Limbic system plasticity after electroacupuncture intervention in knee osteoarthritis rats.

Knee osteoarthritis (KOA) is characterized by debilitating pain. Electroacupuncture (EA), a traditional Chinese medical therapy, has shown promise in KOA pain management. This study investigated the therapeutic potential of EA in KOA and its impact on limbic system neural plasticity. Sixteen rats were randomly assigned into two groups: EA group and sham-EA group. EA or sham-EA interventions were administered at acupoints ST32 (Futu) and ST36 (Zusanli) for three weeks. Post-intervention resting-state fMRI was scanned, assessing parameters including Amplitude of low frequency fluctuations (ALFF), regional homogeneity (ReHo), functional connectivity (FC) and nodal characterizations of network within limbic system. The results showed that EA was strategically directed towards the limbic system, resulting in discernible alterations in neural activity, FC, and network characteristics. Our findings demonstrate that EA had a significant impact on the limbic system neural plasticity in rats with KOA, presenting a novel nonpharmacological approach for KOA treatment.

Tuina therapy promotes behavioral improvement and brain plasticity in rats with peripheral nerve injury and repair.

INTRODUCTION: Tuina is currently one of the popular complementary and alternative methods of rehabilitation therapy. Tuina can improve patients' pain and mobility function. However, the underlying physiological mechanism remains largely unknown, which might limit its further popularization in clinical practice. The aim of this study is to explore the short-term and long-term changes in brain functional activity following Tuina intervention for peripheral nerve injury repair. METHODS: A total of 16 rats were equally divided into the intervention group and the control group. Rats in the intervention group received Tuina therapy applying on the gastrocnemius muscle of the right side for 4 months following sciatic nerve transection and immediate repair, while the control group received nerve transection and repair only. The block-design functional magnetic resonance imaging scan was applied in both groups at 1 and 4 months after the surgery. During the scan, both the injured and intact hindpaw was electrically stimulated according to a "boxcar" paradigm. RESULTS: When stimulating the intact hindpaw, the intervention group exhibited significantly lower activation in the somatosensory area, limbic/paralimbic areas, pain-regulation areas, and basal ganglia compared to the control group, with only the prefrontal area showing higher activation. After 4 months of sciatic nerve injury, the control group exhibited decreased motor cortex activity compared to the activity observed at 1 month, and the intervention group demonstrated stronger bilateral motor cortex activity compared to the control group. CONCLUSION: Tuina therapy on the gastrocnemius muscle of rats with sciatic nerve injury can effectively alleviate pain and maintain the motor function of the affected limb. In addition, Tuina therapy reduced the activation level of pain-related brain regions and inhibited the decreased activity of the motor cortex caused by nerve injury, reflecting the impact of peripheral stimulation on brain plasticity.

Modulation of Brain Network Topological Properties in Knee Osteoarthritis by Electroacupuncture in Rats.

Introduction: Osteoarthritis is a chronic, ongoing disease that affects patients, and pain is considered a key factor affecting patients, but the brain changes during the development of osteoarthritis pain are currently unclear. In this study, we used electroacupuncture (EA) to intervene the rat model of knee osteoarthritis and analyzed the changes in topological properties of brain networks using graph theory. Methods: Sixteen SD rat models of right-knee osteoarthritis with anterior cruciate ligament transection (ACLT) were randomly divided into electroacupuncture intervention group and control group. The electroacupuncture group was intervened on Zusanli (ST36) and Futu (ST32) for 20 min each time, five times a week for 3 weeks, while the control group was applied sham stimulation. Both groups were measured for pain threshold. The small-world properties and node properties of the brain network between the two groups after the intervention were statistically analyzed by graph theory methods. Results: The differences are mainly in the changes in node attributes between the two groups, such as degree centrality, betweenness centrality, and so on in different brain regions (P<0.05). Both groups showed no small-world characteristics in the brain networks of the two groups. The mechanical thresholds and thermal pain thresholds were significantly higher in the EA group than in the control group (P<0.05). Conclusion: The study demonstrated that electroacupuncture intervention enhanced the activity of nodes related to pain circuit and relieved pain in osteoarthritis, which provides a complementary basis for explaining the effect of electroacupuncture intervention on pain through graphical analysis of changes in brain network topological properties and helps to develop an imaging model for pain affected by electroacupuncture.

Effect of aging on the cerebral metabolic mechanism of electroacupuncture treatment in rats with traumatic brain injury.

