Agomelatine Prevents Amyloid Plaque Deposition, Tau Phosphorylation, and Neuroinflammation in APP/PS1 Mice.

Agomelatine, an agonist of melatonergic MT1 and MT2 receptors and a selective 5-hydroxytryptamine 2C receptor antagonist, is widely applied in treating depression and insomnia symptoms in several neurogenerative diseases. However, the neuroprotective effect of agomelatine in Alzheimer's disease (AD) is less known. In this study, a total of 30 mice were randomly divided into three groups, namely, wild type (WT), APP/PS1, and agomelatine (50 mg/kg). After 30 days, the Morris water maze was performed to test the cognitive ability of mice. Then, all mice were sacrificed, and the hippocampus tissues were collected for ELISA, Western blot, and immunofluorescence analysis. In this study, we found that agomelatine attenuated spatial memory deficit, amyloid-ß (Aß) deposition, tau phosphorylation, and neuroinflammation in the hippocampus of APP/PS1 mice. Further study demonstrated that agomelatine treatment upregulated the protein expression of DHCR24 and downregulated P-Akt, P-mTOR, p-p70s6k, Hes1, and Notch1 expression. In summary, our results identified that agomelatine could improve cognitive impairment and ameliorate AD-like pathology in APP/PS1 mice via activating DHCR24 signaling and inhibiting Akt/mTOR and Hes1/Notch1 signaling pathway. Agomelatine may become a promising drug candidate in the therapy of AD.

Melatonin receptor stimulation by agomelatine prevents Aß-induced tau phosphorylation and oxidative damage in PC12 cells.

PURPOSE: As a novel antidepressant drug, agomelatine has good therapeutic effect on the mood disorder and insomnia in Alzheimer's disease (AD). Recent studies have shown the neuroprotective function of agomelatine, including anti-oxidative and anti-apoptosis effect. However, it remains unclear whether agomelatine exerts neuroprotection in AD. Thus, the neuroprotective effect of agomelatine against amyloid beta 25-35 (Aß25-35)-induced toxicity in PC12 cells was evaluated in this study. METHODS: The concentration of malondialdehyde (MDA), LDH, and ROS was investigated to evaluate oxidative damage. The expression of P-tau, tau, PTEN, P-Akt, Akt, P-GSK3ß, and GSK3ß proteins was assessed by Western blotting. Our results demonstrated that Aß25-35 significantly increased the content of MDA, LDH, and ROS. Meanwhile, Aß25-35 upregulated the expression of P-tau and PTEN as well as downregulated P-Akt and P-GSK3ß expression. These effects could be blocked by agomelatine pretreatment. Furthermore, luzindole, the melatonin receptor (MT) antagonist, could reverse the neuroprotective effect of agomelatine. CONCLUSION: The results demonstrated that antidepressant agomelatine might prevent the tau protein phosphorylation and oxidative damage induced by Aß25-35 in PC12 cells by activating MT-PTEN/Akt/GSK3ß signaling. This study provided a novel therapeutic target for AD in the future.

The Synergy of Aging and LPS Exposure in a Mouse Model of Parkinson's Disease.

Aging is an inevitable physiological challenge occurring in organisms over time, and is also the most important risk factor of neurodegenerative diseases. In this study, we observed cellular and molecular changes of different age mice and LPS-induced Parkinson disease (PD) model. The results showed that behavioral performance and dopaminergic (DA) neurons were declined, accompanied by increased expression of pro-inflammatory factors (TLR2, p-NF-kB-p65, IL-1ß and TNF-α), as well as pro-oxidative stress factor gp91phox in aged mice compared with young mice. Aging exaggerated inflammatory M1 microglia, and destroyed the balance between oxidation and anti-oxidation. The intranasal LPS instillation induced PD model in both young and aged mice. The poor behavioral performance and the loss of DA neurons as well as TLR2, p-NF-kB-p65, IL-1ß, TNF-α, iNOS and gp91phox were further aggravated in LPS-aged mice. Interestingly, the expression of Nrf2 and HO-1 was up-regulated by LPS only in young LPS-PD mice, but not in aged mice. The results indicate that the synergy of aging process and LPS exposure may prominently aggravate the DA neurons loss caused by more serious neuroinflammation and oxidative stress in the brain.

