南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (4): 593-599.doi: 10.12122/j.issn.1673-4254.2021.04.17

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钙蛋白酶激活可促进大鼠透析相关性腹膜纤维化

李 芳，洪 雪，蒋建平   

  • 出版日期:2021-04-20 发布日期:2021-04-30

Calpain activation promotes dialysis-associated peritoneal fibrosis in rats

  • Online:2021-04-20 Published:2021-04-30

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摘要： 目的 观察钙蛋白酶（CAPN）激活在腹膜纤维化中的表达和作用。方法 将24只SD雄性大鼠随机分为4组：对照组、生理盐水MDL28170（CAPN抑制剂）组、腹膜透析组、腹膜透析+MDL28170组。8周后处死大鼠，观察各组大鼠腹膜厚度、组织中CAPN的活化状态以及纤维连接蛋白(FN)和胶原蛋白I（COL I）蛋白的表达。体外培养原代大鼠腹膜间皮细胞，分为对照组、单纯MDL28170组、转化生长因子（TGF-β）诱导组、MDL28170联合TGF-β干预组，其中MDL28170联合TGF-β干预组按照施加的MDL28170浓度再分为30、20、10 μmol/L组。采用Western blot和免疫荧光检测CAPN活化状态、FN和COL-I在腹膜间皮细胞中的表达。结果 与对照组相比，腹膜透析组大鼠腹膜厚度增加（P<0.05），腹膜组织CAPN活化程度、FN和COL-I的表达均增加（P<0.05）。应用MDL28170干预后，大鼠腹膜纤维化程度改善，腹膜厚度变薄（P<0.01），FN和COL-I的表达减少（P<0.01）。体外实验与体内实验结果相符，MDL28170与TGF-β共同孵育的腹膜间皮细胞α-SMA、FN和COL-I的表达量较TGF-β组均减少（P<0.05）。结论 腹膜透析模型大鼠腹膜CAPN的活性、FN和COL-I的表达明显增加，CAPN的活化可以促进腹膜纤维化，抑制其活化可以减轻腹膜纤维化程度。

关键词: 钙蛋白酶；纤维化；腹膜透析；转化生长因子

Abstract: Objective To explore the role of calpain activation in the progression of peritoneal fibrosis. Methods Twenty- four male Sprague-Dawley rats were randomized equally into control group, MDL28170 (a calpain inhibitor)+normal saline group, peritoneal dialysis (PD) model group and PD + MDL28170 group. In the latter two groups, the rats received daily intraperitoneal injections of 100 mL/kg of 4.25% glucose PD solution, and those in PD+MDL28170 group and MDL28170 saline group received daily infusion of 4 mg/kg MDL28170 every other day. Eight weeks later, the rats were euthanized for pathological examination of the parietal peritoneum, and the visceral peritoneum was used for examining the activation status of calpain and the expressions of fibronectin (FN) and collagen I (COL-I). Calpain activation and expressions of FN, COL-I and α-SMA were also examined using Western blotting and immunofluorescence assay in primary cultures of rat peritoneal mesothelial cells treated with MDL28170, transforming growth factor-β (TGF-β), or both. Results Compared with the control rats, the rats in PD model group showed significantly increased peritoneal peritoneum thickness, calpain activation in the peritoneal tissue, and expressions of FN and COL-I (P<0.05). Treatment with MDL28170 significantly alleviated associated peritoneal fibrosis, decreased the thickness of the peritoneum (P<0.05), and reduced the expressions of FN and COL- I in the rats with daily PD (P<0.05). In the in vitro experiment, the expressions of FN and COL- I were also significantly lower in rat peritoneal mesothelial cells treated with both MDL28170 and TGF-β than in the cells treated with TGF-β alone (P<0.05). Conclusion Peritoneal calpain activity and expressions FN and COL-I all increase significantly in rat models of PD-associated peritoneal fibrosis. Calpain activation can promote peritoneal fibrosis, and inhibition of calpain can alleviate peritoneal fibrosis.

Key words: calpain; fibrosis; peritoneal dialysis; transforming growth factor-β