制何首乌对CTLA-4抗体诱导的PD-1-/-小鼠肝损伤模型的改善作用

肝脏 ›› 2022, Vol. 27 ›› Issue (1): 33-37.

制何首乌对CTLA-4抗体诱导的PD-1^-/-小鼠肝损伤模型的改善作用

王昱, 张春盼, 王艳, 刘立伟, 李轲鑫, 马子坤, 贾继东, 王伽伯, 赵新颜

100050 首都医科大学附属北京友谊医院肝病中心,国家消化系统疾病临床医学研究中心(王昱,王艳,刘立伟,李轲鑫,马子坤,贾继东,赵新颜),感染科(张春盼);首都医科大学中医药学院(王伽伯)

收稿日期:2021-11-13 出版日期:2022-01-31 发布日期:2022-02-11
通讯作者: 赵新颜,Email:zhao_xinyan@ccmu.edu.cn;王伽伯,Email:jiabo_wang@ccmu.edu.cn
基金资助:
国家自然科学基金项目(81900526,82074112);中国肝炎防治基金会王宝恩肝纤维化研究基金(2021060)

Improvement effect of polygoni multiflori radix praeparatac in PD-1 knockout mice with liver injury induced by anti-CTLA-4

WANG Yu¹, ZHANG Chun-pan², WANG Yan¹, LIU Li-wei¹, LI Ke-xin¹, MA Zi-kun¹, JIA Ji-dong¹, WANG Jia-bo³, ZHAO Xin-yan¹

1. Liver Research Center,Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center of Digestive Diseases,Beijing 100050, China;
2. Department of Infectious Diseases, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China;
3. School of Chinese Medicine, Capital Medical University, Beijing 100069, China

Received:2021-11-13 Online:2022-01-31 Published:2022-02-11
Contact: ZHAO Xin-yan, Email: zhao_xinyan@ccmu.edu.cn; WANG Jia-bo, Email: jiabo_wang@ccmu.edu.cn

摘要/Abstract

摘要： 目的探讨制何首乌对CTLA-4抗体诱导PD-1^-/-小鼠肝损伤的作用特点及其免疫学机制。方法 10只SPF级C57BL/6品系的PD-1^-/-小鼠随机分为对照组(n=4)和制何首乌组(n=6)。两组小鼠均于实验前3 d、前1 d及实验开始后每周腹腔注射CTLA-4抗体,同时分别给予0.9% NaCI溶液和何首乌连续灌胃42 d,第43天处死后留血和肝组织,进行生化、病理、免疫组织化学染色及肝组织流式细胞检测。结果制何首乌组小鼠ALT、AST水平较对照组有降低趋势为(43.2±15.5)U/L比(32.8±6.7)U/L和(32.3±7.5)U/L比(23.8±14.1)U/L但差异均无统计学意义(均P>0.05)。对照组小鼠肝脏病理表现为多个大小不等的炎症坏死灶,主要以巨噬细胞和CD4⁺T细胞浸润为主;而制何首乌组的炎症坏死灶数量显著减少,为121.5(104.0,147.3)比24(17.5,61.5),(P=0.014),肝组织浸润的巨噬细胞、CD4⁺T细胞数量亦明显减少。流式细胞检测显示,与对照组相比,制何首乌组肝脏内巨噬细胞比例显著降低(2.39±0.77)%比(1.37±0.28)%,(P=0.028),中性粒细胞比例显著降低(0.58±0.11)%比(0.34±0.14)%,(P=0.025),且其分泌的TNF-α比例显著降低(30.15±10.69)%比(11.56±6.05)%,(P=0.012);CD4⁺ T及CD8⁺ T细胞比例虽没有显著变化,但CD4⁺T细胞活化比例显著降低(26.20±3.47)%比(16.74±6.95)%,(P=0.044),CD8⁺ T细胞分泌颗粒酶B比例显著降低(77.05±4.23)%比(59.70±10.81)%,(P=0.020)。结论制何首乌通过抑制巨噬细胞募集、中性粒细胞和效应性T细胞活化,对CTLA-4抗体诱导PD-1^-/-小鼠肝损伤模型起到改善和保护作用。

关键词: 制何首乌, PD-1, CTLA-4, 免疫细胞浸润, 巨噬细胞, CD4⁺T细胞

Abstract: Objective To investigate the effects of the polygoni multiflori radix praeparata (PMRP) in programmed-cell-death-1 (PD-1) knockout mice with liver injury induced by anti-cytotoxic-T-lymphocyte-associated protein-4 (anti-CTLA-4) and its immunological mechanisms.Methods C57BL/6-PD-1^-/- mice (n=10) were divided into the control group (n=4) and the PMRP group (n=6). Anti-CTLA-4 was injected intraperitoneally 3 days before experiment, 1 day before experiment and once a week after the experiment started. Normal saline and PMRP were respectively given to the control group and PMRP group with gastric infusion for 42 days. Blood and liver samples were collected on the 43th day, then hematoxylin-eosin (H&E), staining, immunohistochemical stains (IHC) and flow cytometry were performed.Results Compared with the control group, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the PMRP group showed a decreased tendency (43.2±15.5 vs 32.8±6.7, P=0.216 and 32.3±7.5 vs 23.8±14.1, P=0.315, respectively). Pathological features of the control group were the existence of multiple inflammatory necrotic foci, macrophages and CD4⁺ T cells were the main infiltrating cells. The number of inflammatory necrotic foci in the PMRP group [121.5(104.0,147.3) vs 24(17.5,61.5), P=0.014] was significantly lower, the numbers of macrophages and CD4⁺ T cells were also significantly decreased. Compared with control group, flow cytometry showed that the proportions of macrophages (2.39±0.77% vs 1.37±0.28%, P=0.028) and neutrophils (0.58±0.11% vs 0.34±0.14%, P=0.025) were significantly decreased in PMRP group. The proportion of neutrophils secreting TNF-α significantly decreased in PMRP group (30.15±10.69% vs 11.56±6.05%, P=0.012). Compared with control group, the proportions of CD4⁺ T and CD8⁺ T cells did not change significantly, however the proportions of activated CD4⁺ T cells and CD8⁺T cells which secreted granzyme B significantly decreased in the PMRP group (26.20±3.47% vs 16.74±6.95%, P=0.044 and 77.05±4.23 % vs 59.70±10.81%, P=0.020, respectively).Conclusion PMRP can improve liver function in PD-1^-/- mice with liver injury induced by anti-CTLA-4, the mechanisms are inhibiting macrophages recruitment, neutrophils and effective T cells activation.

Key words: Polygonum multiflorum radix praeparato, Programmed-cell-death-1, Cytotoxic-T-lymphocyte-associated protein-4, Immune cells infiltration, Macrophages, CD4⁺T cells

王昱, 张春盼, 王艳, 刘立伟, 李轲鑫, 马子坤, 贾继东, 王伽伯, 赵新颜. 制何首乌对CTLA-4抗体诱导的PD-1^-/-小鼠肝损伤模型的改善作用[J]. 肝脏, 2022, 27(1): 33-37.

WANG Yu, ZHANG Chun-pan, WANG Yan, LIU Li-wei, LI Ke-xin, MA Zi-kun, JIA Ji-dong, WANG Jia-bo, ZHAO Xin-yan. Improvement effect of polygoni multiflori radix praeparatac in PD-1 knockout mice with liver injury induced by anti-CTLA-4[J]. Chinese Hepatolgy, 2022, 27(1): 33-37.

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