The Experimental Study Of Effects Of Ginkgo Biloba Extract On L-Type Calcium Channel Currents And Apoptosis In Rat Retinal Ganglion Cells

Objects:1. To find a method that could be used to screen the effective concentration of ginkgo biloba extract (GBE) in preventing cultured rat retinal neuronal cells (RNCs) and depurated retinal ganglion cells (RGCs) from injury.2. To observe the effect of GBE on cell apoptosis of cultred depurated rat RGCs and investigate the underlying mechanisms.3. To investigate the effect of GBE on L-type calcium channels in acutely dissociated rat RGCs by using whole-cell patch-clamp techniques so as to provide evidence in the cellular level for developing GBE in retinal protection.Methods:1. Rat RNCs mixed culture and rat depurated RGCs culture.2. By using MTT chromatometry, the effective concentration of GBE was explored in cultured RNCs and depurated RGCs respectively.3. The depurated RGCs were cultured for two days in the normal medium, and then GBE at different concentrations (500 ug/ml,100 ug/ml and 10 ug/ml) or brain-derived neurotrophic factor (BDNF,50 ng/ml) was added to the cultured medium. The cells were collected after 24 h GBE or BDNF treatment. Flow cytometer was employed to detect the cell apoptosis.4. Individual RNC was dissociated by enzymatic and mechanical methods.5. Effects of GBE on L-type calcium channels were studied by using whole-cell patch-clamp techniques.Results:1. MTT chromatometry was used to assess the protective effect of GBE on RNCs. The values of OD from the groups whose concentration are 1000μg/ml(GBE),500μg/ml(GBE),100μg/ml(GBE),10μg/ml(GBE),1μg/ml(GBE),0.1μg/ml(GBE),0.01μg/ml(GBE),50ng/ml(BDNF) and zero, respectively are 0.57±0.09,0.74±0.10, 0.74±0.12,0.57±0.07,0.58±0.10,0.46±0.10,0.34±0.07,0.81±0.06,0.35±0.06, respectively.The difference in the values of OD from the groups whose concentration are 1000μg/ml(GBE),500μg/ml(GBE),100μg/ml(GBE),10μg/ml(GBE),1μg/ml(GBE),0.1μg/ml(GBE),50ng/ml(BDNF), respectively and the control group whose concentration is zero is statistically significant (p＜0.01).2,Similarly, MTT chromatometry was used to assess the protective effect of GBE on RGCs. The values of OD from the groups whose concentration are 1000μg/ml(GBE),500μg/ml(GBE),100μg/ml(GBE),10μg/ml(GBE),1μg/ml(GBE)＞0.1μg/ml(GBE),0.01μg/ml(GBE),50ng/ml(BDNF) and zero, respectively are 0.72±0.03,0.52±0.09,0.43±0.11,0.32±0.03,0.32±0.04,0.30±0.05,0.28±0.05, 0.78±0.08,0.25±0.04, respectively. The difference in the value of OD from the groups whose concentration are 1000μg/ml(GBE),500μg/ml(GBE),100μg/ml(GBE)＞10μg/ml(GBE),1μg/ml(GBE),50ng/ml(BDNF), respectively and the control group whose concentration is zero is statistically significant (p＜0.01).3,Cell apoptosis of RGCs is detected by flow cytometer. The rate of apoptosis from high-dose group(whose concentration of GBE is 500ug/ml), middle-dose group(whose concentration of GBE is 100ug/ml), low-dose group (whose concentration of GBE is 10ug/ml), Brain-derived Neurotrophic Factor (BDNF) group (whose concentration of BDNF is 50ng/ml) and control group (whose concentration is zero) are 0.42±0.10,0.32±0.12,0.53±0.13,0.51±0.14,12.15±1.98. The difference in the rate of apoptosis from low-dose group, middle-dose group, high-dose group, BDNF group and control group is statistically significant (p＜0.01).4,GBE at concentrations of 0.1μg/ml, 1μg/ml,10μg/ml,100μg/ml, and 500μg/ml decreased the current amplitudes L-type calcium channels to 101.22±18.74%,91.13±5.03%(p＜0.01),7.53±30.53%(p＜0.01),32.26±15.13% (p＜0.01), and 6.13±6.77%(p＜0.01) of control respectively. The difference in calcium channel currents from the difference of GBE is statistically significant (p＜0.01).Conclusion:1. The effective concentration related to protective effects of GBE on RNCs from rats cultured in vitro are 0.1μg/ml,1μg/ml10μg/ml,100μg/ml,500μg/ml, 1000μg/ml.2. The effective concentration related to protective effects of GBE on depurated RGCs from rats are 1μg/ml10μg/ml,100μg/ml,500μg/ml,1000μg/ml.3. GBE in low-dose, middle-dose and high-dose exhibits inhibitory effect on apoptosis of RGCs. Inhibitory effects of GBE in middle-dose is most significantly.4. GBE whose concentration are 0.1μg/ml,1μg/ml,10μg/ml,100μg/ml and 500μg/ml exhibits inhibitory effect on calcium channel currents in a concentration dependent manner.5. GBE exhibits protective effect on RGCs from rats. The mechanisms may be related to inhibit calcium influx so as to inhibit apoptosis of cells.