School of Medicine, Shihezi University, Shihezi, 832008, Xinjiang, China; Department of Hepatobiliary Surgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, 832008, Xinjiang, China.
School of Medicine, Shihezi University, Shihezi, 832008, Xinjiang, China.
Mol Biochem Parasitol. 2022 Mar;248:111455. doi: 10.1016/j.molbiopara.2022.111455. Epub 2022 Jan 10.
The study aimed to investigate the expression of cytokeratin and apoptosis-related molecules in the livers of two types of hepatic echinococcosis mice models and to preliminarily explore the relationship between the expression of cytokeratin and apoptosis in echinococcosis related liver injury. We established a mouse model infected by Echinococcus granulosus and Echinococcus multilocularis and observed the expression of cytokeratin and apoptosis related proteins in the two types of hepatic echinococcosis tissues during different stages by immunohistochemical staining. A co-culture model was established using normal hepatocytes and different concentrations of E. granulosus and E. multilocularis protoscoleces. Cell Counting Kit-8 was used to detect cell proliferation, flow cytometry was used to detect hepatocyte apoptosis, and western blot was used to quantify cytokeratin and apoptosis-related proteins, such as caspase3, caspase9, Bcl-2, and Bax. Surgical specimens were obtained from patients with hepatic echinococcosis to analyze the expressions of cytokeratin, caspase3, caspase9, Bcl-2, and Bax by western blot. The expressions of cytokeratin and caspase3 were analyzed by immunohistochemistry. The qRT-PCR method was used to determine the expression of CK8 and CK18 in the liver tissues. In vivo experiments showed that compared to that in the control group, the cytokeratin and caspase3 proteins in the liver tissues of the two types of hepatic echinococcosis were strongly expressed around the lesions of liver echinococcosis; there was a difference between cytokeratin expression of the two different echinococcosis parasites in the liver. Echinococcus granulosus and Echinococcus multilocularis in the co-culture model in vitro could promote the expression of CK, caspase3, caspase9, and Bax protein, decrease the expression of Bcl-2, promote hepatocyte apoptosis, and inhibit cell proliferation; in clinical samples, we found that compared with that in the normal tissues, the expression of cytokeratin, caspase3, caspase9, and Bax in echinococcus tissues was high, but that in Bcl-2 was low. Furthermore, the expression of CK8 and CK18 mRNA were higher in echinococcus tissues than that in the normal tissues and immunohistochemistry analysis also showed that cytokeratin and caspase3 levels were higher in echinococcus tissues than that in the normal tissues. The expression of cytokeratin and apoptosis-related molecules, reflecting liver damage, is high in the liver and is caused due to hepatic echinococcosis. This study provides the first evidence of cytokeratin could be useful for evaluating liver tissue damage caused by echinococcus infection.
本研究旨在探讨两种肝包虫病小鼠模型中细胞角蛋白和凋亡相关分子的表达，并初步探讨细胞角蛋白与包虫病相关肝损伤中凋亡的关系。我们建立了感染细粒棘球蚴和泡型包虫的小鼠模型，通过免疫组织化学染色观察两种肝包虫病组织在不同阶段细胞角蛋白和凋亡相关蛋白的表达。建立了用不同浓度的细粒棘球蚴和泡型包虫原头蚴与正常肝细胞共培养的模型。用细胞计数试剂盒-8 检测细胞增殖，用流式细胞术检测肝细胞凋亡，用 Western blot 定量分析细胞角蛋白和凋亡相关蛋白，如 caspase3、caspase9、Bcl-2 和 Bax。从肝包虫病患者手术标本中分析细胞角蛋白、caspase3、caspase9、Bcl-2 和 Bax 的 Western blot 表达。用免疫组织化学分析细胞角蛋白和 caspase3 的表达。用 qRT-PCR 法测定肝组织 CK8 和 CK18 的表达。体内实验结果表明，与对照组相比，两种肝包虫病肝组织病变周围细胞角蛋白和 caspase3 蛋白表达较强；两种不同包虫寄生虫在肝内的细胞角蛋白表达存在差异。体外共培养模型中细粒棘球蚴和泡型包虫能促进 CK、caspase3、caspase9 和 Bax 蛋白的表达，降低 Bcl-2 的表达，促进肝细胞凋亡，抑制细胞增殖；在临床标本中，我们发现与正常组织相比，包虫组织中细胞角蛋白、caspase3、caspase9 和 Bax 的表达较高，而 Bcl-2 的表达较低。此外，包虫组织中 CK8 和 CK18mRNA 的表达高于正常组织，免疫组织化学分析也显示包虫组织中细胞角蛋白和 caspase3 的水平高于正常组织。细胞角蛋白和凋亡相关分子的表达反映了肝损伤，在肝中较高，是由肝包虫病引起的。本研究首次提供了细胞角蛋白可用于评估包虫感染引起的肝组织损伤的证据。
