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T-006, a small-molecule compound derived from tetramethylpyrazine (TMP), has potential for the treatment of neurological diseases. In order to investigate the effect of T-006 prophylactic treatment on an Alzheimer's disease (AD) model and identify the target of T-006, we intragastrically administered T-006 (3 mg/kg) to Alzheimer's disease (AD) transgenic mice (APP/PS1-2xTg and APP/PS1/Tau-3xTg) for 6 and 8 months, respectively. T-006 improved cognitive ability after long-term administration in two AD mouse models and targeted mitochondrial-related protein alpha-F1-ATP synthase (ATP5A). T-006 significantly reduced the expression of phosphorylated-tau, total tau, and APP while increasing the expression of synapse-associated proteins in 3xTg mice. In addition, T-006 modulated the JNK and mTOR-ULK1 pathways to reduce both p-tau and total tau levels. Our data suggested that T-006 mitigated cognitive decline primarily by reducing the p-tau and total tau levels in 3xTg mice, supporting further investigation into its development as a candidate drug for AD treatment.

T-006, a new derivative of tetramethylpyrazine, has been recently found to protect against 6-hydroxydopamine (6-OHDA)-induced neuronal damage and clear α-synuclein (α-syn) by enhancing proteasome activity in an α-syn transgenic Parkinson's disease (PD) model. The effect of T-006 on the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced PD model, however, has not been tested and T-006's neuroprotective mechanisms have not been fully elucidated. In this study, we further investigated the neuroprotective and neurogenic effects of T-006 and explored its underlying mechanism of action in both cellular and animal PD models. T-006 was able to improve locomotor behavior, increase survival of nigra dopaminergic neurons and boost striatal dopamine levels in both MPTP- and 6-OHDA-induced animals. T-006 treatment restored the altered expressions of myocyte enhancer factor 2D (MEF2D), peroxisome proliferator-activated receptor γ (PPARγ) co-activator 1α (PGC1α) and NF-E2-related factor 1/2 (Nrf1/2) via modulation of Akt/GSK3β signaling. T-006 stimulated MEF2, PGC1α and Nrf2 transcriptional activities, inducing Nrf2 nuclear localization. Interestingly, T-006 promoted endogenous adult neurogenesis toward a dopaminergic phenotype by activating brain-derived neurotrophic factor (BDNF) and cAMP responsive element-binding protein (CREB) in 6-OHDA rats. Our work demonstrated that T-006 is a potent neuroprotective and neuroregenerative agent that may have therapeutic potential in the treatment of PD.

Sepsis is a life-threatening organ dysfunction with devastating consequences, prominent among which is lung damage. Memantine, an N-methyl-D-aspartic acid receptor (NMDAR) antagonist, is able to alleviate acute lung injury (ALI). Nitric oxide (NO) suppresses NLRP3 inflammasome activation against lipopolysaccharide (LPS)-induced septic shock. MN-08, a novel nitrate derivative of memantine, possesses both the ability to antagonize NMDAR and release NO. In the present study, we aimed to investigate the protective effects of MN-08 against LPS-induced systemic inflammation and septic lung injury in mice, and to explore the underlying mechanisms of MN-08 in LPS-induced mice and THP-1 macrophages. MN-08 significantly increased the survival rate of septic mice, alleviated LPS-induced sepsis in mice via improving systemic inflammatory response syndrome and immune dysfunction, and attenuated pulmonary injury and inflammatory infiltration. More importantly, the therapeutic benefit of MN-08 for sepsis was greater than that of memantine and dexamethasone. Mechanistically, MN-08 exerted anti-inflammatory activity through inhibiting nuclear translocation of NF-κB, activation of the MAPK signaling pathway and the signaling transduction of STAT3/NF-κB. In addition, MN-08 suppressed NLRP3 inflammasome activation. Taken together, our studies demonstrate that MN-08 may be a promising therapeutic agent for sepsis-induced acute lung injury.

Polydatin is the major active ingredient of Polygonum cuspidatum Sieb. Et Zucc. A recent study indicated that polydatin could protect against substantia nigra dopaminergic degeneration in rodent models associated with Parkinson's disease. However, mechanisms that underlie the neuroprotection of polydatin have not been fully elucidated. In the current study, the neuroprotective effects and detailed mechanisms of action of polydatin were investigated in Parkinson's disease-related cellular models. Polydatin dose- and time-dependently prevented neurotoxicity caused by 1-methyl-4-phenylpyridinium ion (MPP+) in primary cerebellar granule neurons. Moreover, we found that polydatin enhanced the activity of the transcription factor myocyte enhancer factor 2D (MEF2D) at both basal and pathological conditions using luciferase reporter gene assay. Additionally, western blot analysis revealed that polydatin could downregulate glycogen synthase kinase 3β (GSK3β), which is a negative regulator of MEF2D. Molecular docking simulations finally suggested an interaction between polydatin and a hydrophobic pocket within GSK3β. All these results suggest that polydatin prevents MPP+-induced neurotoxicity via enhancing MEF2D through the inhibition of GSK3β and that treatment with polydatin is worthy of further anti-Parkinson's disease study in future.

