Therapeutic efficacy of Schistosoma japonicum cystatin on sepsis-induced cardiomyopathy in a mouse model

Abstract

Background: Myocardial dysfunction is one of the most common complications of multiple organ failure in septic shock and significantly increases mortality in patients with sepsis. Although many studies having confirmed that helminth-derived proteins have strong immunomodulatory functions and could treat inflammatory diseases, there is no report on the therapeutic effect of Schistosoma japonicum-produced cystatin (Sj-Cys) on sepsis-induced cardiac dysfunction.

Methods: A model of sepsis-induced myocardial injury was established by cecal ligation and puncture (CLP) in mice. Upon CLP operation, each mouse was intraperitoneally treated with 10 µg of recombinant Sj-Cys (rSj-Cys). Twelve hours after CLP, the systolic and diastolic functions of the left ventricular were examined by echocardiography. The levels of myoglobin (Mb), cardiac troponin I (cTnI), N-terminal pro-Brain Natriuretic peptide (NT-proBNP) in sera, and the activity of myeloperoxidase (MPO) in cardiac tissues were examined as biomarkers for heart injury. The heart tissue was collected for checking pathological changes, macrophages and pro-inflammatory cytokine levels. To address the signaling pathway involved in the anti-inflammatory effects of rSj-Cys, myeloid differentiation factor 88 (MyD88) was determined in heart tissue of mice with sepsis and LPS-stimulated H9C2 cardiomyocytes. In addition, the therapeutic effects of rSj-Cys on LPS-induced cardiomyocyte apoptosis were also detected. The levels of M1 biomarker iNOS and M2 biomarker Arg-1 were detected in heart tissue. The pro-inflammatory cytokines TNF-α and IL-6, and regulatory cytokines IL-10 and TGF-β were measured in sera and their mRNA levels in heart tissue of rSj-Cys-treated mice.

Results: After rSj-Cys treatment, the sepsis-induced heart malfunction was largely improved. The inflammation and injury of heart tissue were significantly alleviated, characterized as significantly decreased infiltration of inflammatory cells in cardiac tissues and fiber swelling, reduced levels of Mb, cTnI and NT-proBNP in sera, and MPO activity in heart tissue. The therapeutic efficacy of rSj-Cys is associated with downregulated pro-inflammatory cytokines (TNF-α and IL-6) and upregulated regulatory inflammatory cytokines (IL-10 and TGF-β), possibly through inhibiting the LPS-MyD88 signal pathway.

Conclusions: RSj-Cys significantly reduced sepsis-induced cardiomyopathy and could be considered as a potential therapeutic agent for the prevention and treatment of sepsis associated cardiac dysfunction.

Keywords: Cystatin; Immunoregulation; Myocardial dysfunction; Schistosoma japonicum.

Fig. 1

Treatment of rSj-Cys improved the sepsis-induced myocardial malfunction. Representative M-mode echocardiograms obtained from mice 12 h after treatment of sham-operation, sham + rSj-Cys, CLP and CLP + rSj-Cys, respectively (a). The improved left ventricular systolic function evaluated by EF and FS after treatment with rSj-Cys (c). The improved CLP-induced left ventricular diastolic dysfunction was shown as representative transmitral Doppler images. The E wave represents peak early-diastolic transmitral velocity, and the A wave indicates peak late-diastolic transmitral velocity (b). The changes of the E/A ratio were used to assess the alteration in left ventricular diastolic function (d). (n = 6 mice per group). Data are presented as the mean ± SE. ***P < 0.001

Fig. 2

a Histopathological and morphological variations in the cardiac tissue of mice following sham surgery, sham + rSj-Cys, CLP and CLP + rSj-Cys (n = 6 mice per group, 200× magnification). Treatment with rSj-Cys reduced CLP-sepsis induced myocardial injury in the BALB/c mice. b The levels of cTnI, NT-proBNP, Mb in sera and MPO activity in heart tissue were significantly reduced in CLP mice treated with rSj-Cys. The data are presented as the mean ± SE. **P < 0.01, ***P < 0.001. Scale-bars: 50 μm

Fig. 3

Treatment with rSj-Cys reduced the pro-inflammatory cytokine (TNF-α and IL-6) and boosted regulatory cytokine (IL-10 and TGF-β) levels in sera (a) and the similar mRNA expression pattern observed in heart tissues (b) of mice with CLP-induced sepsis 12 h after treatment. The mRNA expression level of M1 macrophage marker (iNOS) was reduced and that of M2 macrophage marker (Arg-1) increased in heart tissues (b). The data are shown as the mean ± SE for each group (n = 3 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

a Incubation with rSj-Cys inhibited the pro-inflammatory cytokines TNF-α and IL-6 and stimulated regulatory cytokines IL-10 and TGF-β released by LPS-induced H9C2 cells. The levels of these cytokines in the supernatant were measured by ELISA 24 h after incubation. The results are shown as the mean ± SE for each group (n = 3 per group). b rSj-Cys reduced LPS-induced cardiomyocyte apoptosis measured by flow cytometry. Representative flow cytometry images showed the reduced cardiomyocyte apoptosis in rSj-Cys + LPS co-incubated H9C2 cells. The normal H9C2 cells in blank medium or medium with rSj-Cys were used as controls. Data are expressed as mean ± SE from three independent experiments) (n = 3 per group). rSj-Cys treatment suppressed the expression of MyD88 in the myocardial tissues of mice with CLP-induced sepsis (c) (n = 6 mice per group) and in LPS-incubated H9C2 cells (d) (n = 3 per group) measured by western blot. β-actin was measured as a control. The density ratio of MyD88/β-actin is shown on the right. The results are shown as the density mean ± SE for each group. *P < 0.05, **P < 0.01, ***P < 0.001