Duodenal‐jejunal bypass surgery activates eNOS and enhances antioxidant system by activating AMPK pathway to improve heart oxidative stress in diabetic cardiomyopathy rats

Figures

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  FIGURE 1. DJB improves general metabolic characteristics. (A) The flow chart of animal experiments. (B) Body weight. (C) Food intake. (D) (FBG). (E) Curves of OGTT before surgery. (F) AUC of OGTT before surgery. (G) Curves of OGTT 12 weeks after surgery. (H) AUC of OGTT 12 weeks after surgery. (I) HOMA‐IR 12 weeks after surgery. Data are presented as mean ± SD. *p < .05, **p < .01 ***p < .001 CON vs. SHAM; #p < .05, ##p < .01 ###p < .001 DJB vs. SHAM. n = 10 per group. AUC, area under the curve; CON, control group; DJB, duodenal‐jejunal bypass; FBG, fasting blood glucose; HFD, high‐fat diet; HOMA‐IR, homeostatic model assessment of insulin resistance; OGTT, oral glucose tolerance test; STZ, streptozotocin.

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  FIGURE 2. Effect of DJB on cardiac remodeling in diabetic cardiomyopathy rats. (A) H&E staining images of left ventricle (10×, 40×). (B) Masson staining of rat hearts, with dark blue staining indicating collagen fibers (10×, 40×). (C) Sirius red staining of collagen in rat hearts (10×, 40×). (D, E) Immunohistochemical staining of collagen 1 and collagen 3 (10×, 40×). (F) WGA staining images (40×). (G) Cardiomyocyte diameter. (H) Heart mass as a percentage of body weight. *p < .05. **p < .01. ***p < .001. CON, control group; DJB, duodenal‐jejunal bypass; H&E, hematoxylin and eosin; WGA, wheat germ agglutinin.

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  FIGURE 3. DJB ameliorates the cardiac AMPK/eNOS pathway and antioxidative system in diabetic cardiomyopathy rats and improves oxidative stress in the heart. (A) ROS fluorescence staining in the heart of three groups of rats (40×). (B) Analysis results of the relative ROS fluorescence intensity. (C) Protein levels of antioxidative system‐related factors and pathway in the heart of rats; β‐Actin was used as an internal reference. (D) Protein levels of AMPK pathway‐related molecules in the heart of rats; β‐Actin was used as an internal reference. (E) Relative mRNA levels of antioxidative system‐related factors and Sirt1 in the heart of rats; β‐Actin was used as a reference gene. (F) Phosphorylation levels of eNOS, (G) relative protein content, and (H) NO content in the heart muscle of rats 12 weeks after surgery. Data are presented as mean ± SD.*p < .05. **p < .01 ***p < .001; n = 10 in each group. AMPK, adenosine monophosphate‐activated protein kinase; CON, control group; DJB, duodenal‐jejunal bypass; eNOS, endothelial nitric oxide synthase; HO‐1, heme oxygenase‐1; NO, nitric oxide; NRF2, nuclear factor erythroid 2‐related factor 2; ROS, reactive oxygen species; SOD2, superoxide dismutase 2.

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  FIGURE 4. Activation of the AMPK pathway can improve eNOS activity and antioxidant system in H9C2 cells in the HG + PA environment. (A) ROS staining in H9C2 cells, shown by green fluorescence (10×). (B) Protein levels of antioxidative system‐related factors were analyzed in H9C2 cells; β‐Actin was used as an internal reference. (C) Relative mRNA levels of antioxidation system‐related factors and Sirt1 in rats; β‐Actin served as a reference gene. (D) Protein levels of AMPK pathway‐related molecules in H9C2 cells; β‐Actin was used as an internal reference. (E) Phosphorylation levels of eNOS in H9C2 cells; β‐Actin was used as an internal reference. (F) Relative protein content and (G) NO content in H9C2 cells. Data are presented as mean ± SD. *p < .05. **p < .01. ***p < .001 n = 3 in each group. AMPK, adenosine monophosphate‐activated protein kinase; CON, control group; DJB, duodenal‐jejunal bypass; eNOS, endothelial nitric oxide synthase; HG, high glucose; HO‐1, heme oxygenase‐1; NO, nitric oxide; NRF2, nuclear factor erythroid 2‐related factor 2; PA, palmitate; ROS, reactive oxygen species; SOD2, superoxide dismutase 2.

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  FIGURE 5. Effects of CON, SHAM, and DJB group sera on the AMPK/eNOS pathway and antioxidant system in H9C2 cells in the HG + PA environment. (A) ROS staining in H9C2 cells, shown by green fluorescence (10×). (B) Protein levels of AMPK pathway‐related molecules in H9C2 cells; β‐Actin was used as an internal reference. (C) Relative mRNA levels of antioxidation system‐related and Sirt1 genes in rats; β‐Actin served as a reference gene. (D) Protein levels of antioxidative system‐related factors in H9C2 cells; β‐Actin was used as an internal reference. (E) Phosphorylation levels of eNOS in H9C2 cells; β‐Actin was used as an internal reference. (F) Relative protein content and (G) NO content in H9C2 cells. Data are presented as mean ± SD. *p < .05. **p < .01. ***p < .001; n = 3 in each group. AMPK, adenosine monophosphate‐activated protein kinase; CON, control group; DJB, duodenal‐jejunal bypass; DM, diabetes mellitus; eNOS, endothelial nitric oxide synthase; HG, high glucose; HO‐1, heme oxygenase‐1; NO, nitric oxide; NRF2, nuclear factor erythroid 2‐related factor 2; PA, palmitate; ROS, reactive oxygen species; SOD2, superoxide dismutase 2.

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  FIGURE 6. Improvement of oxidative stress in H9C2 cells in the HG + PA environment by the serum of DJB rats depends on the activation of the AMPK pathway to a certain extent. (A) ROS staining in H9C2 cells, shown by green fluorescence (10×). (B) Relative mRNA levels of antioxidation system‐related genes in rats. β‐Actin served as a reference gene. (C) Relative protein content and (D) protein levels of AMPK pathway‐related molecules in H9C2 cells; β‐Actin was used as an internal reference. (E) Phosphorylation levels of eNOS in H9C2 cells; β‐Actin was used as an internal reference. (F) Relative protein content and (G) protein levels of antioxidative system‐related factors in H9C2 cells; β‐Actin was used as an internal reference control. (H) NO content in H9C2 cells. Data are presented as mean ± SD. *p < .05. **p < .01 ***p < .001; n = 3 in each group. AMPK, adenosine monophosphate‐activated protein kinase; CON, control group; DJB, duodenal‐jejunal bypass; eNOS, endothelial nitric oxide synthase; HG, high glucose; HO‐1, heme oxygenase‐1; NO, nitric oxide; NRF2, nuclear factor erythroid 2‐related factor 2; PA, palmitate; ROS, reactive oxygen species; SOD2, superoxide dismutase 2.