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| 甘西鼠尾草总酚酸提取物抗嘌呤霉素氨基核苷诱导的足细胞氧化应激损伤 |
| 刘翔1,任红旗1,2*,杨阳3,戴德淑1,柳云1,李向阳4,颜超4,华慧4,刘瀛4,戴春5,尹忠诚5,汤仁仙4 |
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(1. 解放军97医院徐州医学院附属淮海医院肾内科, 徐州 221004;
2. 南京军区南京总医院国家肾脏疾病临床医学研究中心, 全军肾脏病研究所, 南京 210016;
3. 解放军97医院徐州医学院附属淮海医院药剂科, 徐州 221004;
4. 徐州医学院感染与免疫实验室, 徐州 221004;
5. 徐州医学院附属医院肾内科, 徐州 221000
*通信作者) |
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| 摘要: |
| 目的 研究甘西鼠尾草总酚酸提取物(SPE)在体内和体外对嘌呤霉素氨基核苷(PAN)肾病大鼠足细胞以及PAN诱导小鼠足细胞氧化应激损伤的作用。 方法 (1)建立PAN肾病大鼠动物模型, 给予SPE和他克莫司干预, 分别在第5、10、15、21天留取肾组织标本, WT1染色计数足细胞数目, 免疫荧光观察8-羟基脱氧鸟苷(8-OHdG)荧光强度。(2)体外采用PAN致足细胞损伤模型, PAN作用小鼠足细胞24 h, 分别加入含SPE、丹酚酸B(SalB)、迷迭香酸(RA)及他克莫司的培养基培养6、12、24、48 h, 观察足细胞骨架相关蛋白F-actin的表达, 流式细胞仪分析细胞内活性氧(ROS)荧光强度。 结果 (1)肾小球WT1细胞计数结果显示, PAN组第5天时足细胞数已开始下降, 第15天达(14.4±0.7)个/肾小球切面, 较正常组(37.2±1.5)个/肾小球切面减少(P<0.05), SPE组与阳性对照组(他克莫司组)各时间点足细胞数量高于PAN组;第15天时, 阳性对照组肾小球WT1细胞计数与SPE高剂量组较为接近(P>0.05)。第5天时PAN组大鼠肾组织8-OHdG荧光强度较正常组增强, 第10天上升至高峰, 而后开始减弱, 第15天时仍高于正常组;给予药物干预后, 大鼠肾组织的8-OHdG荧光强度降低, 其中阳性对照组与SPE高剂量组8-OHdG荧光强度较为接近。(2)体外研究发现, PAN作用24 h后, F-actin几乎完全解聚, 少数细胞尚有残存的被切断的丝状结构, 给予SPE、SalB、RA及他克莫司治疗后, PAN诱导的足细胞损伤明显减弱, 细胞内重新出现极性分布的微丝。与正常组相比, PAN作用小鼠足细胞24 h后ROS荧光强度增加(P<0.05)。给予药物干预后足细胞内ROS荧光强度降低, 24 h SPE低剂量组、SalB高剂量组和RA高剂量组与阳性对照组足细胞内ROS的荧光强度降低程度相近(P>0.05), 24 h SalB降低足细胞内ROS荧光强度效果优于RA, 且与药物剂量呈正相关。 结论 本研究从体内及体外证实, SPE对PAN所致足细胞氧化应激损伤具有保护作用。 |
| 关键词: 嘌呤霉素氨基核苷 足细胞 甘西鼠尾草 总酚酸提取物 丹酚酸B 迷迭香酸 8-羟基脱氧鸟苷 活性氧 |
| DOI：10.16781/j.0258-879x.2016.03.0322 |
| 投稿时间：2015-08-25修订日期：2015-09-29 |
| 基金项目:南京军区医学创新课题(MS042). |
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| Salvia przewalskii extract of total phenolic acids against puromycin aminonucleoside-induced oxidative stress in podocytes in vitro and in vivo |
| LIU Xiang1,REN Hong-qi1,2*,YANG Yang3,DAI De-shu1,LIU Yun1,LI Xiang-yang4,YAN Chao4,HUA Hui4,LIU Ying4,DAI Chun5,YIN Zhong-cheng5,TANG Ren-xian4 |
(1. Department of Nephrology, No. 97 Hospital of PLA, Huaihai Hospital Affiliated to Xuzhou Medical College, Xuzhou 221004, Jiangsu, China;
2. National Clinical Research Center for Kidney Diseases, PLA Institute of Kidney Disease, General Hospital, PLA Nanjing Military Area Command, Nanjing 210016, Jiangsu, China;
3. Department of Pharmacy, No. 97 Hospital of PLA, Huaihai Hospital Affiliated to Xuzhou Medical College, Xuzhou 221004, Jiangsu, China;
4. Laboratory of Infection and Immunity, Xuzhou Medical College, Xuzhou 221004, Jiangsu, China;
5. Department of Nephrology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221000, Jiangsu, China
*Corresponding author) |
| Abstract: |
