南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (6): 1126-1134.doi: 10.12122/j.issn.1673-4254.2024.06.13

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重组日本血吸虫半胱氨酸蛋白酶抑制剂对急性肝损伤小鼠的保护作用及机制

鲁玲君1(), 杨小迪2, 张华平3, 梁媛1, 石秀兰4, 周鑫1()   

  1. 1.山西医科大学病理生理教研室，山西 太原 030002
    2.蚌埠医科大学人体寄生虫学教研室//安徽省感染与免疫重点实验室，安徽 蚌埠 233030
    3.鹤壁职业技术学院医学检验教研室，河南 鹤壁 458030
    4.太原市第三人民医院内镜室，山西 太原 030012
  • 收稿日期:2024-02-17 出版日期:2024-06-20 发布日期:2024-07-01
  • 通讯作者: 周鑫 E-mail:lxx72308102@163.com;xinxin6633@yeah.net
  • 作者简介:鲁玲君，在读硕士研究生，E-mail: lxx72308102@163.com
  • 基金资助:
    山西省回国留学人员科研资助项目(2020-074);山西省应用基础研究计划(201901D111196);山西省科技厅基础研究计划(202303021221127);山西省回国留学人员科研资助项目(2020-175)

Recombinant Schistosoma japonicum cystatin alleviates acute liver injury in mice by inhibiting endoplasmic reticulum stress, inflammation and hepatocyte apoptosis

Lingjun LU1(), Xiaodi YANG2, Huaping ZHANG3, Yuan LIANG1, Xiulan SHI4, Xin ZHOU1()   

  1. 1.Department of Pathology and Physiology, Shanxi Medical University, Taiyuan 030002, China
    2.Department of Human Parasitology, Anhui Provincial Key Laboratory of Infection and Immunology, Bengbu Medical University, Bengbu 233030, China
    3.Department of Medical Laboratory, Hebi Vocational and Technical College, Hebi 458030, China
    4.Department of Endoscopy, Taiyuan Third People's Hospital, Taiyuan 030012, China
  • Received:2024-02-17 Online:2024-06-20 Published:2024-07-01
  • Contact: Xin ZHOU E-mail:lxx72308102@163.com;xinxin6633@yeah.net

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摘要：

目的 探讨重组日本血吸虫半胱氨酸蛋白酶抑制剂对LPS/D-GalN诱导的急性肝损伤小鼠的保护作用及机制。 方法 将72只雄性C57BL/6J小鼠（6~8周龄）随机分为正常对照组、LPS/D-GaIN模型组、LPS/D-GaIN+rSj-Cys治疗组和rSj-Cys对照组（n=18）。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，每组随机挑选8只小鼠处死，收集小鼠血清及肝组织，进行后续检测，每组剩余10只分别在3、6、12、24 h观察其生存情况，并计算生存率。检测血清丙氨酸转移酶（ALT）、天冬氨酸转移酶（AST）水平，苏木精-伊红（HE）染色观察各组小鼠肝脏组织病理形态，采用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肝组织巨噬细胞表面标记物CD68表达水平，采用免疫组化和免疫印迹法检测肝组织Bax、Bcl-2蛋白水平，采用TUNEL检测肝细胞凋亡情况，采用免疫印迹法检测肝组织内质网应激相关蛋白表达水平。 结果 模型组小鼠12 h生存率为30%，rSj-Cys治疗组小鼠12 h生存率为80%。模型组小鼠24 h生存率为10%，rSj-Cys治疗组小鼠24 h生存率为60%；与正常对照组相比，LPS/D-GaIN模型组小鼠血清中AST、ALT、IL-6、TNF-α含量均显著上升（P<0.01），病理结构损伤严重，肝脏巨噬细胞标志物CD68表达明显增强（P<0.01），促凋亡蛋白Bax表达显著增加（P<0.01），抗凋亡蛋白Bcl-2表达显著降低（P<0.01），肝细胞凋亡水平显著增加，肝组织内质网应激相关信号通路GRP78、CHOP、NF-κB p-p65的蛋白表达水平显著上调（P<0.05或P<0.01）；而LPS/D-GaIN+rSj-Cys治疗组小鼠转氨酶AST、ALT和炎症因子IL-6、TNF-α水平显著下降（P<0.01），肝脏病理损伤程度减轻，肝脏巨噬细胞标志物CD68表达明显降低（P<0.01），Bax表达显著降低（P<0.01），Bcl-2表达显著增强（P<0.01），肝组织内质网应激相关信号通路GRP78、CHOP、NF-κB p-p65的蛋白表达水平下调（P<0.05或P<0.01）；rSj-Cys对照组与正常对照组相比，各指标无统计学差异（P>0.05）。 结论 rSj-Cys通过抑制内质网应激，减轻炎症和肝细胞凋亡缓解LPS/D-GalN引起的小鼠急性肝损伤。

关键词: 急性肝损伤, 日本血吸虫半胱氨酸蛋白酶抑制剂, 内质网应激, 炎症, 凋亡

Abstract:

Objective To investigate the protective effect of recombinant Schistosoma japonicum cystatin (rSj-Cys) against acute liver injury induced by lipopolysaccharide (LPS) and D-GalN in mice. Methods Adult male C57BL/6J mice with or without LPS/D-GaIN-induced acute liver injury were given intraperitoneal injections of rSj-Cys or PBS 30 min after modeling (n=18), and serum and liver tissues samples were collected from 8 mice in each group 6 h after modeling. The survival of the remaining 10 mice in each group within 24 h was observed. Serum levels of ALT, AST, TNF‑α and IL-6 of the mice were measured, and liver pathologies was observed with HE staining. The hepatic expressions of macrophage marker CD68, Bax, Bcl-2 and endoplasmic reticulum stress (ERS)-related proteins were detected using immunohistochemistry or immunoblotting, and TUNEL staining was used to detect hepatocyte apoptosis. Results The survival rates of PBS- and rSj-Cys-treated mouse models of acute liver injury were 30% and 80% at 12 h and were 10% and 60% at 24 h after modeling, respectively; no death occurred in the two control groups within 24 h. The mouse models showed significantly increased serum levels of AST, ALT, IL-6 and TNF-α and serious liver pathologies with increased hepatic expressions of CD68 and Bax, lowered expression of Bcl-2, increased hepatocyte apoptosis, and up-regulated expressions of ERS-related signaling pathway proteins GRP78, CHOP and NF-κB p-p65. Treatment of the mouse models significantly lowered the levels of AST, ALT, IL-6 and TNF‑α, alleviated liver pathologies, reduced hepatic expressions of CD68, Bax, GRP78, CHOP and NF‑κB p-p65, and enhanced the expression of Bcl-2. In the normal control mice, rSj-Cys injection did not produce any significant changes in these parameters compared with PBS. Conclusion rSj-Cys alleviates LPS/D-GalN-induced acute liver injury in mice by suppressing ERS, attenuating inflammation and inhibiting hepatocyte apoptosis.

Key words: acute liver injury, Schistosoma japonicum cystatin, endoplasmic reticulum stress, inflammatory response, apoptosis