Abstract

   The present study was designed to examine the efficacy and protection
   mechanisms of sea buckthorn sterol (SBS) against acute liver injury
   induced by carbon tetrachloride (CCl[4]) in rats. Five-week-old male
   Sprague-Dawley (SD) rats were divided into six groups and fed with
   saline (Group BG), 50% CCl[4] (Group MG), or bifendate 200 mg/kg (Group
   DDB), or treated with low-dose (Group LD), medium-dose (Group MD), or
   high-dose (Group HD) SBS. This study, for the first time, observed the
   protection of SBS against CCl[4]-induced liver injury in rats and its
   underlying mechanisms. Investigation of enzyme activities showed that
   SBS-fed rats exhibited a significant alleviation of inflammatory
   lesions, as evidenced by the decrease in cyclooxygenase-2 (COX-2),
   prostaglandin E2 (PGE2), and gamma-glutamyl transpeptidase (γ-GT). In
   addition, compared to the MG group, the increased indices (superoxide
   dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), total
   antioxidant capacity (T-AOC), and total protein (TP)) of lipid
   peroxidation and decreased malondialdehyde (MDA) in liver tissues of
   SBS-treated groups showed the anti-lipid peroxidation effects of SBS.
   Using the wide range of targeted technologies and a combination of
   means (UPLC-MS/MS detection platform, self-built database, and
   multivariate statistical analysis), the addition of SBS was found to
   restore the expression of metabolic pathways (e.g., L-malic acid,
   N-acetyl-aspartic acid, N-acetyl-l-alanine, etc.) in rats, which means
   that the metabolic damage induced by CCl[4] was alleviated.
   Furthermore, transcriptomics was employed to analyze and compare gene
   expression levels of different groups. It showed that the expressions
   of genes (Cyp1a1, Noct, and TUBB6) related to liver injury were
   regulated by SBS. In conclusion, SBS exhibited protective effects
   against CCl[4]-induced liver injury in rats. The liver protection
   mechanism of SBS is probably related to the regulation of metabolic
   disorders, anti-lipid peroxidation, and inhibition of the inflammatory
   response.

   Keywords: acute liver injury, metabolomics, carbon tetrachloride, sea
   buckthorn sterol

1. Introduction

   Sea buckthorn is a kind of small tree or deciduous shrub in the
   Elaeagnaceae family [[38]1]. Previous studies have shown that sea
   buckthorn contains many types of chemical components and rich bioactive
   substances, offering more than 190 active ingredients [[39]2]. In
   addition, 103 active ingredients have been found in sea buckthorn fruit
   oil [[40]3]. Sea buckthorn also has anti-inflammatory, anti-cancer,
   anti-aging, immunological modification, and anti-oxidation effects, as
   well as the inhibition of cholesterol absorption and the reducing of
   blood lipids [[41]4]. The wide usage of sea buckthorn in healthy food,
   wine, and natural medicine makes it a natural product with ecological,
   social, and economic benefits [[42]5].

   The liver is an important organ for protein synthesis, degradation, and
   detoxification, as well as amino acid metabolism. Nevertheless, the
   liver is easily damaged by various exogenous materials, causing acute
   or chronic liver diseases [[43]6]. Therefore, it is desired to study
   the liver protection mechanism.

   CCl[4] is commonly used in a mouse model of liver injury, which is
   responsible for oxidative stress and lipid peroxidation through the
   cytochrome P450-mediated generation of the highly reactive CCl[3],
   leading to eventual cellular damage characterized by hepatocellular
   necrosis [[44]7]. The biochemical and histological events of
   corresponding damage induced by CCl[4] are quite similar to liver
   cirrhosis in humans. Through oral or gavage feedings, reproducible and
   obvious acute liver injury can be induced, helping in the study of
   pathogenic mechanisms or protection mechanisms related to the liver.
   [MATH: <mrow><mi>β</mi></mrow> :MATH]
   -Sitosterol, a naturally occurring sterol molecule, is a relatively
   mild to moderate antioxidant and exerts beneficial effects in vitro by
   decreasing the level of reactive oxygen species. A previous study
   [[45]8] evaluated the antioxidant potential of
   [MATH: <mrow><mi>β</mi></mrow> :MATH]
   -Sitosterol in 1,2-dimethylhydrazine (DMH)-induced colon
   carcinogenesis. There was another study that suggested that phytosterol
   esters have [[46]9] effects on countering hypercholesterolemia-related
   changes in the brain by decreasing the cholesterol levels, increasing
   the phospholipid levels, and increasing the level of antioxidant
   enzymes.

