Xuebijing injection protects against sepsis-induced myocardial injury by regulating apoptosis and autophagy via mediation of PI3K/AKT/mTOR signaling pathway in rats

Objective: Apoptosis and autophagy are significant factors of sepsis induced myocardial injury (SIMI). XBJ improves SIMI by PI3K/AKT/mTOR pathway. Present study is devised to explore the protective mechanism of XBJ in continuous treatment of SIMI caused by CLP. Methods: Rat survival was first recorded within 7 days. Rats were randomly assigned to three groups: Sham group, CLP group, and XBJ group. The animals in each group were divided into 12 h group, 1 d, 2 d, 3 d and 5 d according to the administration time of 12 hours, 1 day, 2 days, 3 days or 5 days, respectively. Echocardiography, myocardial injury markers and H&E staining were used to detect cardiac function and injury. IL-1β, IL-6 and TNF-α in serum were measured using ELISA kits. Cardiomyocyte apoptosis was assayed by TUNEL staining. Apoptosis and autophagy related proteins regulated by the PI3K/AKT/mTOR signaling pathway were tested using western blot. Results: XBJ increased the survival rate in CLP-induced septic Rat. First of all, the results of echocardiography, H&E staining and myocardial injury markers (cTnI, CK, and LDH levels) showed that XBJ could effectively improve the myocardial injury caused by CLP with the increase of treatment time. Moreover, XBJ significantly decreased the levels of serum inflammatory cytokines IL-1β, IL-6 and TNF-α in SIMI rats. Meanwhile, XBJ downregulated the expression of apoptosis-related proteins Bax, Cleaved-Caspase 3, Cleaved-Caspase 9, Cytochrome C and Cleaved-PARP, while upregulated the protein levels of Bcl-2 in SIMI rats. And, XBJ upregulated the expression of autophagy related protein Beclin-1 and LC3-II/LC3-I ratio in SIMI rats, whereas downregulated the expression of P62. Finally, XBJ administration downregulated the phosphorylation levels of proteins PI3K, AKT and mTOR in SIMI rats. Conclusions: Our results showed that XBJ has a good protective effect on SIMI after continuous treatment, and it was speculated that it might be through inhibiting apoptosis and promoting autophagy via, at least partially, activating PI3K/AKT/mTOR pathway in the early stage of sepsis, as well as promoting apoptosis and inhibiting autophagy via suppressing PI3K/AKT/mTOR pathway in the late stage of sepsis.


