Ruiqin Yao

Quercetin Attenuates Cell Apoptosis in Focal Cerebral Ischemia Rat Brain Via Activation of BDNF–TrkB–PI3K/Akt Signaling Pathway

Many studies have demonstrated that apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Neuroprotective effect of quercetin has been shown in a variety of brain injury models including ischemia/reperfusion. It is not clear whether BDNF–TrkB–PI3K/Akt signaling pathway mediates the neuroprotection of quercetin, though there has been some reports on the quercetin increased brain-derived neurotrophic factor (BDNF) level in brain injury models. We therefore first examined the neurological function, infarct volume and cell apoptosis in quercetin treated middle cerebral artery occlusion (MCAO) rats. Then the protein expression of BDNF, cleaved caspase-3 and p-Akt were evaluated in either the absence or presence of PI3K inhibitor (LY294002) or tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) by immunohistochemistry staining and western blotting. Quercetin significantly improved neurological function, while it decreased the infarct volume and the number of TdT mediated dUTP nick end labeling positive cells in MCAO rats. The protein expression of BDNF, TrkB and p-Akt also increased in the quercetin treated rats. However, treatment with LY294002 or K252a reversed the quercetin-induced increase of BDNF and p-Akt proteins and decrease of cleaved caspase-3 protein in focal cerebral ischemia rats. These results demonstrate that quercetin can decrease cell apoptosis in the focal cerebral ischemia rat brain and the mechanism may be related to the activation of BDNF–TrkB–PI3K/Akt signaling pathway.

Quercetin protects oligodendrocyte precursor cells from oxygen/glucose deprivation injury in vitro via the activation of the PI3K/Akt signaling pathway.

The aim of this study was to investigate the protection of quercetin (QUE) on oligodendrocyte precursor cells (OPCs) from oxygen/glucose deprivation (OGD)-induced injury in vitro and explore whether the PI3K/Akt signaling pathway contributed to the protection provided by quercetin. The OGD condition was induced by including 2mM sodium dithionite (Na(2)S(2)O(4)) in glucose-free DMEM medium. The concentration of QUE in this study ranged from 3μM to 81μM. OPCs were identified by immunocytochemical staining. Cell viability was analyzed using the water soluble tetrazolium salt-8 (WST-8) and lactate dehydrogenase assay (LDH). The morphological changes of the nucleus were measured using Hoechst 33258 nuclear staining, and the ratio of apoptotic cells was determined by FITC annexin V- and propidium iodide (PI) flow cytometry assay kit. In addition, the levels of pro-apoptotic proteins such as cleaved-caspase-3 and Bax and the anti-apoptotic proteins p-Akt and Bcl-2 were quantified using western blotting. The results showed that the OPC cell survival rate was significantly increased by incubation in conditioned medium supplemented with QUE as measured by the WST-8 assay, while the LDH release rate was significantly decreased as analyzed by the LDH assay. Furthermore, apoptosis assay showed that the apoptosis ratio of OPCs was also dramatically reduced by QUE. Western blotting showed that the expression levels of Bax and cleaved-caspase-3 proteins were down-regulated, while Bcl-2 and p-Akt were up-regulated. Further study showed that the increase in p-Akt by QUE was reduced by the PI3K inhibitor LY294002. These results indicated that QUE effectively protected OPCs from OGD-induced injury and that the mechanism might be related to the activation of the PI3K/Akt signaling pathway.

Quercetin improves hypoxia-ischemia induced cognitive deficits via promoting remyelination in neonatal rat.

Myelination failure is associated with perinatal cerebral hypoxia-ischemia (PHI) induced brain injury in premature infants. How to efficiently promote remyelination is crucial for improving cognitive deficits caused by brain injury. Here, we demonstrated that quercetin (Que), a kind of flavonoids, significantly improved cognitive deficits and the behavior of PHI-rat in Morris water maze and open field tasks. After administration of Que to PHI-rat, the number of neogenetic Olig2⁺ oligodendrocyte progenitor cells (OPCs) was evidently increased in the subventricular zone. Additionally, in corpus callosum (CC), the expression of MBP (myelin basic protein) was increased, and the myelin sheaths reached normal level at 30 days with more compact while less damaged myelin sheaths and more mature oligodendrocytes (OLs) repopulating the CC compared with PHI groups. In a word, our findings indicated that Que could remarkably improve both cognition performance and myelination in the context of PHI-induced brain injury by promoting the proliferation of OPCs and strengthening survival of OLs in vivo.

bFGF Protects Pre-oligodendrocytes from Oxygen/Glucose Deprivation Injury to Ameliorate Demyelination

