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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24h,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、48h,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24h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24h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,24hSPE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),24hSalB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。
Objective To investigate the effect of total phenolic acids extracted from Glycyrrhiza glabra on the oxidative stress in podocytes induced by puromycin aminopterithymidine (PAN) nephropathy in rats and PAN-induced podocytes in vitro and in vivo. Methods (1) The establishment of animal model of PAN nephropathy rats, given SPE and tacrolimus intervention, respectively, in the first 5,10,15,21 days to take the kidney tissue samples, WT1 staining number of podocytes, immunofluorescence observation 8- Hydroxy-deoxyguanosine (8-OHdG) Fluorescence intensity. (2) PAN-induced podocyte injury model was established in vitro. PAN-treated mouse podocytes were cultured for 24h with SPE, salvianolic acid B (SalB), rosmarinic acid (RA) and tacrolimus , 12, 24, 48h respectively. The expression of F-actin in podocyte cytoskeleton was observed, and the fluorescence intensity of intracellular reactive oxygen species (ROS) was analyzed by flow cytometry. Results (1) Glomerulus WT1 cell count results showed that the number of podocytes in the PAN group began to decline on the 5th day, reaching (14.4 ± 0.7) / glomerular section on the 15th day compared with that of the normal group (37.2 ± 1.5) (P <0.05). The number of podocytes in SPE group and positive control group (tacrolimus group) at each time point was higher than that in PAN group. On the 15th day, the number of glomerular WT1 cells in the positive control group was significantly correlated with SPE high-dose group is closer (P> 0.05). On the fifth day, the fluorescence intensity of 8-OHdG in renal tissue of PAN group was higher than that of normal group, and reached the peak on the 10th day, then began to decrease, and still higher than that of the normal group on the 15th day. After the drug intervention, the 8 -OHdG fluorescence intensity decreased, the positive control group and SPE high-dose group 8-OHdG fluorescence intensity is relatively close. (2) In vitro studies showed that F-actin was almost completely depolymerized after PAN was treated for 24 h. Some of the cells still had residual filamentous structure. After the treatment with SPE, SalB, RA and tacrolimus, PAN-induced Podiatric injury was significantly weakened, and intracellular re-emergence of micro-filaments of a polar distribution. Compared with the normal group, the fluorescence intensity of ROS in podocytes treated with PAN increased 24h (P <0.05). The fluorescence intensity of ROS in podocytes decreased after 24h intervention, and the levels of ROS decreased in 24hSPE low dose group, SalB high dose group and RA high dose group compared with the positive control group (P> 0.05) The fluorescence intensity of intracellular ROS was better than that of RA, and it was positively correlated with the dose of ROS. CONCLUSIONS: This study demonstrates both in vivo and in vitro studies that SPE has a protective effect on oxidative stress induced by PAN in podocytes.