Aging modulates microglia phenotypes in neuroinflammation of MPTP-PD mice.

As the crucial etiological factor, aging-related microglia activation promotes the development of Parkinson's disease (PD). However, the molecular and functional changes of aged-microglia and their contribution to neurodegeneration in PD are only partially understood, which was investigated in our study. Female C57BL/6 mice were randomly divided into four groups, included young-control group, young-MPTP group, aged-control group and aged-MPTP group. Pole test and adhesive removal test were firstly performed. ELISA assay was used to detect the content of interleukin-1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α) in brain tissue. Then we tested the expression of tyrosine hydroxylase (TH), p-nuclear transcription factor (NF-κB), toll-like receptor2 (TLR2), arginase-1 (arg-1), inducible nitric oxide synthase (iNOS) by western blot and immunofluorescence analysis. Our results showed that aging promoted M1 microglia activation and inhibited M2 microglia activation in SN in MPTP-PD model, accompanied by the elevation of proinflammatory cytokine (TNF-α and IL-1ß). Consequently, aging significantly aggravated motor dysfunction and dopaminergic neuron loss in SN. Besides, compared with young-MPTP group, the protein expression of TLR2 and p-NF-κB-p65 increased obviously in aged-MPTP group. The results revealed that aging aggravated inflammatory response by modulated microglia phenotypes transition in SN in PD, and contributed to further understand the pathogenesis of PD.

The inhibition of CB1 receptor accelerates the onset and development of EAE possibly by regulating microglia/macrophages polarization.

Cannabinoid 1 receptor (CB1R) regulates the neuro-inflammatory and neurodegenerative damages of experimental autoimmune encephalomyelitis (EAE) and of multiple sclerosis (MS). The mechanism by which CB1R inhibition exerts inflammatory effects is still unclear. Here, we explored the cellular and molecular mechanisms of CB1R in the treatment of EAE by using a specific and selective CB1R antagonist SR141716A. Our study demonstrated that SR141716A accelerated the clinical onset and development of EAE, accompanied by body weight loss. SR141716A significantly up-regulated the expression of toll like receptor-4 (TLR-4) and nuclear factor-kappaB/p65 (NF-κB/p65) on microglia/macrophages of EAE mice as well as levels of inflammatory factors (TNF-α, IL-1ß, IL-6) and chemokines (MCP-1, CX3CL1), accompanied by the shifts of cytokines from Th2 (IL-4, IL-10) to Th1 (IFN-γ)/Th17 (IL-17) in the spinal cords of EAE mice. Similar changes happened on splenic mononuclear cells (MNCs) except chemokine CX3CL1. Consistently, SR141716A promoted BV-2 microglia to release inflammatory factors (TNF-α, IL-1ß, IL-6) while inhibited the production of IL-10 and chemokines (MCP-1, CX3CL1). Furthermore, when splenic CD4+ T cells co-cultured with SR141716A-administered BV-2 microglia, the levels of IL-4 and IL-10 were decreased while production of IL-17 and IFN-γ increased significantly. Our research indicated that inhibition of CB1R induced M1 phenotype-Th17 axis changed of microglia/macrophages through TLR-4 and NF-κB/p65 which accelerated the onset and development of EAE. Therefore, CB1R may be a promising target for the treatment of MS/EAE, but its complexity remains to be carefully considered and studied in further clinical application.

Rho kinase II interference by small hairpin RNA ameliorates 1­methyl­4­phenyl­1,2,3,6­tetrahydropyridine­induced parkinsonism in mice.

