舒洛地特对高糖作用下人腹膜间皮细胞炎症及纤维化因子表达影响

Effect of sulodexide on high glucose induced inflammation and fibrosis in human peritoneal mesothelial cells

目的研究高糖对人腹膜间皮细胞(human peritoneal mesothelial cells,HPMCs)糖萼组成成分透明质酸(hyaluronic acid,HA)、核心蛋白聚糖(decorin,DCN)和炎症及纤维化因子(白细胞介素6,interleukin-6,IL-6);转化生长因子-β1,transforming growth factor-β1,TGF-β1)表达的影响,以及观察舒洛地特(sulodexide,SLX)对上述因子表达的影响。方法将HPMCs随机分为以下各组:1.对照组;2.高糖组:(1)不同浓度组:1.5%,2.5%,4.25%葡萄糖作用48h;(2)不同时间组:2.5%葡萄糖作用不同时间(0,6,12,24,48,72h);3.高糖+SLX组:2.5%高糖+不同浓度舒洛地特(50μg/ml,400μg/ml,800μg/ml,1200μg/ml)作用48h。ELISA法检测各组上清液中HA、DCN、IL-6及TGF-β1的蛋白表达情况。结果 1.与对照组相比,不同浓度的高糖作用下HPMCs HA表达增多[(172.074±3.223)pg/ml比(147.000±1.667)pg/ml,F=20.410,P=0.036],DCN表达增多[(2.435±0.150)ng/ml比(1.021±0.138)ng/ml,F=219.555,P<0.001],IL-6表达增多[(67.981±1.655ng/l)比(45.637±1.143ng/l),F=160.674,P<0.001],TGF-β1表达增多[(204.691±2.829)ng/l比(112.716±7.484)ng/l,F=1692.284,P<0.001];不同时间的高糖作用下HPMCs HA表达增多[(205.986±2.746)pg/ml比(147.000±1.667)pg/ml,F=1225.533,P<0.001],DCN表达增多[(1.422±0.134)ng/ml比(1.021±0.138)ng/ml,F=112.124,P=0.001],IL-6表达增多[(68.078±2.944)ng/l比(45.637±1.143)ng/l,F=69.690,P<0.001],TGF-β1表达增多[(194.816±1.854)ng/l比(112.716±7.484)ng/l,F=1160.819,P<0.001]。2.与高糖组相比,SLX作用后能明显上调HA[(196.119±2.260)pg/ml比(172.074±3.223)pg/ml,F=120.845,P=0.013]和DCN[(3.227±0.067)ng/ml比(2.859±0.175)ng/ml,F=15.413,P=0.008]的表达,明显下调IL-6[(67.927±2.467)ng/l比(75.371±2.386)ng/l,F=50.643,P=0.002]及TGF-β1[(232.778±31.029)ng/l比(292.347±3.857)ng/l,F=21.458,P=0.002]的表达。结论 HPMCs存在HA和DCN的表达;高糖刺激可以反应性上调HPMCs的HA和DCN表达,但其表达不足以保护腹膜,而补充舒洛地特后HA和DCN表达显著上调,具有修复受损糖萼的作用;高糖可以显著上调IL-6及TGF-β1的表达,舒洛地特可以抑制高糖刺激下HPMCs IL-6和TGF-β1表达,从而抑制炎症和纤维化,保护腹膜功能。

Objective To explore the effects of high glucose on the expression of glycocalyx including hyaluronic acid（HA） and decorin（DCN）, inflammatory and fibrosis factors including IL-6 and TGF-β1 in human peritoneal mesothelial cells（HPMCs）, and to observe the effects of sulodexide（SLX） on these changes in HPMCs. Methods HPMCs were randomly assigned into control group, high glucose group and high glucose ＋ SLX group. In the high glucose group, HPMCs were divided into different concentrations of glucose subgroups（HPMCs stimulated with 1.5%, 2.5% and 4.25% glucose for 48 hours） and different stimulation time subgroups（HPMCs stimulated with 2.5% glucose for 0, 6, 12, 24, 48 and 72 hours）. In the high glucose＋ SLX group, HPMCs were treated with 2.5% glucose and 50, 400, 800, 1200 μg/ml SLX for 48 hours. HA,DCN, IL-6 and TGF-β1 in the cultured media were assayed by ELISA. Results（1）Compared to the control group, different concentrations of high glucose up-regulated the expressions of HA（172.074±3.223 pg/ml vs.147.000 ± 1.667 pg/ml, F=20.410, P=0.036）, DCN（2.435 ± 0.150 ng/ml vs. 1.021 ± 0.138 ng/ml, F=219.555, P=0.000）, IL-6（67.981±1.655 ng/l vs. 45.637±1.143 ng/l, F=160.674, P=0.000） and TGF-β1（204.691±2.829 ng/l vs. 112.716±7.484 ng/l, F=1692.284, P =0.000） in HPMCs. High concentrations of glucose for different hours also up-regulated the expression of HA（205.986 ± 2.746 pg/ml vs. 147.000 ± 1.667 pg/ml, F=1225.533, P=0.000）, DCN（1.422 ± 0.134 ng/ml vs. 1.021 ± 0.138 ng/ml, F=112.124, P=0.001）, IL-6（68.078 ± 2.944 ng/l vs.45.637±1.143 ng/l, F=69.690, P=0.000）, and TGF-β1（194.816±1.854 ng/l vs. 112.716±7.484 ng/l, F=1160.819,P =0.000） in HPMCs.（2） Compared to the high glucose group, treatment of SLX further increased the expression of HA（196.119±2.260 pg/ml vs. 172.074±3.223 pg/ml, F=120.845, P=0.013） and DCN（3.227±0.067 ng/ml vs. 2.859±0.175 ng/ml, F=15.413, P=0.008）, but down-regulated the expression of IL-6（67.927±2.467 ng/l vs. 75.371 ± 2.386 ng/l, F=50.643, P=0.002） and TGF-β1（232.778 ± 31.029 ng/l vs. 292.347 ± 3.857 ng/l, F=21.458, P=0.002）. Conclusion HPMCs expressed HA and DCN. Treatment of SLX further increased the high glucose induced up-regulation of HA and DCN, which may repair the injured activity of glycocalyx.SLX could reverse the high glucose induced up-regulation of IL-6 and TGF-β1 in HPMCs, which may protect peritoneal function through the inhibition of peritoneal inflammation and fibrosis.