Preliminary research on pyroptosis NLRP3<b>/</b>Caspase<b>-</b>1 pathway in mouse model of hepatic alveolar echinococcosis infection_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

NIE Ru ^1,2,3 , LIU Chuanchuan ^1,2 , LI Wendeng ^1,2 , PANG Mingquan ^1,2 , YIN Fengjiao ^1,2,3 , YE Gengbo ^1,2,3 , WANG Zhixin ^1,2 ,  FAN Haining ^1,2

1. Department of Hepatopancreatobiliary Surgery, Affiliated Hospital of Qinghai University, Xining 810001, P. R. China;
2. Qinghai Research Key Laboratory for Echinococcosis, Xining 810001, P. R. China;
3. Graduate School of Qinghai University, Xining 810016, P. R. China;

Corresponding author：

FAN Haining, Email: fanhaining@medmail.com.cn

Keywords：

alveolar echinococcosis; liver; pyroptosis

DOI：

10.7507/1007-9424.202302007

Video：

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Objectives To observe the expression of key proteins in the NLRP3/Caspase-1 pathway of pyroptosis in the mouse model of hepatic Echinococcus multilocularis (Em) infection and explore its correlation. Methods Twenty-five BALB/c mice were randomly divided into the control group and the infected group. The infected group was injected with 0.2 mL suspension of protoscolex (including 3 000 protoscoleces) injected under the liver capsule to establish a model of secondary infection with hepatic Em. The control group was treated without any treatments and conventional feeding was conducted. The mice were sacrificed at 1, 2, 3, and 5 months after infection. The liver was harvested and observed for gross morphology. HE staining and transmission electron microscopy were performed to observe the histopathological changes. The expressions of key proteins in the NLRP3/Caspase-1 pathway of pyroptosis and the IL-1β, a downstream factor of pyroptosis in the liver were detected by immunohistochemistry, Western blot and ELISA. Results Compared with the control group, the cystic lesions on the surface of liver tissues in the infected group mice gradually increased and protruded from the liver surface with the extension of infection time. HE staining showed various pathological changes such as inflammatory cell infiltration and fibrous hyperplasia in the liver lesions to varying degrees. After 2 months of Em infection, transmission electron microscope observation showed that the cell membrane of hepatocytes were broken and discontinuous, conforming to the "punching" phenomenon of pyroptosis. The results of ELISA showed that the concentration of IL-1β in liver homogenate of mice after 1, 2, 3 and 5 months of Em infection were significantly higher than that of the control group, and the difference was statistically significant (F=127.2, P<0.05). Immunohistochemical examination showed that the positive cell ratios of Caspase-1 and NLRP3 in liver of mice infected with Em at 1, 2, 3 and 5 months, were higher than that of the control group, and the difference were statistically significant (F=114.6, P<0.05; F=85.89, P<0.05). The Western blot results showed that the relative expression levels of Caspase-1, Xiaopi D, and NLRP3 proteins in the liver of infected mice showed a trend of first increasing (the expression of Caspase-1 and GSDMD reached their peak at 1 month of infection, while the expression of NLRP3 reached its peak at 2 months of infection) and then decreasing. There were statistically significant differences between the infection groups at different time points and the control group, as well as comparison between the infection groups at different time points there were also statistically significant differences (all P<0.05). Conclusion It is feasible to establish mouse Em infection model by “skin incision and liver puncture through abdominal muscle layer”. There is a new type of programmed cell death, pyroptosis, after Em infection in mouse liver. It may play a role in inflammation amplification through pyroptosis NLRP3/Caspase-1 pathway.

