Treatment with rSj-Cys alleviated sepsis-induced myocardial malfunction
Left ventricular function was examined by echocardiography 12 hours after CLP surgery. As shown in Fig. 1A and B, CLP-induced sepsis caused significant reduction of left ventricular systolic function in mice characterized by reduced EF and FS compared with sham operation group (ANOVA: F(3,23) = 63.07, P < 0.0001; F(3,23) = 21.08, P < 0.0001). In contrast, rSj-Cys treatment dramatically reversed the sepsis-induced decrease in the left ventricular EF and FS to the similar level of mice with sham surgery control (ANOVA: F(3,23) = 63.07, P < 0.0001; F(3,23) = 21.08, P < 0.0001), indicating that treatment with rSj-Cys reduced the sepsis-induced myocardial systolic malfunction in mice. In addition, administration of rSj-Cys in sham surgery mice did not markedly alter left ventricular EF and FS compared with sham group without treatment.
To assess the left ventricular diastolic function, E/A ratio was calculated from Doppler-derived mitral valve inflow measurements. The results showed that sepsis mice displayed a significant decline in E/A ratio values compared to sham control mice (ANOVA: F(3,23) = 10.99, P < 0.0002) (Fig. 1C+D). Treatment with rSj-Cys significantly recovered the E/A ratio to the similar level of mice with sham surgery or sham + rSj-Cys (ANOVA: F(3,23) = 10.99, P < 0.0002) (Fig. 1C+D). These results indicate that treatment with rSj-Cys also improves sepsis-induced diastolic malfunction in mice. No significant difference was observed between sham group and sham with rSj-Cys treated group in terms of E/A ratio(ANOVA: F(3,23) = 10.99, P < 0.0002) (Fig. 1D).
Treatment of rSj-Cys reduced sepsis-induced heart pathological abnormality
The morphological structures and pathology of the myocardial tissues of mice 12 hours after CLP surgery were determined by H&E staining. The results showed that the sham surgery group and sham with rSj-Cys group had no significant inflammatory cell infiltration with normal appearance of the myofibrillar structure (Fig. 2A). However, the heart tissue in mice with CLP showed significant swollenness, myocardial fiber arrangement disorder and highly recruited inflammatory cell infiltration. Of note, tissue sections from the CLP + rSj-Cys group mice showed significantly improved muscle fiber structure with reduced inflammatory cell infiltration compared with CLP group without rSj-Cys treatment (Fig. 2A). The pathological results indicate that rSj-Cys effectively alleviates CLP-induced cardiac lesion and inflammation in mice.
Administration of rSj-Cys reduced sepsis-induced heart injury
In myocardial injury, the release of Mb, cTnI, NT-proBNP and MPO into sera or heart tissue is usually used as biomarkers to evaluate the ischemic severity of heart injury [29, 30, 31]. Compared with the sham group, the CLP group showed markedly increased MPO activity in the myocardial tissue homogenate and elevated levels of cTnI, Mb, NT-proBNP in sera (ANOVA: F(3,23) =32.10, P < 0.0001; F(3,23) = 61.36, P < 0.0001; F(3,23) = 10.42, P < 0.0002; F(3,23) = 42.79, P < 0.0001) (Fig.2B ). After being treated with rSj-Cys, the MPO activity in the myocardial homogenate and cTnI, Mb, NT-proBNP in sera were significantly reduced in mice with sepsis compared with CLP mice without treatment (ANOVA: F(3,23) =32.10, P < 0.0001; F(3,23) = 61.36, P < 0.0001; F(3,23) = 10.42, P < 0.0002; F(3,23) = 42.79, P < 0.0001) (Fig. 2B), while the level of cTnI, NT-proBNP, Mb and MPO remained at low levels in mice with sham surgery and there was no significant difference between sham surgery groups and sham with treatment of rSj-Cys. The increased MPO activity in heart tissue of CLP-induced sepsis mice is correlated with the increased inflammatory cell infiltration, especially neutrophils, in the heart tissue (Fig. 2B).
The reduced sepsis-caused heart injury in mice treated with rSj-Cys was associated with reduced pro-inflammatory cytokines and increased regulatory cytokines.
