Jujuboside A Attenuates Sepsis-Induced Cardiomyopathy By Inhibiting Inammation and Regulating Autophagy

Background: Jujuboside A (JuA), as a main effective component of Jujubogenin, which was extracted from the seed of Ziziphus jujuba Mill, has long been known as a sedative-hypnotic drug. The aim of the current study was to investigate the potential effect of JuA on sepsis-induced cardiomyopathy (SIC) induced by lipopolysaccharide (LPS) in mouse models. Method: Wide type C57BL/6J mice were randomly divided into four groups: ddH 2 O+control, ddH 2 O+JuA, LPS+NS and LPS +JuA, and the cardiac function of septic mice were detected by echocardiography. Moreover, the survival rate at each time point was calculated for 7 days. ELISA assays were used to analyze inammatory factors in serum. Furthermore, Western blotting, ow cytometry and TUNEL staining were performed to assess cell apoptosis and transmission electron microscopy detecting the number of autophagosomes. Finally, the expression of autophagy-related and oxidative stress-related proteins was analyzed by western blotting and immunohistochemistry staining. Results: Results showed that JuA pretreatment signicantly improved the survival rate and cardiac function, and suppressed systemic inammatory response in septic mice. Further study revealed that JuA could decrease cell apoptosis and enhanced autophagy. Moreover, JuA pretreatment also signicantly decreased oxidative stress and nitrodative stress, as evidenced by downregulating iNOS and gp91 expression in vivo. In addition, the autophagy inhibitor 3 ‐ MA signicantly abolished the effect of JuA on autophagic activity in SIC. Conclusion: In conclusion, the ndings indicated that JuA enhanced autophagy blocking inammasome-mediated cardiomyocyte apoptosis and suppress myocardial iNOS and gp91 expression to improve cardiac function of SIC in septic mice.


Introduction
Sepsis, which is a common condition often caused by a dysregulated host response to infection, can lead to life-threatening organ dysfunction as reported previously [1,2]. Severe sepsis and septic shock are major healthcare problems of morbidity and mortality worldwide annually [3,4]. Sepsis-induced cardiomyopathy (SIC) is a global but reversible cardiac dysfunction caused by sepsis and has long been a research hotspot in the pathogenesis of sepsis [5][6][7], such as dysregulation of in ammatory mediators, oxidative stress, mitochondrial dysfunction, autonomic nervous system dysregulation et al. [8][9][10].
Autophagy is the major intracellular degradation system, which maintains protein quality and organelle function by degrading the damaged or dysfunctional cellular components such as endoplasmic reticulum, mitochondria and peroxisomes, as well as eliminating intracellular pathogens in the lysosome [11,12]. Autophagy is a process of self-degradation, which that is important for balancing energy sources during critical periods of development and in response to nutritional stress [13][14][15].
Autophagy has been demonstrated to be involved in numerous physiological processes, such as the starvation response, cell growth control and innate immunity [16][17][18]. However, the purpose of autophagy is not simply to eliminate materials, but instead as a dynamic recycling system that generates new building blocks and energy for cellular repair and homeostasis. Previous studies have found that autophagy is associated with apoptosis and autophagy is mobilized in the early stage of sepsis through increased accumulation of autophagic vacuoles and increased expression of autophagy-associated proteins [19][20][21][22][23][24].
Jujuboside A (JuA) is a main effective component of Jujubogenin, isolated from the seed of Ziziphus jujuba Mill var spinosa (Bunge) Hu ex H F Chou (Ziziphus), and traditionally used as a folk medicine, due to its anxiolytic and sedative effects [25][26][27]. Recently, previous studies have reported that JuA have the notable neuroprotective activities in dementia diseases such as Alzheimer's disease via antiin ammation, anti-apoptosis, anti-oxidant and neuro-protection [26,[28][29][30]. However, there was almost rarely study showed the potential effect of JuA on cardiac disease. Han et al. indicated that JuA can signi cantly reduce the damage of isoproterenol to H9c2 cells via activating the phosphoinositide 3kinase/AKT/mammalian target of the rapamycin pathway [31]. In addition, Wan et.al reported that pretreatment with JuA can reverse norepinephrine-induced decrease in cell viability and increase in H9c2 apoptosis by regulating the MAPK and AKT signaling pathways [32]. However, to the best of our knowledge, the above two researches were only focused attention to H9c2 cells but not animal disease model; moreover, whether JuA has the potential therapeutic effect on SIC has not been explained, or/and the underlying mechanism remains to be resolved.
This study aims to examine the protective effects of JuA on SIC and to clarify the underlying mechanism of JuA. In the present study, we demonstrated that JuA signi cantly attenuated the cardiac function and reduced the in ammatory response in SIC. JuA treatment effectively weaken the apoptosis through enhancing the autophagy of myocardium, involving in suppressing oxidative and nitrosative stress in sepsis-mice model.