Acute lung injury (ALI) is a clinically severe respiratory disorder, and effective therapy is urgently needed. MN-08, a novel synthetic N-methyl-d-aspartate receptor (NMDAR) antagonist, was investigated for its effect on lipopolysaccharide (LPS)-induced ALI. In vitro, the protective effect of MN-08 on inflammatory response, oxidative stress, and tight junctions (TJs) structure was explored in LPS-induced RAW 264.7 cells and A549 cells. MN-08 markedly decreased (p < 0.001) the levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and reactive oxygen species (ROS), whereas it moderately upregulated (p < 0.05) heme oxygenase (HO)-1 protein expression in LPS-induced RAW 264.7 cells. Moreover, MN-08 significantly inhibited (p < 0.001) cell apoptosis and improved (p < 0.001) protein expression of TJs in LPS-induced A549 cells. In vivo, the therapeutic effect of MN-08 was evaluated in the LPS-induced ALI model through intratracheal instillation in BALB/c mice. MN-08 administration dramatically attenuated (p < 0.001) pulmonary pathological changes and reduced (p < 0.001) the levels of glutamate, myeloperoxidase (MPO), malondialdehyde (MDA), and number of cells in BALF, whereas it increased (p < 0.05) superoxide dismutase (SOD) and glutathione (GSH) activities in ALI mice. Furthermore, MN-08 markedly blocked the mitogen-activated protein kinases (MAPKs)/nuclear translocation of nuclear factor-κB (NF-κB) signaling pathways in RAW 264.7 cells and lung tissues. These results indicate that MN-08 exhibits lung protection in an LPS-induced ALI model via anti-inflammatory and anti-oxidative activities.

Pulmonary arterial hypertension (PAH) is a rare, progressive pulmonary vascular disease with limited therapeutic options. Pulmonary circulation resistance, pulmonary vascular remodeling, and over-activated NMDARs (N-methyl-d-aspartate receptors) play vital roles in the pathogenesis of PAH. In the present study, we aimed to evaluate the efficacy and molecular mechanism of MN-08, a dual-functional memantine nitrate derivative, in experimental animal models of PAH. MN-08 showed a high degree of accumulation in the lungs and dilated pulmonary arterial rings ex vivo by releasing nitric oxide. MN-08 did not lower systemic blood pressure. MN-08 attenuated right ventricular systolic pressure and right ventricular hypertrophy, inhibited pulmonary arterial remodeling, alleviated glutamate-NMDARs dysregulation, and improved survival rates in monocrotaline-induced PAH rats. More importantly, the therapeutic benefit of MN-08 for PAH was greater than that of sildenafil. Moreover, MN-08 can reduce right ventricular systolic pressure in U46619-induced acute PAH rats. Mechanistically, MN-08 suppressed proliferation of pulmonary arterial smooth muscle cells exposed to human platelet-derived growth factor-BB by regulating the cell cycle and expression of NMDAR1, AKT (serine/threonine kinase Akt), and ERK (extracellular signal-regulated kinase) 1/2. In conclusion, our studies demonstrated that MN-08 may be a promising therapeutic agent for PAH.

Alzheimer's disease (AD) is a leading cause of dementia in elderly individuals and therapeutic options for AD are very limited. Over‐activation of N‐methyl‐D‐aspartate (NMDA) receptors, amyloid β (Aβ) aggregation, a decrease in cerebral blood flow (CBF), and downstream pathological events play important roles in the disease progression of AD. In the present study, MN‐08, a novel memantine nitrate, was found to inhibit Aβ accumulation, prevent neuronal and dendritic spine loss, and consequently attenuate cognitive deficits in 2‐month‐old APP/PS1 transgenic mice (for a 6‐month preventative course) and in the 8‐month‐old triple‐transgenic (3×Tg‐AD) mice (for a 4‐month therapeutic course). In vitro, MN‐08 could bind to and antagonize NMDA receptors, inhibit the calcium influx, and reverse the dysregulations of ERK and PI3K/Akt/GSK3β pathway, subsequently preventing glutamate‐induced neuronal loss. In addition, MN‐08 had favorable pharmacokinetics, blood‐brain barrier penetration, and safety profiles in rats and beagle dogs. These findings suggest that the novel memantine nitrate MN‐08 may be a useful therapeutic agent for AD., Memantine nitrate MN‐08 reverses cognitive impairment by antagonizing NMDA receptor and releasing NO in AD mice. MN‐08 antagonizes NMDA receptor, inhibits calcium influx, attenuates the dysregulations of ERK and PI3K/Akt/GSK3β, reduces Aβ accumulation, and consequently prevents neuronal and dendritic spine loss. Moreover, MN‐08 binds NMDA receptor to release NO, dilates blood vessel and increases cerebral blood flow. These findings suggest that MN‐08 may be a useful therapeutic agent for AD.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of upper and lower motor neurons that results in skeletal muscle atrophy, weakness and paralysis. Oxidative stress plays a key role in the pathogenesis of ALS, including familial forms of the disease arising from mutation of the gene coding for superoxide dismutase (SOD1). We have used the SOD1G93A ALS mouse model to investigate the efficacy of 2-[[(1,1-dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a novel tetramethylpyrazine derivative armed with a powerful free-radical scavenging nitrone moiety. TBN was administered to mice by intraperitoneal or intragastric injection after the onset of motor deficits. TBN slowed the progression of motor neuron disease as evidenced by improved motor performance, reduced spinal motor neuron loss and the associated glial response, and decreased skeletal muscle fiber denervation and fibrosis. TBN treatment activated mitochondrial antioxidant activity through the PGC-1α/Nrf2/HO-1 pathway and decreased the expression of human SOD1. These findings suggest that TBN holds promise as a therapeutic agent for ALS.

