Total flavonoids from Semen Cuscutae inhibits osteoblast apoptosis in hormone-induced femoral head avascular necrosis

Zhao Yu; Xue Yun; Huang Jiajun; Wu Diyou; Yang Bin; Huang Junqing

doi:10.12307/2026.195

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.