Effect Of Xiaoyaosan On Depressed Model Rats With Syndrome Of Liver Qi Stagnation And Spleen Deficiency:Role Of Striatal A_2AR—ERK—NF-?B Signaling Pathway

1.ObjectiveDepression is a serious threat to public health and medical finance.The syndrome of liver qi stagnation and spleen deficiency is one of the most important syndrome types of depression.Abnormal mood and nerve injury caused by liver qi stagnation and spleen deficiency are unsolved clinical problems.Xiaoyaosan(XYS)has significant clinical efficacy in the treatment of depression with liver-stagnation and spleen-deficiency syndrome.It is a representative prescription of the mainstream treatment(liver-soothing and spleen-strengthening method),but the mechanism is still unclear.Some scholars believe that the abnormal striatum may be a biomarker of neurodegenerative diseases,and may also be the intrinsic pathological basis of disease phenotypes.The striatum is closely related to depression and involves in the regulation of cognition,emotion and behavior,which may be the key to the prevention and treatment of the syndrome of liver qi stagnation and spleen deficiency.A large number of previous studies have shown that adenosine receptor(AR)specific aggregation in the striatum can play a neuroprotective role by regulating abnormal synaptic plasticity,which is of great significance in the improvement of major depression and the regulation of cognition,learning,memory and sleep intention.AR may be the major candidate target for the prevention and treatment of depression with liver qi stagnation and spleen deficiency syndrome.Therefore,we proposed a scientific hypothesis:XYS plays a neuroprotective role by inhibiting the striatal A_2AR—ERK—NF-?B pathway to reduce the proinflammatory activity of striatal microglia,thus achieving the purpose of anti-depression.And conduct research on it.2.MethodsBased on the team's previous research,this study evaluated the neuroprotective,anti-depression effect of XYS by using the liver qi stagnation and spleen deficiency rats model induced by chronic restraint stress(CRS)for21 days.96 SPF male SD rats,weighing 180?200g,were randomly divided into 8 groups.(1)Normal group:normal rats;(2)CRS Model group:CRS rats;(3)Negative control group:normal rats+XYS;(4)Xiaoyaosan treatment group:CRS rats+XYS;(5)A_2AR antagonist treatment group:CRS rats+SCH 58261;(6)A_2AR antagonist+Xiaoyaosan treatment group:CRS rats+SCH 58261+XYS;(7)A_2AR agonist model group:normal rats+CGS 21680;(8)Xiaoyaosan treatment A_2AR agonist model group:normal rats+CGS21680+XYS.CRS replicates the rats model of liver qi stagnation and spleen deficiency for 21 days,and the body weight were measured every 7 days.In addition to the normal group,rats were given different drugs from day 1 to 21of stressing.In order to avoid the interferences or bias of these behavioral tests,we designed the experimental process strictly.Open field test(OFT)experiment was done in 22nd day,Elevated plus maze test(EPMT)experiment,Y maze test(YMT)experiment,Sucrose preference test(SPT)experiment and Forced swimming test(FST)experiment were conducted on the 23rd day,24th day,25th day and 26th day.On 27th day,the experimental animals were euthanized by peritoneal injection of pentobarbital sodium,and arterial blood and brain tissue were collected.Golgi staining,transmission electron microscopy,western blot(WB),immunofluorescence and kit detection were used to determine the pathological changes in brain tissue and serum of liver qi stagnation and spleen deficiency rats.The details are as follows:(1).Experiment 1:Evaluation of the model of liver qi stagnation and spleen deficiency in rats and the preventive effect of XYS.The rats with liver qi stagnation and spleen deficiency syndrome were cloned by CRS for 21days.(1)Depression-like behaviors were detected by OFT,EPMT,SPT and FST,and spatial memory disorders were detected by YMT,so as to comprehensively evaluate liver depression and the prevention and treatment effect of XYS in rats with liver qi stagnation and spleen deficiency syndrome.(2)Body weight,daily ration and serum D-xylose content of rats were measured to determine the metabolic rate of liver qi stagnation and spleen deficiency rats.Thus to evaluate the state of liver stagnation and the effects of prevention and treatment of XYS on rats with liver qi stagnation and spleen deficiency syndrome.