Angiotensin-(1–7) ameliorates sepsis-induced cardiomyopathy by alleviating inflammatory response and mitochondrial damage through the NF-κB and MAPK pathways

Fig. 1. Clinical correlation between plasma Ang II, Ang II/Ang-(1–7), and Ang-(1–7) levels and SIC patients. A–C Ang-(1–7), Ang II, Ang II/Ang-(1–7), expression levels in the peripheral plasma samples of HC (n = 12), non-SIC (n = 18), and SIC (n = 16) patients. D Correlation matrix of plasma Ang-(1–7), Ang II, and Ang II/Ang-(1–7) levels with biochemical indices and clinical scores in the SIC patients (n = 16). The matrix values included the Spearman correlation coefficient (p < 0.05). NS: no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2. Ang-(1–7) reduces systemic inflammatory response, alleviates myocardial injury, and increases survival in SIC mice. A Schematic representation of the animal experiments. Mice were injected with Ang-(1–7) (or same saline volume), IP, daily for 3 days. 30 min after administering the compounds (or saline) for 3 days of the administration, the animals in the corresponding groups were again IP injected by LPS (10 mg/kg) or saline. Echocardiography images were captured 12 h later and mice were sacrificed for further experiments. B–F Serum Ang II, IL-6, cTnT, IL-1β, and TNF-α levels (n = 6). G M-mode echocardiography representative images. H Quantitative assessment of LVEF, LVEDV, LVESV, LVID; s, LVID; d, LVFS in every mouse group (n = 4). I Representative images of HE staining. J Histopathological scores of the heart (n = 6). K mRNA levels of BNP in the myocardial samples (n = 4). L Influence of Ang-(1–7) pretreatment on the 3rd day of survival of septic mice (n = 12). NS: no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3. Ang-(1–7) attenuates inflammatory response, macrophage infiltration, and oxidative stress in myocardial tissues of the SIC mice. A Representative immunofluorescence images of macrophage infiltration in myocardial tissue of each group (20 μm; × 400, scale bar). B Number of F4/80+ macrophages (n = 4). C–G The IL-6, Ang II, TNF-α, Ang-(1–7), and IL-1β protein concentrations in the myocardial specimens (n = 6). H–J TNF-α, IL-1β, and IL-6 mRNA levels in myocardial tissue samples (n = 4). K Images showing the DHE staining in myocardial tissues (50 μm; × 200, scale bar). L Relative fluorescence intensity of the DHE procedure (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4. Ang-(1–7) maintains the mitochondrial dynamic equilibrium of cardiomyocytes in SIC mice, reverses their structural dysfunction and damage, and alleviates cardiomyocyte apoptosis. A Representative TEM image of myocardial mitochondria. B Flameng score of TEM images of mitochondria of cardiomyocytes (n = 4). C MMP in myocardial tissue specimens of mice was detected using JC-1 staining (n = 4). D Mitochondrial ATP concentration in myocardial tissues (n = 4). E TUNEL staining images of the myocardial cellular apoptosis (× 200, scale bar: 50 μm). F No. of TUNEL positive cells (n = 4). G Western blot images of Bcl-2, cleaved-caspase-3, Bax, Drp1, Mfn2, and caspase-9 in myocardial tissues. (H–M) Quantification of the caspase-9, Drp1, cleaved-caspase-3, Bcl -2, Mfn2, and Bax proteins concentrations in myocardial specimens (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5. Ang-(1–7) suppresses the upregulation of the NF-κB and MAPK pathways in the myocardial tissues of SIC mice. A Western blot images of p-IκBα, IκBα, p-P65, and P65 in the myocardial specimens. B Western blot images of p-P38, p-JNK, JNK, p-ERK, P38, ERK in the myocardial tissues. C–G Quantifying the p-P38/P38, p-IκBα/IκBα, p-ERK/ERK, p-P65/P65, and p-JNK/JNK concentrations in myocardial tissues (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6. Ang-(1–7) inhibits inflammatory response, mitochondrial dysfunctioning, and oxidative stress, in LPS-induced H9c2 cells. A The H9c2 cell viability, after being pretreated with varying concentrations of Ang-(1–7) (10–6, 10–7, 10–8 mol/L) for 12 h, was measured using the CCK8 assay (n = 6). B The H9c2 cell viability after Ang-(1–7) (10–6, 10–7, 10–8 mol/L) pretreatment for 1 h before LPS stimulation for 12 h was measured using the CCK8 assay (n = 6). C IL-1β protein concentration in the H9c2 cells (n = 6). D–F The IL-1β, TNF-α, and IL-6 mRNA expression levels in H9c2 cells (n = 4). G, H TNF-α and IL-6 protein concentrations in H9c2 cells (n = 6). I, J Quantitative analysis and flow cytometry histograms depicting the ROS production in H9c2 cells (n = 6). K, L Quantitative analysis and flow cytometry plots describing the ratio of monomer JC-1 cells (representing mitochondrial damage) in every group (n = 4). Cells with monomeric JC-1 were green in the P2 region, representing damaged mitochondria. B *p < 0.05 than control, #p < 0.05 than LPS mice. D–L Ang-(1–7) intervention using the pretreatment concentration of 10–6 mol/L for 1 h, and LPS activation of 1 ug/mL for 12 h. NS: no…

