The Molecular Mechanism Of Interleukin 6-induced Osteoblast-like Differentiation Of Human Vascular Smooth Muscle Cell

Objective:Rheumatoid arthritis(RA)is a systemic disease with chronic inflammation of synovial joints as the main clinical manifestation.Cardiovascular disease-related death is the first cause of death in RA patients.The mechanism of vascular calcification in RA patients is unclear.Existing studies suggest that vascular smooth muscle cells(VSMC)play a key role in the occurrence and development of vascular calcification.Studies have found that IL6 plays an important role in inducing smooth muscle vascular calcification,but the mechanism is unknown.This study aimed to explore the transmembrane signal transduction mechanism of IL6 inducing osteogenic differentiation and calcification of human umbilical artery smooth muscle cells(HUASMC).Methods: 1.HUASMC were cultured in vitro and stimulated with IL6,IL6+soluble IL6receptor(s IL6R),IL6+s IL6R+soluble gp130(sgp130),and a blank control.Alizarin red and Von Kossa staining was used to detect the level of calcification.Western blotting was used to detect the expression of calcification-related molecules TNAP,OPN,BMP2 and the transcription factor Runx2,and immunofluorescence was used to detect the expression of membrane IL6R(m IL6R).2.HUASMC were cultured in vitro and stimulated with IL6,IL6+soluble IL6 receptor(s IL6R),IL6+s IL6R+soluble gp130(sgp130),and a blank control.Western blotting was used to detect the expression of Jak2,p-Jak2,Stat3,p-Stat3.3.HUASMC were cultured in vitro and stimulated with IL6+soluble IL6 receptor(s IL6R),IL6+s IL6R+ CP690550,and a blank control.Western blotting was used to detect the expression of Jak2、p-Jak2、Stat3、p-Stat3 and the transcription factor Runx2.Results:1.Compared with the blank group and IL6 group stimulation,the calcium salt deposition of Alizarin Red and Von Kossa staining in the IL6+s IL6 R group was significantly increased.At the same time,the expression of TNAP and OPN were significantly increased after stimulation for 12 hours and OPN after stimulation for 72 hours.The expression of BMP2 increased significantly after 12 hours of stimulation and Runx2 after 72 hours of stimulation.After adding excessive sgp130 and antagonizing the s IL6 R pathway,the expression of BMP2 and Runx2 decreased compared with the IL6+s IL6 R group,and the expression of TNAP and OPN decreased to a certain extent.2.Compared with the blank group and IL6 group stimulation,The expression of Jak2、p-Jak2、Stat3、p-Stat3 in the IL6+s IL6 R group was significantly increased.After adding excessive sgp130 and antagonizing the s IL6 R pathway,the expression of ak2、p-Jak2、Stat3、p-Stat3 decreased compared with the IL6+s IL6 R group.3.Adding 40 u M CP690550 antagonistic Jak expression,the expression of Jak2,p-Jak2,Stat3,p-Stat3 and transcription factor Runx2 is significantly less than that of IL6+s IL6 R group.Conclusion: IL6 mainly up-regulates the expression of key molecules BMP2 and Runx2 for osteogenic differentiation of umbilical artery smooth muscle cells through the trans signaling pathway mediated by s IL6 R,induces osteogenic differentiation of umbilical artery smooth muscle cells,and promotes osteocyte matrix proteins TNAP and OPN.Expression and calcium salt deposition make the umbilical artery smooth muscle cells osteogenic differentiation and calcification.