Sophocarpine Attenuates LPS-Induced Liver Injury and Improves Survival of Mice through Suppressing Oxidative Stress, Inflammation, and Apoptosis

Septic liver injury/failure that is mainly characterized by oxidative stress, inflammation, and apoptosis led to a great part of terminal liver pathology with limited effective intervention. Here, we used a lipopolysaccharide (LPS) stimulation model to simulate the septic liver injury and investigated the effect of sophocarpine on LPS-stimulated mice with endotoxemia. We found that sophocarpine increases the survival rate of mice and attenuates the LPS-induced liver injury, which is indicated by pathology and serum liver enzymes. Further research found that sophocarpine ameliorated hepatic oxidative stress indicators (H2O2, O2∙−, and NO) and enhanced the expression of antioxidant molecules such as superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). In addition, sophocarpine also attenuated regional and systematic inflammation and further reduced apoptosis of hepatocytes. Mechanistic evidence was also investigated in the present study as sophocarpine inhibited hepatic expression of the CYP2E/Nrf2 pathway during oxidative stress, inactivated p38/JNK cascade and NF-κB pathway, and, meanwhile, suppressed PI3K/AKT signaling that reduced apoptosis. Conclusively, the present study unveiled the protective role of sophocarpine in LPS-stimulated oxidative reaction, inflammation, and apoptosis by suppressing the CYP2E/Nrf2/ROS as well as PI3K/AKT pathways, suggesting its promising role in attenuating inflammation and liver injury of septic endotoxemia.


Introduction
The liver plays a key role in immunological homeostasis and metabolism [1] while these crucial functions are usually impaired by lipopolysaccharide (LPS), inflammatory factors, and pathogens [2,3]. LPS presents the major component of endotoxin in gram-negative bacteria and causes uncontrolled production of inflammatory mediators and oxidative stress, resulting in acute liver injury (and failure) [4]. LPS-induced liver injury in mice has been employed as a model for molecular pathological research [5], simulating the course of liver damage and failure in septic endotoxemia or sometimes septic shock or death [2,6]. Liver failure is characterized by hepatic encephalopathy and disorder of protein synthesis [6]. Though specific mechanism remains controversial, consensus has been well reached that hepatic inflammatory oxidative stress and apoptosis might be the crucial mechanism.
Sepsis progression and septic liver dysfunction present complex pathophysiological alterations, [7], including processes like releasing of reactive oxygen species (ROS), nitrogen species (RNS), inflammation, and apoptosis. Characterized by the imbalance of endogenous enzymatic activity, such as catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) [8,9], oxidative damage could be reflected by CYP2E1, which promotes the production of ROS during its catalytic cycle and may be the main contributor to oxidative stress and liver injury [10][11][12]. Thus, antioxidant compounds have been considered a promising treatment against ROS-induced liver injury or failure [13].

Materials and Methods
2.1. Cell Culture and Reagents. The hepatic stellate cells (HSCs) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA) and cultured in DMEM media supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco, Waltham, MA, USA) and 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA) in a humidified incubator containing 5% CO 2 and 95% air at 37°C. The HSCs were incubated with LPS (100 ng/mL) in the absence or presence of sophocarpine at the concentration of 1 μM and 2 μM about 24 h. These cells were used for the further studies.

Animals.
Eight-week-old C57 BL/6 male mice, about 22.1 g per mouse, were employed for further study. And these mice were bought from the Experimental Animal Centre of Second Military Medical University (Shanghai, China) and were approved by the Animal Care and Use Committee of Changhai Hospital, Second Military Medical University (Shanghai, China). They were placed in a specific pathogenfree (SPF) room with sawdust bedding at a temperature of 25-26°C and a relative humidity of~50% and light 12 h/day, and water and food were free to access. The authors confirmed that all animals received human care, and all animal experiments were conducted in accordance with the relevant guidelines and regulations. In this study, the normal mice were starved about 16 hours and they were divided randomly into four groups: con (control), LPS-induced group, and LPS-induced mice pretreated with sophocarpine (30 mg/ kg body weight per day and 60 mg/kg body weight per day). For the establishment of liver injury in LPS-administrated mice with endotoxemia, the mice were injected intraperitoneally with LPS (5 mg/kg body weight) and we prepared 30 mice for each group above. Sophocarpine was administered orally once at 16:00~17:00 every day for 24 days. Meanwhile, we marked the number of dead mice for each group.

Analyses of Liver Function.
Performed as the indicators of hepatic function, serum and liver levels of glutathione (GSH), alanine transaminase (ALT), alkaline phosphatase (ALP), and aspartate transaminase (AST) were analyzed by employing the biochemical kits from R&D Systems (Minneapolis, MN, USA).