Novel therapeutic targets are required for the treatment of Parkinson's disease (PD). Previous studies suggest that the Rho/Rho­associated, coiled­coil­containing protein kinases (ROCKs) signaling pathway may be a promising therapeutic target in PD. To elucidate the importance of ROCKII in the pathogenesis of dopaminergic (DA) neuron loss and to investigate the efficacy of ROCK inhibitors in PD, ROCKII expression in the substantia nigra (SN) of mice was silenced through the injection of a lentivirus­based small hairpin RNA system. Empty lentivirus vectors served as controls. Mice were subsequently challenged with 1­methyl­4­phenyl­1,2,3,6­tetrahydropyridine (MPTP). The expression levels and activity of ROCKII were elevated in tyrosine hydroxylase­positive neurons and in cluster of differentiation (CD) 11b­positive microglia within the SN of MPTP­treated mice, which was accompanied by an increased level of expression of inducible nitric oxide synthase (iNOS) and activation of the Toll­like receptor (TLR)2/nuclear factor (NF)­κB signaling pathway in M1 microglia. ROCKII interference (RI) significantly improved movement disorder and attenuated DA neuron loss induced by MPTP. In addition, RI inhibited the activation of M1 microglia in the SN, exhibiting reduced activity of the TLR2/NF­κB signaling pathway and decreased expression levels of iNOS and inflammatory factors, including interleukin (IL)­1ß and IL­6. The results of the present study verify that ROCKII participates in the loss of DA neurons induced by MPTP and suggest that ROCKII inhibition may be a promising therapeutic target for PD.

Changes of synapses in experimental autoimmune encephalomyelitis by using Fasudil.

The ROCK signaling pathway is involved in numerous fundamental cellular functions such as cell migration, apoptosis, inflammatory responses, and neurite outgrowth. Previous studies demonstrate that Fasudil exhibited therapeutic potential of experimental autoimmune encephalomyelitis (EAE) possibly through immune-modulation and anti-inflammation. In this study, we observed the effect of Fasudil on synaptic protection of EAE mice. Fasudil ameliorated the clinical severity of EAE and inhibited Rho kinase (ROCK), especially ROCK II, in brain and spinal cord of EAE mice. Protein extracts from spinal cord of Fasudil-treated EAE mice promoted the formation of neurite outgrowth when co-cultured with primary neurons, indicating that peripheral administration of Fasudil can enter the central nervous system (CNS) and exhibited its biological effect on the formation of neurite outgrowth. Synapse-related molecule synaptophysin was enhanced, and CRMP-2, AMPA receptor, and GSK-3ß were declined in spinal cord of Fasudil-treated mice. Neurotrophic factor BDNF and GDNF as well as immunomodulatory cytokine IL-10 in spinal cord were elevated in Fasudil-treated mice, while inflammatory cytokine IL-17, IL-1ß, IL-6, and TNF-α were obviously inhibited, accompanied by the decrease of inflammatory M1 iNOS and the increase of anti-inflammatory M2 Arg-1, providing a microenvironment that contributes to synaptic protection. Our results indicate that Fasudil treatment protected against synaptic damage and promoted synaptic formation, which may be related with increased neurotrophic factors as well as decreased inflammatory microenvironment in the CNS of EAE mice.

Safflower Yellow regulates microglial polarization and inhibits inflammatory response in LPS-stimulated Bv2 cells.

Activated microglia, especially polarized M1 cells, produce pro-inflammatory cytokines and free radicals, thereby contributing directly to neuroinflammation and various brain disorders. Given that excessive or chronic neuroinflammation within the central nervous system (CNS) exacerbates neuronal damage, molecules that modulate neuroinflammation are candidates as neuroprotective agents. In this study, we provide evidence that Safflor yellow (SY), the main active component in the traditional Chinese medicine safflower, modulates inflammatory responses by acting directly on BV2 microglia. LPS stimulated BV2 cells to upregulate expression of TLR4-Myd88 and MAPK-NF-κB signaling pathways and to release IL-1ß, IL-6, TNF-α, and COX-2. However, SY treatment inhibited expression of TLR4-Myd88 and p-38/p-JNK-NF-κB, downregulated expression of iNOS, CD16/32, and IL-12, and upregulated CD206 and IL-10. In conclusion, our results demonstrate that SY exerts an anti-inflammatory effect on BV2 microglia, possibly through TLR-4/p-38/p-JNK/NF-κB signaling pathways and the conversion of microglia from inflammatory M1 to an anti-inflammatory M2 phenotype.

Role of Rho Kinase and Fasudil on Synaptic Plasticity in Multiple Sclerosis.

