6-Shogaol Mitigates Sepsis-Associated Hepatic Injury through Transcriptional Regulation

Sepsis-associated liver dysfunction presents a significant public health problem. 6-Shogaol is the key bioactive component in dry ginger, which has antioxidant and anti-inflammation capacity. The present study aims to investigate the preventive effect of 6-shogaol on sepsis-induced liver injury. 6-Shogaol was administered to mice for 7 consecutive days before being intraperitoneally injected with lipopolysaccharide (LPS). After 24 h, mice were sacrificed, and biochemical and transcriptomic analyses were performed. Our results demonstrated that 6-shogaol prevented LPS-induced impairment in antioxidant enzymes and elevation in malondialdehyde level in the liver. The hepatic inflammatory response was significantly suppressed by 6-shogaol through suppressing the MAPK/NFκB pathway. RNA-sequencing data analysis revealed that 41 overlapped genes between the LPS vs. control group and 6-shogaol vs. LPS group were identified, among which 36 genes were upregulated, and 5 genes were downregulated for the LPS vs. control group. These overlapped genes are enriched in inflammation-related pathways, e.g., TNF and NFκB. The mRNA expression of the overlapped genes was also verified in the LPS-induced BRL-3A cell model. In summary, 6-shogaol shows great potential as a natural chemopreventive agent to treat sepsis-associated hepatic disorders.


Introduction
Sepsis is a systematic inflammatory response caused mainly by bacteria. It is one of the most life-threatening illnesses among intensive care patients worldwide [1]. Clinically, the progression of sepsis starts from early sepsis, severe sepsis, septic shock, to multi-organ failure [1,2]. Lipopolysaccharide (LPS) is the major component of endotoxin in Gramnegative bacteria. LPS-injected mice model is a very common acute sepsis model [3,4]. The liver is one of the main target organs affected in this model since it plays a crucial role in immunological homeostasis and metabolism [5]. Liver damage and dysfunction in sepsis an independent risk factor for sepsis-induced death [6,7]. It has been demonstrated that patients with sepsis and early liver dysfunction exhibited significantly elevated mortality and poor prognosis [8]. Since sepsis is characterized by overwhelming immune response, combating the inflammation might reduce liver dysfunction and lower the mortality of sepsis [9].
A growing number of studies have indicated that phytochemicals from diets can help manage inflammatory processes; thus, the food industry has increasingly focused on developing functional food from natural sources to prevent inflammation and reduce risks for related diseases. Ginger (Zingiber officinale) is consumed worldwide as a flavoring spice and as a traditional medicinal herb. Ginger is reported to possess antioxidative, anti-inflammatory, and antitumor properties [10][11][12]. 6-Shogaol is a major bioactive component derived from dry ginger. A number of studies have demonstrated the strong anti-inflammatory property of 6-shogaol [13][14][15][16]. For instance, 6-shogaol can inhibit the production of proinflammatory cytokines through regulation of NFκB and phosphorylation

Histological Analysis
The liver tissues were fixed in 10% (v/v) formalin solution and embedded in paraffin wax. Then, the tissues were sliced (3 µm) and subjected to hematoxylin-eosin (HE) staining. All of the sections were observed under an Olympus BX60 microscope (Tokyo, Japan). The histological damage was scored in a blind manner based on lymphocytic cell infiltration (0-2), hydropic degeneration (0-2), and blood vessels congestion (0-2).

RNA-Sequencing Detection
Total RNA was isolated from liver tissues (3 samples per group) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality was verified using Invitrogen Qubit 2.0 fluorometer. cDNA libraries preparation was subsequently performed with reagents supplied in Hieff NGS TM MaxUp Dual-mode mRNA Library Prep Kit for Illumina according to the manufacturer's instructions. The final purified libraries were quality evaluated, quantified, and sequenced using the BGI MGI 2000 platform according to the manufacturer's instructions.

