Recombinant Schistosoma japonicum cystatin alleviates acute liver injury in mice by inhibiting endoplasmic reticulum stress, inflammation and hepatocyte apoptosis

doi:10.12122/j.issn.1673-4254.2024.06.13

Abstract

Abstract:

Objective To investigate the protective effect of recombinant Schistosoma japonicum cystatin (rSj-Cys) against acute liver injury induced by lipopolysaccharide (LPS) and D-GalN in mice. Methods Adult male C57BL/6J mice with or without LPS/D-GaIN-induced acute liver injury were given intraperitoneal injections of rSj-Cys or PBS 30 min after modeling (n=18), and serum and liver tissues samples were collected from 8 mice in each group 6 h after modeling. The survival of the remaining 10 mice in each group within 24 h was observed. Serum levels of ALT, AST, TNF‑α and IL-6 of the mice were measured, and liver pathologies was observed with HE staining. The hepatic expressions of macrophage marker CD68, Bax, Bcl-2 and endoplasmic reticulum stress (ERS)-related proteins were detected using immunohistochemistry or immunoblotting, and TUNEL staining was used to detect hepatocyte apoptosis. Results The survival rates of PBS- and rSj-Cys-treated mouse models of acute liver injury were 30% and 80% at 12 h and were 10% and 60% at 24 h after modeling, respectively; no death occurred in the two control groups within 24 h. The mouse models showed significantly increased serum levels of AST, ALT, IL-6 and TNF-α and serious liver pathologies with increased hepatic expressions of CD68 and Bax, lowered expression of Bcl-2, increased hepatocyte apoptosis, and up-regulated expressions of ERS-related signaling pathway proteins GRP78, CHOP and NF-κB p-p65. Treatment of the mouse models significantly lowered the levels of AST, ALT, IL-6 and TNF‑α, alleviated liver pathologies, reduced hepatic expressions of CD68, Bax, GRP78, CHOP and NF‑κB p-p65, and enhanced the expression of Bcl-2. In the normal control mice, rSj-Cys injection did not produce any significant changes in these parameters compared with PBS. Conclusion rSj-Cys alleviates LPS/D-GalN-induced acute liver injury in mice by suppressing ERS, attenuating inflammation and inhibiting hepatocyte apoptosis.

Key words: acute liver injury, Schistosoma japonicum cystatin, endoplasmic reticulum stress, inflammatory response, apoptosis

Lingjun LU, Xiaodi YANG, Huaping ZHANG, Yuan LIANG, Xiulan SHI, Xin ZHOU. Recombinant Schistosoma japonicum cystatin alleviates acute liver injury in mice by inhibiting endoplasmic reticulum stress, inflammation and hepatocyte apoptosis[J]. Journal of Southern Medical University, 2024, 44(6): 1126-1134.

Figures/Tables 8

Fig.1 Effect of rSj-Cys on survival of the mice after acute liver injury.

Fig.2 Effects of rSj-Cys on serum ALT and AST levels in mice with acute liver injury. ##P<0.01 vs Control group; **P<0.01 vs LPS/D-GaIN group.

Fig.3 Effect of rSj-Cys on serum IL-6 and TNF-α levels in mice with acute liver injury. ##P<0.01 vs Control group; **P<0.01 vs LPS/D-GaIN group.

Fig.4 Pathological changes in the liver of the mice in different groups (HE staining, original magnification: ×200). A: Control group; B: LPS/D-GalN group; C: LPS/D-GalN+rSj-Cys group; D: rSj-Cys group.

Fig.5 　Effect of rSj-Cys on CD68 expression in liver tissue of mice with acuteliver injury (Immunohistochemistry, ×400). A: Blank control group; B: LPS/DGalNgroup; C: LPS/D-GalN+rSj-Cys group; D: rSj-Cys control group; ##P<0.01 vs normal group; **P<0.01 vsLPS/D-GaIN group.

Fig.6 Effect of rSj-Cys on Bax and Bcl-2 expression in the liver tissue of mice with acute liver injury. #P<0.05 vs normal group; ##P<0.01 vs normal group; *P<0.05 vs normal group; **P<0.01 vs model group.

Fig.7 　Effect of rSj-Cys on hepatocyte apoptosis in the livertissue of mice with acute liver injury detected by TUNELstaining (×200). Normal cell nuclei are stained blue andapoptotic cell nuclei are stained red. ##P<0.01 vs Controlgroup; **P<0.01 vs LPS/D-GaIN group.

