序列相似性家族3A在高糖诱导的人脐静脉内皮细胞氧化损伤中的作用

Role of Family with Sequence Similarity 3A in High Glucose-induced Oxidative Damage of Human Umbilical Vein Endothelial Cells

目的评估序列相似性家族3A(Fam3A)在高糖诱导的人脐静脉内皮细胞(HUVECs)氧化损伤中的作用。方法将HUVECs分为对照组和高糖组,分别给予5.5 mmol/L葡萄糖的内皮细胞培养基(ECM)和33.3 mmol/L葡萄糖的ECM培养,RT-q PCR法检测Fam3A mRNA的表达,ELISA法检测Fam3A蛋白的表达。分别用siNT和siFam3A转染对照组和高糖组HUVECs,检测细胞内活性氧簇(ROS)含量、ATP水平、线粒体氧耗率(OCR)和P-p38蛋白表达情况。结果培养24h后,高糖组Fam3A mRNA与对照组的相对表达量为2.52±0.19,差异有统计学意义(t=13.296,P=0.000);Fam3A蛋白浓度为(173.82±33.28)pg/ml,明显高于对照组的(39.45±33.78)pg/ml(t=4.907,P=0.006)。siNT高糖组细胞内ROS含量为(8217±794)RFU,明显高于siNT对照组的(3982±398)RFU(t=15.109,P=0.002);siFam3A高糖组细胞内ROS含量为(11 910±1 001)RFU,明显高于siFam3A对照组的(4171±402)RFU(t=9.705,P=0.010)和siNT高糖组(t=4.026,P=0.048)。以siNT对照组ATP合成量为基线,siNT高糖组、siFam3A对照组和siFam3A高糖组细胞内ATP合成相对值分别为(61.2±5.6)%、(94.6±8.4)%和(29.7±2.7)%,siNT高糖组明显低于siNT对照组(t=12.001,P=0.007),siFam3A高糖组明显低于siFam3A对照组(t=20.742,P=0.002)和siNT高糖组(t=18.814,P=0.003)。siNT高糖组的线粒体OCR为(0.57±0.05)pMO_2/(μg蛋白·min),明显低于siNT对照组的(1.12±0.09)pMO_2/(μg蛋白·min)(t=6.804,P=0.021);siFam3A高糖组的线粒体OCR为(0.31±0.03)pMO_2/(μg蛋白·min),明显低于siFam3A对照组的(1.01±0.09)pMO_2/(μg蛋白·min)(t=19.876,P=0.003),也明显低于siNT高糖组(t=21.444,P=0.002)。以siNT对照组为基线,siNT高糖组、siFam3A对照组和siFam3A高糖组的P-p38相对表达量分别为2.239±0.353、0.816±0.120和1.160±0.185,siNT高糖组明显高于siNT对照组(t=6.075,P=0.026),siFam3A高糖组明显高于siFam3A对照组(t=6.242,P=0.024),低于siNT高糖组(t=9.686,P=0.010)。结论高糖可以诱导HUVECs高表达Fam3A。敲低Fam3A基因表达可加重高糖引起的ATP合成减少及线粒体OCR降低,同时促进高糖时ROS生成增多。Fam3A可能是通过p38 MAPK信号通路调节高糖诱导的HUVECs内ROS生成。

Objective To investigate the role of family with sequence similarity 3A(Fam3A) in highglucose-induced damage of human umbilical vein endothelial cells(HUVECs).Methods HUVECs were divided into control group and high glucose group,which were cultured in endothelial cell medium(ECM) containing 5.5 mmol/L of glucose and ECM containing 33.3 mmol/L of glucose,respectively.Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of Fam3A,whereas the protein expression of Fam3A was detected by enzyme-linked immunosorbent assay.HUVECs in control group and high glucose group were transfected with siNT and siFam3A,respectively,and the levels of reactive oxygen species(ROS),ATP,mitochondrial oxygen consumption rate(OCR),and P-p38 protein were detected.Results After HUVECs had been cultured for 24 h,the relative mRNA expression of Fam3A between high glucose group and control group was2.52±0.19(t=13.296,P=0.000).The Fam3A protein level was(173.82±33.28) pg/ml in the high glucose group,which was significantly higher than that [(39.45±33.78) pg/ml] in the control group(t=4.907,P=0.006).The intracellular ROS content in siNT-high glucose group was(8217±794) RFU,which was significantly higher than that [(3982±398) RFU]in siNT control group(t=15.109,P=0.002).The intracellular ROS content of siFam3A high glucose group was(11 910±1 001) RFU,significantly higher than that[(4171±402) RFU]of siFam3A control group(t=9.705,P=0.010) and than that of siNT high glucose group(t=4.026,P=0.048).The relative amounts of ATP synthesis in siNT high glucose group,siFam3A control group and siFam3A high glucose group were(61.2±5.6) %,(94.6±8.4) %,and(29.7±2.7) % of the siNT control group respectively;thus,it was significantly lower in siNT high glucose group than in siNT control group(t=12.001,P=0.007) and was also significantly lower in siFam3A high glucose group than in siFam3A control group(t=20.742,P=0.002) and in siNT high glucose group(t=18.814,P=0.003).The mitochondrial OCR was(0.57±0.05) pMO_2/(μg protein·min) in siNT high glucose group,significantly lower than that [(1.12±0.09) pMO_2/(μg protein·min) ] of siNT control group(t=6.804,P=0.021).The mitochondrial OCR of siFam3A high glucose group was(0.31±0.03) pMO_2/(μg protein·min),significantly lower than that [(1.01±0.09) pMO_2/(μg protein · min) ] of siFam3A control group(t=19.876,P=0.003),which was significantly lower than that of siNT high glucose group(t=21.444,P=0.002).The relative expression of P-p38 in siNT high glucose group,siFam3A control group,and siFam3A high glucose group was 2.239±0.353,0.816±0.120,and 1.160±0.185,respectively;thus,it was significantly higher in the siNT high glucose group than in siNT control group(t=6.075,P=0.026);in addition,it was significantly higher in the siFam3A high glucose group than in siFam3A control group(t=6.242,P=0.024) and significantly lower than in siNT high glucose group(t=9.686,P=0.010).Conclusions High glucose can induce high expression of Fam3A in HUVECs.Knockdown of Fam3A gene expression can exacerbate the decrease of ATP synthesis and mitochondrial OCR caused by high glucose and promote the generation of ROS in high glucose.Fam3A may regulate high glucose-induced ROS production in HUVECs via the p38 MAPK signaling pathway.