补肾活血中药复方对高糖及糖基化终末产物条件下视网膜Mller细胞的影响

Effect of Bushen Huoxue Compound on Retinal Muller Cells in High Glucose or AGEs Conditions

目的应用血清药理学方法探讨补肾活血中药复方对体外培养的视网膜Mller细胞在高糖及糖基化终末产物(advanced glycation end products,AGEs)干预条件下乳酸脱氢酶(lactate dehydrogenase,LDH)漏出量、血管内皮生长因子(vascular endothelial growth factor,VEGF)以及VEGF mRNA表达的影响。方法以改进酶消化法培养SD大鼠视网膜M(u|¨)ller细胞,将纯化的视网膜M(u|¨)ller细胞分别置于正常、高糖(50 mmol/L)以及AGEs(50 mg/L、100 mg/L)条件下进行培养,并以补肾活血中药复方含药血清进行干预,以酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)测定VEGF、LDH漏出量,以反转录-聚合酶链法(reverse transcription-polymerase chain reaction,RT-PCR)测定VEGF mRNA相对表达量。结果高糖组及高、低AGEs组VEGF和VEGF mRNA的表达量均较正常对照组升高(P<0.01);高AGEs组LDH漏出量较正常对照组及高糖组增加(P<0.01);补肾活血中药复方含药血清能减少高糖组及高、低AGEs组LDH漏出量、VEGF与VEGF mRNA的表达量(P<0.05,P<0.01),对正常条件下LDH漏出量、VEGF及VEGF mRNA均无明显影响(P>0.05)。结论 AGEs能明显降低视网膜M(u|¨)ller细胞膜的稳定性,高糖及AGEs均可致视网膜M(u|¨)ller细胞VEGF mRNA、VEGF的表达上调;补肾活血中药复方含药血清能增强高糖及AGEs条件下视网膜M(u|¨)ller细胞膜的稳定性、抑制VEGF mRNA的转录、进而减少VEGF蛋白表达,可能是其防治糖尿病性视网膜病变(diabetic retinopathy,DR)的重要作用机制之一。

Objective To explore the effect of Bushen Huoxue Compound（BHC） on lactate dehydrogenase（LDH） leakage,expressions of vascular endothelial growth factor（VEGF） and VEGF mRNA in retinal Muller cells under high glucose condition or advanced glycosylation end products（AGEs）condition by using serum pharmacological method.Methods The retinal Muller cells of 5-7 days postnatal Sprague Dawley（SD） rats were cultured with modified enzyme-digestion method.Purified retinal Muller cells were cultured in normal conditions,high glucose condition（50 mmol/L）or AGEs（50 mg/L and100 mg/L） conditions,and BHC-containing serum was added to culture medium.The LDH leakage and VEGF expressions were measured by enzyme-linked immunosorbent assay（ELISA）.In addition,the relative expression of VEGF mRNA was tested by reverse transcription polymerase chain reaction（RTPCR）.Results Compared with the normal control group,expressions of VEGF and VEGF mRNA were significantly increased in the high glucose group,the low dose AGEs group and the high dose AGEs group（all P 〈0.01）.The LDH leakage was obviously increased in the high dose AGEs group,when compared with the normal control group and the high glucose group（P 〈0.01）.The LDH leakage,expressions of VEGF and VEGF mRNA were obviously decreased by BHC-containing serum both in high glucose and AGEs conditions（P〈0.05,P 〈0.01）.BHC-containing serum had no significant effect on the LDH leakage and expressions of VEGF and VEGF mRNA in normal conditions（P 〉0.05）.Conclusions AGEs intervention could obviously lower the stability of Muller cell membrane.Up-regulated expressions of VEGF and VEGF mRNA in cultured Muller cells could be induced by AGEs or high glucose.BHC-containing serum could stabilize the stability of Muller cell membrane,inhibit the transcription of VEGF mRNA and decrease the protein expression of VEGF,which might be one of important mechanisms for preventing and treating diabetic retinopathy.