| Objective To determine the effect of Salvia przewalskii extract of total phenolic acids (SPE) on puromycin aminonucleoside (PAN)-induced oxidative stress in podocytes of rats in vivo and the effect of SPE on PAN-induced oxidative stress in podocytes of mice in vitro. Methods (1) Nephropathy rat model was established by PAN and was given intervention with SPE and tacrolimus.The renal tissue samples were obtained for WT1 staining to calculate the number of podocytes on the 5th, 10th, 15th and 21st day. The intensities of 8-hydroxy-2'-deoxyguanine (8-OHdG) were evaluated by immunofluorescence. (2)The podocytes of mice were exposed to PAN for 24 h in vitro, and then SPE, salvianolic acid B (SalB), rosmarinic acid (RA) or tacrolimus were added for 6, 12, 24, and 48 h culture. Then the cytoskeleton distribution of podocytes, indicated by F-actin, was observed by fluorescence microscopy, and the intracellular reactive oxygen species (ROS) production was measured by flow cytometry. Results (1)Decrease of podocytes per glomerular volume as measured by counting WT1-positive cells was started on day 5 in each group except normal control (NC) group, and on day 15 glomerular podocytes in PAN group was significantly less than that in the NC group ([14.4±0.7]/glomerular volume vs [37.2±1.5]/glomerular volume, P<0.05). The numbers of glomerular podocytes in SPE group and positive group (tacrolimus group) were more than that in PAN group at all time points. The glomerular podocyte count of high-dose SPE group was similar to that of positive group on day 15 ([21.7±1.0]/glomerular volume vs [23.6±1.2]/glomerular volume, P>0.05). After injection of PAN, 8-OHdG intensities were increased in each group except normal control group on day 5; and the intensities peaked on day 10 and then began to decrease, but still higher than that of the normal control group on day 15. The intensities of 8-OHdG in renal tissue was decreased after intervention, and those of the tacrolimus and high-dose SPE groups were similar. (2) In vitro study found that F-actin of podocytes was almost completely disrupted 24 h after PAN treatment, with disrupted filamentous structure. After the treatment with tacrolimus, SPE, SalB and RA, the PAN induced injury of podocytes was lessened, with reappeared polarity distribution of intracellular microfilaments. Compared with NC group, the ROS production in podocytes was significantly increased in PAN group (P<0.05).After treatment of podocyte with drugs, the ROS production was decreased. The cellular ROS production of positive control group was similar to those in tacrolimus group, low-dose SPE group, high-dose SalB group and RA group at 24 h. Compared with RA, SalB had a better efficacy in reducing ROS, and the reducing effect had a positive relation with drug dose. Conclusion Our study suggests that SPE can protect podocytes from PAN-induced oxidant stress in vivo and in vitro. |
| Key words: puromycin aminonucleoside podocytes Salvia przewalskii Maxim. extract of total phenolic acids salvianolic acid B rosmarinic acid 8-hydroxy-2'-deoxyguanine reactive oxygen species |
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