SONFH is a progressive bone disorder caused by excessively administrating glucocorticoids and resulted in vascular damage, mechanism stress damage, intraosseous pressure increasing, adipocyte dysfunction, apoptosis, and coagulation dysfunction5. In our study, we first identified the biological function of SONFH, which includes programmed necrotic cell death, intracellular lipid transport, necrotic cell death, lymph vessel morphogenesis, hydrogen peroxide catabolic process, and lymph vessel development. It has been reported that the final step in osteonecrosis is vascular insufficiency to the femoral head, resulting in apoptosis and necrosis16. Several recent studies have reported that apoptosis relates to the pathogenesis of osteonecrosis of the femoral head17,18. Besides, previous studies have shown that steroid treatment implied the intra-osteoblastic lipid droplets pathology and corresponded to low bone mass with increased bone marrow adiposity19,20. Around lymph vessel morphogenesis and development, several studies have indicated that different stem and progenitor cells reside in distinct cellular niches in bone marrow, such as hematopoietic stem cells occupy a perivascular niche and early lymphoid progenitors occupy an endosteal niche21.
Besides, we screened significant pathways related to SONFH including leishmania infection, glycosaminoglycan biosynthesis chondroitin sulfate, and T cell receptor signaling pathway. Several studies proved that mice infected with Leishmania showed osteonecrosis. In addition, the histopathological analysis demonstrated that mononuclear cells infiltrated in plasma cells richly as well as parasitism of intra-medullary and extra-medullary macrophages intensely, also with bone necrosis areas and discrete cartilaginous tissue involvement22,23. Okazaki previously reports that the toll-like receptor (TLR) 4 signaling pathway, which induces inflammatory status, contributes to the pathogenesis of non-traumatic ONFH in rats24–26. In Okazaki’s another study, it has shown that corticosteroid treatment after the administration of TLR7 or TLR9 ligands causes ONFH in rats, whereas corticosteroids alone failed to induce ONFH in healthy animals. In addition, IRF7 and NF-κB are activated in the liver induced by corticosteroid treatment to trigger the development of ONFH27. Taken together, normalization of inflammatory status when treating underlying inflammatory diseases may potentially prevent ONFH in the future.
In addition, Gessner’s study has illustrated that the differential expressed IL-9 between the susceptible and resistant mice which infected with Leishmania 28. Moreover, Geng’s study also reveals that the production of IL-9 may trigger the cartilage degeneration and destruction in ONFH patients. Il-9 promotes cartilage degeneration, and the effect of IL-9 on cartilage is alleviated by blocking JAK-STAT signaling pathway in a human primary chondrocyte culture model 29. Furthermore, Chen’s study has demonstrated IL-21 promotes cartilage degradation by activating cartilage inflammation through JAK-STAT signaling pathway in ONFH patients 30. The studies above indicated that immune-related genes act as the critical role in ONFH progression through modulating the cartilage degeneration and destruction.
Furthermore, the IPA was used to further investigate the canonical pathway and molecule function of SONFH. The results revealed that 9 pathways were activated, which includes interferon signaling, production of nitric oxide and reactive oxygen species in macrophages, iNOS signaling, Fcγ receptor-mediated phagocytosis in macrophages and monocytes, TREM1 signaling, Gαi signaling, neuroinflammation signaling pathway, Tec kinase signaling, and Dendritic cell maturation. In contrast, Sirtuin signaling pathway was suppressed. Besides, the molecule function analysis showed that oxidative stress, microvascular injury and intravascular coagulation, immune inflammation, and myeloid cells movement were significantly involved in SONFH. Here, we found the production of nitric oxide and reactive oxygen species in macrophages showed the strongest correlation with SONFH based on the highest score. Macrophages play the proinflammatory promoter in necrotic bone, Naga Suresh Adapala et al have reported that the numbers of proinflammatory M1 macrophages are increased in the repair bone tissue, which reveals high expression of proinflammatory cytokines IL-1β, TNF-α, and IL-6 and the pattern recognition receptor TLR431. Another study has proved that TNF-a-mediated alteration of M1/M2 macrophage polarization plays a vital role in the pathogenesis of steroid-induced osteonecrosis, with a dominant position that M1 macrophages in early stage and M2 macrophages in the late stage of osteonecrosis32. In our result, production of nitric oxide and reactive oxygen species in macrophages was activated. Generally, macrophages maintain organism homeostasis by receptor-mediated recognition and phagocytic uptake of pathogenic damaged or apoptotic host cells. The necrosis bone tissues are degraded by phagocytosis, which activated by proteolytic enzymes and oxidative burst through the formation of reactive oxygen species (ROS) and nitric oxide (NO). The reaction of superoxide with NO results in the formation of peroxynitrite, which interacts with lipids, DNA, and proteins via direct oxidative reactions or indirect radical-mediated mechanisms33. All the studies suggest that oxidative stress significantly associates with necrosis bone may inducing macrophages polarization. In the present study, the oxidative stress related pathways were strongly related to the SONFH, and total 12 genes, such as ARG2, IFNGR1, which were associated with the production of nitric oxide and reactive oxygen species in macrophages pathway. Our finding consists of previous studies. ARG2 plays a vital role in nitric oxide and polyamine metabolism through repressing nitric oxide synthesis and inhibiting inflammatory genes levels in macrophages to interrupt M1 macrophage phagocytosis activity34,35.
Based on the above research, the SONFH related candidate genes were filtered by VarElect online tool and then overlapped with the candidate genes from MalaCards database. ACP5, TNF, MMP8 were identified as the hub genes of SONFH.
Tartrate-resistant acid phosphatase 5 (ACP5 ), a metalloprotein enzyme that belongs to the acid phosphatase family and is known to be expressed by osteoclasts. Furthermore, it has been demonstrated that ACP5 acts as a classic marker for bone resorption and osteoclast differentiation 36. In Yin’s study, ACP5 has increased in human ONFH tissues, and high expression of miR-410 and low expression of Wnt-11 inhibit ACP5 and MMP9 expression in ONFH rats37. Fang’s study has shown the effects of TNFα on proliferation, angiogenesis, and osteogenesis, and osteogenesis of rat bone mesenchymal stem cells (rMSCs)38. TNF-α plays as a mediator of bone destruction by stimulating osteoclastogenesis39–41.
Several studies have indicated that MMPs degrades and modifies the components of extracellular matrix and basement membrane and push forward an immense influence on cancer invasion and metastasis37,42−46. Previous studies have demonstrated that SNPs in the MMP8 and MMP9 genes associates with risk of osteonecrosis of the femoral head in the Chinese Han population43,45−47. Jiang’s study has shown a remarkable association between rs11225394 in MMP-8 gene and an increased risk of ONFH and a significant association between MMP-8 rs2012390 and the decreased risk of ONFH44. In Chen’s study, MMP-8 rs11225394 and MMP-8 rs2012390 are risky and protective factors of alcohol-induced ONFH46. Du et al have speculated that polymorphisms of MMP-8 might have an effect on the inflammation or circulatory impairment of the femoral head43.
To further study the function and mechanism of hub genes in SONFH, the 372 miRNAs of ACP5, 795 miRNAs of TNF, 4 miRNAs of MMP8 were screened. And hsa-miR-7845-5p, hsa-miR-6772-3p, hsa-miR-5010-3p, hsa-miR-4653-5p, hsa-miR-1587 were intersected in ACP5, TNF, MMP8. And the lncRNAs were predicted by combination of miRNAs and miRNAs. Then, 7 miRNAs, and ACP5, TNF, MMP8 and lncRNAs.