甘草干姜汤对顺铂所致巨噬细胞毒性的保护作用

doi:10.19803/j.1672-8629.20220716

摘要/Abstract

摘要： 目的研究甘草干姜汤（LDGD）对顺铂所致巨噬细胞毒性的保护作用及机制。方法体外培养小鼠巨噬细胞RAW264.7,噻唑蓝（MTT）比色法检测不同浓度顺铂（1、2、3、4、6 μg·mL^-1）和LDGD（5、10、15、20 mg·mL^-1）对RAW264.7细胞活力的影响,考察顺铂（2、3、4 μg·mL^-1）和LDGD（5、10、20 mg·mL^-1）单用、序贯和同时作用对RAW264.7细胞的毒性作用;流式细胞术测定顺铂（4 μg·mL^-1）和LDGD（5、10、20 mg·mL^-1）单用及不同方式联合作用对RAW264.7细胞凋亡、细胞活性氧（ROS）及线粒体膜电位的影响。雄性C57/BL6J小鼠24只,随机分为4组：对照组、模型组、顺铂组（4 mg·kg^-1）、联用组（顺铂4 mg·kg^-1+LDGD 5 g·kg^-1）,每组6只。除对照组外其他各组接种小鼠肺腺癌细胞（LLC）后第2天,联用组小鼠连续灌胃给药LDGD 20 d,顺铂组和联用组每3 d腹腔注射顺铂,荷瘤21 d后处死小鼠,测定甘草干姜汤与顺铂联用对小鼠肿瘤体积、体重、脏器指数、脾脏T淋巴细胞分型、血清中肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）等免疫因子水平及肿瘤切片中巨噬细胞浸润等的影响。结果与对照组相比,顺铂呈剂量依赖性抑制小鼠巨噬细胞RAW264.7的细胞活力（P<0.01）,而LDGD各剂量组均能促进RAW264.7的细胞活力（P<0.01）;LDGD能降低顺铂对RAW264.7细胞活力的抑制作用（P<0.01）,降低顺铂引起的RAW264.7细胞凋亡（P<0.01）;LDGD能够逆转顺铂所致RAW264.7细胞的ROS水平升高及线粒体膜电位下降（P<0.01）;顺铂组和联用组均能显著抑制荷瘤小鼠肿瘤的生长;顺铂能导致小鼠体重显著下降,肝脏指数显著升高,肾脏、脾脏和胸腺指数显著下降;联用组能使胸腺指数恢复到正常水平,肝肾和脾脏指数向对照组水平恢复;LDGD能在顺铂作用的基础上进一步提高小鼠脾脏CD3⁺、CD4⁺、CD8⁺T淋巴细胞的水平;同时LDGD提高了肿瘤组织中的巨噬细胞浸润和小鼠血清TNF-α含量,降低了血清IL-6含量。结论 LDGD可能通过减少ROS产生和稳定线粒体膜电位抑制顺铂诱导的小鼠巨噬细胞RAW264.7细胞凋亡,降低顺铂的细胞毒性;LDGD与顺铂联用,抗肿瘤同时能降低顺铂造成的小鼠肝肾和免疫器官损伤,促进巨噬细胞功能,进一步提高机体免疫水平。

关键词: 顺铂, 甘草干姜汤, 活性氧, 线粒体膜电位, 细胞凋亡, 小鼠肺腺癌, 巨噬细胞, 肿瘤坏死因子-α, 白细胞介素6

Abstract: Objective To investigate the protective effect and mechanism of licorice and dried ginger decoction (LDGD) against cisplatin-induced cytotoxicity in macrophages. Methods The effects of different concentrations of cisplatin（1、2、3、4、6 μg·mL^-1）, LDGD (5、10、20 mg·mL^-1), the sequential administration and coadministration of cisplatin and LDGD on the cell viability of mouse macrophage RAW264.7 cells cultured in vitro were detected by MTT assay. The apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) of RAW264.7 cells were analyzed by flow cytometry. Twenty-four male C57/BL6J mice were randomly divided into four groups (n=6): the control group, model group, cisplatin (4 mg·kg^-1) group and combination group (4 mg·kg^-1+LDGD 5 g·kg^-1). One day after inoculation with Lewis lung carcinoma (LLC), the mice in each group except those in the control group were given LDGD by intragastric administration for 20 days. The cisplatin group and the combination group were intraperitoneally injected with cisplatin every 3 days. The changes of tumor volume, body weight, visceral indexes, spleen T lymphocyte typing, immune factors of serum TNF-α, IL-6 and macrophage infiltration in tumor sections were determined after 21 days. Results Compared with the control group, cisplatin dose-dependently inhibited the cells' viability of RAW264.7 (P<0.01), while LDGD promoted the cell viability (P<0.01). LDGD could reduce the inhibitory effect of cisplatin on RAW264.7 cell viability (P<0.01). LDGD could reduce the apoptosis rate of RAW264.7 induced by cisplatin (P<0.01). Cisplatin induced an increase of ROS and a decrease of mitochondrial membrane potential (MMP) in RAW264.7 cells, but LDGD reversed the cisplatin - induced increase of ROS and the decrease of MMP (P<0.01). Both the cisplatin group and the combination group could significantly inhibit the growth of tumors, and cisplatin could lead to a significant increase in the liver indexes of mice, and a significant decrease in the indexes of body weight, kidneys, spleens and thymuses. Thymus indexes were restored to normal in the combination group, so were the organ indexes. LDGD could improve the levels of CD3⁺, CD4⁺ and CD8⁺ T lymphocytes in the spleen of mice following the use of cisplatin. LDGD increased macrophage infiltration in tumor tissues and TNF-α contents, but reduced IL-6 contents in mice serum. Conclusion LDGD inhibits cisplatin-induced apoptosis and reduces cisplatin-induced cytotoxicity in mouse macrophage RAW264.7 cells by reducing ROS production and stabilizing MMP. LDGD can mitigate the damage to the liver, kidney and immune organs caused by cisplatin, promote macrophage function, and improve the body's immunity.

Key words: cisplatin, licorice and dried ginger decoction, reactive oxygen species, mitochondrial membrane potential, apoptosis, Lewis lung carcinoma, macrophage cells, TNF-α, IL-6

中图分类号:

R969

辛雨濛, 卢天公, 孙震晓. 甘草干姜汤对顺铂所致巨噬细胞毒性的保护作用[J]. 中国药物警戒, 2023, 20(6): 661-670.

XIN Yumeng, LU Tiangong, SUN Zhenxiao. Licorice and dried ginger decoction protects macrophages from cisplatin-induced cytotoxicity[J]. Chinese Journal of Pharmacovigilance, 2023, 20(6): 661-670.

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