Grape Seed Extract Attenuates Demyelination in Experimental Autoimmune Encephalomyelitis Mice by Inhibiting Inflammatory Response of Immune Cells.

OBJECTIVE: To examine the anti-inflammatory effect of grape seed extract (GSE) in animal and cellular models and explore its mechanism of action. METHODS: This study determined the inhibitory effect of GSE on macrophage inflammation and Th1 and Th17 polarization in vitro. Based on the in vitro results, the effects and mechanisms of GSE on multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE) mice model were further explored. The C57BL/6 mice were intragastrically administered with 50 mg/kg of GSE once a day from the 3rd day to the 27th day after immunization. The activation of microglia, the polarization of Th1 and Th17 and the inflammatory factors such as tumor necrosis factor- α (TNF- α), interleukin-1 ß (IL-1 ß), IL-6, IL-12, IL-17 and interferon-γ (IFN-γ) secreted by them were detected in vitro and in vivo by flow cytometry, enzyme linked immunosorbent assay (ELISA), immunofluorescence staining and Western blot, respectively. RESULTS: GSE reduced the secretion of TNF-α, IL-1 ß and IL-6 in bone marrow-derived macrophages stimulated by lipopolysaccharide (P<0.01), inhibited the secretion of TNF-α, IL-1 ß, IL-6, IL-12, IL-17 and IFN-γ in spleen cells of EAE mice immunized for 9 days (P<0.05 or P<0.01), and reduced the differentiation of Th1 and Th17 mediated by CD3 and CD28 factors (P<0.01). GSE significantly improved the clinical symptoms of EAE mice, and inhibited spinal cord demyelination and inflammatory cell infiltration. Peripherally, GSE downregulated the expression of toll-like-receptor 4 (TLR4) and Rho-associated kinase (ROCKII, P<0.05 or P<0.01), and inhibited the secretion of inflammatory factors (P<0.01 or P<0.05). In the central nervous system, GSE inhibited the infiltration of CD45+CD11b+ and CD45+CD4+ cells, and weakened the differentiation of Th1 and Th17 (P<0.05). Moreover, it reduced the secretion of inflammatory factors (P<0.01), and prevented the activation of microglia (P<0.05). CONCLUSION: GSE had a beneficial effect on the pathogenesis and progression of EAE by inhibiting inflammatory response as a potential drug and strategy for the treatment of MS.

Neuroprotective effect of hyperoside in MPP+/MPTP -induced dopaminergic neurodegeneration.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the pathological loss of nigrostriatal dopaminergic neurons, which causes an insufficient release of dopamine (DA) and then induces motor and nonmotor symptoms. Hyperoside (HYP) is a lignan component with anti-inflammatory, antioxidant, and neuroprotective effects. In this study, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active neurotoxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) were used to induce dopaminergic neurodegeneration. The results showed that HYP (100 µg/mL) reduced MPTP-mediated cytotoxicity of SH-SY5Y cells in vitro, and HYP [25 mg/(kg d)] alleviated MPTP-induced motor symptoms in vivo. HYP treatment reduced the contents of nitric oxide (NO), H2O2, and malondialdehyde (MDA), as well as the mitochondrial damage of dopaminergic neurons, both in vitro and in vivo. Meanwhile, HYP treatment elevated the levels of neurotrophic factors such as glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, and recombinant cerebral dopamine neurotrophic factor in vivo, but not in vitro. Finally, Akt signaling was activated after the administration of HYP in MPP+/MPTP-induced dopaminergic neurodegeneration. However, the blockage of the Akt pathway with Akt inhibitor did not abolish the neuroprotective effect of HYP on DA neurons. These results showed that HYP protected the dopaminergic neurons from the MPP+- and MPTP-induced injuries, which did not rely on the Akt pathway.

[Acupuncture ameliorates neurological function by suppressing microglia polarization and inflammatory response after cerebral ischemia in rats].

