Background: Pseudo-ginsenoside-Rh2 (pseudo-G-Rh2), a novel derivative of ginsenoside Rh2, is reported to exert a pro-apoptotic effect on various malignancies. However, whether this anti-cancer action of pseudo-G-Rh2 involves autophagy remains to be determined and explored.

Objective: The objective of this study was to investigate the pseudo-G-Rh2-induced apoptosis and autophagy and the underlying mechanism.

Methods: In the present study, the MTT assay was used for evaluating cell viability, and the lactate dehydrogenase (LDH) assay was performed to assess cell toxicity. Autophagy evaluation was performed using monodansylcadaverine (MDC) staining and transmission electron microscopy (TEM). The levels of autophagy-associated and apoptosis-associated proteins were determined using Western blotting. The Annexin V-FITC/propidium iodide (PI) assay was used to assess apoptosis.

Results: The Annexin V-FITC/PI assay revealed that the percentage of apoptotic cells in HepG2 cells at concentrations 0, 20, 40, and 60 μM was 3.75%±1.37%, 5.70%±1.04%, 12.30%±2.10%, and 34.26%±4.73%, respectively. Pseudo-G-Rh2 was observed to significantly increase the expressions of BAX, cleaved-caspase-3, and cleaved-caspase-9, while it decreased the Bcl-2 expression. MDC and TEM analysis revealed that pseudo-G-Rh2 at concentrations 20, 40, and 60 μM significantly facilitated the accumulation of autophagosomes and autolysosomes within the HepG2 cells. Moreover, pseudo-G-Rh2 significantly increased the expressions of LC3 II/LC3 I and Beclin-1 and decreased the expression of p62. The Annexin V-FITC/PI assay also revealed that in comparison to the pseudo-G-Rh2 group, the concurrent treatment with pseudo-G-Rh2 and an autophagy inhibitor (CQ or 3-MA) significantly induced distinct apoptosis. In addition, pseudo-G-Rh2 activated AMPK and inhibited the PI3K/Akt/mTOR pathway in a concentration-dependent manner. Pseudo- G-Rh2 is similar to the current patents, which enhanced its anti-cancer activity by combining with autophagy inhibitors.

Conclusion: Pseudo-G-Rh2 could induce protective autophagy in HepG2 cells, at least in part, via AMPK and the PI3K/Akt/mTOR pathway.

背景：据报道，人参皂苷 Rh2 的一种新型衍生物 Pseudo-ginsenoside-Rh2 (pseudo-G-Rh2) 对各种恶性肿瘤具有促凋亡作用。然而，pseudo-G-Rh2的这种抗癌作用是否涉及自噬仍有待确定和探索。

目的：本研究旨在探讨pseudo-G-Rh2诱导的细胞凋亡和自噬及其机制。

方法：在本研究中，MTT 法用于评估细胞活力，乳酸脱氢酶 (LDH) 法用于评估细胞毒性。使用单丹磺酰尸胺 (MDC) 染色和透射电子显微镜 (TEM) 进行自噬评估。使用蛋白质印迹法测定自噬相关蛋白和凋亡相关蛋白的水平。膜联蛋白 V-FITC/碘化丙啶 (PI) 测定用于评估细胞凋亡。

结果：Annexin V-FITC/PI检测显示，0、20、40和60 μM浓度的HepG2细胞中凋亡细胞百分比分别为3.75%±1.37%、5.70%±1.04%、12.30%±2.10%、和 34.26%±4.73%，分别。观察到 Pseudo-G-Rh2 显着增加 BAX、cleaved-caspase-3 和 cleaved-caspase-9 的表达，同时降低 Bcl-2 的表达。MDC 和 TEM 分析显示，浓度为 20、40 和 60 μM 的假 G-Rh2 显着促进了 HepG2 细胞内自噬体和自溶酶体的积累。此外，pseudo-G-Rh2显着增加了LC3 II/LC3 I和Beclin-1的表达，降低了p62的表达。Annexin V-FITC/PI 检测还显示，与伪 G-Rh2 组相比，用假 G-Rh2 和自噬抑制剂（CQ 或 3-MA）同时处理显着诱导明显的细胞凋亡。此外，pseudo-G-Rh2 以浓度依赖性方式激活 AMPK 并抑制 PI3K/Akt/mTOR 通路。Pseudo-G-Rh2与现有专利类似，通过与自噬抑制剂结合增强其抗癌活性。

结论：Pseudo-G-Rh2 可以在 HepG2 细胞中诱导保护性自噬，至少部分通过 AMPK 和 PI3K/Akt/mTOR 途径。