In addition to myelin loss and oligodendrocyte injury, axonal damage is a major cause of irreversible neurological disability in multiple sclerosis (MS). A series of studies have demonstrated that Rho kinase (ROCK) is involved in synaptic plasticity of neurons. Here, we found that ROCK activity in MS serum was elevated compared with serum from healthy controls. In experimental autoimmune encephalomyelitis (EAE), ROCK activity was also increased in serum, spleen, brain and spinal cord. Neuron injury with scratch and TNF-α stimulation induced the up-regulation of ROCK activity. When serum of MS patients was co-cultured with mouse cortical neurons in vitro, MS serum caused neurite shortening and reduction of cell viability, while the addition of Fasudil partially restored synaptic morphology of neurons, revealing that MS sera inhibited neurite outgrowth and synapse formation. The expression of synaptophysin was decreased in MS serum-neurons, and elevated in the presence of Fasudil. In contrast, the expression of phosphorylated collapsin response mediator protein-2 (CRMP-2) was elevated in MS serum-neurons and decreased in the presence of Fasudil. However, the addition of anti-ROCK I/II mixed antibodies in MS serum partially declined ROCK activity, but did not improve neurite outgrowth of neurons, revealing that Fasudil should prevent synaptic damage possibly through inhibiting intracellular ROCK activation mediated with MS serum. Our results indicate that axonal loss in MS may be related to increased ROCK activity. Fasudil could promote synaptogenesis and thus may contribute to preventing irreversible neurological disability associated with MS.

Multitarget intervention of Fasudil in the neuroprotection of dopaminergic neurons in MPTP-mouse model of Parkinson's disease.

Recent studies have demonstrated that activation of the Rho-associated kinase (ROCK) pathway participates in the dopaminergic neuron degeneration and possibly in Parkinson's disease (PD). In the current study, we tried to observe the therapeutic potential of ROCK inhibitor Fasudil against dopaminergic neuron injury in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mouse model of PD, and explore possible molecular mechanisms by enzyme-linked immunosorbent assay (ELISA), western blot and immunofluorescent assays. The results showed that MPTP-PD mice presented motor deficits, dopaminergic neuron loss, activation of inflammatory response and oxidative stress as well as ROCK and glycogen synthase kinase 3ß (GSK-3ß) signaling pathways. The administration of Fasudil exhibited neuroprotective effects against the dopaminergic neurons and improved the motor function recovery in the MPTP-PD mice, accompanied by the suppression of inflammatory responses (IL-1ß, TNF-α, NF-κB-p65 and TLR-2), and oxidative stress (iNOS and gp91Phox), which might be associated with the inhibition of ROCK and GSK-3ß activity. Simultaneously, the administration of Fasudil resulted in the shift from inflammatory M1 to anti-inflammatory/neurorepair M2 microglia. Additionally, Fasudil intervention enhanced the expression of anti-oxidative factors such as NF-E2-related factor 2 (Nrf2), Hmox as well as neurotrophic factor including GDNF. Our observations defined the neuroprotective effects of Fasudil in MPTP-PD mice, and we found a series of novel effector molecules and pathways for explaining the neuroprotective effects against dopaminergic neurons. However, a lot of investigations are warranted to further elucidate the crosstalk among Fasudil, oxidative stress, inflammatory response, GDNF and ROCK/NF-kB/Nrf2 pathways in the therapeutic potential of PD.

The therapeutic potential of Rho kinase inhibitor fasudil derivative FaD-1 in experimental autoimmune encephalomyelitis.

