effects of single intraperitoneal injection of TNF-α on spatial memory function in mice1.1 The effects of TNF-α on the spatial probe test phase of the Morris water maze experiment in miceThe memory process is composed of information acquisition,consolidation and retrieval phases.The place navigation phase of the Morris water maze experiment,the training phase,mainly reflects the acquisition and consolidation of memory,and the spatial probe test phase,the testing phase,mainly reflects the retrival of memory.After 5 days of normal training,mice were injected intraperitoneally with 0.1mg/kg,0.2mg/kg,0.4mg/kg TNF-α 1 hour before the spatial probe experiment on the sixth day to observe the changes in their space exploration behavior.The results showed that compared with the control group,the escape latency of mice was significantly prolong and the number of crossing the platform was significantly reduced after intraperitoneal injection of TNF-α,suggesting that intraperitoneal injection of TNF-αduring the spatial probe test phase can significantly impair the spatial memory retrieval function of mice.1.2 The effects of TNF-α on the place navigation phase of the Morris water maze in miceWhether the increase in TNF-α level will also affect the acquisition and consolidation of learning and memory,the mice were treated with 0.2mg/kg TNF-α in the abdomen on the first,third and fifth days 1 hour before training and the escape latency of mice during the place navigation phase and the number of crossing the platform during the spatial probe test phase were assessed with Morris water maze place navigation experiment.The results showed that compared with the control group,the escape latency and the number of crossing the platform did not change significantly,suggesting that the increase of TNF-α level has no significant effect on the learning and memory acquisition and consolidation phase of the mice.The above research results indicate that the increase of TNF-α level mainly affects the memory retrieval process of mice.2.The effects of single intraperitoneal injection of TNF-α on anxiety-like behavior in miceIn order to explore whether the elevated TNF-α level has an effect on the anxiety-like behaviors of mice,the mice were intraperitoneically injected 0.1,0.2and 0.4mg/kg TNF-α to observe the behavior of mice in the elevated cross maze experiment after 1 hour of injection.The results showed that compared with the control group,the mice in the TNF-α group had no significant difference in the number of entering open arms,the closed arms,the total number of open and closed arms,the total time,and the percentage of the number of open arms in the total number of open and closed arms.The percentage of residence time in the open arm and the residence time in the open arm in the total time in the open and closed arms was significantly reduced,and the time in the closed arm was significantly increased,suggesting that intraperitoneal injection of TNF-α can cause mood disorders in mice.Next,mice were intraperitoneally injected 0.1,0.2and 0.4mg/kg TNF-α 1 hour before the experiment to observe the total distance and the exploration time in the central area in the open field experiment.The results showed that compared with the control group,the total distance of the mice in the open field was not significantly affected by the TNF-α,and the exploration time in the central area of the open field tended to decrease.The above research results show that in addition to learning and memory,the increase of TNF-α level can also cause mice anxiety-like behavior.二、Possible ways of single intraperitoneal injection of TNF-α on cognition and anxiety-like behaviors in mice1.The effects of single intraperitoneal injection of TNF-α on synaptic plasticity in m:iceLTP is an important manifestation of synaptic plasticity.It is considered the physiological basis of learning and memory,and is also closely related to emotional changes.To investigate whether the elevated TNF-α level affects the cognition and anxiety-like behaviors of mice by damaging LTP.Mice were given intraperitoneal injections of 0.1,0.2,0.4 mg/kg TNF-α 1 hour before the in vivo electrophysiological experiment to observe the effects of TNF-α on the average peak potential in the hippocampus DG.The experimental results showed that,compared with the control group,the average peak potential of mice in TNF-α modle group was significantly reduced.It is suggested that the elevated level of TNF-α can cause LTP damage in mice.Next,in order to explore the effects of TNF-α on LTP in mice after different periods of time,the aforementioned results showed that there was damage to LTP of mice after injection of 0.2mg/kg,so 1h,12h,24h,48h before the experiment,0.2mg/kg TNF-α was injected intraperitoneally to observe the effects of TNF-α on the average peak potential in the hippocampal DG of mice.The results showed that compared with the control group,after lh,12h,24h intraperitoneal injection of TNF-α,the average peak potential of mice was significantly reduced,and after 48h,it returned to close to the normal level,suggesting an acute increase in TNF-α level can cause transient changes in mouse LTP.The above research results indicate that increased levels of TNF-α cause LTP damage in mice,which may be closely related to the spatial learning and memory dysfunction and anxiety-like behaviors of mice.2.The effects of single intraperitoneal injection of TNF-α on the number of Nissl bodies in the hippocampus and other different brain regions of miceThe learning and memory process and emotional response are accompanied by nerve cell and synaptic plasticity.Whether the acute increase of TNF-α affects the cognition and anxiety-like behavior of mice by damaging neurons.The Nissl staining method was used to stain the Nissl bodies of the mouse hippocampus,cortex,amygdala,and prefrontal cortex neurons.The foregoing research results showed that there were damage to spatial learning and memory behavior,anxiety-like behaviors,and barriers to LTP of mice after 1 hour of injection of 0.2mg/kg TNF-α.Therefore,the single intraperitoneal injection of 0.2 mg/kg was observed in mice.The results showed that compared with the control group,the number of hippocampal Nissl bodies in the TNF-α group was significantly reduced,and the integrated optical density(IOD)values of Nissl bodies in the cortex,amygdala,and prefrontal cortex did not change significantly,suggesting that intraperitoneal injection of TNF-α can damage hippocampal neurons and cause a significant decrease in the number of neuronal Nissl bodies,but it has no obvious effect on neurons in the cortex,amygdala and prefrontal cortex.The above results indicate that elevated levels of TNF-α can cause significant damage to hippocampal neurons,which may be closely related to the impairment of spatial learning and memory in mice and the appearance of anxiety and the damage of hippocampal LTP.