We have previously designed and synthesized a series of novel memantine nitrates, and some of them have shown neuroprotective effects; however, the detailed mechanisms remain unknown. In this study, we demonstrated that MN-12, one of the memantine nitrates, concentration-dependently protected against glutamate-induced neurotoxicity in rat primary cultured cerebellar granule neurons (CGNs). Western blotting assays revealed that MN-12 might possess neuroprotective effects through the inhibition of ERK pathway and activation of PI3K/Akt pathway concurrently. Moreover, MN-12 concentration-dependently dilated precontracted rat middle cerebral artery through activation of NO-cGMP pathway

Miconazole, a frequently used antifungal drug, has been identified with new functions to promote oligodendrocyte progenitor cells differentiation and to enhance remyelination. However, the neuroregenerative and therapeutic benefit of miconazole on ischemic stroke model have not been tested. In the present study, the effects of miconazole on a rat model of transient middle cerebral artery occlusion were evaluated. Rats received miconazole (10 mg/kg) or saline by intravenous administration for 7 days after stroke. A battery of neurobehavioral assessments, including rotarod test, open-field test, neurological severity score and novel object recognition task were evaluated. The results revealed a significant functional improvement in miconazole-treated rats compared with vehicle-treated control. Animals were sacrificed at 7 and 28 days after stroke. Double immunofluorescence staining for NeuN+/BrdU+, DCX+/BrdU+ and Nestin+/BrdU+ cells indicated miconazole significantly promoted neurogenesis. Western blotting analysis revealed miconazole upregulated the protein expression of brain derived neurotrophic factor, myocyte enhancer factor 2D, synaptophysin, and postsynaptic density protein 95, while downregulated the expression of cyclin-dependent kinase 5. Taken together, miconazole promoted functional recovery on ischemic stroke model via stimulating post-ischemic neurogenesis.

Our previous studies demonstrated that the multifunctional agent TBN, a derivative of tetramethylpyrazine armed with a nitrone moiety, displayed high therapeutic efficacy in experimental ischemic stroke models. However, its molecular mechanisms of action underlying the neuroprotective effect need further exploration. In the present study, we found that TBN had significant activities scavenging free radicals such as ·OH, O 2 ·− and ONOO−, inhibiting Ca2+ overload, maintaining mitochondrial function and preventing neuronal damage in primary cortical cultures. Further, TBN was effective in reducing brain infarction and ameliorating impairment of behavioral functions in the permanent middle cerebral artery occlusion (p-MCAo) rat model. TBN down-regulated the expression of pro-apoptotic factors Bax, while up-regulated the expression of anti-apoptotic factor Bcl-2 and increased the expression of pro-survival factors including p-Akt and p-GSK3β in the peri-infarct cortex of p-MCAo rats. In addition, LY-294002 (a PI3K inhibitor) and MK2206 (an Akt inhibitor) significantly blocked the protective effect of TBN against OGD-induced death of cortical neurons. Taken together, the multifunctional mechanisms including scavenging free radicals, blocking calcium overload, maintaining mitochondrial function and activating the PI3K/Akt/p-GSK3β cell survival pathway were possibly involved in the neuroprotective effects of TBN, making it a promising clinical candidate for the treatment of ischemic stroke.

ADTM, a previously reported novel Danshensu (DSS)/tetramethylpyrazine (TMP) derivative with cardioprotective and antiplatelet aggregative effects, is a promising therapeutic candidate for ischemic heart diseases. In the present study, ADTM increased coronary blood flow and protected myocardium against ischemic injury in dogs. In addition, the relaxing effect of ADTM on rat thoracic aorta and its underlying mechanisms were examined. ADTM relaxed KCl- and phenylephrine-precontracted arotic rings in a concentration-dependent manner. The relaxation by ADTM was greater than that by DSS, TMP and the mixture of DSS and TMP. ADTM induced endothelium-independent relaxation, which couldn't be abolished by removal of endothelium and the preincubation with inhibitors of nitric oxide synthase (L-NAME) and guanylate cyclase (ODQ). Potassium channel blockers including tetraethylammonium, BaCl2 and glibenclamide failed to inhibit the relaxation by ADTM. In addition, cyclooxygenase (COX), muscarine receptor and β-adrenoceptor were not involved in ADTM-induced vasorelaxation. ADTM inhibited contraction induced by CaCl2 and phenylephrine in Ca2+-free buffer, suggesting that ADTM inhibited both extracellular Ca2+ influx and intracellular Ca2+ release. Taken together, the vasorelaxation of ADTM may be possibly involved in its cardioprotection. ADTM may serve as a promising candidate for the treatment of ischemic heart diseases.

