ABSTRACT
OBJECTIVE: This study analyzed how high glucose affects CSF1R and p-ERK1/2 expression in RF/6A cells. METHODS: The cells were cultured as high glucose (HG) and normal control (C) groups, and CSF1R shRNA was introduced. Real time PCR was used to detect the expression of CSF1R and p-ERK1/2 mRNA. Western blot was used to detect the expression of CSF1R and p-ERK1/2 proteins. Cell Counting Kit 8 (CCK-8) method was used to detect cell proliferation, while flow cytometry was used to detect apoptosis in HREC. RESULTS: Real-time PCR showed significantly raised CSF1R mRNA expression in HG. CSF1R inhibition lowered HG + LV shCSF1R CSF1R mRNA levels. Western blotting revealed higher CSF1R and p-ERK1/2 protein expression in HG than in C. Their expression level dropped after CSF1R inhibition. The number of tube-forming cells was higher in HG than in C, which reduced after CSF1R suppression. Inhibiting CSF1R also decreased cell proliferation and raised apoptosis. CONCLUSION: Overall, under high glucose, CSF1R and p-ERK1/2 were highly expressed, leading to reduced cellular activity, and CSF1R inhibition helped alleviate this effect.
Subject(s)
Apoptosis , Blotting, Western , Cell Proliferation , Glucose , MAP Kinase Signaling System , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor , Glucose/pharmacology , Cell Proliferation/drug effects , Apoptosis/drug effects , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , MAP Kinase Signaling System/physiology , Real-Time Polymerase Chain Reaction , Mitogen-Activated Protein Kinase 1/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Flow Cytometry , Animals , Gene Expression Regulation , Cell Line , Microglia/metabolism , Microglia/drug effects , Rats , Signal Transduction , RNA, Small Interfering/geneticsABSTRACT
AIM: To investigate the role of CCR7/p-ERK1/2/VEGF signaling in the mouse model of oxygen-induced retinopathy (OIR). METHODS: Neonatal C57BL/6J mice were evenly randomized into four groups: normoxia, OIR, OIR control (treated with scramble siRNA), and OIR treated (treated with CCR7 siRNA). Normoxia group was not specially handled. Postnatal day 7 (P7) mice in the OIR group were exposed to 75%±5% oxygen for 5d (P7-P12) and then maintained under normoxic conditions for 5d (P12-P17). Mice in the OIR control and OIR treated groups were given injections of scramble or CCR7 siRNA plasmid on P12 before returning to normoxic conditions for 5d (P12-P17). Retina samples were collected from all mice on P17, stained with adenosine diphosphatase (ADPase), and retinal neovascularization (RNV) was assessed. Retinas were also stained with hematoxylin and eosin (H&E) for RNV quantitation. The distribution and expression of CCR7, p-ERK1/2 and vascular endothelial growth factor (VEGF) were assessed via immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: High oxygen promoted retinal neovascularization (P<0.05) and increased the number of endothelial nuclei in new vessels extending from the retina to the vitreous body; CCR7 promoted this process (P<0.05). CCR7 and VEGF mRNA were expressed at higher levels in the OIR and OIR control groups than in the normoxia and OIR treated groups. CCR7, p-ERK1/2, and VEGF protein were expressed in the retinas of mice in the OIR and OIR control groups. Intravitreal injection of CCR7 siRNA significantly reduced CCR7, p-ERK1/2, and VEGF expression in the OIR mouse model (all P<0.05). CCR7 significantly enhanced the neovascularization and non-perfusion areas in the OIR group (P<0.05). CCR7 siRNA significantly reduced levels of p-ERK1/2 and VEGF as compared to OIR controls (P<0.05). CONCLUSION: These results suggest that CCR7/p-ERK 1/2/VEGF signaling plays an important role in OIR. CCR7 may be a potential target for the prevention and treatment of retinopathy of prematurity.
ABSTRACT
AIM: Spinal cord injury (SCI) is a serious condition of the central nervous system and it affects the quality of life and even hampers the day-to-day activity of the patient. In the current study, we investigated the efficacy of intrathecal administration of flavopiridol in an experimental animal model of SCI. The study also aimed at exploring the physiological effects of flavopiridol on neurons, astrocytes and cell cycle regulatory proteins. MATERIAL AND METHODS: In vitro scratch wound experiments were performed on female Sprague-Dawley rats (n=23). A complete hemisection to the right of T10 was made, and flavopiridol solution (200 mM, 0.8 nmol flavopiridol/animal) was delivered topically to the lesion site. Cell viability assay, in vitro scratch injury assay, cell cycle analysis using flow cytometry and behavioural assessments were performed. RESULTS: The local delivery of flavopiridol reduced cavity formation and improved regeneration of neurons with improvement in physiological performance. Flavopiridol also inhibited the migration and proliferation of astrocytes, and at the same time, promoted the survival of neurons. CONCLUSION: Intrathecal administration of flavopiridol can be a promising treatment strategy in patients with SCI and it needs to be validated in patient setting.