Study On Neuroprotective Effects Of ClC-2Chloride Channel In RGC-5Cell Apoptosis

Retinal ganglion cells (RGCs) apoptosis is an important pathological feature ofglaucoma and other eye diseases. The RGC-5cell line is currently used as an invitromodel of glaucoma and optic nerve protection. Glutamate normally functions as themajor excitatory amino acid neurotransmitter in the retina, but at high concentrationsit becomes neurotoxic after the intraocular pressure increased, over-expression ofglutamate will play a large role in RGCs apoptosis. Therefore, a glutamate-inducedRGC-5apoptosis model can be used to investigate RGC apoptosis and opticneuroprotection.Chloride ion is the most abundant negion, participating in a variety of biologicalfunctions through transmembrane transport and ion channels. Chloride channels (ClC)have been shown extensively distributing in mammals organs, tissues and cells. Itplays an important role in many physiological and pathological functions, such asapoptosis, cell proliferation and cell cycle. Voltage-gated chloride channels involvedin the apoptosis process has been widely confirmed. Mitochondrial apoptosis pathwayis one of the main way of apoptosis, which have been shown extensively distributingin eye and play a role in many ophthalmology disease. Bcl-2gene family plays a keyrole in activation on cell apoptosis and regulation, which is the main endogenouspathway of apoptosis of RGCs.Reverse transcription-polymerase chain reaction (RT-PCR) results revealed ClC-2mRNA expressed in RGC-5cells. Cell immunofluorescence suggested that ClC-2protein expressions in RGC-5cells was localized in the cytoplasm.The apoptosis rate of20%is suitable for research at a concentration of1mmol/Lglutamate. The RGC-5cells were allowed to grow for24hours, followed by culturewith serum-free DMEM supplemented with1mmol/L glutamate for24hours. Thepresent experiment analyzed glutamate-induced RGC-5cell apoptosis, showing thatClC-2mRNA and protein expressions significantly increased with ClC-2cDNAtransfection. MTT、flow cytometry cell cycle and FITC/PI analysis detected these viability and apoptosis. After the ClC-2cDNA transfection, MTT assay resultsdemonstrated significantly increased survival rate, flow cytometry and apoptosis ratewas significantly decreased, the cell proportion in cell cycle in G1phase decreased, Sphase increased, which suggests that ClC-2overexpression exhibited a protectiveeffect on apoptosis.ClC-2mRNA and protein expressions were inhibited by RNAi technology.MTT、flow cytometry cell cycle and FITC/PI analysis detected cell viability andapoptosis. After the ClC-2expressions were inhibited, MTT assay resultsdemonstrated significantly decreased survival rate, flow cytometry revealed theapoptosis rate was significantly increased, the cell proportion in cell cycle in G1phaseincreased, S phase decreased, which suggests that the inhibition of ClC-2maypromote the apoptosis of glutamate-induced RGC-5cell.In order to further explore how ClC-2chloride channels inhibits glutamate-induced apoptosis in RGC-5cells, we conduct study on molecular mechanism.Weused cDNA transfection to increase and RNAi technology to decrease the intracellularexpression of ClC-2in RGC-5cells, then to observe the Bcl-2, Bax protein andenzyme activities of Caspase-3/9. Compared with the glutamate-induced group, theBax protein and Caspase-3/9enzyme activities significantly decreased in the ClC-2cDNA transfection+glutamate-induced group, the Bcl-2protein significantlyincreased. In contrast, compared with the glutamate-induced group, Bax protein andCaspase-3/9enzyme activities significantly increased in the ClC-2RNAi transfection+glutamate-induced group, the Bcl-2protein significantly decreased. Our findingssuggest that ClC-2chloride channels play a protective role in glutamate-induced apoptosis viamitochondria-dependent pathways involving Bcl-2, Bax, and caspases-3and-9.In summary, we found that ClC-2has protective effects on glutamate-induced apoptosis ofRGC-5cells by influencing the mitochondrial signaling pathway, involving Bcl-2, Bax, andcaspases-3and-9. Our results suggest that increasing the function of ClC-2channels may result inan improvement of cell survival and a reduction of cell apoptosis. Thus, the neuroprotectiveeffects of ClC-2channels may lead to a novel approach for the treatment of retinopathies, such asglaucoma.