基于SIRT3/HIF-1α/PFKFB3糖代谢相关通路探讨益气养精方调节肺癌生长及血管形成的研究

Tumor glucose metabolism and its angiogenesis and invasion and metastasis are closely related to the pathway SIRT3 / HIF-1α / PFKFB3 regulation of tumor glucose metabolism. Previous studies have shown thatEnhance Qi Nourish Jing Formula can regulate the expression of hypoxia-inducible factors HIF-1α and VEGF, and can inhibit lung cancer cell proliferation, angiogenesis, invasion and metastasis. Therefore, it is speculated that this side may regulate the glycometabolism and angiogenesis of lung cancer through this pathway. In vitro study of human lung cancer cell lines co-cultured with human umbilical vein endothelial cells as the research object, RNAi were used to interfere with the pathway of SIRT3, HIF-1α, PFKFB3 expression was observed in the hypoxic environmentEnhance Qi Nourish Jing Formula intervention Molecular expression of intracellular glucose metabolism pathway and extracellular lactate concentration. In vivo, A549 and H1975 lung cancer xenografts were induced with Enhance Qi Nourish Jing Formula and 3PO (PFKFB3 inhibitors) respectively. Tumor tissues were examined for the expression of relevant pathway factors, tissue lactate concentration and microvessel density. To further explore the Enhance Qi Nourish Jing Formula by regulating the glucose metabolism pathway to affect tumor metabolism and angiogenesis may be revealed from a new perspective of Qi and nourishing, detoxification and anti-cancer treatment of lung cancer mechanism.

肿瘤的糖代谢与其血管生成和侵袭转移有密切关系，相关通路SIRT3/HIF-1α/PFKFB3调控着肿瘤的糖代谢。前期研究表明益气养精方能调控缺氧诱导因子HIF-1α和VEGF的表达，并能抑制肺癌细胞增殖、血管生成和侵袭转移。因而推测该方可能通过此通路调节肺癌的糖代谢和血管形成。拟体外研究以肺癌细胞株、肺癌细胞共培养的脐静脉内皮细胞为研究对象，用RNAi分别干扰通路中SIRT3、HIF-1α、PFKFB3的表达,观察缺氧环境下的益气养精方干预后细胞糖代谢相关通路分子表达和细胞外乳酸浓度。体内研究以表达荧光素酶的A549和H1975的肺癌移植瘤模型，用益气养精方和3PO（PFKFB3抑制剂）分别干预后，取肿瘤组织检测相关通路因子表达、组织乳酸浓度和微血管密度。进一步探讨益气养精方通过调节该糖代谢通路来影响肿瘤代谢和血管形成的可能，从新的角度揭示益气养精、解毒抗癌法治疗肺癌的机制。

肺癌是世界上发病率和死亡率最高的恶性肿瘤之一。益气养精方（YYR）是一种治疗肺癌的常用中药配方，具有良好的临床疗效，但YYR的具体抗癌机制仍不清楚。方法：采用CCK-8法测定YYR对A549细胞的细胞毒作用。用RNAi分别干扰通路中SIRT3、HIF-1α、PFKFB3的表达,观察缺氧环境下的YYR干预后细胞糖代谢相关通路分子表达和细胞外乳酸浓度，使用流式细胞术分析和海马XF96细胞外通量分析仪，以分别研究YYR在正常、PFKFB3低表达和高表达A549细胞中的促凋亡和抗糖酵解能力，此外，YYR的抗癌作用也通过体内裸鼠的致瘤性试验得到证实。结果：YYR对A549细胞具有明显的细胞毒活性。在缺氧状态下，YYR干预后引起SIRT3的上调和HIF-1α的下调，并使得细胞内乳酸和葡萄量产量减少。此外，YYR和PFK15处理还可以降低A549细胞的细胞外酸化率（ECAR）和耗氧率（OCR），并促进细胞凋亡。研究发现，与PFK15类似，YYR可以下调PFKFB3的mRNA和蛋白表达，过度表达PFKFB3可以抑制A549细胞的凋亡，而YYR可以降低过度表达的A549细胞对PFKFB3细胞的凋亡抗性。上述所有结果表明，PFKFB3因其促凋亡和抗糖酵解能力而成为YYR的潜在药物靶点。动物实验证实，YYR可以抑制肿瘤生长，诱导肿瘤细胞凋亡，并下调肿瘤组织中的PFKFB3。结论：我们的研究表明，YYR通过调控SIRT3-HIF-1α-PFKFB3通路下调PFKFB3的表达促进肺癌细胞凋亡并抑制能量代谢，这为YYR作为肺癌治疗的补充或替代药物提供了科学依据。