Abstract: Objective　To evaluate the protective effect of recombinant Schistosoma japonicum cystatin (rSj⁃Cys) against acute kidney injury induced by acute liver failure and unravel the underlying mechanism, so as to provide insights into the clinical therapy of acute kidney injury. Methods　Twenty⁃four male C57BL/6J mice at ages of 6 to 8 weeks were randomly divided into the normal control group, rSj⁃Cys control group, lipopolysaccharide (LPS)/D⁃galactosamine (D⁃GaIN) model group and LPS/D⁃GaIN +rSj⁃Cys treatment group, of 6 mice each group. Mice in the LPS/D⁃GaIN group and LPS/D⁃GaIN + rSj⁃Cys group were intraperitoneally injected with LPS (10 μg/kg) and D⁃GaIN (700 mg/kg), and mice in the LPS/D⁃GaIN + rSj⁃Cys group were additionally administered with rSj⁃Cys (1.25 mg/kg) by intraperitoneal injection 30 min post⁃modeling, while mice in the rSj⁃Cys group were intraperitoneally injected with rSj⁃Cys (1.25 mg/kg), and mice in the normal control group were injected with the normal volume of PBS. All mice were sacrificed 6 h post⁃modeling, and mouse serum and kidney samples were collected. Serum creatinine (Cr) and urea nitrogen (BUN) levels were measured, and the pathological changes of mouse kidney specimens were examined using hematoxylin⁃eosin (HE) staining. Serum tumor necrosis factor (TNF)⁃α and interleukin (IL)⁃6 levels were detected using enzyme⁃linked immunosorbent assay (ELISA), and the expression of inflammatory factors and pyroptosis⁃related proteins was quantified in mouse kidney specimens using immunohistochemistry. In addition, the expression of pyroptosis⁃related proteins and nuclear factor⁃kappa B (NF⁃κB) signaling pathway⁃associated proteins was determined in mouse kidney specimens using Western blotting assay. Results　HE staining showed no remarkable abnormality in the mouse kidney structure in the normal control group and the rSj⁃Cys control group, and renal tubular injury was found in LPS/D⁃GaIN group, while the renal tubular injury was alleviated in LPS/D⁃GaIN+rSj⁃Cys treatment group. There were significant differences in serum levels of Cr (F = 46.33, P < 0.001), BUN (F = 128.60, P < 0.001), TNF⁃α (F = 102.00, P < 0.001) and IL⁃6 (F = 202.10, P < 0.001) among the four groups , and lower serum Cr [(85.35 ± 32.05) µmol/L], BUN [(11.90 ± 2.76) mmol/L], TNF⁃α [(158.27 ± 15.83) pg/mL] and IL⁃6 levels [(56.72 ± 4.37) pg/mL] were detected in the in LPS/D⁃GaIN + rSj⁃Cys group than in the LPS/D⁃GaIN group (all P values < 0.01). Immunohistochemical staining detected significant differences in TNF⁃α (F = 24.16, P < 0.001) and IL⁃10 (F = 15.07, P < 0.01) expression among the four groups , and lower TNF⁃α [(106.50 ± 16.57)%] and higher IL⁃10 expression [(91.83 ± 5.23)%] was detected in the LPS/D⁃GaIN + rSj⁃Cys group than in the LPS/D⁃GaIN group (both P values < 0.01). Western blotting and immunohistochemistry detected significant differences in the protein expression of pyroptosis⁃related proteins NOD⁃like receptor thermal protein domain associated protein 3 (NLRP3) (F = 24.57 and 30.72, both P values < 0.001), IL⁃1β (F =19.24 and 22.59, both P values < 0.001) and IL⁃18 (F = 16.60 and 19.30, both P values < 0.001) in kidney samples among the four groups, and lower NLRP3, IL⁃1β and IL⁃18 expression was quantified in the LPS/D⁃GaIN + rSj⁃Cys treatment group than in the LPS/D⁃GaIN group (P values < 0.05). In addition, there were significant differences in the protein expression of NF⁃κB signaling pathway⁃associated proteins p⁃NF⁃κB p⁃P65/NF⁃κB p65 (F = 71.88, P < 0.001), Toll⁃like receptor (TLR)⁃4 (F = 45.49, P < 0.001) and p⁃IκB/IκB (F = 60.87, P < 0.001) in mouse kidney samples among the four groups, and lower expression of three NF⁃κB signaling pathway⁃associated proteins was determined in the LPS/D⁃GaIN + rSj⁃Cys treatment group than in the LPS/D⁃GaIN group (all P values < 0.01). Conclusion　rSj⁃Cys may present a protective effect against acute kidney injury caused by acute liver failure through inhibiting inflammation and pyroptosis and downregulating the NF⁃κB signaling pathway.

