山慈菇酯提物对4T1乳腺癌免疫微环境的影响

Effects of Ethyl Acetate Extract from Cremastra Appendiculata on 4T1 Breast Cancer Immune Microenvironment

为探究山慈菇酯提物(Cremastra appendiculata,CrAp)体内外抗4T1乳腺癌的作用以及对4T1乳腺癌荷瘤小鼠免疫相关的分子机制的影响。采用四甲基偶氮唑盐微量酶反应比色法(MTT法)检测不同浓度(12 500、1 250、125、12.5、1.25、0.125μg/mL)的CrAp在24、48、72 h对4T1乳腺癌细胞的增殖抑制作用。100只BABL/c雌性小鼠植瘤成功后,随机分成空白对照组、模型组、阿霉素(adriamycin, ADM)阳性对照组、CrAp组以及CrAp+ADM组,每隔7 d小鼠称重一次,绘制体重曲线;每3 d测量小鼠肿瘤长径及短径,绘制肿瘤曲线;剥离癌组织,计算抑瘤率;取癌组织做苏木精-伊红(hematoxylin-eosin staining, HE)染色;ELISA法检测白介素-2(interleukin-2, IL-2)、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、干扰素-γ(interferon-γ, IFN-γ)和白介素-10(interleukin-10, IL-10)含量的变化。TMT分析CrAp调控与4T1乳腺癌免疫相关的差异蛋白。结果表明:在MTT实验中,不同浓度的CrAp抑制率范围为13.48%~74.47%,其中CrAp(125μg/mL)在72 h抑制作用最强,抑制率为74.47%。乳腺癌荷瘤鼠体重明显降低;CrAp能明显增加乳腺癌小鼠的体重(P<0.01),并明显降低乳腺癌肿瘤体积(P<0.01)。癌组织HE染色:模型组肿瘤细胞形态完好,数目较多,坏死区域少见;CrAp及CrAp+ADM组肿瘤细胞破裂以及胞核溶解,密度降低,并存在不同程度的死亡区域,且各给药组增加了乳腺癌中免疫细胞的数量。在癌组织中,与模型组相比,各药物组明显升高IL-2和IFN-γ的含量(P<0.01),而明显降低TNF-α和IL-10的含量(P<0.01)。在体外,CrAp可抑制4T1乳腺癌细胞的增殖。在体内,CrAp能抑制4T1乳腺癌的增长,增加荷瘤小鼠的体重,改善乳腺癌荷瘤小鼠的生存。CrAp提高癌组织中免疫因子IL-2、IFN-γ的表达而降低TNF-α、IL-10的表达。此外,CrAp可以增强ADM抗4T1乳腺癌及免疫调节作用。

In order to explore the effects of ethyl acetate extract from Cremastra appendiculata(CrAp) on 4 T1 breast cancer and its immune mechanism in mice bearing 4 T1 breast cancer in vitro and in vivo, spectrophotometric determination of tetramethylazozolium salt by micro enzyme reaction(MTT assay was used to measure the inhibition effect of CrAp on proliferation of 4 T1 cells with different concentrations(12 500、1250、125、12.5、1.25、0.125 μg/mL) at 24, 48, 72 h. One hundred BABL/c female mice were implanted with tumor, which, after successful implantation, were randomly divided into 5 groups: blank control group, model group, Adriamycin(ADM) positive control group, CrAp group and CrAp+ADM group. The mice were weighed every 7 days to draw the weight change curve. The tumor’s long diameter and short diameter were measured every 3 days to draw the tumor curve. The tumor inhibition rate was calculated by peeling off the tumor of mice. Some cancer tissues were stained with HE. The levels of Interleukin-2(IL-2), Tumor necrosis factor-α(TNF-α), Interferon-γ(IFN-γ) and Interleukin-10(IL-10) were detected by Enzyme-linked immunosorbent assay(ELISA). Results show that in MTT experiment, the inhibitory effect of different concentrations of CrAp ranges from 13.48% to 74.47%, of which CrAp(125 μg/mL) has the strongest inhibitory effect at 72 h, with the inhibitory rate of 74.74%. The weight of model group is significantly lower than that of blank group(P<0.01). At the same time, the weight of CrAp group is significantly greater than that of model group(P<0.01), but the tumor volume of CrAp group is significantly reduced(P<0.01). All drug groups have inhibitory effect on tumor in tumor-bearing mice. Compared with the model group, the tumor cells rupture, the nucleus is dissolved, the density is reduced, and there are different degrees of death areas of CrAp and CrAp+ADM group in HE staining of cancer tissue. And each administration group increases the number of immune cells in breast cancer. In cancer tissues, compared with the model group, the levels of IL-2 and IFN-γ in each drug group are significantly increased(P<0.01), and the levels of TNF-α and IL-10 are significantly decreased(P<0.01). In vitro, CrAp can inhibit the proliferation of 4 T1 breast cancer cells. In vivo, CrAp can inhibit the growth of breast cancer, improve the general behavior of tumor-bearing mice, and increase the weight of tumor-bearing mice. CrAp anti-4 T1 breast cancer may increase the expression of IL-2, IFN-γ and reduce TNF-α, IL-10 in cancer tissue. In addition, CrAp can enhance ADM’s anti-4 T1 breast cancer and immunoregulation effects.