竹节参皂苷Ⅳa调控环状RNA EIF4G2/微小RNA-379-5p对高糖诱导的人肾小管上皮细胞损伤的影响

Effects of chikusetsu saponin Ⅳa on high glucose-induced human renal tubular epithelial cell injury through regulating circEIF4G2/miR-379-5p

目的探讨竹节参皂苷Ⅳa(chikusetsu saponinⅣa,CHS)对高糖诱导的人肾小管上皮细胞(human renal tubular epithelial cells,HK-2)损伤的影响,探讨其机制是否与调控环状RNA EIF4G2(circular RNA EIF4G2,circEIF4G2)和微小RNA-379-5p(microRNA-379-5p,miR-379-5p)表达有关。方法HK-2细胞分为正常糖(normal glucose,NG)组(5.0 mmol/L葡萄糖)、高糖(high glucose,HG)组、高渗(mannitol glucose,MG)组、HG+CHS 25μM组、HG+CHS 50μM组、HG+CHS 100μM组、HG+si-NC组、HG+si-circEIF4G2组、HG+CHS+pcDNA组、HG+CHS+pcDNA-circEIF4G2组。流式细胞术检测细胞凋亡率,试剂盒分析细胞内丙二醛(malondialedhyde,MDA)水平和超氧化物歧化酶(superoxide dismutase,SOD)活性。RT-qPCR测定circEIF4G2和miR-379-5p水平。双荧光素酶报告实验验证circEIF4G2和miR-379-5p靶向关系。结果与NG组比较,HG组HK-2细胞凋亡率[(7.62±0.74)%比(23.81±1.45)%]、MDA水平[(139.17±13)nmol/mL比(637.50±59.93)nmol/mL]、circEIF4G2相对水平[(0.96±0.07)比(3.00±0.15)]显著升高(P<0.05),SOD活性[(165.05±16.52)U/L比(55.77±7.30)U/L]、miR-379-5p相对水平[(1.00±0.00)比(0.14±0.02)]显著降低(P<0.05)。与HG组比较,HG+CHS 25μM组、HG+CHS 50μM组、HG+CHS 100μM组HK-2细胞凋亡率[(23.81±1.45)%比(20.70±1.07)%比(16.78±0.87)%比(11.06±0.77)%]、MDA水平[(637.50±59.93)nmol/mL比(533.36±17.83)nmol/mL比(407.51±34.34)nmol/mL比(276.04±15.15)nmol/mL]、circEIF4G2相对水平[(3.00±0.15)比(2.26±0.10)比(1.75±0.07)比(1.33±0.11)]显著降低(P<0.05),SOD活性[(55.77±7.30)U/L比(79.99±8.95)U/L比(118.67±11.73)U/L比(144.78±8.52)U/L]、miR-379-5p相对水平[(0.14±0.02)比(0.29±0.03)比(0.48±0.06)比(0.75±0.07)]显著升高(P<0.05)。circEIF4G2直接靶向结合miR-379-5p。与HG+si-NC组比较,HG+si-circEIF4G2组HK2细胞凋亡率[(23.93±1.32)%比(13.77±1.18)%]、MDA水平[(642.24±52.33)nmol/mL比(311.91±20.40)nmol/mL]显著降低(P<0.05),SOD活性[(55.41±7.75)U/L比(130.67±13.17)U/L]、miR-379-5p相对水平[(1.00±0.00)比(4.13±0.11)]显著升高(P<0.05)。与HG+CHS+pcDNA组比较,HG+CHS+pcDNA-circEIF4G2组HK2细胞凋亡率[(11.14±0.83)%比(19.74±1.14)%]、MDA水平[(277.89±15.46)nmol/mL比(583.39±10.29)nmol/mL]显著升高(P<0.05),SOD活性[(144.04±13.23)U/L比(62.98±6.14)U/L]显著降低(P<0.05)。结论CHS可减轻高糖诱导的HK-2凋亡和氧化应激损伤,其机制与抑制circEIF4G2/miR-379-5p轴有关。

Objective To explore the effect of chikusetsu saponin Ⅳa(CHS)on high glucoseinduced human renal tubular epithelial cells(HK-2)injury and examine whether or not its mechanism is correlated with the regulation of circEIF4G2 and miR-379-5p expression.Methods HK-2 cells were divided into 10 groups of normal glucose(NG)(5.0 mmol/L glucose),high glucose(HG),HG+CHS 25μM,HG+CHS 50μM,HG+CHS 100μM,HG+si-NC,HG+si-circEIF4G2,HG+CHS+pcDNA and HG+CHS+pcDNA-circEIF4G2.Flow cytometry was performed for detecting apoptotic rate and kit employed for evaluating intracellular malondialdehyde(MDA)level and superoxide dismutase(SOD)activity.The levels of circEIF4G2 and miR-379-5p were assessed by reverse transcription-quantitative polymerase chain reaction(RT-qPCR).And targeting relationship between circEIF4G2 and miR-379-5p was verified by dual lucifase reporting assay.Results Compared with NG group,apoptotic rate[(7.62±0.74)%vs(23.81±1.45)%],MDA level[(139.17±13.60)nmol/mL vs(637.50±59.93)nmol/mL]and relative level of circEIF4G2[(0.96±0.07)vs(3.00±0.15)]spiked markedly in HG group(P<0.05)while SOD activity[(165.05±16.52)U/L vs(55.77±7.30)U/L]and relative level of miR-379-5p[(1.00±0.00)vs(0.14±0.02)]dropped obviously(P<0.05).Compared with HG group,apoptotic rate[(23.81±1.45)%vs(20.70±1.07)%vs(16.78±0.87)%vs(11.06±0.77)%],MDA level[(637.50±59.93)nmol/mL vs(533.36±17.83)nmol/mL vs(407.51±34.34)vs(276.04±15.15)nmol/mL]and relative level of circEIF4G2[(3.00±0.15)vs(2.26±0.10)vs(1.75±0.07)vs(1.33±0.11)]declined markedly in HG+CHS 25/50/100μM group(P<0.05)while SOD activity[(55.77±7.30)U/L vs(79.99±8.95)U/L vs(118.67±11.73)U/L vs(144.78±8.52)U/L]and relative level of miR-379-5p[(0.14±0.02)vs(0.29±0.03)vs(0.48±0.06)vs(0.75±0.07)]spiked obviously(P<0.05).And circEIF4G2 was directly conjugated with miR-379-5p.Compared with HG+si-NC group,apoptotic rate[(23.93±1.32)%vs(13.77±1.18)%]and MDA level[(642.24±52.33)nmol/mL vs(311.91±20.40)nmol/mL]declined markedly in HG+si-circEIF4G2 group(P<0.05)and SOD activity[(55.41±7.75)U/L vs(130.67±13.17)U/L]and relative level of miR-379-5p rose significantly[(1.00±0.00)vs(4.13±0.11)](P<0.05).Compared with HG+CHS+pcDNA group,apoptotic rate[(11.14±0.83)%vs(19.74±1.14)%]and MDA level[(277.89±15.46)nmol/mL vs(583.39±10.29)nmol/mL]spiked markedly in HG+CHS+pcDNA-circEIF4G2 group(P<0.05)while SOD activity dropped markedly[(144.04±13.23)U/L vs(62.98±6.14)U/L,P<0.05].Conclusion Chikusetsu saponinⅣa may alleviate high glucose-induced human renal tubular epithelial cell apoptosis and oxidative stress injury.The mechanism is correlated with a suppression of circEIF4G2/miR-379-5p axis.