Study On Bone Repair And Inflammatory Response Of Icariin In Hormone-induced Necrosis Of The Femoral Head

Part one:Effect of icariin on bone tissue morphology and microstructure in rabbit with early steroid-induced osteonecrosis of the femoral headObjective:Long-term or high dosage of glucocorticoid(GC)has become the major inducement of non-traumatic osteonecrosis of the femoral head(ONFH).However,the mechanism of this disease is complicated.Clinical diagnosis and treatment are still a challenge without specific and effective treatment methods.Icariin(ICA)is an 8-isopentenyl flavonoid glycoside,and it is the main active component of herba epimedii.Modern pharmacological studies have confirmed that ICA has a variety of pharmacological activities,such as enhancing immunity,regulating bone metabolism,promoting bone growth and so on.The purpose of this study is to observe the effects of ICA on bone tissue morphology and microstructure using the rabbit model of early steroid-induced ONFH.Methods:Fifty mature New Zealand rabbits(weighing,2.5-3.0 kg)were randomly divided into control group(n=10),GC group(n=20),and ICA group(n=20).The rabbits of GC and ICA groups were injected with lipopolysaccharide and methylprednisolone,modelling early steroid-induced ONFH.The rabbits in ICA group were gavaged by icariin solution once a day for 6 weeks since the first injection of methylprednisolone,whereas the rabbits of control and GC groups were given normal saline at the same time.Six weeks later,the animals were sacrificed and the femoral head specimens were collected for study:?The morphology of the femoral head specimens of each group were observed with naked eyes;?Bone tissue morphological changes were observed by Micro-CT scan and reconstruction technique.Quantitative analysis was performed to analyze the microstructure parameters of cancellous bone,and the main parameters include trabecular bone volume to total volume(BV/TV),trabecular number(Tb.N),trabecular thickness(Tb.Tn),and trabecular separation(Tb.Sp);? The histopathological characteristics of the femoral head specimens of each group were observed by HE staining,and the empty bone lacuna rate was calculated;?The microstructural changes of a single bone trabeculae in each group were analyzed by synchrotron radiation phase contrast nano-imaging and reconstruction technique.Results:Finally,9,16,and 18 rabbits in the control group,GC group,and ICA group were enrolled the analysis respectively.? The gross observation showed that there were significant differences in the contour of femoral head,collapse,and cartilage surface color among the three groups.? Micro-CT scanning and reconstruction of bone histomorphology showed that the femoral head in the GC group collapsed obviously and the trabeculae were sparse,incomplete and misaligned.While in the icariin group,the collapse was not obvious,the trabecular structure was slightly degenerated and there was no change in the control group.According to the measurement and analysis of the microstructure parameters of cancellous bone,the Tb.N,Tb.Tn and BV/TV in the GC group and ICA group were lower than those in the control group(P<0.05),while the Tb.Sp was higher than that in the control group(P<0.05).The Tb.N,Tb.Tn and BV/TV in the ICA group were higher than those in the GC group(P<0.05),and the Tb.Sp was lower than that in the control group(P<0.05).?In the GC group,HE staining showed that the number of osteocytes decreased,while empty lacunae increased.The adipocytes accumulated between trabeculae,some mashed together into a cyst.In the ICA group,the trabecular structure was relatively intact compared with GC group and no obvious apoptosis of osteocytes was observed.The size and number of adipocytes were almost normal.No ONFH occurred in control group based on the criteria for pathological diagnosis.The incidence was 81.3%(13/16)in the GC group and 66.7%(12/18)in the ICA group,and the difference was not significant(P=0.448).The rate of empty lacunae of osteonecrotic femoral heads in the GC group was 33.1%± 1.4%,which was higher than that in ICA group(18.9%±0.8%)and control group(12.7%±1.5%,P<0.05).?The observation of synchrotron radiation phase contrast imaging showed that the bone trabeculae became thinner and broken in the GC group,and the bone trabecular