Fig.8 Effect of rSj-Cys on expressions of endoplasmic reticulum stress-related signaling pathway proteins in mice with acute liver injury. ##P<0.01 vs Control group; *P<0.05 vs, **P<0.01 vs LPS/D-GaIN group.

References 31

1	Yang WC, Wang YX, Zhang CG, et al. Maresin1 protect against ferroptosis-induced liver injury through ROS inhibition and Nrf2/HO-1/GPX4 activation[J]. Front Pharmacol, 2022, 13: 865689.
2	Lyu Z, Ji XF, Chen G, et al. Atractylodin ameliorates lipopolysaccharide and d-galactosamine-induced acute liver failure via the suppression of inflammation and oxidative stress[J]. Int Immunopharmacol, 2019, 72: 348-57.
3	Pang XH, Qiao Q, Vonglorkham S, et al. Asiatic acid ameliorates acute hepatic injury by reducing endoplasmic reticulum stress and triggering hepatocyte autophagy[J]. Biomedecine Pharmacother, 2020, 129: 110375.
4	Yang WC, Tao KX, Zhang P, et al. Maresin 1 protects against lipopolysaccharide/d-galactosamine-induced acute liver injury by inhibiting macrophage pyroptosis and inflammatory response[J]. Biochem Pharmacol, 2022, 195: 114863.
5	Hu XW, Li XM, Wang AM, et al. Caffeine alleviates acute liver injury by inducing the expression of NEDD4L and deceasing GRP78 level via ubiquitination[J]. Inflamm Res, 2022, 71(10/11): 1213-27.
6	Jiang ZQ, Ma YX, Li MH, et al. 5-Hydroxymethylfurfural protects against ER stress-induced apoptosis in GalN/TNF‑α‑injured L02 hepatocytes through regulating the PERK-eIF2α signaling pathway[J]. Chin J Nat Med, 2015, 13(12): 896-905.
7	Jiao ZH, Liu XN, Ma YJ, et al. Adipose-derived stem cells protect ischemia-reperfusion and partial hepatectomy by attenuating endoplasmic reticulum stress[J]. Front Cell Dev Biol, 2020, 8: 177.
8	Nan B, Yang CY, Li L, et al. Allicin alleviated acrylamide-induced NLRP3 inflammasome activation via oxidative stress and endoplasmic reticulum stress in Kupffer cells and SD rats liver[J]. Food Chem Toxicol, 2021, 148: 111937.
9	Chen ZY, Liu YL, Yang L, et al. MiR-149 attenuates endoplasmic reticulum stress-induced inflammation and apoptosis in nonalcoholic fatty liver disease by negatively targeting ATF6 pathway[J]. Immunol Lett, 2020, 222: 40-8.
10	毕晟, 丁有权, 齐建国. 蠕虫及蠕虫源性免疫调控性分子应用于自身免疫性疾病治疗的研究进展[J]. 细胞与分子免疫学杂志, 2016, 32(9): 1276-9, 1284.
11	Elliott DE, Weinstock JV. Helminth-host immunological interactions: prevention and control of immune-mediated diseases[J]. Ann N Y Acad Sci, 2012, 1247: 83-96.
12	Liu Y, Ye Q, Liu YL, et al. Schistosoma japonicum attenuates dextran sodium sulfate-induced colitis in mice via reduction of endoplasmic reticulum stress[J]. World J Gastroenterol, 2017, 23(31): 5700-12.
