Cun-Gen Ma

Targeting the Shift from M1 to M2 Macrophages in Experimental Autoimmune Encephalomyelitis Mice Treated with Fasudil

We observed the therapeutic effect of Fasudil and explored its mechanisms in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Fasudil, a selective Rho kinase (ROCK) inhibitor, was injected intraperitoneally at 40 mg/kg/d in early and late stages of EAE induction. Fasudil ameliorated the clinical severity of EAE at different stages, and decreased the expression of ROCK-II in spleen, accompanied by an improvement in demyelination and inhibition of inflammatory cells. Fasudil mainly inhibited CD4+IL-17+ T cells in early treatment, but also elevated CD4+IL-10+ regulatory T cells and IL-10 production in late treatment. The treatment of Fasudil shifted inflammatory M1 to anti-inflammatory M2 macrophages in both early and late treatment, being shown by inhibiting CD16/32, iNOS, IL-12, TLR4 and CD40 and increasing CD206, Arg-1, IL-10 and CD14 in spleen. By using Western blot and immunohistochemistry, iNOS and Arg-1, as two most specific markers for M1 and M2, was inhibited or induced in splenic macrophages and spinal cords of EAE mice treated with Fasudil. In vitro experiments also indicate that Fasudil shifts M1 to M2 phenotype, which does not require the participation or auxiliary of other cells. The polarization of M2 macrophages was associated with the decrease of inflammatory cytokine IL-1β, TNF-α and MCP-1. These results demonstrate that Fasudil has therapeutic potential in EAE possibly through inducing the polarization of M2 macrophages and inhibiting inflammatory responses.

Fasudil ameliorates disease progression in experimental autoimmune encephalomyelitis, acting possibly through antiinflammatory effect.

The purpose of this investigation was to further explore the mechanism(s) underlying the amelioration in EAE caused by Fasudil, particularly focusing on anti‐inflammatory effect.

Rho Kinase Inhibitor Fasudil Regulates Microglia Polarization and Function

Macrophages/microglia exhibit phenotypic and functional heterogeneity under physiological and pathological conditions. Owing to this heterogeneity, the polarization of macrophages/microglia is capable of effecting both detrimental and beneficial outcomes in various disease processes. In this study, murine microglial cell line BV-2 and primary microglia were used as cell models to elucidate the polarization of microglia. Using flow cytometry, Western blot, chemical/enzymatic determination, and immunohistochemistry, treatment with LPS primed microglia into the M1 phenotype in both BV-2 cells and primary microglia, while fasudil skewed LPS-stimulated M1 toward M2 microglia, which showed lower NF-κB activity and inflammatory cytokines IL-1ß, IL-6, and TNF-a, and increased anti-inflammatory cytokine IL-10. To examine whether the regulatory role of LPS and fasudil on microglia can occur in vivo, mice were administered LPS (25 µg/10 µl) via nasal instillation every other day for 1 month. The results demonstrated that LPS also triggered iNOS+/CD11b+ M1 microglia in the brain, while fasudil increased Arg-1+/CD11b+ M2 microglia, although the difference did not reach statistical significance. Fasudil-conditioned microglia medium promoted a neuroprotective effect against PC12 neurons, suggesting that fasudil-induced M2 microglia contribute to the survival of neurons. These results indicate a new treatment option whereby fasudil inhibits the inflammatory response by controlling a helpful polarization in microglia/macrophages.

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 Parkinsons 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 3β (GSK-3β) 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-1β, 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-3β 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.

Intranasal delivery of stem cells as therapy for central nervous system disease.

Stem cells, upon entering the CNS, can preferentially migrate into disease foci, where they exert therapeutic effects that compensate for lost tissue, reconstructing damaged neuronal circuitry and establishing in the brain a new microenvironment suitable for cell survival. However, the route of stem cell delivery into the CNS remains a challenge: with systemic administration (e.g., intravenous injection), a fraction of cells may be trapped in other organs than the CNS, while direct CNS injections, e.g., intracerebroventricular or transcranial, are invasive. Intranasal (i.n.) delivery of stem cells, in contrast, can effectively bypass the blood-brain barrier, rapidly enter the CNS, and minimize systemic distribution. I.n. delivery of stem cells may therefore be a safe and non-invasive way of targeting the CNS and would thus be a promising therapeutic option for CNS disease. In this review we discuss the i.n. route for stem cell delivery into the CNS, and the perspectives of i.n. stem cell-based therapy in CNS disease.

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‐β+ 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.

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.

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-1β, 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.

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-1β 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 inhibition of Rho kinase blocks cell migration and accumulation possibly by challenging inflammatory cytokines and chemokines on astrocytes

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by the immune-mediated demyelination and neurodegeneration of the CNS. Our previous studies showed that Rho kinase inhibitor Fasudil can delay onset, and ameliorate severity of EAE, accompanied by the improvement in myelination and the inhibition of inflammatory responses in the CNS. In this study, we found that Fasudil inhibited the migration of T cells indirectly by affecting the production of inflammatory factors and the expression of chemokines in astrocytes functions, indicating that Fasudil treatment reduced inflammatory cytokines such as TNF-α and IL-6, reactive oxygen species (NO) and chemokines like MIP-3α (CCL-20), RANTES (CCL5), MIP-1α (CCL-3) and MCP-1 (CCL2) in vitro, and blocked the chemotaxis of reactive mononuclear cells in EAE mice. Further studies found that Fasudil treatment reduced the infiltration and accumulation of pathogenic T cells into the CNS. Astrocytes expressing GFAP and CCL-20 were inhibited in Fasudil-treated EAE compared with control mice. These results demonstrate that Fasudil alleviates the pathogenesis of EAE possibly by blocking astrocyte-derived chemokine-mediated migration of inflammatory macrophages and pathogenic T cells, and might be used to treat MS.