INTRODUCTION
Sepsis, a potentially fatal host reaction to infection that causes organ failure, is one of the primary diseases that poses a substantial threat to the health of people worldwide [1]. According to an epidemiological assessment conducted in 2020, sepsis has a global incidence and mortality rate of approximately 6.775% AGING and 1.481%, respectively [2]. A significant sign of disease progression in hospitalized septic patients is sepsis-induced multiorgan dysfunction, which has a pooled hospital incidence of 9.3% [3] and an in-hospital mortality greater than 10% [1]. It has drawn more attention in recent years in the field of Intensive Care Unit (ICU) management among the numerous secondary organ dysfunctions associated with sepsis, and myocardial injury is one of the most frequent complications and a significant cause of death in septic patients [4]. Additionally, myocardial injury, which has incidence of between 10 and 70% [4] and the fatality rate of 70-90% [1], predominates in the pathophysiology of sepsis. Although there have been significant improvements in organ function support, stabilization of microcirculation, and anti-infective therapy in recent years [5,6], there are still no satisfactory medications or therapies for sepsis. As a result, finding new cures to treat SIMI is challenging but essential for managing sepsis patients.
The ingredients in XBJ, a Chinese patent medicine approved for the treatment of sepsis and multi-organ dysfunction in China in 2004 Figure 1A) [7]. XBJ acts in both the early and late stages of sepsis by anti-inflammatory, anti-coagulation, immune regulation, vascular endothelial protection, antioxidative stress and other mechanisms [8]. A key response protein target coverage of 90.16% was found in the target association pattern of the phytoconstituents of XBJ with the causative genes of sepsis, demonstrating the dominancy of XBJ in the treatment of sepsis [9]. However, the underlying therapeutic mechanisms of XBJ on sepsis are still unknown, the specific mechanisms of XBJ in SIMI need to be further investigated. The survival rate of XBJ for SIMI was evaluated by the Kaplan-Meier method followed by the log rank test within 7 days. AGING Sepsis triggers autophagy in multiple organs including the heart [10][11][12]. More and more studies concentrating on autophagy had shown that the activation of autophagy can improve SIMI [13,14]. While, some studies have proposed a novel view that the inhibiting of autophagy protects cardiomyocytes from LPS-induced oxidative stress [15,16], which is in conflict with the previous assertion. What's more, autophagy triggered inflammatory responses and apoptosis in infected cells [17]. Inflammatory imbalance represents the most critical basis of sepsis pathogenesis and occurs throughout the whole process of sepsis [18]. Inflammation has been center-staged in the field of sepsis research in the past. It is now clear that sepsis is a multifaceted host response to pathogens, involving the early activation of both pro-and anti-inflammatory responses, along with major changes in nonimmunologic pathways [1]. Prior research has suggested that the onset of sepsis was intimately connected to immune paralysis brought on by lymphocyte apoptosis [19]. What's even more intriguing is that the notion that the majority of sepsis deaths are actually caused by a severely compromised immune response as a result of the widespread apoptosis of immune effector cells is deemed to be more 'fashionable' than before [20]. Nevertheless, as the research project of apoptosis in the pathophysiology of sepsis progressed, apoptosis of non-immune cells such as hepatocytes [21], renal cells [22], vascular endothelial cells [23], and cardiomyocytes [13,14,24] was also finally discovered. As a consequence, the theory of anti-apoptotic mechanisms underlies a quantity of therapeutic concepts used to treat infectious diseases and inflammatory responses, especially SIMI.
The cell proliferation, autophagy, apoptosis, angiogenesis, epithelial-to-mesenchymal transition, and chemoresistance are all regulated by the PI3K/ AKT/mTOR signaling pathway [25]. According to prior study, blocking the PI3K/AKT/mTOR signaling pathway had a protective effect against SIMI; apoptosis that is regulated by PI3K/AKT/mTOR signaling pathway has also been demonstrated to have a significant role in the pathophysiology of SIMI [26]. The study has shown that the PI3K/AKT/mTOR signaling pathway plays an important role in regulating apoptosis and autophagy [27]. Thus, PI3K/AKT/mTOR signaling pathway-regulated apoptosis and autophagy may provide a novel approach to examining XBJ's action mechanism in the treatment of SIMI.
In this study, a septic rat model was created using CLP, the rats were then given XBJ intraperitoneally, and a series of laboratory tests was conducted. The aim here is to find out how XBJ regulates autophagy and apoptosis mediated by PI3K/AKT/mTOR signaling pathway to enhance SIMI.

XBJ increases the survival rate in CLP-induced sepsis rat model
We conducted a survival test in a rat model of sepsis to determine if XBJ has a protective function in CLPinduced sepsis. As shown in Figure 1C, after CLP induction, the survival rate of rats in CLP group began to decline, reaching 80% on 2d, 60% on 3d, 50% on 5d, 40% on 6d and 30% on 7d; whereas, the survival rate of rats in XBJ group started to decrease to 90% on 3d, 80% on 4d, 70% on 6d and 50% on 7d. The rat survival rate in the CLP group was noticeably lower than those in the Sham group (p < 0.05), and the administration of 10ml/kg XBJ significantly improves the survival of CLP rat (p < 0.05). Our results illustrated that rats exposed to CLP do not develop fatal sepsis within 48 hours and XBJ protected rat from lethal infection with CLP. As a result, this might offer a reasonable starting point for sepsis treatment.