Objective: Aging has great influence on the clinical treatment effect of cerebrovascular diseases, and evidence suggests that the effect may be associated with age-related brain plasticity. Electroacupuncture is an effective alternative treatment for traumatic brain injury (TBI). In the present study, we aimed to explore the effect of aging on the cerebral metabolic mechanism of electroacupuncture to provide new evidence for developing age-specific rehabilitation strategies. Methods: Both aged (18 months) and young (8 weeks) rats with TBI were analyzed. Thirty-two aged rats were randomly divided into four groups: aged model, aged electroacupuncture, aged sham electroacupuncture, and aged control group. Similarly, 32 young rats were also divided into four groups: young model, young electroacupuncture, young sham electroacupuncture, and young control group. Electroacupuncture was applied to "Bai hui" (GV20) and "Qu chi" (LI11) for 8 weeks. CatWalk gait analysis was then performed at 3 days pre- and post-TBI, and at 1, 2, 4, and 8 weeks after intervention to observe motor function recovery. Positron emission computed tomography (PET/CT) was performed at 3 days pre- and post-TBI, and at 2, 4, and 8 weeks after intervention to detect cerebral metabolism. Results: Gait analysis showed that electroacupuncture improved the forepaw mean intensity in aged rats after 8 weeks of intervention, but after 4 weeks of intervention in young rats. PET/CT revealed increased metabolism in the left (the injured ipsilateral hemisphere) sensorimotor brain areas of aged rats during the electroacupuncture intervention, and increased metabolism in the right (contralateral to injury hemisphere) sensorimotor brain areas of young rats. Results: This study demonstrated that aged rats required a longer electroacupuncture intervention duration to improve motor function than that of young rats. The influence of aging on the cerebral metabolism of electroacupuncture treatment was mainly focused on a particular hemisphere.

Combined robot motor assistance with neural circuit-based virtual reality (NeuCir-VR) lower extremity rehabilitation training in patients after stroke: a study protocol for a single-centre randomised controlled trial.

INTRODUCTION: Improving lower extremity motor function is the focus and difficulty of post-stroke rehabilitation treatment. More recently, robot-assisted and virtual reality (VR) training are commonly used in post-stroke rehabilitation and are considered feasible treatment methods. Here, we developed a rehabilitation system combining robot motor assistance with neural circuit-based VR (NeuCir-VR) rehabilitation programme involving procedural lower extremity rehabilitation with reward mechanisms, from muscle strength training, posture control and balance training to simple and complex ground walking training. The study aims to explore the effectiveness and neurological mechanisms of combining robot motor assistance and NeuCir-VR lower extremity rehabilitation training in patients after stroke. METHODS AND ANALYSIS: This is a single-centre, observer-blinded, randomised controlled trial. 40 patients with lower extremity hemiparesis after stroke will be recruited and randomly divided into a control group (combined robot assistance and VR training) and an intervention group (combined robot assistance and NeuCir-VR training) by the ratio of 1:1. Each group will receive five 30 min sessions per week for 4 weeks. The primary outcome will be Fugl-Meyer assessment of the lower extremity. Secondary outcomes will include Berg Balance Scale, Modified Ashworth Scale and functional connectivity measured by resting-state functional MRI. Outcomes will be measured at baseline (T0), post-intervention (T1) and follow-ups (T2-T4). ETHICS, REGISTRATION AND DISSEMINATION: The trial was approved by the Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Chinese Traditional Medicine (Grant No. 2019-014). The results will be submitted to a peer-reviewed journal or at a conference. TRIAL REGISTRATION NUMBER: ChiCTR2100052133.

Alteration of brain functional networks induced by electroacupuncture stimulation in rats with ischemia-reperfusion: An independent component analysis.

