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 groups:normal 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.%背景 糖尿病视网膜病变(DR)的早期损害是高糖和缺氧导致的视网膜神经细胞的结构和功能性损伤.研究表明含补肾活血中药的血清能减轻缺氧和高糖状态下神经兴奋性毒性物质谷氨酸的毒性作用,但是否可提高神经兴奋抑制性物质甘氨酸的释放,从而对视网膜神经细胞发挥保护作用尚不清楚. 目的 探讨补肾活血中药血清对体外缺氧条件下纯化培养的视网膜神经节细胞(RGCs)的保护作用. 方法 采用中药或生理盐水对SD大鼠每日灌胃1次,7d后腹主动脉取血离心过滤提取正常大鼠血清和含补肾活血中药的大鼠血清.体外两步法纯化培养SD乳鼠的RGCs,用免疫荧光染色法对培养的RGCs进行鉴定.将RGCs置于96孔培养板中培养72 h后分为正常对照组(20％正常SD大鼠血清培养)、缺氧组(1.0 mmol/L连二亚硫酸钠培养)、正常中药干预组(20％ SD大鼠含药血清培养)、缺氧中药干预组(1.0 mmol/L连二亚硫酸钠+20％SD大鼠含药血清干预).于培养后24、48、72 h用L-8800型全自动氨基酸分析仪测定各组细胞外液中谷氨酸及甘氨酸的质量浓度,用乳酸脱氢酶(LDH)试剂盒测定各组细胞培养上清液LDH的漏出量.结果 培养的细胞荧光染色阳性.细胞培养24 h,正常对照组谷氨酸、甘氨酸质量浓度和LDH的含量分别为(0.080 5±0.001 0)mg/L、(0.055 4±0.001 5) mg/L及(1 626.03±122.10) μmol/(min·L),而缺氧组为(0.022 5±0.001 1)mg/L、(0.014 6±0.001 1)mg/L及(1 458.68±94.23) μmol/(min·L),均明显低于正常对照组,差异均有统计学意义(q=-3.53,P=0.00；q=-2.45,P=0.00；q=-2.98,P=0.02).细胞培养48 h,缺氧组的LDH含量及甘氨酸质量浓度均明显高于正常对照组,差异均有统计学意义(q=2.55,P=0.01；q=4.48,P=0.00)；细胞培养72 h,缺氧组谷氨酸和甘氨酸质量浓度均明显高于正常对照组,差异均有统计学意义(q=2.45,P=0.00；q=3.72,P=0.00).细胞培养48 h、72 h,缺氧中药干预组甘氨酸质量浓度为(0.017 4±0.001 5) mg/L和(0.019 2±0.001 2) mg/L,明显高于缺氧组的(0.016 0±0.001 2)mg/L和(0.018 0±0.000 8) mg/L,差异均有统计学意义(q=2.28,P=0.04；q=2.33,P=0.03).缺氧中药干预组的LDH含量分别为(1 632.94±264.31) μmol/(min·L)和(1 875.00±137.45)μmol/(min·L),明显低于缺氧组的(1 688.49±112.86)μmol/(min · L)和(2 267.86±175.21) μmol/(min·L),差异均有统计学意义(q=-2.95,P=0.02；q=-2.35,P=0.00)；细胞培养24、48和72 h,缺氧中药干预组和缺氧组谷氨酸质量浓度的差异均无统计学意义(P=0.55、0.28、0.46).RGCs中谷氨酸与甘氨酸质量浓度均呈正相关(Kendall、Spearman相关系数分别为0.519和0.696,均P=0.000).结论 缺氧条件下RGCs中谷氨酸与甘氨酸释放量同步增加,可能是细胞对短期缺氧的自身代偿性反应.补肾活血中药能增强缺氧条件下RGCs中甘氨酸的释放,减少LDH的漏出,从而保护RGCs.
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