AbstractOne of the pathological hallmarks of periventricular white matter injury is the vulnerability of pre-oligodendrocytes (preOLs) to hypoxia-ischemia (HI). There is increasing evidence that basic fibroblast growth factor (bFGF) is an important signaling molecule for neurogenesis and neuroprotection in the central nervous system. However, it is unknown whether bFGF protects preOLs from oxygen/glucose deprivation (OGD) damage in vitro and promotes remyelination in HI-induced rats. In this present study, bFGF exerted a protective effect on myelin by increasing the myelin thickness, the number of myelinated axons, and myelin basic protein expression in the HI-induced demyelinated neonatal rat corpus callosum. In vitro, bFGF ameliorated the impaired mitochondria and cell processes induced by OGD to promote the survival of isolated O4-positive preOLs. Additionally, the expression of fibroblast growth factor receptor 3 (FGFR3) was dramatically up-regulated in the preOLs after bFGF administration in vivo and in vitro. Thus, bFGF-stimulated remyelination in HI-induced rats by protecting the preOLs from hypoxic injury, and the mechanism involved may be mediated by FGFR3.

Transplanted miR-219-overexpressing oligodendrocyte precursor cells promoted remyelination and improved functional recovery in a chronic demyelinated model

Oligodendrocyte precursor cells (OPCs) have the ability to repair demyelinated lesions by maturing into myelin-producing oligodendrocytes. Recent evidence suggests that miR-219 helps regulate the differentiation of OPCs into oligodendrocytes. We performed oligodendrocyte differentiation studies using miR-219-overexpressing mouse embryonic stem cells (miR219-mESCs). The self-renewal and multiple differentiation properties of miR219-mESCs were analyzed by the expression of the stage-specific cell markers Nanog, Oct4, nestin, musashi1, GFAP, Tuj1 and O4. MiR-219 accelerated the differentiation of mESC-derived neural precursor cells (NPCs) into OPCs. We further transplanted OPCs derived from miR219-mESCs (miR219-OPCs) into cuprizone-induced chronically demyelinated mice to observe remyelination, which resulted in well-contained oligodendrocyte grafts that migrated along the corpus callosum and matured to express myelin basic protein (MBP). Ultrastructural studies further confirmed the presence of new myelin sheaths. Improved cognitive function in these mice was confirmed by behavioral tests. Importantly, the transplanted miR219-OPCs induced the proliferation of endogenous NPCs. In conclusion, these data demonstrate that miR-219 rapidly transforms mESCs into oligodendrocyte lineage cells and that the transplantation of miR219-OPCs not only promotes remyelination and improves cognitive function but also enhances the proliferation of host endogenous NPCs following chronic demyelination. These results support the potential of a therapeutic role for miR-219 in demyelinating diseases.

miR‐219 attenuates demyelination in cuprizone‐induced demyelinated mice by regulating monocarboxylate transporter 1

Remyelination is limited in patients with multiple sclerosis (MS) due to the difficulties in recruiting proliferating oligodendrocyte precursors (OPCs), the inhibition of OPC differentiation and/or maturation, and/or failure in the generation of the myelin sheath. In vitro studies have revealed that miR‐219 is necessary for OPC differentiation and monocarboxylate transporter 1 (MCT1) plays a vital role in oligodendrocyte maturation and myelin synthesis. Herein, we hypothesized that miR‐219 might promote oligodendrocyte differentiation and attenuate demyelination in a cuprizone (CPZ)‐induced demyelinated model by regulating the expression of MCT1. We found that CPZ‐treated mice exhibited significantly increased anxiety in the open field test. However, miR‐219 reduced anxiety as shown by an increase in the total distance, the central distance and the mean amount of time spent in the central area. miR‐219 decreased the quantity of OPCs and increased the number of oligodendrocytes and the level of myelin basic protein (MBP) and cyclic nucleotide 3′ phosphodiesterase (CNP) protein. Ultrastructural studies further confirmed that the extent of demyelination was attenuated by miR‐219 overexpression. Meanwhile, miR‐219 also greatly enhanced MCT1 expression via suppression of oligodendrocyte differentiation inhibitors, Sox6 and Hes5, treatment with the MCT1 inhibitor α‐cyano‐4‐hydroxycinnamate (4‐CIN) reduced the number of oligodendrocytes and the protein levels of MBP and CNP. Taken together, these results suggest a novel mode of action of miR‐219 via MCT1 in vivo and may provide a new potential remyelination therapeutic target.

Neuroprotection of Ro25-6981 Against Ischemia/Reperfusion-Induced Brain Injury via Inhibition of Autophagy.