In this study, we investigated the cardioprotective mechanisms of action of DT-010, a novel danshensu-tetramethylpyrazine conjugate. DT-010 significantly preserved cell viability and suppressed cell apoptosis in H9c2 cells injured by tert-butylhydroperoxide (t-BHP), iodoacetic acid (IAA) and hypoxia-reoxygenation. In addition, DT-010 pre-treatment reduced the intracellular level of free radicals including superoxide anion (·O2-), hydroxyl radical (·OH) and peroxynitrite anion (ONOO-) after t-BHP exposure. Moreover, DT-010 up-regulated the protein expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and nuclear factor-E2-related factor 2 (Nrf2) as well as mitochondrial transcription factor A (Tfam) and heme oxygenase-1 (HO-1) in H9c2 cells. DT-010 also triggered Nrf2 nuclear translocation. In a rat myocardial ischemia-reperfusion model, DT-010 significantly alleviated myocardial infarction. The results indicated that DT-010 may be a promising candidate for the treatment of cardiovascular diseases, particularly myocardial ischemia and reperfusion injury.

Myocardial ischemia is a primary cause of sudden death worldwide. Numerous active ingredients of traditional Chinese medicines including danshensu (DSS) and tetramethylpyrazine (TMP) have been widely used for the treatment of myocardial ischemia. To enhance their therapeutic efficacy and improve their drugability, in this work, we designed new DSS and TMP conjugates. Their water solubility and protective effects were studied in vitro and in experimental animal models. The new compounds demonstrated higher activities than the positive control agents acetylated danshensu and tetramethylpyrazine conjugate (ADTM) and salvianolic acid B (SAB) in preventing cells from oxidative insult. Among the new compounds, 14, bearing two glycine moieties, was more water soluble. In addition, compound 14 was much more potent in preventing cells from oxidative injury, at least 10- and 20-fold as potent as ADTM and SAB, respectively. The protective effects of compound 14 may be attributed to its anti-radical activity and anti-apoptotic activity. These results suggest that compound 14 is a promising candidate for the treatment of myocardial ischemia.

Background and purpose Cerebral vasospasm and neuronal apoptosis after subarachnoid haemorrhage (SAH) is the major cause of morbidity and mortality in SAH patients. So far, single-target agents have not prevented its occurrence. Memantine, a non-competitive NMDA re3ceptor antagonist, is known to alleviate brain injury and vasospasm in experimental models of SAH. Impairment of NO availability also contributes to vasospasm. Recently, we designed and synthesized a memantine nitrate MN-08, which has potent dual functions: neuroprotection and vasodilation. Here, we have tested the therapeutic effects of MN-08 in animal models of SAH. Experimental approach Binding to NMDA receptors (expressed in HEK293 cells), NO release and vasodilator effects of MN-08 were assessed in vitro. Therapeutic effects of MN-08 were investigated in vivo, using rat and rabbit SAH models. Key results MN-08 bound to the NMDA receptor, slowly releasing NO in vitro and in vivo. Consequently, MN-08 relaxed the pre-contracted middle cerebral artery ex vivo and increased blood flow velocity in small vessels of the mouse cerebral cortex. It did not, however, lower systemic blood pressure. In an endovascular perforation rat model of SAH, MN-08 improved the neurological scores and ameliorated cerebral vasospasm. Moreover, MN-08 also alleviated cerebral vasospasm in a cisterna magna single-injection model in rabbits. MN-08 attenuated neural cell apoptosis in both rat and rabbit models of SAH. Importantly, the therapeutic benefit of MN-08 was greater than that of memantine. Conclusion and implications MN-08 has neuroprotective potential and can ameliorate vasospasm in experimental SAH models.

Neuronal death is among the deleterious pathological changes that occur after cerebral ischemia and can lead to transient or permanent neurological deficits. The tetramethylpyrazine analog T-006 has been shown to be a multifunctional neuroprotective agent; however, its neuroprotective effect and mechanism of action have not been studied in ischemic stroke model rats. This study investigated the neuroprotective effects of T-006 in rat stroke model using a battery of behavioral and molecular biological tests. Results indicated that T-006 treatment significantly improved neurological function and behavior. Double immunofluorescence staining showed that T-006 visibly improved the number of NeuN/BrdU, Nestin/BrdU, and DCX/BrdU cells and induced neuronal regeneration. Western blot analyses indicated that T-006 upregulated neurogenesis-related protein expression of postsynaptic density protein 95, brain-derived neurotrophic factor, synaptophysin, and myelin basic protein. Collectively, these data suggest that T-006 stimulated neurogenesis in rats with middle cerebral artery occlusion and restored neurological functions.