13	Xie H, Wu LQ, Chen XZ, et al. Schistosoma japonicum cystatin alleviates sepsis through activating regulatory macrophages[J]. Front Cell Infect Microbiol, 2021, 11: 617461.
14	Yang HJ, Li HQ, Chen WD, et al. Therapeutic effect of Schistosoma japonicum cystatin on atherosclerotic renal damage[J]. Front Cell Dev Biol, 2021, 9: 760980.
15	褚亮, 李徽徽, 王书书, 等. 源自日本血吸虫的半胱氨酸蛋白酶抑制剂对葡聚糖硫酸钠诱导的小鼠溃疡性结肠炎的影响[J]. 中国血吸虫病防治杂志, 2018, 30(3): 269-72, 338.
16	林子扬, 林泉酉, 刁礼聪, 等. 复方贞术调脂胶囊通过TLR4/NF-κB通路改善脂多糖诱导的小鼠急性肝损伤[J]. 世界科学技术-中医药现代化, 2023, 25(1): 430-7.
17	甘草酸制剂肝病临床应用专家委员会. 甘草酸制剂肝病临床应用专家共识[J]. 临床肝胆病杂志, 2016, 32(5): 844-52. DOI: 10.3969/j.issn.1001-5256.2016.05.004
18	Wang HJ, Chen LY, Zhang XY, et al. Kaempferol protects mice from d-GalN/LPS-induced acute liver failure by regulating the ER stress-Grp78-CHOP signaling pathway[J]. Biomedecine Pharmacother, 2019, 111: 468-75.
19	李燕楠, 杨小迪, 陈思宇, 等. 日本血吸虫重组半胱氨酸蛋白酶抑制剂对小鼠心肌梗死预后的影响及其免疫调节机制[J]. 中国生物制品学杂志, 2022, 35(1): 55-62. DOI: 10.3969/j.issn.1004-5503.2022.1.zgswzpxzz202201011
20	万勇坤, 李徽徽, 左琳, 等. 日本血吸虫半胱氨酸蛋白酶抑制剂干预脂多糖诱导的小鼠脓毒症[J]. 南方医科大学学报, 2018, 38(5): 625-9. DOI: 10.3969/j.issn.1673-4254.2018.05.020
21	Hu TY, Wang JY, Li WX, et al. Endoplasmic reticulum stress in hepatitis B virus and hepatitis C virus infection[J]. Viruses, 2022, 14(12): 2630.
22	Cao YP, He W, Li XP, et al. Rosiglitazone protects against acetaminophen-induced acute liver injury by inhibiting multiple endoplasmic reticulum stress pathways[J]. Biomed Res Int, 2022, 2022: 6098592.
23	Jiang CJ, Li JH. Research progress on mechanism of exercise improving non-alcoholic fatty liver disease through regulating endoplasmic reticulum stress[J]. Sheng Li Xue Bao, 2022, 74(5): 816-26.
24	Remick DG, Ward PA. Evaluation of endotoxin models for the study of sepsis[J]. Shock, 2005, 24(): 7-11.
25	Huang SJ, Xing YY, Liu Y. Emerging roles for the ER stress sensor IRE1α in metabolic regulation and disease[J]. J Biol Chem, 2019, 294(49): 18726-41.
26	Tam AB, Mercado EL, Hoffmann A, et al. ER stress activates NF-κB by integrating functions of basal IKK activity, IRE1 and PERK[J]. PLoS One, 2012, 7(10): e45078.
27	Li HH, Qiu DP, Yuan Y, et al. Trichinella spiralis cystatin alleviates polymicrobial sepsis through activating regulatory macrophages[J]. Int Immunopharmacol, 2022, 109: 108907.
28	谢红. 过继转移r-SjCystatin诱导的巨噬细胞对小鼠脓毒症的保护作用研究[D]. 蚌埠: 蚌埠医学院, 2020.
29	Wang YY, Zhou XQ, Zhao D, et al. Berberine inhibits free fatty acid and LPS-induced inflammation via modulating ER stress response in macrophages and hepatocytes[J]. PLoS One, 2020, 15(5): e0232630.
30	So JS. Roles of endoplasmic reticulum stress in immune responses[J]. Mol Cells, 2018, 41(8): 705-16.
31	刘娅. 日本血吸虫感染对DSS诱导实验性结肠炎的作用与机制研究[D]. 武汉: 武汉大学, 2017.