OBJECTIVE: To observe the effect of acupuncture on microglia polarization and inflammatory reaction in rats with cerebral ischemia-reperfusion injury (CIRI), so as to explore its mechanisms underlying improvement of CIRI. METHODS: Thirty male SD rats were randomly divided into sham operation, model, and acupuncture groups, with 10 rats in each group. The CIRI model was established by occlusion of the middle cerebral artery (MCAO) for 1 h, followed by reperfusion. After modeling, rats in the acupuncture group received manual acupuncture stimulation of "Dazhui" (GV14), "Baihui"(GV20), "Shuigou" (GV26), bilateral "Zusanli" (ST36) and "Fengchi" (GB20) by twirling the needles rapidly for 10 s/acupoint every 10 min, with the needles retained for 20 min. The treatment was conducted once daily for successive 7 days. The neurological function was evaluated according to Longa's method. The state of CIRI was observed after Nissl staining, and the expression levels of Iba-1, iNOS, Arg1, BDNF, GDNF and NeuN in the ischemic cortex tissue were detected by immunofluorescence staining. The contents of TNF-α, IL-6 and IL-10 in the ischemic tissue were assayed by ELISA. The protein expression levels of BDNF, GDNF, TLR4, MyD88 and NF-κB in the ischemic tissues were detected by Western blot. RESULTS: The neurological deficit score on the 24 h and 7th day was considerably higher in the model group than in the sham operation group (P<0.01), and evidently lower on the 7th day in the acupuncture group than in the model group (P<0.01). The number of NeuN positive cells，the area of immunofluorescence dual labelling of Arg1, BDNF and GDNF positive staining, IL-10 content, BDNF and GDNF protein expressions were significantly decreased (P<0.01), and the immunofluorescence dual labelling area of Iba-1 and iNOS, TNF-α and IL-6 contents, the pretein expression levels of TLR4, MyD88 and NF-κB considerably increased (P<0.01) in the model group relevant to the sham operation group. In contrast to the model group, the acupuncture group had a significant increase in the number of NeuN positive cells, the immunofluorescence dual labelling area of Arg1, BDNF and GDNF positive staining, IL-10 content, and BDNF and GDNF protein expressions (P<0.05, P<0.01), and an evident decrease in Iba-1 and iNOS positive staining, contents of TNF-α and IL-6, and the protein expression levels of TLR4, MyD88 and NF-κB (P<0.01, P<0.05). Nissl staining showed a marked reduction in the number of neurons, the nucleus pyknosis and nissl bodies and loose arrangement of the neuronal cells in the model group, which was relatively milder in the acupuncture group. CONCLUSION: Acupuncture intervention can improve neurological function in CIRI rats, which may be related to its effects in regulating the polarization of microglia, reducing inflammatory reaction and increasing the secretion of neurotrophic factors in the brain, inhibiting TLR4/MyD88/NF-κB signaling pathway.

Wuzi Yanzong pill attenuates MPTP-induced Parkinson's Disease via PI3K/Akt signaling pathway.

Wuzi Yanzong Pill (WYP) was found to play a protective role on nerve cells and neurological diseases, however the molecular mechanism is unclear. To understand the molecular mechanisms that underly the neuroprotective effect of WYP on dopaminergic neurons in Parkinson's disease (PD). PD mouse model was induced by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Gait and hanging tests were used to assess motor behavioral function. Immunofluorescence assay was used to determine TH-positive neurons in substantia nigra (SN). Apoptosis, dopamine and neurotrophic factors as well as expression of PI3K/Akt pathway were detected by TUNEL staining, ELISA and western blotting, respectively. First, it was observed that WYP intervention improved abnormal motor function in MPTP-induced PD model, alleviated the loss of TH+ neurons in SN, and increased dopamine content in brain, revealing a potential protective effect. Second, network pharmacology was used to analyze the possible targets and pathways of WYP action in the treatment of PD. A total of 126 active components related to PD were screened in WYP, and the related core targets included ALB, GAPDH, Akt1, TP53, IL6 and TNF. Particularly, the effect of WYP on PD may be medicate through PI3K/Akt signaling pathway and apoptotic regulation. The WYP treated PD mice had higher expression of p-PI3K, p-Akt and Bcl-2 but lower expression of Bax and cleaved caspase-3 than the non-WYP treated PD mice. Secretion of brain-derived neurotrophic factor (BDNF) and cerebral dopamine neurotrophic factor (CDNF) were also increased in the treated mice. WYP may inhibit apoptosis and increase the secretion of neurotrophic factor via activating PI3K/ Akt signaling pathway, thus protecting the loss of dopamine neurons in MPTP-induced PD mice.

The therapeutic potential of bilobalide on experimental autoimmune encephalomyelitis (EAE) mice.

