Effect of ALDH2 on High Glucose‐Induced Cardiac Fibroblast Oxidative Stress, Apoptosis, and Fibrosis

Figure 1. The identification of cardiac fibroblasts by immunofluorescence technique. (a) Nuclei of CFs were stained with DAPI (magnification ×100). (b) The cytoplasm was stained green which showed that CFs can be identified with the positive expression of vimentin (magnification ×100). (c) The merged picture of (a) and (b) (magnification ×100).

Figure 2. The ALDH2 expression of cardiac fibroblasts detected by double-label immunofluorescence-staining technique (magnification ×100) (a). Red fluorescence-positive cells indicated that ALDH2 was expressed in CFs. The mRNA (b) and protein (c) expressions of ALDH2 were detected by RT-PCR and Western blot in three different batches of CFs. GAPDH was used as a loading control.

Figure 3. The OD value of CFs in different experimental groups were measured by MTT method. Data were presented as the mean ± SEM (N = 6). ∗∗P < 0.01 versus NG, ##P < 0.01 versus HG, ^P < 0.05 versus HG + Alda-1.

Figure 4. ROS level of CFs in different experimental groups. (a) Intracellular ROS accumulation in different groups stained by DHE. Representative pictures were shown. (b) Mean fluorescence intensity was detected by ImageJ software. Data were presented as the mean ± SEM (N = 3). ∗∗P < 0.01 versus NG, ##P < 0.01 versus HG, ^^P < 0.01 versus HG + Alda-1.

Figure 5. ALDH2 activity in different experimental groups. Data were presented as mean ± SEM (N = 6). ∗∗P < 0.01 versus NG, #P < 0.05 versus HG, ^^P < 0.01 versus HG + Alda-1.

Figure 6. Expressions of collagen I and collagen III at mRNA level in the different experimental groups. mRNA levels of collagen I (a) and collagen III (b) were quantified by real-time-PCR analysis in the CFs of each experimental group. GAPDH was used as a loading control. Data were presented as the mean ± SEM (N = 3). ∗P < 0.05, ∗∗P < 0.01 versus NG, #P < 0.05, ##P < 0.01 versus HG, ^P < 0.05, ^^P < 0.01 versus HG + Alda-1.

Figure 7. The changes of ALDH2 and 4-HNE protein levels in different experimental groups. (a) Representative blots of ALDH2 and GAPDH in CFs. (b) ALDH2 protein levels in CFs normalized by GAPDH levels and all the data were presented as mean ± SEM (N = 8). (c) Representative blots of 4-HNE and GAPDH in CFs. (d) 4-HNE protein levels in CFs normalized by GAPDH levels and all the data were presented as mean ± SEM (N = 5). ∗P < 0.05, ∗∗P < 0.01 versus NG, ##P < 0.01 versus HG, ^P < 0.05, ^^P < 0.01 versus HG + Alda-1.

Figure 8. Apoptosis of CFs in different experimental groups. (a) CFs were labeled with Annexin V-FITC and propidium iodide (PI) and analyzed by flow cytometry. Representative pictures were shown. Q1, Q2, Q3, and Q4 represent necrosis, early-stage apoptosis, late-stage apoptosis, and normal cells, respectively. (b) Q2 and Q3 were chosen to analyze the change of apoptosis rate in different experimental groups. Data were presented as mean ± SEM (N = 3). ∗∗P < 0.01 versus NG, ##P < 0.01 versus HG, ^^P < 0.01 versus HG + Alda-1.