structure was badly destroyed in the GC group.In the ICA group,the trabecular volume thickness and structural morphology were normal.Conclusion:In the rabbit model of early steroid-induced ONFH,icariin can alleviate the destructive effect of GC on bone tissue structure,maintain bone tissue morphology,stabilize bone microstructure,promote self-repair of injured bone tissue,and reduce osteocyte apoptosis and the rate of empty bone lacunae,delaying the progress of the disease.Part two:Effects of Icariin and glucocorticoid on mRNA expression of rat bone marrow-derived macrophagesObjective:Macrophages play an important role in the occurrence and progression of steroid-induced osteonecrosis of the femoral head.A large number of macrophages will migrate to the necrotic lesion after osteocyte and bone marrow tissue injury.Then,the macrophages will secrete large amounts of pro-inflammatory factors,which further expand the inflammatory reaction and aggravate tissue destruction,leading to the progression of disease.By changing the macrophage function,Icariin could effectively control inflammation and inhibit bone resorption,delaying the progression of osteonecrosis.However,the exact mechanism is still unclear.The purpose of this study is to evaluate the effects of Icariin and glucocorticoid on mRNA expression of rat bone marrow-derived macrophages(BMMs),and to analyze the effects of differentially expressed gene(DEGs)on the function of macrophages through bioinformatics analysis.Methods:Rat BMMs were extracted in vitro and induced by macrophage colony-stimulating factor(M-CSF)for 7 days.It was identified by morphological observation and cellular immunohistochemistry.The cells were randomly divided into control group(n=3),dexamethasone(DEXA)group(n=3)and icariin(ICA)group(n=3).The DEXA and ICA group were treated by dexamethasone and dexamethasone plus icariin for 24h,respectively.Cell viability was determined with CCK-8 method.The mRNA expression profile of the three groups was analyzed using high-throughput sequencing and DEGs were selected.Gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis of DEGs were performed.A protein-protein interaction(PPI)network was established using STRING database followed by identification of key genes.A miRNA-Gene regulatory network was established finally.Results:The BMMs cell activity in the DEXA group and ICA group was lower than that in the control group,while the cell activity in the ICA group was higher than that in the DEXA group.Differentially expressed analysis resulted in a total of 641 DEGs between DEXA and control group,of which 358 were up-regulated and 283 were down-regulated.GO and KEGG functional enrichment analysis showed that these DEGs were involved in heat stress,lipid metabolism,Ubiquitin-dependent protein catabolic process and other biological process,and mainly regulating renin secretion pathways,tight junction,etc.Based on PPI network,6 hub genes were selected:Rp126-psl,Ghr1,LOC500594,LOC688462,LOC683961,Gng4.MiRNA-Gene regulatory network showed that mo-miR-125b-5p,mo-miR-9a-5p,mo-miR-128-3p,mo-miR-290 and mo-miR-382 interacted with DEGs.Differentially expressed analysis resulted in a total of 629 DEGs between DEXA and ICA group,of which 300 were up-regulated and 329 were down-regulated.GO and KEGG functional enrichment analysis showed that these DEGs were involved in cell adhesion,regulation of ion transmembrane transport,Membrane raft polarization process and other biological process,and mainly regulating Calcium signaling pathway,Neuroactive ligand-receptor interaction,etc.Based on PPI network,7 hub genes were selected:LOC688462,LOC683961,Rp126-ps1,LOC500594,Rp128,Gng4,Ghrl.MiRNA-Gene regulatory network showed that mo-miR-125b-5p,mo-miR-9a-5p,mo-miR-128-3p,mo-miR-290 and mo-miR-382 interacted with DEGs.Conclusions:The DEXA can inhibit the cell activity of BMMs,while ICA can alleviate the inhibitory effect.After DEXA and ICA intervention,the gene expression of BMMs changed significantly,and the DEGs played a role in many biological processes and signal pathways of macrophages.It contributes to understand the molecular mechanism of functional changes of macrophages in different micro-environments and its effects in the occurrence and progression of steroid-induced osteonecrosis of the femoral head.