Bioinformatics Analysis
To obtain high-quality clean data for downstream analysis, the low-quality reads and reads containing adapters or ploy-N in raw data were removed. Differential expression of three groups was analyzed using R DESeq package 1.12.4. The threshold value of differentially expressed genes was set by |fold change| > 1.5 and q-value < 0.05.
Hierarchical cluster analysis was performed using R gplots package 2.17.0 to define differential gene expression patterns. GO and KEGG functional pathways were analyzed by R topGO and clusterProfiler packages for differentially expressed genes, respectively. Furthermore, the PPI network was screened based on the STRING database using the R igraph package.

Cell Culture and Treatment
BRL-3A cells, a fibroblast cell line derived from the liver of Rattus norvegicus, were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM complete medium containing 10% FBS at 37 • C in a thermostatic incubator with 5% CO 2 . When the cells were approximately 80% confluent, they were seeded in a 12-well plate and incubated for 24 h for adhesion. For quantitative real-time PCR (qPCR) analysis, the cells were pretreated with 6-shogaol for 12 h before being treated with LPS (0.1 µg/mL) for 5 h. For Western blot analysis, the cells were pretreated with 6-shogaol for 2 h before being treated with LPS (0.1 µg/mL) for 0.5 h. The dose for 6-shogaol was applied at 10 and 20 µM based on MTT assay and previous studies [13,19,20]. According to the MTT assay, 6-shogaol at 20 µM was found to be the highest concentration that did not show cytotoxicity to BRL-3A cells after treatment for 24 h (Supplementary Figure S1).

Quantitative Real-Time PCR Analysis for mRNA Expression
After treatments, total RNA from BRL-3A cells were extracted using a Monarch Total RNA Miniprep Kit (New England BioLabs Inc., Ipswich, MA, USA), and was reversetranscripted using a high-capacity cDNA reverse transcription kit (ThermoFisher Scientific, Waltham, MA, USA). For the hepatic qPCR experiment, total RNA was isolated from the liver tissues using Trizol reagent, and reverse-transcribed using the high-capacity cDNA reverse transcription kit. The qPCR reactions were performed with an SYBR green PCR system in an ABI 7500 thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). Primers are synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). The sequences are shown in Table 1. The cycling conditions were as follows: 95 • C for 1 min, followed by 40 cycles involving denaturing at 95 • C for 15 s and annealing at 60 • C for 60 s. Expression of mRNAs was normalized by the mRNA levels of β-actin, which was used as an internal control. The relative expression levels of mRNAs were analyzed using the 2 −∆∆Ct method.

Western Blot Analysis for Protein Expression
Proteins from BRL-3A cells were extracted using NP-40 lysis buffer with protease and phosphatase inhibitors added. Extraction of liver tissue protein fractions was performed as described by Das et al. [21]. Protein concentration was measured following the instructions of the BCA kit. An equal amount of protein (30 µg) was separated on 10% SDS-PAGE at a constant voltage of 100 V and transferred to nitrocellulose membranes under a current of 250 mA for 1.5 h. After blocking with 5% BSA, the membranes were incubated with primary antibodies overnight at 4 • C, followed by incubation with the HRP-linked secondary antibody at room temperature for 1 h. The blots were incubated with ECL substrate for 1 min. The signal intensities were visualized using the Tanon 5200 automatic chemiluminescence imaging system (Shanghai, China), and quantification of bands was performed using the Image J software (national institutes of health Bethesda, MD, USA).

Statistical Analysis
Results have been presented as means ± SD. Data differences were evaluated with a one-way analysis of variance (ANOVA), followed by Tukey's test using Origin 8.0 (Origin-Lab, Northampton, MA, USA) differences were considered significant at p < 0.05.

