Puerarin Attenuates High-Glucose and High-Lipid-Induced Inflammatory Injury in H9c2 Cardiomyocytes via CAV3 Protein Upregulation

Figure 1. HGHL-Induced CAV3 Protein Expression Downregulation and Morphological Injury in H9c2 Cells. HGHL treatment downregulated CAV3 protein expression in a time-dependent manner, with a significant decrease at 24 hours observed using Western blot analysis (A and B). The data were presented as mean ± SD, with n = 6 per group. Microscopic observation (200× amplification) revealed HGHL treatment resulted in increased morphological damage to H9c2 cardiomyocytes (C). The scale bars in the panel were 50 μm. The statistical significance of the observed effects was indicated by ***P<0.001.

Figure 2. HGHL Provoked Inflammatory Injury in H9c2 Cells. Results showed that treatment with HGHL significantly decreased cell viability and increased cell apoptosis (A and B). Additionally, HGHL treatment resulted in significant morphological changes to cardiomyocytes when compared to the normal group, as indicated by hematoxylin and eosin (H&E) staining (C). Western blot analysis indicated significant upregulation of cleaved-Caspase-3/pro-Caspase-3, and Bax expression, while Bcl-2 showed significant downregulation following HGHL treatment (D-G). Moreover, HGHL treatment resulted in a significant upregulation of IL-6 expression in H9c2 cells (H). The IL-6 release and cell viability assays were performed with n = 6 per group and Western blot analysis with n = 3 per group. Flow cytometry assays were conducted with n = 3 per group. All HE staining images were obtained using microscopy with 200× magnification, and the scale bars in the panel were 50 µm. The statistical significance of the observed effects was indicated by **p<0.01 and ***p<0.001.

Figure 3. Puerarin alleviated HGHL-induced inflammatory injury in H9c2 cells. (A) Chemical Structure of Puerarin. (B) No obvious changes in cell viability with puerarin (0,10−6,10−5,10−4,10−3M) treatment. (C) When cells were treated with similar puerarin concentrations in HGHL-induced cells, cell viability was significantly improved with the increased concentration of puerarin and results show a dose-dependent way. (D) Puerarin groups reduced myocardial cell morphology injury. (E) Puerarin significantly suppressed the HGHL-induced apoptosis. (F-I) Puerarin noticeably inhibited HGHL-induced levels of cleaved-Caspase-3/pro-Caspase-3 and Bax and enhanced the level of Bcl-2. (J) Puerarin markedly suppressed the production of IL-6. This experiment was conducted with six replicates in the IL-6 release and cell viability assays, four replicates in the Western blot, and three replicates in the Flow cytometry assay. The HE staining images were obtained via microscope with 200× amplification and the scale bars in the panel were 50 µm. The statistical significance of the observed effects was indicated by *p <0 0.05, **p<0.01 and ***p<0.001.

Figure 4. Puerarin inhibited the NF-κB and p38MAPK pathways in H9c2 cells. (A and B) Puerarin inhibited the p38MAPK pathway. (C and D) Puerarin inhibited the NF-κB pathway. n = 4 per group for the Western blot. *p < 0.05; **p <0 0.01.

Figure 5. Puerarin positively regulates the expression level of CAV3. (A and B) The protein expression level of CAV3 was significantly downregulated after HGHL treatment. n = 4 per group for the Western blot. *p < 0.05; **p < 0.01.

Figure 6. Puerarin inhibited HGHL-induced inflammatory damage via up-regulated CAV3 expression. (A and B) CAV3 expression was significantly downregulated upon transfection with CAV3siRNA. (C-E) SB203580 significantly decreased phosphorylation levels of p/t-p38MAPK induced by CAV3 siRNA transfection; BAY11-7082 significantly reduced phosphorylation levels of p/t-p38MAPK induced by CAV3 siRNA transfection. (F) CAV3 siRNA significantly improved puerarin-treated IL-6 expression level. (G) The myocardial cells had significant morphological changes after the transfection of CAV3 siRNA. The Western blot analysis was conducted with 6 replicates for each group, while the IL-6 release assay was conducted with 6 replicates per group. All HE staining images were obtained via a microscope with 200× amplification and the scale bars in the panel were 50 µm. The statistical significance of the observed effects was indicated by **p<0.01 and ***p<0.001.