| Heart failure patients often accompanied by significant cognitive dysfunction,which have severely impaired quality of life and increased hospitalization rates and mortality.However,the pathological mechanisms underlying cognitive dysfunction in heart failure are still poorly understood.Brain natriuretic peptide(BNP),as a biomarker for clinical diagnosis of heart failure,plays an important role in the whole pathological process of heart failure.BNP was an independent predictor of cognitive dysfunction and close relationship have found between them.It is worth noting that BNP and its receptors are not only present in the blood circulatory system,but also widely distributed in the central nervous system.Classical studies have found that the cyclic guanosine-protein kinase G(cGMP-PKG)signaling pathway mediated by BNP and its receptor is involved in the regulation of various brain functions,such as cerebral fluid homeostasis,synaptic transmission,blood-brain barrier integrity and neuro-inflammation.Whether the pathway participates in cognitive dysfunction after heart failure is not clear.Brainderived neurotrophic factor(BDNF)is a neurotrophin that promotes neuronal proliferation,survival,and plasticity.Meanwhile,as a novel diagnostic biomarker,it has been gradually studied.Clues suggest that the activation or inhibition of natriuretic peptide receptor A(NPRA)may affect the expression level of BDNF,while the role of BDNF in the genesis of cognitive dysfunction after heart failure and the specific relationship with the natriuretic peptide system are basically a blank area of research.The theory of Traditional Chinese Medicine suggests that "the heart governs blood" and "the heart controlling mental activities".This theory provides a new perspective for the study of the correlation between the heart and the brain.Qiliqiangxin Capsule is a Chinese patent medicine for the treatment of heart failure.Large-scale clinical trials have confirmed its effectiveness in the treatment of heart failure and which was include in the 2014 Chinese chronic heart failure treatment guidelines.Our previous research found that QiliQiangxin Capsules can not only improve the heart function of rats with heart failure after myocardial infarction,but also improve the learning and memory ability of rats with cognitive dysfunction after heart failure which means QiliQiangxin Capsule has special heart and brain protection.Using BNP as the point of this study,we aimed to explore the pathological mechanism of cognitive dysfunction after heart failure induced by BNP and its related cGMP-PKG signaling pathway and the relationship between BNP and BDNF and to elucidate the pharmacological mechanism of Qiliqiangxin Capsule in protecting heart and brain.We used a rat model of infarction-induced heart failure.In order to evaluate cardiac function and cognitive function,echocardiography and morris water maze was performed.To observe the changes of neurons,blood-brain barrier and synapse in heart failure rats,electron microscope was performed.In addition,the changes of three neurotransmitters in brain tissue were detected by liquid chromatography-mass spectrometry.At the same time,in order to investigate the expression of BNP and BNP-related signaling pathway as well as its regulation of BDNF were observed by intracerebroventricular administration of BNP for normal and heart failure rats and siRNA interference of fetal primary neurons.On this basis,the effects of Qiliqiangxin Capsule on the above indicators was examined and molecular docking was employed to predict the potential pharmacoperones of Qiliqiangxin Capsule.Through exploring the pathological mechanism of cognitive dysfunction after heart failure and the pharmacological mechanism of Traditional Chinese Medicine in improving heart failure and cognitive function,our study provides scientific evidence for the prevention and treatment of cognitive dysfunction after heart failure which is important from both theoretical perspective and applications.Objectives:1.Elucidate the characteristics of cognitive dysfunction in rats with heart failure after myocardial infarction and the effect of Qiliqiangxin Capsule on improving the learning and memory abilities of rats via detecting changes in cognitive behavior of rats,ultrastructure and neurotransmitters of the nervous system.2.Reveal the pathogenesis of cognitive dysfunction after heart failure via observing the