(2).Experiment 2:Protective effect of XYS on neuron and neural substructure in liver qi stagnation and spleen deficiency rats.The pathological damage of neurons and neural substructures(dendrite degree,spinal density,ratio of mature and immature spinal column)in hippocampus and striatum of rats with liver qi stagnation and spleen deficiency was detected by Golgi staining.The pathological damage of non-symmetrical synapses in the striatum was observed by transmission electron microscopy,and the expression of BDNF and Occludin in the striatum was detected by WB.Further,to study the pathological alterations in the neuronal damage,atrophy and neural substructure loss of chronic stress induced liver qi stagnation and spleen deficiency syndrome,and to discuss the neuroprotective effect of XYS against stress-induced depression.(3).Experiment 3:Effect of XYS on activation of microglia in striatum in rats with liver qi stagnation and spleen deficiency.The morphology and number of microglia in the striatum of CRS rats were observed using IBA-1,a histological microglia marker,as an immunofluorescence staining index.The expression of i NOS and Arg-1 in striatum microglia was detected by WB.Chronic stress leads to adverse changes in Glutamic acid(Glu)synapses,including reduced extracellular Glu clearance by glial cells,which may lead to synaptic loss and activation of apoptotic pathways.Therefore,Glu levels in the striatum and serum of CRS rats were also measured in experiment 3.So as the regulatory effect of XYS.In experiment 3,the effect of XYS on the activation of microglia in striatum of rats with liver qi stagnation and spleen deficiency was investigated through the above tests.(4).Experiment 4:Effects of XYS on adenosine receptor A_2AR—ERK—NF-?B pathway in striatum of rats with liver qi stagnation and spleen deficiency.The expression of adenosine receptors A_2AR in striatum of rats with liver qi stagnation and spleen deficiency,and the expression of Extracellular regulated protein kinases(ERK)and nuclear factor kappa-B(NF-?B)protein was detected by WB.To study the effect of chronic stress on A_2AR—ERK—NF-?B pathway in striatum of rats with liver qi stagnation and spleen deficiency,and to discuss the effect of XYS on it.(5).Experiment 5:Effect of XYS on adenosine receptor A₁R-A_2AR balance in striatum of rats with liver qi stagnation and spleen deficiency.The activities of adenosine receptor A₁R and A_2AR in striatum of rats with liver qi stagnation and spleen deficiency were detected by immunofluorescence,and the protein expression of A₁R was detected by WB.The adenosine 5'-triphosphate(ATP)content in serum and striatum tissues and the expression of Adenosine 5'-triphosphate synthase(ATPase)protein in striatum were detected to understand the energy metabolism of rats.Furthermore,the effect of chronic stress on A₁R-A_2AR balance in striatum of rats with liver qi stagnation and spleen deficiency was studied,and the effect of XYS on A₁R-A_2AR balance was discussed.3.ResultsThe purpose of this study was to explore the neuroprotective mechanism of Xiaoyaosan on depression rats with liver qi stagnation and spleen deficiency syndrome based on striatum A_2AR—ERK—NF-?B pathway.(1).Experiment 1:The 21-day CRS rat model met the requirements of syndrome model of liver qi stagnation and spleen deficiency syndrome.Animal model of depression plays an important role in the study of depression in traditional Chinese medicine,and an appropriate model can better clarify the mechanism of action of Traditional Chinese medicine.(1)The 21-days CRS can effectively simulate the"liver depression"of depression syndrome,and the model group has significant depression-like behavior(including decreased exploration behavior and hedonic behavior,increased fear-like behavior and despairing behavior),and spatial memory impairment.XYS can effectively relieve liver depression in CRS-induced liver qi stagnation and spleen deficiency rats,which may be closely related to inhibiting the overexpression of A_2AR.(2)The 21-days CRS can effectively simulate the"spleen deficiency"representation of the syndrome of liver qi stagnation and spleen deficiency in depression,and the model group had significant decrease in metabolic rate(including weight loss,reduced daily ration and low serum D-xylose content).XYS can effectively relieve spleen deficiency in CRS-induced liver qi stagnation and spleen deficiency rats,which may be closely related to inhibiting the overexpression of A_2AR.Experiment 1(1)and(2)jointly completed the evaluation of the rat model of liver qi stagnation and spleen deficiency syndrome.21-days CRS rats had typical"liver depression"and"spleen deficiency",which reflected the essence of TCM of this syndrome to a large extent and laid a foundation for further research.