In this study, we investigated the neuroprotective effect of Ro25-6981 against cerebral ischemia/reperfusion injury. Ro25-6981 alone or in combination with rapamycin was intracerebroventricularly administered to rats which suffered transient forebrain ischemia inducing by 4-vessel occlusion and reperfusion. Nissl staining was used to determine the survival of CA1 pyramidal cells of the hippocampus, while immunohistochemistry was performed to measure neuron-specific enolase (NSE) expression. The expression of autophagy-related proteins, such as microtubule-associated protein l light chain 3 (LC3), Beclin 1, and sequestosome 1 (p62), was assessed by immunoblotting. Nissl staining showed that neuronal damage was reduced in the hippocampal CA1 pyramidal layer in rats that received Ro25-6981. The protective effect of Ro25-6981 was dose-dependent, with a significant effect in the middle-dose range. The expression of NSE increased after Ro25-6981 treatment. Ro25-6981 significantly decreased LC3II (which is membrane bound) and Beclin 1, and increased p62. In addition, Ro25-6981 decreased rapamycin-induced neuronal damage and excessive activation of autophagy after I/R. Taken together, the results suggest that Ro25-6981 could suppress ischemic brain injury by regulating autophagy-related proteins during ischemia/reperfusion.

MiR-30a inhibits Th17 differentiation and demyelination of EAE mice by targeting the IL-21R.

T helper cells 17 (Th17) are recognized as key participants in the pathogenesis of chronic autoimmune diseases such as multiple sclerosis (MS). Regulation of Th17 differentiation is a valuable strategy for diagnosis and treatment of these complicated immune disorders. Here, by genome-wide expression profiling of microRNAs (miRs), we screened miR-30a, whose level was greatly decreased during Th17 differentiation and the process of demyelination disease, both in MS patients and experimental autoimmune encephalomyelitis (EAE) mice. Enforced constitutive expression of miR-30a in naïve T cells inhibited their differentiation into Th17, and in vivo overexpression of miR-30a resulted in fewer Th17 and alleviative EAE. Moreover, target prediction analysis and dual luciferase report assay revealed that interleukin-21 receptor (IL-21R) was a direct target of miR-30a, a finding consistent with the results that miR-30a downregulated the expression of IL-21R, while overexpression of IL-21R alleviated the inhibitory effect of miR-30a on Th17 differentiation. Taken together, our findings imply that miR-30a inhibits Th17 differentiation and the pathogenesis of MS by targeting IL-21R.

MiR-384 Regulates the Th17/Treg Ratio during Experimental Autoimmune Encephalomyelitis Pathogenesis

Specific miRNAs are involved in the pathogenesis of multiple sclerosis (MS), during which IL-17-producing CD4+ T helper (Th17) cells accumulate in the central nervous system (CNS). In this study, we identified levels of miR-384 as significantly increased in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Over-expression of miR-384 in vivo led to severe EAE, characterized by exacerbated demyelination, and increased inflammatory cell infiltration of the spinal cord; inhibition of miR-384 reversed these changes. Both the percentage of Th17, and ratio of Th17/regulatory T (Treg), cells were elevated in miR-384-transfected EAE mice, which was consistent with the observed upregulation of expression of IL-17 and the Th17 lineage-specific transcription factor, RORγt. Importantly, transfer of miR-384 overexpressing naïve T cells from wild-type (WT) to Rag1−/− mice, which are deficient in functional autologous T and B cells, led to aggravated EAE pathogenesis, while an miR-384 inhibited group was protected from EAE. Moreover, miR-384 promoted differentiation of naïve T cells into Th17 cells in vitro. Furthermore, target prediction and dual luciferase reporter assays demonstrated that suppressor of cytokine signaling 3 (SOCS3), a gene encoding protein with an established role in Th17 differentiation, was a direct target of miR-384. Our results demonstrate an important role for miR-384 in regulation of the Th17/Treg ratio during the pathogenesis of EAE, indicating that this molecule may have potential as a biomarker and/or therapeutic target in MS.

Olig2 overexpression accelerates the differentiation of mouse embryonic stem cells into oligodendrocyte progenitor cells in vitro

Oligodendrocyte progenitor cells (OPCs) transplantation is receiving considerable attention in the field of regenerative medicine therapy for demyelinating diseases. Although embryonic stem cells (ESCs) have been successfully induced to differentiate into OPCs with cytokines cocktails in vitro, the regulatory roles of many key transcription factors in this process are not clear. Here, we introduced oligodendrocyte lineage transcription factor 2 (Olig2), a basic helix‐loop‐helix transcription factor, into mouse embryonic stem cells (mESCs) to investigate its effects on the differentiation of mESCs into OPCs. The results showed that Olig2 overexpression alone did not affect pluripotency of mESCs, but in the stimulation of differentiating cocktails, Olig2 accelerated mESCs to differentiate into OPCs, shortening the induction time span from normal 21 days to 11 days. Further study demonstrated the Olig2‐mESCs derived OPCs were able to differentiate into C‐type natriuretic peptid (CNP) and Myelin Basic Protein (MBP) positive mature oligodendrocytes (OLs) in vitro, suggesting these induced OPCs might be favorable for myelin regeneration in vivo.