[1]	Xiaoyu CHANG, Hanwen ZHANG, Hongting CAO, Ling HOU, Xin MENG, Hong TAO, Yan LUO, Guanghua LI. Heat stress affects expression levels of circadian clock gene Bmal1 and cyclins in rat thoracic aortic endothelial cells [J]. Journal of Southern Medical University, 2025, 45(7): 1353-1362.
[2]	Bing XIA, Jin PENG, Jiuyang DING, Jie WANG, Guowei TANG, Guojie LIU, Yun WANG, Changwu WAN, Cuiyun LE. ATF3 regulates inflammatory response in atherosclerotic plaques in mice through the NF-κB signaling pathway [J]. Journal of Southern Medical University, 2025, 45(6): 1131-1142.
[3]	Yujia YANG, Lifang YANG, Yaling WU, Zhaoda DUAN, Chunze YU, Chunyun WU, Jianyun YU, Li YANG. Cannabidiol inhibits neuronal endoplasmic reticulum stress and apoptosis in rats with multiple concussions by regulating the PERK-eIF2α-ATF4-CHOP pathway [J]. Journal of Southern Medical University, 2025, 45(6): 1240-1250.
[4]	Yue CHEN, Linyu XIAO, Lü REN, Xue SONG, Jing LI, Jianguo HU. Monotropein improves motor function of mice with spinal cord injury by inhibiting the PI3K/AKT signaling pathway to suppress neuronal apoptosis [J]. Journal of Southern Medical University, 2025, 45(4): 774-784.
[5]	Fei CHU, Xiaohua CHEN, Bowen SONG, Jingjing YANG, Lugen ZUO. Moslosooflavone ameliorates dextran sulfate sodium-induced colitis in mice by suppressing intestinal epithelium apoptosis via inhibiting the PI3K/AKT signaling pathway [J]. Journal of Southern Medical University, 2025, 45(4): 819-828.
[6]	Yi ZHANG, Yu SHEN, Zhiqiang WAN, Song TAO, Yakui LIU, Shuanhu WANG. High expression of CDKN3 promotes migration and invasion of gastric cancer cells by regulating the p53/NF-κB signaling pathway and inhibiting cell apoptosis [J]. Journal of Southern Medical University, 2025, 45(4): 853-861.
[7]	Di CHEN, Ying LÜ, Yixin GUO, Yirong ZHANG, Ruixuan WANG, Xiaoruo ZHOU, Yuxin CHEN, Xiaohui WU. Dihydroartemisinin enhances doxorubicin-induced apoptosis of triple negative breast cancer cells by negatively regulating the STAT3/HIF-1α pathway [J]. Journal of Southern Medical University, 2025, 45(2): 254-260.
[8]	Yu BIN, Ziwen LI, Suwei ZUO, Sinuo SUN, Min LI, Jiayin SONG, Xu LIN, Gang XUE, Jingfang WU. High expression of apolipoprotein C1 promotes proliferation and inhibits apoptosis of papillary thyroid carcinoma cells by activating the JAK2/STAT3 signaling pathway [J]. Journal of Southern Medical University, 2025, 45(2): 359-370.
[9]	Hanjun ZUO, Zhaoda DUAN, Zhao WANG, Tao GUO, Jinsha SHI, Haolong SHI, Juanjuan LI. Gastrodin improves microglia-mediated inflammatory response after hypoxic-ischemic brain damage in neonatal rats via PI3K/AKT pathway [J]. Journal of Southern Medical University, 2024, 44(9): 1712-1719.
[10]	Mingyuan LI, Wei ZHANG, Mengqing HUA. Bardoxolone methyl alleviates acute liver injury in mice by inhibiting NLRP3 inflammasome activation [J]. Journal of Southern Medical University, 2024, 44(9): 1662-1669.
[11]	Shuo LIU, Jing LI, Xingwang WU. Swertiamarin ameliorates 2,4,6-trinitrobenzenesulfonic acid-induced colitis in mice by inhibiting intestinal epithelial cell apoptosis [J]. Journal of Southern Medical University, 2024, 44(8): 1545-1552.
[12]	Xiaofan CONG, Teng CHEN, Shuo LI, Yuanyuan WANG, Longyun ZHOU, Xiaolong LI, Pei ZHANG, Xiaojin SUN, Surong ZHAO. Dihydroartemisinin enhances sensitivity of nasopharyngeal carcinoma HNE1/DDP cells to cisplatin-induced apoptosis by promoting ROS production [J]. Journal of Southern Medical University, 2024, 44(8): 1553-1560.
[13]	Linyu XIAO, Ting DUAN, Yongsheng XIA, Yue CHEN, Yang SUN, Yibo XU, Lei XU, Xingzhou YAN, Jianguo HU. Linarin inhibits microglia activation-mediated neuroinflammation and neuronal apoptosis in mouse spinal cord injury by inhibiting the TLR4/NF-κB pathway [J]. Journal of Southern Medical University, 2024, 44(8): 1589-1598.
[14]	Mengdong ZHENG, Yan LIU, Jiaojiao LIU, Qiaozhen KANG, Ting WANG. Effect of deletion of protein 4.1R on proliferation, apoptosis and glycolysis of hepatocyte HL-7702 cells [J]. Journal of Southern Medical University, 2024, 44(7): 1355-1360.
[15]	Yuanguo WANG, Peng ZHANG. Ferroptosis suppressor genes are highly expressed in esophageal cancer to inhibit tumor cell ferroptosis [J]. Journal of Southern Medical University, 2024, 44(7): 1389-1396.