安正抗瘤方通过PI3K/Akt/mTOR通路抑制糖酵解抗结直肠癌作用机制研究

Study on the Mechanism of Anti-Colorectal Cancer Effects of Anzheng Kangliu Decoction by Inhibiting Glycolysis via PI3K/Akt/mTOR Pathway

    摘要
  • 摘要:
    目的 探讨安正抗瘤方调控磷酯酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(mTOR)信号通路抑制糖酵解抗结直肠癌的作用机制。
    方法 用安正抗瘤方干预人结直肠癌SW620细胞，检测细胞增殖、迁移能力。将42只BALB/c裸鼠随机分为空白对照组、模型组、5-氟尿嘧啶(5-FU)组(0.025 g·kg-1)、安正抗瘤方低剂量组(7.67 g·kg-1)、安正抗瘤方中剂量组(15.34 g·kg-1)、安正抗瘤方高剂量组(30.68 g·kg-1)，通过观察体质量、瘤体体积、肿瘤质量、HE染色、Ki67免疫组织化学染色等指标，检测安正抗瘤方对裸鼠皮下移植瘤的抑制作用。采用高通量转录组测序技术，探讨模型组和安正抗瘤方中剂量组裸鼠肿瘤组织的差异表达基因及通路，探讨安正抗瘤方抗结直肠癌的潜在机制。葡萄糖测试盒、乳酸测试盒检测安正抗瘤方干预后SW620细胞及裸鼠肿瘤组织内葡萄糖消耗量与乳酸生成量的变化，Western blot检测安正抗瘤方干预后SW620细胞及裸鼠肿瘤组织内p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR、己糖激酶2(HK2)、乳酸脱氢酶A(LDHA)蛋白表达水平的变化。
    结果 体外细胞实验显示，与空白对照组相比，安正抗瘤方干预后，SW620细胞增殖和迁移能力均下降(P＜0.01)，葡萄糖消耗量及乳酸生成量均降低(P＜0.05，P＜0.01)，p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR、HK2、LDHA蛋白表达水平均降低(P＜0.05，P＜0.01)。体内动物实验显示，与模型组相比，安正抗瘤方能够抑制裸鼠皮下移植瘤的生长(P＜0.01)，使肿瘤组织坏死程度增加，肿瘤组织内葡萄糖消耗量及乳酸生成量均降低(P＜0.05，P＜0.01)，p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR、HK2、LDHA蛋白表达水平均降低(P＜0.05，P＜0.01)。
    结论 安正抗瘤方具有抑制结直肠癌的作用，其作用机制可能与调控PI3K/Akt/mTOR信号通路抑制糖酵解有关。

     

    Abstract:
    OBJECTIVE To investigate the inhibitory effect mechanism of Anzheng Kangliu Decoction against colorectal cancer by suppressing glycolysis through regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway.
    METHODS Human colorectal cancer SW620 cells were treated with Anzheng Kangliu Decoction, and cell proliferation and migration abilities were assessed. Forty-two BALB/c nude mice were randomly divided into a blank control group, model group, 5-fluorouracil (5-FU) group (0.025 g ·kg-1), Anzheng Kangliu Decoction low-dose group (7.67 g ·kg-1), medium-dose group (15.34 g ·kg-1), and high-dose group (30.68 g ·kg-1). The inhibitory effect of Anzheng Kangliu Decoction on subcutaneous xenograft tumors was evaluated by observing body weight, tumor volume, tumor mass, HE staining, immunohistochemical staining of Ki67 and other indicators. High-throughput transcriptome sequencing was performed to identify differentially expressed genes and pathways in tumor tissues between the model group and the Anzheng Kangliu Decoction medium-dose group, elucidating the potential mechanism of Anzheng Kangliu Decoction against colorectal cancer. Glucose and lactate assay kits were used to measure glucose consumption and lactate production in SW620 cells and tumor tissues after Anzheng Kangliu Decoction intervention. Western blot was employed to detect the expression levels of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA) in SW620 cells and tumor tissues following Anzheng Kangliu Decoction treatment.
    RESULTS In vitro cell experiments demonstrated that, compared with the blank control group, Anzheng Kangliu Decoction intervention significantly inhibited the proliferation and migration of SW620 cells (P < 0.01), reduced glucose consumption and lactate production (P < 0.05, P < 0.01), and downregulated the protein expression levels of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, HK2, and LDHA (P < 0.05, P < 0.01). In vivo animal experiments revealed that, compared with the model group, Anzheng Kangliu Decoction suppressed the growth of subcutaneous xenograft tumors in nude mice (P < 0.01), increased tumor tissue necrosis, decreased glucose consumption and lactate production in tumor tissues (P < 0.05, P < 0.01), and reduced the protein expression levels of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, HK2, and LDHA (P < 0.05, P < 0.01).
    CONCLUSION Anzheng Kangliu Decoction exerts an inhibitory effect on colorectal cancer, and its mechanism may be associated with the suppression of glycolysis through regulation of the PI3K/Akt/mTOR signaling pathway.

     

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