XBJ attenuates the myocardial dysfunction in CLPinduced sepsis rat model
Rats in the CLP group had dilated hearts, as seen on the echocardiographic images, as opposed to rats in the Sham and XBJ group (Figure 2A). According to Echocardiographic findings, up to 12 hours after CLP induction, the decline in cardiac function in septic rats was not statistically significant (p > 0.05) in both the LVEF and LVFS in the CLP group; while there was a significant decrease, which started 1d after CLP (p < 0.05). In septic rats, XBJ significantly enhanced LVEF at 2d, 3d, 5d, and LVFS at 3d, 5d (p <0.05); however, XBJ's effect on LVEF at 12h, 1d, and LVFS at 12d, 1d, 2d was not statistically significant (p > 0.05, Figure 2B, 2C). Overall, the levels of LVEF, and LVFS dropped in the CLP group while the levels of LVEF, and LVFS increased in the XBJ group in a time-dependent way.
To probe the effect of XBJ on sepsis-induced myocardial injury, CLP-induced rats' myocardial tissues were detected by H&E staining. As shown in the Figure 2D, H&E staining revealed that as the septic rats' exposure to CLP was prolonged, the extent of damage to their heart tissue grew progressively more severe. Moreover, the CLP group had noticeable inflammatory cell infiltration compared to the Sham group, interstitial edema, abnormal myocardial cell structure, local necrosis, and indistinct myocardial fiber texture. However, the administration of XBJ obviously ameliorated the foregoing disorder.
These results demonstrated that CLP led to myocardial dysfunction in a time-dependent manner, as well as XBJ improved the myocardial dysfunction in CLP-induced septic rat.

XBJ improves the myocardial markers and inflammation in CLP-induced sepsis rat model
We also measured three myocardial injury markers in serum, cTnI, CK, and LDH, using a fully automated biochemical analyzer to visualize myocardial injury in rats. Overall, the cTnI, CK, and LDH levels in the serum were significantly higher in the CLP group than in the Sham group (p < 0.05), whereas they were markedly lower in the XBJ group than in the CLP group (p < 0.05), with the exception of CK and LDH levels at 12h (p > 0.05, Figure 3A-3C). As a whole, the levels of cTnI, CK, and LDH dropped in the XBJ group while the levels of cTnI, CK, and LDH increased in the CLP group in a time-dependent way.
The inflammatory factors the IL-1β, IL-6 and TNF-α levels in the serum were detected by ELISIA. As depicted in Figure 3D-3F, serum inflammatory factors IL-1β, IL-6 and TNF-α levels were significantly higher in the CLP group than in the Sham group (p < 0.05), whereas they were markedly lower in the XBJ group than in the CLP group (p < 0.05), with the exception of IL-1β at 1d and IL-6 levels at 12h (p > 0.05). These results showed as the septic rats' exposure to CLP was prolonged, the expression level of myocardial inflammatory factors increased in a time-dependent manner, indicating that the degree of myocardial inflammation was gradually aggravated. Additionally, the treatment of XBJ greatly improved the myocardial inflammation in a time-dependent manner.

XBJ ameliorates the cellular apoptosis of heart tissue in CLP-induced sepsis rat model
We measured the expression of the proteins Bax, Bcl-2, Cleaved-Caspase 3, Cleaved-Caspase 9, Cytochrome C, and Cleaved-PARP by western blot and the apoptotic state by TUNEL staining in the myocardium of different groups over the course of five time points (12h, 1d, 2d, 3d and 5d) to explore the effect of XBJ on cardiomyocyte apoptosis during sepsis. We used TUNEL staining ( Figure 4A) to observe that the CLP group's cardiomyocyte apoptosis was clearly apparent when compared to the myocardial structure of the Sham group at the same point of time. Similarly, as the septic rats' exposure to CLP was prolonged, the extent of their cardiomyocyte apoptosis grew progressively more severe.
As shown in Figure 4B, the expression of the proteins Bax, Cleaved-Caspase 3, Cleaved-Caspase 9, Cytochrome C, and Cleaved-PARP were significantly increased, whereas the expression of Bcl-2 was significantly decreased in the myocardium of rat with sepsis as compared with the Sham group at the same point of time. Interestingly, treatment with XBJ significantly reversed the foresaid changes. These results indicated that XBJ alleviated SIMI by promoting apoptosis in the early stage of sepsis, and inhibiting apoptosis in the late stage of sepsis.