Citation： NIE Ru, LIU Chuanchuan, LI Wendeng, PANG Mingquan, YIN Fengjiao, YE Gengbo, WANG Zhixin, FAN Haining. Preliminary research on pyroptosis NLRP3/Caspase-1 pathway in mouse model of hepatic alveolar echinococcosis infection. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2023, 30(6): 666-673. doi: 10.7507/1007-9424.202302007 Copy

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2.	Deplazes P, Rinaldi L, Alvarez Rojas CA, et al. Global distribution of alveolar and cystic echinococcosis. Adv Parasitol, 2017, 95: 315-493.
3.	Casulli A. Recognising the substantial burden of neglected pandemics cystic and alveolar echinococcosis. Lancet Glob Health, 2020, 8(4): e470-e471. doi: 10.1016/S2214-109X(20)30066-8.
4.	Kaste SC, Pratt CB, Cain AM, et al. Metastases detected at the time of diagnosis of primary pediatric extremity osteosarcoma at diagnosis: imaging features. Cancer, 1999, 86(8): 1602-1608.
5.	de Carvalho RVH, Silva ALN, Santos LL, et al. Macrophage priming is dispensable for NLRP3 inflammasome activation and restriction of Leishmania amazonensis replication. J Leukoc Biol, 2019, 106(3): 631-640.
6.	Kihel A, Hammi I, Darif D, et al. The different faces of the NLRP3 inflammasome in cutaneous Leishmaniasis: A review. Cytokine, 2021, 147: 155248. doi: 10.1016/j.cyto.2020.155248.
7.	卢亚奇. NLRP3炎症小体活化在日本血吸虫感染引起的肝纤维化中的作用及机制研究. 华中科技大学. 2018.
8.	王涛. GSDMD/Caspase-1、Caspase-4、Caspase-5等焦亡相关因子在肝多房包虫病的表达及其意义. 青海大学, 2019.
9.	詹希美. 人体寄生虫学. 第2版. 北京: 人民卫生出版社, 2010: 200-202.
10.	陈颖丹, 周长海, 周晓农, 等. 2015年全国人体重点寄生虫病现状调查分析. 中国寄生虫学与寄生虫病杂志, 2020, 38(1): 5-12.
11.	韩帅, 蒉嫣, 伍卫平, 等. 2004-2020年全国棘球蚴病疫情分析. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 475-480.
12.	付世强, 王志鑫, 樊海宁, 等. 175例晚期肝多房棘球蚴病患者临床特征分析. 中国血吸虫病防治杂志, 2019, 31(2): 204-206,209.
13.	艾麦提·牙森, 温浩. 免疫学在包虫病研究中的应用. 新疆医科大学学报, 2020, 43(6): 830-836.
14.	Chen Y, Smith MR, Thirumalai K, et al. A bacterial invasion induces macrophage apoptosis by binding directly to ICE. EMBO J, 1996, 15(15): 3853-3860.
