Soluble RAGE And XAV-939 Inhibite Arterial Calcification Through RAGE-Wnt/β-catenin Signaling Pathway In Db/db Mouse

Objective: To investigate the effects of different concentrations of soluble RAGE(sRAGE) and XAV-939 on the level of arterial calcification and the expression of osteogenic protein in db/db mice, and to further understand the relationship between RAGE protein and Wnt/β-catenin signaling pathway in vivo. Methods :(1) The 8 weeks db/db mice were fed in the SPF room. After one week, the mice were randomly divided into control group,sRAGE group, XAV-939 group, s RAGE+XAV-939 group, and all the mice were given high fat diet(HFD) feeding;(2) the control group received intraperitoneal injection of 100 ul physiological saline(NS); soluble RAGE group(sRAGE group) were divided into two groups: low dose group and high dose group, the low dose group was given sRAGE 0.5ug and high dose group was given sRAGE 2ug through intraperitoneal injection; XAV-939 group were also divided into two groups, The low dose group was XAV-939 0.1mg and high dose group was XAV-939 0.4mg by intraperitoneal injection; soluble RAGE+XAV-939 group(sRAGE+XAV-939 group) received 2ug sRAGE+0.4mg XAV-939 through intraperitoneal injection;(3) Before the experiment 1 days and experiment in 7, 14, 21 and 28 days, each mouse’s weight and random blood glucose were measured and recorded. In 28 days, the mice were killed by cervical dislocation, immediately use scissors to open mouse’s chest, and blood collection from the heart using the 1ml empty needl,get the supernatant after centrifugation, packed in EP tubes,-20℃ stored in the refrigerator, and used the isolated aorta to detection of aortic calcification and the expression of osteogenic protein;(4) The content of AGEs in plasma was measured by ELISA, the general Von Kossa staining and calcium content in aorta were determined. The part of the aorta to make paraffin sections, do elastic fiber staining and immunohistochemistry;(5) Detection of the expression of β-catenin, BMP-2, OPG and Runx2 in aorta by Western blotting.Results:(1) Using sRAGE or XAV-939 in mice, it can reduce the arterial calcification, calcium content, the expression of collagen, and maintain the integrity of elastic fibers;(2) Soluble RAGE can reduce the expression ofβ-catenin, thus inhibiting the Wnt pathway, down regulate the expression of osteogenic protein Runx2 and BMP-2, and showed a dose dependent.Conclusion: After the use of inhibitors sRAGE or XAV-939 in diabetic mice,through the regulation of β-catenin transcriptional activity, down regulated the expression of BMP-2, Runx2, OPG and reduce the aortic calcification. In vivo further verify the RAGE protein by regulating Wnt /β-catenin signaling pathway abnormal activation, so as to promote the function of vascular calcification; and verify the RAGE, β-catenin signaling pathway as a target site in the treatment of diabetic arterial medial calcification.