Recombinant human angiotensin-converting enzyme 2 plays a protective role in mice with sepsis-induced cardiac dysfunction through multiple signaling pathways dependent on converting angiotensin II to angiotensin 1–7

Figure 1. RhACE2 administration improves cardiac function in mice with LPS-induced cardiac dysfunction. (A) Transthoracic echocardiography (M-mode) of mice among three groups (6 per group). (B,C) Comparison of LVEF and LVFS of mice among three groups (6 per group). Data are expressed as mean ± SD. LPS, lipopolysaccharide; rhACE2, recombinant human angiotensin-converting enzyme 2; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; SD, standard deviation.

Figure 2. RhACE2 administration relieved myocardial injury in mice with LPS-induced cardiac dysfunction. (A) Representative pictures of HE-stained heart tissue of mice in three groups (n=6). Magnification, ×200. (B) Myocardial injury score in three groups (n=6). (C) Evaluation of myocardial injury by cTnI in serum from mice in three groups (n=6). Data are expressed as mean ± SD. LPS, lipopolysaccharide; rhACE2, recombinant human angiotensin-converting enzyme 2; HE, hematoxylin and eosin, cTnI, cardiac troponin I; SD, standard deviation.

Figure 3. RhACE2 significantly inhibited activation of NLRP3 inflammasome followed by significant decrease of NLRP3 inflammasome related inflammatory factors in serum and cell pyroptosis in heart tissues in mice with LPS-induced cardiac dysfunction. (A-D) Representative bands of WB and quantitative analysis of pyroptosis-related proteins in three groups (n=6). α-tubulin was used as an internal control. (E) The serum levels of IL-1β in three groups (n=6). (F) The serum levels of IL-18 in three groups (n=6). (G) The serum levels of TNF-α in three groups (n=6). (H) Representative images of cell pyroptosis (indicated by TUNEL positive cells) in heart tissue of mice in three groups (6 per group). Nuclei were depicted by DAPI (blue). The pyroptotic cells were depicted by TUNEL (green). Magnification, ×200. (I) Comparison of pyroptosis cell rates in heart tissue from mice in three groups (6 per group). Data are expressed as mean ± SD. GSDMD, gasdermin D; GSDMD-NT, N-terminal cleavage product of GSDMD; NLRP3, nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3; LPS, lipopolysaccharide; rhACE2, recombinant human angiotensin-converting enzyme 2; IL,…

Figure 4. RhACE2 regulated the changes of angiotensin in mice with LPS-induced cardiac dysfunction, and the levels of Ang II in serum correlated with the levels of NLRP3 expression in heart tissue positively. (A) Ang II levels in serum of mice in three groups (n=6). (B) Ang 1–7 serum levels in serum of mice in three groups (n=6). (C) Ang II levels in serum were positive correlated with levels of NLRP3 expression in heart tissue. Data are expressed as mean ± SD. Ang II, angiotensin II; LPS, lipopolysaccharide; rhACE2, recombinant human angiotensin-converting enzyme 2; Ang 1–7, angiotensin 1–7; NLRP3, nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3; SD, standard deviation.

Figure 5. RhACE2 significantly inhibited phosphorylation of NF-κB, p38 MAPK signaling pathway, and partly recovered activation of AMPK signaling pathway changed in mice with LPS-induced cardiac dysfunction. (A) Representative bands of WB and quantitative analysis of NF-κB, p38 MAPK, and AMPK-α1 signaling pathway related proteins expression in three groups. (B) Quantitative analysis phosphorylation levels of phosphorylation levels of NF-κB in three groups (n=6). (C) Quantitative analysis phosphorylation levels of p38 MAPK in three groups (n=6). (D) Quantitative analysis phosphorylation levels of AMPK-α1 in three groups (n=6). α-tubulin was used as an internal control. Data are expressed as mean ± SD. p-, phosphorylated-; NF-κB, nuclear factor κB; AMPK, AMP-activated protein kinase; MAPK, mitogen-activated protein kinase; LPS, lipopolysaccharide; rhACE2, recombinant human angiotensin-converting enzyme 2; WB, western blot; SD, standard deviation.

Figure 6. A779 administration offset the improve effect of rhACE2 in mice with LPS-induced cardiac dysfunction. (A) Transthoracic echocardiography (M-mode) of mice in each group (6 per group). (B) Comparison of LVEF of mice among four groups (6 per group). (C) Comparison of LVFS of mice among four groups (6 per group). (D) Representative pictures of HE-stained heart tissue of mice in four groups (n=6). Magnification, ×200. (E) Myocardial injury score in four groups (n=6). (F) Evaluation of myocardial injury by cTnI in serum from mice in four groups (n=6). (G) Representative images of cell pyroptosis (indicated by TUNEL labeled cells) in heart tissue of mice in four groups (6 per group). Nuclei were depicted by DAPI (blue). The pyroptotic cells were depicted by TUNEL (green). Magnification, ×200. (H) Comparison of pyroptosis cell rates in heart tissue from mice in four groups (6 per group). (I) WB for NLRP3 expression in heart tissue of mice from four groups (n=6), α-tubulin was used as an internal control. (J) Quantitative analysis of NLRP3 expression in heart tissue of mice from four groups (n=6). Data are expressed as mean ± SD. LPS, lipopolysaccharide; rhACE2, recombinant human…