It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.

目的:探究瞬时受体电位阳离子通道V亚家族成员4(transient receptor potential cation channel sub?family V member 4,TRPV4)在H9C2细胞中缺氧环境下的作用机制.方法:采用H9C2细胞建立缺氧损伤模型,检测缺氧不同时间细胞内乳酸脱氢酶(lactate dehydrogenase,LDH)释放率、丙二醛(malondialdehyde,MDA)含量、活性氧(reactive oxygen species,ROS)水平及过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase,SOD)活性.将细胞分为常氧组(0 h)、HC-067047(TRPV4抑制剂)预处理+常氧(0 h)组、缺氧(24 h)组和HC-067047预处理+缺氧(24 h)组,使用Fluo-4 AM检测细胞内Ca2+含量,JC-1检测线粒体膜电位,Western blot检测TRPV4蛋白水平,RT-qPCR检测TRPV4的mRNA水平,DCFH-DA检测ROS含量,酶标仪检测CAT和SOD活性,CCK-8法检测细胞活力.结果:与0 h组相比,随着缺氧时间的延长,LDH释放率提高(P<0.05),细胞内ROS和MDA含量上升(P<0.05),CAT和SOD活性增强(P<0.05),氧化损伤逐渐加重并在24 h达到顶峰.此外,缺氧后细胞出现钙超载现象,线粒体膜电位下降,TRPV4被激活.使用TRPV4抑制剂HC-067047预处理后,H9C2的氧化损伤明显被抑制,细胞活力上升(P<0.05),并且钙超载现象和线粒体膜电位下降均有所逆转.结论:在心肌细胞缺氧后,TRPV4被激活,Ca2+内流,线粒体膜电位受损,ROS增多,氧化损伤加重,抗氧化系统被激活,CAT和SOD活性上升,进而调控氧化与抗氧化平衡.

OBJECTIVETo explore whether the effect of low-frequency pulsed electromagnetic fields (PEMFs) in promoting osteoblast mineralization and maturation is related to the primary cilia, polycystin2 (PC2) and sAC/PKA/CREB signaling pathway.METHODSWe detected the expression levels of PC2, sAC, PKA, CREB and their phosphorylated proteins in primary rat calvarial osteoblasts exposed to 50 Hz 0.6 mT PEMFs for 0, 5, 15, 30, 60, 90, and 120 min. We blocked PC2 function with amiloride hydrochloride and detected the changes in the activity of sAC/PKA/CREB signal pathway and the mineralization and maturation of the osteoblasts. These examinations were repeated in the osteoblasts after specific knockdown of PC2 via RNA interference and were the co-localization of PC2, sAC, PKA, CREB and their phosphorylated proteins with the primary cilia were using immunofluorescence staining. The expressions of PC2 and the signaling proteins of sAC/PKA/CREB pathway were detected after inhibition of primary ciliation by RNA interference.RESULTSThe expression levels of PC2, sAC, p-PKA and p- CREB were significantly increased in the osteoblasts after exposure to PEMFs for different time lengths (P < 0.01). Blocking PC2 function or PC2 knockdown in the osteoblasts resulted in failure of sAC/PKA/CREB signaling pathway activation and arrest of osteoblast mineralization and maturation. PC2, sAC, p-PKA and p-CREB were localized to the entire primary cilia or its roots, but PKA and CREB were not detected in the primary cilia. After interference of the primary cilia, PEMFs exposure no longer caused increase of PC2 expression and failed to activate the sAC/PKA/CREB signaling pathway or promote osteoblast mineralization and maturation.CONCLUSIONPC2, located on the surface of the primary cilia of osteoblasts, can perceive and transmit the physical signals from PEMFs and promote the mineralization and maturation of osteoblasts by activating the PC2/ sAC/PKA/CREB signaling pathway.

目的 研究松脂醇二葡萄糖苷(pinoresinol diglucoside,PDG)对青年大鼠骨代谢的影响.方法 将实验大鼠按随机数字表达法分为Control组、PDG-25组和PDG-50组.PDG-25组和PDG-50组每天分别按25 mg·kg-1和50 mg·kg-1的剂量灌服PDG,Control组每天灌服等体积蒸馏水.6周后处死所有大鼠,取脏器、血清、股骨、胫骨以及椎骨,用于组织病理学检测、离体骨密度检测、Micro CT成像分析、血清生化指标检测、双荧光标记观察以及股骨骨组织蛋白表达分析.结果 各组大鼠主要脏器的组织病理学结果均未见明显异常;与Control组相比,PDG-25组和PDG-50组的离体骨密度均明显增高;Micro CT结果显示,PDG-50组的骨体积分数、骨小梁数以及骨小梁厚度均明显升高,但骨小梁分离度明显下降;PDG-25组和PDG-50组荧光标记间距明显增大;PDG-25组和PDG-50组血清OPG含量明显提高,但RANKL含量明显降低;PDG-25组和PDG-50组BMP-2、Runx-2、OSX和OPG表达量明显增多,RANKL表达量明显减少.结论 25 mg·kg-1和50 mg·kg-1的PDG可能通过促进骨形成和抑制骨吸收来提高青年大鼠骨质量与骨强度.