华支睾吸虫感染对小鼠肝纤维化和免疫调节功能的影响

doi:10.12140/j.issn.1000-7423.2023.06.015

中国寄生虫学与寄生虫病杂志 ›› 2023, Vol. 41 ›› Issue (6): 760-765.doi: 10.12140/j.issn.1000-7423.2023.06.015

华支睾吸虫感染对小鼠肝纤维化和免疫调节功能的影响

赵磊¹(), 李佳¹, 莫刚¹, 李醇¹, 黄国洋¹, 彭小红¹^,²^,^*()

1 广西教育厅病原生物学重点实验室，桂林 541004
2 桂林医学院附属医院广西肝脏损伤与修复分子医学重点实验室，广西桂林 541004

收稿日期:2023-06-21 修回日期:2023-07-18 出版日期:2023-12-30 发布日期:2023-12-26
通讯作者: * 彭小红（1982-），男，博士，副教授，从事寄生虫与宿主免疫相关研究。E-mail：pxh815@163.com
作者简介:赵磊（1995-），男，博士研究生，从事寄生虫与宿主免疫相关研究。E-mail：zll431@163.com
基金资助:
国家自然科学基金(82060376);广西基础医学协同创新研究生联合培养基地建设项目(桂学位[2020]7号)

Effect of Clonorchis sinensis infection on hepatic fibrosis and immune regulation in mice

ZHAO Lei¹(), LI Jia¹, MO Gang¹, LI Chun¹, HUANG Guoyang¹, PENG Xiaohong¹^,²^,^*()

1 Guangxi University Key Laboratory of Pathogenic Biology, Guilin 541004, China
2 Guangxi Key Laboratory of Molecular Medicine in Liver Injury and Repair, the Affiliated Hospital of Guilin Medical University, Guilin 541004, China

Received:2023-06-21 Revised:2023-07-18 Online:2023-12-30 Published:2023-12-26
Contact: * E-mail: pxh815@163.com
Supported by:
National Science Foundation of China(82060376);Guangxi Province Postgraduate Co-training Base for Cooperative Innovation in Basic Medicine (Gui Xue Wei [2020]7)