6-Shogaol Improved Hepatic Function, Oxidative Stress, and Inflammation
In order to examine the hepatoprotective effect of 6-shogaol in vivo, animal experiments were performed. We observed that LPS induced liver injury in mice characterized by elevated ALT and AST levels (Figure 1a,b, p < 0.05). 6-Shogaol significantly protected the liver function by reducing these levels (p < 0.05). As shown in Figure 1c-e, LPS treatment evoked oxidative stress in the liver, presented by decreased SOD and CAT activities, and elevated MDA levels (p < 0.05). Treatment with 6-shogaol enhanced the cellular antioxidant defense system by elevating SOD and CAT activities (p < 0.05 for SOD). Moreover, hepatic MDA level was markedly attenuated by 6-shogaol treatment (Figure 1e, p < 0.05). LPS-induced liver injury was confirmed by the morphological alterations of the liver by HE staining. As shown in Figure 1f, liver tissue sections from LPS-exposed mice displayed lymphocytic infiltration, cellular hydropic degeneration, and congested blood vessels. Obvious reductions in these pathological alterations were observed in the 6-shogaol-treated group, i.e., hepatic sections were nearly normal except for moderate hydropic degeneration. The histological score for the 6-shogaol group decreased 38.6%, compared with LPS alone (p < 0.01). These data suggest that 6-shogaol treatment may improve LPS-induced liver function, oxidative stress, and inflammation.  were administered with the vehicle. On day 8, mice in LPS and 6-shogaol groups received an i.p. injection of LPS (10 mg/kg). After 24 h, the mice were sacrificed: (a) serum ALT activity; (b) serum AST activity; (c) hepatic SOD activity; (d) hepatic CAT activity; (e) hepatic MDA level; (f) histological examination of liver tissues by HE staining. LPS group showed lymphocytic infiltration (arrow), hydropic degeneration (thick arrow), and congested blood vessels (arrow with dot tail). Data are presented as mean ± SEM (n = 8). # p < 0.05, ## p < 0.01 versus control group; * p < 0.05 versus LPS group, ** p < 0.01 versus LPS group. LPS, lipopolysaccharide; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde.

6-Shogaol Suppressed MAPK/NFκB Pathway in the Liver
Activation of MAPK/NFκB is crucial for inflammatory response. Therefore, we next evaluated the protein expression of JNK, ERK, IkBα, and NFκB p65 ( Figure 2). LPS activated MAPK by significantly elevating the phosphorylation of JNK and ERK, thereby caused degradation of IκBα and triggered the phosphorylation of p65 (p < 0.01). All these changes were markedly reversed by treatment with 6-shogaol (p < 0.05 and p < 0.01). These results indicate that 6-shogaol regulated hepatic inflammation through MAPK/NFκB pathway.

Transcriptional Response in the Liver after 6-Shogaol Treatment
To further investigate the target genes regulated by 6-shogaol, RNA sequencing was performed. As shown in Figure 3, LPS-and LPS/6-shogaol-treated mice showed differential gene profiles in the liver. Using the cutoff false discovery rate < 0.05 and fold change > 1.5 (upregulated) or < 0.67(downregulated), we identified 41 overlapped genes between the LPS vs. control group and 6-shogaol vs. LPS group, among which 36 genes were upregulated, and 5 genes were downregulated for LPS vs. control group (Table 2). We performed hierarchical cluster analysis for the overlapped genes and observed distinctly different expression patterns (Figure 3a). All of these genes are differentially expressed in the opposite patterns in 6-shogaol vs. LPS group, e.g., Ptgs2, Mmp9, Ccl20 are upregulated by LPS, whereas downregulated in the 6-shogaol group.

Transcriptional Response in the Liver after 6-Shogaol Treatment
To further investigate the target genes regulated by 6-shogaol, RNA sequencing was performed. As shown in Figure 3, LPS-and LPS/6-shogaol-treated mice showed differential gene profiles in the liver. Using the cutoff false discovery rate <0.05 and fold change >1.5 (upregulated) or <0.67(downregulated), we identified 41 overlapped genes between the LPS vs. control group and 6-shogaol vs. LPS group, among which 36 genes were upregulated, and 5 genes were downregulated for LPS vs. control group (Table 2). We performed hierarchical cluster analysis for the overlapped genes and observed distinctly different expression patterns (Figure 3a). All of these genes are differentially expressed in the opposite patterns in 6-shogaol vs. LPS group, e.g., Ptgs2, Mmp9, Ccl20 are upregulated by LPS, whereas downregulated in the 6-shogaol group.

