高糖条件下高表达ARNO对人肾小球内皮细胞通透性的影响及其作用机制

Effects and mechanism of high ARNO expression on the permeability of human renal glomerular endothelial cells under high glucose condition

目的探讨高糖条件下高表达ARNO对人肾小球内皮细胞(HRGEC)通透性的影响及其作用机制。方法体外培养HRGEC,分别观察不同时间梯度(15、30、45和60 min)和浓度梯度(10、20、30和40 mmol/L)高糖作用对ARNO蛋白表达和内皮细胞通透性的影响。分别采用ARNOsiRNA和Arf6siRNA重组慢病毒载体感染HRGEC,获得沉默ARNO和Arf6的HRGEC细胞株,观察ARNO和Arf6基因沉默对内皮细胞通透性的影响。结果时间梯度实验中,与高糖对照组相比,高糖15、30、45、60 min组HRGEC ARNO表达明显升高(分别为0.670±0.051、0.960±0.106、0.716±0.026、0.531±0.030vs.0.242±0.029),差异有统计学意义(P<0.05),与高糖15 min组相比,高糖30 min组ARNO表达升高(P<0.05),与高糖30min组相比,高糖45 min组ARNO表达回落(P<0.05),与高糖45 min组相比,高糖60 min组ARNO表达降低(P<0.05)。与高糖对照组相比,高糖15、30、45、60 min组内皮细胞通透性明显增加(分别为1.196±0.004、1.399±0.012、1.301±0.052、1.184±0.030vs.1.000),差异有统计学意义(P<0.05),与高糖15 min组相比,高糖30 min组通透性增加(P<0.05),与高糖30 min组相比,高糖45 min组通透性下降(P<0.05),与高糖45 min组相比,高糖60 min组通透性下降(P<0.05)。浓度梯度实验中,与正常糖组相比,高糖20、30、40 mmol/L组HRGEC ARNO表达明显升高(分别为0.632±0.031、0.927±0.041、1.183±0.098 vs. 0.169±0.033),差异有统计学意义(P<0.05),且随高糖浓度增加,ARNO表达呈递增趋势,差异均有统计学意义(P<0.05)。与正常糖组相比,高糖10、20、30、40 mmol/L组内皮细胞通透性明显增加(分别为1.147±0.015、1.237±0.023、1.351±0.015、1.444±0.019 vs. 1.000),差异有统计学意义(P<0.05),且随高糖浓度增加,内皮细胞通透性呈递增趋势,差异均有统计学意义(P<0.05)。转染慢病毒后,与正常糖未转染组及正常糖空载体组相比,正常糖ARNO siRNA组ARNO mRNA转录明显降低(0.255±0.056 vs.1.000、1.183±0.297),ARNO蛋白表达明显降低(0.088±0.005vs.0.246±0.011、0.237±0.009);正常糖Arf6siRNA组Arf6 mRNA转录明显降低(0.314±0.090 vs. 1.000、1.140±0.236),Arf6蛋白表达明显降低(0.690±0.012 vs. 0.917±0.009、0.919±0.009),差异均有统计学意义(P<0.05)。沉默内皮ARNO后,与高糖未转染组及高糖空载体组相比,高糖ARNO siRNA组ARNO蛋白表达明显降低(0.572±0.021vs.0.915±0.005、0.916±0.012),Arf6活性明显降低(0.263±0.007vs.0.484±0.014、0.490±0.008),细胞通透性也明显降低(0.718±0.017vs.1.000、0.978±0.040),差异均有统计学意义(P<0.05)。此外,沉默内皮Arf6后,与高糖未转染组及高糖空载体组相比,高糖Arf6siRNA组Arf6蛋白表达明显降低(0.673±0.015 vs. 0.932±0.020、0.899±0.022),内皮细胞通透性也明显降低(0.768±0.050 vs. 1.000、0.978±0.040),差异均有统计学意义(P<0.05)。结论高糖诱导的HRGEC高通透性与ARNO/Arf6信号活化有关,抑制ARNO的表达及Arf6活性可抑制糖尿病肾病肾小球内皮的高通透性。

