■雌激素缺乏是绝经后骨质疏松症的主要原因。Eldecalcitol(ED-71)是临床上用于治疗绝经后骨质疏松症的新型活性维生素D类似物。我们旨在研究EphrinB2-EphB4和RANKL/RANK/OPG信号是否在ED-71介导的骨质疏松症过程中协同作用。
■体内,对去卵巢(OVX)大鼠每天一次口服30ng/kgED-71,共8周.HE染色,Masson染色和免疫荧光染色用于评估骨量,骨形成,破骨细胞生成相关因子与EphrinB2,EphB4,RANKL和OPG的表达。体外,H2O2刺激用于模拟骨质疏松症的细胞环境。免疫荧光,实时定量PCR(qRT-PCR),采用酶联免疫吸附法(ELISA)和WesternBlot检测EphrinB2、EphB4、RANKL和OPG的表达。在成骨细胞中,通过EphB4小干扰RNA(siRNA)转染敲低EphB4。LY294002(PI3K抑制剂)或ARQ092(AKT抑制剂)用于阻断PI3K/AKT途径。建立了成骨细胞和破骨细胞的间接共培养体系。通过qRT-PCR和WesternBlot检测破骨细胞相关因子的mRNA和蛋白表达。
■ED-71增加了OVX大鼠的骨量并减少了破骨细胞的数量。此外,ED-71促进EphrinB2、EphB4的表达,降低成骨细胞RANKL/OPG比值。当破骨细胞与ED-71处理的成骨细胞间接共培养时,破骨细胞的生成受到抑制。EphB4在成骨细胞中表达沉默后,ED-71抑制P-PI3K和P-AKT的表达并增加RANKL/OPG的比率。这逆转了ED-71对破骨细胞的抑制作用。因此,在ED-71抑制的破骨细胞中,EphB4是影响成骨细胞分泌RANKL和OPG的关键因素。EphB4通过激活成骨细胞中的PI3K/AKT信号传导抑制RANKL/OPG比率。
■ED-71通过EphrinB2-EphB4-RANKL/OPG轴抑制破骨细胞生成,改善去卵巢大鼠的骨量。PI3K/AKT通路参与这一过程。
UNASSIGNED: Estrogen deficiency is the main reason of postmenopausal osteoporosis. Eldecalcitol (ED-71) is a new active vitamin D analogue clinically used in the treatment of postmenopausal osteoporosis. We aimed to investigate whether EphrinB2-EphB4 and RANKL/RANK/OPG signaling cooperate in mediating the process of osteoporosis by ED-71.
UNASSIGNED: In vivo, the ovariectomized (OVX) rats were administered orally with 30 ng/kg ED-71 once a day for 8 weeks. HE staining, Masson staining and Immunofluorescence staining were used to evaluate bone mass, bone formation, osteoclastogenesis associated factors and the expression of EphrinB2, EphB4, RANKL and OPG. In vitro, H2O2 stimulation was used to simulate the cell environment in osteoporosis. Immunofluorescence, quantitative real time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western Blot were applied to detect the expression of EphrinB2, EphB4, RANKL and OPG. In osteoblasts, EphB4 was knocked down by EphB4 small-interfering RNA (siRNA) transfection. LY294002 (PI3K inhibitor) or ARQ092 (AKT inhibitor) was used to block PI3K/AKT pathway. An indirect co-culture system of osteoblasts and osteoclasts was established. The mRNA and protein expression of osteoclastogenes is associated factors were tested by qRT-PCR and Western Blot.
UNASSIGNED: ED-71 increased bone mass and decreased the number of osteoclasts in OVX rats. Moreover, ED-71 promoted the expression of EphrinB2, EphB4, and decreased the RANKL/OPG ratio in osteoblasts. Osteoclastogenesis was restrained when osteoclasts were indirectly co-cultured with ED-71-treated osteoblasts. After silencing of EphB4 expression in osteoblasts, ED-71 inhibited the expression of P-PI3K and P-AKT and increased the ratio of RANKL/OPG. This reversed the inhibitory effect of ED-71 on osteoclastogenes. Therefore, in ED-71-inhibited osteoclastogenes, EphB4 is a key factor affecting the secretion of RANKL and OPG by osteoblasts. EphB4 suppressed the RANKL/OPG ratio through activating PI3K/AKT signaling in osteoblasts.
UNASSIGNED: ED-71 inhibits osteoclastogenesis through EphrinB2-EphB4-RANKL/OPG axis, improving bone mass in ovariectomized rats. PI3K/AKT pathway is involved this process.