Inflammatory demyelination in the central nervous system (CNS) is a hallmark of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Besides MS disease-modifying therapy, targeting myelin sheath protection/regeneration is currently a hot spot in the treatment of MS. Here, we attempt to explore the therapeutic potential of Bilobalide (BB) for the myelin protection/regeneration in EAE model. The results showed that BB treatment effectively prevented worsening and demyelination of EAE, accompanied by the inhibition of neuroinflammation that should be closely related to T cell tolerance and M2 macrophages/microglia polarization. BB treatment substantially inhibited the infiltration of T cells and macrophages, thereby alleviating the enlargement of neuroinflammation and the apoptosis of oligodendrocytes in CNS. The accurate mechanism of BB action and the feasibility of clinical application in the prevention and treatment of demyelination remain to be further explored.

Hydroxyfasudil alleviates demyelination through the inhibition of MOG antibody and microglia activation in cuprizone mouse model.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system characterized by oligodendrocyte loss and progressive neurodegeneration. The cuprizone (CPZ)-induced demyelination is widely used to investigate the demyelination/remyelination. Here, we explored the therapeutic effects of Hydroxyfasudil (HF), an active metabolite of Fasudil, in CPZ model. HF improved behavioral abnormality and reduced myelin damage in the corpus callosum. Splenic atrophy and myelin oligodendrocyte glycoprotein (MOG) antibody were observed in CPZ model, which were partially restored and obviously inhibited by HF, therefore reducing pathogenic binding of MOG antibody to oligodendrocytes. HF inhibited the percentages of CD4+IL-17+ T cells from splenocytes and infiltration of CD4+ T cells and CD68+ macrophages in the brain. HF also declined microglia-mediated neuroinflammation, and promoted the production of astrocyte-derived brain derived neurotrophic factor (BDNF) and regeneration of NG2+ oligodendrocyte precursor cells. These results provide potent evidence for the therapeutic effects of HF in CPZ-induced demyelination.

Neuroprotection and CD131/GDNF/AKT Pathway of Carbamylated Erythropoietin in Hypoxic Neurons.

Carbamylated erythropoietin (CEPO), an EPO derivative, is attracting widespread interest due to neuroprotective effects without erythropoiesis. However, little is known about molecular mechanisms behind CEPO-mediated neuroprotection. In primary neurons with oxygen-glucose deprivation (OGD) and mice with hypoxia-reoxygenation, the neuroprotection and possible molecular mechanism of CEPO were performed by immunohistochemistry and immunocytochemistry, Western blot, RT-PCR, and ELISA. The comparisons were analyzed by ANOVA followed by unpaired two-tailed Student's t test. Both CEPO and EPO showed the neuroprotective effects in OGD model and hypoxic brain. CEPO did not trigger JAK-2 but activated AKT through glial cell line-derived neurotrophic factor (GDNF). It has been shown that CEPO acts upon a heteroreceptor complex comprising both the EPO receptor and the common ß receptor subunit (ßcR, also known as CD131). The blockage of CD131 reduced CEPO-mediated GDNF production, while GFR receptor blockage and GDNF neutralization inhibited CEPO-induced neurogenesis. Addition of GDNF to cultured neurons increased phosphorylation of AKT. CEPO protects neurons possible through the CD131/GDNF/AKT pathway.

Multitarget Therapeutic Effect of Fasudil in APP/PS1transgenic Mice.

INTRODUCTION: Therapeutic strategies targeting Alzheimer's disease-related molecule ß- amyloid (Aß), Tau protein and ß-site amyloid precursor protein cleaving enzyme (BACE) have been recently explored. However, the treatment effect for single target is not ideal. Based on multiaspect roles of Rho kinase inhibitor Fasudil on neuroprotection, neurorepair and immunomodulation, we observed therapeutic potential of Fasudil and explored possible mechanisms in amyloid precursor protein/ presenilin-1 transgenic (APP/PS1 Tg) mice, an animal model of Alzheimer's disease. METHODS: APP/PS1 Tg mice were treated with Fasudil (25 mg/kg/day) for 2 months by intraperitoneal injection. Mouse behavior tests were recorded every day. The expression of Aß deposition, Tau protein phosphorylation, BACE and postsynaptic density 95 (PSD-95) in hippocampus was assayed. The levels in the brain of Toll-like receptors (TLRs)-nuclear factor kappa B/p65（NF-κB/p65)- myeloid differentiation primary response gene 88 (MyD88) inflammatory cytokine axis were measured. RESULTS: Fasudil treatment ameliorated learning and memory deficits, accompanied by reduced Aß deposition, Tau protein phosphorylation, and BACE expression, as well as increased PSD-95 expression in hippocampus. Fasudil intervention also inhibited TLR-2/4, p-NF-κB/p65, MyD88, interleukin-1beta, interleukin-6 and tumor necrosis factor-α for TLRs-NF-κB-MyD88 inflammatory cytokine axis and the induction of interleukin-10. CONCLUSION: Fasudil exhibited multitarget therapeutic effect in APP/PS1 Tg mice. The study provides preclinical evidence that Fasudil treatment ameliorated memory deficits in APP/PS1 Tg mice, accompanied by the reduction of Aß deposition and Tau protein phosphorylation, the decrease of BACE and the increase of PSD-95, as well as inhibition of TLRs-NF-κB-MyD88 inflammatory cytokine axis. However, these results still need to be repeated and confirmed before clinical application.