三、The intervention effects of LW-AFC on anxiety-like behavior in TNF-α model mice and possible ways1.The effects of etanercept on anxiety-like behavior of TNF-α model micePrevious studies have shown that restoring the balance of the NIM network is the basis for LW-AFC to play a pharmacological role.In the NIM network,the immune system mainly regulates the nervous system function through the cytokine network,while the regulatory effect of LW-AFC on the anxiety-like behavior caused by the acute increase of TNF-α in the cytokine network in mice remains unclear.The above results indicated that elevated TNF-α level could induce anxiety-like emotions in mice.Next,the TNF-α receptor antagonist etanercept was used to investigate whether direct intervention of TNF-α could improve anxiety-like behaviors in TNF-α model mice,providing a reference for possible pharmacological pathways of LW-AFC affecting anxiety-like behaviors in TNF-α model mice.In order to explore whether direct intervention of TNF-α can improve anxiety-like behaviors in TNF-α model mice.In this study,the elevated plus maze experiment was used,and the TNF-α receptor antagonist etanercept was given to mice by intraperitoneal injection of 2.5mg/kg three days and 25mg/kg in the lateral ventricle one day before the experiment,respectively,to observe the behavioral changes of etanercept administration of TNF-α model mice.The experimental results show that compared with the control group,the mice in the intraperitoneal injection of TNF-α group entering the open arm,the closed arms,the total number of open arms and closed arms,the total time,and the percentage of time in open arms in the total time of open and closed arms had no significant difference.The time to enter the open arm was significantly reduced,and the time to enter the closed arm was significantly increased.Compared with the model group,mice in the intraperitoneal injection and lateral ventricle injection etanercept group had no significant difference in the number of entering open arms,the closed arms,the total number of open and closed arms,the total time,and the percentage of the number of open arms in the total number of open and closed arms.The time to enter the open arm and the percentage of time in open arms in the total time in open and closed arms were significantly increased,and the time to enter the closed arm was significantly reduced,suggesting peripheral or central administration of TNF-α receptor antagonists can improve mood disorders in mice caused by elevated TNF-α levels.The above research results show that the use of TNF-α receptor antagonists can directly intervene and improve the anxiety-like behavior of mice caused by TNF-α.2.The effects of LW-AFC on anxiety-like behavior in TNF-α model miceIn order to explore the effect of LW-AFC on anxiety-like behavior of TNF-α model mice and possible ways,the elevated plus maze experiment was used.Mice were given LW-AFC by continuous intragastric administration 2 weeks before the experiment and etanercept was continuous intraperitoneally injected 3 days before the experiment to observe the improvement effects of LW-AFC on the anxiety-like behavior of TNF-α model mice in the elevated plus maze.The experimental results showed that compared with the control group,the mice in the control group given TNF-α and etanercept showed no significant difference in the number of entering open arms,closed arms,the total number of open and closed arms,and the total time.The residence time of the mice in the open arm,the percentage of the number of entering the open arm in the total number of entries,and the percentage of time in the open arm in the total time in the open and closed arms were significantly reduced.The mice in the TNF-α group were significantly increased in the closed arm time;and compared with the TNF-α model group,there was no significant difference in the number of open arms,the number of closed arms,the total number of open and closed arms,and the total time in the LW-AFC and etanercept groups mice.The time in the open arms as a percentage of the total time in open arm and close arm increased significantly,and the time in the closed arm was significantly reduced,suggesting that the preventive administration of LW-AFC can improve anxiety-like behaviors in model mice.The above research results show that LW-AFC can improve the anxiety caused by elevated TNF-α levels in:mice,and regulating the binding of TNF-α to its receptor may be one of the ways for LW-AFC to exert pharmacological effects.3.The effects of LW-AFC on the number of Nissl bodies in different brain areas in TNF-αmodel miceThe foregoing experiments show that TNF-α can damage the hippocampal neurons in mice,in order to explore whether LW-AFC can improve the anxiety-like behavior of mice by improving the neuronal damage of TNF-α model mice.Nissl staining was used to observe the changes of LW-AFC on the neuron IOD value of model mice.The experimental results showed that compared with the control group,the number of hippocampal Nissl bodies in the model group was significantly reduced,and the IOD values of the cortex and amygdala Nissl bodies showed a downward trend,while the IOD values of Nissl bodies in the prefrontal cortex were not obvious changes.Compared with the TNF-α model group,the IOD values of hippocampus of the LW-AFC administration group increased significantly,suggesting that the level of TNF-αincreased can cause hippocampal neuron damage,and LW-AFC can improve the neuron damage of model mice.The above research results show that LW-AFC can improve the damage of hippocampal neurons in mice caused by elevated TNF-α levels,which may also be one of the ways LW-AFC can improve anxiety in model mice.The following conclusions are obtained through the above research:1.Acute elevation of TNF-α can cause spatial learning and memory dysfunction and anxiety-like behavior in mice.2.Acute elevation of TNF-α can cause damage to hippocampal LTP and neuron.3.LW-AFC can improve anxiety and neuron damage in TNF-α model mice.