Gene Functional Analysis
Functional analysis was performed for the overlapped differentially expressed genes to examine whether they have biological relevance. These genes were classified into gene ontology (GO) terms such as positive regulation of immune system process, cellular response to lipopolysaccharide, and positive regulation of biological process (Figure 3b). Pathway enrichment analysis revealed that 21 overlapped genes were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways including the TNF signaling pathway and NFκB signaling pathway (Figure 3c,d). In particular, Ccl20, Cxcl1, Mmp9, Vcam1, Sele, Lif, Ptgs2, and Pgam5 were enriched in the TNF signaling pathway; Vcam1, Ptgs2, Lbp were enriched in NFκB signaling pathway. Except for Pgam5, all the above genes were upregulated in the LPS group, which were significantly decreased by 6-shogaol. The detailed information for the pathway enrichment was listed in Table 3. Expression of representative differentially expressed genes was evaluated by qPCR analysis.
The results show similar expression patterns, as observed with RNA sequencing, which validated our transcriptome data (Figure 4). These results revealed that the overlapped differentially expressed genes were mainly focused on inflammation-related functions.

Protein-Protein Interaction (PPI) Network for the Overlapped Genes
PPI network of the overlapped differentially expressed genes is shown in Figure 5. In the network, nodes represent proteins and edges represent interactions between two proteins. The size of proteins indicates node degree value, i.e., the larger the node is, the more important it is in the network. The color of the nodes indicates fold change for different comparison groups. We observed 14 differentially expressed genes encoding proteins were involved in the network, among which, NOS2, VCAM1, E-Selectin, MMP9, PTGS2, CXCL1 are important hub proteins. These proteins were linked primarily to inflammation and cell adhesion.

Protein-Protein Interaction (PPI) Network for the Overlapped Genes
PPI network of the overlapped differentially expressed genes is shown in Figure 5. In the network, nodes represent proteins and edges represent interactions between two proteins. The size of proteins indicates node degree value, i.e., the larger the node is, the more important it is in the network. The color of the nodes indicates fold change for different comparison groups. We observed 14 differentially expressed genes encoding proteins were involved in the network, among which, NOS2, VCAM1, E-Selectin, MMP9, PTGS2, CXCL1 are important hub proteins. These proteins were linked primarily to inflammation and cell adhesion.

6-Shogaol Suppressed the mRNA Expression of Overlapped Genes in LPS-Induced BRL-3A Cells
To evaluate whether 6-shogaol has the same effect in vitro, the mRNA expression of the regulated genes was also analyzed in LPS-induced BRL-3A cells. The results show that the differentially expressed genes exhibited similar expression patterns in LPS-treated BRL-3A cells ( Figure 6). 6-Shogaol dose dependently and effectively reversed the upregulation/downregulation by LPS. 6-Shogaol at high dose markedly decreased mRNA expression of Ptgs2 to the level even slightly lower than the control group (p < 0.01). Moreover, it significantly elevated Usp50 mRNA expression by over twofold, compared to the LPS-treated group (p < 0.01). For mRNA expression of Lcn2, Nos2, Ccl20, and Cxcl1, 6shogaol at a high dose could induce approximately 50% reduction, compared to the LPStreated group (p < 0.05).

6-Shogaol Suppressed the mRNA Expression of Overlapped Genes in LPS-Induced BRL-3A Cells
To evaluate whether 6-shogaol has the same effect in vitro, the mRNA expression of the regulated genes was also analyzed in LPS-induced BRL-3A cells. The results show that the differentially expressed genes exhibited similar expression patterns in LPS-treated BRL-3A cells ( Figure 6). 6-Shogaol dose dependently and effectively reversed the upregulation/downregulation by LPS. 6-Shogaol at high dose markedly decreased mRNA expression of Ptgs2 to the level even slightly lower than the control group (p < 0.01). Moreover, it significantly elevated Usp50 mRNA expression by over twofold, compared to the LPS-treated group (p < 0.01). For mRNA expression of Lcn2, Nos2, Ccl20, and Cxcl1, 6-shogaol at a high dose could induce approximately 50% reduction, compared to the LPS-treated group (p < 0.05).

