Combination of Dendrobium Mixture and Metformin Curbs the Development and Progression of Diabetic Cardiomyopathy by Targeting the lncRNA NEAT1

Figures

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  Figure 1. NEAT1 is downregulated in patients with diabetes mellitus and H9c2 cells maintained in high-glucose medium. A, Expression of NEAT1 in patients with diabetes mellitus (DM); B, Expression of NEAT1 in H9c2 cells maintained in high-glucose (HG) medium; C-E, mRNA expression levels of Nrf2, MDA, and HO-1 in patients with DM; F-H, mRNA expression levels of Nrf2, MDA, and HO-1 in H9c2 cells maintained in HG medium; I-L, Western blotting of Nrf2, MDA, and HO-1 in patients with DM; M-P, Western blotting of Nrf2, MDA, and HO-1 in H9c2 cells maintained in HG medium. *p<0.05, **p<0.01, ***p<0.001, Mann-Whitney U test.

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  Figure 2. NEAT1 expression in H9c2 cells maintained in high-glucose medium is upregulated by combination treatment with dendrobium mixture and metformin, which was accompanied by a significant decrease in apoptosis. A-D, The combination of dendrobium mixture (DN) and metformin (Met) promoted the expression of NEAT1 and Nrf2 pathway-related genes in H9c2 cells maintained in high-glucose (HG) medium. The combination of dendrobium mixture and Met inhibited apoptosis (E-I) and promoted the proliferation (J) of H9c2 cells maintained in HG medium. *p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis test followed by Dunn’s multiple comparisons test (A-I), Mann-Whitney U test (J).

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  Figure 3. Upregulation of NEAT1 inhibited the apoptosis of myocardial cells. Transfection of pcDNA3.1-NEAT1 upregulated the mRNA expression of NEAT1 (A) and Nrf2 protein levels (B-C) and inhibited apoptosis (D) in H9c2 cells maintained in high-glucose (HG) medium. ***p<0.001, Mann-Whitney U test.

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  Figure 4. NEAT1 regulates the expression of Nrf2 by targeting miR-23a-3p. A, Identification of a binding site between NEAT1 and miR-23a-3p; B, Dual luciferase reporter assay confirming binding between NEAT1 and miR-23a-3p; C, Identification of a binding site between Nrf2 and miR-23a-3p; D, Dual luciferase reporter assay confirming binding between Nrf2 and miR-23a-3p; E, The expression of miR-23a-3p is upregulated in patients with diabetes mellitus (DM); F-G, The expression levels of miR-23a-3p are negatively correlated with the levels of NEAT1 and Nrf2. *p<0.05, **p<0.01, Mann-Whitney U test.

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  Figure 5. pc-NEAT1 upregulated the expression of NEAT1 and Nrf2 pathway-related genes. A-D, Transfection of pcDNA3.1-NEAT1 upregulated the expression of NEAT1 and Nrf2 pathway-related genes and E, decreased blood glucose levels in rats with diabetic cardiomyopathy (DCM). **p<0.01, ***p<0.001, Mann-Whitney U test.

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  Figure 6. Treatment with the combination of dendrobium mixture and metformin improved the morphology of heart tissues in rats with streptozotocin-induced diabetes mellitus. A-C, Hematoxylin and eosin staining of the heart tissue of the rats in each group; D, Treatment with dendrobium mixture and metformin improved myocardial cell size in streptozotocin-induced diabetic rats; and E-F, upregulated the expression of NEAT1 and Nrf2. *p<0.05, ***p<0.001, Kruskal-Wallis test followed by Dunn’s multiple comparisons test. DCM, diabetic cardiomyopathy.

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  Figure 7. Proposed mechanism of action for the combination of dendrobium mixture and metformin in the treatment of diabetic cardiomyopathy. NEAT1, acting as a sponge of miR-23a-3p, can regulate the expression of MDA and HO-1 by targeting Nrf2.