Acacetin improves endothelial dysfunction and aortic fibrosis in insulin-resistant SHR rats by estrogen receptors.

The aim of the work was to investigate the effects of acacetin on endothelial dysfunction and aortic fibrosis in insulin-resistant SHR rats and explore its mechanism. Seven-week-old male spontaneously hypertensive rats (SHR) were selected to establish a rat model of hypertension with insulin resistance induced by 10% fructose. The nuclear factor kappa B p65 (NF-κB p65) and Collagen I were observed by Immunohistochemistry. Immunofluorescence was used to observe estrogen receptor-alpha (ERα), estrogen receptor-beta (ERß), and G protein-coupled receptor 30 (GPR30). Western blotting was used to detect interleukin (IL-1ß), Arginase 2 (ARG2), Nostrin, endothelial nitric oxide synthase (eNOS), TGF-ß, Smad3, ERK pathway proteins such as p-c-Raf, p-MEK1/2, p-ERK, ERK, p-P90RSK and p-MSK1. We found that acacetin did have an improvement on endothelial dysfunction and fibrosis. Meanwhile, it was also found to have a significant effect on the level of estrogen in this model by accident. Then, the experiment of uterine weight gain in mice confirmed that acacetin had a certain estrogen-like effect in vivo and played its role through the estrogen receptors pathway. In vitro experience HUVEC cells were stimulated with 30 mM/L glucose and 100 mM/L NaCl for 24 h to establish the endothelial cell injury model. HUVEC cells were treated with 1 µM/L estrogen receptors antagonist (ICI 182780) for 30 min before administration. Cell experiments showed that acacetin could reduce the apoptosis of HUVEC cells, the levels of inflammatory cytokines and the expression of TGF-ß, Collagen I and Smad3 in endothelial cell injury model. After treatment with ICI 182780, the improvement of acacetin was significantly reversed. The results showed that acacetin relieved endothelial dysfunction and reduced the aortic fibrosis in insulin-resistant SHR rats by reducing the release of inflammatory factors and improving vasodilatory function through estrogen signaling pathway.

[Impact and related mechanisms of glucose fluctuations on aortic fibrosis in type 1 diabetic rats].

Objective: To investigate the impact and related mechanisms of glucose fluctuations on aortic fibrosis in rats with type 1 diabetes mellitus. Methods: After injection of streptozotocin (STZ), male Sprague Dawley (SD) (8-12 weeks) rats (n=24) were randomly divided into three groups in accordance with the random number table: controlled STZ-induced diabetes (C-STZ) group (n=8); uncontrolled STZ-induced diabetes (U-STZ) group (n=8); STZ-induced diabetes with glucose fluctuations (STZ-GF) group (n=8). After three weeks, rats were sacrificed and aorta was obtained, aortic fibrosis was detected by Masson trichrome staining. The expression of collagen type 1 (collagen Ⅰ) was tested by immunofluorescence. The expression of runt-related transcription factor 2 (Runx2) was tested by immunohistochemistry. The mRNA levels of collagen Ⅰ and Runx2 were detected by quantitative real-time PCR (qRT-PCR). The protein expressions of collagen Ⅰ, Runx2 and nuclear factor (NF)-κB were determined by Western blot. Primary rat aortic smooth muscle cells (VSMCs) were cultured in three conditions: normal glucose (NG), high glucose (HG) and glucose fluctuations (GF). Cells in GF group were incubated for 72 hours with glucose alternating between 5.5 and 25 mmol/L every 12 hours. TPCA-1, the inhibitor of NF-κB, the expression of collagenⅠin different groups of cells was tested by immunofluorescence. The protein expressions of collagen Ⅰ, Runx2 and NF-κB were also determined by Western blot. Results: (1) The quantitative ratios of the area of fibrosis in the C-STZ group, U-STZ group, STZ-GF group were (8.42±0.10)%, (21.30±0.74)% and (44.39±1.09)% (P<0.05), respectively. The means of integral optical density (IOD) of collagenⅠ in the three groups were 11.92±0.88, 50.04±3.56 and 77.52±2.69, respectively (P<0.05). The mRNA levels of collagenⅠ in the three groups were 1.00±0.10, 2.02±0.28 and 2.83±0.33, respectively (P<0.05). The protein expressions of collagenⅠ in the three groups were 1.05±0.03, 2.06±0.32 and 4.93±0.25, respectively (P<0.05). (2) The average IOD of Runx2 in the three groups were 150.00±7.35, 204.84±2.32 and 391.48±7.13, respectively (P<0.05). The mRNA levels of Runx2 in the three groups were 1.02±0.02, 1.27±0.04 and 2.18±0.12, respectively (P<0.05). The protein expressions of Runx2 in the three groups were 1.03±0.01, 2.34±0.36 and 4.52±0.75, respectively (P<0.05). (3) The protein expressions of NF-κB in the three groups were 1.02±0.01, 1.96±0.13 and 2.64±0.21, respectively (P<0.05). (4) In vitro, application of inhibitor of NF-κB reversed glucose fluctuations-induced upregulation of protein levels of Col Ⅰ and Runx2 (P<0.05). Conclusion: Glucose fluctuations could aggravate aortic fibrosis through activating Runx2 via NF-κB signaling pathways.