Animal, Sepsis model and treatment
Wild-type (WT) C57BL/6 male mice (8-10weeks old, 20-25g) were purchased from Slac Laboratory (Shanghai, China) and were fed a standard rodent diet with free access to food and tap water. All experimental procedures were in accordance with the guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (8th Edition, 2011), and all protocols were approved by the Institute's Animal Ethics Committee of Shanghai Jiao Tong University. All the mice were kept at constant temperature (21 ± 2°C) in a light and dark cycle with relative humidity of 50±5%, and free access to food and water. The experimental mice were randomly divided into 4 groups (10 mice To investigate the potential effects, mice were given JuA (20mg/kg, 0.2ml, p.o.) or the equal volume of saline (0.2ml, p.o.) for consecutive 7 days. On the last day, all the mice were given a single intraperitoneal injection of LPS (20mg/kg) or the same volume of saline to mimic sepsis-induced cardiomyopathy. After 12 hours, cardiac function was detected by M-mode echocardiography and all the mice were sacri ced to harvest hearts.
The survival rate was observed using other 20 mice in each group. These LPS/saline-induced mice were returned to their cages after JuA/NS-treatment and were closely monitored for up to 7 days, as well as given ad libitum access to food and water.

Cell Lines and Cell Culture
The cardiomyoblast cell line H9c2 was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modi ed Eagle's medium supplemented with 10% fetal bovine serum (Gibco, CA, USA) and antibiotics (100 U/mL penicillin and 100 U/mL streptomycin) and maintained at 37°C in 5% CO2. And the cells were passaged every 2-3 days to maintain growth. For in vitro treatment, the cells were pretreated with JuA (0 µ M and 20 µ M) or an equal volume of PBS for 1 h, and then were treated with LPS (20µg/mL) for 24 h. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany) according to the manufacturer's instructions to assess apoptosis in heart tissues. Diphenyl phenylindole (DAPI) staining was used to stain the cell nucleus. The apoptosis index was de ned as the number of TUNEL-positive myocytes/the total number of myocytes stained with DAPI. Images were obtained using the Leica laser uorescence microscope at a magni cation of ×200 and analyzed with ImageJ software (ImageJ version 1.43r; NIH).

Transmission electron microscopy (TEM)
The heart tissues were harvested to determine the quanti cation of autophagosomes. The heart tissues were xed with 2.5% glutaraldehyde and stored at 4°C. According to the manufacturer's instructions, each specimen was performed by a series of manipulation, including xation, dehydration, embedding, curing, biopsy, and dyeing. The autophagosomes were observed by a transmission electron microscope (JEOL, Tokyo). Random sections were imaged and analyzed by two technicians blinded to the experiment. The mean number (and standard deviation) of autophagosomes per eld was calculated.

Cytokine measurement
At the predetermined time points, these mice were anesthetized by iso urane, the plasma samples were obtained from blood after centrifugation for 15 minutes at 3000g at 4°C, and stored at -80°C until use in various biochemical assays. The concentrations of TNF-α, IL-6, IL-1β, IL-18 were measured by ELISA kits according to the manufacturer's instructions (Sangon Biotech, Shanghai, China). OD450 was calculated by subtracting the background, and standard curves were plotted. Values are expressed as pg/ml of total protein.

Flow Cytometry
Flow cytometry was used to determine the apoptosis rate in LPS-induced H9c2 cells. Apoptotic cells were differentiated from viable or necrotic cells by the combined application of annexin V (AV)-uorescein isothiocyanate (FITC) and propidium iodide (PI) according to the manufacture of the Annexin V-FITC cell apoptosis kit (C1062, Beyotime Biotechnology). Cells were washed twice and adjusted to a concentration of 10 6 cells/ml. AV-FITC (5 µl) and PI (10 µl) were added to 195 µl of binding buffer in each sample and incubated for 20 min at room temperature in the dark. Then without washing, samples were analyzed by using ow cytometry. Each experiment was performed at least in three independent experiments.

