The Role And Mechanism Of Suspension For Less Mechanical Load In Alleviating The Pathological Ossification Of Ankylosing Spondylitis Via MiR-103/ROCK1/β-catenin Pathway

BACKGROUND:Ankylosing spondylitis(AS)is a common immune rheumatic disease involving the sacroiliac and axial joints.Inflammatory lesions and osteophyte formation in AS are the principal factors restricting activity.Enthesitis with secondary pathological ossification is the main cause of disability in patients with AS.Recent studies have shown that excessive mechanical load in axial weight-bearing joints can accelerate ossification at AS-inflamed attachment points,and Wnt/β-catenin is an important pathway for AS pathological ossification.Rehabilitation training is considered to be a significant means for AS treatment,moreover sling exercise therapy(SET)is a novel therapeutic exercise that reduces load bearing to adjust the body’s biomechanical balance.In addition,mouse tail suspension is widely recognized as a typical model for reducting mechanical load.Under reduced mechanical load,mechanical microRNA-103(miR-103)can inhibit the proliferation and differentiation of osteoblasts,and ROCK/MAPK are the main signal pathway for cells to respond to mechanical load.The bioinformatics predicts that miR-103 can combine with the 3’-UTR of ROCK1.The role and mechanism of suspension for less mechanical load in alleviating the pathological ossification of ankylosing spondylitis through mechanical pathway have not been reported to date.OBJECTIVE:To explore whether tail suspension to reduce mechanical load could delay the pathological ossification associated with attachment point inflammation due to AS.METHODS:24-week-old female BALB/c mice were immune induced with proteoglycans(PGs)as a model for AS.The PGs-induced mice were divided into four groups:PGs-induced natural crawling(PG),tail suspension for no load bearing(PG+NLB),tail suspension for partial load bearing(PG+PLB),and vertical full load bearing(PG+FLB)(n=10 per group).The(PG+FLB)and PG groups were used as the conditional control and model control respectively.The(PG+NLB)and(PG+PLB)groups were tail suspended for 12 weeks after model induction,and the vertical(PG+FLB)mice were forcibly kept upright for 12 weeks.Pathological sections based on hematoxylin and eosin staining intensity and bone morphological changes from microCT analysis of the sacroiliac joint were recorded.Real-time RT-PCR,immunohistochemical staining,and western blot(WB)analysis were carried out to explore the expression of osteogenesis-related genes and mechanical proteins.RESULTS:Using RT-PCR,the data showed that the mechanosensory miR-103 increased in response to less mechanical load.We found that expression of the osteogenesis genes BMP2,Runx2,and OCN was significantly reduced in the two groups with reduced load bearing(PG+NLB)and(PG+PLB)compared to the natural crawling group(PG),while expression of these genes was increased in the upright group(PG+FLB).Consistently,in groups with reduced mechanical load(PG+NLB)and(PG+PLB),proteins with mechanical functions,including ROCK1,pERK1/2 MAPK,andβ-catenin,were reduced compared to the PG controls,as suggested by Western Blotting analysis.However,the immunohistochemical staining expression of Wnt/β-catenin pathway antagonist Dickkopf-1(DKK1)is completely opposite to activation of Wnt/β-catenin pathway.CONCLUSION:In summary,tail suspension can inhibit the activation of mechanical ROCK1 kinase and p-ERK1/2 MAPK signaling pathway by up-regulating mechanically sensitive miR-103,thereby inhibiting the classic osteogenic Wnt/β-catenin pathway.The mechanism clarified that tail suspension for less mechanical load can alleviate heterotopic bone formation at the inflamed attachment point associated with AS.