Although therapeutic potential of fasudil in EAE is promising, action mechanism and clinical limitations are still not fully understood and resolved. In this study, we observed the therapeutic potential of a novel Rho kinase (ROCK) inhibitor FaD-1, a fasudil derivative, and explored possible mechanism in MOG35-55-induced EAE. Experimental autoimmune encephalomyelitis (EAE) was induced by myelin oligodendrocyte glycoprotein (MOG35-55) immunization. The pathology of spinal cord was measured by immunohistochemistry and neurological impairment was evaluated using clinical scores. FaD-1, as a novel ROCK inhibitor, inhibited the expression of ROCK II that is mainly expressed in the CNS. We show here that FaD-1 ameliorates the neurological defects and the severity of MOG-induced EAE in mice, accompanied by the protection of demyelination and the inhibition of neuroinflammation in spinal cord of EAE. In addition, FaD-1 dampened TLR2 and TLR4 signaling as well as Th1 (IFN-γ) and Th17 (IL-17) responses in spinal cord of EAE. FaD-1 also prevented the expression of iNOS and production of inflammatory cytokine IL-1ß, IL-6, and TNF-α which are specific markers for M1 inflammatory microglia/macrophages. This study highlights the therapeutic potential of FaD-1 as a ROCK inhibitor for the treatment of human autoimmune diseases with both inflammatory and autoimmune components.

Fasudil regulates T cell responses through polarization of BV-2 cells in mice experimental autoimmune encephalomyelitis.

AIM: Fasudil, a selective Rho kinase (ROCK) inhibitor, has been shown to alleviate the severity of experimental autoimmune encephalomyelitis (EAE) via attenuating demyelination and neuroinflammation. The aim of this study was to investigate the effects of fasudil on interactions between macrophages/microglia and T cells in a mice EAE model. METHODS: Mouse BV-2 microglia were treated with IFN-γ and fasudil. Cell viability was detected with MTT assay. BV-2 microglia polarization was analyzed using flow cytometry. Cytokines and other proteins were detected with ELISA and Western blotting, respectively. Mice were immunized with MOG35-55 to induce EAE, and then treated with fasudil (40 mg/kg, ip) every other day from d 3 to d 27 pi. Encephalomyelitic T cells were prepared from the spleen of mice immunized with MOG35-55 on d 9 pi. RESULTS: Treatment of mouse BV-2 microglia with fasudil (15 µg/mL) induced significant phenotype polarization and functional plasticity, shifting M1 to M2 polarization. When co-cultured with the encephalomyelitic T cells, fasudil-treated BV-2 microglia significantly inhibited the proliferation of antigen-reactive T cells, and down-regulated IL-17-expressing CD4(+) T cells and IL-17 production. Furthermore, fasudil-treated BV-2 microglia significantly up-regulated CD4(+)CD25(high) and CD4(+)IL-10(+) regulatory T cells (Tregs) and IL-10 production, suggesting that the encephalomyelitic T cells had converted to Tregs. In EAE mice, fasudil administration significantly decreased both CD11b(+)iNOS(+) and CD11b(+)TNF-α(+) M1 microglia, and increased CD11b(+)IL-10(+) M2 microglia. CONCLUSION: Fasudil polarizes BV-2 microglia into M2 cells, which convert the encephalomyelitic T cells into Tregs in the mice EAE model.

[Surgical strategy for the treatment of thoracolumbar metastatic tumor and its clinical outcomes].

OBJECTIVE: To evaluate surgical strategy and clinical outcomes for the treatment of thoracolumbar metastatic tumor. METHODS: From January 2009 to December 2010,42 patients with thoracolumbar metastatic tumor were treated surgically. Among the patients, 30 patients were male, and 12 patients were female, ranging in age from 28 to 76 years old, with an average age of 56.8 years old. Twenty-five patients had metastatic tumor in thoracic vertebraes, and 17 patients had metastatic tumor in lumbar vertebraes. Thirty-four patients had metastatic tumor in 1 segment, 6 patients had metastatic tumor in 2 segments and 2 patients had metastatic tumor in 3 segments. Two patients had no symptoms and 40 patients had back or leg pain. Eighteen patients had neurologic deficits, and 5 patients had injuries of A degree, 3 patients had injuries of B degree, 4 patients had injuries of C degree, 6 patients had injuries of D degree according to ASIA grading system. The operation goal was made according to Tomita evaluation. The surgical procedures included pallative decompression, tumor curettage and total vertebrectomy, which were decided based on Tomita classification. The pain, spinal cord function,part control of tumor,survival rate and conditions of internal fixation were evaluated at 1 week, 3 months, 6 months, 1 year and 2 years after operation. RESULTS: One patient died in the operation. Pain relief was obtained in 38 patients after operation. Among 18 patients suffering from spinal cord compromise, 17 patients improved 1 to 4 grades after surgery according to the ASIA grading system. All the patients were followed up and the duration ranged from 24 to 48 months, with a mean time of 34.2 months. Five patients got recurrence. The postoperative survival rates at 3 months, 6 months, 1 year and 2 years were 95.2%, 85.7%, 58.2%, 37.6% respectively. CONCLUSION: According to Tomita system, the different surgical treatments can be selected for patients with spinal metastatic tumors, which can relieve pain, improve the neurological status and spine stabilization, maintain local control, improve quality of life.

