摘要：
Objective Clinical treatment can delay the development of renal interstitial fibrosis , but it can not reverse renal dysfuntion.The article was to discuss the influence of recombinant human erythropoietin ( rHuEPO ) on inflammatory factors in the process of renal interstitial fibrosis and its possible mechanism . Methods The vitro cultured HK-2 cells were randomized into 7 groups:the blank control group , rHuEPO control group ( addition of 20U/mL rHuEPO), albumin stimulation group (addition of 5mg/mL albumin), 5mg/mL rHuEPO intervention group (5mg/mL albumin +5U/mL rHuEPO), 10 U/mL rHuEPO intervention group (5mg/mL albumin +10 U/mL rHuEPO), 20U/mL rHuEPO intervention group (5mg/mL albumin +20U/mL rHuEPO), and Rho inhibi-taion group (addition of 5mg/mL albumin 30min after 10μmol/L Y27632), 24 h acting time for each group.We observed the changes of cell morphology in each group .Reverse transcription polymerase chain reaction ( RT-PCR) was used to evaluate the mRNA levels of RhoA, ROCK1 and IL-6 , and ELISA was applied to measure the levels of supernatant TNF-αand IL-6 protein. Results The form of pebbles or paving stone was observed in blank control group and rHuEPO intervention groups , a long and thin spindle change with the appearance of fibre cells in albumin stimulation group , the transformation to pebbles in 5, 10, 20 mg/mL rHuEPO intervention groups , the form of oval and slightly increased intercellular space in Rho inhibitaion group .Compared with the blank control group , the expressions of RhoA mRNA, ROCK1 mRNA and IL-6 mRNA significantly increased in the albumin stimulation group (P<0.05), while significantly reduced in 5, 10, 20 mg/mL rHuEPO intervention groups (P<0.05), which was in negative relation with the rHuEPO concentrations .Compared with the albumin stimulation group , the expressions of ROCK 1 mRNA and IL-6 mRNA reduced in Rho inhibtation group (P<0.05), while there was no significant difference as to the expression of RhoA mRNA .ELISA results showed:compared with blank control group , the expressions of supernatant TNF-α([452.32 ±33.23] ng/L vs [1347.54 ±41.52] ng/L), IL-6 protein([884.62 ±0.73] pg/L vs [95.12 ±0.32]pg/LP<0.05) increased significantly.Compared with albumin stim-ulation group, the expressions of TNF-αin 5, 10, 20 mg/mL rHuEPO intervention groups and Rho inhibitation group reduced signifi-cantly([1003.32 ±3.42] ng/L, [821.32 ±21.32] ng/L, [590.15 ±7.68] ng/L, [488.13 ±65.03] ng/L vs [1 347.54 ± 41.52]ng/L,P<0.05), while the expressions of IL-6 mRNA reduced accordingly in 5, 10, 20 mg/mL rHuEPO intervention groups and Rho inhibitation group reduced significantly ([656.68 ±0.55] pg/L, [422.35 ±0.22] pg/L, [217.32 ±0.35] pg/L, [309.49 ±0.21] pg/L vs [884.62 ±0.73]pg/L,P<0.05).Moreover, there was significant statistical difference among 5, 10, 20 mg/mL rHuEPO intervention groups(P<0.05). Conclusion RHuEPO can inhibit the transdifferentiation process of HK-2 cells in-duced by albumin by suppressing inflammation factors , and the mechanism may be involved in RhoA/ROCK signaling pathway .%目的：临床治疗虽可延缓肾间质纤维化进展，却无法逆转肾功能丧失。探讨重组人促红细胞生成素（ recombi-nant human erythropoietin ， rHuEPO）对肾间质纤维化过程中炎症因子的影响及其可能作用机制。方法将体外培养的HK-2细胞随机分为7组：空白对照组、rHuEPO对照组（20 U／mL rHuEPO）、清蛋白刺激组（5 mg／mL清蛋白）、5 U／mL rHuEPO干预组（5 mg／mL清蛋白＋5 U／mL rHuEPO）、10 U／mL rHuEPO干预组（5 mg／mL清蛋白＋10 U／mL rHuEPO）、20 U／mL rHuEPO干预组（5 mg／mL清蛋白＋20 U／mL rHuEPO）、Rho激酶抑制组（10μmol／L Y27632＋5 mg／mL清蛋白），各组均作用24 h。观察各组细胞形态的变化；RT-PCR检测各组细胞RhoA、ROCK1 mRNA及白细胞介素-6因子（ interleukin-6， IL-6） mRNA含量水平；ELISA检测细胞上清液中肿瘤坏死因子（ tumor necrosis factor ， TNF-α）、IL-6蛋白的含量表达。结果空白对照组、rHuEPO干预组显示鹅卵石或者铺路石样形态，清蛋白刺激组表现出细长梭状改变，呈现纤维细胞样外观。5、10、20 U／mL rHuEPO干预组细胞向鹅卵石样复转，Rho激酶抑制组细胞形态呈椭圆形、细胞间隙稍增大；与空白对照组比较，清蛋白刺激组RhoA、ROCK1 mRNA及IL-6 mRNA显著升高（P＜0．05），而5、10、20 U／mL rHuEPO干预组逐渐下调（P＜0．05），且与rHuEPO浓度负相关；与清蛋白刺激组比较，Rho激酶抑制组ROCK1 mRNA、IL-6 mRNA表达下调（P＜0．05），但RhoA mRNA表达差异无统计学意义（P＞0．05）。 ELISA结果显示：清蛋白刺激组上清液TNF-α、IL-6蛋白[（1347．54±41．52） ng／L、（884．62±0．73） pg／L]表达较空白对照组[（452．32±33．23） ng／L，（95．12±0．32） pg／L]显著增高（P＜0．05），5、10、20 U／mL rHuEPO干预组、Rho激酶抑制组TNF-α表达[（1003．32±3．42）、（821．32±21．32）、（590．15±7．68）、（488．13±65．03）ng／L）]较清蛋白刺激组[（1347．54±41．52） ng／L]下降（P ＜0．05）、IL-6蛋白表达[（656．68±0．55）、（422．35±0．22）、（217．32±0．35）、（309．49±0．21）pg／L]亦较清蛋白刺激组[（884．62±0．73）pg／L]下降（P＜0．05），5、10、20 U／mL rHuEPO干预组组间两两比较差异均有统计学意义（P＜0．05）。结论 rHuEPO可能通过减少炎症因子的产生来抑制清蛋白诱导的HK-2细胞转分化过程，其作用机制部分涉及RhoA／ROCK信号通路。