Analyses of H 2 O 2 and O •
2 − Production and ROS Levels in Liver of Mice. Hepatic levels of O • 2 − were measured using the chemiluminescence method [26]. Firstly, the weighed liver tissues of mice were homogenized in lysis buffer, pH 7.4, containing 10 mM EDTA as well as 20 mM HEPES. The samples were centrifuged for 10 min at 1000g, and, then, the aliquot of samples was incubated with a Krebs-HEPES buffer, pH 7.4, containing 5 mM lucigenin (Sigma, Shanghai, China) about 2 min at 37°C. Next, light emission data were obtained on a M200 PRO multifunctional microplate reader (TECAN, Switzerland), and the results were showed as mean light unit (MLU) min/mg protein. Levels of O • 2 − were measured by adding SOD (350 U/mL) to the medium according to the manufacturer's instruction (R&D Systems, Minneapolis, MN, USA). In addition, liver tissues were homogenized in normal saline, and the samples were treated with equal volume of cold methanol for 60 min in a 4°C icebox. Then, the samples were centrifuged for half an hour at 10000g and we obtained the supernatant for H 2 O 2 evaluation using the biochemical kits from the R&D Systems (Minneapolis, MN, USA). Protein concentration was measured using the Bradford method, and BSA was employed as the standard.
2.5. Determination of IL-1β, TNF-α, and IL-16 by ELISA. The weighed liver tissues were put in a cold PBS buffer (pH 7.0) containing 0.002% sodium acid, 0.1 mg/mL soybean trypsin inhibitor, 2 mM PMSF, 10 nM EDTA, and 1.0 mg/mL BSA. The tissues were homogenated, and, then, the samples were incubated for 2 h in a 4°C refrigerator. For further assays, the supernatants were collected by centrifugation at 12000g for 10 min. IL-1β, TNF-α, and IL-16 levels in the supernatant of the serum and liver were measured using ELISA kits (Sigma, Shanghai, China).
2.6. Hematoxylin-Eosin Staining. The liver tissues from mice were fixed in 10% formalin, and the fixed specimens were processed to paraffin blocks, sectioned (5 μm), and stained with hematoxylin-eosin (H&E) for histological analysis according to the standard protocols [27]. In this study, the sections were observed in a blind manner [27].
2.7. Reverse Transcription Polymerase Chain Reaction (RT-PCR). The reverse transcription polymerase chain reaction (RT-PCR) and the quantitative real-time PCR (Q-PCR) were performed as previously described [20]. Total RNA was extracted from liver tissues and HSCs using TRIzol reagent from Thermo Fisher Scientific (Waltham, MA, USA). The cDNA was obtained by reverse transcription in a 20 μL reaction containing 2 μg of total RNA, oligo (dT), and reverse transcription premix.
2.8. Immunoblot Analysis. The liver tissues or the HSCs were lysed in RIPA Buffer (1 mM EDTA pH 8.0, 50 mM Tris-HCl pH 8.0, 2% SDS, and 5 mM DTT), and their protein concentration was decided by the BCA assay (Beyotime Inc., Shanghai, China). The total protein (about 30 μg) was separated by a SDS-PAGE gel and transferred to PVDF (polyvinylidene fluoride) membranes (Invitrogen, CA, USA)= and blocked with 5% nonfat dry milk in PBST (phosphate-buffered saline with Tween), pH 7.5. The membranes were immunoblotted with primary antibodies for 4 hours or overnight at 4°C. The primary antibodies were all purchased from Cell Signaling Technology (MA, USA), and they were diluted at 1 : 1000 in the immunoblot analysis. Secondary antibodies with horseradish peroxidase were used in this study. The protein bands were determined by an enhanced chemiluminescence kit (Pierce, Rockford, USA). The corresponding semiquantitative analysis was based on optical density with ImageJ software.
2.9. Determination of the Apoptotic Cells by TUNEL. We determined the apoptosis of HSCs using TUNEL methods as previously described [19]. Briefly, the TUNEL and DAPI, which were from Sigma (Shanghai, China), were used to detect the apoptosis of cultured cells and the apoptotic cells could be TUNEL-positive. Then, the TUNEL-positive HSCs were calculated under a Carl Zeiss microscope (Axio Observer A1, Jena, Germany).

Statistical
Analysis. Data were shown as the mean ± SEM. Student's t-test was performed for comparisons between two groups, and one-way ANOVA test was employed for comparisons among several groups. Log-rank test was used for survival data. P value < 0.05 was considered to be statistically significant.

