| Objective1.Build the induced membrane technology model of rats to evaluate the effects of total flavonoids of Rhizoma Drynariae(TFRD)on the blood vessel and its important regulatory pathways(HIF1α/EGF)in the bone graft area.2.To determine the effect of different doses of TFRD on the expression of non coding RNA(MiR-18a-5p)that may regulate HIF1α in the induced membrane bone graft area,and further verify whether HIF1α is the target gene of MiR-18a-5p.Methods1.The effect of TFRD on the HIF1α/VEGF pathway regulating angiogenesis and osteogenesis in the induced membrane area.Sixty male SD rats were randomly divided into 5 groups:control group,model group,TFRD low dose group,TFRD medium dose group and TFRD high dose group,with 12 rats in each group.A 4 mm large bone defect was constructed.Except the control group,all groups were treated with induction membrane technology.Each rat was constructed with a 4mm bone defect.Except the control group,each group was treated with induced membrane technique.(1)X-ray,femoral micro-CT,hematoxylin eosin(HE)and safranin O-Fast green staining were used to detect the imaging and histological repair effect of large bone defects.(2)Angiography was used to detect the spatial distribution of blood vessels,CD31 and Emcn fluorescent double staining were used to detect the location and quantification of type-H vessels,RT-PCR and Western blot were used to detect the level of HIF1α/VEGF pathway factors.2.The effect of HIF1α/VEGF pathway on EPCs and the intervention effect of TFRD on this pathway.Endothelial progenitor cells(EPCs)derived from rat bone marrow were extracted.EPCs were divided into four groups according to different intervention methods:control group,si-HIF1α(Silence sequence)group,si-HIF1α+TFRD group and TFRD group.CCK8 method was used to detect the activity of EPCs in each group,scratch test and Transwell chamber method were used to detect the invasion and migration ability of EPCs,tube formation test was used to detect the angiogenesis ability of EPCs,RT-PCR and Western blot were used to detect the expression level of HIF1α/VEGF pathway factors.3.The effect of TFRD on the non coding RNA(miRNA)that may target HIF1α in the bone graft area.Bioinformatics software predicted the non-coding RNA(miRNA)that may target HIF1α,and RT-PCR verified the expression of this miRNA in the bone graft area under the intervention of TFRD at different doses.4.The effect of MiR-18a-5p on EPCs and the intervention effect of TFRD.According to different intervention methods,EPCs were divided into five groups:control group,MiR-18a-5p overexpression(MiR-18a-5p mimic)group,MiR-18a-5p inhibitor group,MiR-18a-5p mimic+TFRD group and TFRD group.CCK8 method was used to detect the activity of EPCs in each group,scratch test and Transwell chamber method were used to detect the invasion and migration ability of EPCs,and tube formation test was used to detect the angiogenesis ability of EPCs.5.Determination of MiR-18a-5p target gene and intervention effect of TFRD(1)According to the different intervention methods,EPCs were divided into two groups:control group and TFRD group.TFRD group was cultured in EGM-2 medium containing TFRD.After 48h of culture,the mRNA and protein expressions of MiR-18a-5p and HIF1 in the two groups cells were detected.(2)Double-Luciferase Reporter Assay System was used to confirm whether HIF1αwas the target gene of MiR-18a-5p.(3)According to different intervention methods,EPCs were divided into five groups:control group,MiR-18a-5p mimic group,MiR-18a-5p inhibitor group,MiR-18a-5p mimic+TFRD group and TFRD group.The expression of downstream HIF1α/VEGF pathway-related factors was detected by RT-PCR,Western blot and immunofluorescence.Results1.The effect of TFRD on the HIF1α/VEGF pathway and its angiogenesisosteogenesis function in the bone graft area.The X-ray score showed that the induced membrane treatment group(model,low.medium and high dose group)was significantly higher than the control group without induced membrane treatment.The scores in the low,medium,and high dose groups were,significantly higher than that in the model group,while the scores in the medium and high dose groups were significantly higher than that in the low dose group.Micro-CT results showed that the femoral bone defect in the control group could not be repaired,and callus at the defect end was seen in the other groups.Quantitative evaluation of bone volume fraction(BV/TV)in the defect area showed that the BV/TV in the induced membrane treatment group(model,low,medium and high dose groups)was significantly higher than that in the control group.The BV/TV of low,middle and high dose TFRD group was significantly higher than that in model group.The BV/TV in the medium and high dose TFRD groups was significantly higher than that in the low dose group.HE and safranin O-Fast green staining showed that the defect area in the control group was mainly filled with fiber and muscle tissue,and new bone