The Changes In Oxidative And Inflammatory Mechanism On Brain-heart Disorder Following Stress And Optogenetic Stimulation To Hippocampal Dentate Gyrus And Treatment By Liqi-somatic Therapy

ObjectiveThe formation of depression is driven by somatization symptoms.In addition to the core manifestations of low mood and anhedonia,patients often have multiple somatization symptoms and even the main complaint.Among somatization symptoms,cardiovascular disease associated with depression is the leading cause of death in the Chinese population.Surprisingly,the comorbidity rate of cardiovascular disease and depression has reached 20?50%.Depression has been proven to be a new independent risk factor for cardiovascular disease,affecting the occurrence,development and prognosis of cardiovascular disease.The traditional Chinese medicine Fructus Aurantii has been reported to treat depression and cardiovascular disease.However,the mechanism of its effect on comorbidities remains unclear.The cardioprotection and antidepressant effects of fructus aurantii and its absorbent ingredients were studied for the first time in this paper to clarify the mechanism of fructus aurantii in the treatment of the comorbidity of cardiovascular disease and depression.To provide experimental basis for the follow-up development of targeted prevention and treatment of depression associated with cardiovascular by traditional Chinese medicine.Methods and Results1.To determine the content of naringin,neohesperidin,Naringin,Hesperidin,meranzin hydrate and nobiletin in decoction of fructus aurantii by LC-MS/MS.We draw standard curve of each compounds and investigate the specificity,linearity range,limit of quantitation,limit of determination,precision,repeatability,stability and recovery ect.Results:The method was precision,stability,repeatability and the recovery test results conformed to the requirements of the methodology.The active absorbent components and extraction methods were relatively stable in the three batches of fructus aurantii.The contents of 6 components were as follows:hesperidin 10.74 mg/g,nehesperidin 86.03 mg/g,naringin 165.98 mg/g,brassica naringin 29.64 mg/g,citrinin 10.67 mg/g and MH 3.73 mg/g,respectively.2.The CUMS model is selected.The sucrose preference test was used to detect the sucrose preference rate of mice.The spontaneous activity and immobility time of mice were detected by forced swimming test and tail suspension test.The feeding delay time and food consumption of mice were detected by novel environment inhibitory feeding behavior test.The expression of inflammatory factor(IL-6 iNOS TNF-?)in serum was measured by ELISA kit,The expression of CK-MB?IL-6?INF-??iNOS?TNF-??pPI3K?pAKT?pNF-?B in the heart of mice and the expression of PKA,PSD95,PCREB,BDNF Synapsin1 in the hippocampus of mice were detected by Western-blot.The behavioral results showed that the CUMS model group showed significant depression-like behavioral changes compared with the blank control group.The expression of iNOS TNF-? and IL-6 in serum were decreased in both FRA group and MH group;In the heart,the expression of CK-MB?IL-6?INF-??iNOS?TNF-??pPI3K?pAKT?pNF-?B was reduced,and the expression of eNOS and IL-10 was increased;In the hippocampus,the expression of PKA,PSD95,PCREB,BDNF and Synapsin1 were increased.3.GluR-Cre mice were microinjected with a specific inhibitory virus and photostimulation in the hippocampus.The sucrose preference test was used to detect the sucrose preference rate of mice.The spontaneous activity and immobility time of mice were detected by forced swimming test and tail suspension test.The expression of eNOS?pNF-?B in the heart of mice and the expression of PKA?PSD95?GluR1?Synapsin1 in the hippocampus of mice were detected by Western-blot.The behavioral results showed that the model group showed significant depression-like behavioral changes compared with the blank control group.Both FRA group and MH group could reverse the depression-like behavior;In the heart,the expression of eNOS?pNF-?B was reduced;In the hippocampus,the expression of PKA?PSD95?GluR1?Synapsin1 and Synapsin1 were increased.4.Learned helplessness(LH)and lipopolysaccharide(LPS)induced mouse models were selected to reveal the antidepressant-like effects of Fructus Aurantii in behavioral tests.By detecting the expression of PCREB,CREB,PKA,BDNF,PSD95,Synapsin1,GLUR1,GAD67 and NR1 in the hippocampus of mice,the levels of synaptic molecules in the hippocampus and the potential molecular mechanisms were determined.We found FRA,like ketamine,reversed