Naringin inhibits vascular endothelial cell apoptosis via endoplasmic reticulum stress and mitochondrialmediated pathways and promotes intraosseous angiogenesis in ovariectomized rats

In this study, to investigate the effects of naringin on vascular endothelial cell (VEC) function, proliferation, apoptosis, and angiogenesis, rat VECs were cultured in vitro and randomly divided into four groups: control, serumstarved, lowconcentration naringin treatment, and highconcentration naringin treatment. MTT assay was used to detect cell proliferation while Hoechst 33258 staining and flow cytometry were used to detect apoptosis. Changes in the expression of apoptosisassociated proteins [GRP78, CHOP, caspase12, and cytochrome c (Cyt.c)] were detected using western blotting. JC1 staining was employed to detect changes in mitochondrial membrane potential. Intracellular caspase3, 8, and 9 activity was determined by spectrophotometry. ELISA was used to detect endothelin (ET), and a Griess assay was used to detect changes in the expression of nitric oxide (NO) in culture medium. The study further divided an ovariectomized (OVX) rat model of osteoporosis randomly into four groups: OVX, shamoperated, lowconcentration naringin treatment (100 mg/kg), and highconcentration naringin treatment (200 mg/kg). After 3 months of treatment, changes in serum ET and NO expression, bone mineral density (BMD), and microvessel density of the distal femur (using CD34 labeling of VECs) were determined. At each concentration, naringin promoted VEC proliferation in a time and dosedependent manner. Naringin also significantly reduced serum starvationinduced apoptosis in endothelial cells, inhibited the expression of GRP78, CHOP, caspase12, and Cyt.c proteins, and reduced mitochondrial membrane potential as well as reduced the activities of caspase3 and 9. Furthermore, naringin suppressed ET in vitro and in vivo while enhancing NO synthesis. Distal femoral microvascular density assessment showed that the naringin treatment groups had a significantly higher number of microvessels than the OVX group, and that microvascular density was positively correlated with BMD. In summary, naringin inhibits apoptosis in VECs by blocking the endoplasmic reticulum (ER) stress and mitochondrialmediated pathways. Naringin also regulates endothelial cell function and promotes angiogenesis to exert its antiosteoporotic effect.