The Role And Mechanism Of Primary Cilia/PGE2-related Signal Pathway In Low-magnitude High-frequency Vibration-induced Effects In Osteoblasts

Osteoporosis(OP)is a systemic bone disease characterized by decreased bone mass and degeneration of bone microstructure,resulting in decreased bone strength,increased fragility and increased risk of fracture.In China,the total prevalence rate of people over 50 years old is 20.7% for women and 14.4% for men.Osteoporosis has become a major health problem in China,which is one of the major diseases endangering the life and health of the elderly.The traditional treatment method is drug therapy,but drug therapy has the disadvantages of long course,large side effects,high cost and poor compliance.As a non drug and non-invasive therapy,low-magnitude high-frequency vibration(LMHFV;frequency 10-100 hz,intensity < 0.5g;G: acceleration of gravity)has achieved a good effect in the treatment of osteoporosis.LMHFV has the advantages of noninvasive,comfortable,easy to operate,safe and effective.LMHFV can improve the mechanical properties and density of long bone,microstructure and cancellous bone,so as to reduce the occurrence of brittle fracture.LMHFV treatment can significantly increase muscle mass,inhibit skeletal muscle atrophy,promote skeletal muscle regeneration,improve the function of skeletal muscle,thus reducing the risk of falls.In addition,LMHFV can improve peripheral blood flow.Although LMHFV has been proved to be effective in promoting osteogenesis and preventing osteoporosis,its mechanism is still unclear.The research focus and difficulties are mainly focused on two aspects: 1.What kind of structure can sense the mechanical stress and transfer the mechanical stimulation into the biochemical signal of bone tissue cells.2.How does the bone tissue cell transmit the biological signals it transduces to the nucleus or other cells.Primary cilia are highly conserved organelles in cell evolution.Primary cilia can feel extracellular chemical signals(such as various growth factors and hormones)and mechanical signals like an antenna,so as to regulate intracellular signal transduction and gene expression.Primary cilia play an important role in the development and metabolism of skeletal muscle.The maintenance of the normal structure of the primary cilia depends on the forward and reverse transport in the cilia,which is closely related to the cilia transport protein(IFT).Once the expression of IFT is inhibited,the formation of cilia will be hindered.The role of primary cilia under the action of LMHFV is unclear.We speculate that the primary cilia can sense the vibration stress and play an important role in the process of LMHFV induced osteogenesis and treatment of osteoporosis.Prostaglandins(PGs)are important regulators of bone metabolism.Prostaglandin E2(PGE2)is the most expressed PG in human body.PGE2 is associated with OP related bone formation,fracture healing,heterotopic ossification,and inflammation related bone resorption.The production of PGE2 is mainly induced by cyclooxygenase(COX2).It was found that many kinds of mechanical stimulation(such as continuous dynamic compressive stress,compressive stress and FSS)could increase the expression of PGE2 in bone cells.PGE2 works by coming out of the cell in the way of autocrine / paracrine and binding to related receptors.There are four kinds of receptors in PGE2,namely EP1,EP2,EP3 and EP4.These receptors mediate different intracellular signal transduction pathways through different G proteins,and play different or even antagonistic physiological roles.EP2 and EP4 are believed to mediate the synthesis and metabolism of PGE2 on bone,but the details of PGE2 related signaling pathway and receptor behavior under mechanical stress are not clear.At present,the relationship between primary cilia and PGE2 signaling pathway,especially under the action of LMHFV,is not clear.This study will take this as a starting point to study whether osteoblasts can sense low load mechanical vibration through primary cilia,activate PGE2-related signal pathway,activate osteoblast function;whether PGE2-related signal pathway can affect the formation and function of primary cilia,and regulate Osteoblasts’ perception of mechanical stress.This will deepen the application of low load mechanical vibration therapy in osteoporosis,and provide theoretical basis and guidance for the development of new drugs(activators or inhibitors of related signaling pathways)to promote fracture healing or prevent heterotopic ossification.Objectives:To investigate whether LMHFV can affect the differentiation,maturation and calcification of MC3T3-E1 cells,and the role of primary cilia and PGE2 signaling pathway in this process.Methods:1.The morphology of primary cilia was observed by immunofluorescence and confocal laser microscopy.2.Alizarin red staining was used to identify the osteogenic differentiation ability of the cultured cells.3.Chloral hydrate(CH)and IFT88 shRNA inhibited the expression of primary cilia.4.DNA quantitative identification of cell value-added ability.5.CCK8 method was used to detect cell viability.6.The effect of LMHFV on the content of PGE2 in OBs was measured by ELISA.7.Western blotting was used to detect the expression of COL-1,OCN,OPN,COX2,IFT88,mPGES1,EP1-4 and histone H3.8.Real time PCR was used to detect the effects of COL-1,Runx-2,COX2,IFT88,mPGES1,EP1-4 and GAPDH mRNA expression levels.Results:1.MC3T3-E1 cells express primary cilia: the primary cilia were labeled with anti acetylated α-tubulin,and the nuclei of mc3t3-e1 s were observed by DAPI.It was observed by confocal laser microscopy that DAPI stained the nucleus blue.MC3T3-E1 cells have primary cilia,which are small and bright processes on the top of cell