(2).Experiment 2:XYS had protective effect on neurons and neural substructures of liver qi stagnation and spleen deficiency rats.In rats with depression syndrome of liver qi stagnation and spleen deficiency,21-days CRS resulted in neuron damage in hippocampal CA3 region and striatum(including dendrite dendrization,decreased spine density,and low mature/immature spines ratio),pathological damage to ultrastructure of asymmetrical synapses in the striatum,and low protein expression of BDNF and Occludin.XYS can effectively alleviate the damage of neurons and neural substructures in CRS or A2AR agonist induced liver qi stagnation and spleen deficiency rats,and may be related to the inhibition of A_2AR overexpression.(3).Experiment 3:XYS had a certain inhibitory effect on the activation of microglia in striatum of rats with liver qi stagnation and spleen deficiency.CRS by 21 days can induce microglia proliferation(increased expression of IBA-1)and pro-inflammatory activation(high expression of i NOS and low expression of ARG-1)in striatum of rats with depression syndrome of liver qi stagnation and spleen deficiency,and higher Glu content in tissues and serum may be due to decreased removal of extracellular Glu by glial cells.XYS can effectively relieve the pro-inflammatory activity of microglia in striatum of CRS-induced liver qi stagnation and spleen deficiency rats,and may be related to inhibiting the overexpression of A_2AR.(4).Experiment 4:XYS reduced the proinflammatory activity of striatum microglia by inhibiting the A_2AR—ERK—NF-?B pathway.Chronic stress at 21 days of CRS resulted in dysregulation of the number of AR receptors in the striatum,a decrease in A₁R and a large increase in A_2AR,which first activates the A_2AR—ERK—NF-?B signaling pathway,then resulting in pro-inflammatory activation of microglia,and finally leading to nerve injury.XYS can regulate AR receptor,inhibit A_2AR—ERK—NF-?B pathway,reduce the pro-inflammatory activation of microglia,and ultimately play a neuroprotective role in depression.(5).Experiment 5:XYS can regulate the striatum A₁R-A_2AR imbalance caused by CRS and improve ATP deficiency.Chronic stress of CRS at 21 days resulted in dysregulation of the number of ARs in the striatum,with a decrease in A₁R and a large increase in A_2AR.XYS is beneficial to the recovery of A₁R-A_2AR equilibrium,and energy metabolism abnormality(low ATP content and decreased ATPase activity).4.Conclusion(1).This study took the syndrome of liver qi stagnation and spleen deficiency as the research object,and successfully replicated the rat model of liver qi stagnation and spleen deficiency by using the method of 21-days CRS.The model rats had symptoms similar to depression syndrome:(1)"liver depression"-like symptoms included depression-like behaviors and spatial memory disorders,and(2)"spleen deficiency"-like symptoms included weight loss,reduced daily intake,and reduced metabolic rate.The success of liver qi stagnation and spleen deficiency rats was also confirmed by XYS.(2).XYS has protective effect on neurons and neural substructures of liver qi stagnation and spleen deficiency rats.Specifically,XYS alleviates CRS-induced neuronal damage in hippocampal CA3 region and striatum(including dendrite dendrization,decreased spine density and low mature/immature spines ratio),alleviates the ultrastructural pathological changes of asymmetric protrusion in striatum,and increases neuronutrition,thus playing a neuroprotective role in anti-depression.(3).XYS has a certain inhibitory effect on proinflammatory activation of microglia in striatum of rats with liver qi stagnation and spleen deficiency.XYS can effectively reduce the proliferation and proinflammatory activity of microglia in striatum induced by CRS,thus alleviating nerve injury.(4).XYS attenuates the proinflammatory activity of striatum microglia by inhibiting the A_2AR—ERK—NF-?B pathway.(5).The regulation of AR(maintain the balance of A1R-A2AR)may provide a potential target for XYS in the prevention and treatment of depression syndrome of liver qi stagnation and spleen deficiency.XYS can increase A₁R and decrease A_2AR.XYS can improve energy metabolism abnormality(insufficient ATP and low expression of ATPase).In conclusion,XYS can reduce the proinflammatory activity of striatum microglia by inhibiting the striatum A_2AR—ERK—NF-?B pathway,play a neuroprotective role,and ultimately achieve the purpose of antidepressant.