Study On The Effect Of Complement C3a On Bone Metabolism Through Receptors C3aR And RAGE In Chronic Inflammatory Diseases

【Objectives】Earlier studies using APPswe transgenic mice showed that β-amyloid peptide(Aβ)can regulate bone metabolism through the receptor for advanced glycation end products(RAGE)depending on age.Both Aβ and C3 a are RAGE ligands.Complement C3 a can affect bone metabolism through the multi-ligand receptor RAGE and its specific receptor C3 a R.C3 a is the product of the complement cascade in the inflammatory response.APPswe transgenic mice are models of chronic inflammatory degenerative diseases(Alzheimer’s disease).The expression of complement C3 a in APPswe transgenic mice is increased.In this study,APPswe transgenic mice,gene knockout mice(C3aR-/-,RAGE-/-)were used as models,and osteoblasts and osteoclasts were induced and cultured in vitro to explore the effects of complement C3 a through multiple receptors in chronic inflammatory diseases.The mechanism of bone metabolism provided a theoretical basis for the treatment of inflammatory bone metabolism diseases.【Methods】In order to compare the differences of bone resorption and formation in the mice of different genotypes,bone trabecular morphology and bone structure parameters were analyzed by Micro-CT scanning;acid phosphatase staining(TRAP)and alkaline phosphatase staining(ALP)were also used to detect the number of osteoclasts and osteoblasts in femoral tissue;immunohistochemical staining was used to analyze the expression of C3 a R and RAGE protein in bone tussue.Osteoblasts and osteoclasts were induced and cultured in vitro,and the addition of complement C3 a,inflammatory factor IL-1β stimulation and C3 a R activation and inhibition experiments were performed.RTPCR and Western Blot were used to detect the effect of complement and its receptors on bone formation and resorption under inflammatory conditions in vitro.【Results】1.Micro-CT and trabecular bone microstructure morphometric parameters showed that middle-aged and elderly chronic inflammation group mice(APPswe)had osteoporosis;gene knockout mice C3 a R-/-,RAGE-/-had high trabecular bone density and bone mass.ALP staining results showed that,compared with WT mice,the positive rate of ALP in femoral tissue of APPswe mice was significantly reduced in 6 and 12 months,indicating that their bone resorption was reduced.The results of femur TRAP staining showed that the positive rate of TRAP staining in aged C3 a R-/-and RAGE-/-mice was lower than that in WT mice’s femoral tissue at the same age,indicating that bone resorption was decreased;on the contrary,bone resorption was enhanced in aged APPswe transgenic mice.2.The expression of C3 a and RAGE protein in femurs by immunohistochemical staining showed that,compared with WT mice,the expression of RAGE protein in bone tissue of C3 a R-/-mice was increased;so as the expression of C3 a R protein in bone tissue of RAGE-/-mice;compared with WT mice,RAGE and C3 a R proteins were highly expressed in the bone tissue of APPswe transgenic mice.3.The addition of complement C3 a and inflammatory factors IL-1β,and C3 a R activation/inhibition experiments showed that complement C3 a and inflammatory factors promoted the differentiation of osteoblasts and osteoclasts in vitro,and upregulated the expression of C3 a R,RAGE,bone formation and bone resorption marker genes.C3 a R activation could stimulate both osteoblast differentiation and osteoclast differentiation;C3aR inhibitors could inhibit osteoblast and osteoclast differentiation.Inflammation and C3 a R stimulation could up-regulate the gene and protein expression of C3 a R and RAGE in osteoclasts and osteoblasts.【Conclusion】1.C3 a R plays an indispensable role in bone metabolism.C3 a R can promote the differentiation of osteoblast and osteoclast.2.Under inflammatory conditions,complement C3 a can up-regulate the expression of C3 a R and RAGE in osteoblasts induced from C3H10T1/2 cell line and osteoclasts induced from RAW264.7 cell line,and promote the differentiation of osteoblasts and osteoclasts in vitro through C3 a R to maintain the high transport state of bone metabolism.