Key words: Schistosoma japonicum, Cystatin, Acute liver failure, Acute kidney injury, Pyroptosis, Nuclear factor?κB

摘要： 目的　探究重组日本血吸虫半胱氨酸蛋白酶抑制剂（rSj⁃Cys）对急性肝衰竭所致急性肾损伤的保护作用及其机制，为急性肾损伤临床治疗提供科学依据。方法　将24只雄性C57BL/6J小鼠（6～8周龄）随机分为正常对照组、rSj⁃Cys对照组、脂多糖（LPS）⁃D⁃氨基半乳糖（D⁃GaIN）模型组和LPS/D⁃GaIN+rSj⁃Cys治疗组，每组6只小鼠。LPS/D⁃GaIN组和LPS/D⁃GaIN+rSj⁃Cys组小鼠均腹腔注射LPS（10 μg/kg）和D⁃GaIN（700 mg/kg）造模；造模后30 min，LPS/D⁃GaIN+rSj⁃Cys组及rSj⁃Cys对照组小鼠均腹腔注射rSj⁃Cys（1.25 mg/kg），正常对照组小鼠注射等体积PBS。造模6 h后，处死各组小鼠，收集小鼠血清及肾组织，检测血清肌酐（Cr）、尿素氮（BUN）含量。苏木精⁃伊红（HE）染色观察各组小鼠肾脏组织病理形态，采用酶联免疫吸附试验（ELISA）检测小鼠血清炎性因子肿瘤坏死因子（TNF）⁃α和白细胞介素（IL）⁃6表达，采用免疫组化法检测肾组织炎性因子和焦亡相关蛋白表达水平，采用免疫印迹法检测肾组织焦亡相关蛋白和核因子⁃κB（NF⁃κB）信号通路蛋白表达水平。结果　HE染色显示，正常对照组和rSj⁃Cys对照组小鼠肾脏结构未见明显异常，LPS/D⁃GaIN组小鼠肾组织出现肾小管损伤，而LPS/D⁃GaIN+rSj⁃Cys治疗组小鼠肾小管损伤减轻。4组小鼠血清Cr（F = 46.33，P < 0.001）、BUN（F = 128.60，P < 0.001）、TNF⁃α（F = 102.00，P < 0.001）和IL⁃6水平（F = 202.10，P < 0.001）差异均有统计学意义，LPS/D⁃GaIN+rSj⁃Cys治疗组小鼠血清Cr[（85.35 ± 32.05） µmol/L]、BUN [（11.90 ± 2.76） mmol/L]、TNF⁃α [（158.27 ± 15.83） pg/mL]和IL⁃6水平[（56.72 ± 4.37） pg/mL]均低于LPS/D⁃GaIN组（P均< 0.01）。4组小鼠肾组织TNF⁃α（F = 24.16，P < 0.001）和IL⁃10表达水平（F = 15.07，P < 0.01）差异均有统计学意义；LPS/D⁃GaIN+rSj⁃Cys治疗组肾组织TNF⁃α表达水平[（106.50 ± 16.57）%]低于LPS/D⁃GaIN组（P < 0.01），IL⁃10表达水平[（91.83 ± 5.23 ）%]高于LPS/D⁃GaIN组（P < 0.01）。免疫印迹法及免疫组化染色结果均表明，各组小鼠肾脏焦亡相关蛋白NLRP3（F = 24.57、30.72，P均 < 0.001）、IL⁃1β （F =19.24、22.59，P 均< 0.001）和IL⁃18表达水平（F = 16.60、19.30，P均< 0.001）差异均有统计学意义，LPS/D⁃GaIN+rSj⁃Cys治疗组小鼠NLRP3、IL⁃1β和IL⁃18 蛋白表达水平均低于LPS/D⁃GaIN组（P 均< 0.05）。各组小鼠肾脏NF⁃κB信号通路相关蛋白p⁃NF⁃κB p⁃p65 /NF⁃κB p65（F = 71.88，P < 0.001）、TLR4（F = 45.49，P < 0.001）和p⁃IκB/IκB表达水平（F = 60.87，P < 0.001）差异均有统计学意义，LPS/D⁃GaIN+rSj⁃Cys治疗组小鼠肾脏NF⁃κB信号通路相关蛋白表达水平均低于LPS/D⁃GaIN组（P均< 0.01）。结论　rSj⁃Cys可通过抑制炎症和焦亡下调NF⁃κB通路，从而发挥对急性肝衰竭所致的急性肾损伤的保护作用。

关键词: 日本血吸虫, 半胱氨酸蛋白酶抑制剂, 急性肝衰竭, 急性肾损伤, 焦亡, 核因子κB