Motor dysfunction is the major sequela of ischemic stroke. Motor recovery after stroke has been shown to be associated with remodeling of large-scale brain networks, both functionally and structurally. Electroacupuncture (EA) is a traditional Chinese medicine application that has frequently been recommended as an alternative therapy for ischemic stroke and is reportedly effective for alleviating motor symptoms in patients. In the present study, the effect of EA on the alterations of functional resting state networks (RSNs) was explored after middle cerebral artery occlusion/reperfusion (MCAO/R) injury using resting-state functional MRI. Rats were randomly assigned to three groups, including the sham group, MCAO/R group and MCAO/R+EA group. The ladder rung walking test was conducted prior to and after modeling to assess behavioral changes. RSNs were identified based on the independent component analysis (ICA) performed on the fMRI data from groups. EA treatment effectively reduced the occurrence of contralateral forelimb foot faults. Furthermore, our results suggested the disrupted function of the whole-brain network following ischemic stroke and the modulatory effect of acupuncture. The sensorimotor network (SMN), interoceptive network (IN), default mode network (DMN) and salience network (SN) were related to the therapeutic effect of EA on stroke recovery. Collectively, our findings confirmed the effect of EA on motor function recovery after cerebral ischemia reperfusion and shed light on the assessment of EA intervention-induced effects on brain networks. This study provides neuroimaging evidence to explain the therapeutic effects of EA in ischemic stroke and will lay the groundwork for further studies.

Surface-based map plasticity of brain regions related to sensory motor and pain information processing after osteonecrosis of the femoral head.

Pain is one of the manifestations of hip disorder and has been proven to lead to the remodeling of somatotopic map plasticity in the cortex. However, most studies are volume-based which may lead to inaccurate anatomical positioning of functional data. The methods that work on the cortical surface may be more sensitive than those using the full brain volume and thus be more suitable for map plasticity study. In this prospective cross-sectional study performed in Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, China, 20 patients with osteonecrosis of the femoral head (12 males and 8 females, aged 56.80 ± 13.60 years) and 20 healthy controls (9 males and 11 females, aged 54.56 ± 10.23 years) were included in this study. Data of resting-state functional magnetic resonance imaging were collected. The results revealed that compared with healthy controls, compared with the healthy controls, patients with osteonecrosis of the femoral head (ONFH) showed significantly increased surface-based regional homogeneity (ReHo) in areas distributed mainly in the left dorsolateral prefrontal cortex, frontal eye field, right frontal eye field, and the premotor cortex and decreased surface-based ReHo in the right primary motor cortex and primary sensory cortex. Regions showing significant differences in surface-based ReHo values between the healthy controls and patients with ONFH were defined as the regions of interests. Seed-based functional connectivity was performed to investigate interregional functional synchronization. When the areas with decreased surface-based ReHo in the frontal eye field and right premotor cortex were used as the regions of interest, compared with the healthy controls, the patients with ONFH displayed increased functional connectivity in the right middle frontal cortex and right inferior parietal cortex and decreased functional connectivity in the right precentral cortex and right middle occipital cortex. Compared with healthy controls, patients with ONFH showed significantly decreased cortical thickness in the para-insular area, posterior insular area, anterior superior temporal area, frontal eye field and supplementary motor cortex and reduced volume of subcortical gray matter nuclei in the right nucleus accumbens. These findings suggest that hip disorder patients showed cortical plasticity changes, mainly in sensorimotor- and pain-related regions. This study was approved by the Medical Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine (approval No. 2018-041) on August 1, 2018.

Electroacupuncture treatment improves motor function and neurological outcomes after cerebral ischemia/reperfusion injury.

Electroacupuncture (EA) has been widely used for functional restoration after stroke. However, its role in post-stroke rehabilitation and the associated regulatory mechanisms remain poorly understood. In this study, we applied EA to the Zusanli (ST36) and Quchi (LI11) acupoints in rats with middle cerebral artery occlusion and reperfusion. We found that EA effectively increased the expression of brain-derived neurotrophic factor and its receptor tyrosine kinase B, synapsin-1, postsynaptic dense protein 95, and microtubule-associated protein 2 in the ischemic penumbra of rats with middle cerebral artery occlusion and reperfusion. Moreover, EA greatly reduced the expression of myelin-related inhibitors Nogo-A and NgR in the ischemic penumbra. Tyrosine kinase B inhibitor ANA-12 weakened the therapeutic effects of EA. These findings suggest that EA can improve neurological function after middle cerebral artery occlusion and reperfusion, possibly through regulating the activity of the brain-derived neurotrophic factor/tyrosine kinase B signal pathway. All procedures and experiments were approved by the Animal Research Committee of Shanghai University of Traditional Chinese Medicine, China (approval No. PZSHUTCM200110002) on January 10, 2020.

Effectiveness of theta and gamma electroacupuncture for post-stroke patients on working memory and electrophysiology: study protocol for a double-center, randomized, patient- and assessor-blinded, sham-controlled, parallel, clinical trial.