membrane.The primary cilia and cytoplasmic microtubule networks are located near the nucleus.2.Chloral hydrate(CH)injury and removal of primary cilia: using CCK-8 analysis,we confirmed that the cell viability was not affected by pretreatment at 4 m M CH,and the cell viability was significantly reduced when the concentration of CH exceeded 8 mM or the exposure time of CH exceeded 48 hours.The recovery period of 24 hours after CH treatment is enough to restore the cell vitality to the original state.The results showed that pretreatment with 4mch for 24 hours and then recovery for 24 hours was a safe and effective method to remove primary cilia.3.Primary cilia regulate the differentiation,maturation and mineralization of OB induced by LMHFV: Western blotting was used to detect the main bone matrix protein COL-1 and the two most common non collagen bone matrix proteins OPN and OCN.The results showed that the expression of COL-1,OPN and OCN in LMHFV group was significantly increased,and the expression of these proteins was significantly decreased after CH pretreatment.RT-PCR was used to detect the main bone matrix COL-1,early differentiation related transcription factors COX2 and Runx-2.The results showed that the expression of COL-1,OPN and OCN genes in LMHFV group increased significantly,but after CH pretreatment,the expression of these genes decreased significantly.ALP staining was used to detect the maturation of osteoblasts,and alizarin red staining was used to detect the mineralization of osteoblasts.However,after CH pretreatment,the area of staining area of both dyes decreased significantly.4.LMHFV caused the morphological changes of primary cilia in OBS: the primary cilia were labeled with anti acetylated α-tubulin.The results showed that the receiving strength of treated bone cells was 0.25 g,the frequency was 35 Hz,once a day.After 20 minutes of LMHFV treatment for 5 days,the primary cilia of cells became shorter and fewer.5.LMHFV activated COX2-mPGEs-1-PGE2 signal pathway of OBS: Western blotting was used to detect the protein expression of COX2 and mPGES1 after LMHFV treatment,and the level of PGE2 was measured by ELISA.The results showed that the expression of COX2,mPGES1 and PGE2 were all increased after LMHFV treatment.LMHFV activated a amplified signal cascade reaction,and the best effect was achieved at 30 minutes vibration.6.LMHFV increased EP4 expression level: Western blotting detected the expression of four known PGE2 receptors(EP1,EP2,EP3 and EP4): after LMHFV treatment,EP4 expression in OBS increased significantly.This suggests that EP4 may be involved in the osteogenesis of LMHFV to OBs.7.The activation of COX2-PGE2-EP4 signaling pathway induced by LMHFV requires the existence of primary cilia: firstly,the expression of primary cilia is effectively inhibited by IFT88 shRNA.Primary cilia are essential for the activation of COX2-PGE2 pathway induced by LMHFV: Western blotting was used to detect the expression level of COX2 and mPGES1 protein,and ELISA was used to detect the level of extracellular PGE2.The expression level of COX2 and mPGES1 in LMHFV treated cells(LMHFV+NC group)was significantly higher than that in nc-shRNA treated cells,but the expression level of COX2 in IFT88 targeted shRNA transfected and LMHFV treated cells(LMHFV+shRNA group)was significantly lower.Similarly,compared with LMHFV+NC group,the expression level of PGE2 was significantly lower in LMHFV+shRNA group.Fluorescence staining showed that EP4 was Co located with primary cilia.The increase of EP4 expression induced by LMHFV requires the presence of primary cilia.Results RT-PCR showed that IFT88 silencing did not affect the expression of EP1-3 mRNA,which significantly prevented the increase of EP4 mRNA expression induced by LMHFV.Western blotting showed that the increase of EP4 protein expression in LMHFV+shRNA group was significantly lower than that in LMHFV+NC group.8.COX2-PGE2-EP4 pathway is involved in the repair of primary cilia: AH23848,an EP4 antagonist,blocks the effect of EP4.24 hours after LMHFV(0.25 g,35Hz,30min)treatment,Western blotting was used to detect the protein expression level of IFT88.The results showed that antagonizing EP4 significantly blocked the increase of IFT88 expression after LMHFV treatment.Laser confocal microscopy was used to observe the effect of EP4 antagonist on the self-healing of primary cilia of osteoblasts.The results showed that after LMHFV induced morphological changes of primary cilia,EP4 antagonists blocked the self repair of primary cilia in OBS.9.Blocking COX2-PGE2-EP4 signaling pathway can inhibit the osteogenic effect of LMHFV: OBS was treated with celecoxib(CEL),a COX2 antagonist,or AH23848,an EP4 inhibitor.Western blotting was used to detect the main proteins of Runx-2,OCN,OPN and COL-1.The results showed that the expression levels of Runx-2,OCN and COL-1 in LMHFV group were significantly higher than those in the control group.However,when AH23848 or celecoxib were added,they decreased to the control level.ALP staining was used to detect the maturation of osteoblasts,and alizarin red staining was used to detect the mineralization of osteoblasts.After AH23848 or celecoxib treatment,the area of staining area decreased significantly.Conclusions:1.LMHFV promotes osteogenic differentiation,maturation and mineralization of ob.2.The primary cilia of OB participate in the signal transduction of mechanical stimulation of LMHFV,and LMHFV has an effect on the morphology of primary cilia.3.LMHFV can activate COX2– PGE2 – EP4 signaling pathway through primary cilia,and promote osteogenic differentiation,maturation and mineralization of OBs.4.COX2 – PGE2 – EP4 signaling pathway plays an important role in the self repair of primary cilia damaged by LMHFV.