The inflammatory response of RA and the activation of the immune system leading to abnormal bone tissue metabolism, which can lead to increasing bone resorption, reduced bone formation, decreased bone density, and osteoporosis and fractures12,13. This disorder is most typical among women and the elderly, uncontrolled active RA leads to osteoporosis, joint destruction, impaired mobility and cardiovascular disease and other complications14. In the experiments, we immunized DBA / 1 mice with Col II collagen in CFA to establish a CIA mouse model, which has both immunological and pathological features of RA. The pathological changes in Fig. 1 are inflammatory cell infiltration, proliferative synovitis, bone erosion with destruction of articular cartilage, and the condition worsens with time, prompting that our RA model was successfully established. Severe OP leads to compression fractures and deformities in the spine, as well as nerve compression, affecting lower limb activities15. As shown in Fig. 4, An x-ray of the CIA mouse revealed deformities in the spinal and X-ray transmittance increased with time. Bone density decreased over time, leading to osteoporosis and increased risk of fragility fracture. Generalized bone loss is the leading cause of fracture in RA patients. This loss of bone density occurs quietly and continuously. Studies have shown that the destruction of RA articular cartilage and bone, and secondary osteoporosis is nearly related to the formation and activation of osteoclasts16–18. Osteoblast (OB) and osteoclast (OC) are the two main types of cells that maintain dynamic balance of bone. Under normal circumstances, OB and OCs are in a dynamic equilibrium state in vivo. Therefore, bone formation and resorption are in a balanced state, which is determined by the balance of OPG and RANKL19,20. RANKL-RANK-OPG is a molecular bridge between bone and the immune system, RANKL receptor, nuclear factor kappa-B receptor activator (RANK) is expressed in monocyte-macrophage osteoclast precursor cells and mature osteoclasts, after RANKL binds to RANK, it induces OC precursor cells to differentiate into OC, and also promotes OC's bone resorption activity, inhibits OC apoptosis and prolongs OC life21,22. Osteoprotegerin (OPG), a soluble decoy receptor of RANKL, can specifically bind to RANKL, thereby competitively inhibiting RANKL-RANK binding and inhibiting RANKL activity23. OPG is also a member of the tumor necrosis factor receptor family, which can block tumor necrosis factor, induce osteoblast apoptosis in vivo, and reduce the bone loss of tumor necrosis factor-transgenic mice24. In the experiments, from the results in Fig. 3, the RANK of mice in the CIA model group increased with time, and OPG decreased with time. The process of homeostasis is broken. Osteoclast activity prevails, which can result in RA.
The immune system, including CD4 + T cells and inflammatory cytokines, is a crucial regulator of bone formation and bone resorption balance. Th17 cells are CD4 + T cells, secreted explicitly under the control of the nuclear transcription factor ROR-γt of IL-17. IL-17 is a cytokine promoting and inducting of T lymphocytes, and promoting immune response and inflammation development25. Tyagi et al 26 demonstrated that IL-17 can directly stimulate OC differentiation, inhibit bone matrix mineralization, inhibit OB differentiation, increase the secretion of RANKL by OB. Moreover, in the collaborative culture of bone marrow stromal cells with RANKL and M-CSF, the differentiation of bone marrow stromal cells towards OC could be induced. In the experiments, from the results in Fig. 2, IL-17 serum level, and spleen expression of mice in the CIA model group increased with time, and the condition became more serious. Treg cell is a subgroup of CD4 + T cells with immunosuppressive activity, inhibiting T cell over-activation and OC formation, inducing immune incompetence, and maintaining autoimmune tolerance, which inhibits the inflammatory response to relieve RA symptoms 27,28. Transcription regulator Foxp3 is regarded as specific lineage-specific marker for Treg cells and its sustained expression is a critical factor in keeping Treg repressing activity 29. More studies have found that the change of the transcription factor RORγt / Foxp3 balance in T cells fundamentally determines the shift of Th17 / Treg differentiation, and then affects the occurrence and progress of RA30. As shown in Fig. 3, nuclear transcription factor ROR-γt mRNA expression of spleen in CIA model group increased over time, IL-17 secreted by TH17 cells also increased, Treg transcription factor Foxp3 mRNA expression of spleen in CIA model group decreased over time. The reason may be the decrease of CD4 + Treg, which makes the negative immune regulation mechanism disorder in RA patients, and cannot inhibit the excessive activation of T cells, coupled with the powerful inflammatory cells cytokine IL-7, also able to up-regulate the expression of RANKL in OC, and increases the Osteoclast Precursor cells (OCPs) expression of RANK, increasing the sensitivity of OCPs to RANKL, potentiates the osteoclastogenic activity, thereby disrupting the balance of RANKL/OPG in synovial fluid and aggravating bone destruction and resorption31–33. Resulting in RA and secondary OP.