Quantitative reverse transcription PCR (q-PCR) analysis
Total RNA of myocardial tissues was isolated using TRIzol Reagent (Invitrogen). The Concentration of mRNA was determined using absorbance at 260 and 280 nm. The sample RNA was reversely transcribed to cDNA with reverse transcription reagent kit (Takara BIO) according to the manufacturer's instructions. q-PCR was performed using a SYBR® Premix Ex TaqTM Perfect Real Time Kit (Takara BIO) in the 7300 System SDS Software (Roche Applied Science). The primer sequences utilized for real-time PCR are presented in Table S2. Results from 7300 System SDS Software are presented as Ct values, normalized against GAPDH, and shown as 2 −∆∆Ct .

Cell viability assay
Cell Counting Kit-8 (CCK8) was applied to assess the number of viable cells. Cells were seeded in a 96well plate at a concentration of 1 × 105 cells/mL, and were allowed to acclimatize overnight. and then cultured in complete medium in the presence or absence of LPS and JuA (20µM). The cells were pretreated with various concentrations of the JuA (0 µM to 20 µM) for 1 h, and then treated with LPS 20 µg/mL for 24 h. The OD450 was tested at 3h, 6h, 12h, 24 h using a microtiter plate reader (SkanIt Software 5.0, RE, ver. 5.0.0.42). The results were calculated using the following formula: Cell viability = (Treatment Group OD − Blank Group OD)/ (Control Group OD -Blank Group OD) [33].

Statistical analysis
All data are presented as mean ± standard deviation at least three experiments. Statistical analysis was performed using SPSS software (SPSS Inc., Chicago, IL). Unpaired t-tests were used to determine the statistical differences between 2 groups in each analysis. A one-way analysis of variance (ANOVA) was used for multiple comparisons. Survival was analyzed with Kaplan-Meier survival curves and compared with the log-rank test. A p value of less than 0.05 was considered signi cant. P<0.05 was considered to be statistically signi cant.

JuA treatment attenuated LPS-induced cardiac dysfunction and ameliorated survival rate in sepsis mice
To investigate whether JuA treatment protected against sepsis, mice were received JuA (20mg/kg, p.o.) for 7days before LPS injection in LPS+JuA group. Subsequently, cardiac function of these sepsis mice was detected by echocardiography as shown in Figure1. As expected, mice injected LPS for 12 hours displayed signi cant cardiac dysfunction with decreased LVEF, LVFS and increased LVESV, LVEDV, LVESD, LVEDD compared with that in the control group. Conversely, pre-treatment with JuA signi cantly improved cardiac function in LPS-treated mice by increased LVEF, LVFS and decreased LVESV, LVEDV, LVESD, LVEDD compared with the cardiac function of those with saline-treated sepsis mice ( Figure 1A-E).
Furthermore, the survival rate was analyzed in LPS+saline group and LPS+JuA group. The mortality rate in the LPS group was 50-70%. Signi cantly, pre-treatment (20mg/kg by p.o. for 7 days) with JuA improved survival rate of sepsis mice ( Figure 1F), compared with the group of LPS + NS. Therefore, these results showed that early treatment of JuA in sepsis could promote the cardiac function and prognosis of these septic mice.

JuA treatment suppressed cardiac in ammation and apoptosis in LPS-induced sepsis mice
To investigate the protective effects of JuA in vitro, further experiments were performed to examine the effects of JuA on the in ammation in septic mice. ELISA showed that the level of the pro-in ammatory cytokines TNF-α, IL-1β and IL-18 were increased in the LPS-treated mice compare with the control group, which on the contrary were largely reduced in LPS + JuA group by JuA treatment (Figure 2A). Moreover, JuA treatment signi cantly inhibited the mRNA expression of TNF-α, IL-1β and IL-18 in the heart of LPStreated mice ( Figure 2B), whilst elevated levels of IL-10, which was also re ected by decreased pathology scores compared with saline-treated mice ( Figure 2B). Furthermore, to evaluate the effects of JuA on cardiac apoptosis, a TUNEL assay was performed ( Figure 2C). Signi cantly, there were more TUNEL-positive cardiomyocytes detected in the heart of LPS-treated mice than that of the control group, in contrast, lower percentages of apoptotic cells were found in LPS + JuA group compared with the LPS+NS group ( Figure 2C). Western blotting showed that the levels of cleaved caspase-3 were signi cantly increased in the group of LPS-treated mice compared with the control group, but decreased in the LPS + JuA group ( Figure 2D). Further detection also revealed that JuA pretreatment downregulated the expression of Bax but promoted Bcl-2 induced by LPS( Figure 2D). These results demonstrated that pretreatment with JuA had a cardioprotective effects against in ammation and apoptosis in sepsis mice.

