Effects And Mechanisms Of Exercise Mediated LncRNA H19 On Bone Metabolism

Research backgroundOsteoporosis is a kind of metabolic disease that reduces bone mass and destroys bone microstructure.Weightlessness,long-term bed rest and aging can lead to the continuous decline of bone mass and induce the occurrence of osteoporosis.Osteoporotic fracture will bring great challenge to social medicine and economic development,so osteoporosis has gradually become a clinical public health problem to be solved urgently.Appropriate exercise training can effectively reduce the risk of osteoporotic fracture,which has become a research hotspot in the treatment of osteoporosis.Exercise is a comprehensive embodiment of various mechanical stimuli,which can regulate bone metabolism by regulating the balance between bone resorption and bone formation.Recent studies have found that long non-coding RNA H19 plays an important role in the regulation of bone metabolism,but there is still confusion about the regulatory role and mechanism of various mechanical stimuli induced by exercise in bone metabolism.Therefore,it is very important to explore the pathway and mechanism of H19-mediated exercise in regulating bone metabolism.In addition,it is also of great clinical significance to further improve the effectiveness of exercise in the prevention and treatment of osteoporosis.Research methodEffect of knocking out lncRNA H19 from osteoblasts on bone mass and expression of bone metabolism related genes in mice1.OSX Cre tool mice and H19fl/+ mice were used for hybridization to obtain the mouse model with specific knockout H19 in osteoblasts.In this study,H19fl/fl(WT)was selected as the control group,and Osx cre,H19fl/fl(CKO)were selected as the experimental group.Both WT mice and CKO mice with different weeks of age(6W,10 W and 13W)and different genders(female and male)were selected for continuous observation of the changes of bone microstructure and biomechanical indexes,8mice in each group.After determining the phenotypic time node according to age and genders,the bone metabolism related indicators of CKO mice were detected.2.After the mice were killed under anesthesia,the femur and tibia of the mice were quickly separated.The right femur was fixed and MicroCT scanning was performed,in which the detection indicators were BMD,BV/TV,Tb.Th,Tb.N,Tb.SP,Ct Th et al.Biomechanical indexes of the left femur were tested,including maximum load,breaking load,bending strength,elastic modulus and failure strain.The right femur was stained with HE and TRAP by paraffin section.The serum was used to detect the bone formation marker P1 NP and bone absorption marker DPD.Total RNA extracted from the left tibia was detected by RT-qPCR and RNAseq.The expressions of total proteins(including OSX,ATF4 and RANKL et al.)extracted from right tibia were detected by WB.Part Ⅱ: Effects of treadmill and suspension intervention on bone mass and bone metabolism related indicators in H19 osteoblast knockout mice1.13-week-old male WT and CKO mice were randomly divided into WT quiet group(WT),WT treadmill exercise group(WT+T),CKO quiet group(CKO),CKO treadmill exercise group(CKO+T),with 8 mice in each group.The mice began to exercise for 5 days from the 7th week of age.The speed started from 10 m/min and increased by 2 m/min every day until 18 m/min.The exercise intervention was carried out for 30 minutes every day with the treadmill slope of 0°.At the age of 8-13 weeks,a 6-week formal treadmill intervention was carried out at a speed of 18m/min,a slop of 5°,5 days a week,1 hour a day(5 min warm-up and 55 min formal exercise).2.13-week-old male WT and CKO mice were randomly divided into WT quiet group(WT),WT suspension group(WT+S),CKO quiet group(CKO),CKO suspension group(CKO+S),with 8 mice in each group.The mice began to carry out a 7-day continuous tail suspension intervention from the 12th week of age,and always kept their forelimbs on the ground and kept 30 ° with the horizontal ground.In addition,the mice were kept drinking and eating freely during the suspension intervention.3.After the running platform and suspension intervention,the mice were killed under anesthesia the next day,and the femur and tibia of the mice were quickly separated after blood collection.The right femur was fixed and MicroCT scanning was completed.The detection indicators were BMD,BV/TV,Tb.Th,Tb.N,Tb.SP,Ct Th.Biomechanical indexes of the left femur were tested,including maximum load,breaking load,bending strength,elastic modulus and failure strain.The right femur was stained with HE,TRAP and immunofluorescence using paraffin sections.The serum was used to detect the bone formation marker P1NP and bone absorption marker DPD.Total RNA extracted from the left tibia was detected by RT-q PCR and RNAseq.The expressions of total proteins(including OSX,ATF4 and RANKL et al.)extracted from right tibia were detected by WB.Part 3: lncRNA H19 mediates mechanical stretch to regulate the expression of downstream genes in osteoblasts1.The primary osteoblasts of newborn WT and CKO mice were extracted and cultured in vitro.Flexcell-5000 traction system was used for mechanical traction of cells.The traction intervention program was sinusoidal traction intervention with 6%deformation intensity,4 