Shangguan Wen-Ji, Zhang Yue-Hui, Li Zhan-Chun, Tang Lu-Min, Shao Jiang, Li He
Department of Traditional Chinese Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P.R. China.
Department of Orthopedic Surgery, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, P.R. China.
Int J Mol Med. 2017 Dec;40(6):1741-1749. doi: 10.3892/ijmm.2017.3160. Epub 2017 Sep 28.
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, serum‑starved, low‑concentration naringin treatment, and high‑concentration 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 apoptosis‑associated proteins [GRP78, CHOP, caspase‑12, and cytochrome c (Cyt.c)] were detected using western blotting. JC‑1 staining was employed to detect changes in mitochondrial membrane potential. Intracellular caspase‑3, ‑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, sham‑operated, low‑concentration naringin treatment (100 mg/kg), and high‑concentration 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 dose‑dependent manner. Naringin also significantly reduced serum starvation‑induced apoptosis in endothelial cells, inhibited the expression of GRP78, CHOP, caspase‑12, and Cyt.c proteins, and reduced mitochondrial membrane potential as well as reduced the activities of caspase‑3 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 mitochondrial‑mediated pathways. Naringin also regulates endothelial cell function and promotes angiogenesis to exert its anti‑osteoporotic effect.
在本研究中，为了探究柚皮苷对血管内皮细胞（VEC）功能、增殖、凋亡和血管生成的影响，体外培养大鼠VEC，并将其随机分为四组：对照组、血清饥饿组、低浓度柚皮苷处理组和高浓度柚皮苷处理组。采用MTT法检测细胞增殖，采用Hoechst 33258染色和流式细胞术检测凋亡。使用蛋白质印迹法检测凋亡相关蛋白[GRP78、CHOP、半胱天冬酶 - 12和细胞色素c（Cyt.c）]表达的变化。采用JC - 1染色检测线粒体膜电位的变化。通过分光光度法测定细胞内半胱天冬酶 - 3、 - 8和 - 9的活性。采用ELISA法检测内皮素（ET），采用Griess法检测培养基中一氧化氮（NO）表达的变化。该研究还将骨质疏松症的去卵巢（OVX）大鼠模型随机分为四组：OVX组、假手术组、低浓度柚皮苷处理组（100 mg/kg）和高浓度柚皮苷处理组（200 mg/kg）。治疗3个月后，测定血清ET和NO表达、骨密度（BMD）以及股骨远端微血管密度（使用VEC的CD34标记）的变化。在每个浓度下，柚皮苷均以时间和剂量依赖性方式促进VEC增殖。柚皮苷还显著降低血清饥饿诱导的内皮细胞凋亡，抑制GRP78、CHOP、半胱天冬酶 - 12和Cyt.c蛋白的表达，并降低线粒体膜电位以及降低半胱天冬酶 - 3和 - 9的活性。此外，柚皮苷在体外和体内均抑制ET，同时增强NO合成。股骨远端微血管密度评估显示，柚皮苷处理组的微血管数量明显高于OVX组，且微血管密度与BMD呈正相关。总之，柚皮苷通过阻断内质网（ER）应激和线粒体介导的途径抑制VEC凋亡。柚皮苷还调节内皮细胞功能并促进血管生成，以发挥其抗骨质疏松作用。