JuA treatment reduced LPS-induced in ammatory response and apoptosis in H9c2 cells
Considering the protective effects of JuA in vivo, we further veri ed the role of JuA in vitro. As shown in Figure 3, H9c2 cells were used to explore the protective effect of JuA on LPS-induced cardiomyocyte damage. Apoptosis in H9c2 cells was detected by ow cytometry, which showed that JuA pretreatment could decrease H9c2 cell apoptosis with LPS condition ( Figure 3A). Western blot also con rmed that JuA not only decreased Bax expression but also increased Bcl2 expression in LPS-treated H9c2 (Figure3B). In addition, when LPS decreased cell viability in vitro, JuA pretreatment could block this effect (Figure3C). Immuno uorescence showed that JuA could also prevent LPS-induced cardiomyocyte apoptosis in vitro according to TUNEL staining (Figure3D). These results demonstrated that JuA could suppress the apoptosis induced by LPS-treatment.

JuA enhances cardiomyocyte autophagy in LPS-induced sepsis mice
Increasing evidence have found that autophagy plays a critical role in cell repairment in sepsis and impairment of autophagy may contribute to myocardial dysfunction and trigger apoptotic death in cardiomyocyte of sepsis [34]. We next investigated whether JuA could induce autophagy, which in turn contributed to the therapeutic effects of JuA on LPS-induced myocardial injury. As shown in Figure 4A, TEM analysis observed more autophagosomes and messy myocardium in the LPS group compared with that in the control group. And JuA treatment further increased the number of autophagosomes and attenuated the structure of myocardium in the heart of septic mice. Meanwhile, western analysis revealed that JuA pretreatment promoted autophagy-related protein Beclin1 and LC3II expression, while decreased the expression of p62 protein in the heart of LPS-induced sepsis mice compared with the control group ( Figure 4B-E), revealing the activation of autophagy in response to myocardial injury caused by LPS and JuA. These results demonstrated that JuA could enhance autophagy pathway to attenuate myocardial injury.
JuA treatment downregulated the production of iNOS and gp91 in the heart of LPS-induced septic mice Oxidative damage was known as consequences of sepsis and may contribut to myocardial dysfunction. To explore the mechanisms underlying the potential role of JuA against LPS-induced sepsis, we subsequently assessed JuA in regulating sepsis-induced oxidative stress and nitrosative stress. Compared with the LPS group, mice pretreatment with JuA displayed a reduction of iNOS (a mediator of endoplasmic reticulum-stress apoptosis pathway) in the heart of sepsis mice ( Figure 5A-B). Moreover, western blot analysis con rmed that JuA signi cantly downregulatd the expression of gp91phox (the major component of NADPH oxidase responsible for the generation of superoxide anions) in LPSchallenged mice (P < 0.01, Figure 5A-C).
Furthermore, immunohistochemistry staining in the heart of septic mice indicated that JuA could decrease the expression of iNOS and gp91phox ( Figure 5D). In all, these results demonstrated the attenuation of myocardial injury by JuA may due to the inhibited role against oxidative stress, which facilitated cardiomyocyte survival and improved cardiac function.
Inhibition of autophagy reversed the suppressive effect of JuA on LPS-induced in ammation and apoptosis in sepsis mice To establish the autophagy in the protective role of JuA against sepsis, 3-MA (20 mg/kg) was administered intraperitoneally 6 h before LPS injected. However, inhibition of autophagy with 3-MA pretreatment reversed the protective effect of JuA on LPS-induced cardiomyocyte in ammation, as evidenced by the pathways involving apoptosis and oxidative stress and nitrosative stress. As shown in