was not formed.The area of new bone and new cartilage in the bone defect area of the low,midium and high dose groups was significantly larger than that of the model group.The area of new bone and new cartilage in the bone defect area of the midium and high dose groups was significantly larger than that of the low dose groupgroup.The angiographic results showed that there was no blood vessel in the femoral defect area of the control group rats who were not treated with induced membrane technique.Vascular formation was found in the defect repair area of the right femur of the rats in each group treated with induced membrane technique.The percentage of vascular area in the low,medium and high dose groups was larger than that in the model group,while the vascular area in the medium and high dose groups was larger than that in the low dose group.Immunofluorescence double staining showed that there were also type-H vessels with high expression of CD31 and Emcn in the bone graft area of the rats in each group.The area of type-H vessels in the low,medium and high dose groups was significantly higher than that in the model group,and the medium and high dose groups were significantly higher than that in the low dose group.RT-PCR results showed that the transcription levels of HIF1α and VEGF in the low,medium and high dose groups were higher than those in the model group.Compared with the low dose group,the medium and high dose groups had higher HIFla and VEGF transcription levels.In addition,the transcription level of angiogenesis-osteogenesis factor(SLIT3)in the low,medium and high dose groups was significantly higher than that in the model group,while the transcription level of SLIT3 in the medium and high dose groups was significantly higher than that in the low dose group.Western blot results showed that the protein levels of HIF1α,VEGF and SLIT3 in the low,medium and high dose groups were significantly higher than those in the model group,and the expression levels of these three proteins in the medium and high dose groups were also significantly higher than those in the low dose group.2.The effect of HIF1α/VEGF pathway on EPCs and the intervention effect of TFRD.CCK8 results showed that compared with the control group(Control)without transfection,the activity of EPCs in TFRD group was significantly higher,while that in si-HIF1α group was significantly lower.Compared with the si-HIF1α group,the activity of EPCs in the si-HIF1α+TFRD group was higher.Scratch test showed that the area of cell invasion in the scratch area of TFRD group was significantly larger than that in the control group without transfection(Control),while the area of invasion in the scratch area of si-HIF1α group was significantly smaller than that in the control group without transfection(Control),and the area of invasion of EPCs in si HIF1α+TFRD group was significantly larger than that in si-HIF1α group.Transwell chamber assay showed that the number of migrated cells in TFRD group was significantly higher than that in control group(Control)without transfection,while the number of migrated cells in si HIF1α group was significantly lower than that in control group(Control)without transfection,and the number of migrated cells in si HIF1α+TFRD group was significantly higher than that in si-HIF1α group.The tubular formation test found that the total tubular length of TFRD group was significantly greater than that in the control group(Control)without transfection,while the total tubular length of si-HIF1α group was significantly smaller than that in the control group(Control)without transfection,and the total tubular length of EPCs in siHIF1α+TFRD group was significantly greater than that of si-HIF1α group.RT-PCR results showed that the expression of HIF1α,VEGF and SLIT3 mRNA in TFRD group was significantly higher than that in control group,while the expression of HIF1α,VEGF and SLIT3 mRNA in si-HIF1 group was significantly lower than that in control group.At the same time,the mRNA expression of HIF1α,VEGF and SLIT3 in si-HIF1α+TFRD group was significantly higher than that in si-HIF1α group.Western blot results showed that the protein expressions of HIF1α,VEGF and SLIT3 in TFRD group were significantly higher than those in the control group,while the protein expressions of HIF1α,VEGF and SLIT3 in si-HIFα group were significantly lower than those in the control group.At the same time,the protein expression of HIF1α,VEGF and SLIT3 in si-HIF1α+TFRD group was significantly higher than that in si HIF1α group.3.The effect of TFRD on the expression of non coding RNA(miRNA)that may target HIF1α in the induced membrane bone graft area.According to the predicting results of TargetScan7.2,miRDB and miRmap,MiR-18a-5p can be recognized by three databases together,and the combination score is high.According to literature reports,MiR-18a-5p is a non coding RNA closely related to angiogenesis and has the potential to target HIF1α.RT-PCR results showed