Calpain-1、caspase-3表达在氟致肾细胞凋亡中作用

Asscociation of calpain-1 and caspase-3 protein and mRNA Expressions with apoptosis of renal cells in rats with chronic fluorosis

目的观察氟对大鼠肾脏calpain-1、caspase-3蛋白及其mRNA表达和肾小管上皮细胞凋亡的影响。方法健康SD大鼠36只,随机分为对照组、低氟组和高氟组3组,每组12只,雌雄各半,分别饮用<1、5、50 mg/L氟化钠的自来水,连续8个月;定期检查大鼠氟斑牙发生情况,实验期末,收集大鼠24 h尿液测尿氟含量;应用免疫组化和原位杂交法检测肾组织中calpain-1和caspase-3蛋白及mRNA表达;原位末端脱氧核苷酸转移酶标记法(TUNEL)及流式细胞仪检测肾小管上皮细胞凋亡率。结果染氟组大鼠出现不同程度氟斑牙表现,且高氟组大鼠氟斑牙程度重于低氟组;与对照组比较,染氟组大鼠尿氟含量明显升高,低、高氟组大鼠肾小管上皮细胞中calpain-1和caspase-3表达[分别为(77.00±7.60)、(83.62±7.78)和(62.33±5.86)、(66.65±3.87)]升高(P<0.05),呈剂量效应关系;肾组织中mRNA表达与蛋白表达水平一致;与对照组比较,低、高剂量染氟组大鼠肾小管上皮细胞凋亡率[(19.76±2.21)%、(24.58±2.57)%]均增加(P<0.05);肾小管上皮细胞中calpain-1表达与caspase-3表达呈正相关(r=0.699,P<0.05)。结论 Calpain-1、caspase-3基因表达升高引起的肾小管上皮细胞凋亡是慢性氟中毒大鼠肾脏病变的机制之一。

Objective To investigate calpain-1 and caspase-3 protein and mRNA expressions in renal tubular epithelial cells and the impact of the expressions on apoptosis of renal cells in rats with chronic fluorosis. Methods Thirty-six healthy Sprague-Dawley rats were randomly divided into 3 groups （6 males and 6 females in each group） ： a control group with sodium fluoride of less than 1 mg/L in drinking water,two dose（ low and high）groups with sodium fluoride of 5 and 50 mg/L in drinking water. At the end of the 8-month consecutive treatment,the occurrence of dental fluorosis in the rats was observed;the fluorine content in 24-hour urine samples was detected;the expressions of calpain-1 and caspase-3 protein and mRNA were determined with immunohistochemistry and in-situ hybridization （ISH） ;the apoptosis of renal tubular epithelial cells was detected with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） and flow cytometry. Results Dental fluorosis was observed in the rats of low and high fluoride dose groups, with a higher detection rate of dental fluorosis in the high dose group than in the low dose group. Compared to the control group,the rats in fluoride dose groups had a significantly increased urine fluoride content. The expressions of calpain-1 and caspase-3 in renal tubular epithelial cells were increased in the rats of low dose group（77. 00 ± 7.60 and 62. 33 ± 5.86） and high dose group（83.62 ±7.78 and 66.65±3.87） ,with a dose-effect manner;the expression of mRNA in the renal cells was the same as that of the protein. Compared to that of the control group,the apoptosis rate of renal tubular epithelial cells was significantly increased in the rats of low dose group （ 19. 76 ± 2. 21% ） and high dose group （24. 58 ± 2. 57% ） （P 〈0. 05）. There was a correlation between the expressions of calpain-1 and caspase-3 protein and mRNA in renal tubular epithelial cells （ r = 0. 699,P 〈 0. 05）. Conclusion The apoptosis of renal tubular epithelial cells induced by the increase in the expressions of calpain-1,caspase-3 proteins and mRNA contributes to kidney damage in the rats with chronic fluorosis.