Synergistic and Superimposed Effect of Bone Marrow-Derived Mesenchymal Stem Cells Combined with Fasudil in Experimental Autoimmune Encephalomyelitis.

Bone marrow-derived mesenchymal stem cells (MSCs) are the ideal transplanted cells of cellular therapy for promoting neuroprotection and neurorestoration. However, the optimization of transplanted cells and the improvement of microenvironment around implanted cells are still two critical challenges for enhancing therapeutic effect. In the current study, we observed the therapeutic potential of MSCs combined with Fasudil in mouse model of experimental autoimmune encephalomyelitis (EAE) and explored possible mechanisms of action. The results clearly show that combined intervention of MSCs and Fasudil further reduced the severity of EAE compared with MSCs or Fasudil alone, indicating a synergistic and superimposed effect in treating EAE. The addition of Fasudil inhibited MSC-induced inflammatory signaling TLR-4/MyD88 and inflammatory molecule IFN-γ, IL-1ß, and TNF-α but did not convert M1 microglia to M2 phenotype. The delivery of MSCs enhanced the expression of glial cell-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) compared with that of Fasudil. Importantly, combined intervention of MSCs and Fasudil further increased the expression of BDNF and GDNF compared with the delivery of MSCs alone, indicating that combined intervention of MSCs and Fasudil synergistically contributes to the expression of neurotrophic factors which should be related to the expression of increased galactocerebroside (GalC) compared with mice treated with Fasudil and MSCs alone. However, a lot of investigation is warranted to further elucidate the cross talk of MSCs and Fasudil in the therapeutic potential of EAE/multiple sclerosis.

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.

Protective effect of a novel Rho kinase inhibitor WAR-5 in experimental autoimmune encephalomyelitis by modulating inflammatory response and neurotrophic factors.

The Rho-kinase (ROCK) inhibitor Fasudil has proven beneficial in experimental autoimmune encephalomyelitis (EAE). Given the small safety window of Fasudil, we are looking for novel ROCK inhibitors, which have similar or stronger effect on EAE with greater safety. In this study, we report that WAR-5, a Y-27632 derivative, alleviates the clinical symptoms, attenuates myelin damage and reduces CNS inflammatory responses in EAE C57BL/6 mice at an extent similar to Fasudil, while exhibits less vasodilator and adverse reaction in vivo. WAR-5 inhibits ROCK activity, and selectively suppresses the expression of ROCK II in spleen, brain and spinal cord of EAE mice, especially in spinal cord, accompanied by decreased expression of Nogo. WAR-5 also regulates the imbalance of Th1/Th17 T cells and regulatory T cells, inhibits inflammatory microenvironment induced with NF-κB-IL-1ß pathway. Importantly, WAR-5 converts M1 toward M2 microglia/macrophages that are positively correlated with BDNF and NT-3 production. Taken together, WAR-5 exhibits therapeutic potential in EAE by more selectively inhibits ROCK II, with a greater safety than Fasudil, and is worthy of further clinical study to clarify its clinical value.

The lack of CD131 and the inhibition of Neuro-2a growth by carbamylated erythropoietin.

