新藤黄酸增强仑伐替尼在肝细胞癌中的敏感性及其机制研究

目的 探究新藤黄酸（gambogenic acid，GNA）协同仑伐替尼对肝细胞癌的抑制、促进肝细胞癌的凋亡作用及其机制。方法 采用H22肝癌细胞建立H22移植瘤小鼠模型，将模型复制成功的小鼠随机分为模型组（生理盐水），GNA组（20 mg/kg），仑伐替尼组（10 mg/kg），GNA（20 mg/kg）+仑伐替尼（10 mg/kg）组，每组10只，各组小鼠给予相应药物连续灌胃2周，每日1次；比较各组小鼠的肿瘤质量和体质量，观察GNA联合仑伐替尼对肿瘤生长的作用；苏木精-伊红染色观察GNA联合仑伐替尼对移植瘤模型小鼠各脏器的影响；TUNEL染色检测移植瘤模型小鼠肿瘤组织中细胞凋亡情况；Western blot检测移植瘤模型小鼠肿瘤组织中孕烷X受体（pregnane X receptor，PXR）、P-糖蛋白（P-glycoprotein，P-gp）、乳腺癌耐药蛋白（breast cancer resistant protein，BCRP）、细胞色素P450 3A4酶（cytochrome P450 3A4，CYP3A4）、P53、B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2-associated X，Bax）、cleaved-caspase-3蛋白的表达水平。结果 与模型组比较，GNA+仑伐替尼组肿瘤生长得到明显抑制（P＜0.05），并且优于GNA组和仑伐替尼组（P＜0.05）；给药后小鼠的主要器官（心、肝、脾、肺、肾）均未出现明显病变；与模型组比较，各给药组均发生明显的细胞凋亡，GNA+仑伐替尼组细胞凋亡较仑伐替尼组更加明显；GNA+仑伐替尼组肿瘤组织中PXR、P-gp、BCRP、CYP3A4、Bcl-2的蛋白表达水平显著降低（P＜0.05），P53、Bax、cleaved-caspase-3蛋白表达水平显著上升（P＜0.05）。结论 GNA与仑伐替尼联用可以发挥协同作用，GNA增强仑伐替尼对肝细胞癌的敏感性，其分子机制可能与下调核受体PXR的表达水平，从而影响代谢酶CYP3A4,转运体P-gp,BCRP和凋亡蛋白P53、Bax、Bcl-2、cleaved-caspase-3的表达有关。

Mechanism of Gambogenic Acid Enhancing the Sensitivity of Lenvatinib in Hepatocellular Carcinoma

Objective To investigate the synergistic effect of gambogenic acid (GNA) and lenvatinib in inhibiting hepatocellular carcinoma (HCC) and promoting HCC apoptosis and related mechanism. Methods H22 hepatoma cells were used to establish a mouse xenograft tumor model, and after successful modeling, the mice were randomly divided into model group (normal saline), GNA group (20 mg/kg), lenvatinib group (10 mg/kg), and GNA (20 mg/kg)+lenvatinib (10 mg/kg) group, with 10 mice in each group. The mice in each group were given the corresponding drug by gavage once a day for 2 consecutive weeks. Tumor weight and body weight were compared between groups to observe the effect of GNA combined with lenvatinib on tumor growth; HE staining was used to observe the effect of GNA combined with lenvatinib on visceral organs in the mice with xenograft tumor; TUNEL staining was used to observe cell apoptosis in tumor tissue of the mice with xenograft tumor; Western blot was used to measure the protein expression levels of pregnane X receptor(PXR), P-glycoprotein(P-gp), breast cancer resistant protein(BCRP), cytochrome P450 3A4(CYP3A4),P53,B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax), and cleaved-caspase-3 in tumor tissue of the mice with xenograft tumor. Results Compared with the model group, GNA combined with lenvatinib significantly inhibited tumor growth (P<0.05) and had a better effect than GNA or lenvatinib used alone (P<0.05).After administration, no obvious lesions were observed in the major organs (heart, liver, spleen, lung, and kidney) of the mice. Compared with the model group, all administration groups showed significant cell apoptosis, and the GNA+lenvatinib group showed more significant cell apoptosis than the lenvatinib group. The GNA+lenvatinib group had significant reductions in the protein expression levels of PXR,P-gp,BCRP,CYP3A4, and Bcl-2 (P<0.05) and significant increases in the protein expression levels of P53,Bax, and cleaved-caspase-3 (P<0.05) in tumor tissue. Conclusion GNA combined with lenvatinib can exert a synergistic effect and enhance the sensitivity of HCC to lenvatinib, possibly by downregulating the expression of the nuclear receptor PXR and affecting the expression of the metabolic enzyme CYP3A4, the transporters P-gp and BCRP, and the apoptotic proteins P53,Bax,Bcl-2, and cleaved-caspase-3.