XBJ enhances the autophagy of heart tissue in CLPinduced sepsis rat model
Autophagosome development is marked by the protein LC3, and the ratio of LC3 II to LC3 I indicates how many autophagosomes are active (positive correlation). Beclin-1 and LC3-II play the vital role to the promotion of autophagy. On the contrary, P62 is regarded as a negative regulator of autophagic activity. To explore the effect of XBJ on cardiomyocyte autophagy during sepsis, we measured LC3-II/LC3-I ratio and the expression of the proteins Beclin-1 and P62 by western blot in the myocardium of different groups over the course of five time points (12h, 1d, 2d, 3d and 5d). As shown in Figure  5, the expression of the protein Beclin-1 (except for 12h) and LC3-II/LC3-I ratio (except for 12h) were significantly decreased, whereas the expression of P62 was significantly increased in the CLP group as compared with the Sham group at the same point of time. However, treatment with XBJ significantly augmented the expression level of the protein Beclin-1 (except for 12h) and LC3-II/LC3-I ratio (except for 12h), as well as reduced the expression of P62 (except for 12h) in the myocardium as compared with the CLP group. These results revealed that XBJ alleviated SIMI by inhibiting autophagy in the early stage of sepsis, and enhancing autophagy in the late stage of sepsis.
XBJ inhibits the PI3K/AKT/mTOR signaling pathway of heart tissue in CLP-induced sepsis rat model By measuring the levels of PI3K, AKT, and mTOR in cardiac tissues, the molecular mechanism involving the therapeutic function of XBJ through the regulation of apoptosis and autophagy was further studied. As shown in Figure 6A, the expression of the proteins p-AKT, p-PI3K, and p-mTOR were considerably decreased in the myocardium of the CLP-treated rats at 12h compared to the Sham group, while XBJ dramatically increased these proteins' levels in CLPtreated rats at this time. Interestingly, the expression of the proteins p-AKT, p-PI3K and p-mTOR were significantly upregulated in the myocardium of rat with sepsis as compared with the Sham group at the same point of time (1d, 2d, 3d and 5d). But, treatment with XBJ significantly downregulated the expression level of the proteins p-AKT, p-PI3K and p-mTOR in the myocardium as compared with the CLP group (1d, 2d, 3d and 5d).
The results of the immunofluorescence assay also demonstrated that the expression of the proteins p-AKT ( Figure 6B) and p-PI3K ( Figure 6C) were upregulated in the CLP group at the same time as compared to the Sham group, whereas it was downregulated in the XBJ group as compared to the CLP group at the same time. These findings suggested that XBJ can safeguard the myocardium in septic rats by activating the PI3K/AKT /mTOR signaling pathway in the early stages of sepsis and inhibiting it in the latter stages.

