Expression Of MRNA And LncRNA In Femur Of Ovariectomized Rats And The Intervention Effects Of Chinese Medicine

Objective1.To study the expression profiles of mRNA and lncRNA in the distal femur of ovariectomized rats by high-throughput sequencing.Functional and bioinformatics analysis of differentially expressed mRNA and lncRNA were performed to explore the potential role in postmenopausal osteoporosis;2.To investigate the changes of pathways enriched by differentially expressed mRNA in ovariectomized rats and the regulation of Gushukang;3.To study the changes of target genes and related pathways of differentially expressed lncRNA in ovariectomized rats and the regulation of Gushukang.Methods:1.Study on the expression of femur mRNA and lncRNA in ovariectomized rats24 male SD rats(6-month-old)were randomly divided into sham operation group(sham,12)and model group(OVX,12).A rat model of postmenopausal osteoporosis was established by ovariectomy.Sham sham-operated rats were used as controls.High-throughput sequencing and differential analysis were performed to establish mRNA and lncRNA expression profile in the distal femoral bone tissue.The results of differential expression were verified by Real-time PCR.Finally,functional and bioinformatics analysis of differentially expressed mRNA and lncRNA were performed to explore the potential role of them in postmenopausal osteoporosis.2.Gushukang inhibits osteocyte apoptosis and enhances BMP-2/Smads signaling pathway in ovariectomized rats.(1)Effect of Gushukang on BMD and bone microarchitectural property in ovariectomized rats48 SD rats aged 6 months were randomly divided into 4 groups:sham operation group(sham,12),model group(OVX,12),Chinese medicine group(GSK,12),alendronate group(ALN,12).After 12 weeks,drug intervention was performed.The dose was converted according to human and rat body surface area.The dose in the GSK group was 0.27 g/kg,and the dose in the ALN group was 1.02 mg/kg.The sham group and the OVX group were given an equal volume of normal saline.After 12 weeks,?BMD of the whole rat and the lumbar was measured after anesthesia,?BMD of isolated femur was detected,?microCT of femur distal was detected,and ?HE staining of femur distal was performed.(2)Effect of Gushukang on BMP2/Smads signaling pathway in ovariectomized ratsThe femurs of each group were taken.The left femur was fixed with 10%neutral paraformaldehyde for 24 hours for ALP staining and Trap staining.The right femur was frozen by liquid nitrogen for qRT-PCR and western blot.(3)Apoptosis of osteocyte in the bone of ovariectomized rats and the intervention effect of GushukangThe left femur of the rats in the above experiment was fixed with 10%neutral paraformaldehyde for 24 hours for TUNEL staining.The right femur was frozen by liquid nitrogen for qRT-PCR and western blot.Results1.Study on the expression of femur mRNA and lncRNA in ovariectomized rats(1)Establishment of ovariectomized modelsBMD was measured in the two groups after 12 weeks of modeling.Compared with the sham group,the BMD of the whole rat and lumbar spine in the OVX group were significantly decreased(P<0.05).BMD measurements of isolated femurs showed a significant reduction in the BMD of the whole femur,femoral head,proximal femur,femoral shaft,and distal femur compared with the sham group(P<0.05).Compared with the sham group,the levels of BV/TV,BS/TV and Tb.N in the OVX group were significantly lower than those in the sham group,and the Tb.Sp level was significantly increased(P<0.05).Although there was no statistical difference,the Tb.Th level in the OVX group showed a downward trend.HE staining showed that the trabecular bone of the distal femur was thinner,the number of trabecular bone was significantly reduced,the arrangement was sparse,and the trabecular space was relatively wide in OVX group,which was consistent with the three-dimensional image of microCT.These results indicate that the bone mass of the femur of OVX rats is significantly reduced compared with the sham group,and the bone microstructure at the distal femur is significantly deteriorated.(2)Differential expression analysis of transcriptsDifferential expression analysis of mRNA,lncRNA and TUCP in sham group and OVX group revealed that a total of 440 mRNAs(160 up-regulated and 280 down-regulated),17 IncRNAs(9 up-regulated and 8 down-regulated)and 21 TUCPs(11 up-regulated and 10 down-regulated)were differentially expressed.Bioinformatics analysis of differentially expressed mRNA revealed that the signal pathways enriched mainly include osteoclast differentiation,antigen processing and presentation,Hippo signaling pathway,estrogen signaling pathway,phagocytosis,RNA transport,ribosome,ubiquitin Guided proteolysis,mTOR signaling pathway.Target gene prediction of lncRNA and TUCP found that multiple differentially expressed transcripts were predicted to the BMP family.KEGG enrichment analysis revealed that lncRNA and TUCP have regulatory effect on ribosomal pathways,endocytosis,mucin type 0-glycan biosynthesis,long-term depression,Fc ? R-mediated phagocytosis,thyroid hormone signaling pathway,vascular smooth muscle contraction,neuroactive ligand-receptor interaction,cytosolic DNA sensing Pathway,RIG-I-like receptor signaling pathway,TRP channel inflammatory mediator regulation.2.Gushukang inhibits osteocyte apoptosis and enhances BMP-2/Smads signaling pathway in ovariectomized rats.During the experiment,6 rats died due to various accidents,and the number of experimental animals finally included in the study was 42.