Recombinant human erythropoietin (EPO), a glycohormone, is one of the leading biopharmaceutical products, while carbamylated erythropoietin (CEPO), an EPO derivative, is attracting widespread interest due to its neuroprotective effects without erythropoiesis in several cells and animal models. However, exogenous EPO promotes an angiogenic response from tumor cells and is associated with tumor growth, but knowledge of CEPO on tumor growth is lacking. Here we show that CEPO, but not EPO, inhibited Neuro-2a growth and viability. As expected, CEPO--unlike EPO--did not activate JAK-2 either in primary neurons or in Neuro-2a cells. Interestingly, CEPO did not induce GDNF expression and subsequent AKT activation in Neuro-2a cells. Before CEPO/EPO treatment, glial cell line-derived neurotrophic factor (GDNF) neutralization and GFR receptor blocking decreased the viability of EPO-treated Neuro-2a cells but did not influence CEPO-treated Neuro-2a cells. As compared to primary neurons, the expression of CD131, as a receptor complex binding to CEPO, is almost lacking in Neuro-2a cells. In BABL/C-nu mice, CEPO did not promote the growth of Neuro-2a cells nor extended the survival time compared to mice treated with EPO. The results indicate that CEPO did not promote tumor growth because of lower expression of CD131 and subsequent dysfunction of CD131/GDNF/AKT pathway in Neuro-2a cells, revealing its therapeutic potential in future clinical application.

Fasudil mediates cell therapy of EAE by immunomodulating encephalomyelitic T cells and macrophages.

Although Fasudil has shown therapeutic potential in EAE mice, the mechanism of action are still not fully understood. Here, we examined the immunomodulatory effect of Fasudil on encephalitogenic mononuclear cells (MNCs), and tested the therapeutic potential of Fasudil-treated MNCs in active EAE. Fasudil inhibited expression of CCL20 on T cells and migration of T cells, decreased CD4(+) IFN-γ(+) and CD4(+) IL-17(+) T cells, but increased CD4(+) IL-10(+) and CD4(+) TGF-ß(+) T cells. Fasudil reduced expression of CD16/32 and IL-12, while elevating expression of CD206, CD23, and IL-10. Fasudil also decreased levels of iNOS/NO, enhanced levels of Arg-1, and inhibited the TLR-4/NF-κB signaling and TNF-α, shifting M1 macrophage to M2 phenotype. These modulatory effects of Fasudil on T cells and macrophages were not altered by adding autoantigen MOG35-55 to the culture, i.e., autoantigen-independent. Further, we observed that, in vitro, Fasudil inhibited the capacity of encephalitogenic MNCs to adoptively transfer EAE and reduced TLR-4/p-NF-κB/p65 and inflammatory cytokines in spinal cords. Importantly, Fasudil-treated encephalitogenic MNCs exhibited therapeutic potential when injected into actively induced EAE mice. Together, our results not only provide evidence that Fasudil mediates the polarization of macrophages and the regulation of T cells, but also reveal a novel strategy for cell therapy in MS.

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.

Emodin azide methyl anthraquinone derivative induced G0/ G1 arrest in HER2/neu-overexpressing MDA-MB-453 breast cancer cells.

PURPOSE: Our previous data have shown that emodin azide methyl anthraquinone derivative (AMAD) triggered mitochondrial- dependent cell apoptosis involving caspase-8-mediated Bid cleavage, and induced proteasomal degradation of HER2/neu by blocking Her2/neu binding to Hsp90. In the present study, we futher investigated the effect of this compound on the cell cycle and related molecular mechanisms in HER2/neu-overexpressing MDA-MB-453 breast cancer cells. METHODS: The cell cycle distribution was tested by flow cytometry. The expression of cell cycle-related proteins was determined by Western blot analysis; DNA agarose gel electrophoresis was used to examine the apoptosis of MDAMB- 453 cells induced by emodin AMAD. RESULTS: After MDA-MB-453 cells were treated with different concentrations of emodin AMAD for 24 hrs, cells were arrested in G0/G1 phase, and the expression of G0/G1 related proteins c/Myc, Cyclin D1, CDK4 and p-Rb changed. DNA fragmentation appeared on the agarose gel in a concentration- dependent manner. CONCLUSION: Emodin AMAD induced G0/G1 arrest in Her2/ neu-overexpressing MDA-MB-453 cancer cells. This G0/G1 arrest was associated with decreasing protein expression of c-Myc, Cyclin D1, CDK4, and p-Rb.

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.

Intranasal delivery of FSD-C10, a novel Rho kinase inhibitor, exhibits therapeutic potential in experimental autoimmune encephalomyelitis.