DISCUSSION
In the present study, we imitated the septic conditions of human using the rat model of CLP and confirmed that XBJ treatment alleviates myocardial injury in rat sepsis models. The mechanism by which XBJ relieves SIMI is to decrease the release of pro-inflammatory cytokines, attenuate cardiomyocyte apoptosis, and enhance autophagy. XBJ exerted these above effects by inhibiting the phosphorylation of the PI3K/AKT/ mTOR pathway. In Figure 7, the precise mechanism is displayed. Early evidence has demonstrated that XBJ administration improved survival rates and protected against myocardial injury in mice models of sepsis [28]. Our results revealed that the 7 days' mortality of septic rats given XBJ treatment compared favorably to CLP, which is consistent with the previous experiment [29]. In addition, in our study, LVEF and LVFS values were not observed statistically after 12 hour of CLP induction in rats, indicating that cardiac function may not be unimpaired, which may be the result of the compensatory state of the heart in the early stage of sepsis, while it was significantly reduced after 1 day (Figure 2A-2C); H&E staining ( Figure 2D) showed that the degree of cardiac tissue damage induced by CLP was serious; the serum levels of myocardial injury markers cTnI, CK, and LDH ( Figure 3A-3C) were significantly increased in CLP-induced rats. The results showed the rat model of sepsis was successfully established using CLP. Meanwhile, a study showed that XBJ attenuated the myocardial dysfunction/injury and systemic inflammatory response of the CLP-induced septic rat [30]. Another study found that XBJ drastically enhanced cardiac function in septic mice in 24h after CLP [30]. In our study, the outcomes of echocardiography, H&E staining and the serum levels of myocardial injury markers showed that XBJ improved significantly cardiac dysfunction in septic rats after CLP.
What's more, sepsis induces systemic inflammation [31]. It is known from the current literature [32,33] that two pathophysiological processes-exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs)-are closely related to the development of sepsis. During sepsis, the pathogen-associated molecular patterns trigger systemic inflammatory responses, leading to an excess production of proinflammatory cytokines that are responsible for coagulopathy, such as interleukin IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) [34]. Studies have shown inflammation in multiple organs in experimental sepsis induced by LPS or CLP, including the heart, the kidney, the liver, and the lung [35][36][37][38]. Researchers have also consistently observed elevated circulating levels of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α in septic patients [39]. It has been reported that pretreatment with XBJ reduces the serum levels of TNF-α and IL-6 in a dose-dependent manner [40]. In addition, it has been also reported that XBJ effectively reduced circulating IL-1β, IL-6, and TNF-α in CLP rats within 12 h after sepsis [41]. In our study, the plasma levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α were markedly elevated in CLP group rats at 12 hours to 5 days, also demonstrating once again the successful establishment of a CLPinduced sepsis model. However, treatment with XBJ significantly reduced these inflammatory factor levels ( Figure 3D-3F), suggesting that XBJ administration alleviates sepsis-induced systemic inflammation in CLP rats.
Apoptosis that is caused by an excessive inflammatory response plays an essential role in SIMI as well as the pathophysiological process that leads to sepsis [42]. Apoptosis is a tightly regulated form of cell death that is vital in both embryo implantation and development and turnover of tissues during maturation [43]. During sepsis-induced immunosuppression, apoptosis plays a pivotal role in tissues/organs [44]. Numerous fundamental investigations have shown that lowering cardiomyocyte apoptosis can protect against cardiac dysfunction in sepsis [45,46]. Additionally, the key biological functional modules including cell apoptosis of XBJ for treating sepsis were identified [30]. By examining the levels of apoptosis-related gene expression in sepsis patients, Weber SU et al. revealed that apoptosis was sparked in the early stages of the condition [47]. In our result, TUNEL staining ( Figure  4A) proved that XBJ dramatically reduced cardiomyocyte apoptosis in the myocardium as likened to the CLP group. Previous studies have found that CLP induced the over-expression of Bax, Cleaved-Caspase 3, Cleaved-Caspase 9 and Cytochrome C and the lowexpression of Bcl-2 in rat heart tissue [14,48,49]. Our current findings indicate that the myocardium in sepsis rat does not express more pro-apoptotic genes in response to stimulation during the first 12 hours of CLP exposure. The explanation for this may be that when rats were first exposed to CLP, Bax was unable to trigger the opening of mitochondrial outer membrane permeability and was unable to stimulate a variety of pro-apoptotic proteins, which lead to anti-apoptotic proteins predominated in the body. Generally speaking, XBJ reduced the expression of pro-apoptotic proteins and increased the expression of anti-apoptotic proteins during the early 12h of CLP-induced sepsis, while these indexes were reversed during the late 1 day to 5 days ( Figure 4B). These results showed that XBJ alleviated SIMI by promoting apoptosis in the early stage of sepsis, and inhibiting apoptosis in the late stage of sepsis.
