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Abstract：
Introduction: Cervical cancer (CC) ranks as the fourth most prevalent malignant tumor among women worldwide, and is the fourth leading cause of cancer-related mortality. GuiErBai (GEB), a compound preparation developed by our research team, is derived from the ancient Chinese medicine of the Miao nationality and is comprised of podophyllotoxin (PTOX), imperatorin, isoimperatorin, and A. dahurica alkaloids. These individual components have demonstrated notable efficacy in tumor treatment. However, the specific anti-tumor effect of the compound Chinese medicine GEB in the context of CC has yet to be validated. Methods: HeLa and SiHa cell lines were utilized for in vitro experiments and treated with 5 mg/mL and 10 mg/mL GEB concentrations, respectively. The cell cycle changes after GEB treatment were assessed using flow cytometry. Transmission electron microscopy was employed to observe autophagic bodies and apoptotic bodies, while MDC staining evaluated the occurrence of autophagy. CCK-8 was used to observe the effect of GEB on cell proliferation, and Transwell assays assessed cell migration and invasion. Western blotting detected cell cycle and apoptosis-related protein expression, along with the expression level of autophagy-related protein LC3I/II. Changes in ROS and mitochondrial membrane potential in cervical cancer cells following GEB treatment were determined using ROS detection and mitochondrial membrane potential detection kits. For the in vivo experiment, a nude mouse model of cervical cancer transplantation based on HeLa cells was established. Experimental animals were divided into negative control, positive control, high-dose GEB (10 mg/mL), and low-dose GEB (5 mg/mL) groups. Results: In HeLa and SiHa cell lines, the G0/G1 phase of tumor cells significantly decreased (p < 0.001), while the G2/M phase increased notably (p < 0.001) following various GEB treatments. Electron microscopy showed GEB promoted apoptotic body and autophagosome formation in both cell lines. Compared to untreated HeLa and SiHa cells, GEB-treated cells exhibited significantly reduced caspase3 protein expression, and substantially increased autophagy-related protein LC3I/II expression. GEB treatment significantly reduced migration and invasion capabilities in both cell lines (p < 0.001), while ROS content and mitochondrial membrane potential were significantly elevated (p < 0.001). GEB effectively inhibited cervical cancer cell proliferation, with the optimal concentration being 10 mg/mL. A successful nude mouse model of cervical cancer transplantation was established using HeLa cells. Post-GEB treatment, the tumor volume and weight in nude mice significantly decreased (p < 0.001), with diminished expression of CD34, VEGF, and caspase3 proteins in tumor tissues. Discussion: GEB exhibits a robust antitumor effect against cervical cancer, both in vitro and in vivo, in a concentration-dependent manner, by regulating autophagy and apoptosis of tumor cells.
摘要：
简介：宫颈癌（CC）是全球女性中第四大最常见的恶性肿瘤，并且是癌症相关死亡率的第四大原因。贵尔白（GEB），我们研究小组开发的一种复合制剂，来源于苗族古代中药，由鬼臼毒素（PTOX）组成，欧周素,异欧前胡素,和A.dahuria生物碱。这些单独的组分已证明在肿瘤治疗中具有显著的功效。然而,复方中药GEB在CC背景下的具体抗肿瘤作用尚待验证。方法：HeLa和SiHa细胞系用于体外实验，并用5mg/mL和10mg/mLGEB浓度处理，分别。使用流式细胞术评估GEB处理后的细胞周期变化。透射电镜观察自噬体和凋亡体,而MDC染色评价自噬的发生。CCK-8用于观察GEB对细胞增殖的影响。和Transwell测定评估细胞迁移和侵袭。Westernblotting检测细胞周期和凋亡相关蛋白的表达,自噬相关蛋白LC3I/II的表达水平。使用ROS检测和线粒体膜电位检测试剂盒测定GEB治疗后宫颈癌细胞中ROS和线粒体膜电位的变化。对于体内实验，建立了基于HeLa细胞的宫颈癌移植裸鼠模型。实验动物分为阴性对照,阳性对照,高剂量GEB（10mg/mL），和低剂量GEB（5mg/mL）组。结果：在HeLa和SiHa细胞系中，肿瘤细胞G0/G1期显著降低(p<0.001),而G2/M期在各种GEB治疗后显著增加(p<0.001)。电子显微镜显示GEB促进两种细胞系的凋亡小体和自噬体形成。与未经处理的HeLa和SiHa细胞相比，GEB处理的细胞表现出显著降低的caspase3蛋白表达,并显著增加自噬相关蛋白LC3I/II的表达。GEB处理显著降低两种细胞系的迁移和侵袭能力（p<0.001），而ROS含量和线粒体膜电位显著升高(p<0.001)。GEB能有效抑制宫颈癌细胞增殖,最佳浓度为10mg/mL。使用HeLa细胞成功建立了宫颈癌移植的裸鼠模型。GEB治疗后，裸鼠的肿瘤体积和重量显着降低（p<0.001），随着CD34，VEGF的表达减少，和肿瘤组织中的caspase3蛋白。讨论：GEB对宫颈癌具有强大的抗肿瘤作用，在体外和体内，以浓度依赖的方式，通过调节肿瘤细胞的自噬和凋亡。