BACKGROUND: Practitioners of complementary and alternative medicine have suggested that electroacupuncture (EA) could improve post-stroke cognitive impairment, based on the clinical evidence. This study protocol is aimed at showing the effectiveness of theta and gamma EA for post-stroke patients on working memory (WM) and electrophysiology. METHODS: After assessing their eligibility, 66 patients with stroke will be enrolled from two Chinese medicine hospitals and randomly divided into theta frequency EA group, gamma frequency EA group, and sham-EA group according to the ratio of 1:1:1. All patients will receive 20 sessions of EA procedures for 4 weeks. Patients in three groups will receive EA at two same acupoints in the head: Baihui (GV20) and Shenting (GV24). The frequency of the three groups of EA is set as follows: 6 Hz (theta-EA group), 40 Hz (gamma-EA group), and no current through the electrodes (sham EA). Patients and assessors will be blinded throughout the entire study. The primary outcome is the performance accuracy of 1-back task which is a frequently used measure of WM in cognitive neuroscience research contexts. Secondary outcome measures will include the response time of 1-back task, the Rivermead Behavioral Memory Test, Trail Making Test, Loewenstein Occupational Therapy Cognitive Assessment Scale, modified Barthel Index, and electroencephalogram (EEG) signals during 1-back tasks. A blinding index will be assessed. Data will be statistically analyzed by one-way ANOVA, at 5% of significance level. DISCUSSION: We expect this double-center, randomized, patient- and assessor-blinded, sham-controlled, parallel, clinical trial to explore the effectiveness of theta and gamma EA therapy, compared with sham EA, for post-stroke WM. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2000031995 . Registered on 17 April 2020.

Functional Organization of Brain Network in Peripheral Neural Anastomosis Rats after Electroacupuncture: An ICA and Connectome Analysis.

Acupuncture is a mild therapy in rehabilitation practice of peripheral nerve injury. Previous studies confirmed the deep participation of brain plasticity in the process of functional restoration. The therapeutic effect of acupuncture is also believed to be closely associated with brain plasticity, especially in the hypothalamus and limbic system. But the fuzzy neural mechanism somehow limits the application or improvement of this therapy. There is little information about the effect of acupuncture on topological properties of brain networks. Instead of functional segregation approach, we utilized graph theory method to analyze the large-scale and distributed properties of information processing. We first established rat model of sciatic nerve injury and performed rehabilitation therapy of electroacupuncture for 120 days. Meanwhile, we used independent component analysis to extract seven sub-networks from the whole brain. Then measurements of graph theory were calculated in each sub-network as well as the whole brain network. We found no significant difference of any measurement in whole brain network among intervention group, model group and normal group. But the assortativity, hierarchy, small-world properties of sub-network displayed significant differences among three groups. It induces changes of neural plasticity in several sub-networks instead of whole brain network. We attributed the changes to the enhancement of the short-term compensatory adaptation and the reduction of the long-term overacting regional information transmission. The present study may shed light on the vague distinction of large-scale property of brain networks after electroacupuncture, which leads to a better understanding of this ancient traditional Chinese therapy.

Brain Metabolism in Rats with Neuropathic Pain Induced by Brachial Plexus Avulsion Injury and Treated via Electroacupuncture.

PURPOSE: Brain organisation is involved in the mechanism of neuropathic pain. Acupuncture is a common clinical practise in traditional Chinese medicine for the treatment of chronic pain. This study explored electroacupuncture's effects on brain metabolism following brachial plexus avulsion injury (BPAI)-induced pain. METHODS: A total of 32 female rats were randomised into a normal group, model group, sham electroacupuncture group, and electroacupuncture group. A pain model was included via right BPAI. The electroacupuncture intervention at cervical "Jiaji" points (C5-7) was performed for 11 weeks. The mechanical withdrawal threshold of the non-injured (left) forepaw was measured at the baseline and on days 3, 7, 14, 21, 28, 56, 84, and 112 subsequent to BPAI. Positron emission tomography (PET) was applied to explore metabolic changes on days 28, 84, and 112. RESULTS: After electroacupuncture, the mechanical withdrawal threshold of the left forepaws was significantly elevated and the effect persisted until 4 weeks after the intervention ceased (p<0.05 or p<0.001). In the sensorimotor-related brain regions, standardised uptake values in the bilateral somatosensory and motor cortices were observed in the electroacupuncture group. Metabolism particularly increased in the right somatosensory cortex. Metabolism changes also occurred in the pain-related brain regions and emotion- and cognition-related brain regions. CONCLUSION: The present study demonstrated the beneficial effects of electroacupuncture for relieving BPAI-induced neuropathic pain in rats. Electroacupuncture intervention might inhibit maladaptive plasticity in brain areas governing multidimensional functions, especially in sensorimotor- and cognition-related cortices.