Overactivation of N-methyl-D-aspartate (NMDA) receptors has been associated with neurodegenerative disorders such as Alzheimer's disease (AD), cerebral vascular disorders and amyotrophic lateral sclerosis (ALS). We have previously designed and synthesized a series of memantine nitrate and some of them have shown vessel dilatory effects and neuroprotective effects; however, the detailed mechanisms have not been elucidated. In this study, we further demonstrated that memantine nitrate-06 (MN-06), one of the novel compounds derived from memantine, possessed significant neuroprotective effects against glutamate-induced excitotoxicity in rat primary cerebellar granule neurons (CGNs). Pretreatment of MN-06 reversed the activation of GSK3b and the suppression of phosphorylated Akt induced by glutamate. In addition, the neuroprotective effects of MN-06 could be abolished by LY294002, the specific phosphatidylinositol 3-kinase (PI3-K) inhibitor. Ca2+ imaging shown that pretreatment of MN-06 prevented Ca2+ influx induced by glutamate. Moreover, MN-06 might inhibit the NMDA-mediated current by antagonizing NDMA receptors, which was further confirmed by molecular docking simulation. Taken together, MN-06 protected against glutamate-induced excitotoxicity by blocking calcium influx and attenuating PI3-K/Akt/GSK-3b pathway, indicating that MN-06 might be a potential drug for treating neurodegenerative disorders.

Oxidative stress is one of the major secondary injury mechanisms after traumatic brain injury (TBI). 2-[[(1,1-Dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a derivative of the clinically used anti-stroke drug tetramethylpyrazine armed with a powerful free radical-scavenging nitrone moiety, has been demonstrated promising therapeutic efficacy in ischemic stroke and Parkinson's models. The present study aims to investigate the effects of TBN on behavioral function and neuroprotection in rats subjected to TBI. TBN (90 mg/kg) was administered twice daily for 7 days by intravenous injection following TBI. TBN improved neuronal behavior functions after brain injury, including rotarod test and adhesive paper removal test. Compared with the TBI model group, TBN treatment significantly protected NeuN-positive neurons, while decreased glial fibrillary acidic protein (GFAP)-positive cells. The number of 4-hydroxynonenal (4-HNE)-positive and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells around the damaged area after TBI were significantly decreased in the TBN treatment group. In addition, TBN effectively reversed the altered expression of Bcl-2, Bax and caspase 3, and the down-regulation of nuclear factor erythroid-derived 2-like 2 (Nrf-2) and hemeoxygenase-1 (HO-1) proteins expression stimulated by TBI. In conclusion, TBN improves neurobehavioral functions and protects neurons against TBI. This protective effect may be achieved by anti-neuronal apoptosis, alleviating oxidative stress damage and up-regulating Nrf-2 and HO-1 expression.

Cerebral vasospasm is one of the deleterious complications after subarachnoid hemorrhage (SAH), leading to delayed cerebral ischemia and permanent neurological deficits or even death. Free radicals and oxidative stress are considered as crucial causes contributing to cerebral vasospasm and brain damage after SAH. Tetramethylpyrazine nitrone (TBN), a derivative of the clinically used anti-stroke drug tetramethylpyrazine armed with a powerful free radical scavenging nitrone moiety, has been reported to prevent brain damage from ischemic stroke. The present study aimed to investigate the effects of TBN on vasospasm and brain damage after SAH. Two experimental SAH models were used, a rat model by endovascular perforation and a rabbit model by intracisternal injection of autologous blood. The effects of TBN on SAH were evaluated assessing basilar artery spasm, neuronal apoptosis, and neurological deficits. TBN treatment significantly attenuated vasospasm, improved neurological behavior functions and reduced the number of apoptotic neurons in both the SAH rats and rabbits. Mechanistically, TBN suppressed the increase in 3-nitrotyrosine and 8-hydroxy-2-deoxyguanosine immuno-positive cells in the cortex of SAH rat brain. Western blot analyses indicated that TBN effectively reversed the altered expression of Bcl-2, Bax and cytochrome C, and up-regulated nuclear factor erythroid-derived 2-like 2 (Nrf2) and hemeoxygenase-1 (HO-1) protein expressions. In the in vitro studies, TBN inhibited H2O2-induced bEnd.3 cell apoptosis and reduced ROS generation. Additionally, TBN alleviated the contraction of rat basilar artery rings induced by H2O2 ex vivo. In conclusion, TBN ameliorated SAH-induced cerebral vasospasm and neuronal damage. These effects of TBN may be attributed to its anti-oxidative stress effect and up-regulation of Nrf2/HO-1.

