Supplementary Material for: Glucose Fluctuations Promote Aortic Fibrosis through the ROS/p38 MAPK/Runx2 Signaling Pathway

Aim: Glucose fluctuations may be responsible for, or further the onset of arterial hypertension, but the exact mechanisms remain unclear. The purpose of this study was to investigate the mechanisms behind and related to aortic fibrosis and aortic stiffening induced by glucose fluctuations. Methods: Sprague-Dawley rats were injected with streptozotocin (STZ) and randomly divided into three treatment groups: controlled STZ-induced diabetes (C-STZ); uncontrolled STZ-induced diabetes (U-STZ); and STZ-induced diabetes with glucose fluctuations (STZ-GF). After 3 weeks, rat blood pressure (BP) was tested, and aortic fibrosis was detected by using the Masson trichrome staining technique. Levels of p38 mitogen-activated protein kinase (p38 MAPK), runt-related transcription factor 2 (Runx2), collagen type 1 (collagen I), and NADPH oxidases were determined by Western blot.Rat vascular smooth muscle cells in vitro were used to explore underlying mechanisms. Results: The systolic BP of diabetic rats in the C-STZ, U-STZ, and STZ-GF groups was 127.67 ± 6.53, 150.03 ± 5.24, and 171.63 ± 3.53 mm Hg, respectively (p< 0.05). The mean BP of diabetic rats in the three groups was 91.20 ± 10.07, 117.29 ± 4.28, and 140.58 ± 2.14 mm Hg, respectively (p< 0.05). The diastolic BP of diabetic rats in the three groups was 73.20 ± 12.63, 101.93 ± 5.79, and 125.37 ± 4.62 mm Hg, respectively (p< 0.05). The ratios of fibrosis areas in the aortas of the three groups were 11.85 ± 1.23, 29.00 ± 0.87, and 48.36 ± 0.55, respectively (p< 0.05). The expressions of p38 MAPK, Runx2, and collagen I were significantly increased in the STZ-GF group. In vitro, applications of inhibitors of reactive oxygen species (ROS) and p38 MAPK successfully reversed glucose fluctuations that would have possibly induced aortic fibrosis. Conclusions: Blood glucose fluctuations aggravate aortic fibrosis via affecting the ROS/p38 MAPK /Runx2 signaling pathway.