摘要/Abstract

摘要：

为进一步了解小鼠感染华支睾吸虫过程中的肝纤维化情况和免疫调节变化，将BALB/c小鼠随机分成感染组和对照组，每组30只，感染组小鼠胃饲200 μl含150个华支睾吸虫囊蚴的生理盐水，对照组小鼠灌胃同体积生理盐水，于感染后第1、2、4、8和16周随机各取6只，称体质量后取肝、脾和血清。取小鼠肝组织，石蜡切片后进行苏木素-伊红（HE）染色和Masson染色，观察胶原纤维沉积情况。小鼠肝组织总RNA逆转录后qRT-PCR检测肌成纤维细胞标志α-肌动蛋白（ACTA2）基因的相对转录水平，蛋白质免疫印迹（Western blotting）检测肝组织ACTA2蛋白的相对表达量。取小鼠脾组织，称重并计算每10 g体质量的脾质量（脾脏指数，mg/10 g）。流式细胞术分析小鼠脾CD4⁺ T细胞、辅助性T1（Th1）细胞、Th2细胞、Th17细胞和调节性T（Treg）细胞的动态变化。取小鼠血清，使用流式细胞术检测肿瘤坏死因子（TNF）、白细胞介素6（IL-6）、γ干扰素（IFN-γ）、IL-2、IL-4、IL-10和IL-17A等细胞因子的分泌水平。使用GraphPad Prism 9.0软件进行数据分析。组间比较采用双向方差分析，两两比较采用t检验。结果显示，小鼠感染华支睾吸虫囊蚴后体质量降低、脾指数升高，感染后各时期肝组织皆可见炎症病灶。HE染色结果显示，小鼠感染华支睾吸虫囊蚴后第1周，肝胆管周围可见华支睾吸虫幼虫，幼虫周围出现炎症细胞浸润。Masson染色结果显示，小鼠感染华支睾吸虫囊蚴后各时期，胶原纤维沉积相对面积均高于对照组（F = 20.190，P < 0.05）。qRT-PCR结果显示，感染后第2、4和16周，感染组小鼠肝组织ACTA2基因相对转录水平均高于对照组（t = 2.042、2.475、1.634，均P < 0.01）；第8周低于对照组（t = 2.758，P < 0.05）。Western blotting检测结果显示，感染后各时期，感染组小鼠肝脏ACTA2蛋白相对表达量均高于对照组（F = 3.225，P < 0.01）。流式细胞术结果显示，感染组小鼠脾CD4⁺ T细胞占比在感染后第2周降低至（13.4 ± 1.8）%，16周升高至（22.4 ± 1.5）%；Th1细胞占比在感染后第2周升至（16.9 ± 5.3）%，第16周降低至（3.9 ± 2.6）%；Th2细胞占比在感染后第4周降低至（2.3 ± 0.6）%，第4周后逐渐升高；Th17细胞占比在第8周时升至（5.3 ± 3.4）%，第16周下降至（2.4 ± 1.4）%；Treg细胞占比在第4周降至（7.3 ± 1.5）%，第16周升至（13.9 ± 1.2）%。感染组小鼠血清TNF水平在感染后第1周时升至（35.16 ± 11.28） pg/ml，第8周起降至正常水平[（8.98 ± 1.66） pg/ml]；血清IL-6水平在感染后各时期均高于对照组（t = 2.095，P < 0.05）；血清IL-2水平在感染后第8周降至（0.09 ± 0.18） pg/ml，但在第16周升高至（3.81 ± 2.79） pg/ml；感染组小鼠血清IFN-γ、IL-4、IL-10和IL-17A水平随着感染时间延长而逐渐升高（F = 8.726、8.068、6.795、14.840，均P < 0.05）。本研究结果提示，CD4⁺ T细胞和血清细胞因子的动态变化与华支睾吸虫诱导肝纤维化的发生发展密切相关。

关键词: 华支睾吸虫, 肝纤维化, 脾CD4⁺ T细胞, 动态变化

Abstract:

