重组日本血吸虫半胱氨酸蛋白酶抑制剂可通过调节炎症微环境对“二次打击”脓毒症小鼠发挥治疗作用

doi:10.12122/j.issn.1673-4254.2025.01.14

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (1): 110-117.doi: 10.12122/j.issn.1673-4254.2025.01.14

重组日本血吸虫半胱氨酸蛋白酶抑制剂可通过调节炎症微环境对“二次打击”脓毒症小鼠发挥治疗作用

多文娟¹^,²^,⁴(), 王怡祥¹^,²(), 王家兴¹, 许鑫龙¹, 李林献¹, 杨栋臣¹, 申启利³, 杨立春³, 刘晓静¹, 金启旺¹^,², 褚亮³(), 杨小迪¹^,²()

^1.蚌埠医科大学基础医学院，安徽蚌埠 233030
^2.安徽省感染与免疫重点实验室，安徽蚌埠 233030
^3.蚌埠医科大学第二附属医院普外科，安徽蚌埠 233030
^4.南京紫金医院，江苏南京 210000

收稿日期:2024-09-15 出版日期:2025-01-20 发布日期:2025-01-20
通讯作者: 褚亮,杨小迪 E-mail:18332762023@163.com;1506214487@qq.com;chew8151@163.com;yxd_qf@bbmc.edu.cn
作者简介:多文娟，硕士，E-mail: 18332762023@163.com
王怡祥，在读硕士研究生，E-mail: 1506214487@qq.com
第一联系人：（多文娟、王怡祥并列第一作者）
基金资助:
国家级大学生创新训练项目(202210367068);蚌埠医科大学自然科学重点(2023byzd028);蚌埠医科大学研究生科研创新计划项目(Byycx22011);省级大学生创新训练项目(S202310367045)

Schistosoma japonicum cystatin has protective effects against "two-hit" sepsis in mice by regulating the inflammatory microenvironment

Wenjuan DUO¹^,²^,⁴(), Yixiang WANG¹^,²(), Jiaxing WANG¹, Xinlong XU¹, Linxian LI¹, Dongchen YANG¹, Qili SHEN³, Lichun YANG³, Xiaojing LIU¹, Qiwang JING¹^,², Liang CHU³(), Xiaodi YANG¹^,²()

^1.School of Basic Medical Sciences, Bengbu Medical University, Bengbu 233030, China
^2.Anhui Provincial Key Laboratory of Infection and Immunity, Bengbu 233030, China
^3.Department of General Surgery, Second Affiliated Hospital of Bengbu Medical University, Bengbu 233030, China
^4.Nanjing Zijin Hospital, Nanjing 210000, China

Received:2024-09-15 Online:2025-01-20 Published:2025-01-20
Contact: Liang CHU, Xiaodi YANG E-mail:18332762023@163.com;1506214487@qq.com;chew8151@163.com;yxd_qf@bbmc.edu.cn

摘要/Abstract

摘要：

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。

关键词: 重组日本血吸虫半胱氨酸蛋白酶抑制剂, 脂多糖, 盲肠结扎穿刺, 脓毒症, 免疫调节因子

Abstract:

Objective 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. Conclusion rSj-Cystatin has a protective effect against "two-hit" sepsis in mice by regulating the inflammatory microenvironment.

Key words: Schistosoma japonicum cysteine protease inhibitors, lipopolysaccharide, cecal ligation and puncture, sepsis, immunoregulatory cytokines

多文娟, 王怡祥, 王家兴, 许鑫龙, 李林献, 杨栋臣, 申启利, 杨立春, 刘晓静, 金启旺, 褚亮, 杨小迪. 重组日本血吸虫半胱氨酸蛋白酶抑制剂可通过调节炎症微环境对“二次打击”脓毒症小鼠发挥治疗作用[J]. 南方医科大学学报, 2025, 45(1): 110-117.

Wenjuan DUO, Yixiang WANG, Jiaxing WANG, Xinlong XU, Linxian LI, Dongchen YANG, Qili SHEN, Lichun YANG, Xiaojing LIU, Qiwang JING, Liang CHU, Xiaodi YANG. Schistosoma japonicum cystatin has protective effects against "two-hit" sepsis in mice by regulating the inflammatory microenvironment[J]. Journal of Southern Medical University, 2025, 45(1): 110-117.

图/表 9

表1 肝脏严重程度分级

Tab. 1 Liver injury scoring criteria

Hepatocyte edema, congestion, inflammatory cell infiltration	Index
None	0
0%-25%	1
25%-50%	2
50%-70%	3
75%-100%	4

表2 肺脏严重程度分级

Tab. 2 Lung injury scoring criteria

Alveolar hemorrhage and septum thickening, inflammatory cell infiltration	Index
None	0
0%-25%	1
25%-50%	2
50%-70%	3
75%-100%	4

表3 肾脏严重程度分级

Tab. 3 Kidney injury scoring criteria

Injured renal tubules and shrunk glomerulu	Index
None	0
0%-25%	1
25%-50%	2
50%-70%	3
75%-100%	4

图1 各实验组小鼠72 h生存率

Fig.1 Survival of the mice within 72 h in each group (n=10; **P<0.01, ***P<0.001).

图2 各组小鼠的病理检查情况

Fig.2 Histopathological staining and pathological injury scoring of mice in each group. ***P<0.001 vs Group C.

图3 各实验组小鼠脾脏CD3+CD4+CD25+Foxp3+T细胞的比例

Fig.3 Proportion of CD3+CD4+CD25+Foxp3+ T cells in the spleen of mice in each experimental group. Representative three-dimension scatter diagrams of CD3+CD4+CD25+Foxp3+ T cells in the spleen of mice in each group. The percentage of CD3+CD4+CD25+Foxp3+ T cells in the splenocytes of mice in each group, *P<0.05, **P<0.01 vs Group C.

图4 各实验组小鼠血清中细胞因子的变化

Fig.4 Changes of serum levels of cytokines in each group. **P<0.01, ***P<0.001 vs Group C.

图5 各实验组小鼠肝脏、肺脏和肾脏组织局部炎症因子分泌水平

Fig.5 Levels of inflammatory factors in the liver, lung, and kidney tissues of the mice in each group. *P<0.05, **P<0.01, ***P<0.001 vs Group C.

图6 各实验组小鼠肝脏、肺脏和肾脏组织中M1型和M2型标志物iNOS和Arg-1表达水平

Fig.6 Expression of M1 and M2 type biomarkers iNOS and Arg-1 in the liver, lungs, and kidney tissues in each group. *P<0.05, **P<0.01, ***P<0.001 vs Group C.

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重组日本血吸虫半胱氨酸蛋白酶抑制剂可通过调节炎症微环境对“二次打击”脓毒症小鼠发挥治疗作用

Schistosoma japonicum cystatin has protective effects against "two-hit" sepsis in mice by regulating the inflammatory microenvironment

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