Parkinson's disease (PD) is a complex neurodegenerative disorder with multifactorial pathologies, including progressive loss of dopaminergic (DA) neurons, oxidative stress, mitochondrial dysfunction, and increased monoamine oxidase (MAO) enzyme activity. There are currently only a few agents approved to ameliorate the symptoms of PD; however, no agent is able to reverse the progression of the disease. Due to the multifactorial pathologies, it is necessary to develop multifunctional agents that can affect more than one target involved in the disease pathology. We have designed and synthesized a series of new multifunctional anti-Parkinson's compounds which can protect cerebral granular neurons from 1-methyl-4-phenylpyridinium (MPP+) insult, scavenge free radicals, and inhibit monoamine oxidase (MAO)/cholinesterase (ChE) activities. Among them, MT-20R exhibited the most potent MAO-B inhibition both in vitro and in vivo. We further investigated the neuroprotective effects of MT-20R using a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. In vivo, MT-20R alleviated MPTP-induced motor deficits, raised the striatal contents of dopamine and its metabolites, and restored the expression of tyrosine hydroxylase (TH) and the number of TH-positive DA neurons in the substantia nigra. Additionally, MT-20R enhanced the expression of Bcl-2, decreased the expression of Bax and Caspase 3, and activated the AKT/Nrf2/HO-1 signaling pathway. These findings suggest that MT-20R may be a novel therapeutic candidate for treatment of PD.

TBN, a novel tetramethylpyrazine derivative armed with a powerful free radical-scavenging nitrone moiety, has been reported to reduce cerebral infarction in rats through multi-functional mechanisms of action. Here we study the therapeutic effects of TBN on non-human primate model of stroke. Thirty male Cynomolgus macaques were subjected to stroke with 4 hours ischemia and then reperfusion. TBN were injected intravenously at 3 or 6 hours after the onset of ischemia. Cerebral infarction was examined by magnetic resonance imaging at 1 and 4 weeks post ischemia. Neurological severity scores were evaluated during 4 weeks observation. At the end of experiment, protein markers associated with the stroke injury and TBN treatment were screened by quantitative proteomics. We found that TBN readily penetrated the blood brain barrier and reached effective therapeutic concentration after intravenous administration. It significantly reduced brain infarction and modestly preserved the neurological function of stroke-affected arm. TBN suppressed over-expression of neuroinflammatory marker vimentin and decreased the numbers of GFAP-positive cells, while reversed down-regulation of myelination-associated protein 2′, 3′-cyclic-nucleotide 3′-phosphodiesterase and increased the numbers of NeuN-positive cells in the ipsilateral peri-infarct area. TBN may serve as a promising new clinical candidate for the treatment of ischemic stroke.

Oxidative stress plays a crucial role in neurological diseases, resulting in excessive production of reactive oxygen species, mitochondrial dysfunction and cell death. In this work, we designed and synthesized a series of tetramethylpyrazine (TMP) derivatives and investigated their abilities for scavenging free radicals and preventing against oxidative stress-induced neuronal damage in vitro. Among them, compound 22a, consisted of TMP, caffeic acid and a nitrone group, showed potent radical-scavenging activity. Compound 22a had broad neuroprotective effects, including rescuing iodoacetic acid-induced neuronal loss, preventing from tert-butylhydroperoxide (t-BHP)-induced neuronal injury. Compound 22a exerted its neuroprotective effect against t-BHP injury via activation of the phosphatidyl inositol 3-kinase (PI3K)/Akt signaling pathway. Furthermore, in a rat model of permanent middle cerebral artery occlusion, compound 22a significantly improved neurological deficits, and alleviated the infarct area and brain edema. In conclusion, our results suggest that compound 22a could be a potential neuroprotective agent for the treatment of neurological disease, particularly ischemic stroke.

Chronic cerebral hypoperfusion is an important risk factor for vascular dementia and other brain dysfunctions, for which there are currently no effective medications available. We investigated the neuroprotective and axonal outgrowth promoting effects of tetramethylpyrazine nitrone (TBN) in a permanent bilateral occlusion of the common carotid arteries (2VO) rat model and in primary hippocampal neurons exposed to oxygen glucose deprivation (OGD). At 6th week after 2VO, TBN increased the time spent in novel arms in the Y-maze test and improved the discrimination ratio in object reorganization task. TBN attenuated axonal damage, and reduced oxidative DNA injury and lipid peroxidation in white matter. TBN also attenuated the neuronal apoptosis and ameliorated accumulation of astrocytes in parietal cortex and CA1 region of hippocampus. Western blot analyses indicated that TBN increased Bcl-2 expression, decreased Bax and Caspase 3 expressions, and upregulated the phosphorylation levels of high-molecular weight neurofilament (p-NFH), Akt (p-Akt) and glycogen synthase kinase-3β (p-GSK3β) in hippocampus at 6th week after chronic hypoperfusion. In vitro, TBN rescued hippocampal neuronal viability and axonal elongation from OGD damage. The p-Akt and p-GSK3β upregulation by TBN was abolished by a specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002, resulting in suppression of axonal outgrowth. Collectively, the results showed that TBN alleviated white matter lesion and impairment of cortex and hippocampus, attenuated oxidative damage and enhanced axonal outgrowth through the regulation of PI3K/Akt/GSK3β signaling pathway, leading to improved cognitive deficit in a rat chronic hypoperfusion model.