Autophagy is one of the mechanisms involved in the pathophysiology of SIMI, and a homeostasis process involving self-digestion for energy [50]. Autophagy contributes to the preventive function during infectious diseases [51]. Autophagosome formation and maturation are regulated by several core autophagy gene (ATG) proteins in a highly controlled manner. Beclin-1 is a mammalian cell homolog of the yeast autophagyrelated gene ATG6, and mediates the initiation stage of autophagy [52]. A study showed that injection of a cell-permeable Tat-Beclin-1 peptide to activate autophagy improved cardiac function, attenuated inflammation, and rescued the phenotypes caused by Beclin-1 deficiency in LPS-challenged mice [35]. Microtubuleassociated protein LC3, another key protein in autophagy, is encoded by a mammalian ortholog of yeast Atg8 that is ubiquitous in mammalian tissues and cultured cells. It has two forms, LC3-I and LC3-II [53]. The autophagic vesicle membrane is where LC3-II is located after LC3-I is typically transformed to LC3-II during the development of an autophagosome [54]. P62 functions in autophagy as a selective junction protein by modifying ubiquitinated proteins and delivering them to the proteasome for destruction [55]. However, whether autophagy is inhibited or activated throughout the pathophysiological process of sepsis is still a topic of debate. A study showed that the pathophysiological process of sepsis' organ failure is mostly due to blocked autophagy [11]. Other studies showed increased autophagy in the early phases of sepsis followed by a decline near late-stage organ failure in the mouse CLP sepsis model [56,57]. Several basic studies presenting various therapeutic methods have determined that SIMI can be improved by mediating autophagy [13,58,59]. Since autophagy is a dynamic and intricate process, it is challenging for us to record consistent and uniform autophagic changes over the course of numerous static time periods. Therefore, the dynamic time changes (1h, 1d, 2d, 3d and 5d) during sepsis were investigated in the present study to clarify the mechanism of SIMI. The results showed that compared with the CLP group, Beclin-1 expression was low, LC3-II/LC3-I ratio decreased, and P62 accumulation was evident at 12 h of XBJ intervention, and all data were reversed at 1 to 5 days (Figure 5), demonstrated XBJ alleviated SIMI by inhibiting autophagy in the early stage of sepsis, and enhancing autophagy in the late stage of sepsis.
We will further investigate XBJ's impact on the PI3K/AKT/mTOR pathway after identifying its cardioprotective role of regulating apoptosis and autophagy during sepsis. Regulation of PI3K/AKT/mTOR pathway is mediated in the mechanism of apoptosis and autophagy, which has been confirmed in animal experiments [60,61]. According to a recent systemic pharmacological analysis, XBJ can affect sepsis by acting on alterations in a number of genes involved in the PI3K/AKT/mTOR signaling pathway [8,9]. Previous research has found that when applied to sepsis, XBJ blocked the phosphorylation of the PI3K/AKT/mTOR signaling pathway [62]. However, there have been two divergent voices on the road to sepsis mitigation research. The first one is that the improvement of SIMI is through the activation of PI3K/AKT/mTOR signaling pathway [63,64], which is in line with our findings at 12h that administration of XBJ therapy increased the AGING phosphorylation levels of PI3K, AKT and mTOR in CLP-induced sepsis, suggesting by activation of the PI3K/AKT/mTOR pathway. Study effectively inhibited hypoxia and reoxygenation-induced autophagy and apoptosis via, at least partially, activating the PI3K/AKT/mTOR pathways. The second one is that blocking the PI3K/AKT/mTOR signaling pathway had a protective effect against sepsis [26,27], which is in line with our findings at 1d, 2d, 3d and 5d that administration of XBJ therapy suppressed p-PI3K, p-AKT and p-mTOR, showing by inhibition of the PI3K/AKT/mTOR pathway. Meanwhile, the PI3K/AKT/ mTOR signaling pathway was blocked during sepsis, to trigger autophagy and block apoptosis [27]. Based on our results and previous literature we speculated that XBJ can safeguard the myocardium in septic rats by activating the PI3K/AKT /mTOR signaling pathway in the early stages of sepsis and inhibiting it in the latter stages. Therefore, we speculated that in CLP-induced septic rats, XBJ implementation may potentially result in inhibiting apoptosis and promoting autophagy via activating PI3K/AKT/mTOR pathway in the early stage of sepsis, as well as promoting apoptosis and inhibiting autophagy via, at least partially, suppressing PI3K/AKT/mTOR pathway in the late stage of sepsis. The specific mechanism of this regulation still needs to be proved by further studies. Figure 7 presents a diagram of the molecular mechanism by which XBJ protects SIMI.
The innovative point of the present study is the fact that we utilized five time points to monitor the long-term changing regularity of SIMI and XBJ's myocardial protective mechanisms in sepsis, which is more in accord with the dynamic and intricate features of sepsis. Although there are important discoveries revealed by these studies, there are also limitations. First, the precise molecular mechanism by which XBJ affects the PI3K/AKT/mTOR pathway in sepsis remains unknown. Second, we have to point out that we do not look into how changes to the PI3K/AKT/mTOR pathway XBJ work to affect cardiomyocyte apoptosis and autophagy during sepsis in further detail.