In order to further understand the liver fibrosis and immune regulatory changes in mice infected with Clonorchis sinensis, BALB/c mice were randomly divided into 2 groups with 30 mice in each group. The infected group were administered 200 μl normal saline with 150 C. sinensis metacercariae through oral gavage and the control group received 200 μl normal saline. Six mice were randomly selected for dissection at 1, 2, 4, 8 and 16 weeks. The paraffin sections of the liver tissue were stained with hematoxylin-eosin (HE) and Masson to observe the progression of fibrosis. The relative transcription level and actin alpha 2 (ACTA2) expression in the liver were detected by qRT-PCR and western blotting separately. The spleen tissue were weighed for calculating the spleen index (spleen weight per 10 g weight, mg/10 g). The flow cytometry was performed to detect the dynamical changes of CD4⁺ T cells, helper T1 (Th1) cells, Th2 cells, Th17 cells and regulatory T (Treg) cells in spleen. The secretion levels of tumor necrosis factor (TNF), interleukin-6 (IL-6), interferon-γ (IFN-γ), IL-2, IL-4, IL-10, and IL-17A were quantified by cytometric bead array. The data was analyzed by GraphPad Prism 9.0. Two-way ANOVA was used for groups comparison and t test was used for pairwise comparison. The results showed that the mice weight decreased and spleen index increased after infected with C. sinensis and the inflammatory lesions were visible in liver tissue. HE staining showed that C. sinensis larvae and inflammatory cells could be found around the hepatobiliary duct at week 1 after infection. Masson staining revealed that the relative area of collagen fiber deposition was higher than the control group after infection (F = 20.190, P < 0.05). The qRT-PCR results showed that the hepatic ACTA2 relative transcription level of the infected group were higher than the control group at week 2, 4 and 16 after infection (t = 2.042, 2.475, 1.634; all P < 0.01) but lower than the control group at week 8 (t = 2.758, P < 0.05). Western blotting results showed that the hepatic ACTA2 protein relative expression level of the infected group were higher than the control group (F = 3.225, P < 0.01). The flow cytometry results showed that the percentage of splenic CD4⁺ T cells decreased to (13.4 ± 1.8)% at week 2 after infection and increased to (22.4 ± 1.5)% at week 16. The percentage of Th1 cells increased to (16.9 ± 5.3)% at week 2 and decreased to (3.9 ± 2.6)% at week 16. The percentage of Th2 cells decreased to (2.3 ± 0.6)% at week 4 and increased gradually after week 4. The percentage of Th17 cells increased to (5.3 ± 3.4)% at week 8 and decreased to (2.4 ± 1.4)% at week 16. The percentage of Treg cells decreased to (7.3 ± 1.5)% at week 4 and increased to (13.9 ± 1.2)% at week 16. The serum TNF content of infected group mice increased to (35.16 ± 11.28) pg/ml at week 1 after infection and decreased to (8.98 ± 1.66) pg/ml at week 8. The serum IL-6 content was higher than the control group after infection (t = 2.095, P < 0.05). The serum IL-2 content decreased to (0.09 ± 0.18) pg/ml at week 8 and increased to (3.81 ± 2.79) pg/ml at week 16 after infection. The content of serum IFN-γ, IL-4, IL-10 and IL-17A increased gradually after infection (F = 8.726, 8.068, 6.795, 14.840; all P < 0.05). As a conclusion, the dynamic changes of CD4⁺ T cells and serum cytokines were closely related to the hepatic fibrosis induced by C. sinensis.

Key words: Clonorchis sinensis, Hepatic fibrosis, Splenic CD4⁺ T cells, Dynamic change

中图分类号:

R532.23

赵磊, 李佳, 莫刚, 李醇, 黄国洋, 彭小红. 华支睾吸虫感染对小鼠肝纤维化和免疫调节功能的影响[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(6): 760-765.

ZHAO Lei, LI Jia, MO Gang, LI Chun, HUANG Guoyang, PENG Xiaohong. Effect of Clonorchis sinensis infection on hepatic fibrosis and immune regulation in mice[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(6): 760-765.

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小鼠组别	感染后时间/周	体质量/g	肝湿重/g	脾湿重/mg	脾指数/mg·(10 g)^-1
感染组	1	22.55 ± 0.96	1.20 ± 0.12	173.3 ± 28.1	7.69 ± 1.22
	2	23.38 ± 1.22	1.43 ± 0.25	210.0 ± 42.9	8.72 ± 1.05
	4	25.40 ± 0.85	1.76 ± 0.66	240.0 ± 101.7	9.43 ± 3.88
	8	25.97 ± 1.56	1.56 ± 0.36	181.7 ± 45.8	6.95 ± 1.49
	16	25.99 ± 2.23	1.20 ± 0.15	118.8 ± 15.5	4.59 ± 0.62
对照组	1	22.70 ± 1.32	1.02 ± 0.35	088.6 ± 6.9	3.98 ± 0.36
	2	23.12 ± 0.88	1.03 ± 0.03	087.1 ± 7.6	3.75 ± 0.38
	4	25.64 ± 1.13	1.02 ± 0.03	106.0 ± 18.2	4.16 ± 0.85
	8	24.80 ± 0.80	1.03 ± 0.05	091.7 ± 9.8	3.69 ± 0.41
	16	27.41 ± 1.34	1.13 ± 0.09	091.3 ± 9.9	3.35 ± 0.26

华支睾吸虫感染对小鼠肝纤维化和免疫调节功能的影响

Effect of Clonorchis sinensis infection on hepatic fibrosis and immune regulation in mice

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