Alzheimer's disease is a progressive neurodegenerative disorder, characterized by irreversible impairment of memory and cognitive function. The exact causes of Alzheimer's disease still remain unclear and current single target drugs could only offer limited therapeutic effect to the patients. We have previously reported that T-006, a promising anti-Alzheimer's compound derived from Chinese medicinal component tetramethylpyrazine, might protect neurons through inhibiting the overproduction of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS). In this study, we further investigated the neuroprotective effects, as well as the molecular pathways involved, of T-006 against glutamate-induced excitotoxicity in rat cerebellar granule neurons (CGNs). T-006 was also found to promote neuronal differentiation in both PC12 cells and primary cultured rat cortical neurons. The results showed that the pretreatment of T-006 (0.01-1 μM) might prevent glutamate-induced neuronal loss in a concentration-dependent manner. T-006 is found to inhibit the over-activation of NMDAR and ensued calcium overload caused by glutamate. The following activation of phosphorylated extracellular signal-regulated kinase (ERK) were also abolished. Moreover, T-006 concurrently prevented the suppression of phosphorylated protein kinase B (Akt) and glycogen synthase kinase 3β (GSK3β). T-006 was also found to promote neurite outgrowth in PC12 cells and primary cortical neurons. In our study, T-006 (0.1-3 μM) dose-dependently stimulated neurite outgrowth in PC12 cells and the efficacy was comparable to nerve growth factor (NGF). Moreover, co-treatment of T-006 and NGF revealed that T-006 could robustly potentiate the NGF-induced neuritogenesis. Further signal transduction studies indicated that T-006 rapidly up-regulated phosphorylation of ERK but did not activate tyrosine kinase receptor A (Trk A). These findings offer deeper understanding of the anti-neurodegenerative activity of T-006 and provide insight into its possible therapeutic potential for AD treatment in light of the multipotent nature of T-006.

Ischemic stroke resulting from obstruction of blood vessels is an enormous public health problem with urgent need for effective therapy. The co-administration of thrombolytic/antiplatelet agent and neuroprotective agent improves therapeutic efficacy and agent possessing both thrombolytic/antiplatelet and antiradical activities provides a promising strategy for the treatment of ischemic stroke. We have previously reported a novel compound, namely TBN, possessing both antiplatelet and antiradical activities, showed significant neuroprotective effect in a rat stroke model. We herein report synthesis of a series of new pyrazine derivatives, and evaluation of their biological activities. Their mechanisms of action were also investigated. Among these new derivatives, compound 21, armed with two nitrone moieties, showed the greatest neuroprotective effects in vitro and in vivo. Compound 21 significantly inhibited ADP-induced platelet aggregation. In a cell free antiradical assay, compound 21 was the most effective agent in scavenging the three most damaging radicals, namely OH, O 2 · - and ONOO−.

Free radical-mediated neuronal cell damage is an important pathological process in ischemic stroke. We have previously reported a novel dual-functional agent, 2-[[(1,1-dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a derivative of tetramethylpyrazine armed with anitrone moiety. In this report, we further evaluate TBN'stherapeutic parameters in a rat middle cerebral artery occlusion (MCAO) model. Its abilities to cross the blood-brain barrier, scavenge free radicals, and inhibit Ca(2+) influx were also investigated. TBN showed significant activity in both the transient MCAO (t-MCAO) and permanent MCAO (p-MCAO) stroke models in the rat. The therapeutic time window is 8 hr in the t-MCAO model. TBN readily crossed the blood-brain barrier and in vitro had strong activity in neutralizing ·OH, O(-)(2)·, and ONOO(-) and significantly decreased intracellular Ca(2+) concentration. TBN is a promising new treatment forischemic stroke, with multiple mechanisms of action. It blocks Ca(2+) overload and neutralizes ·OH, O(-)(2)·, and ONOO(-).

A series of celastrol analogues were synthesized, and their neuroprotective effect against t-BHP-induced cytotoxicity was investigated in neuronal PC12 cells. Their effects on Hsp70 protein expression were quantified by Western blot analysis. The study found that compound CL12 is more effective than the parent celastrol against t-BHP-induced cytotoxicity. CL12 up-regulates Hsp70 protein expression dose-dependently. These results suggest that CL12 is a potential candidate for the intervention of neurodegenerative diseases.

AL-1 is a novel andrographolide derivative synthesized by conjugating andrographolide and alpha lipoic acid. AL-1 has been found to increase insulin secretion, decrease blood glucose level and protect β-cell mass and function in alloxan-induced diabetic mouse model. However, the protective mechanism of AL-1 on high glucose-induced pancreatic β-cell injury is still not clear. In the present study, we found that AL-1 reduced reactive oxygen species (ROS) and nitric oxide (NO) generation induced by high glucose in RIN-m cells, and which elevated the activities of superoxide dismutase (SOD) and catalase (CAT). In addition, AL-1 increased the expression of NF-E2-related factor 2 (Nrf2), thioredoxin-1 (Trx-1) and heme oxygenase-1 (HO- 1) proteins in RIN-m cells. These results suggest that AL-1 prevented RIN-m cells from high glucose-induced oxidative damage via upregulation of Nrf2 signaling pathway.

