A Study On The Protective Effect Of BSHX Decoction And Its Mechanisms Of Protection On RGCs Cultured In High Glucose Concentration Or (and) Hypoxic Conditions

Objective:To investigate the protective effect of BuShenHuoXue (means tonifying kidney and activating blood circulation,BSHX) decoction on retinal ganglion cells and retinal Miiller cells co-cultured in vitro under high glucose concentration or (and) hypoxic conditions. In addition, molecular mechanism of protective action was also studied.Methods:1. Serum with BSHX decoction was obtained by the serum pharmacology method of traditional Chinese medicine 2. The retinal Miiller cells and retinal ganglion cells of newborn-rats were purified and cultured using the modified enzyme-digesting method by means of a two-step filtration process.The retinal ganglion cells were then planted onto culture plates coated with retinal Miiller cells. The two types of cells were co-cultured in a ratio of 1:25 to make the co-culture model of retinal ganglion cells and retinal Miiller cells 3. Glucose (final concentration of 50mmol/L) or sodium dithionite (final concentration 1.0mmol/L) was added to the co-culture cells to make the high glucose concentration or hypoxic experimental models respectively (ie. high glucose or hypoxic groups). The high glucose concentration and hypoxic group was made by adding glucose (final concentration of 50mmol/L) and sodium dithionite (final concentration 1.0mmol/L) simultaneously; Each group was treated by adding 20% serum BSHX decoction.4. After 24hrs, 48hrs and 72hrs of serum BSHX decoction treatment, extracellular lactate dehydrogenase (LDH) leakage and glutamine synthetase (GS) activity of the co-culture cells was measured by enzyme-linked immunosorbent assay (ELISA); Glutamate up-take activity of the co-culture cells was determined with intracellular [3H]labeled D,L-glutamate concentration by isotopic techniques;The content of the glutamate in the extracellular fluid was detected with a full-automatic biochemical analyzer. Data was analyzed with SPSS 13.0.Results:1. The co-culture model of RGCs and RMCs was established successfully; 2. The LDH leakage of co-cultured cells in the high glucose concentration group,hypoxic group and the combination of high glucose concentration and hypoxic group was significantly increased (P<0.05) compared with the control group,suggesting that membrane stability decreased in these conditions. The serum of BSHX decoction can noticeably reduced the pathological damage; 3. The release of glutamate of co-culture cells of the hypoxic group and the combination of high glucose and hypoxic group increased significantly in comparison to the control group in 48h time point(Ps<0.05); The release of glutamate from co-culture cells of the high glucose group decreased significantly compared with the control group at 48hrs (Ps<0.05); the serum of BSHX decoction can therefore significantly decrease the release of glutamate of co-culture cells of the control group,the hypoxic group (at 24hrs,48hrs,and 72hrs) and of hypoxic group and the high glucose and hypoxic group (at 48hrs,72hrs time points)(Ps<0.05).4.The glutamate up-take activity of co-culture cells of the hypoxic group and the high glucose and hypoxic group were significantly reduced compared with the control group (P<0.05) at 24hrs,48hrs and 72hrs, the glutamate up-take activity of co-culture cells in the high glucose group strengthened compared to the control group (P<0.05) at 24hrs,48hrs, but was lower than the control group (P<0.05) at 72hrs; the serum of BSHX decoction can remarkably increase the glutamate up-take activity of co-culture cells in the control group and the hypoxic group at 24hrs and 48hrs time points, the high glucose concentration group at 48hrs time point and the high glucose and hypoxia group at 72hr time point(P<0.05); 5. The GS activity of RMCs of the hypoxic group was significantly decreased compared to that of the control group (P<0.05) after 48h treatment. The GS activity of RMCs of the high glucose group was increased compared to the control group (P<0.05) at 24hrs,48hrs and72hr time points. The results showed that the serum of BSHX decoction can enhance GS activity of RMCs in hypoxic conditions and high glucose concentration and hypoxic conditions, and this function gradually strengthen with time;Conclusion:1. Retinal Miiller cells can nourish and support retinal ganglion cells in the condition of in vitro co-culture model; 2. The high glucose or (and) hypoxic decrease the membrane stability and increase the membrane permeability of the co-culture cells of RMCs and RGCs; the serum of BSHX decoction can help to improve the membrane stability of co-culture cells of RMCs and RGCs in the different conditions. The serum BSHX acts to maintain normal cell physiological state. It decreases the release of Glu and thus the accumulation of Glu extracellularly.3. The high glucose or(and) hypoxia can cause Glu metabolism dysfunction in the co-culture cells of RMCs and RGCs. This is mainly due to the reduced activity of GS, therefore increasing the release of Glu and decreasing its uptake. The serum of BSHX decoction can reduce the release of Glu, strengthen GS activity of RMCs, enhance uptake and transformation of Glu, reduce the accumulation of Glu extracellularly so as to reduce the neural excitability glutamic acid toxic; this may be one of the protection mechanisms of BSHX to the retinal ganglion cells under high glucose concentration/hypoxic conditions.