Regulation of Th1/Th2 immune balance by shikonin via STING in Lewis lung carcinoma mice

Objective To investigate the effects of shikonin (SK) on Th1/Th2 immune balance in Lewis lung cancer-bearing mice via STING.Methods A lung cancer tumor-bearing mouse model was established. Mice were randomly divided into six groups: normal control (NC), model (PBS), low-dose SK (SK-L), medium-dose SK (SK-M), high-dose SK (SK-H), medium-dose SK plus STING inhibitor (SK-M+C176), and control (PTX), with six mice per group. Administration began on day 7 post-tumor implantation, with alternate-day dosing. Mice were sacrificed on day 28 post-implantation to record survival and tumor progression, plotting tumor volume growth curves and tumor-bearing mouse survival curves. Calculate spleen and thymus indices. Flow cytometry detects STING expression in splenic dendritic cells (DCs), surface molecules MHCII, CD80, CD86, chemokine receptors CXCR4, CCR5, CCR7, and T cell subset expression levels (CD3+T, CD4+T, CD8+T, Th1, Th2). Detect concentrations of IFN-β, IL-12, IFN-γ, TNF-α, IL-4, VEGF, Gzms-B and PF in mouse ocular blood serum via ELISA.Results Compared with the model group, the SK medium and high dose group delayed tumor growth and prolonged survival in mice (P<0.01); increased spleen and thymus indices (P<0.01); upregulated STING, surface molecules MHCII, CD80, CD86, and chemokines CXCR4 and CCR7 expression in splenic dendritic cells (DCs), while downregulating CCR5 expression (P<0.01); increased the proportion of CD4+T cells and the CD4+T/CD8+T ratio in the spleen; upregulated Th1 expression while downregulating Th2 expression in splenic CD4+T lymphocytes, promoting Th1/Th2 balance (P<0.01); Increased serum concentrations of IFN-β, IL-12, IFN-γ, TNF-α, Gzms-B, and PF in mice while decreasing IL-4 and VEGF levels (P<0.05); Compared with the SK-M group, the SK-M+C176 group reversed the suppression of tumor progression in Lewis tumor-bearing mice by SK and its promotion of STING, DC, and Th1 cell activation (P<0.05).Conclusion SK delays tumor growth and prolongs survival in tumor-bearing mice by enhancing immune cell functions to exert antitumor immune effects. This mechanism may involve upregulating STING expression in DCs, promoting DC maturation and migration, enhancing antigen presentation capacity, and thereby shifting the Th1/Th2 balance toward Th1 dominance.

目的 探讨紫草素（shikonin, SK）通过STING通路对Lewis肺癌荷瘤小鼠Th1/Th2免疫平衡的影响。方法 构建肺癌荷瘤小鼠模型，将小鼠随机分为6组：正常对照组（normal control, NC）、模型组（PBS）、低剂量紫草素组（SK-L）、中剂量紫草素组（SK-M）、高剂量紫草素组（SK-H）、中剂量紫草素联合STING抑制剂组（SK-M+C176）及阳性对照组（PTX），每组6只小鼠。于肿瘤植入后第7日开始给药，隔日给药1次。于肿瘤植入后第28日处死小鼠，记录存活情况与肿瘤进展状况，绘制肿瘤体积生长曲线及荷瘤小鼠生存曲线；计算脾脏指数与胸腺指数。采用流式细胞术检测脾脏树突状细胞（dendritic cells, DCs）中STING的表达水平、表面分子MHCII、CD80、CD86及趋化因子受体CXCR4、CCR5、CCR7的表达水平，以及T细胞亚群的表达水平（CD3+T细胞、CD4+T细胞、CD8+T细胞、Th1、Th2）。采用酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测小鼠眼静脉血清中IFN-β、IL-12、IFN-γ、TNF-α、IL-4、VEGF、Gzms-B及PF的浓度。结果 与模型组相比，SK中、高剂量组可延缓小鼠肿瘤生长并延长生存期（P<0.01）；升高脾脏指数与胸腺指数（P<0.01）；上调脾脏树突状细胞中STING、表面分子MHCII、CD80、CD86及趋化因子CXCR4、CCR7的表达，下调CCR5的表达（P<0.01）；增加脾脏中CD4+T细胞占比及CD4+T/CD8+T细胞比值；上调脾脏CD4+T淋巴细胞中Th1的表达，下调Th2的表达，促进Th1/Th2免疫平衡（P<0.01）；升高小鼠血清中IFN-β、IL-12、IFN-γ、TNF-α、Gzms-B及PF的浓度，降低IL-4与VEGF的水平（P<0.05）；与SK-M组相比，SK-M+C176组可逆转SK对Lewis肺癌荷瘤小鼠肿瘤进展的抑制作用，以及其对STING、树突状细胞及Th1细胞活化的促进作用（P<0.05）。结论 紫草素可通过增强免疫细胞功能发挥抗肿瘤免疫作用，延缓荷瘤小鼠肿瘤生长并延长其生存期。其机制可能涉及上调树突状细胞中STING的表达，促进树突状细胞成熟与迁移，增强抗原呈递能力，进而使Th1/Th2免疫平衡向Th1优势方向偏移。