Study On The Effects Of Paeonol On Chronic Stress-induced Neuronal Injury And Its Mechanism

According to the data released by the World Health Organization?WHO?in2016,depression is one of the most prevalent psychiatric disorders,accompanied by symptoms including a depressed mood,reduced motivation,loss of interest or pleasure,suicidal thoughts,and hopelessness,with more than 350 million people of all ages suffering from the disease.Antidepressant drugs have been widely used to treat depression and have been recommended in clinical guidelines as the preferred treatment for the moderate and severe depression.However,anti-depressants only provide a complete remission of symptoms in approximately 50%of patients with major depression,as well as causing side effects,as sedation and weight gain.Although monoamine systems?including serotonin,norepinephrine,and dopamine systems?have long been the focus of research and treatment for depression,the development of new therapeutic targets and drugs has become one of the most important directions for discovery.More and more evidence shows that the two most important factors inducing depression are chronic stress and psychological trauma that can damage neurons.Protecting neurons from injury may provide new drug targets for the prevention and treatment of future depression.In China,the clinical application of polyphenols has been used for thousands of years.Polyphenols are often easily absorbed by the gastrointestinal tract and quickly cross the blood-brain barrier.Paeonol(2'-hydroxy-4'-methoxyacetophenone,C₉H₁₀O₃)is a typical polyphenolic substance and is a phenolic acid compound extracted from the famous Cortex Moutan.It has been determined that Paeonol has a variety of clinical therapeutic effects.Recent studies have found that Paeonol can reduce lipopolysaccharide-induced depression-like behavior in mice,but its cellular and molecular mechanisms are still poorly understood.Therefore,in this study,we used Paeonol to treat animal models of depression induced by chronic stress,aiming to further understand the cellular and molecular mechanisms of its effects,and provide experimental basis for the development of new drugs for potential prevention and treatment of depression.In this study,we induced a rat model of depression through chronic unpredictable mild stress?CUMS?.The behavioral assessment was performed using open field test?OFT?,elevated plus maze test?EPMT?,forced swimming test?FST?,and sucrose preference test?SPT?to analyze the effects of Paeonol on CUMS-induced exploration activity decline,anxiety,despair,etc.Golgi-cox staining combined with Sholl analysis software,ImageJ software and RECONSTRUCT three-dimensional reconstruction software were used to analyze the dendritic morphological changes of neurons,including dendrite length,number of dendritic branches,dendritic spine density and types.On the basis of this effect,the potential molecular mechanisms of Paeonol on CUMS-induced neuronal damage in brain-related regions were further investigated by immunofluorescence double-labeling and western blotting.Our results are shown below:1.Paeonol can prevent CUMS-induced behavioral injury in rats.CUMS caused Rats to show lower activity and less traveled distance in Open Field Tests?P<0.05for both?,less time in open arms in EPMT?P<0.05?,less sucrose consumption in SPT?P<0.05?,and more immobile time in FST?P<0.01?.Significant differences were also identified between the control group and the fluoxetine-treated group?P<0.05?or the CUMS group and the high-dose Paeonol-treated group?P<0.05?in activity in the open field test,and between the CUMS group and the fluoxetine-treated?P<0.01?,low-dose Paeonol-treated?P<0.001?,or high-dose Paeonol-treated group?P<0.01?in immovable time in the forced swimming test,as well as between the CUMS group and the fluoxetine-treated group?P<0.05?or the CUMS group and the high-dose Paeonol-treated group?P<0.05?in sucrose consumption in the sucrose preference test.These data suggest that Paeonol treatment significantly attenuated CUMS-induced behavioral damages.2.Paeonol significantly ameliorated CUMS-induced neuronal damage in the relevant brain regions of rats.In this study,we conducteddetailed studies on the length and number of branches of neurons in the relevant brain regions of the medial frontal lobe,amygdala,hippocampus,and nucleus accumbens,and the densities and types of neuron dendritic spines.Among them,on the same neuron,we made statistics on the lengths of the basal dendrites and the total dendrites of the neurons,respectively,and counted the number of intersections on different segments.Dendritic spine density and type percentages were calculated in different dendritic regions,including basal dendrites,apical proximal and distal dendrites,respectively.At the same time,according to the size of the dendritic spines measured by the RECONSTRUCT three-dimensional reconstruction software,the dendritic spines are automatically divided into three types:mushroom/branched?MB,width>0.6?m or“branch”?,stubby?ST,length:width ratio?LWR??1 and length<1?m?,and