changes of BNP and BNP-related signal pathway and BDNF in brain tissue of rats with heart failure and fetal primary hippocampal and cortex neurons.3.Reveal the pharmacodynamic mechanism of Qiliqiangxin Capsule in improving cognitive dysfunction after heart failure via observing the effects of Qiliqiangxin Capsule on BNP and BNP-related signal pathway in the brain of rats and predicting targeted binding ability of the drug ingredients to NPRA.Methods:1.A heart failure rat model after myocardial infarction was built by ligating the left anterior descending coronary artery.Then the rats were randomly divided into sham,model,QLQX and enalapril group based on the 24h postoperative ECG.Intragastric administration were treated from day 2 onwards on alternate days for 8 weeks.2.Morris water maze was used to observe the changes in the learning and memory abilities of rats in each group and spatial acquisition trial,probe trial and reverse platform test were conducted.3.Transmission electron microscopy was utilized to morphology of neurons and the tight junctions of blood-brain barrier.Quantitative analysis was applied in the number of synapses,average distance of the synaptic cleft and postsynaptic density(PSD).4.Untargeted lipid profiling was performed to detect the changes of acetylcholine,amino acid neurotransmitters and monoamine neurotransmitters with a total of 13 neurotransmitters in the cerebral cortex tissue of each group.5.Western blot was used to detect the protein levels of BNP,NPRA,cGMP,PKG and BDNF in the cerebral cortex of rats.6.Intragastric administration of BNP were treated from day 5 onwards on alternate days for 14 days.Then morris water maze was used to observe the changes in the learning and memory abilities of rats in the group of sham,sham+BNP,model and model+BNP,and spatial acquisition trial,probe trial and reverse platform test were conducted.7.Primary cultured hippocampal and cerebral cortex neurons were prepared and treated with BNP to assess its effect on axonal growth of normal neurons.siRNA-NPRA fetal neuron model was established to visualize the changes in BDNF expression.8.Mass spectrometry and molecular docking was employed to analyze the ability of the compounds contained in Qiliqiangxin Capsule to target NPRA and the possible pharmacodynamic substances were analyzed.Results:1.Cardiac function and structure:In comparison with the sham group,the ejection fraction(EF)and fractional shortening(FS)were significantly reduced,the left ventricular endsystolic internal diameter(LVIDs),left ventricular end-diastolic internal diameter(LVIDd),left ventricular end-systolic volume(LVESV)and left ventricular end-diastolic volume(LVEDV)were markedly elevated(P<0.001),and the left ventricular end-systolic anterior wall thickness(LVAWs),left ventricular end-diastolic anterior wall thickness(LVAWd)(P<0.001),left ventricular end-systolic posterior wall thickness(LVPWs)(P<0.05)were significantly thinner in the model group.Compared with the nodel group,Qiliqiangxin Capsule markedly improved EF and FS,decreased LVIDs,LVIDd,LVESV and LVEDV,and and thickened LVPWs(P<0.05).In the infarct region of the failure heart,cardiac cell necrosis was easily observed.The formation of fibrous scar caused wall thinning.A large number of cardiac cell necrosis,collagen deposition,cardiomyocyte hypertrophy,intercellular space widening,and disorderly arrangement were found in infarct border zone.2.Cognition evaluated by Morris water maze:No statistical difference was observed in spatial acquisition trial between sham and model group(P>0.05).In probe trial,the number of times of passing over platform in heart failure rats was significantly lower than that in the sham group.In reverse platform test,the average escape latency and total distance in model group were significantly increased than that in the sham group(P<0.01).Compared with rats in the model group,the Qiliqiangxin Capsule group had a significantly higher number of times in probe trial and decreased average escape latency and total distance in the reverse platform test(P<0.05).3.Ultrastructural changes of brain:The changes in cerebral structure such as neurons pyknosis,netosis,nuclear condensation and hyperchromatism,evaginations of the inner nuclear membrane,semi-open structure of tight junctions,significantly reduced synapses,widened synaptic gap and thinner PSD were observed in model rats(P<0.001).Compared with rats in