| 四君子汤调节铁死亡增强小鼠三阴性乳腺癌免疫治疗效果的研究 |
| Sijunzi Decoction regulates ferrotptosis of cancer cell and enhances effect of immunotherapy in mice with triple negative breast cancer. |
| DOI： |
| 中文关键词: 四君子汤 三阴性乳腺癌 免疫治疗 免疫检查点抑制剂 铁死亡 |
| 英文关键词: Sijunzi Decoction Triple negative breast cancer Immunotherapy Immune checkpoint inhibitors ferroptosis |
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| 中文摘要: |
| 目的 应用三阴乳腺癌小鼠荷瘤模型研究四君子汤增强抗PD-1抗体免疫治疗三阴乳腺癌小鼠的效果及其机制。方法 建立TNBC移植瘤小鼠模型,将小鼠随机分为4T1组(对照组),四君子汤治疗组、抗PD-1抗体组、四君子汤抗PD-1抗体联合治疗组,每组6只。四君子汤治疗组小鼠按动物与人体表比换算灌胃四君子汤,对照组小鼠灌胃等体积的0.9%氯化钠溶液,每周5次,连续3周,比较小鼠肿瘤大小。并建立荷瘤小鼠模型通过生存分析比较各组小鼠生存时间。通过BODIPY-C11染色进行脂质过氧化评估各组小鼠肿瘤细胞脂质过氧化水平,并通过测量MDA确认脂质过氧化水平。并通过7-氨基放线菌素D(7-AAD)染色检测细胞死亡比例。通过实时定量PCR检测四君子汤对铁死亡相关基因表达。并进一步通过体外实验分析四君子汤有效成分人参皂苷Rb1对4T1癌细胞脂质过氧化和铁死亡的影响。结果 四君子汤联合抗PD-1治疗组肿瘤体积较抗PD-1抗体单药治疗组小鼠肿瘤体积有明显差异(P<0.01)。四君子汤联合抗PD-1治疗组中位生存期较抗PD-1抗体治疗组有明显延长 34天 vs.25天 (P<0.05)。四君子汤联合抗PD-1治疗组脂质过氧化水平明显高于抗PD-1单药治疗组(P>0.05)。四君子汤联合抗PD-1治疗组癌细胞死亡率明显高于抗PD-1单药治疗组。四君子汤组铁死亡抑制基因NRF2,GPX4,SLC7A11表达水平显著低于对照组(P<0.05),四君子汤联合PD-1抗体铁死亡抑制基因也低于PD-1抗体单药治疗组(P<0.05)。但四君子汤组铁死亡促进基因TFRC在各组较对照组无明显差异。人参皂苷Rb1单体作用于乳腺癌癌细胞,与对照组相比,癌细胞脂质过氧化水平未见明显变化。但是人参皂苷Rb1单体联合IFN-γ作用于乳腺癌细胞,诱导乳腺癌细胞脂质过氧化水平明显高于单用IFN-γ(P<0.05)。人参皂苷Rb1干预组4T1癌细胞铁死亡抑制基因NRF2,GPX4,SLC7A11表达水平显著低于对照组(P<0.05)。结论 四君子汤可以通过促进癌细胞铁死亡增强抗PD-1抗体免疫治疗小鼠三阴性乳腺癌的效果,其机制可能与四君子汤降低癌细胞铁死亡抑制基因表达有关。 |
| 英文摘要: |
| Objective To study the effect and mechanism of Sijunzi decoction(SJZD) on anti-PD-1 antibody immunotherapy in mice with triple negative breast tumor bearing model. Methods TNBC transplanted tumor mouse model was established, and the mice were randomly divided into 4T1 group (control group), Sijunzi decoction treatment group, anti-PD-1 antibody group and SJZD with anti-PD-1 antibody combined treatment group, with 6 mice in each group. Mice in SJZD treatment group were administrated with SJZD, and mice in control group were administrated with 0.9% sodium chloride solution of the same volume, 5 times a week for 3 weeks, to compare the tumor size of mice. A tumor-bearing mouse model was also established and the survival time of each group was compared by survival analysis. Lipid peroxidation was evaluated by BODIPY-C11 staining, and lipid peroxidation was confirmed by MDA measurement. The proportion of cell death was detected by 7-amino-actinomycin D (7-AAD) staining. The expression of genes related to iron death in SJZD was detected by real-time quantitative PCR. The effect of ginsenoside Rb1 on lipid peroxidation and iron death of 4T1 cancer cells was analyzed by in vitro experiment. Results The tumor volume of SJZD combined with anti-PD-1 treatment group was significantly different from that of anti-PD-1 antibody monotherapy group (P<0.01). The median survival of SJZD combined with anti-PD-1 treatment group was significantly longer than that of anti-PD-1 antibody treatment group by 34 days vs.25 days (P<0.05). The lipid peroxidation level of SJZD combined with anti-PD-1 treatment group was significantly higher than that of anti-PD-1 monotherapy group (P>0.05). The cancer cell death rate of SJZD combined with anti-PD-1 treatment group was significantly higher than that of anti-PD-1 monotherapy group. The expression levels of ferroptosis suppressor genes NRF2, GPX4 and SLC7A11 in SJZD group were significantly lower than those in control group (P<0.05), and ferroptosis suppressor genes in SJZD combined with PD-1 antibody were also lower than those in PD-1 antibody monotherapy group (P<0.05). However, there was no significant difference in iron death promoting gene TFRC in SJZD group compared with control group. Ginsenoside Rb1 was also used to treat breast cancer cells, and the lipid peroxidation level of cancer cells did not change significantly compared with the control group. However, the level of lipid peroxidation induced by ginsenoside Rb1 combined with IFN-γ in breast cancer cells was significantly higher than that induced by IFN-γ alone (P<0.05). The expression levels of iron death suppressor genes NRF2, GPX4 and SLC7A11 in 4T1 cancer cells in ginsenoside Rb1 intervention group were significantly lower than those in control group (P<0.05). Conclusion SJZD can enhance the effect of anti-PD-1 antibody immunotherapy on triple negative breast cancer in mice by promoting the ferroptosis of cancer cells, and its mechanism may be related to the decrease of the expression of ferroptosis inhibitory gene in cancer cells. |
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