(1)Effect of Gushukang on BMD and bone microarchitectural property in ovariectomized rats?The BMD measurement found that the BMD of the whole rat and lumbar of the OVX group were significantly lower than those of the sham group(P<0.05).Compared with the OVX group,the BMD of the GSK group and the ALN group were significantly increased(P<0.05).? The BMD measuremen of the isolated femur showed that the BMD of the femoral head femur,proximal femur,femoral shaft,distal femur and femur was significantly lower in the OVX group than in the sham group(P<0.05).In the GSK group and the ALN group,the BMD of the femoral head femur,proximal femur,femoral shaft,distal femur,and femur was significantly higher than that of the OVX group(P<0.05).Among them,the BMD of the distal femur of the GSK group was higher than that of the ALN group,although there was no statistical difference.? MicroCT scan of the distal femur showed that BV/TV,BS/TV,Tb.Th and Tb.N levels were significantly decreased and Tb.Sp levels were increased in the OVX group compared with the sham group(P<0.05).Tb.Sp levels were significantly reduced in the GSK and ALN groups,while other parameters were increased(P<0.05).Moreover,there was no statistical difference between the GSK and ALN groups.These data indicate that both GSK and ALN can increase trabecular bone volume.? HE staining of the distal femur showed that the trabecular bone was thinned and fractured,the number of trabecular bone was reduced,the trabecular bone gap was widened in OVX group,and the trabecular bone was relatively thick and connected,the trabecular bone gap was narrowed in GSK group and ALN group.It was found that the changes in femoral trabecular structure showed by HE staining were consistent with the three-dimensional reconstruction by microCT,(2)Effect of Gushukang on BMP2/Smads signaling pathway in ovariectomized rats? ALP staining showed that the number of ALP-positive osteoblasts in the trabecular bone of the distal femur of the OVX group was reduced,the staining was light,gray or even grayish white.Trap staining showed that the number of TRAP-positive osteoclasts increased,and the red staining was obvious or even merged in OVX group.Compared with the OVX group,the ALP-positive osteoblasts in the GSK group and the ALN group stained darker,the number of osteoclasts was less,and the staining was light red.? qRT-PCR analysis showed that the mRNA expression of BMP-2,Osterix and Runx2 was significantly down-regulated in the OVX group compared with the sham group(P<0.05).There was no significant change in the Smadl and Smad5 genes between the sham and OVX groups.Interestingly,treatment with GSK caused a significant up-regulation of BMP-2,Osterix and Runx2 mRNA levels,whereas only Runx2 mRNA levels was up-regulated(P<0.05)in the ALN group.? Western blot analysis showed that the protein expression levels of BMP-2,Smadl,p-Smadl,p-Smad5 and Runx2 were down-regulated in the OVX group compared with the sham group(P<0.05),Smad5 and Osterix also were downward in the OVX group,but there was no statistical difference.These indicated that the BMP-2/Smads signaling pathway was inhibited in the ovariectomized rat model.Protein expression levels of BMP-2,p-Smadl,p-Smad5,Osterix,and Runx2 were elevated in the GSK group compared with the OVX group(P<0.05).The expression levels of BMP-2,p-Smadl,Osterix and Runx2 were elevated in the ALN group compared to the OVX group.(3)Apoptosis of osteocyte in the bone of ovariectomized rats and the intervention effect of Gushukang? TUNEL staining and apoptotic rate measurement showed that there were more TUNEL-positive osteocytes in the OVX group,and TUNEL-positive osteocytes in the GSK group were significantly reduced compared with the OVX group(P<0.05).? qRT-PCR detection showed that the expression level of Bcl-xL decreased(P<0.05),Bak increased(P<0.05)in OVX group and the expression level of Bcl-xL was significantly increased,the Bak level was significantly decreased(P<0.05)in the GSK group compared with the OVX group,.?Western blot analysis showed that compared with sham group,Bcl-xL proteinlevel was significantly decreased(P<0.05),Bak level was significantly increased(P<0.05)in OVX group,Bcl2 level was decreased,but has no stati.stically significant,which is consistent with the gene level test.Compared with the OVX group,Bcl-xL protein levels were elevated and Bak levels were decreased in the GSK group(P<0.05).Conclus ions1.There were 440 differentially expressed mRNAs(160 up-regulated and 280 down-regulated)in the femur of ovariectomized rats,and pathway enrichment analysis revealed that most of the pathways were closely related to apoptosis.2.There were 17 lncRNAs(9 up-regulated and 8 down-regulated)and 21 TUCPs(11 up-regulated and 10 down-regulated)differentially expressed in the femur of ovariectomized rats,and target gene prediction revealed multiple differentially expressed transcripts were predicted to the BMP family.3.Gushukang can improve the BMD and bone microstructural characteristics of the femur of ovariectomized rats by activating the BMP-2/Smads signaling pathway reducing the apoptosis rate.