CONCLUSIONS
Here, we presented strong support for a putative mechanism through which XBJ defends against SIMI caused by CLP. It has been found that CLP induced myocardial injury and dysfunction at a time-dependent manner. Additionally, based on our results, the possible molecular mechanism of XBJ in the treatment of CLPinduced SIMI speculated that XBJ's cardioprotective advantages may be attributable to its ability to suppress apoptosis and promote autophagy via, at least partially, activating PI3K/AKT/mTOR pathway in the early stage of sepsis, as well as promoting apoptosis and inhibit autophagy via suppressing PI3K/AKT/mTOR pathway in the late stage of sepsis.

Animal experiment
All animal procedures were carried out in accordance with the regulations of the Animal Protection Committee of Ningxia Medical University, and all experimental procedures were approved by the Ethics Committee of the General Hospital of Ningxia Medical University. 6-8 weeks male specific pathogen free Sprague-Dawley rats (220 ± 20 g), were purchased from and housed at the Ningxia Medical University laboratory animal center. Rats were raised in plastic cages with temperature and humidity-controlled room (22.8±2.0° C and 50%~60%, respectively) with a 12/12 hours light/dark cycle and allowed ad libitum access to food and water.

Polymicrobial sepsis caused by CLP
Polymicrobial sepsis was generated in rat by CLP surgery as previously described [65]. To put it simply, the rats were fixed on the operating table after anesthesia (4% phenobarbital (40 mg/kg i.p.)), the abdominal hair was removed for routine disinfection, the abdominal cavity was opened to expose the cecum, and the distal 50% was ligation. A "penetrating" operation with 22-gauge needle was then performed on the ligation of the cecum. Abdominal incision and intestinal manipulation with neither ligation nor puncture were performed in shamoperated animals. All rats were subcutaneous injected with lactated Ringer's solution (30 ml/kg) after injury for fluid resuscitation and housed alone. Postoperative pain was managed by antibiotic Imipenem/Cilastin (20 mg/kg s.c.) and analgesia flurbiprofen axetil injection (5 mg/kg i.v.).

