Rapamycin is a new immunosuppressant that has a primarily anti-inflammatory effect and selectively inhibits the activation of T helper (Th)-cell subsets. It is widely used to treat autoimmune disease. We studied the mechanism of rapamycin action against experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice, a classic animal model of multiple sclerosis. Rapamycin significantly inhibited the development of EAE by decreasing both clinical scores and inflammatory cell infiltration into the spinal cord. Furthermore, rapamycin reversed EAE symptoms in mice showing the initial signs of paralysis. Rapamycin, is a mammalian target of rapamycin (mTOR) inhibitor. By measuring the downstream markers phospho-mTOR (p-mTOR)/mTOR and phospho-signal transducer and activator of transcription 3 (p-STAT3)/STAT3, we showed that rapamycin suppressed the mTOR-STAT3 pathway in EAE mice. The mTOR-STAT3 signaling pathway is important for Th1 and Th17 cell responses. We found that rapamycin-treated mice had reduced proportions of Th1 and Th17 cells, as well as lower mRNA expression for the transcription factors T-bet and RoRγt in EAE mouse splenocytes. To evaluate Th1 and Th17 cell function, we examined expression of their specific cytokines in the peripheral immune system and central nervous system. Rapamycin treatment reduced protein and mRNA levels of interferon (IFN)-γand interleukin (IL)-17 in splenocytes, and reduced IFN-γ and IL-17 mRNA levels in the spinal cords of EAE mice. These findings suggest that rapamycin treatment inhibits the mTOR-STAT3 pathway in EAE mice, thereby promoting immunosuppression. This study may provide new insight into the mechanism controlling rapamycin effects in multiple sclerosis.
Background
Vascular dementia (VAD) is the second most common type of dementia lacking effective treatments. Pentoxifylline (PTX), a nonselective phosphodiesterase inhibitor, displays protective effects in multiple cerebral diseases. In this study, we aimed to investigate the therapeutic effects and potential mechanisms of PTX in VAD.
Methods
Bilateral common carotid artery stenosis (BCAS) mouse model was established to mimic VAD. Mouse behavior was tested by open field test, novel object recognition test, Y-maze and Morris water maze (MWM) tests. Histological staining, magnetic resonance imaging (MRI) and electron microscopy were used to define white matter integrity. The impact of PTX on microglia phagocytosis, peroxisome proliferator-activated receptors-γ (PPAR-γ) activation and Mer receptor tyrosine kinase (Mertk) expression was assessed by immunofluorescence, western blotting and flow cytometry with the application of microglia-specific Mertk knockout mice, Mertk inhibitor and PPAR-γ inhibitor.
Results
Here, we found that PTX treatment alleviated cognitive impairment in novel object recognition test, Y-maze and Morris water maze tests. Furthermore, PTX alleviated white matter injury in corpus callosum (CC) and internal capsule (IC) areas as shown by histological staining and MRI analysis. PTX-treatment group presented thicker myelin sheath than vehicle group by electron microscopy. Mechanistically, PTX facilitated microglial phagocytosis of myelin debris by up-regulating the expression of Mertk in BCAS model and primary cultured microglia. Importantly, microglia-specific Mertk knockout blocked the therapeutic effects of PTX in BCAS model. Moreover, Mertk expression was regulated by the nuclear translocation of PPAR-γ. Through modulating PPAR-γ, PTX enhanced Mertk expression.
Conclusions
Collectively, our results demonstrated that PTX showed therapeutic potentials in VAD and alleviated ischemic white matter injury via modulating Mertk-mediated myelin clearance in microglia.
We studied the role of Sirtuin 3 (SIRT3) in microglial cell migration in ischemic stroke. We used a middle cerebral artery occlusion (MCAO) model of focal ischemia. We then applied lentivirus-packaged SIRT3 overexpression and knock down in microglial N9 cells to investigate the underlying mechanism driving microglial cell migration. More microglial cells appeared in the ischemic lesion side after MCAO. The levels of SIRT3 were increased in macrophages, the main source of microglia, after ischemia. CX3CR1 levels were increased with SIRT3 overexpression. SIRT3 promoted microglial N9 cells migration by upregulating CX3CR1 in both normal and glucose deprived culture media. These effects were G protein-dependent. Our study for the first time shows that SIRT3 promotes microglia migration by upregulating CX3CR1.
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