Pathological changes in the formation of Echinococcus granulosus cysts in sheep liver at different stages

doi:10.12140/j.issn.1000-7423.2024.03.006

Abstract

Abstract:

Objective To observe the pathological characteristics of hepatocyte damage, local inflammation response, and the composition of cyst fibrin in liver of Echinococcus granulosus infected sheep during the cyst formation at different stages. Methods Livers with significant E. granulosus cysts were collected from slaughtered Altai sheep in an abattoir in Urumqi City, Xinjiang, China. From the sampled livers, cysts were separated and assigned into the groups of development stage, formation stage and mature stage according to the morphological features of the cysts. The cysts of all groups and the lesion tissues at the junction with healthy liver tissues were used to prepare paraffin sections. Liver tissues from healthy sheep were used as the control. The morphological features and changes of the hepatocytes and liver tissues around the cysts were observed by hematoxylin-eosin (HE) staining, and the fibrosis process of the cyst wall at different formation stages was observed by Masson staining. Transmission electron microscope was used to observe the ultrastructural changes of hepatocytes. Immunohistochemical staining was used to observe the cysts and surrounding tissues at different cyst-formation stages for the changes of inflammatory cells CD3⁺ T lymphocytes (CD3⁺), CD25⁺ regulatory T cells (CD25⁺), CD56⁺ NK cells (CD56⁺), CD14⁺ monocyte macrophages (CD14⁺) and basophilic granulocytes CD63 (CD63), as well as the changes of expression of fibrotic proteins collagen type I (COL1), COL3, alpha-smooth muscle actin (a-SMA), calcium-binding protein A4 (S100A4), matrix metalloproteinase 2 (MMP2) and tumour necrosis factor-alpha (TNF-α), and vascular endothelial growth factor receptor-3 (VEGFR-3). One-way ANOVA analysis was used for comparison between groups, and the LSD-t test was used for pairwise comparison. Results The HE staining results showed that the liver exhibited significant inflammatory reaction bands and inflammatory cell clusters in the cyst wall during the encapsulation development stage. The inflammatory cells spread to the liver parenchyma in the periphery of the cyst, and the hepatocytes around the encapsulation atrophied, degenerated, and damaged. During the formation of the encapsulation, the cyst wall became thinner, the number of inflammatory cells decreased, the inflammatory reaction bands became thinner, the encapsulation cavity enlarged. In the maturation stage, fibrous tissue proliferation occurred in the encapsulation wall to form the cuticle, and the number of inflammatory cells decreased. The outer wall of the cyst, known as the stratum corneum, was formed by the proliferation of fibrous tissue. The number of inflammatory cells decreased, resulting in a significant reduction in the inflammatory response. Masson’s staining showed that a large amount of fibrous tissue was produced in the periphery of the cyst wall during the period of cyst development and showed extensive growth into the surrounding liver tissue. The cyst grew and developed during the period of cyst formation, the inflammatory response was attenuated, the fibrous tissue of the cyst wall matured, and a dense, fibrous cyst wall was formed during the period of cyst maturation. Transmission electron microscopy showed that as the cyst developed and formed, the mitochondria of the hepatocytes gradually enlarged and increased in number, and the lipid droplets in the hepatocytes increased in number and size. Immunohistochemical staining showed that as the cyst increases, the inflammatory response zone on the cyst wall becomes thinner, the range of positive expression of inflammatory cells decreases, and the expression level decreases. The mean optical density of the CD3⁺ inflammatory cells at the cyst developmental stage, cyst formation stage, and cyst maturation stage were 0.171 ± 0.009, 0.132 ± 0.009, 0.120 ± 0.006 (F = 1.640, P > 0.05); CD25⁺ cells were 0.302 ± 0.012, 0.174 ± 0.009, and 0.080 ± 0.005 (F = 49.051, P < 0.01); CD56⁺ cells were 0.219 ± 0.008, 0.209 ± 0.009, and 0.118 ± 0.004 (F = 126.411, P < 0.01), CD14⁺ cells were 0.140 ± 0.027, 0.096 ± 0.012, 0.090 ± 0.017 (F = 3.954, P > 0.05); CD63 cells were 0.318 ± 0.007, 0.096 ± 0.013, 0.086 ± 0.011 (F = 307.442, P < 0.01). The positive expression range and expression level of fibroin increased, and the mean optical density of COL1 were 0.139 ± 0.029, 0.157 ± 0.022, 0.186 ± 0.014 (F = 2.136, P > 0.05); COL3 were 0.109 ± 0.014， 0.144 ± 0.008， 0.206 ± 0.008 (F = 42.116, P < 0.01); α-SMA were 0.255 ± 0.008, 0.283 ± 0.009, 0.301 ± 0.022 (F = 5.106, P < 0.05); MMP2 were 0.155 ± 0.002, 0.172 ± 0.011, 0.185 ± 0.008 (F = 7.853, P < 0.05); S100A4 were 0.210 ± 0.012, 0.248 ± 0.004, 0.258 ± 0.007 (F = 18.137 3, P < 0.05). TNF-α was expressed in the surrounding tissue of the cyst, the positive expression range increased, and the expression level increased. the mean optical density of TNF-α were 0.115 ± 0.016, 0.263 ± 0.003, 0.267 ± 0.006 (F = 145.627, P < 0.01). VEGFR-3 VEGFR-3 was expressed in the cyst wall, with the highest expression during the formation of the cyst, and the mean optical density at 3 stages were 0.248 ± 0.009, 0.357 ± 0.045, 0.268 ± 0.004 (F = 9.423, P < 0.05). The mean optical density values in the cyst phase were higher than those in healthy livers (all P < 0.01). Conclusion As the cyst develops, the cyst and its surrounding liver tissue show varying degrees of hepatocellular damage, cyst wall structural changes and fibrotic response, enhanced liver mitochondrial metabolism, lipid metabolism affected, as well as the cyst-surrounding inflammatory cytokines and liver fiber seen in the inflammatory zone of cyst wall.