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.

Impact and related mechanisms of glucose fluctuations on aortic fibrosis in type 1 diabetic rats / 中华心血管病杂志

Objective: To investigate the impact and related mechanisms of glucose fluctuations on aortic fibrosis in rats with type 1 diabetes mellitus. Methods: After injection of streptozotocin (STZ), male Sprague Dawley (SD) (8-12 weeks) rats (n=24) were randomly divided into three groups in accordance with the random number table: controlled STZ-induced diabetes (C-STZ) group (n=8); uncontrolled STZ-induced diabetes (U-STZ) group (n=8); STZ-induced diabetes with glucose fluctuations (STZ-GF) group (n=8). After three weeks, rats were sacrificed and aorta was obtained, aortic fibrosis was detected by Masson trichrome staining. The expression of collagen type 1 (collagen Ⅰ) was tested by immunofluorescence. The expression of runt-related transcription factor 2 (Runx2) was tested by immunohistochemistry. The mRNA levels of collagen Ⅰ and Runx2 were detected by quantitative real-time PCR (qRT-PCR). The protein expressions of collagen Ⅰ, Runx2 and nuclear factor (NF)-κB were determined by Western blot. Primary rat aortic smooth muscle cells (VSMCs) were cultured in three conditions: normal glucose (NG), high glucose (HG) and glucose fluctuations (GF). Cells in GF group were incubated for 72 hours with glucose alternating between 5.5 and 25 mmol/L every 12 hours. TPCA-1, the inhibitor of NF-κB, the expression of collagenⅠin different groups of cells was tested by immunofluorescence. The protein expressions of collagen Ⅰ, Runx2 and NF-κB were also determined by Western blot. Results: (1) The quantitative ratios of the area of fibrosis in the C-STZ group, U-STZ group, STZ-GF group were (8.42±0.10)%, (21.30±0.74)% and (44.39±1.09)% (P<0.05), respectively. The means of integral optical density (IOD) of collagenⅠ in the three groups were 11.92±0.88, 50.04±3.56 and 77.52±2.69, respectively (P<0.05). The mRNA levels of collagenⅠ in the three groups were 1.00±0.10, 2.02±0.28 and 2.83±0.33, respectively (P<0.05). The protein expressions of collagenⅠ in the three groups were 1.05±0.03, 2.06±0.32 and 4.93±0.25, respectively (P<0.05). (2) The average IOD of Runx2 in the three groups were 150.00±7.35, 204.84±2.32 and 391.48±7.13, respectively (P<0.05). The mRNA levels of Runx2 in the three groups were 1.02±0.02, 1.27±0.04 and 2.18±0.12, respectively (P<0.05). The protein expressions of Runx2 in the three groups were 1.03±0.01, 2.34±0.36 and 4.52±0.75, respectively (P<0.05). (3) The protein expressions of NF-κB in the three groups were 1.02±0.01, 1.96±0.13 and 2.64±0.21, respectively (P<0.05). (4) In vitro, application of inhibitor of NF-κB reversed glucose fluctuations-induced upregulation of protein levels of Col Ⅰ and Runx2 (P<0.05). Conclusion: Glucose fluctuations could aggravate aortic fibrosis through activating Runx2 via NF-κB signaling pathways.