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Title: [Protective effect of recombinant Schistosoma japonicum cystatin against acute kidney injury associated with acute liver failure in mice].
Author: Xue Y, Yang X, Zhang H, Zhang T, Chen W, Chang X, Wang Y.
Journal: Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi; 2023 Oct 10; 35(4):331-339. PubMed ID: 37926467.
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). CONCLUSIONS: 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. ［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。.

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