Viewing multiple sclerosis (MS) as both neuroinflammation and neurodegeneration has major implications for therapy, with neuroprotection and neurorepair needed in addition to controlling neuroinflammation in the central nervous system (CNS). While Fasudil, an inhibitor of Rho kinase (ROCK), is known to suppress experimental autoimmune encephalomyelitis (EAE), an animal model of MS, it relies on multiple, short-term injections, with a narrow safety window. In this study, we explored the therapeutic effect of a novel ROCK inhibitor FSD-C10, a Fasudil derivative, on EAE. An important advantage of this derivative is that it can be used via non-injection routes; intranasal delivery is the preferred route because of its efficient CNS delivery and the much lower dose compared with oral delivery. Our results showed that intranasal delivery of FSD-C10 effectively ameliorated the clinical severity of EAE and CNS inflammatory infiltration and promoted neuroprotection. FSD-C10 effectively induced CNS production of the immunoregulatory cytokine interleukin-10 and boosted expression of nerve growth factor and brain-derived neurotrophic factor proteins, while inhibiting activation of p-nuclear factor-κB/p65 on astrocytes and production of multiple pro-inflammatory cytokines. In addition, FSD-C10 treatment effectively induced CD4(+) CD25(+) , CD4(+) FOXP3(+) regulatory T cells. Together, our results demonstrate that intranasal delivery of the novel ROCK inhibitor FSD-C10 has therapeutic potential in EAE, through mechanisms that possibly involve both inhibiting CNS inflammation and promoting neuroprotection.

The triterpenoid pristimerin induces U87 glioma cell apoptosis through reactive oxygen species-mediated mitochondrial dysfunction.

It has become evident that some of the natural or synthetic triterpenoids are natural proteasome inhibitors that have great potential for use in cancer prevention and treatment. However, the mechanisms for the antitumor activity of triterpenoids remain to be elucidated. In the present study, we investigated the anticancer activities of a natural triterpenoid, pristimerin, and the signaling pathways affected. Pristimerin was found to possess potent cytotoxic effects, inducing apoptosis and inhibiting proliferation in U87 human glioma cells. Hoechst 33258 staining and Annexin V/PI double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. Moreover, western blotting assay revealed that this apoptotic induction was associated with activated caspase-9, caspase-3, PARP cleavage and downregulation of Bcl-xl/Bax in a concentration-dependent manner. Pristimerin also increased the generation of reactive oxygen species and induced the subsequent release of cytochrome c from the mitochondria into the cytosol. Additionally, pristimerin downregulated EGFR protein expression and inhibited downstream signaling pathways in U87 cells. Our results suggest that pristimerin may have potential as a new targeting therapeutic strategy in the treatment of EGFR-overexpressing gliomas.

[Effect of Fasudil on miroglia and astrocytes in experimental autoimmune encephalomyelitis mice].

AIM: To explore the therapeutic effect of Fasudil and its possible mechanisms in experimental autoimmune encephalomyelitis (EAE) mice, mainly focusing on the roles of microglia and astrocytes in the treatment. METHODS: Female adult C57BL/6 mice were immunized with MOG35-55 to induce chronic EAE. Fasudil was injected on day 3 p.i. (early Fasudil treatment), or at the onset of EAE (late Fasudil treatment). Normal saline was injected in other mice as EAE controls in a similar manner. Clinical score and body mass were recorded every other day. The expressions of iNOS on microglia and p-NF-κB/p65 on astrocytes were measured by immunohistochemistry and Western blotting. The levels of IL-1ß and TNF-α in spinal cord homogenate were determined by ELISA. RESULTS: Fasudil delayed onset and ameliorated the severity of EAE. Fasudil inhibited the expression of iNOS on microglia and p-NF-κB/p65 on astrocytes in spinal cords, accompanied by the inhibition of inflammatory factors IL-1ß and TNF-α. CONCLUSION: Fasudil exhibits therapeutic effect on EAE, possibly through inhibiting inflammatory molecules on microglia and astrocyte.

[Effect of Fasudil on the phenotype conversion of LPS-stimulated BV-2 microglia].

AIM: To explore the effect of Fasudil on LPS-stimulated BV-2 microglia in inflammatory reaction and phenotype conversion. METHODS: The routinely cultured BV-2 microglia in vitro were divided into PBS control group, PBS plus Fasudil treatment group, LPS stimulation group and LPS plus Fasudil group. We determined the production of NO by Griess reaction, the level of TNF-α by ELISA, and analyzed the M1 and M2 phenotypes of microglia by flow cytometry. RESULTS: The treatment of LPS lead to the characteristics of M1 phenotype in BV-2 microglia. Fasudil inhibited the production of NO and the release of TNF-α in LPS-stimulated BV-2 microglia. Interestingly, Fasudil transformed inflammatory M1 cells to anti-inflammatory M2 cells. CONCLUSION: Fasudil shows an anti-inflammatory effect, which may be associated with the conversion of inflammatory M1 microglia to anti-inflammatory M2 cells.