Sophocarpine Increases the Survival Rate and
Attenuates the LPS-Induced Liver Injury. The data in our study suggested that the 16-day survival rate was 73.3% (22 out of 30) and 76.7% (22 out of 30) in sophocarpine-pretreated group in a dose-dependent manner; meanwhile, the 16-day survival rate was 30.0% (9 out of 30) in the sepsis group ( Figure 1(a)). Compared to the sepsis group, the 16-day survival rate was higher in the sophocarpine-treated group (P < 0 001); in the sham group (30 mice), the survival rate was 100% on the 16th day. In a word, pretreatment of mice with sophocarpine before LPS injection remarkably decreased lethality in contrast to LPS-caused sepsis animals.
It has been reported that LPS-induced liver dysfunction may be assessed by serum liver-specific enzymes including AST, ALT, and ALP, and the morphological alterations of the liver may be observed by H&E staining. Firstly, we found that sophocarpine (30 mg/kg and 60 mg/kg per day) recovered destructive damage of hepatocytes significantly in LPS-induced septic liver injury (Figures 1(b) and 1(c)). Then, AST, ALT, and ALP levels in sepsis mice were higher than sham (normal) group, and sophocarpine significantly decreased AST, ALT, and ALP levels in the serum and liver of sepsis mice (Figures 2(a)-2(f)). Combined with the survival rate in Figure 1(a), the data revealed that sophocarpine showed its protective role in sepsis and sepsis-related acute liver injury via downregulating ALT, AST, and ALP expression.    ROS signaling in the LPS-induced liver of mice by Western blot. As shown in Figure 3, the results demonstrated that SOD1 and Nrf2 expression was dramatically downregulated in the LPS-induced liver, compared with the normal mice. After injection of sophocarpine, data presented that the levels of SOD1 and Nrf2 were elevated markedly by sophocarpine in a dose-dependent manner in endotoxic mice (Figures 3(a) and 3(b)). Moreover, we investigated oxidative stress-associated protein including ROS, CYP2E, P38, JNK, and STAT3 in mice. The data showed that ROS, CYP2E, P38, STAT3, and JNK were increased in LPSinduced mice (Figures 3(c) and 3(d)). However, the expression of CYP2E and ROS, as well as the phosphorylation of P38, STAT3, and JNK, was significantly inhibited by sophocarpine in a dose-dependent manner (Figures 3(c) and  3(d)). Thus, sophocarpine protected against endotoxemia via improving ROS-mediated oxidative stress in the liver of sepsis model animals.  Similarly, we found that LPS enhanced the protein expression of TNF-α and IL-1β, and LPS also upregulated expression of IκBα protein and phosphorylation of NF-κB (Figures 3(h) and 3(i)). Contrarily, sophocarpine downregulated the expression of the above proteins in inflammation of the LPS-induced liver of mice (Figures 3(h) and 3(i)). The data indicated that sophocarpine may ameliorate LPSinduced liver injury by suppressing inflammation responses.

Sophocarpine Suppresses Apoptosis in Liver of LPS-Induced Mice.
To explore the effects of sophocarpine on apoptosis, we analyzed the PI3K/AKT pathway-related apoptosis progressing. The data proved that LPS significantly promoted the expression of PI3K and AKT, which were restored to the normal levels by administration of sophocarpine at 60 mg/kg per day. Furthermore, apoptosis-associated proteins were analyzed by Western blot in this section. Then, we found that Bcl-xL, Cyto-c, Apaf1, and cleaved caspase-9 and caspase-3 were increased by LPS dramatically (Figures 4(a) and 4(b)), indicating that LPS may promote apoptosis development in the liver of mice. After administrating sophocarpine, LPSinduced apoptosis in the liver may be significantly inhibited by depressing the expression of the related proteins above (Figures 5(a) and 5(b)).
To verify the vital role of apoptosis in the progression of LPS-induced acute liver injury, we analyzed the mRNA expression of the apoptosis-associated genes above by realtime PCR. The results showed that sophocarpine markedly downregulated the mRNA levels of PI3K and AKT (Figures 6(a) and 6(b)), and sophocarpine also reduced the mRNA levels of Bad, Bax-xL, Cyto-c, Apaf1, caspase-9, caspase-3, and caspase-6 in the liver of LPS-caused liver failure (Figures 6(c)-6(i)). Thus, sophocarpine attenuated liver injury by repressing the expression of apoptosis-related genes at both mRNA and protein levels.
3.6. Sophocarpine Improves Injury of LPS-Treated HSCs by Suppressing Apoptosis. To study the potential role of sophocarpine on LPS-stimulated hepatic stellate cells (HSCs), we pretreated HSCs with LPS. Then, the LPS-stimulated HSCs were subjected to sophocarpine incubation in order to determine whether sophocarpine may improve the liver injury by regulating apoptosis. In the present study, we found that LPS significantly elevated apoptosis-related gene  expression, including PI3K and AKT, as well as Cyto-C, Apaf1, caspase-9, and caspase-3 (Figures 4(a)-4(f)). However, sophocarpine obviously inhibited this gene expression (Figures 4(a)-4(f)). Subsequently, sophocarpine may attenuate HSC damage and apoptosis in a dose-dependent manner (Figure 4(g)). These in vitro data suggested that sophocarpine could improve HSCs injury by suppressing PI3K/AKTassociated apoptosis.

