Network Pharmacology-Based Prediction and Verification of Flavonoids from Elsholtiza densa Benth. for Treating Cervical Cancer

Objective: Elsholtzia, as a folk medicine, has been widely used in Miao, Yi, and Lahu nationalities and Tibetan and Chinese medicines in China. We aimed to elucidate the possible mechanisms of action of flavonoids of Elsholtzia densa Benth. (FIE) on apoptosis in HeLa human cervical cancer cells in vitro. Methods: Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to detect the chemical constituents responsible for the effects of FIE. Network pharmacology was employed to predict the active constituents and their targets to explore the mechanism of action of FIE in treating cervical cancer. Cell cycle and apoptosis were detected by flow cytometry. The intracellular reactive oxygen species (ROS) level and the mitochondrial membrane potential were detected in HeLa cells treated with FIE. RT-PCR assays quantified the expression of apoptosis-related genes. Results and Discusssion: A total of 36 and 109 components were identified by GC-MS and LC-MS, respectively. Six active components, including 5-hydroxy-6,7-dimethoxyflavone, baicalein, cirsimaritin, epicatechin, luteolin, and 5,7-dihydroxy-6-methoxy2-phenylchromen-4-onem, were predicted by network pharmacology. The active components may affect the proliferation and apoptosis of HeLa cells through various cancer pathways, such as NF-kB and PI3K-Akt signaling pathways. FIE significantly inhibited colony formation in HeLa cells. We observed an increase in the total population of apoptotic cells and ROS content in HeLa cells. Further, the mitochondrial membrane potential of HeLa cells was significantly decreased. In HeLa cells, the expression levels of apoptosis-related genes, such as cytochrome C, caspase-3, caspase-9, and bax, were increased, while that of bcl-2 was decreased. Moreover, the ultrastructure of the cells exhibited apoptotic characteristics. Numerous vacuoles and the autophagic structure of the damaged mitochondrial organelles were observed in the cytoplasm of HeLa cells. Conclusions: Flavonoids of Elsholtzia densa Benth. showed clear inhibitory activity in HeLa cells and induced HeLa cell apoptosis through the mitochondrial endogenous apoptotic pathway.