To understand the mechanism underlying the improvement of sepsis caused cardiac dysfunction with treatment of rSj-Cys, the levels of pro-inflammatory cytokines (TNF-α, IL-6) and regulatory cytokines (IL-10, TGF-β) were measured in sera, and the mRNA levels measured in heart tissue of experimental mice. The results showed that the inflammatory cytokines (TNF-α, IL-6) were dramatically increased in the sera of CLP-induced sepsis mice compared to that in mice with sham surgery only or sham + rSj-Cys (ANOVA: F(3,23) =18.39, P < 0.0001; F(3,23) =361.3, P < 0.0001) (Fig. 3A). Treatment with rSj-Cys significantly reduced the production of TNF-α and IL-6 in CLP-induced sepsis mice compared with CLP mice without treatment (ANOVA: F(3,23) =18.39, P < 0.0001; F(3,23) =361.3, P < 0.0001) (Fig. 3A). However, there was no significant difference of TNF-α and IL-6 levels in sera of mice between the sham group and sham + rSj-Cys group (ANOVA: F(3,23) =18.39, P < 0.0001; F(3,23) =361.3, P < 0.0001) (Fig. 3A). The reduced TNF-α and IL-6 levels were correlated with the increased IL-10 and TGF-β levels in sera of CLP-induced sepsis mice treated with rSj-Cys compared with CLP group without treatment (Fig. 3A). The IL-10 and TGF-β levels were significantly lower in CLP-induced sepsis mice than that in sham surgery or sham + rSj-Cys mice (ANOVA: F(3,23) = 9.032, P < 0.0006; F(3,23) = 9.789, P < 0.0004) (Fig. 3A). The mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-6) and regulatory cytokines (IL-10 and TGF-β) detected in hear tissues showed the similar pattern as measured in sera (Fig. 3B). The results suggested that CLP-induced sepsis mice stimulated the secretion of pro-inflammatory cytokines (TNF-α and IL-6), but inhibited the regulatory immune pathway (lower IL-10 and TGF-β). Treatment of rSj-Cys was able to significantly inhibit the activation of pro-inflammatory pathway possibly through activating regulatory immune pathway. Interestingly, the mRNA level of M1 macrophage marker iNOS was significantly reduced and M2 macrophage maker Arg-1 significantly increased in heart tissues of rSj-Cys-treated sepsis-mice (ANOVA: F(3,11) =4.967, P < 0.0311; F(3,11) = 77.27, P < 0.0001) (Fig. 3B), indicating the more macrophages were shifted from M1 to M2, the alternatively activated macrophages, after being treated with rSj-Cys.
The inhibitory effect of rSj-Cys on LPS-induced inflammatory response in H9C2 cardiomyocytes
LPS is blamed to be the major component to cause cardiac dysfunction in sepsis through inducing the innate immune inflammatory response [32]. In this study, we identified that LPS significantly induced H9C2 cardiomyocytes to release pro-inflammatory cytokines IL-6 and TNF-α (ANOVA: F(3,11) =20.78, P < 0.0004; F(3,11) =18.53, P < 0.0006), but inhibited the release of regulatory cytokines TGF-β and IL-10 (ANOVA: F(3,11) =25.67, P < 0.0002; F(3,11) =14.41, P < 0.0014) compared with cells treated with PBS (Fig. 4A). After being treated with rSj-Cys, the LPS-induced IL-6 and TNF-α were reduced to the level of medium control (ANOVA: F(3,11) =20.78, P < 0.0004; F(3,11) =18.53, P < 0.0006), and TGF-β and IL-10 were significantly boosted compared to cells without rSj-Cys treatment (ANOVA: F(3,11) =25.67, P < 0.0002; F(3,11) =14.41, P < 0.0014) (Fig. 4A). The rSj-Cys alone has no significant effect on the innate immune response of normal cardiomyocytes.
rSj-Cys reduced LPS-induced cardiomyocyte apoptosis.
To further determine whether rSj-Cys reduced LPS-induced cardiomyocyte apoptosis, H9C2 cells were incubated with LPS alone or with rSj-Cys. The flow cytometry results revealed that incubation with LPS induced 14.4% H9C2 cells apoptosis, whereas co-incubation with rSj-Cys significantly reduced LPS-induced apoptosis to the similar level without LPS (ANOVA: F(3,11) =22.68, P < 0.0003) (Fig. 4B). There was no remarkable difference in apoptotic rate between rSj-Cys alone group and blank control group.
rSj-Cys suppressed the activation of MyD88 in LPS-stimulated H9C2 cells in vitro and CLP-induced cardiac tissues in vivo
MyD88 is a crucial molecule involved in the inflammatory TLR signaling pathway. To determine if MyD88 is involved in the therapeutic effect of rSj-Cys on the sepsis-induced inflammation and damage of cardiomyocytes, we detected the expression of MyD88 in heart tissue of mice with CLP-induced sepsis treated with rSj-Cys in vivo, and in LPS-stimulated H9C2 cells co-incubated with rSj-Cys in vitro. The elevated level of MyD88 was observed in cardiac tissue 12 h after CLP surgery compared with sham surgery control (ANOVA: F(3,11) =8.823, P < 0.0064) (Fig. 4C). Treatment with rSj-Cys significantly reduced the expression of MyD88 in cardiac tissue of sepsis mice compared with mice without treatment (ANOVA: F(3,11) =8.823, P < 0.0064) (Fig. 4C). There was no effect of rSj-Cys on the expression of MyD88 in normal mice (sham control). At the similar pattern, the expression of MyD88 was increased in cardiomyocytes incubated with LPS for 24h. Co-incubation with rSj-Cys significantly suppressed the expression of MyD88 in LPS-stimulated cardiomyocytes (ANOVA: F(3,11) =8.550, P < 0.0071) (Fig. 4D).