that compared with the model group,the expression of MiR-18a-5p in the low,medium and high dose groups was lower,and the level of MiR-18a-5p in the medium and high dose groups was significantly lower than that in the low dose group.4.The effect of MiR-18a-5p on EPCs and the intervention effect of TFRD.CCK8 results showed that compared with the control group(Control)without miRNA transfection,the activity of EPCs in the MiR-18a-5p mimic group was lower,while the activity of EPCs in the MiR-18a-5p inhibitor group and TFRD group was significantly higher.Compared with MiR-18a-5p mimic group,the activity of EPCs in MiR-18a-5p mimic+TFRD group was higher.The scratch test showed that compared with the control group(Control)without miRNA transfection,the cell invasion area in the scratch area of the MiR-18a-5p MiR-18a-5p mimic group was significantly smaller,while the cell invasion area in the scratch area of the MiR-18a-5p inhibitor group and TFRD group was significantly larger.Compared with MiR-18a-5p mimic group,the invasive area of EPCs in MiR-18a-5p mimic+TFRD group was also significantly larger.Transwell chamber assay showed that the number of migrated cells in the MiR-18a-5p mimic group was significantly less than that in the control group(Control)without miRNA transfection,while the number of migrated cells in the MiR-18a-5p inhibitor group and TFRD group was significantly higher than that in the control group(Control),and the number of invasive cells in the MiR-18a-5p mimic+TFRD group was also significantly higher than that in the MiR-18a-5p mimic group.The tubule formation test found that the total tubule length of the MiR-18a-5p mimic group was significantly smaller than that in the control group(Control)without miRNA transfection,while the total tubule length in the MiR-18a-5p inhibitor group and TFRD group was significantly greater than that in the control group(Control)without miRNA transfection,and the total tubule length of the MiR-18a-5p mimic+TFRD group was also significantly greater than that in the MiR-18a-5p mimic group.5.Determination of MiR-18a-5p target gene and intervention effect of TFRD.RT-PCR results showed that compared with the control group,the expression of MiR-18a-5p in TF-RD group was significantly decreased,while HIF1α mRNA was significantly up-regulated.Western blot also found that the expression of HIF1α protein in TFRD group was significantly up-regulated compared with the control group.This was consistent with the in vivo results.Double-Luciferase Reporter Assay showed that MiR-18a-5p mimic could inhibit the expression of luciferase in HIF1α-WT cells,while no significant luciferase change was found in HIF1α-MUT cells.RT-PCR results showed that the mRNA levels of HIF1α,VEGF and SLIT3 in the MiR-18a-5p mimic group were significantly lower than those in the control group,while the mRNA levels of HIF1α,VEGF and SLIT3 in the MiR-18a-5p inhibitor group and TFRD group were significantly higher than those in the control group.At the same time,the levels of HIF1α,VEGF and SLIT3 mRNA in MiR-18a-5p mimic+TFRD group were significantly higher than those in MiR-18a-5p mimic group.Western blot results showed that the protein levels of HIF1α,VEGF and SLIT3 in the MiR-18a-5p mimic group were significantly lower than those in the control group,while the protein levels of HIF1α,VEGF and SLIT3 in the MiR-18a-5p inhibitor group and TFRD group were significantly higher than those in the control group.At the same time,the protein levels of HIFla,VEGF and SLIT3 in MiR-18a-5p mimic+TFRD group were significantly higher than those in MiR-18a-5p mimic group.The immunofluorescence results showed that the number of HIF1α-positive cells in the MiR-18a-5p mimic group was significantly lower than that in the control group,while the number of HIF1α-positive cells in the MiR-18a-5p inhibitor group and TFRD group was significantly higher than that in the control group.At the same time,the number of HIF1α-positive cells in MiR-18a-5p mimic+TFRD group was significantly higher than that in MiR-18a-5p mimic group.Conclusion1.TFRD can up regulate the expression of HIF1α/VEGF signaling pathway factors in the induced membrane bone graft area in a dose-dependent manner,thereby promoting angiogenesis and its osteogenic ability.2.After TFRD intervention,MiR-18a-5p in the bone graft area showed significantly low expression,and MiR-18a-5p inhibitor could promote the proliferation,migration and tube formation of EPCs in vitro,indicating that MiR-18a-5p could regulate angiogenesis in the bone graft area.3.HIF la is the target gene of MiR-18a-5p.MiR-18a-5p inhibits the proliferation,migration and angiogenesis of EPCs by targeting HIF1α.4.TFRD can promote function of EPCs in bone graft area,and its mechanism is related to inhibit the expression of MiR-18a-5p,reducing the degradation of target gene HIF1α,and then up regulating HIF1α/VEGF signaling pathway. |
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