Objective To explore the effects and mechanism of high expression of ARNO(ARF nucleotide-binding-site opener) on the permeability of human renal glomerular endothelial cells(HRGECs) under high glucose conditions. Methods HRGECs were cultured in vitro, and ARNO expression and endothelial permeability were detected in high glucose time gradient(15, 30, 45 and 60 min) and concentration gradient(10, 20, 30 and 40 mmol/L) experiments, respectively. Cell lines of HRGECs with ARNO and Arf6 gene silencing were obtained by infecting HRGECs with ARNO si RNA and Arf6 si RNA recombinant lentivirus vectors, respectively. The effects of ARNO and Arf6 gene silencing on endothelial permeability were observed. Results In the time gradient experiments, compared with control group, ARNO expressions increased significantly in the groups with high glucose for 15, 30, 45 and 60 min(0.670±0.051, 0.960±0.106, 0.716±0.026, 0.531±0.030 vs. 0.242±0.029. P<0.05). Compared with high glucose 15 min group, ARNO expression increased in high glucose 30 min group(P<0.05);Compared with high glucose 30 min group, ARNO expression decreased in high glucose 45 min group(P<0.05);Compared with high glucose 45 min group, ARNO expression decreased in high glucose 60 min group(P<0.05). Compared with control group, endothelial permeability increased significantly in the groups with high glucose for 15, 30, 45 and 60 min(1.196±0.004, 1.399±0.012, 1.301±0.052, 1.184±0.030 vs. 1.000, P<0.05). Compared with high glucose 15 min group, endothelial permeability increased in high glucose 30 min group(P<0.05);Compared with high glucose 30 min group, endothelial permeability decreased in high glucose 45 min group(P<0.05);Compared with high glucose 45 min group, endothelial permeability decreased in high glucose 60 min group(P<0.05). In the concentration gradient experiments, compared with normal concentration glucose group, ARNO expressions increased significantly in the groups with high glucose of 20, 30 and 40 mmol/L(0.632±0.031, 0.927±0.041, 1.183±0.098 vs. 0.169±0.033, P<0.05), and the ARNO expression level was increased along with the increase of glucose concentration(P<0.05). Compared with normal concentration glucose group, endothelial permeability increased significantly in the groups with high glucose of 10, 20, 30 and 40 mmol/L(1.147±0.015, 1.237±0.023, 1.351±0.015, 1.444±0.019 vs. 1.000, P<0.05), and the endothelial permeability increased along with the increase of glucose concentration(P<0.05). After transfection with lentivirus, compared with normal concentration glucose untransfected group and normal concentration glucose empty vector group, ARNO m RNA transcription reduced significantly(0.255±0.056 vs. 1.000, 1.183±0.297), and ARNO protein expression also reduced significantly(0.088±0.005 vs. 0.246±0.011, 0.237±0.009) in normal concentration glucose ARNO si RNA group(P<0.05). In addition, compared with those groups, Arf6 m RNA transcription was reduced significantly(0.314±0.090 vs. 1.000, 1.140±0.236), and Arf6 protein expression was also reduced significantly(0.690±0.012 vs. 0.917±0.009, 0.919±0.009) in the normal concentration glucose Arf6 si RNA group(P<0.05). After silencing ARNO, compared with high-glucose untransfected group and high-glucose empty vector group, the ARNO protein expression was reduced significantly(0.572±0.021 vs. 0.915±0.005, 0.916±0.012), Arf6 activity was reduced significantly(0.263±0.007 vs. 0.484±0.014, 0.490±0.008), and endothelial permeability was also decreased(0.718±0.017 vs. 1.000, 0.978±0.040) in high-glucose ARNO si RNA group(P<0.05). Besides, after silencing Arf6, compared with high-glucose untransfected group and high-glucose empty vector group, Arf6 protein expression was reduced significantly(0.673±0.015 vs. 0.932±0.020, 0.899±0.022), and endothelial permeability was also decreased(0.768±0.050 vs. 1.000, 0.978±0.040) in high-glucose Arf6 si RNA group(P<0.05). Conclusion High-glucose induced high permeability of HRGECs is related to the activation of ARNO/Arf6 signal, and inhibition of ARNO expression and Arf6 activity may inhibit the glomerular endothelial hyperpermeability in diabetic nephropathy, and is expected to be a new direction for the treatment of glomerular endothelial dysfunction in diabetic nephropathy.