Discussion
Nowadays, the mechanism of acute liver injury (or failure) still has not been completely investigated and required further study for promising clinical strategies [28]. Researches proposed LPS-induced acute liver injury, possibly derived from endotoxemia, was related to the inflammatory-associated Kupffer cells as well as inflammatory mediators including TNF-α, IL-1β, nitric oxide, and superoxide [29]. Furthermore, LPS extends acute liver injury by modulating the oxidative stress and production of free radical, protein synthesis, and apoptosis of hepatocytes [30]. In this study, we found that ALT, AST, and ALP were upregulated in the serum and liver of LPS-induced mice and these three indicators above were dramatically downregulated by sophocarpine (Figures 2(a)-2(f)).
Oxidative stress that is associated with cellular metabolism in the O 2 environment has been regarded as a balance between prooxidant and antioxidant [31]. Based on the cellular microenvironment, the prooxidation process generates ROS including hydrogen peroxide (H 2 O 2 ) and superoxide radical (O • 2 − ) [32]. The prooxidants H 2 O 2 , O • 2 − , and NO are the main sources of ROS production according to the diverse stress conditions [33,34]. Previous studies have demonstrated that the ROS plays a vital role in septic shock and organ failure [35]. Also, excessive ROS is expressed in LPS- induced liver injury and antioxidant agents seem to be a good choice to reverse the liver injury [35]. As a main antioxidant and O • 2 − scavenger, SOD may react with ROS and NO [36]. CAT may reduce ROS production by degrading H 2 O 2 into oxygen and water [37]. Additionally, GSH can protect the liver and other organs against oxidative stress by decreasing the levels of H 2 O 2 and lipid hydroperoxide [38]. In the current study, we found that LPS significantly elevated the levels of H 2 O 2 , O • 2 − , and NO, which were downregulated nearly to normal levels by sophocarpine in a dose-dependent manner. Meanwhile, sophocarpine obviously upregulated the activity and expression of endogenous antioxidants, such as SOD, CAT, and GSH, suggesting that sophocarpine shows its potential antioxidative by blocking ROS-mediated signaling.
As an essential sensor of redox status in ROS process, cellular Nrf2 often binds to the cytoskeletal-anchoring protein under normal conditions [39]. As the main contributor, CYP2E always promotes the production of ROS in liver injury and ethanol-induced oxidative stress [11]. In the present study, LPS decreased the levels of CYP2E, Nrf2, and ROS, which may be restored to normal by sophocarpine administration, indicating that sophocarpine possesses the activity to attenuate oxidative stress resulting in the improvement of LPStriggered acute liver injury.
It has been reported that PI3K/Akt signaling plays vital roles in survival and antiapoptosis of cells by modulating its downstream targets, including caspase-9, caspase-3, and Bad [14,34,36]. Meanwhile, NF-κB signaling has been demonstrated to be an important regulator in apoptosis of cancer cells [34,36]. However, it remains unknown about the role of PI3K/Akt signaling in the liver of LPS-induced mice. In this study, sophocarpine significantly downregulated the expression of PI3K and phosphorylation of AKT, implying that the chemical can inhibit the expression of activated AKT. Furthermore, we found that sophocarpine reduced the expression of Bcl-xL, which was an antiapoptotic gene. Sophocarpine downregulated the expression of cleaved caspases substantially. Consequently, the data indicate that sophocarpine suppresses the activation of PI3K/AKT signaling resulting in apoptosis of liver injury induced by LPS.
NF-κB signaling is a central regulator of inflammatory cytokines, such as TNF-α and IL-1β, which are key factors in inflammation responses [40]. In the current study, we found that LPS dramatically upregulated content and activity of TNF-α, IL-1β, and IL-6 in the serum and liver of mice. Thus, LPS-induced liver failure may be generated by promoting the inflammation responses including the mentioned inflammatory signaling and cytokines, which can be inhibited by sophocarpine in a dose-dependent manner.
In conclusion, sophocarpine shows the activity against oxidative stress and inflammation in the LPS-induced liver injury of mice. Moreover, sophocarpine suppresses the liver injury in LPS-induced mice with endotoxemia through blocking the inflammatory pathway NF-κB, contributing to downregulation of proinflammatory cytokines. Meanwhile, sophocarpine affects apoptosis in the liver by inhibiting the PI3K/AKT-associated signaling. These findings suggest sophocarpine might be a novel and promising agent to improve inflammation, apoptosis, and oxidative responses in the liver of mice with endotoxemia.

Conflicts of Interest
The authors declare no conflicts of interests.