丹参酮Ⅰ、二甲双胍和阿司匹林联合用药对黑素瘤模型小鼠的抑瘤作用

Inhibitory effect of combination of tanshinoneⅠ, metformin and aspirin on malignant melanoma model mice

目的研究丹参酮Ⅰ(TanⅠ)、二甲双胍(Met)和阿司匹林(Asp)联合应用对黑素瘤模型小鼠的抑瘤作用及可能机制。方法 C57BL/6小鼠右前腋下sc接种B16F10细胞制备黑素瘤模型。根据体质量分为8组,即模型组、TanⅠ组(20 mg·kg^(-1),ip)、Asp组(210 mg·kg^(-1),饮用)、Met组(70 mg·kg^(-1),饮用)、Asp+Met组、TanⅠ+Asp组、TanⅠ+Met组和TanⅠ+Asp+Met组,每组10只。每只小鼠摄水量约7 m L·d^(-1),连续给药18 d。隔天称量小鼠体质量,每天测量皮下肿瘤直径。给药结束后将小鼠处死,测瘤质量,计算抑瘤率。制备脾和肝组织切片,HE染色观察病理变化。流式细胞术检测肿瘤组织中CD8+T,CD4+T和Treg细胞百分比。ELISA检测肿瘤组织中白细胞介素6(IL-6)、IL^(-1)β和肿瘤坏死因子α(TNF-α)含量。结果实验期间,各组小鼠体质量(包括瘤质量)均呈上升趋势;与模型组比较,TanⅠ+Asp+Met组体质量增加减慢(P<0.01)。实验结束时,各组存活小鼠肝和脾组织均未见明显病理改变,小鼠存活数分别为:模型组8/10,TanⅠ组8/10,Asp组7/10,Met组7/10,TanⅠ+Asp组8/10,TanⅠ+Met组8/10,Asp+Met组7/10,TanⅠ+Asp+Met组5/10。与模型组比较,TanⅠ,Asp和Met单用组及两两联用组肿瘤体积和瘤质量无明显变化;TanⅠ+Asp+Met组肿瘤体积和瘤质量较模型组明显降低(P<0.01,P<0.05),抑瘤率为46.2%。与模型组比较,TanⅠ,Asp和Met单用组及两两联用组肿瘤组织中CD8+T淋巴细胞百分比无明显变化,TanⅠ+Asp+Met组CD8+T淋巴细胞百分比增加(P<0.05)。TanⅠ+Met组肿瘤组织中IL-6,IL^(-1)β和TNF-α含量较模型组升高(P<0.01,P<0.05,P<0.05),TanⅠ+Asp+Met组IL-6含量升高(P<0.01)。结论 TanⅠ+Asp+Met联用可抑制黑素瘤模型小鼠肿瘤生长,其机制可能与增加肿瘤组织CD8+T淋巴细胞百分比和IL-6含量有关,但副作用可能性较大。

OBJECTIVE To explore the antitumor effects of combined tanshinoneⅠ（TanⅠ）, metformin（Met） and aspirin（Asp） on malignant melanoma in mice and the possible mechanisms. METHODS C57 BL/6 mice were injected with 0.1 m L B16 F10 cells（2.8×109 L^-1） to establish the subcutaneous transplantation tumor model at the right forelimbs axillary. Then, the mice were divided into 8 groups according to body mass, including model group, TanⅠgroup（20 mg·kg^-1, ip）, Asp group（210 mg·kg^-1, orally in drinking water）, Met group（70 mg·kg^-1, orally in drinking water）, Asp＋Met group, TanⅠ＋Asp group,TanⅠ＋Met group and TanⅠ＋Asp＋Met group, 10 mice in each group. Each mouse drank about 7 m L of water every day for a total of 18 d. The mouse body mass was measured every other day and the tumor diameter was calculated every day. The mice were sacrificed after treatment, the tumor mass was measured and the tumor inhibitory rates were counted. The histopathological changes of the liver and spleen were observed with HE staining. The percentage of lymphocytes in the tumor tissue such as CD8＋T, CD4＋T and Treg cells was detected by flow cytometry. Inflammatory factors such as interleukin-6（IL-6）, IL^-1β and tumor necrosis factor-α（TNF-α） were detected by ELISA. RESULTS The body mass（including tumor mass） of mice in different groups increased during the experiment, but that of TanⅠ＋Asp＋Met group increased more slower than in model group（P〈0.01）. At the end of the experiment, no lesions were seen in any liver or spleen tissue by pathological observation, and the number of survivors was 8/10（model group）, 8/10（TanⅠgroup）, 7/10（Asp group）, 7/10（Met group）, 8/10（TanⅠ＋Asp group）,8/10（Tan Ⅰ ＋ Met group）, 7/10（Asp ＋ Met group） and 5/10（Tan Ⅰ ＋ Asp ＋ Met group）, respectively.Compared with model group, there were no obvious changes in tumor volume or tumor mass in TanⅠ,Asp and Met groups and other two-two joint groups, but the tumor volume and tumor mass in TanⅠ＋Asp＋ Met group were significantly decreased（P〈0.01, P〈0.05）, and the tumor inhibitory rate in this group was 46.2%. Compared with the model group, the percentage of CD8＋T cells increased（P〈0.05）in TanⅠ＋ Asp＋ Met group, but there were no significant changes in other groups. The contents of IL-6,IL^-1β and TNF-α in tumor tissue of TanⅠ＋Met group were much higher than in model group（P〈0.01,P〈0.05, P〈0.05） and the content of IL-6 increased in TanⅠ＋Asp＋Met group（P〈0.01）. CONCLUSION Combination of TanⅠ, Asp and Met can effectively inhibit the growth of melanoma in mice, which may be related to the increasing percentage of CD8＋T lymphocytes and IL-6 in tumor tissue. However there are possibly some side effects.