Atorvastatin ameliorated myocardial fibrosis in db/db mice by inhibiting oxidative stress and modulating macrophage polarization

Figure 1 Comparison of echocardiographic indices of cardiac systolic and diastolic function between each group. A: Representative pictures of cardiac echocardiography in each group; B–E: db/db mice received daily oral gavage of sterilized water (DM group) showed a significant increase in left ventricular end-diastolic diameter (LVIDd) and left ventricular internal dimension in systole (LVIDs), accompanied by a significant reduction in left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF), while db/db mice that received daily oral gavage of 10 mg/kg/d atorvastatin group showed decreased levels of LVIDd, and augmented levels of LVFS and LVEF. The DM+MET group had decreased levels of LVIDd and LVIDs, but no effects on LVFS and LVEF. Data represent the means ± SD (n = 5). ^aP < 0.05 compared with db/db mice received daily oral gavage of sterilized water group. ^bP < 0.05 compared with C57BL/6 mice. DM: db/db mice received daily oral gavage of sterilized water; DM + ATO: db/db mice received daily oral gavage of 10 mg/kg/d atorvastatin; DM + MET: db/db mice received daily oral gavage of 200 mg/kg metformin; CON: C57BL/6 mice; LVEF: Left ventricular ejection fraction; LVFS: Left ventricular fractional shortening; LVIDs: Left ventricular internal dimension in systole; LVIDd: Left ventricular end-diastolic diameter.

Figure 2 Histological changes in the myocardium. A: Hematoxylin and eosin staining was performed for each group. db/db mice that received daily oral gavage of sterilized water (DM group) had disorganized and broken myocardial fibers and irregular nuclei, which were relieved in db/db mice by daily oral gavage of 10 mg/kg/d atorvastatin (DM + ATO) or in db/db mice by daily oral gavage of 200 mg/kg metformin (DM + MET) group, especially in the DM + ATO group; B: Masson staining was performed for each group. DM group displayed deposition of collagen in the myocardial interstitium, which was relieved in the DM + ATO or DM + MET group, especially the DM + ATO group. Scale bar, 100 μm. DM: db/db mice received daily oral gavage of sterilized water; DM + ATO: db/db mice received daily oral gavage of 10 mg/kg/d atorvastatin; DM + MET: db/db mice received daily oral gavage of 200 mg/kg metformin; CON: C57BL/6 mice; H&E: Hematoxylin and eosin staining.

Figure 3 Expression of markers of cardiac injury and indicators of inflammation in the serum of each group. A–C: Serum creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH) and troponin (cTnI) were measured using the automatic biochemical instrument. Compared with C57BL/6 mice (CON group), db/db mice that received daily oral gavage of sterilized water (DM group) had elevated serum CK-MB, LDH and cTnI. db/db mice that received daily oral gavage of 10 mg/kg/d atorvastatin (DM + ATO) or db/db mice that received daily oral gavage of 200 mg/kg metformin (DM + MET) group had decreased serum CK-MB, LDH and cTnI; D–F: The levels of transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in the serum samples were detected by ELISA. Compared with CON group, the serum levels of TGF-β1, TNF-α, and IL-1β were markedly elevated. In the DM + ATO and DM + MET groups, these indicators were markedly lower. Data represent the means ± SD (n = 5). ^aP < 0.05 compared with db/db mice received daily oral gavage of sterilized water group. ^bP < 0.05 compared with C57BL/6 mice. DM: db/db mice received daily oral gavage of sterilized water; DM + ATO: db/db mice received daily oral gavage of 10 mg/kg/d atorvastatin; DM + MET: db/db mice received daily oral gavage of 200 mg/kg metformin. CON: C57BL/6 mice.

Figure 4 Immunohistochemical staining and relative gene expression of transforming growth factor-β1, tumor necrosis factor-α, and interleukin-1β in each group. A–C: Immunohistochemistry staining of TGF-β1, TNF-α, and IL-1β in each group; D–F: relative gene expression of TGF-β1, TNF-α, and IL-1β in each group. Compared with C57BL/6 mice (CON group), the expression of TGF-β1, TNF-α and IL-1β in db/db mice that received daily oral gavage of sterilized water (DM) group was markedly elevated, and daily oral gavage of 10 mg/kg/d atorvastatin (DM + ATO) or 200 mg/kg metformin (DM + MET) alleviated these manifestations. The DM + MET group showed no effects on IL-1β mRNA expression in the myocardium. Data represent the means ± SD (n = 5). ^aP < 0.05 compared with db/db mice received daily oral gavage of sterilized water group. ^bP < 0.05 compared with C57BL/6 mice. DM: db/db mice received daily oral gavage of sterilized water; DM+ATO: db/db mice received daily oral gavage of 10 mg/kg/d atorvastatin; DM+MET: db/db mice received daily oral gavage of 200 mg/kg metformin; CON: C57BL/6 mice; TGF-β1: Transforming growth factor-β1; TNF-α: Tumor necrosis factor-α; IL-1β: Interleukin-1β.

Figure 5 Levels of superoxide dismutase activity and malondialdehyde content and immunofluorescence staining of macrophages. A and B: SOD activity was decreased and MDA content was significantly increased in db/db mice that received daily oral gavage of sterilized water (DM group). db/db mice that received daily oral gavage of 10 mg/kg/d atorvastatin (DM + ATO) or 200 mg/kg metformin (DM + MET) group had markedly decreased MDA content and enhanced SOD activity; C and E: Compared with C57BL/6 mice (CON group), iNOS⁺ in the DM group were markedly increased, and the DM + ATO group had reduced the expression of M1 macrophages. The DM + MET group showed no effects on expression of M1 macrophages in the myocardium of db/db mice; D and F: Compared with the CON group, CD206⁺ in the DM group were markedly decreased, and the DM + ATO and DM + MET groups had increased expression of M2 macrophages in the myocardium. Data represent the means ± SD (n = 5). ^aP < 0.05 compared with db/db mice received daily oral gavage of sterilized water group. ^bP < 0.05 compared with C57BL/6 mice. iNOS: Inducible nitric oxide synthase; MDA: Malondialdehyde; SOD: Superoxide dismutase; DM: db/db mice received daily oral gavage of sterilized water; DM + ATO: db/db mice received daily oral gavage of 10 mg/kg/d atorvastatin; DM + MET: db/db mice received daily oral gavage of 200 mg/kg metformin; CON: C57BL/6 mice.

Figure 6 Graphical abstract. MDA: Malondialdehyde; SOD: Superoxide dismutase; TGF-β1: Transforming growth factor-β1; TNF-α: Tumor necrosis factor-α; IL-1β: Interleukin-1β.