filopodia/thin?FT,length>1?m or LWR>1?.It is not difficult to understand from the analysis that chronic stress can generally induce shortened neuron dendrites,reduced number of dendritic branches,and reduced density of dendritic spines in each brain region,while use of fluoxetine or Paeonol can prevent or ameliorate these alterations.However,from the analysis of different types of spines,it can be found that although chronic stress has different effects on different brain regions,the overall effect on different types of spines was not the same:a more consistent phenomenon was that chronic stress can reduce the density of the mushroom/branched spines,but it was often accompanied by an increase in the density of the filopodia/thin spines;the use of the same fluoxetine or Paeonol can prevent or attenuate these changes.Therefore,Paeonol alleviated the neuronal damage in the relevant brain regions induced by chronic stress,probably by reducing the loss of neuronal dendrites,increasing the density of functional dendritic spines-mushroom/branch type,and thus improving behavioral performance.3.Paeonol treatment can effectively alleviate the disturbance of CUMS-induced cofilin1 activity and its redistribution in neurons.Cofilin1 is one of the most important regulators of actin filament remodeling that constitutes the neuronal dendritic spine and can be activated by dephosphorylation on Ser3 or release from the membrane proteins into the cytoplasm.Overactivation or inactivation of cofilin1 can result in remodeling of actin and loss of dendrites and synapses.Our results showed that chronic stress induced increased levels of cofilin1 in the mPFC and hippocampus,but decreased in the amygdala;induction of p-cofilin1 levels increased in the amygdala and nucleus accumbens and decreased in the mPFC,whereas there was no significant change in the hippocampus.At the same time,more distribution of cofilin1in the cytoplasm was also induced by chronic stress in some brain regions such as the hippocampus.These changes are usually relieved after treatment with fluoxetine or Paeonol.These suggest that cofilin1 was activated or inactivated mostly by altering its expression level and phosphorylation,but also by regulating binding to membrane proteins.On the other hand,activation or inactivation of cofilin1 was not the same in different brain regions.Thees area-specific changes of cofilin1 activity maybe affect the remodeling of actin,and then change the morphology of dendrites and dendritic spines.4.Paeonol treatment significantly ameliorated CUMS-induced changes in Rho GTPase?Rac1,cdc42,and RhoA?levels.As a molecular switch,Rho GTPase can mediate a variety of intracellular and extracellular signal molecules,acting via a variety of signaling pathways on downstream of target proteins,including cofilin1.Our data showed that CUMS generally altered the expression of GTPase:it decreased the levels of Rac1 and RhoA in mPFC,increased the levels of Rac1 and RhoA in the amygdala and hippocampus,decreased the Rac1 level but increased cdc42 and RhoA levels in the nucleus accumbens.Treatment with fluoxetine or different concentrations of Paeonol can alleviate the changes above.These results suggest that GTPase-cofilin1 signaling pathway may be involved in the physiological process of Paeonol to relieve CUMS-induced neuronal dendritic spines loss and dendritic atrophy.Our results also suggest that BDNF in some brain regions,such as the hippocampus,may affect the GTPase-cofilin1 signaling pathway via TrkB receptors.5.Paeonol significantly alleviated CUMS-induced alteratioons of GSK3?and calcineurin?CaN?levels in some brain regions.Studies have shown that GSK3?and calcineurin may be involved in chronic stress-induced cofilin1 disturbance and behavioral impairment.Our results revealed that in some brain regions,such as the amygdala,chronic stress reduced CaN levels,and the use of Paeonol?high or low dose?can prevent its decline;chronic stress increased GSK3?levels,however,fluoxetine or high-dose Paeonol treatment can alleviate the increase of GSK3?levels.These showed that CaN and GSK3?were also involved in the physiological process of Paeonol in relieving chronic stress-induced neuronal dendritic spines loss and dendritic atrophy in some brain regions such as the amygdala.In summary,Paeonol significantly reduced CUMS-induced behavioral impairment.These behavioral impairments may be causally related to the loss of dendritic spines and dendritic atrophy in the relevant brain regions.Consistent with these dendritic morphological changes was the disruption of cofilin1 activity,accompanied by changes in the expression of Rho GTPase in different brain regions,and changes in BDNF and/or GSK3?/calcineurin.The disruptions or changes above can be alleviated by Paeonol treatment.Our data suggest that Paeonol may attenuate CUMS-induced synaptic and dendritic loss in different brain regions via various signaling pathways such as GTPase-cofilin1 or GSK3?/calcineurin,and ultimately improve behavioral deficits.Our experiments also suggest that Paeonol may be a potential drug for the treatment of depression.