Acupuncture Induces Reduction in Limbic-Cortical Feedback of a Neuralgia Rat Model: A Dynamic Causal Modeling Study.

Background: Neuropathic pain after brachial plexus avulsion remained prevalent and intractable currently. However, the neuroimaging study about neural mechanisms or etiology was limited and blurred. Objective: This study is aimed at investigating the effect of electroacupuncture on effective connectivity and neural response in corticolimbic circuitries during implicit processing of nociceptive stimulus in rats with brachial plexus pain. Methods: An fMRI scan was performed in a total of 16 rats with brachial plexus pain, which was equally distributed into the model group and the electroacupuncture group. The analysis of task-dependent data determined pain-related activation in each group. Based on those results, several regions including AMY, S1, and h were recruited as ROI in dynamic causal modeling (DCM) analysis comparing evidence for different neuronal hypotheses describing the propagation of noxious stimuli in regions of interest and horizontal comparison of effective connections between the model and electroacupuncture groups. Results: In both groups, DCM revealed that noxious stimuli were most likely driven by the somatosensory cortex, with bidirectional propagation with the hypothalamus and amygdala and the interactions in them. Also, the 3-month intervention of acupuncture reduced effective connections of h-S1 and AMY-S1. Conclusions: We showed an evidence that a full connection model within the brain network of brachial plexus pain and electroacupuncture intervention reduces effective connectivity from h and AMY to S1. Our study for the first time explored the relationship of involved brain regions with dynamic causal modeling. It provided novel evidence for the feature of the organization of the cortical-limbic network and the alteration caused by acupuncture.

Diffusional plasticity induced by electroacupuncture intervention in rat model of peripheral nerve injury.

Electroacupuncture (EA) is an adjuvant therapy for peripheral nerve injury (PNI). Both peripheral and central alterations contribute to the rehabilitation process. We employed diffusion tensor imaging (DTI) to investigate the diffusion plasticity of afferent and efferent pathways caused by EA in model of peripheral nerve injury and reparation. Twenty-four rats were divided into three groups: normal group, model group and intervention group. Rats of the model group and the intervention group underwent sciatic nerve transection and anastomosis. EA intervention was performed on the intervention group at ST-36 and GB-30 for three months. Gait assessment and DTI were conducted at days post-operative (DPO) 30, 60 and 90. We selected corticospinal tract, spinothalamic tract and internal capsule as regions of interest and analyzed diffusion metrics including fractional anisotropy (FA), axial diffusivity (AD) and radial diffusivity (RD). FA values and RD values displayed significant differences or obvious tendency while AD values maintained a stable level. RD values displayed better indicative performance than FA in internal capsule. The intervention group presented significant correlation between RD values and Regularity Index (RI) during the intervention period. The effect of EA on peripheral nerve injury repairing rats appeared to be accelerated recovery process of sensory and motor neural pathway. We proposed that RD was a potential in vivo indicator for structural plasticity caused by EA and PNI.

Cortical remodeling after electroacupuncture therapy in peripheral nerve repairing model.

Electroacupuncture (EA) is an alternative therapy for peripheral nerve injury (PNI). The treatment relies on post-therapeutic effect rather than real-time effect. We utilized fMRI to clarify the resting-state alteration caused by sustained effect of EA on peripheral nerve repairing model. Twenty-four rats were divided equally into three groups: normal group, model group and intervention group. Rats of the model and intervention group underwent sciatic nerve transection and direct anastomosis. EA intervention at ST-36 and GB-30 was conducted continuously for 4 months on the intervention group. Behavioral assessments and fMRI were performed 1 month and 4 months after surgery. Intervention group showed significant improvement on the gait parameters max contact mean intensity (MCMI) and thermal withdrawal latency (TWL) than model group. EA-related sustained effects of amplitude of low frequency fluctuations (ALFF) could be described as a remolding pattern of somatosensory area and sensorimotor integration regions which presented higher ALFF in the contralateral hemisphere and lower in the ipsilateral hemisphere than model group. Interhemispheric functional connectivity (FC) analysis showed a significantly lower FC after EA therapy between the largest significantly different clusters in bilateral somatosensory cortices than the model group 4 months after surgery(p < 0.05). And the model group presented significantly higher FC than the normal group at both two time-points (p < 0.01). The sustained effect of EA on peripheral nerve repairing rats appeared to induce both regional and extensive neuroplasticity in bilateral hemispheres. We proposed that such EA-related effect was a reverse of maladaptive plasticity caused by PNI.