15.	Kayagaki N, Warming S, Lamkanfi M, et al. Non-canonical inflammasome activation targets caspase-11. Nature, 2011, 479(7371): 117-121.
16.	Shi J, Gao W, Shao F. Pyroptosis: Gasdermin-Mediated Programmed Necrotic Cell Death. Trends Biochem Sci, 2017, 42(4): 245-254.
17.	Qiu S, Liu J, Xing F. 'Hints' in the killer protein gasdermin D: unveiling the secrets of gasdermins driving cell death. Cell Death Differ, 2017, 24(4): 588-596.
18.	Ivanov K, Garanina E, Rizvanov A, et al.Inflammasomes as targets for adjuvants. Pathogens, 2020, 9(4): 252. doi: 10.3390/pathogens9040252.
19.	陈蔡松, 张耀刚, 王志鑫, 等. Nod样受体蛋白3炎症小体在寄生虫病中的研究进展. 中国寄生虫学与寄生虫病杂志, 2020, 38(3): 390-394.
20.	Sborgi L, Rühl S, Mulvihill E, et al. GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death. EMBO J, 2016, 35(16): 1766-1778.
21.	Karmakar M, Minns M, Greenberg EN. et al. N-GSDMD trafficking to neutrophil organelles facilitates IL-1β release independently of plasma membrane pores and pyroptosis. Nat Commun, 2020, 11(1): 2212. doi: 10.1038/s41467-020-16043-9.
22.	Chao-Yang G, Peng C, Hai-Hong Z. Roles of NLRP3 inflammasome in intervertebral disc degeneration. Osteoarthritis Cartilage, 2021, 29(6): 793-801.
23.	潘徐彪, 王宏宾, 王志鑫, 等. NLRP3炎性小体在肝泡型包虫病中表达水平的研究. 中华肝胆外科杂志, 2020, 26(10): 753-756.
24.	Parola M. Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues. Mol Aspects Med, 2019, 65: 37-55.
25.	张伟, 贾继东. 肝纤维化的发病机制及治疗新靶点. 临床肝胆病杂志, 2017, 33(3): 409-412.
26.	张力, 茆军, 张立, 等. 细胞焦亡与组织纤维化. 基础医学与临床, 2018, 38(12): 1766-1770.
27.	Alegre F, Pelegrin P, Feldstein AE. Inflammasomes in liver fibrosis. Semin Liver Dise, 2017, 37(2): 119-127.
28.	Niu F, Chong S, Qin M, et al. Mechanism of fibrosis induced by echinococcus spp. Diseases, 2019 , 7(3): 51. doi: 10.3390/diseases7030051.
29.	张金辉, 温浩, 刘章锁, 等. 原发性肝泡球蚴动物模型的建立. 中国局解手术学杂志, 2000, 9(1): 11-13.
30.	卞志远, 马少波, 夏海洋, 等. 新型肝穿刺建立原发性肝多房棘球蚴病小鼠模型. 中国人兽共患病学报, 2013, 29(5): 452-455.
31.	周启锋, 任利, 张灵强, 等. 肝泡状棘球蚴病动物模型的研究进展. 临床肝胆病杂志, 2017, 33(11): 2247-2250.
32.	吴亮亮, 杨凌菲, 宋涛. 不同方式建立肝多房棘球蚴感染SD大鼠模型病灶的超声及病理表现. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 549-552.