Fig. 7. Ang-(1–7) maintained mitochondrial dynamic equilibrium in H9c2 cardiomyocytes and alleviated LPS-induced apoptosis. A Western blot images of Drp1, Mfn2, caspase-9, cleaved caspase-3, Bcl-2, and Bax in H9c2 cells. B–G Quantification of the concentrations of cleaved caspase-3, Drp1, Mfn2, Bcl-2, caspase-9, and Bax proteins in H9c2 cells (n = 4). H, I Flow cytometry plots and quantitative analysis depicting the proportion of H9c2 apoptotic cells (n = 4). J Before LPS stimulation of H9c2 cells for 12 h, cells in each group were pretreated using differing concentrations of A-779 (10–5,10–6,10–7 mol/L) for 2 h, using Ang-(1–7) (10–6 mol/L) for 1 h and then with CCK8 assay for cell viability (n = 6). *p < 0.05 than the control group, #p < 0.05 than LPS group, &p < 0.05 in comparison to LPS + Ang-(1–7) group. K, L) The IL-6 and TNF-α protein concentrations in H9c2 cells (n = 6). M–P IL-6, Bcl-2, TNF-α, and the Bax mRNA levels in the H9c2 cells (n = 4). K–P The A-779 intervention was pretreatment with 10–5 mol/L for 2 h, the Ang-(1–7) intervention included 10–6 mol/L pretreatment for 1 h, and LPS activation of 1 µg/mL for 12 h. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8. Ang-(1–7) inhibits LPS-induced activation of NF-κb and MAPK pathways in H9c2 cardiomyocytes. A Western blotting images of the p-P65, p-IκBα, P65, and IκBα in H9c2 cells. B Western blots of p-JNK, p-P38, p-ERK, P38, ERK, and JNK in H9c2 cells. C–G Quantifying the concentrations of p-IκBα/IκBα, p-ERK/ERK, p-JNK/JNK, p-P38/P38, and p-P65/P65 proteins in H9c2 cells (n = 4). ***p < 0.001, **p < 0.01, *p < 0.05

Fig. 9. Schematic diagram showing the protective effect of Ang-(1–7) on SIC mice and its underlying mechanism. LPS can induce RAAS activation and increase Ang II secretion in blood, tissues, and cells. LPS and Ang II can activate NF-κB and MAPK pathways and generate a large number of inflammatory mediators. Excessive production of proinflammatory factors aggravates oxidative stress, mitochondrial damage, and dynamic imbalance in cardiomyocytes, and further leads to increased apoptosis of cardiomyocytes. Ang-(1–7) may regulate inflammation, reduce oxidative stress, maintain mitochondrial homeostasis, and alleviate mitochondrial structural and functional damage through NF-κB and MAPK pathways