Objectives: To evaluate the protective effect of Schistosoma japonicum cystatin (rSj-Cystatin) in a mouse mode of "two-hit" sepsis.
Methods: Sixty male C57BL/6 mice randomized equally into sham-operated group, protein group, "two-hit" modeling group, and protein intervention group. In the former two groups, the mice received an intraperitoneal injection of 100 μL PBS followed by exposure of the cecum and then by intraperitoneal injection of 100 μL PBS or 25 μg rSj-Cystatin 30 min later; In the latter two groups, 100 μL PBS containing LPS (5 mg/kg) was injected intraperitoneally 24 h before cecal ligation and puncture (CLP), and 100 μL PBS or 25 μg rSj-Cystatin were injected 30 min after CLP. At 12 h after rSj-Cystatin treatment, 6 mice from each group were sacrificed for detection of TNF-α, IL-6, IL-10, TGF-β, iNOS and Arg-1 in the serum, spleen, liver, lung and kidney tissues using ELISA, for examinations of liver, lung and kidney pathologies with HE staining, and for analysis of CD3+CD4+CD25+Foxp3+ T cell percentage in the spleen using flow cytometry. The remaining mice were observed for general condition and 72-h survival.
Results: The 72-h survival rates in the 4 groups were 100%, 100%, 0% and 20%, respectively, showing significant differences between the latter two groups. The mouse models of "two-hit" sepsis exhibited obvious tissue pathologies and significant elevations of TNF-α and IL-6 in both the serum and tissue homogenate, which were significantly ameliorated by rSj-Cystatin treatment. Treatment with rSj-Cystatin also increased IL-10 and TGF-β levels and spleen CD3+CD4+CD25+Foxp3+ T cell percentage. The septic mouse models also showed increased iNOS levels in all the detected tissues and a decreased Arg-1 level in the kidney, and these changes were obviously improved by rSj-Cystatin treatment.
Conclusions: rSj-Cystatin has a protective effect against "two-hit" sepsis in mice by regulating the inflammatory microenvironment.
目的: 探讨重组日本血吸虫半胱氨酸蛋白酶抑制剂（rSj-Cystatin）对“二次打击”脓毒症小鼠的干预作用。方法: 64只C57BL/6雄性小鼠随机分为假手术组（A组）、蛋白组（B组）、“二次打击”造模组（C组）、蛋白干预组（D组），16只/组。A组和B组小鼠首先腹腔注射PBS（100 μL/只），24 h后开腹探查盲肠，但不进行盲肠结扎和穿刺（CLP），术后30 min，分别腹腔注射PBS（100 μL/只）或含25 μg rSj-Cystatin 蛋白的PBS（100 μL/只）；C组和D组小鼠首先腹腔注射含LPS（5 mg/kg）的PBS（100 μL/只），24 h后行 CLP手术，术后30 min，分别腹腔注射PBS（100 μL/只）或含25 μg rSj-Cystatin 蛋白的PBS（100 μL/只）。每组随机抽取6只，造模12 h后取小鼠血清、脾、肝、肺和肾组织。肝、肺和肾组织HE染色观察其病理损伤并进行评分；酶联免疫吸附实验（ELISA）检验血清和肝、肺、肾组织匀浆中肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、白细胞介素-10（IL-10）和转化生长因子-β（TGF-β）水平以及组织中巨噬细胞极化标志物iNOS和Arg-1水平；流式细胞术检测脾脏CD3+CD4+CD25+Foxp3+T的比例变化；剩余小鼠造模后记录72 h生存率并观察小鼠状态。结果: 与A、B组（100%）相比，C组（0%）72 h生存率降低，经rSj-Cystatin蛋白治疗后，D组生存率较C组增高（20%）。与A、B两组相比，C组肝、肺、肾组织切片病理损伤加重，血清和组织匀浆中TNF-α、IL-6水平明显升高（P<0.05）；经蛋白治疗后，病理损伤程度减轻，血清和组织匀浆中TNF-α、IL-6水平降低（P<0.05），调节因子IL-10、TGF-β水平明显升高（P<0.05），同时CD3+CD4+CD25+Foxp3+T比例升高（P<0.05）；与A、B两组相比，C组各器官组织中iNOS水平均升高，而Arg-1水平呈差异性，仅在肾中呈下降趋势（P<0.01）；与C组相比，D组iNOS水平下降（P<0.05），Arg-1水平升高（P<0.001）。结论: rSj-Cystatin可通过调节炎症微环境进而对 “二次打击”脓毒症起到明显的治疗作用。.
Keywords: Schistosoma japonicum cysteine protease inhibitors; cecal ligation and puncture; immunoregulatory cytokines; lipopolysaccharide; sepsis.