Key words: Echinococcus granulosus, Cyst, Pathology, Inflammatory cell, Fibrosis, Sheep

CLC Number:

R532.32

HOU Mengdan, JIGU Xiaoan, LIU Weiwei, QIU Meiling, HU Meihe, LI Kunlei, JIAYINAER Jikesanbayi, ZHAI Shaohua. Pathological changes in the formation of Echinococcus granulosus cysts in sheep liver at different stages[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2024, 42(3): 316-324.

Figures/Tables 7

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

References 20

[1]	Wen H, Vuitton L, Tuxun T, et al. Echinococcosis: advances in the 21st century[J]. Clin Microbiol Rev, 2019, 32(2):e00075-18.
[2]	Wu WP, Wang H, Wang Q, et al. A nationwide sampling survey on echinococcosis in China during 2012-2016[J]. Chin J Parasitol Parasit Dis, 2018, 36(1): 1-14. (in Chinese)
	(伍卫平, 王虎, 王谦, 等. 2012—2016年中国棘球蚴病抽样调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 1-14.)
[3]	McManus DP. Immunodiagnosis of sheep infections with Echinococcus granulosus: in 35 years where have we come?[J]. Parasite Immunol, 2014, 36(3): 125-130.
[4]	Li K, Shahzad M. Epidemiology of cystic echinococcosis in China (2004-2016)[J]. Travel Med Infect Dis, 2020, 33: 101466.
[5]	Xiao GL, Zhong Q, Xie WH, et al. Epidemiological investigation on echinococcosis in Kashgar of Xinjiang from 2014 to 2017[J]. China Anim Health Insp, 2019, 36(5): 1-5. (in Chinese)
	(肖国亮, 钟旗, 谢文辉, 等. 2014—2017年新疆喀什地区绵羊包虫病流行病学调查[J]. 中国动物检疫, 2019, 36(5): 1-5.)
[6]	Kodama Y, Fujita N, Shimizu T, et al. Alveolar echinococcosis: MR findings in the liver[J]. Radiology, 2003, 228(1): 172-177.
[7]	Yu YY, Jiang L, Wang H, et al. Hepatic transferrin plays a role in systemic iron homeostasis and liver ferroptosis[J]. Blood, 2020, 136(6): 726-739.
[8]	Wang H, Yu Q, Wang MK, et al. Hepatic macrophages play critical roles in the establishment and growth of hydatid cysts in the liver during Echinococcus granulosus sensu stricto infection[J]. PLoS Negl Trop Dis, 2023, 17(11): e0011746.
[9]	Tsuchida T, Friedman SL. Mechanisms of hepatic stellate cell activation[J]. Nat Rev Gastroenterol Hepatol, 2017, 14(7): 397-411.
[10]	Wu HY, Xiang DB. Advances in vascular endothelial growth factor in tumour angiogenesis[J]. Chin J Clin Oncol Rehabil, 2012, 19(5): 470-471. (in Chinese)