A Longitudinal Mapping Study on Cortical Plasticity of Peripheral Nerve Injury Treated by Direct Anastomosis and Electroacupuncture in Rats.

OBJECTIVE: We used functional magnetic resonance imaging to provide a longitudinal description of cortical plasticity caused by electroacupuncture (EA) of sciatic nerve transection and direct anastomosis in rats. METHODS: Sixteen rats in a sciatic nerve transection and direct anastomosis model were randomly divided into intervention and control groups. EA intervention in the position of ST-36, GB-30 was conducted continuously for 4 months in the intervention group. Functional magnetic resonance imaging and gait assessment were performed every month after intervention. RESULTS: The somatosensory area was more activated in the first 2 months and then deactivated in the rest 2 months when EA was applied. The pain-related areas had the same activation pattern as the somatosensory area. The limbic/paralimbic areas fluctuated more during the EA intervention, which was not constantly activated or deactivated as previous studies reported. We attributed such changes in somatosensory and pain-related areas to the gradual reduction of sensory afferentation. The alterations in limbic/paralimbic system might be associated with the confrontation between the upregulating effect of paresthesia or pain and the downregulating effect of EA intervention through the autonomic nerve system. The gait analysis showed significantly higher maximum contact mean intensity in the intervention group. CONCLUSIONS: The alterations in the brain brought about by the long-term therapeutic effect of EA could be described as a synchronized activation pattern in the somatosensory and pain-related areas and a fluctuating pattern in the limbic/paralimbic system.

Novel cinnamamide-dibenzylamine hybrids: Potent neurogenic agents with antioxidant, cholinergic, and neuroprotective properties as innovative drugs for Alzheimer's disease.

By using fragments endowed with interesting and complementary properties for the treatment of Alzheimer's disease (AD), a novel series of cinnamamide-dibenzylamine hybrids have been designed, synthesized, and evaluated biologically. In vitro assay indicated that most of the target compounds exhibited a significant ability to inhibit ChEs, strong potency inhibitory of self-induced ß-amyloid (Aß) aggregation and to act as potential antioxidants and biometal chelators. A Lineweaver-Burk plot and molecular modeling study showed that compound 7f targeted both the CAS and PAS of AChE. In addition, compound 7f could chelate metal ions, reduce PC12 cells death induced by oxidative stress and penetrate the blood-brain barrier (BBB). Overall, all of these outstanding in vitro results in combination with promising in vivo outcomes highlighted derivative 7f as the lead structure worthy of further investigation.

Rational Design and Multibiological Profiling of Novel Donepezil-Trolox Hybrids against Alzheimer's Disease, with Cholinergic, Antioxidant, Neuroprotective, and Cognition Enhancing Properties.

A novel series of donepezil-trolox hybrids were designed, synthesized, and evaluated as multifunctional ligands against Alzheimer's disease (AD). Biological assays showed that these derivatives possessed moderate to good inhibitory activities against acetylcholinesterase (AChE) and monoamine oxidase B (MAO-B) as well as remarkable antioxidant effects. The optimal compound 6d exhibited balanced functions with good inhibition against hAChE (IC50 = 0.54 µM) and hMAO-B (IC50 = 4.3 µM), significant antioxidant activity (41.33 µM IC50 by DPPH method, 1.72 and 1.79 trolox equivalent by ABTS and ORAC methods), excellent copper chelation, and Aß1-42 aggregation inhibition effect. Furthermore, cellular tests indicated that 6d has very low toxicity and is capable of combating oxidative toxin (H2O2, rotenone, and oligomycin-A) induced neurotoxicity. Most importantly, oral administration of 6d demonstrated notable improvements on cognition and spatial memory against scopolamine-induced acute memory deficit as well as d-galactose (d-gal) and AlCl3 induced chronic oxidative stress in a mouse model without acute toxicity and hepatotoxicity. In summary, both in vitro and in vivo results suggested that 6d is a valuable candidate for the development of a safe and effective anti-Alzheimer's drug.