水飞蓟素对小鼠肾缺血再灌注损伤的保护作用

Protective effects of silymarin on renal ischemia-reperfusion injury

目的 探讨水飞蓟素（silymarin）预处理对小鼠肾脏缺血再灌注损伤（I/R）的保护作用及机制.方法 雄性C57BL/6小鼠24只,随机均分为3组：假手术组（Sham组）、缺血再灌注对照组（I/R组）、水飞蓟素＋缺血再灌注（silymarin＋ I/R）组,每组8只.Sham仅切开腹腔,游离两侧肾蒂,但不阻断任何动脉;silymarin＋ I/R组以100 mg/kg剂量silymarin每天灌胃方式给药;I/R组每天生理盐水＋0.1％乙醇灌胃,连续7d至手术前.silymarin＋ I/R、I/R组用无损伤微动脉夹夹闭双侧肾蒂45 min后,松开动脉夹恢复血流灌注24h后,处死小鼠.应用血生化检测小鼠血清肌酐及尿素氮水平,高碘酸-无色品红（PAS）染色观察肾脏组织损伤,检测各组肾脏髓过氧化物酶（MPO）活性、免疫荧光检测CD68的表达、酶联免疫吸附试验（ELISA）检测肾组织炎性因子的分泌、原位缺口末端标记法（TUNEL）检测细胞凋亡、Western blot检测裂解的（Cleaved）-半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3、B细胞淋巴瘤/白血病-2（bcl-2）及bcl-2相关X蛋白（bax）蛋白表达.结果 silymarin＋ I/R、I/R组肌酐及尿素氮均高于Sham[（4.4±0.2）、（291.7±17.7） mg/L,P＜0.05],I/R组肌酐及尿素氮[（22.7±1.2）、（1 295.7±64.2）mg/L]明显高于silymarin＋ I/R[（14.0±3.9）、（571.7±17.0） mg/L,P＜0.05];PAS染色检测肾肾小管细胞损伤Sham、silymarin＋ I/R组明显低于I/R组,I/R＋ silymarin组小鼠肾小管坏死评分[（1.75±0.16）分]与I/R组[（4.17±0.29）分]比较减少,差异有统计学意义（P＜0.05）;silymarin＋ I/R组MPO活性（31.8±5.4） U/g与I/R组（52.0±6.3） U/g比较减少（P＜0.05）;免疫荧光染色法显示silymarin＋ I/R组CD68表达比I/R组减少（P＜0.05）;ELISA结果显示炎性因子的分泌silymarin＋ I/R组与I/R组比较减少（P＜0.05）;TUNEL检测细胞凋亡显示Sham、silymarin＋I/R组细胞凋亡数量明显少于I/R组（P＜0.05）;Western blot检测显示：I/R组Cleaved-Caspase-3、bax表达明显高于Sham、silymarin＋ I/R组,bcl-2表达量低于silymarin＋ I/R组（P＜0.05）.结论 水飞蓟素通过抑制肾缺血再灌注过程中炎性因子的表达及免疫细胞的浸润,减少肾脏组织损伤,抑制细胞凋亡,从而达到保护肾功能的作用.

Objective To explore the protective effect of silymarin on renal ischemia-reperfusion （I/R） injury.Methods Twenty-four male C57BL/6 mice were randomly divided into three groups （n =8）：Sham,I/R,silymarin ＋ I/R.In Sham group,the animals were subjected to only incision of abdominal cavity and separation of bilateral renal pedicle.In silymarin ＋ I/R group,the animals were administered with 100 mg/kg dose of silymarin daily intragastrically.In I/R group,the animals were administered with normal saline ＋ 0.1% ethanol daily intragastrically.Seven consecutive days before surgery,the animals in silymarin ＋ I/R group and I/R group were subjected to a standard renal ischemia to induce acute kidney injury （AKI） after 45 min of bilateral renal artery clamping.Serum creatinine （SCr） and blood urea nitrogen （BUN） were determined.The renal injury was observed by distribution of periodate schiff （PAS） staining.Myeloperoxidase （MPO） activity in renal tissues was measured.The expression of CD68 was detected by immunofluorescence.The secretion of inflammatory factors in renal tissues was measured by enzyme-linked immunosorbent assay （ELISA）.The apoptosis was tested by TdT-mediated dUTP nick end labeling （TUNEL）.The expression of Cleaved-Caspase-3,B-cell lymphoma/Leukemia-2 （bcl-2） and bax proteins was detected by Western blotting.Results SCr and BUN levels in I/R group [（22.7 ± 1.2）,and （1 295.7 ± 64.2） mg/L] were significantly higher than in silymarin ＋ I/R group [（14.0 ± 3.9）,and （571.7±17.0） mg/L,P〈0.05] and Sham group [（4.4 ±0.2）,and （291.7 ±17.7） mg/L] （P〈0.05）.Renal tubular injury scores in Sham,and silymarin ＋ I/R groups （1.75 ± 0.16） were significantly lower than in I/R group （4.17 ±0.29）.The MPO activity in I/R group [（52.0 ±6.3） U/g] was higher than in silymarin ＋ I/R group [（31.8 ± 5.4） U/g] （P 〈 0.05）.The expression of CD68 and secretion of inflammatory factors in I/R group were significantly increased as compared with sham group and silymarin ＋ I/R group （P 〈 0.05）.The number of apoptotic cells in silymarin ＋ I/R group was less than in I/R group （P 〈 0.05）.The Cleaced-Caspase-3 and bax expression in I/R group was higher than in sham group and silymarin ＋ I/R group,but bcl-2 expression in I/R group was lower than in silymarin ＋ I/R group （P 〈 0.05）.Conclusion Silymarin reduced expression of inflammatory factors and neutrophil and macrophage infiltration in the process of renal I/R,alleviated renal tubular injury,and inhibited apoptosis so as to achieve protective effects on renal I/R injury.