	(吴华英, 向德兵. 血管内皮生长因子在肿瘤血管生成的研究进展[J]. 中国肿瘤临床与康复, 2012, 19(5): 470-471).
[11]	Xie ZZ, Huang Y, Yu XB. Research progress on the mechanism of liver fibrosis caused by parasitic infection[J]. J Trop Med, 2013, 13(9): 1161-1165. (in Chinese)
	(谢芝芝, 黄艳, 余新炳. 寄生虫感染致肝纤维化机制的研究进展[J]. 热带医学杂志, 2013, 13(9): 1161-1165.)
[12]	Li J, Xu SF, Li YY, et al. Effects of xiaochaihu decoction on expressions of TIMP-2 and MMP-2 in rats with hepatic fibrosis[J]. Liaoning J Tradit Chin Med, 2018, 45(5): 1066-1068, 1119. (in Chinese)
	(李晋, 徐尚福, 李远洋, 等. 小柴胡汤对肝纤维化大鼠肝脏MMP-2、 TIMP-2表达的影响[J]. 辽宁中医杂志, 2018, 45(5): 1066-1068, 1119.)
[13]	Zhang YF. Efficiency and safety of 6MV-X ray for treating hydatid disease[D]. Urumqi: Xinjiang Medical University, 2021: 38. (in Chinese)
	(张月芬. 6MV-X线对棘球蚴病的有效性及安全性的临床和实验研究[D]. 乌鲁木齐: 新疆医科大学, 2021: 38.)
[14]	Sun HY, Sun C, Tian ZG, et al. NK cells in immunotolerant organs[J]. Cell Mol Immunol, 2013, 10(3): 202-212.
[15]	Parola M, Pinzani M. Liver fibrosis: pathophysiology, pathogenetic targets and clinical issues[J]. Mol Aspects Med, 2019, 65: 37-55.
[16]	Bosch M, Sánchez-álvarez M, Fajardo A, et al. Mammalian lipid droplets are innate immune hubs integrating cell metabolism and host defense[J]. Science, 2020, 370(6514): eaay8085.
[17]	Zhao XY. Study on cell proliferation around two kinds of Echinococcus in mouse liver at different time points[D]. Shihezi: Shihezi University, 2021: 13. (in Chinese)
	(赵雪源. 小鼠肝内两种棘球蚴不同时间点周围细胞增殖的探究[D]. 石河子: 石河子大学, 2021: 13.)
[18]	Pervin M, Hasan I, Kobir MA, et al. Immunophenotypic analysis of the distribution of hepatic macrophages, lymphocytes and hepatic stellate cells in the adult rat liver[J]. Anat Histol Embryol, 2021, 50(4): 736-745.
[19]	Guo YR, Sun R, Li W, et al. Establishment of sensitization model based on the activation of basophil in mice[J]. J Shandong Univ Health Sci, 2019, 57(3): 31-37. (in Chinese)
	(郭岩乳, 孙蓉, 李伟, 等. 基于小鼠嗜碱性粒细胞活化致敏性模型的建立[J]. 山东大学学报(医学版), 2019, 57(3): 31-37.)
[20]	Yang B, Luo Q, Kang Q, et al. Tumor necrosis factor-α and transforming growth factor-β1 balance liver stem cell differentiation in cholestatic cirrhosis[J]. J South Med Univ, 2018, 38(4): 375-383.
	(杨博, 罗庆, 康权, 等. TNF-α、 TGF-β1在胆汁淤积性肝硬化中平衡调控肝脏干细胞分化[J]. 南方医科大学学报, 2018, 38(4): 375-383.)