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    黄连汤联合辐射对Lewis肺癌荷瘤小鼠抑瘤作用的研究

    Study on the Antitumor Effect of Huanglian Decoction Combined with Radiation on Mice with Lewis Lung Cancer

      摘要
    • 摘要:
      目的  研究中药复方黄连汤联合X射线辐射对Lewis肺癌荷瘤小鼠的抑瘤作用及机制。
      方法 选取60只SPF级雄性C57BL/6小鼠，随机分为空白组，模型组，辐射组，辐射联合黄连汤低、中、高剂量组，每组10只。除空白组外，其余各组在小鼠右腋下注射Lewis细胞构建肺癌荷瘤小鼠模型，模型建立成功后，辐射各组用4 Gy X射线局部照射肿瘤部位；辐射联合黄连汤低、中、高剂量组在照射后分别灌胃5.78、11.57、23.14 g·kg−1的黄连汤0.2 mL·d−1，空白、模型与辐射组分别灌胃等体积的生理盐水，连续干预7 d后处死小鼠，取肿瘤组织样本。HE观察肿瘤组织病理学变化；免疫组化检测肿瘤组织中Ki67表达；免疫荧光检测肿瘤细胞凋亡情况；qRT-PCR检测肿瘤组织中EGFR、HIF-1α mRNA的表达水平；Western blotting检测肿瘤组织中EGFR、HIF-1α、Bcl-2、Bax和Caspase-3蛋白的表达水平。
      结果 与模型组相比，辐射组小鼠肿瘤质量减轻(P<0.05)，肿瘤细胞排列疏松，细胞间隙模糊，出现片状出血性坏死现象，凋亡细胞数量上升(P<0.01)，EGFR、HIF-1α mRNA表达升高，EGFR、HIF-1α、Bax、Bcl-2和Caspase-3表达升高，Ki67蛋白表达降低(P<0.05或P<0.01)。与辐射组相比，辐射联合黄连汤中、高剂量组小鼠肿瘤质量减轻(P<0.05或P<0.01)，小鼠肿瘤组织均出现坏死现象，凋亡细胞数量显著上升(P<0.01)，EGFR、HIF-1α mRNA表达降低，EGFR、HIF-1α、Bcl-2和Ki67蛋白表达降低，Bax、Caspase-3蛋白表达升高(P<0.01)。
      结论 黄连汤联合辐射可以有效抑制Lewis肺癌荷瘤小鼠的肿瘤生长，其机制可能与调控EGFR、HIF-1α信号通路所介导细胞凋亡有关。

       

      Abstract:
      OBJECTIVE To study the antitumor effect and mechanism of Huanglian decoction combined with X-ray radiation on mice with Lewis lung cancer.
      METHODS The 60 SPF grade male C57BL/6 mice were randomly divided into blank group, model group, radiation group, radiation combined with Huanglian decoction low dose, medium dose and high dose groups, with 10 mice in each group. In addition to the blank group, Lewis cells were injected into the right armpit of mice in all other groups to construct a lung cancer tumor bearing mouse model. After model successfully established, the tumor site was irradiated locally with 4 Gy X-ray in each radiation group. After irradiation, the radiation combined with Huanglian decoction low dose, medium dose and high dose groups were given 0.2 mL·d−1 of Huanglian decoction by gavage at doses of 5.78, 11.57 and 23.14 g·kg−1, respectively, whereas those in the blank, model and radiation group were given equal volume of normal saline. After continuous intervention for 7 d, the mice were killed and tumor tissue samples were collected. HE staining was used to observe the histopathological changes of tumor. The expression of Ki67 in tumor tissues was detected by immunohistochemistry. The apoptosis of tumor cells was detected by immunofluorescence. qRT-PCR was used to detect the expression levels of EGFR and HIF-1α mRNA in tumor tissues. The expression levels of EGFR, HIF-1α, Bcl-2, Bax and Caspase-3 proteins in tumor tissues were detected by Western blotting.
      RESULTS Compared with the model group, the tumor weight of mice in radiation group was reduced(P<0.05), the tumor cells were arranged loose, the cell gap was blurred, the phenomenon of patchy hemorrhagic necrosis appeared, the apoptotic cells were increased(P<0.01), and the mRNA expression of EGFR and HIF-1α were increased. The protein expressions of EGFR, HIF-1α, Bax, Bcl-2 and Caspase-3 were increased while Ki67 was decreased(P<0.05 or P<0.01). Compared with the radiation group, the tumor weight of mice in the radiation combined with medium dose and high dose groups of Huanglian decoction were decreased(P<0.05 or P<0.01), necrosis were observed in tumor tissues of mice, apoptotic cells were increased significantly(P<0.01), EGFR and HIF-1α mRNA expression decreased, and EGFR, HIF-1α, Bcl-2 and Ki67 protein expression were decreased, while the expression of Bax and Caspase-3 protein were increased(P<0.01).
      CONCLUSION Huanglian decoction combined with radiation can effectively inhibit the tumor growth of mice with Lewis lung cancer, and the mechanism may be related to the regulation of EGFR and HIF-1α signaling pathway mediated apoptosis.

       

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