h and 0.5 HZ for 3 consecutive days.2.After the traction intervention,ALP and fluorescence staining were performed respectively,and total protein and total RNA were extracted for WB and RT-PCR detection,respectively.Research resultsPart Ⅰ1.After specific knockout of H19 expression in osteoblasts,bone mass and bone biomechanical indexes of male mice decreased significantly at 13 weeks of age.2.In male 13 week old CKO mice,the number of bone trabeculae decreased,adipocytes increased,TRAP staining area increased,osteogenic differentiation gene expression decreased,and bone resorption gene expression increased.3.GO analysis of RNAseq differential gene in male 13 week old mice with specific knockout of H19 in osteoblasts showed that bone remodeling,ion channel activation,glucose and lipid metabolism and inflammatory response signal pathway were enriched.Part Ⅱ1.After conditional knockout of H19 expression in osteoblasts,bone density and trabecular thickness decreased.The content of DPD in serum increased and the content of P1 NP decreased.The number of adipocytes increased and the secretion of TRAP increased.Bone formation genes(OPG,ALP,ATF4,OCN)decreased significantly,and bone resorption(RANKL)increased significantly.2.After treadmill exercise intervention,WT and CKO mice showed increased bone mass,weakened TRAP staining,thickened bone trabeculae,and more regular arrangement of bone tissue cells.The fat content of WT mice decreased after treadmill exercise intervention,the expression of bone formation(ALP,ATF4,RUNX2)factors increased,and the expression of bone resorption(RANKL,C-FOS)was inhibited.KEGG enrichment analysis showed that cell adhesion molecules,synapses,c AMP signal pathway and PI3K Akt signal pathway were enriched after treadmill exercise intervention in WT mice;However,after treadmill exercise intervention,the bone formation(OSX)of CKO mice was up-regulated,while the expression of bone resorption factor(RANKL,FTO,NFATC1,TRACP and C-FOS)d was decreased.The KEGG enrichment analysis of different genes showed that after treadmill exercise intervention,CKO mice were involved in carbohydrate and lipid metabolism,inflammatory response,synaptic and cell adhesion molecules and other signal pathways.3.After suspension intervention,the thickness of bone trabeculae and the content of P1NP in serum of WT mice can be significantly reduced,the TRAP staining area increases,and the expression of bone formation(OPG,OSX,ALP,ATF4 and OCN)factors is inhibited,while the expression of bone resorption(RANKL,C-FOS and CTSK)factors is significantly increased.In addition,the KEGG analysis of WT mice after suspension intervention showed that neurodegenerative diseases,gap junctions,ECM receptors and other signal pathways were enriched;after suspension intervention in CKO mice,there was no significant change in bone microstructure,and the expression of bone morphogenetic factor(OSX)increased.However,after suspension intervention in CKO mice,KEGG analysis showed that synapse,gap node,arachidonic acid,B cell receptor,p53,Wnt,VEGF and other signal pathways were involved.4.After specific knockout of lncRNA H19 in osteoblasts,the expression of EFNB1 was significantly reduced,but mechanical stimuli such as treadmill and suspension intervention may regulate the level of bone formation through the expression of EFNB1 and OSX.Part Ⅲ1.Compared with the osteoblasts of WT,the activity of ALP in OB from CKO mice was decreased,and the proliferation of osteoblasts was weakened.In addition,the expression of bone formation related factors(OPG,ALP,OSX and RUNX2)was significantly decreased,and the expression of bone resorption factor(TRACP)was increased.In addition,the expression of cell adhesion factor(EFNB1)was decreased.2.The activity of ALP was enhanced after WT primary cells were stimulated by traction.The expression of cell adhesion factor(EFNB1)and bone formation factor(OCN,OPG and OSX)increased,while the expression of bone resorption genes(NFATC1 and TRACP)decreased significantly.Immunofluorescence staining showed that the expression of ATF4 and RUNX2 increased,while the expression of C-FOS and RANKL decreased.3.ALP activity was enhanced after CKO primary osteoblasts were stimulated by distraction.The expression of bone morphogenetic factors(ALP,OPG and RUNX2)and cell adhesion factor(EFNB1)increased,while the expression of bone resorption gene(TRACP)decreased significantly.Immunofluorescence staining showed that the expression of ATF4 and RUNX2 increased,while the expression of RANKL decreased significantly.Conclusion1.Conditional knockout of lncRNAH19 in osteoblasts,bone formation is inhibited,while bone resorption is promoted,resulting in poor bone mass and bone biomechanical properties.2.Conditional knockout of lncRNAH19 in osteoblasts can up regulate bone formation under moderate intensity treadmill exercise or mechanical stretch stimulation,indicating that adaptive mechanical stimulation can promote bone formation;however,there was no significant change in bone mass after tail suspension intervention,indicating that lncRNAH19 may be a regulatory target for the occurrence of disuse osteoporosis.3.lncRNAH19 may become a double-edged sword regulating bone metabolism balance through regulating the expression of EFNB1.