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

The influence of rHuEPO on inflammatory factors in the process of renal interstitial fibrosis and its possible mechanism

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(recombinant human erythropoietin,r Hu EPO)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、r Hu EPO对照组(20 U/m L r Hu EPO)、清蛋白刺激组(5 mg/m L清蛋白)、5 U/m L r Hu EPO干预组(5 mg/m L清蛋白+5 U/m L r Hu EPO)、10 U/m L r Hu EPO干预组(5 mg/m L清蛋白+10 U/m L r Hu EPO)、20 U/m L r Hu EPO干预组(5 mg/m L清蛋白+20 U/m L r Hu EPO)、Rho激酶抑制组(10μmol/L Y27632+5 mg/m L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Rho A、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、r Hu EPO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/m L r Hu EPO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Rho A、ROCK1 mRNA及IL-6 mRNA显著升高(P<0.05),而5、10、20 U/m L r Hu EPO干预组逐渐下调(P<0.05),且与r Hu EPO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Rho A 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[(1 347.54±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/m L r Hu EPO干预组、Rho激酶抑制组TNF-α表达[(1 003.32±3.42)、(821.32±21.32)、(590.15±7.68)、(488.13±65.03)ng/L)]较清蛋白刺激组[(1 347.54±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/m L r Hu EPO干预组组间两两比较差异均有统计学意义(P<0.05)。结论 r Hu EPO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Rho A/ROCK信号通路。

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（ r Hu EPO） on inflammatory factors in the process of renal interstitial fibrosis and its possible mechanism.Methods The vitro cultured HK-2 cells were randomized into 7groups： the blank control group,r Hu EPO control group（ addition of20 U / m L r Hu EPO）,albumin stimulation group（ addition of 5mg / m L albumin）,5mg / m L r Hu EPO intervention group（ 5mg / m L albumin ＋ 5U / m L r Hu EPO）,10 U / m L r Hu EPO intervention group（ 5mg /m L albumin ＋ 10 U / m L r Hu EPO）,20 U / m L r Hu EPO intervention group（ 5mg / m L albumin ＋ 20 U / m L r Hu EPO）,and Rho inhibitaion group（ addition of 5mg / m L albumin 30 min 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 Rho A,ROCK1 and IL-6,and ELISA was applied to measure the levels of supernatant TNF-α and IL-6 protein. Results The form of pebbles or paving stone was observed in blank control group and r Hu EPO 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 / m L r Hu EPO intervention groups,the form of oval and slightly increased intercellular space in Rho inhibitaion group. Compared with the blank control group,the expressions of Rho A 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 / m L r Hu EPO intervention groups（ P〈0. 05）,which was in negative relation with the r Hu EPO concentrations. Compared with the albumin stimulation group,the expressions of ROCK1 mRNA and IL-6 mRNA reduced in Rho inhibtation group（ P〈0. 05）,while there was no significant difference as to the expression of Rho A 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 stimulation group,the expressions of TNF-αin 5,10,20 mg / m L r Hu EPO intervention groups and Rho inhibitation group reduced significantly（ [1 003. 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 / m L r Hu EPO 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 / m L r Hu EPO intervention groups（ P〈0. 05）. Conclusion RHu EPO can inhibit the transdifferentiation process of HK-2 cells induced by albumin by suppressing inflammation factors,and the mechanism may be involved in Rho A / ROCK signaling pathway.