Factors as predictors for thoracic and thoracolumbar/lumbar structural curves in adolescent idiopathic scoliosis.

BACKGROUND: Recent studies have demonstrated that the Lenke system is relatively efficient and consistent in classifying scoliosis curves. Basically, fusion should include the main curve and the structural minor curve. The criteria for defining the structural minor curve were established to help guide these decision-making process. The present study was designed to investigate predictors of the structural curve, and see whether it was possible to prevent the formation of the structural curve by interfering with influencing factors to decrease the fusion level. METHODS: Age, gender, Cobb angle, Perdriolle rotation, Risser sign and the number of vertebrae included in the curve, brace treatment, and curve location were recorded in 145 idiopathic scoliosis patients from July 2001 to January 2007. The patients were divided into two groups: structural and non-structural groups. Demographics and baseline characteristics were compared between the two groups as an initial screen. Logistic regression was used to analyze factors affecting the minor curve to become the structural curve. RESULTS: Compared with the non-structural group, the structural group had a higher Cobb angle ((51.34 ± 13.61)° vs. (34.20 ± 7.21)°, P < 0.001), bending angle ((33.94 ± 9.92)° vs. (8.46 ± 5.56)°, P < 0.001) and curve rotation ((23.25 ± 12.86)° vs. (14.21 ± 8.55)°, P < 0.001), and lower flexibility ((33.48 ± 12.53)% vs. (75.50 ± 15.52)%, P < 0.001). There was no significant difference in other parameters between the two groups. The results of the Logistic regression analysis showed that the Cobb angle (OR: 9.921, P < 0.001) and curve location (OR: 4.119, P = 0.016) were significant predictors of structural curve in adolescent idiopathic scoliosis. Every 10° change of Cobb angle increased the possibility of turning the minor curve into the structural curve by 10-fold. And thoracic curve showed, on the average, the possibility of becoming the structural curve about 4-fold more often than did the thoracolumbar/lumbar curve. CONCLUSIONS: Curve severity and curve location affect the minor curve's structural features in adolescent idiopathic scoliosis.

Factors affecting curve flexibility in skeletally immature and mature idiopathic scoliosis.

BACKGROUND: The determination of factors affecting curve flexibility is important in idiopathic scoliosis patients with regard to the Risser sign. The objective of this retrospective study was to identify factors affecting curve flexibility in patients with skeletally immature and mature idiopathic scoliosis. METHODS: The records of all patients with idiopathic scoliosis who received surgical treatment from July 2001 to August 2008 at our hospital were screened. The Risser sign was used to separate the patients into a skeletally mature group (Risser grade = 5) and skeletally immature group (Risser grade < 5). Data recorded and compared were flexibility (%), bending angle (°), apical vertebral rotation (°), Cobb angle (°), curve location, prior use of brace treatment, and number of vertebrae in the curve. RESULTS: The study cohort consisted of 217 patients (34 males, 183 females) in the Risser grade < 5 group and 124 (21 males, 103 females) in the Risser grade = 5 group. Multiple linear regression analysis revealed that the Cobb angle and the curve location significantly affected curve flexibility in the Risser grade < 5 group, whereas in the Risser grade = 5 group, Cobb angle and age significantly affected flexibility. CONCLUSIONS: Cobb angle and curve location influence main curve flexibility in skeletally immature adolescent idiopathic scoliosis, and Cobb angle and age influence curve flexibility in skeletally mature adult scoliosis. Measurement of these values may aid in the evaluation of treatment options and preoperative planning.