1. Casulli A, Barth TFE, Tamarozzi F. Echinococcus multilocularis. Trends Parasitol, 2019, 35(9): 738-739.
2. Deplazes P, Rinaldi L, Alvarez Rojas CA, et al. Global distribution of alveolar and cystic echinococcosis. Adv Parasitol, 2017, 95: 315-493.
3. Casulli A. Recognising the substantial burden of neglected pandemics cystic and alveolar echinococcosis. Lancet Glob Health, 2020, 8(4): e470-e471. doi: 10.1016/S2214-109X(20)30066-8.
4. Kaste SC, Pratt CB, Cain AM, et al. Metastases detected at the time of diagnosis of primary pediatric extremity osteosarcoma at diagnosis: imaging features. Cancer, 1999, 86(8): 1602-1608.
5. de Carvalho RVH, Silva ALN, Santos LL, et al. Macrophage priming is dispensable for NLRP3 inflammasome activation and restriction of Leishmania amazonensis replication. J Leukoc Biol, 2019, 106(3): 631-640.
6. Kihel A, Hammi I, Darif D, et al. The different faces of the NLRP3 inflammasome in cutaneous Leishmaniasis: A review. Cytokine, 2021, 147: 155248. doi: 10.1016/j.cyto.2020.155248.
7. 卢亚奇. NLRP3炎症小体活化在日本血吸虫感染引起的肝纤维化中的作用及机制研究. 华中科技大学. 2018.
8. 王涛. GSDMD/Caspase-1、Caspase-4、Caspase-5等焦亡相关因子在肝多房包虫病的表达及其意义. 青海大学, 2019.
9. 詹希美. 人体寄生虫学. 第2版. 北京: 人民卫生出版社, 2010: 200-202.
10. 陈颖丹, 周长海, 周晓农, 等. 2015年全国人体重点寄生虫病现状调查分析. 中国寄生虫学与寄生虫病杂志, 2020, 38(1): 5-12.
11. 韩帅, 蒉嫣, 伍卫平, 等. 2004-2020年全国棘球蚴病疫情分析. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 475-480.
12. 付世强, 王志鑫, 樊海宁, 等. 175例晚期肝多房棘球蚴病患者临床特征分析. 中国血吸虫病防治杂志, 2019, 31(2): 204-206,209.
13. 艾麦提·牙森, 温浩. 免疫学在包虫病研究中的应用. 新疆医科大学学报, 2020, 43(6): 830-836.
14. Chen Y, Smith MR, Thirumalai K, et al. A bacterial invasion induces macrophage apoptosis by binding directly to ICE. EMBO J, 1996, 15(15): 3853-3860.
15. Kayagaki N, Warming S, Lamkanfi M, et al. Non-canonical inflammasome activation targets caspase-11. Nature, 2011, 479(7371): 117-121.
16. Shi J, Gao W, Shao F. Pyroptosis: Gasdermin-Mediated Programmed Necrotic Cell Death. Trends Biochem Sci, 2017, 42(4): 245-254.
17. Qiu S, Liu J, Xing F. 'Hints' in the killer protein gasdermin D: unveiling the secrets of gasdermins driving cell death. Cell Death Differ, 2017, 24(4): 588-596.
18. Ivanov K, Garanina E, Rizvanov A, et al.Inflammasomes as targets for adjuvants. Pathogens, 2020, 9(4): 252. doi: 10.3390/pathogens9040252.
19. 陈蔡松, 张耀刚, 王志鑫, 等. Nod样受体蛋白3炎症小体在寄生虫病中的研究进展. 中国寄生虫学与寄生虫病杂志, 2020, 38(3): 390-394.
20. Sborgi L, Rühl S, Mulvihill E, et al. GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death. EMBO J, 2016, 35(16): 1766-1778.
21. Karmakar M, Minns M, Greenberg EN. et al. N-GSDMD trafficking to neutrophil organelles facilitates IL-1β release independently of plasma membrane pores and pyroptosis. Nat Commun, 2020, 11(1): 2212. doi: 10.1038/s41467-020-16043-9.
22. Chao-Yang G, Peng C, Hai-Hong Z. Roles of NLRP3 inflammasome in intervertebral disc degeneration. Osteoarthritis Cartilage, 2021, 29(6): 793-801.
23. 潘徐彪, 王宏宾, 王志鑫, 等. NLRP3炎性小体在肝泡型包虫病中表达水平的研究. 中华肝胆外科杂志, 2020, 26(10): 753-756.
24. Parola M. Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues. Mol Aspects Med, 2019, 65: 37-55.
25. 张伟, 贾继东. 肝纤维化的发病机制及治疗新靶点. 临床肝胆病杂志, 2017, 33(3): 409-412.
26. 张力, 茆军, 张立, 等. 细胞焦亡与组织纤维化. 基础医学与临床, 2018, 38(12): 1766-1770.
27. Alegre F, Pelegrin P, Feldstein AE. Inflammasomes in liver fibrosis. Semin Liver Dise, 2017, 37(2): 119-127.
28. Niu F, Chong S, Qin M, et al. Mechanism of fibrosis induced by echinococcus spp. Diseases, 2019 , 7(3): 51. doi: 10.3390/diseases7030051.
29. 张金辉, 温浩, 刘章锁, 等. 原发性肝泡球蚴动物模型的建立. 中国局解手术学杂志, 2000, 9(1): 11-13.
30. 卞志远, 马少波, 夏海洋, 等. 新型肝穿刺建立原发性肝多房棘球蚴病小鼠模型. 中国人兽共患病学报, 2013, 29(5): 452-455.
31. 周启锋, 任利, 张灵强, 等. 肝泡状棘球蚴病动物模型的研究进展. 临床肝胆病杂志, 2017, 33(11): 2247-2250.
32. 吴亮亮, 杨凌菲, 宋涛. 不同方式建立肝多房棘球蚴感染SD大鼠模型病灶的超声及病理表现. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 549-552.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Preliminary research on pyroptosis NLRP3/Caspase-1 pathway in mouse model of hepatic alveolar echinococcosis infection

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