| Background Sepsis is a systemic response to infection, and may lead to septic shock which is one of the primary causes of death in the intensive care unit in many countries. The mortality would be much higher in septic patients who developed septic shock. LPS, a component of the outer membrane of Gram-negative bacteria, induces expression of tumor necrosis factor cardiomyocyte TNF-a production, which is responsible for myocardial depression induced by endotoxemia. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. During endotoxemia, LPS can cause excessive oxidative stress. In response to LPS, NADPH oxidase 2(Nox2)-derived ROS activates ERK1/2 and p38 MAPK by phosphorylation, leading to proinflammatory cytokine production and cardiac dysfunction. Thus, inhibition of local inflammation and oxidative stress may prevent endotoxin-induced cardiac dysfunction. Tanshinone ⅡA (Tan ⅡA), which is a member of the major lipophilic components extracted from Salvia miltiorrhiza, has been shown possessing anti-inflammatory and anti-oxidative properties. TⅡA has been clinically used in Asian countries for the prevention and treatment of ischemic heart disease. In 2014, the issue of Science Translational Medicine reported that Tanshinone ⅡA potently induced inflammation resolution in vivo both by induction of neutrophil apoptosis and by promoting reverse migration of neutrophils. Pharmacologic pretreatment with TⅡA can significantly reduce lethality in LPS-treated mice and attenuate LPS-induced acute lung injury. Therefore, it is possible that TⅡA will attenuate heart dysfunction through inhibition of the NADPH oxidase2-mediated inflammatory responses during endotoxemia. To test this hypothesis, we pretreated mice with TⅡA followed by LPS challenge. We observed that TⅡA pretreatment significantly improved cardiac dysfunction following LPS challenge. The mechanisms by which TⅡA attenuated cardiac dysfunction in endotoxemia may involve downregulation of the NADPH oxidase2-related ERK1/2 and p38MAPK signaling pathway.Methods 1. In vivo study. Neonatal cardiomyocytes were prepared from mice born within 24 h. After 48 h of cell culture, cardiomyocytes were incubated with various concentrations of TⅡA (0.1,1,10 μM) for 24 h followed by LPS stimulation for 4 h. The supernatants were collectedand and the concentrations of TNF-a were measured by ELISA.2. Adult mice (male, aged 2-3 months) were treated with LPS (10 mg/kg, i.p.) to induce endotoxemia. After adjusting to the environment, mice were randomly divided into four groups:CON, TⅡA, LPS and LPS+TⅡA groups. In the LPS and LPS+TⅡA groups, mice received 10 mg/kg LPS intraperitoneally to induce endotoxemia, mice in the other two groups received saline instead of LPS in the same manner and served as controls. To evaluate the effects of TⅡA on LPS-induced cardiac dysfunction, mice were pretreated with TⅡA 10 mg/kg intraperitoneally once a day for consecutive 3 days. In all groups, measurements were made at 1 h or 6 h after LPS or saline administration.3. Survival study.30 mice were also randomized in LPS and LPS plus TⅡA groups (n=15), the animals were carefully monitored for 120 hours for mortality assessment.Results1. Treatment with the indicated concentrations of TⅡA for 24 h did not affect the viability. LPS (10 μg/mL,4 h) significantly induced TNF-a secretion (164.0±1.73 pg/mL). TⅡA remarkably inhibited LPS-induced TNF-a production in a dose-dependent manner in cultured cardiac myocytes.2.LPS-induced sepsis in mice produced a significantly higher mortality compared with CON mice (53.3% vs.13.3%). TⅡA administration was able to increase the survival rate by 40% in treated animals and reduced mortality in LPS challenged mice (P< 0.05).3. At 6 hours after challenge, LPS administration dramatically decreased ejection fraction (EF%) by 48.3%, fractional shortening (FS%) by 41.3%, stroke volume (SV) by 58.16%, and CO by 66.1% respectively, when compared with saline-treated control mice (P< 0.05). However, the LPS-induced depression of cardiac systolic function was significantly attenuated by TⅡA. TⅡA pretreatment increased EF% by 42.3%, FS% by 30.1%, SV by 60.94% and CO 77.6%, when compared with the LPS alone group (P< 0.05).4. Myocardial TNF-α and IL-1β concentrations were extremely low in mice from control and TⅡA alone groups. LPS induced a significant increase in TNF-α and IL-1β concentrations at 6 h after LPS administration, which was inhibited by pretreatment with TⅡA (P< 0.05).5. LPS treatment was associated with a significant increase in MDA expression compared to saline treated mice. In contrast, TⅡA pretreatment resulted in an attenuation of the MDA level by 20.9% compared with LPS-treated mice without TⅡA administration. The cardiac SOD activity was decreased in the LPS group when compared with the control group, whereas pretreatment with TⅡA significantly elevated SOD activity by 78.3%(P<0.05). Compared with LPS group, GSH-Px activity in LPS+TⅡA group tended to increase, but the difference was not significant (P> 0.05).6. The iNOS protein was extremely low in control mice. In contrast to control mice, LPS administration significantly increased cardiac iNOS expression (P<0.05). Interestingly, TⅡA pretreatment decreased the LPS-induced iNOS upregulation by 39.6% compared with LPS-treated mice that did not receive TⅡA (P<0.05). On the other hand, LPS challenge resulted in a significant decrease in eNOS level by 47.3% compared with control mice. However, TⅡA administration markedly increased cardiac eNOS expression by 65.0% in LPS-challenged mice as compared to vehicle treated mice (P<0.05).7. A marked increase in myocardial ROS production was also observed in the LPS group compared with control group, which was inhibited by pretreatment with TⅡA. Western blot analysis showed that the protein expression of Nox2 was significantly upregulated in LPS-treated mice. However, pretreatment of TⅡA markedly reduced the increase in Nox2 expression induced by LPS. LPS treatment led to a significant increase in phosphorylation levels of ERK1/2 and p38 MAPK. Pretreatment with TⅡA effectively reversed the LPS-induced increases in ERK1/2 and p38 phosphorylation.Conclusion TⅡA played a crucial role in the prevention of LPS-induced cardiac dysfunction in mice and in the inhibition of inflammatory response. TⅡA protected cardiac function against LPS challenge and the mechanisms may involved the downregulation of Nox2-related ERK1/2 and p38MAPK signaling pathway. |
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