Abstract: BACKGROUND: The PI3K/Akt pathway, as an intracellular signal transduction pathway, plays a crucial role in regulating cell proliferation, growth, and metabolism. Semen Cuscutae, a traditional Chinese medicine used for kidney tonification, possesses potential bone-protective effects due to its total flavonoids; however, the specific molecular mechanisms remain unclear.
OBJECTIVE: To investigate the protective effect of total flavonoids from Semen Cuscutae against dexamethasone-induced osteoblast apoptosis and its potential molecular mechanisms through a combination of network pharmacology and in vitro experiments.
METHODS: Network pharmacology was used to predict potential targets and signaling pathways for Semen Cuscutae in the treatment of dexamethasone-induced femoral head avascular necrosis. hFOB1.19 cells were cultured in vitro and treated with different concentrations of total flavonoids from Semen Cuscutae (0, 25, 50, 100, 200, 400, and 600 μg/mL) for 24 hours. Cell viability was assessed using the cell counting kit-8 assay to select the optimal treatment dose for subsequent experiments. hFOB1.19 cells were divided into six groups: control, dexamethasone, total flavonoids from Semen Cuscutae at 100, 200, 
400 μg/mL plus 2 μmol/L dexamethasone, and total flavonoids from Semen Cuscutae plus LY294002 groups (400 μg/mL total flavonoids from Semen Cuscutae and 10 μmol/L LY294002, a PI3K inhibitor). Cell viability was determined using the cell counting kit-8 assay, apoptosis rate was measured by flow cytometry, apoptosis-related protein expression was assessed by immunofluorescence, reactive oxygen species levels were quantified using the DCFH-DA probe, and expression of proteins related to the PI3K/Akt pathway was analyzed by western blot assay after treatment,.
RESULTS AND CONCLUSION: (1) Network pharmacology results indicated that the potential targets of Semen Cuscutae may be related to the regulation of tumor necrosis factor, p53, interleukin-6, serine/threonine protein kinase 1, vascular endothelial growth factor A, caspase-3, interleukin-1β, hypoxia-inducible factor 1α expression, and the PI3K/Akt pathway. (2) CCK-8 results showed that total flavonoids from Semen Cuscutae promoted the proliferation of hFOB1.19 cells within a certain range and alleviated the inhibitory effect of dexamethasone on the proliferation of hFOB1.19 cells. (3) Total flavonoids from Semen Cuscutae inhibited dexamethasone-induced apoptosis and increased reactive oxygen species levels, reduced the expression of cleaved caspase-3 and Bax, and decreased reactive oxygen species levels, while increased the protein expression of Bcl-2. (4) Compared with the dexamethasone group, total flavonoids from Semen Cuscutae enhanced the expression of p-PI3K and p-Akt proteins. (5) The PI3K inhibitor LY294002 reversed the effects of total flavonoids from Semen Cuscutae on apoptosis and reactive oxygen species levels in dexamethasone-treated cells. The results indicate that total flavonoids from Semen Cuscutae can attenuate dexamethasone-mediated apoptosis in hFOB1.19 cells, and the mechanism may be related to the activation of the PI3K/Akt pathway. 

Key words: total flavonoids from Semen Cuscutae, network pharmacology, osteoblasts, apoptosis, PI3K/Akt signaling pathway