氧化应激被认为与航天引起的骨丢失密切相关;然而,机制难以捉摸,没有有效的对策。使用随机定位机模拟的暴露于微重力的培养的大鼠颅骨成骨细胞,这项研究解决了以下假设:微重力诱导的初级纤毛缩短会导致氧化应激,而初级纤毛保护会阻止氧化应激和成骨损失。发现微重力会诱导氧化应激(表现为活性氧(ROS)和丙二醛产生水平增加,和抗氧化酶活性降低),在NG中生长的成骨细胞中完全复制,原代纤毛消失(通过干扰RNA的转染产生),表明初级纤毛的缩短可能导致氧化应激。氧化应激伴随着线粒体功能障碍(表现为线粒体ROS增加和线粒体膜电位降低)和细胞内Ca2+过载,发现后者是由于Ca2通道瞬时受体电位香草酸4(TRPV4)的活性增加所致,TRPV4激动剂GSK1016790A引起的Ca2+流入也证明了这一点。补充HC-067047,TRPV4的特异性拮抗剂,减轻微重力诱导的线粒体功能障碍,氧化应激,和成骨损失。尽管发现TRPV4位于初级纤毛中并在NG中以低水平表达,微重力引起的初级纤毛缩短导致TRPV4水平和Ca2流入增加。当初级纤毛被miR-129-3p过表达或补充天然类黄酮莫苏黄酮保护时,微重力诱导的TRPV4表达增加,线粒体功能障碍,氧化应激,骨生成损失均得到预防。我们的数据揭示了一种新的机制,即初级纤毛可作为TRPV4表达的控制器。微重力对初级纤毛的损伤导致TRPV4的表达增加和通道过度活跃,引起细胞内Ca2+过载和氧化应激,初级纤毛保护可能是对抗微重力诱导的成骨细胞氧化应激和成骨潜能丧失的有效对策。
Oxidative stress has been considered to be closely related to spaceflight-induced bone loss; however, mechanism is elusive and there are no effective countermeasures. Using cultured rat calvarial osteoblasts exposed to microgravity simulated by a random positioning machine, this study addressed the hypotheses that microgravity-induced shortening of primary cilia leads to oxidative stress and that primary cilium protection prevents oxidative stress and osteogenesis loss. Microgravity was found to induce oxidative stress (as represented by increased levels of reactive oxygen species (ROS) and malondialdehyde production, and decreased activities of antioxidant enzymes), which was perfectly replicated in osteoblasts growing in NG with abrogated primary cilia (created by transfection of an interfering RNA), suggesting the possibility that shortening of primary cilia leads to oxidative stress. Oxidative stress was accompanied by mitochondrial dysfunction (represented by increased mitochondrial ROS and decreased mitochondrial membrane potential) and intracellular Ca2+ overload, and the latter was found to be caused by increased activity of Ca2+ channel transient receptor potential vanilloid 4 (TRPV4), as also evidenced by TRPV4 agonist GSK1016790A-elicited Ca2+ influx. Supplementation of HC-067047, a specific antagonist of TRPV4, attenuated microgravity-induced mitochondrial dysfunction, oxidative stress, and osteogenesis loss. Although TRPV4 was found localized in primary cilia and expressed at low levels in NG, microgravity-induced shortening of primary cilia led to increased TRPV4 levels and Ca2+ influx. When primary cilia were protected by miR-129-3p overexpression or supplementation with a natural flavonoid moslosooflavone, microgravity-induced increased TRPV4 expression, mitochondrial dysfunction, oxidative stress, and osteogenesis loss were all prevented. Our data revealed a new mechanism that primary cilia function as a controller for TRPV4 expression. Microgravity-induced injury on primary cilia leads to increased expression and overactive channel of TRPV4, causing intracellular Ca2+ overload and oxidative stress, and primary cilium protection could be an effective countermeasure against microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts.