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.	盧亞奇. NLRP3炎癥小體活化在日本血吸蟲感染引起的肝纖維化中的作用及機制研究. 華中科技大學. 2018.
8.	王濤. GSDMD/Caspase-1、Caspase-4、Caspase-5等焦亡相關因子在肝多房包蟲病的表達及其意義. 青海大學, 2019.
9.	詹希美. 人體寄生蟲學. 第2版. 北京: 人民衛生出版社, 2010: 200-202.
10.	陳穎丹, 周長海, 周曉農, 等. 2015年全國人體重點寄生蟲病現狀調查分析. 中國寄生蟲學與寄生蟲病雜志, 2020, 38(1): 5-12.
11.	韓帥, 蕢嫣, 伍衛平, 等. 2004-2020年全國棘球蚴病疫情分析. 中國寄生蟲學與寄生蟲病雜志, 2022, 40(4): 475-480.
12.	付世強, 王志鑫, 樊海寧, 等. 175例晚期肝多房棘球蚴病患者臨床特征分析. 中國血吸蟲病防治雜志, 2019, 31(2): 204-206,209.
13.	艾麥提·牙森, 溫浩. 免疫學在包蟲病研究中的應用. 新疆醫科大學學報, 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.	陳蔡松, 張耀剛, 王志鑫, 等. Nod樣受體蛋白3炎癥小體在寄生蟲病中的研究進展. 中國寄生蟲學與寄生蟲病雜志, 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, 王宏賓, 王志鑫, 等. NLRP3炎性小體在肝泡型包蟲病中表達水平的研究. 中華肝膽外科雜志, 2020, 26(10): 753-756.
24.	Parola M. Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues. Mol Aspects Med, 2019, 65: 37-55.
25.	張偉, 賈繼東. 肝纖維化的發病機制及治療新靶點. 臨床肝膽病雜志, 2017, 33(3): 409-412.
26.	張力, 茆軍, 張立, 等. 細胞焦亡與組織纖維化. 基礎醫學與臨床, 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.	張金輝, 溫浩, 劉章鎖, 等. 原發性肝泡球蚴動物模型的建立. 中國局解手術學雜志, 2000, 9(1): 11-13.
30.	卞志遠, 馬少波, 夏海洋, 等. 新型肝穿刺建立原發性肝多房棘球蚴病小鼠模型. 中國人獸共患病學報, 2013, 29(5): 452-455.
31.	周啟鋒, 任利, 張靈強, 等. 肝泡狀棘球蚴病動物模型的研究進展. 臨床肝膽病雜志, 2017, 33(11): 2247-2250.
32.	吳亮亮, 楊凌菲, 宋濤. 不同方式建立肝多房棘球蚴感染SD大鼠模型病灶的超聲及病理表現. 中國寄生蟲學與寄生蟲病雜志, 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. 盧亞奇. NLRP3炎癥小體活化在日本血吸蟲感染引起的肝纖維化中的作用及機制研究. 華中科技大學. 2018.
8. 王濤. GSDMD/Caspase-1、Caspase-4、Caspase-5等焦亡相關因子在肝多房包蟲病的表達及其意義. 青海大學, 2019.
9. 詹希美. 人體寄生蟲學. 第2版. 北京: 人民衛生出版社, 2010: 200-202.
10. 陳穎丹, 周長海, 周曉農, 等. 2015年全國人體重點寄生蟲病現狀調查分析. 中國寄生蟲學與寄生蟲病雜志, 2020, 38(1): 5-12.
11. 韓帥, 蕢嫣, 伍衛平, 等. 2004-2020年全國棘球蚴病疫情分析. 中國寄生蟲學與寄生蟲病雜志, 2022, 40(4): 475-480.
12. 付世強, 王志鑫, 樊海寧, 等. 175例晚期肝多房棘球蚴病患者臨床特征分析. 中國血吸蟲病防治雜志, 2019, 31(2): 204-206,209.
13. 艾麥提·牙森, 溫浩. 免疫學在包蟲病研究中的應用. 新疆醫科大學學報, 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. 陳蔡松, 張耀剛, 王志鑫, 等. Nod樣受體蛋白3炎癥小體在寄生蟲病中的研究進展. 中國寄生蟲學與寄生蟲病雜志, 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, 王宏賓, 王志鑫, 等. NLRP3炎性小體在肝泡型包蟲病中表達水平的研究. 中華肝膽外科雜志, 2020, 26(10): 753-756.
24. Parola M. Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues. Mol Aspects Med, 2019, 65: 37-55.
25. 張偉, 賈繼東. 肝纖維化的發病機制及治療新靶點. 臨床肝膽病雜志, 2017, 33(3): 409-412.
26. 張力, 茆軍, 張立, 等. 細胞焦亡與組織纖維化. 基礎醫學與臨床, 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. 張金輝, 溫浩, 劉章鎖, 等. 原發性肝泡球蚴動物模型的建立. 中國局解手術學雜志, 2000, 9(1): 11-13.
30. 卞志遠, 馬少波, 夏海洋, 等. 新型肝穿刺建立原發性肝多房棘球蚴病小鼠模型. 中國人獸共患病學報, 2013, 29(5): 452-455.
31. 周啟鋒, 任利, 張靈強, 等. 肝泡狀棘球蚴病動物模型的研究進展. 臨床肝膽病雜志, 2017, 33(11): 2247-2250.
32. 吳亮亮, 楊凌菲, 宋濤. 不同方式建立肝多房棘球蚴感染SD大鼠模型病灶的超聲及病理表現. 中國寄生蟲學與寄生蟲病雜志, 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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