the behavioral deficits both in LPS and LH at 1 day after a single administration.FRA was also capable of increasing the expressions of PKA/CREB/BDNF signaling in hippocampus.Consistent with ketamine,FRA up-regulated the expressions of GABAergic receptor(GAD67)and glutamatergic receptor 1(GluR1)in mouse hippocampus both exposed to LPS and LH.Moreover,synaptic proteins such as postsynaptic density-95(PSD95)and synapsin1 were also up-regulated by a single dose of FRA both in LH and LPS-induced models,like ketamine.Finally,metadoxine(an antagonist of CREB)inhibited the antidepressant effects of FRA in tail suspension test and forced swimming test in LPS-induced mice,which also blocked the phosphorylation of CREB and the expressions of neurotransmitters and synaptic molecules.5.Merazin hydrate(MH),an essential ingredient of Fructus aurantii,has been identified to have an antidepressant-like effect.Here,in lipopolysaccharide-induced mice,we identified that a single administration of MH recovered depressive behaviors and downregulated the expressions of neuronal nitric oxide synthase(nNOS)in the hippocampus after 1 day.Activation of nNOS by L-arginine led to depressive behaviors,and inhibition of nNOS contributed to antidepressive behaviors.Notably,MH only reversed the expression of nNOS's downstream NF-?B and not the CREB/BDNF pathway in the hippocampus,and MH's antidepressant-like effects were prevented by Asatone(an agonist of NF-?B)and not H89(an antagonist of CREB).MH also normalized the expressions of GFAP and IB-1 in dentate gyrus in the hippocampus and inflammatory factors such as IL-1?,IL-10,and TNF-? in serum.6.Neural plasticity and neurogenesis have been identified to contribute to pathophysiology of depression.Previous studies found Fructus Aurantii,a Chinese herb,was capable of relieving depressive symptoms in rodents by chronic treatment.However,the underlying mechanisms of FRA's antidepressant effects was still elusive,especially in relation to neurons and synapses in brain regions.Here,we tested the depression-like measures,neurogenesis factors including Ki67 and Brdu cell and spine density in hippocampus after FRA treatment in chronic mild stress(CUMS)mice.we also measured the relationship between protein kinase A(PKA)and neurogenesis.We found that FRA reversed the depression-like symptoms after chronic treatment,which also reversed the expressions of PKA and its downstream molecules including synaptic proteins in hippocampus.Moreover,FRA also increased the number of Ki67 and Brdu positive cells in hippocampus.Furthermore,Disruption of PKA activity blocked the antidepressant effects of FRA,which also inhibited the increasing expressions of synaptic proteins and Brdu positive cell,but Ki67 was no change,which were different with escitalopram increasing expressions both of Brdu and Ki67 significantly.These findings aid in understanding the antidepressant effects of FRA requires neurogenesis in the hippocampus and PKA plays critical role in the FRA's antidepressant process.Conclusion In this study,the LC-MS/MS method was established to simultaneously determine the content of six components in FRA Decoction.The method is reproducible,accurate and reliable,and can provide a basis for the quality control of FRA Decoction.After determining the dosage,we clarified that the FRA and its absorption component,MH,played an anti-apoptotic effect and improved depressive behavior.Inflammation,oxidative stress and synaptic plasticity may be involved in this process.Based on the research foundation of early animal experiments,we applied the methods of modern medical research to the treatment of comorbid diseases,and comprehensively used new research methods such as optogenetics and pharmacological techniques to explore the molecular mechanism of the treatment of comorbidities from different angles.FRA and its absorption component MH can improve the depression phenotype of optogenetic stimulated mouse models,and can also improve the downstream PKA/NF-?B inflammation pathway and the expression of synaptic proteins(PSD95,Synapsin1)to produce resistance Myocardial apoptosis.In rapid antidepressant research,the rapid antidepressant effect of FRA depends on the PKA/CREB/BDNF pathway,which subsequently regulates downstream synaptic transmission and promotes the generation of hippocampal neurons.The antidepressant effect of its absorption component MH relies on the inhibition of nitric oxide-mediated and downstream ERK/NF-?B,and has nothing to do with the antidepressant pathway PKA/CREB/BDNF of FRA.