6-Shogaol Inhibited NFκB and MAPK Pathway in BRL-3A Cells
We next investigated the effect of 6-shogaol on protein expressions of MAPK and NFκB pathways in BRL-3A cells. As shown in Figure 7a-c, LPS significantly activated the MAPK pathway, with an 8.2-fold and 1.9-fold increase in p-JNK/JNK and p-ERK/ERK expressions, respectively, compared to the control cells. Moreover, phosphorylation of IκBα was markedly elevated in LPS-treated cells (p < 0.01), which promoted degradation of IκBα and activation of NFκB (Figure 7a,d,e). 6-Shogaol evidently blocked the activation of MAPK and NFκB pathways, the expression levels of p-JNK/JNK, p-ERK/ERK, p-IκBα/IκBα, and p-p65/p65 were reduced to the level comparable to or even lower than the control cells (p < 0.01). These results are in accordance with those in the in vivo study.

6-Shogaol Inhibited NFκB and MAPK Pathway in BRL-3A Cells
We next investigated the effect of 6-shogaol on protein expressions of MAPK and NFκB pathways in BRL-3A cells. As shown in Figure 7a-c, LPS significantly activated the MAPK pathway, with an 8.2-fold and 1.9-fold increase in p-JNK/JNK and p-ERK/ERK expressions, respectively, compared to the control cells. Moreover, phosphorylation of IκBα was markedly elevated in LPS-treated cells (p < 0.01), which promoted degradation of IκBα and activation of NFκB (Figure 7a,d,e). 6-Shogaol evidently blocked the activation of MAPK and NFκB pathways, the expression levels of p-JNK/JNK, p-ERK/ERK, p-IκBα/IκBα, and p-p65/p65 were reduced to the level comparable to or even lower than the control cells (p < 0.01). These results are in accordance with those in the in vivo study.

Discussion
A murine model of LPS-induced inflammatory liver injury aims to replicate sepsisinduced acute hepatic injury in humans. It is widely used to investigate potential therapeutics for the treatment of acute hepatic failure [22][23][24]. In the present study, administra-