Edaravone (EDA) is clinically used for treatment of acute ischemic stroke in Japan and China due to its potent free radical-scavenging effect. However, it has yet to be determined whether EDA can attenuate iodoacetic acid- (IAA-) induced neuronal deathin vitro. In the present study, we investigated the effect of EDA on damage of IAA-induced primary cerebellar granule neurons (CGNs) and its possible underlying mechanisms. We found that EDA attenuated IAA-induced cell injury in CGNs. Moreover, EDA significantly reduced intracellular reactive oxidative stress production, loss of mitochondrial membrane potential, and caspase 3 activity induced by IAA. Taken together, EDA protected CGNs against IAA-induced neuronal damage, which may be attributed to its antiapoptotic and antioxidative activities.

Andrographolide is the most active constituent of the medicinal plant Andrographis paniculata. Previously, we synthesized a novel andrographolide derivative AL-1, conjugating andrographolide with lipoic acid. Although the antioxidative and/or anti-inflammatory activity of AL-1 contributes to its cytoprotective effects, whether AL-1 can improve insulin resistance and the mechanisms responsible for its action have not been elucidated.We investigated the anti-hyperlipidaemic and anti-hyperglycaemic effects of AL-1 in a high-fat diet/streptozocin-induced animal diabetic model. In addition, we investigated the effect of AL-1 on the NF-κB signalling pathway in rat islet derived insulinoma cells (RIN-m cells) with a focus on the link between reactive oxygen species-associated inflammation and insulin resistance.AL-1, at doses of 40 and 80 mg · kg(-1), had a significant hypoglycaemic effect; it significantly reduced the level of cholesterol and increased HDL. AL-1 also reduced the homeostasis model assessment of insulin resistance and enhanced insulin sensitivity. In addition, AL-1 improved the morphology of pancreatic islets and their function. Furthermore, AL-1 suppressed high glucose-induced phosphorylation of p65 and IκBα in RIN-m cells.AL-1 has a hypoglycaemic effect and improves insulin resistance in type 2 diabetic rats. It protected islet from high glucose-induced oxidative damage by down-regulating the NF-κB signalling pathway. Further investigations of AL-1 as a promising new agent for treatment and/or prevention of diabetes are warranted.

Pancreatic β-cell dysfunction and death are important feature of diabetes mellitus. Beta-cell protection has demonstrated clinical benefits in the treatment of this disease. In the present study, andrographolide derivatives with nitric oxide (NO)-releasing capability were synthesized and their protective effects against tert-butyl hydroperoxide (t-BHP) induced cell damage were investigated in RIN-m cells. Compound 6b was found to release a moderate amount of NO and was more potent than its natural parent andrographolide in inhibiting cell apoptosis. These findings suggested that andrographolide derivatives with NO-releasing capacity may be a potential therapy for diabetes.

A series of (R)-3,4-dihydroxyphenyllactic acid Danshensu (DSS) derivatives were synthesized, and their cardioprotective effects were evaluated in vitro and in vivo. Among the new derivatives, compound 14 showed significant protective effects in cultured myocardial cells and in the rat model of myocardial ischemia. The therapeutic efficacy of compound 14 was significantly higher than that of its parent compound DSS, and amlodipine, a first-line treatment for angina pain. Compound 14 potently scavenged free radicals, significantly decreased the levels of LDH and MDA, and inhibited the leakage of CK in animal model of ischemia. We had previously found that compound 14 activated PI3K/Akt/GSK-3β and Nrf2//Keap1/heme oxygenase-1 (HO-1) signaling pathways in H9c2 cells. These results suggest that compound 14 has a unique mechanism of action, that is, multifunctional. Compound 14 may be a new potential therapy for ischemic heart diseases.

We have synthesized new 10-hydroxycamptothecin (HCPT) analogs and evaluated their anticancer activity in cell culture and in experimental animal tumor model. Although the new analogs were less potent against L1210 leukemia cells in vitro, some of them were more efficacious against L1210 leukemia in vivo compared to the parent HCPT.

We have designed and synthesized a series of novel apocynin analogues, and evaluated their biological activity. Compound 10 , an apocynin dimer analogue, compound 12 , the lipoic acid (LA) and apocynin conjugate, were the most potent in protecting cells from lipopolysaccharide (LPS)-induced cytotoxicity, had significant activity scavenging ROS induced by LPS, and greatly decreased LPS-induced P67 phox protein expression. SAR analysis suggests that modification of apocynin can increase its activity. Our results demonstrate that arming apocynin with a powerful antioxidant such as lipoic acid is a valid strategy to design new apocynin analogues with enhanced biological activity.