the model group,the ultrastructure of the brain in the Qiliqiangxin Capsule group was significantly improved.Moreover,the number of synapses,synaptic space and PSD thickness were significantly improved.4.The results of neurotransmitter:A marked reduction in glutamic acid,aspartic acid(P<0.05)and y-aminobutyric acid(P<0.01)of cerebral cortex was observed in heart failure rats.No statistical differences in acetylcholine and monoamine neurotransmitters(P>0.05).There was no significant difference between model group and Qiliqiangxin Capsule group(P>0.05).5.Changes of the expression of brain BNP and related signal pathways in rats with heart failure:The expression of BNP,NPRC were significantly higher,the NPRA,BDNF(P<0.05),cGMP,PKG(P<0.01)were lower in the model group than in the sham group.6.Changes of cognitive function in rats after intracerebroventricular injection of BNP:The Evans blue assay was used to confirm that the injection site was correct.In spatial acquisition trial,significantly shortened average escape latency(P<0.05)and increased trend of total distance(P=0.068)on the first day was showed in sham+BNP group.There was no statistical difference in the results of the remaining two days and probe trial(P>0.05).In comparison with the sham group,there was no statistical difference in spatial acquisition trial of model group(P>0.05).In probe trial,the number of times of passing over platform in heart failure rats was significantly lower than that in the sham group(P<0.05).In reverse platform test,the average escape latency and total distance in model group were significantly increased than that in the sham group(P<0.01).Compared with model group,model+BNP group did not reveal any significant differences(P>0.05).7.Effects of BNP and NPRA-siRNA on neuronal cells:Primary cultured hippocampal and cerebral cortex neuron was prepared,and the purities of them reached above 97.75%.After BNP intervention,the number of axon bifurcation were increasing significantly.SiRNA interference of NPRA was performed on neurons.Compared with the normal group and the siRNA blank group,the NPRA protein in the neurons of the NPRA-siRNA group was significantly reduced,indicating that the interference was successful.The neuronal injury,sparse synaptic connections and significantly decreased expression level of BDNF were observed in NPRA-siRNA group.8.Effects of Qiliqiangxin capsule on BNP and BNP-related signal pathway:Compared with heart failure rats,significantly increased expression levels of NPRA(P<0.05),cGMP(P<0.05),PKG(P<0.01),BDNF(P<0.01)proteins were showed in Qiliqiangxin Capsule group.There was no significance in NPRC(P>0.05).9.Mass spectrometric detection and molecular docking results of Qiliqiangxin Capsule:A total 47 small molecules were identified by mass spectrometry.Astragaloside Ⅳ showed the strongest binding affinity with NPRA,which was predominantly expressed in the complementary to shape of the catalytic pocket of NPRA and the 5 short hydrogen bonds forms with NPRA.The strong combination of Astragaloside Ⅳ and NPRA is conducive to the stable combination.This study provides the basis for further investigations into the study on the components of Traditional Chinese Medicine targeting NPRA.Conclusions:1.Cognitive dysfunction performances such as mild impaired learning and memory was observed in heart failure rats,Neurons pyknosis,blood-brain barrier disruption and significantly reduced synapses and amino acid neurotransmitters suggested that damage to brain structures and reduced amino acid neurotransmitters were important pathogenesis of cognitive dysfunction.2.BNP enabled protective effects in cognitive function of normal rats and synapse formation of normal neuronal cells.A marked decrease in the expression of NPRA induced inhibition of the BNP-related cGMP-PKG signaling pathway.BNP failed to exert protection in the brain of heart failure rats,which caused reduced BDNF expression,neuronal damage,synaptic suppression and cognitive dysfunction.3.The results showed that Qiliqiangxin Capsule remarkably improved cognitive function and protected the brain structure by activating NPRA and BNP-related cGMP-PKG signaling pathway.Astragaloside Ⅳ showed the highest binding ability to NPRA,and it is speculated that it may pass through the damaged blood-brain barrier and then bind to the NPRA receptor to exert the main effects. |
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