| Retinal ganglion cells (RGCs) apoptosis is an important pathological feature of glaucoma and other eye diseases. The RGC-5 cell line is currently used as an in vitro model of glaucoma. Glutamate normally functions as the major excitatory amino acid neurotransmitter in the retina,but at high concentrations it becomes neurotoxic after the intraocular pressure increased,Over-expression of glutamate plays a large role in RGCs apoptosis. Therefore, a glutamate-induced RGC-5 apoptosis model can be used to investigate RGC apoptosis and optic neuroprotection.Chloride ion is the most abundant negion, participating in a variety of biological functions through transmembrane transport and ion channels.Chloride channels (ClC) have been shown extensively distributing in mammal's organs, tissues and cells.It plays an important role in many physiological and pathological functions,such as apoptosis.The transforming growth factorβ(TGF-β)is a kind of multifunctional protein of cellular signal transmission,which have been shown extensively distributing in eye and play a role in many ophthalmology disease.The TGF-β/Smad signaling pathways play a role in many clinical diseases and are involved in apoptosis in a variety of cells. TGF-βsignaling transfers from the cell membrane to the nucleus and subsequently regulates Smads family proteins. Eight Smad proteins are expressed in mammals, and Smad2, Smad3, Smad4, and Smad7 participate in signal transduction of TGF-β.Reverse transcription (RT)-PCR results revealed ClC-2, ClC-3, and ClC-5,TGF-β1, TGF-β2 mRNA expressed in RGC-5 cells. TGF-p 1 gene expression was significantly less than TGF-β2.Therefore, TGF-β2 represented the detection index in the present study.Immunohistochemical staining suggested that ClC-3 protein expression in RGC-5 cells was localized in the cytoplasm.The apoptosis rate of 20% is a suitable for research at a concentration of 1 mmol/L glutamate. The RGC-5 cells were allowed to grow for 24 hours, followed by culture with serum-free DMEM supplemented with 1 mmol/L glutamate for 24 hours.The present experiment analyzed glutamate-induced RGC-5 cell apoptosis, showing that ClC-3 mRNA and protein expressions significantly increased with ClC-3 cDNA transfection. MTT,DNA ladder assay and flow cytometry analyses detected the cell viability and apoptosis.After the ClC-3 cDNA transfection,MTT assay results demonstrated significantly increased survival rate,flow cytometry and the DNA ladder assay revealed the apoptosis rate was significantly decreased,which suggests that ClC-3 overexpression exhibited a protective effect on apoptosis.Subsequently, ClC-3 mRNA and protein expressions were inhibited by RNAi technology.MTT,DNA ladder assay and flow cytometry analyses detected cell viability and apoptosis.After the ClC-3 expressions were inhibited,MTT assay results demonstrated significantly decreased survival rate,flow cytometry and the DNA ladder assay revealed the apoptosis rate was significantly increased,which suggests that the inhibition of ClC-3 may promote the apoptosis of glutamate-induced RGC-5 cell.The apoptosis rate was significantly increased after the ClC-3 mRNA and protein expressions were inhibited by RNAi technology,then Reverse transcription (RT)-PCR and western blot results revealed Smad2, Smad3, Smad4, and Smad7 mRNA and protein expression increased to varying degrees, suggesting that these molecules contribute to cellular apoptosis.In the present study, overexpression of ClC-3 chloride channel inhibited apoptosis in glutamate-induced RGC-5 cells. Inhibition of ClC-3 chloride channel expression promoted apoptosis, and TGF-β/Smad signaling pathways could play a role in this process. These results demonstrated a role for the ClC-3 chloride channel in TGF-β/Smad signaling pathways, suggesting a novel target for optic nerve and function protection.All my experiments offered new ideas for further study of the pathogenesis of glaucoma and development of neuroprotection.
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