Drug treatment and sample collection
Rats were randomly divided into 3 groups: (1) Sham group; (2) CLP group; and (3) XBJ group. Three groups of rats after Sham or CLP were randomly divided into 12 hours, 1 day, 2 days, 3 days and 5 days groups (n = 6/group). The animals in each group were treated with drugs for 12 hours, 1 day, 2 days, 3 days or 5 days, respectively. One hour after CLP, rats in XBJ group were treated with XBJ (10 ml/kg s.c.), while rats in sham or CLP group were treated with the same volume of normal saline. See Figure 1B for details.

Animals
under anesthesia were used for echocardiographic evaluation. Blood samples in heparinized tubes were collected from the heart, centrifuged at 3500 rpm/min for 10 min at room temperature, and the supernatant serum was aspirated and stored at -80° C for the subsequent analyses. Then, the rats were euthanized by excessive anesthesia administration. The heart tissues were stored in 4% paraformaldehyde at 4° C for Hematoxylin and Eosin (H&E) staining, TdT mediated dUTP biotin nick-end labeling (TUNEL) staining and Immunofluorescence assay, and cryopreserved in liquid nitrogen for protein western blot.

Survival curve
Another thirty rats were also subjected to the CLP or sham procedure as mentioned above to investigate survival. After the CLP or sham surgery, the survival of the rat was observed every 12 h up to 7 days.

Echocardiography
Cardiac function was assessed using an Ultra High Resolution Small Animal Ultrasound Imaging System (Vevo®2100 Imaging System, Visualsonics, Toronto, Canada) with a 15-MHz transducer. After the induction of general anaesthesia with 4% pentobarbital (40 mg/kg i.p.), hearts were imaged in 2-D mode in the parasternal long-axis view prior to M-mode imaging positioned perpendicular to the interventricular septum and posterior left ventricular wall. Heart rate was measured over 3 consecutive cycles. The left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) parameters were calculated by the software of Vevo770TM imaging system.

Biochemical detection
The enzyme activity of cardiac troponin I (cTnI), creatine kinase (CK), lactate dehydrogenase (LDH) levels in serum was measured using a quick, convenient, and sensitive corresponding assay kit based on the protocol. Serum concentrations of these factors were detected by an Automatic Biochemical Analyzer (Bio Majesty JCA-BM6010, JEOL Ltd., Japan).

Histopathological (H&E) staining
4 μm sections were stained using a H&E Staining Kit (Biotopped, Beijing, China), according to the manufacturer's instructions. Morphological changes in myocardial tissues were observed by H&E under a light microscope. Morphological changes in myocardial tissues were observed at 200 × magnification under a light microscope (Leica, USA). Three hearts were analyzed per group.

TUNEL staining
Extensive DNA degradation is the signature of the late stage of apoptosis. Visualization of apoptotic cardiomyocytes was performed on left ventricular tissue cross sections (4 μm thick) using One-step TUNEL Apoptosis Detection Kit (Beyotime, Beijing, China) and according to the manufacturer's procedure. TUNEL staining changes in myocardial tissues were observed with a fluorescence microscope (MF43-N, Mshot, China) to obtain representative fluorescence images.

Statistical analysis
All values described in the text and figures are presented as mean ± standard deviation (SD). The Kaplan-Meier method was applied to assess survival followed by the log rank test. One-way analysis of variance (ANOVA) test was used to compare among multiple groups, followed by Tukey's test after a homogeneity test for variance and Tamhane T2's test after a heterogeneity test for variance. SPSS 24.0 software was used to analyze the data. p < 0.05 in twotailed testing was considered statistically significant. In all the resulting graphs, # indicates p < 0.05 (vs. the Sham group) and * indicates p < 0.05 (vs. the CLP group).

AUTHOR CONTRIBUTIONS
ZJF and YLS designed the study. BCF and LJ performed most of the experiments and wrote the manuscript. HSW, XZX, MX and KXF performed the in vivo experiments and analyzed the data.