Discussion
In this study, we found that JuA, a main effective component of jujubogenin extracted from the seed of Ziziphus jujuba Mill var spinosa (Bunge) Hu ex H F Chou (Ziziphus) [35,36], signi cantly ameliorates LPS-induced myocardial injury by enhancing autophagy. These results in present study were consistent with Wan et.al, who reported that JuA played a protective role on norepinephrine-induced decreased cell viability and increased apoptosis of H9c2 [37] and Han et al, JuA could notably reduce the damage cause by isoproterenol via promoting the phosphorylation of PI3K, Akt, and mTOR and inhibiting LC3 conversion to attenuate the injury of H9c2 [31], which indicated that JuA may be a potential choice for the treatment of heart diseases.
In the present study, mice were subjected to LPS to construct sepsis animal model. The results of this study showed that the cardioprotective effect of JuA against sepsis-induced myocardial injury was revealed by ameliorating the cardiac function and reducing levels of the in ammatory cytokines TNF-α, IL-1β and IL-18, as well as alleviating cardiomyocyte apoptosis in vitro and in vivo. Furthermore, our results demonstrated that JuA increases autophagic activity in the heart of sepsis mice. However, this action of JuA was abolished by 3-MA in cardiomyocytes treated with LPS, suggesting that regulating autophagy by JuA may be essential in maintaining cardiac function in response to sepsis injury. Furthermore, the cardioprotective effects of JuA against sepsis-induced cardiomyopathy were associated with oxidative stress and nitrodative stress signaling was strongly involved in these protective effects.
JuA is a natural product isolated from the seeds of Zizyphus jujuba, possessing numerous biological effects [31], which is widely available for treating symptoms of insomnia and anxiety [36,38]. Previous studies have reported that JuA exerts anti-injury effects, cardioprotective and neuroprotective activity via anti-in ammatory and antioxidative effects in animal models of dementia in vivo [29,31,39]. As reported, sepsis-induced cardiac dysfunction is a complication of severe sepsis and septic shock characterized by an invertible myocardial depression [40,41], as evidenced by the major hemodynamic characteristics with decreased ejection fraction and LV systolic dysfunction [42]. The possible bene cial effects of JuA on sepsis-induced cardiac dysfunction have not yet been fully demonstrated. In this study, the results detected from CCK8 assay indicated that JuA (0-20 µM) did not exhibit a cytotoxic effect on H9c2 cells. Moreover, Zhu et al. reported that JuA effectively reversed the decreased cell viability caused by norepinephrine and reduced norepinephrine-induced H9c2 cell apoptosis [37]. Consistently in the present study, we found that pre-treatment with JuA in mice with SIC behaved with increased LVEF, LVFS values and decreased LVESV and LVEDV values by echocardiography. To the best of our knowledge, the present study is the rst to report that JuA was signi cantly attenuated the cardiac function in mice with SIC.
Notably, the in ammatory response is the initial process in the hallmark development of SIC [43,44]. Mayer et al. reports that excessive levels of releasing pro-in ammatory mediators result in the in ammatory response in sepsis, while the compensatory anti-in ammatory reaction fails to suppress the immune response, resulting an imbalance between pro-in ammatory response and anti-in ammatory response occurred during infection [45,46]. TNF-α, IL-1 and IL-6 are the main in ammatory mediators which could lead to myocardial depression in sepsis [47]. Accumulating studies have reported that treatment with anti-in ammatory antibodies could improve cardiac function in patients with septic shock [48,49]. In this study, the in ammatory factors of TNF-α, IL-1β and IL-18 were increased in the serum of septic mice and in the supernatant of LPS-stimulated H9c2. Moreover, JuA pretreatment ameliorated the cardiac dysfunction of SIC by reducing the expression of in ammatory factor both in vivo and in vitro, suggesting that the cardioprotective effects of JuA are in connection with its anti-in ammatory activity in septic mice, which was consistent with the previous reported studies [50,51]. Furthermore, we rstly demonstrated that JuA exhibited inhibitory effects on LPS-induced in ammation and possessed the potentially protective role in the treatment of LPS-induced sepsis.
In the present study, C57BL/6 mice and H9c2 cells were exposed to LPS to establish myocardial toxicity models, and apoptosis has been extensively implicated as a determining process in myocardial depression of SIC [34,52]. Previous studies have found that JuA may protect against norepinephrine-induced apoptosis of cardiomyocytes via modulation of the mitogen-activated protein kinase and AKT signaling pathways [37]. Positively, we found that JuA effectively reversed the decreased cell viability caused by LPS and inhibited apoptosis induced by LPS both in vivo and in vitro, as evidenced by decreased TUNEL-positive cardiomyocytes by TUNEL staining and decreased H9C2 cell apoptosis detected by Flow Cytometry. In addition, the expression of caspase enzymes and Bax/Bcl-2 ratio was measured to demonstrate the molecular basis of the antiapoptotic effects of JuA. JuA treatment attenuated the expression of cleaved caspase-3 and decreased Bax/Bcl-2 ratio both in vivo and in vitro. These data indicate that JuA ameliorated sepsis-induced myocardial dysfunction by reducing myocardial apoptosis both in vivo and in vitro.
