[Protective effect of recombinant adult serine protease inhibitor from Trichinella spiralis on sepsis-associated acute kidney injury in mice]

Objective: To investigate the protective effect of recombinant adult serine protease inhibitor from Trichinella spiralis (TsadSPI) on sepsis-associated acute kidney injury in mice.

Methods: A total of 18 male BALB/c mice were randomly divided into the sham-operation group, the model group, and the TsadSPI treatment group, of 6 mice in each group. Sepsis-associated acute kidney injury was modeled in the model group and TsadSPI treatment group by cecal ligation puncture (CLP), while mice in the sham-operation group were only given exploratory laparotomy without ligation or perforation of the cecum. After 30 min of CLP, mice in the sham-operation group and the model group were intraperitoneally injected with PBS (100 μL), and mice in the TsadSPI treatment group were intraperitoneally injected with PBS (100 μL) containing TsadSPI (2 μg). At 12 h following modeling, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr) and urea nitrogen (BUN) were measured to assess the liver and kidney functions, and the changes of the mouse kidney structure were observed using HE staining. In addition, the serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and transforming growth factor (TGF)-β were measured using an enzyme-linked immunosorbent assay (ELISA), and the myeloid differentiation factor 88 (MyD88) and nuclear factor kappa-B (NF-κB) p65 expression was determined in kidney tissues using immunohistochemical staining.

Results: At 12 h following CLP, there were significant differences in the serum levels of ALT (F = 41.031, P < 0.001), AST (F = 54.757, P < 0.001), Cr (F = 24.142, P < 0.001) and BUN (F = 214.849, P < 0.001) among the three groups, and higher levels of ALT, AST, Cr and BUN were measured in model group than in the sham-operation group (P < 0.001), while lower ALT, AST, Cr and BUN levels were found in the TsadSPI treatment group than in the model group (P < 0.001). HE staining showed severe mouse kidney injuries following CLP, and TsadSPI treatment resulted in remarkable alleviation of the injury. ELISA measured significant differences in the TNF-α (F = 47.502, P < 0.001) and IL-6 levels (F = 222.061, P < 0.001) among the three groups, and showed a remarkable reduction in the TNF-α and IL-6 levels in the TsadSPI treatment group as compared to those in the model group (P < 0.001). In addition, there were significant differences in serum IL-10 (F = 16.227, P < 0.001) and TGF-β levels (F = 52.092, P < 0.001) among the three groups, and higher IL-10 and TGF-β levels were seen in the TsadSPI treatment group than in the model group (P < 0.001). Immunohistochemical staining showed greater MyD88 expression and a higher nuclear positive rate of NF-κB p65 in kidney tissues in the model group than in the TsadSPI treatment group.

Conclusions: TsadSPI may reduce the MyD88 expression and nuclear positive rate of NF-κB p65 in mouse kidney tissues to up-regulate the expression of immunomodulatory factors and down-regulate the expression of pro-inflammatory cytokines, thereby protecting sepsis-associated acute kidney injury.

［摘要］ 目的 探讨旋毛虫重组成虫丝氨酸蛋白酶抑制剂 (TsadSPI) 对小鼠脓毒症急性肾损伤的保护作用。方法 BALB/c雄性小鼠随机分为假手术组、模型组、TsadSPI治疗组, 每组6只, 共18只。模型组和TsadSPI治疗组小鼠采用盲肠结扎穿孔术 (CLP) 构建脓毒症急性肾损伤模型, 假手术组小鼠剖腹探查, 不结扎、不穿孔盲肠。术后30 min, 假手术组和模型组小鼠腹腔注射100 μL PBS溶液, TsadSPI治疗组小鼠腹腔注射100 μL含有2 μg TsadSPI蛋白的PBS溶液。造模后12 h, 检测小鼠血清丙氨酸氨基转移酶 (ALT)、天门冬氨酸氨基转移酶 (AST)、肌酐 (Cr) 和尿素氮 (BUN) 水平以评价肝肾功能, HE染色观察小鼠肾脏结构改变, 应用酶联免疫吸附试验 (ELISA) 检测小鼠血清中肿瘤坏死因子 (TNF) -α、白细胞介素 (IL) -6、IL-10、转化生长因子 (TGF) -β水平, 采用免疫组化染色评估肾脏组织中髓样分化因子88 (MyD88)、核因子-κB p65 (NF-κB p65) 表达水平。结果 术后12 h, 3组小鼠血清ALT (F = 41.031, P < 0.001)、AST (F = 54.757, P < 0.001)、C (r F =24.142, P < 0.001) 和BUN水平 (F = 214.849, P < 0.001) 差异均有统计学意义, 模型组小鼠ALT、AST、Cr和BUN水平均显著高于假手术组 (P < 0.001), 而TsadSPI治疗组小鼠ALT、AST、Cr和BUN水平均显著低于模型组 (P < 0.001)。HE染色结果显示, 小鼠CLP术后肾脏损伤严重, TsadSPI治疗后损伤明显缓解。ELISA检测结果表明, 3组小鼠血清TNF-α (F =47.502, P < 0.001) 和IL-6水平 (F = 222.061, P < 0.001) 差异均有统计学意义, TsadSPI治疗组小鼠TNF-α、IL-6水平较模型组显著下降 (P < 0.001) ; 3组小鼠血清IL-10 (F = 16.227, P < 0.001) 和TGF-β水平 (F = 52.092, P < 0.001) 差异均有统计学意义, TsadSPI治疗组小鼠血清IL-10、TGF-β水平较模型组均显著升高 (P < 0.001)。免疫组化染色结果表明, 模型组小鼠肾脏组织内MyD88表达水平及NF-κB p65核阳性率显著高于TsadSPI治疗组。结论 TsadSPI可减少小鼠肾脏组织内MyD88表达及NF-κB p65核阳性率, 进而上调免疫调节因子、下调促炎因子水平, 从而保护脓毒症造成的肾脏损伤。.

Keywords: Acute kidney injury; Myeloid differentiation factor 88; Sepsis; Serine protease inhibitor; Trichinella spiralis; nuclear factor kappa-B.