Discussion
A murine model of LPS-induced inflammatory liver injury aims to replicate sepsisinduced acute hepatic injury in humans. It is widely used to investigate potential therapeutics for the treatment of acute hepatic failure [22][23][24]. In the present study, administration of LPS results in an impaired structure of the liver and obvious histological changes including lymphocytic infiltration, cellular hydropic degeneration, and congested blood vessels. These histological changes were markedly ameliorated by 6-shogaol treatment. In addition, 6-shogaol evidently reduced serum ALT and AST activities. These results demonstrated the potency of 6-shogaol in treating inflammatory liver injuries. The present study further investigated the possible protection mechanism offered by oral administration of 6-shogaol, which is not yet reported by other reports.
The impairment of the antioxidant defense system is a critical step in LPS-induced injury. Evidence has shown that LPS insult is characterized by a change in the level of tissue antioxidant enzyme and increment of ROS production in the liver [25]. Restoring the antioxidant system may be a good choice to reverse liver injury [26]. As a main O 2− scavenger, SOD may react with ROS and NO, and produce H 2 O 2 , which is degraded into oxygen and water by CAT. In the current study, we found a modulation of the antioxidant system in the LPS-induced mice consuming 6-shogaol, with the normalization of SOD and CAT activities. The enhanced antioxidant system may help prevent LPS-induced liver damage.
LPS-induced liver injury was highly associated with inflammatory response [27]. A previous study has proposed that 6-shogaol is beneficial in suppressing inflammatory response in LPS-induced macrophages [17]. Our present study demonstrated that 6-shogaol possesses a similar effect in LPS-challenged hepatocytes. NFκB signaling is a central regulator of an inflammatory response, which regulates inflammatory cytokines and mediator genes expression [28]. In resting cells, the NFκB is binding with its inhibitory proteins IκBα. With exposure to proinflammatory stimuli, IκBα becomes phosphorylated and degraded. Then, the liberated NFκB is translocated to the nuclei and induces the transcription of target genes. In the present study, LPS induced the degradation of IκBα and significantly elevated phosphorylated p65 protein expression (Figures 2 and 6), which indicated that the NFκB pathway is activated. 6-Shogaol significantly inhibits hepatic inflammatory injury by suppressing the activation of the NFκB pathway. MAPK pathway has been proposed to respond to cytokines and stress, which are always related to the inflammation signaling pathway [29]. Particularly, MAPKs play an important role in modulating the NFκB pathway [30]. Therefore, protein expressions of MAPK are detected. LPS induces activation of MAPKs via phosphorylating JNK and ERK. Additionally, we confirmed the inhibitory effect of 6-shogaol on MAPKs. These results suggest that the protective effect of 6-shogaol on LPS-injured hepatocytes is related to the suppression of the MAPK/NFκB pathway.
We further investigated targeted genes involved in 6-shogaol treatment by applying RNA-sequencing analysis. We observed that 6-shogaol alleviates LPS-induced hepatic inflammation by altering a batch of gene expression, most of which were involved in the TNF and NFκB signaling pathways by KEGG pathway analysis. This is consistent with our results that 6-shogaol inhibits the NFκB pathway both in vivo and in vitro by Western blot analysis (Figures 2 and 7).
Lipocalin-2 (LCN-2) in the liver shows a protective role against pernicious conditions, such as infection, inflammation, and other forms of cellular stress [31]. There is a rapid and sustained LCN2 production in injured hepatocytes, which is regulated by the NFκB pathway [32,33]. As expected, LPS-induced a 785-fold increase in the expression level of Lcn2 in the liver of mice by RNA sequencing. This elevation was markedly reversed by 6-shogaol treatment. These results suggest that LCN2 is a potential biomarker for hepatic inflammation, as well as a viable clinical target for treating sepsis-related hepatic injuries.
NO is a critical factor in the pathogenesis of inflammatory diseases. The majority of NO production under the inflammatory state derives from inducible nitric oxide synthase (NOS2) [34]. Ptgs2 encodes the protein expression of cyclooxygenase-2 (COX-2), which is responsible for the synthesis of prostaglandin E2. Elevation in NOS2 and Ptgs2 expression by LPS via the NFκB pathway is well documented in various in vivo and in vitro models [35][36][37]. We proved that NOS2 and Ptgs2 play critical roles in hepatocyte inflammation evoked by LPS. These elevations could be effectively attenuated by treatment of 6-shogaol, which is consistent with other studies [17,38].
Adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1) and E-selectin, have been shown to involve in the inflammation response promoted by TNF-α via activating the NFκB signaling pathway [39,40]. Chemokines are also downstream targets of the NFκB pathway [41,42]. They play an important role in the pathogenesis of hepatic diseases. Ccl20 and Cxcl1 mediate LPS-induced liver injury and are potential drivers of inflammation and fibrosis in alcoholic hepatitis [43][44][45]. All of the aforementioned genes were downregulated by 6-shogaol, which is rarely reported by other researchers.
Matrix metalloproteinases (MMPs) are responsible for the degradation and remodeling of the extracellular matrix proteins. They participate in many pathological processes, such as inflammation and tumor invasion. Mmp9 is a downstream effector of the NFκB pathway [46]. Elevation in Mmp9 expression was frequently observed in reported LPSinduced cell models [47,48]. However, Zhang et al. demonstrated that overexpression of Mmp9 plays a protective role against LPS-induced inflammation in osteoblasts [49]. In the present study, we observed that Mmp9 was upregulated in LPS-treated mice/cells and remarkably reduced by 6-shogaol treatment. Therefore, future investigations are warranted to safely include Mmp9 into viable clinical treatments for sepsis-related inflammation.

Conclusions
Our findings demonstrated, for the first time, that 6-shogaol shows the preventive effect in sepsis-associated acute liver injury via enhancing antioxidant defense system and suppressing MAPK/NFκB induced inflammatory responses. Transcriptomic analysis revealed that the efficacy of 6-shogaol involves the regulation on Lcn2, Nos2, Ccl20, Cxcl1, Mmp9, Vcam1, Sele, Ptgs2, and Usp50 mRNA expression. 6-Shogaol shows great potential as a natural chemopreventive agent and may be used in the future to treat sepsis-associated hepatic disorders Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nu13103427/s1, Figure S1: Effect of 6-shogaol on cell viability. BRL-3A cells were treated with different concentrations of 6-shogaol for 24 h, and cell viability was measured using an MTT assay.

Conflicts of Interest:
The authors declare no conflict of interest.