Another signi cant nding was that JuA could enhance autophagy in the heart of LPS-induced sepsis mice. Previous studies have shown that autophagy, a primary mechanism for maintaining cellular homeostasis, plays an important role in the regulation of sepsis [21,53,54]. Autophagy is de ned as a major intracellular degradation system, promoting cellular survival by controlling the degradation of proteins and organelles, including the formation of double-membraned autophagosomes and proteolytic degradation after delivery to lysosomes [55,56]. Many previous studies have shown that autophagy possessed an important role in the process of myocardial dysfunction, such as ischemia/reperfusion injury or cardiac hypertrophy [57][58][59][60]. Moreover, apoptosis has been extensively regarded as the decisive process in SIC [61-63] and activation of autophagy has been initially observed in sepsis, followed by a subsequent stage of myocardial impairment [64-66]. In the present study, autophagy was activated in SIC and the LPS-treated H9c2 cells, as revealed by the increasing number of autophagosomes in vivo, the aggravation of cell apoptosis in vitro, and the increased expression of LC3II and Beclin1, decreased expression of p62 consistent with previous studies [67-69]. Recent investigations suggest that appropriate autophagy modulation has a potential role to improve cardiac function by regulating mitochondria and attenuating in ammation in SIC; moreover, the role of autophagy during the pathogenesis of sepsis has been under intensive exploration in recent years [62,65,66]. Previous studies have shown that multiple medicine and bioactive molecules exert cardioprotective effects by regulating autophagy in sepsis [70][71][72][73][74][75]. In this study, we explored the potential role of JuA in SIC. Western blotting obtained from the present study demonstrated that autophagic activity was enhanced by JuA pretreatment both in vivo and in vitro, as shown by increasing the expression levels of LC3II and Beclin1, decreasing SQSTM1/p62 expression. Moreover, TEM data illustrated the number of autophagosomes in the heart of septic mice were signi cantly decreased followed by JuA treatment. 3-Methyladenine (3-MA), an autophagy inhibitor, exerts a cardioprotective effect in a lethal model of murine endotoxemia and polymicrobial sepsis by inducing autophagy [76,77]. In our study, we found that sepsis mice treated with 3-MA reversed the JuA-induced upregulation of autophagy, accompanied by an increase in the expression of Bax/Bcl-2 ratio and cleaved caspase3, indicating that JuA treatment may play a protective role in sepsis by increasing autophagy.
The increasing evidence has indicated that sepsis-induced cardiac injury was a consequence of uncontrolled in ammation [78], mitochondrial dysfunction [79,80] . To investigate the mechanisms underlying the potential effect of JuA on LPS-induced sepsis, this study subsequently demonstrated JuA in regulating sepsis-induced oxidative stress and nitrosative stress. As expected, this study demonstrated a potentially protective role of JuA in the pathophysiology of SIC by suppressing the expression of iNOS and gp91, acting by western blot and immunohistochemistry in the heart of sepsis mice and LPS-induced cardiomyocytes. In addition, the data in this study identi ed a potentially novel mechanism that links autophagy with abnormal oxidative stress and myocardial dysfunction. Importantly, autophagy inhibition (3-MA) neutralized the effect of JuA in ameliorating oxidative stress revealed by the expression of iNOS and gp91 and increasing the apoptosis in the heart of sepsis mice. Taken together, it can thus be suggested that the suppression of oxidative stress in myocardium by JuA could associate with the autophagy of cardiomyocyte, which might contribute to the alleviation of myocardial dysfunction during sepsis.

Limitation
However, there are still several limitations exist in this study. Firstly, sepsis-induced myocardiopathy was established by injected LPS intraperitoneally, but mice with cecal ligation and puncture (CLP)-induced sepsis was not constructed in this study. Secondly, the H9C2 rat cardiomyoblast cell line were used in in vitro experiment. However, whether H9C2 cells can accurately mimic the hypertrophic responses of primary cardiomyocytes has not yet been fully determined. Furthermore, H9c2 cells and primary neonatal mouse cardiomyocytes have the functions of differentiation and proliferation, while the adult mouse cardiomyocytes are terminally differentiated cells and have no differentiation and proliferation function at the same time [87]. H9c2 cells can't fully mimic the primary neonatal mouse cardiomyocytes and the adult cardiomyocytes. Furthermore, ROS level in the myocardium should be detected to further clarify the oxidative stress induced by sepsis. In addition, this study did not investigate the effect of JuA at multiple time points or in in vitro cell models. Since the current experimental settings may have also produced bias in the results, further extensive research is needed before a reliable conclusion can be drawn. These problems should be considered and resolved in future experiments.