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

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

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。

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-αsignificantly 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.