Bone is a constantly metabolizing organ, and bone remodeling is a dynamic balance process of bone resorption and bone formation. OC absorbs old bone and Osteoblast OB forms new bone, respectively. Bone remodeling relies on the precise coordination of the two kinds of cells to keep in balance. OC can regulate the functions of OB in both positive and negative ways, while OB can adjust OC through OPG/RANKL/RANK signaling system. RANKL and OPG are a pair of important bioactive proteins in regulating the balance of bone metabolism, which are widely expressed in lymphoid tissues. RANKL is a member of tumor necrosis factor(TNF) superfamily, which activates downstream related molecules after binding to its receptor RANK to enhance osteoclast differentiation and activity, then leads to increased bone resorption. RANKL is highly expressed in the thymus gland, lungs and lymph nodes, and low expressed in the spleen and bone marrow. A variety of cytokines, hormones, growth factors can regulate the expression of RANKL. RANK and OPG are also members of TNF Receptor superfamily. RANKL binds to RANK on the surface of osteoclasts to induce the accumulation of TNF receptor-related factor (TRAF6) in osteoclasts and promotes the differentiation and activation of osteoclasts, while the apoptosis of osteoclasts was inhibited. OPG has two forms including monomer and dimer, and is mainly expressed in osteoblasts and vascular cells. It can competitively bind RANK, so the ratio of RANKL/OPG plays a key role in the process of bone remodeling. Our study mainly detected serum RANKL and OPG in children with SLE.
The expression of RANKL, OPG and RANK is regulated by different hormones and cytokines, including Parathormone(PTH), 1,25(OH)2VitD3, prostaglandins, TNF-α, Interleukin (IL)-6 and M-CSF, etc. The high expression of estrogen could increase the expressions of transforming growth factor (TGF) -β and OPG, which inhibited RANKL signal transduction, and then promoted bone formation. On the contrary, the low expression of estrogen could significantly increase the expressions of IL-1, IL-6, TNF-α and other pro-inflammatory factors, which increased the expression of RANKL and promoted bone resorption. At present, the most commonly used method for bone mineral density(BMD) assessment is dual energy x-ray absorptiometry (DXA). But local DXA is insufficient to reflect the condition, and the whole body DXA is difficult to perform in children because of the high dose of radiation and coordination problem. PTH, 25 (OH)VitD3 and osteocalcin and can only represent the body endocrine levels, so that new and simple biomarkers are needed, which are the reflection of bone metabolic state. Fracture risk assessment should be performed on all patients with long-term oral GC and should also be regularly assessed during treatment. RANKL and OPG play a key role in the balance of osteoblasts and osteoclasts, which are very important in the regulation of bone metabolism in vivo and believed to be related to the reduction of bone mineral density in patients with SLE. A number of studies have shown that the RANKL/OPG ratio is decreased in chronic diseases such as type 1 diabetes, juvenile idiopathic arthritis, and nephrotic syndrome[8, 9, 10, 11], which may represent the negative balance of bone remodeling. Therefore, the detection of RANKL and OPG may become new sensitive biomarkers to evaluate bone metabolism in children.
It is reported that annual incidence of SLE in adults ranges from 0.3 to 31.5/100,000[3, 4], and 30 to 70/100,000 in China. The incidence in children ranges from 0.36 to 2.50/ 100,000, and the prevalence ranges from 1.89 to 25.70/100,000. Although the incidence of SLE in children is not high, the vast majority of children with SLE need lifelong treatment, and the mortality is still high in young patients. GC is widely used in patients with inflammatory, autoimmune and allergic diseases, and is one of the most common and irreplaceable drugs for SLE at present. With the application of GC and other immunosuppressants, the mortality of SLE patients has decreased significantly in recent decades. More than 80% of patients with SLE need long-term duration[15, 16] so that GIOP is more common. GC stimulates RANKL and inhibit OPG, which promotes osteoclast differentiation and osteolysis, while it induces osteocyte apoptosis and inhibits osteocyte generation. Studies have shown that bone loss caused by the GC can be roughly divided into two stages, bone mineral density loss fast which is about 6 ~ 12% in the first year of treatment, and about 3% a year later. At the later stages, GC increased apoptosis of osteoblasts and osteocytes, and then the expression of RANKL decreased, while the number of osteoclasts decreased by apoptosis and autophagy. In addition, control of inflammation and reduction of GC dosage at the later stage also resulted in less bone loss. A study found that OPG, RANKL and RANKL/OPG were significantly increased in the SLE group, and OPG level was related to the activity of the disease. Studies on children with SLE also showed that RANKL level was not related to disease activity. Therefore our study selected the patients with disease activity controlled to reduce the interference of inflammation. The results showed that serum RANKL and OPG concentrations in inactive state of disease are also significantly different with normal children. It suggested that dynamic balance regulation of bone metabolism still exists in the remission state of the disease, partially verifying the above conclusion that RANKL is not related to disease activity, but the effect of the active stage of SLE on bone still needs to be further explored and clarified. Studies on lupus nephritis (LN) showed that OPG levels in active LN were increased, and OPG levels were significantly decreased when disease activity was controlled, suggesting that OPG levels may be increased by inflammatory stimulation, but decreased by GC. Our study showed that RANKL expression was increased and OPG expression was decreased in the remission state in children with SLE, and respectively they were positively and negatively correlated with duration of SLE and accumulated dose of GC. It speculated that bone remodeling process could keep active by the action of GC. Since the durations of the disease in our patients are all less than 2 years, longer follow-up and observation are needed. One limitation in this part is absence of patients with other rheumatic diseases on corticosteroid therapy, so we will try to collect more patients, which would have been helpful to better clarify whether or not elevated serum RANKL/OPG ratios are primarily related to disease rather than corticosteroid. Another limitation is that we have not done multivariate analysis because of small sample size, and more patients will be enroll our research in the future.
At present, the correlation between BMD and RANKL and OPG levels is still inconclusive, but the correlation of increased RANKL/OPG ratio and BMD has been confirmed in many childhood diseases[8, 9, 10, 11]. Since SLE may lead to decreased bone mineral density, the effect of RANKL and OPG on bone mineral density in SLE active stage is not clear. In our study, all the 40 children with SLE were in remission, so the chronic inflammation in SLE had little effect on RANKL and OPG. Meanwhile, the correlation between GC accumulation and RANKL/OPG suggested that GC played a very important role in regulating the balance of RANKL and OPG. Considering the amount of radiation and the difficulty of operation, whole-body BMD was not performed in these cases, which is a limitation and the correlation between RANKL/OPG and BMD cannot be determined. So we've done animal and cell study to find out the machanisms of the bone loss by GC in SLE model.
Vitamin D is an important indicator of bone metabolism, which regulates the balance of calcium and phosphorus. It is also associated with a number of non-skeletal diseases, including cardiovascular disease, cancer, autoimmune disease and diabetes. Serum 25(OH)VitD3 is considered to be the best indicator of vitamin D status. Low vitamin D level is a risk factor of osteoporosis. Our study also found that children in SLE with have low vitamin D level, which is correlated with accumulated dose of GC, RANKL and OPG. It speculated that GC may participate in bone remodeling process in SLE mediated by the transformation of RANKL/OPG, which characterized by low levels of vitamin D status. But the specific regulatory mechanism still needs further research.