GPR55 Inhibits Inflammatory Factor TNF-α-Mediated RhoA/ROCK2/IκBα/p65 Signaling Pathway to Alleviate Diabetic Cardiomyopathy in Rats

Figures

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  Figure 1. Body weight (a) and blood glucose levels (b) of rats. Data are presented as mean ± SD (n = 8 rats per group). **p < 0.01 and ***p < 0.001 versus the Control group. C, healthy control group; M, untreated diabetic model group; LD, diabetic rats treated with low-dose O-1602; HD, diabetic rats treated with high-dose O-1602.

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  Figure 2. Oxidative stress levels. (a and b) MDA and SOD levels in rat heart tissue; (c and d) MDA and SOD levels in cell line H9C2 (n = 8). ** p < 0.01 and *** p<0.001.

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  Figure 3. Diabetes-mediated alterations in cardiac histopathology. (a–d) Representative hematoxylin and eosin staining images showing pathological alterations in rat myocardial tissue. (e–h) Representative Masson’s trichrome staining images illustrating myocardial collagen deposition and fibrosis. (i–l) Representative periodic acid–Schiff staining images showing glycogen deposition in rat myocardial tissue. Scale bar = 0.5 mm.

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  Figure 4. Diabetes-mediated changes in cardiac pathology. (a–c) Levels of TNF-α, 1L-1β, and 1L-6 in rat serum. (d–f) Levels of TNF-α, 1L-1β, and 1L-6 in cell line H9C2 supernatants. (n = 8). ** p < 0.01 and *** p<0.001.

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  Figure 5. Effects of O-1602 on high-glucose-induced injury in H9C2 cells and diabetes-associated myocardial apoptosis. (a) Viability of H9C2 cells in the normal-control (CK), mannitol osmotic-control (MA), high-glucose (HG), and low-, medium-, and high-dose O-1602 groups (LD, MD, and HD, respectively). (b) Percentages of normal, apoptotic, and necrotic H9C2 cells in the same experimental groups. (c–f) Representative TUNEL-stained rat myocardial sections from the control C, diabetic-model, low-dose O-1602 (LD), and high-dose O-1602 (HD) groups, respectively. TUNEL-positive apoptotic nuclei are shown in green, and total nuclei are counterstained with DAPI in blue. Scale bar = 0.2 mm. Data are presented as mean ± SD (n = 8). ***p < 0.001.

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  Figure 6. Effects of O-1602 on the expression of NF-κB- and RhoA/ROCK-related proteins in H9c2 cells. (a) Densitometric quantification of total p65 protein expression normalized to GAPDH. (b) Densitometric quantification of phosphorylated p65 (p-p65) normalized to total p65. (c) Densitometric quantification of total IκBα protein expression normalized to GAPDH. (d) Densitometric quantification of phosphorylated IκBα (p-IκBα) normalized to total IκBα. (e) Densitometric quantification of RhoA protein expression normalized to GAPDH. (f) Densitometric quantification of ROCK2 protein expression normalized to GAPDH. (g) Representative Western blot images of p65, p-p65, IκBα, p-IκBα, RhoA, ROCK2, and GAPDH in the indicated experimental groups. Data are presented as the mean ± SD from three independent biological experiments per condition. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparison test. **p < 0.01 and ***p < 0.001 versus the CK group; #p < 0.05, ##p < 0.01, and ###p < 0.001 versus the HG group; ns, not statistically significant.

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  Figure 7. Effects of O-1602 on NF-κB- and RhoA/ROCK-related protein expression in cardiac tissue from diabetic rats. (a) Densitometric quantification of total p65 protein expression normalized to GAPDH. (b) Densitometric quantification of phosphorylated p65 (p-p65) normalized to total p65. (c) Densitometric quantification of total IκBα protein expression normalized to GAPDH. (d) Densitometric quantification of phosphorylated IκBα (p-IκBα) normalized to total IκBα. (e) Densitometric quantification of RhoA protein expression normalized to GAPDH. (f) Densitometric quantification of ROCK2 protein expression normalized to GAPDH. (g) Representative Western blot images of p65, p-p65, IκBα, p-IκBα, RhoA, ROCK2, and GAPDH in the indicated experimental groups. Data are presented as the mean ± SD; n = 8 animals per group, or the actual number analyzed when samples were lost or excluded. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparison test. **p < 0.01 and ***p < 0.001 versus the healthy control group; #p < 0.05, ##p < 0.01, and ###p < 0.001 versus the untreated diabetic group; ns, not statistically significant.

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  Figure 8. Schematic representation of the proposed molecular mechanism involving the TNF-α/TNFR–RhoA/ROCK2–NF-κB signaling axis in cardiomyocytes. Binding of extracellular TNF-α to TNFR activates the RhoA/ROCK2 signaling pathway, thereby promoting downstream NF-κB activation. In the cytoplasm, the NF-κB heterodimer, composed of p65 and p50, is retained in an inactive state through its association with IκBα. Phosphorylation of IκBα generates p-IκBα, which is subsequently targeted for proteasome-mediated degradation, resulting in the release and phosphorylation of p65. Activated p-p65 then translocates into the nucleus, where it promotes the transcription of inflammatory and proapoptotic mediators, including IL-1β, and contributes to cardiomyocyte apoptosis. The blue boxes represent the proinflammatory cytokines TNF-α and IL-1β, and the blue dashed connector denotes extracellular cytokine-associated inflammatory signaling. The Orange membrane-associated structure represents TNFR, whereas the green box denotes the RhoA/ROCK2 signaling module. The orange dashed enclosure represents the cytoplasmic NF-κB complex containing p65, p50, and IκBα. The light-green and pink circles represent p65 and…