Protection and mechanism of bushenhuoxue on retinal ganglian cells under the hypoxic condition in vitro / 中华实验眼科杂志

ABSTRACT

Background The early disorder of diabetic retinopathy (DR) is the damage of retinal neural cells induced by high glucose and lack of oxygen.Previous studies show that bushenhuoxue serum can enhance the activity of glutamine synthetase (GS) in Müller cells under hypoxia,and glutamate-mediated retinal excitotoxicity also can be reduced by bushenhuoxue serum intervention.However,whether the concentration of glycine can be increased by bushenhuoxue serum is not clear.Objective This study was to investigate the protective effects of bushenhuoxue serum on retinal ganglion cells (RGCs) under hypoxia.Methods The Sprague Dawley (SD) rat serum containing bushenhuoxue was prepared.The RGCs of newborn SD rats were purified and identified by a twostep immunopanning procedure.After 72 hours,all RGCs were cultured in 96-well plates and divided into four groupsnormal control group (cultured in adult SD rats normal serum),bushenhuoxue group (cultured in bushenhuoxue serum),hypoxia group (cultured in 1 mmol/L sodium dithionite); hypoxia + bushenhuoxue intervention group (cultured in bushenhuoxue serum+sodium dithionite).Glutamate and glycine contents in the extracellular fluid were detected by L-8800 automatic amino acid analyzer,and the content of lactate dehydrogenase (LDH) was assayed using LDH kits in 24,48 and 72 hours after culture.Results Cultured cells showed the green fluorescence under the immnofluorescence microscope.The contents of glutamate,glycine and LDH in the extracellular fluid were (0.0805±0.0010)mg/L,(0.0554±0.001 5)mg/L and (1 626.03 ±122.10)μmol/(min · L) in the normal control group in 24 hours after culture,and those in the hypoxia group were (0.022 5±0.001 1) mg/L,(0.014 6±0.001 1)mg/L and (1 458.68±94.23)μμmol/(min · L),showing significant reducing in the hypoxia group (q =-3.53,P =0.00 ; q =-2.45,P =0.00 ; q =-2.98,P =0.02).Compared with the normal control group,LDH and glycine contents in the extracellular fluid were significant raised in the hypoxia group 48 hours after culture (q =2.55,P =0.01 ;q =4.48,P =0.00).Seventy two hours after culture,the glutamate and glycine contents in the hypoxia group were higher than those of the normal control group (q =2.45,P =0.00 ;q =3.72,P =0.00).In 48 and 72 hours of culture,the contents of glycine were (0.017 4±0.001 5) and (0.019 2±0.001 2) mg/L in the hypoxia+bushenhuoxue intervention group,which were significantly higher than (0.016 0±0.001 2) and (0.018 0±0.000 8) mg/L in the hypoxiagroup (q=2.28,P=0.04;q=2.33,P=0.03),but the LDH level were (1 632.94±264.31) and (1 875.00±137.45)μmol/(min · L) in the hypoxia+ bushenhuoxue intervention group,which were lower than (1 688.49 ± 112.86) and (2 267.86 ± 175.21) μmol/(min · L) of the hypoxia group (q =-2.95,P =0.02 ; q =-2.35,P=0.00).No significant differences were seen in the glutamate content 24,48 and 72 hours after culture (P=0.55,0.28,0.46).A positive correlation was seen between the glutamate content and glycine content in the extracellular fluid (Kendall coefficient =0.519,Spearman coefficient =0.696,both at P =0.000).Conclusions The release levels of glutamate and glycine increase in the hypoxia RGCs,which probably is a compensatory response of RGCs.Bushenhuoxue serum can protect RGCs against injury by increasing the release of glycine and decreasing the LDH leakage from RGCs.