   Extensive studies have shown that sea buckthorn fermentation liquid
   protects against alcoholic liver disease. The underlying protection
   mechanism may involve regulations of liver lipid metabolism and
   oxidative stress [[47]10]. Dietary supplements that contain sea
   buckthorn for hyperlipidemia treatment are useful in minimizing the
   oxidative damage caused by lipid peroxidation [[48]11]. In addition, a
   previous work [[49]12] indicated that pretreatment with sea buckthorn
   protects against LPS/d-GalN-induced liver injury in mice by suppressing
   the TLR4-NF-κB signaling pathway, which means that sea buckthorn may be
   a promising drug for the prevention of acute live injury. Furthermore,
   sea buckthorn inhibits CYP3A down-regulation during immune liver
   injury, primarily through the transcriptional regulation and
   post-translational nitration of enzyme proteins. This mechanism is also
   applicable to alcoholic liver injury and non-alcoholic fatty liver
   [[50]13]. On the other hand, many investigations demonstrate that SO[2]
   exposure causes a significant change in the glutathione redox system of
   rats. This change might be one of the possible mechanisms by which
   SO[2]inhalation leads to lipid peroxidation. Sea buckthorn seed oil was
   found to be protective against oxidative damage induced by SO[2]
   [[51]14]. Furthermore, sea buckthorn berry juice can alleviate symptoms
   of oxidative stress and tissue damage by preventing the destruction of
   antioxidant enzymes and inhibiting lipid peroxidation [[52]15]. The
   extract of Hippophae rhamnoides as well as vitamin E can protect the
   liver against nicotine-induced oxidative stress [[53]16].

   The studies mentioned above have shown that sea buckthorn has multiple
   functions, especially in liver protection and anti-oxidation. However,
   the existing studies focus mainly on sea buckthorn oil, sea buckthorn
   berry juice, and sea buckthorn fermentation liquid, which are crude
   extracts of sea buckthorn. There are surprisingly few studies
   investigating the liver protection mechanism of specific components of
   sea buckthorn. In addition, previous related works barely use different
   types of newer techniques, such as transcriptomics and metabonomics, to
   comprehensively analyze the action mechanisms of sea buckthorn in
   CCl[4]-induced liver injury rats.

   In the present study, we focus on analyzing the liver protection
   mechanism of sterols extracted from sea buckthorn for the first time.
   In this study, sea buckthorn sterol pre-treatment was used. The
   pretreatment experiment is a common way to study the protective effect
   of a substance that needs to be analyzed [[54]17]. Additionally, a
   variety of research methods (e.g., transcriptomics and metabonomics)
   were employed, which made it possible for us to carefully and
   accurately explain the protection mechanisms of sea buckthorn. In our
   previous studies, we reported that sea buckthorn regulates inflammatory
   factors and transaminases in the blood of CCl[4]-induced liver injury
   rats [[55]18]. The present work further demonstrates the protective
   effects of sea buckthorn sterol (SBS) on the liver, and the
   corresponding protection mechanisms are comprehensively analyzed from
   multiple aspects.

2. Materials and Methods

2.1. Animals, Reagents, and Drugs

   Sixty five-week-old male Sprague-Dawley (SD) rats, of SPF grade,
   provided by Chongqing, China, Tengxin Biotechnology Co., Ltd. (License:
   SCXK[Shan] 2018-001) were used in this study. All animal studies were
   approved by the animal ethics committee of Qinghai University. The rats
   anesthetized by intraperitoneal injection of 20% urethane (0.5 mL/100
   g) and then euthanized by bloodletting of the celiac artery (Audit Form
   No.: 2019-1). The SBS was extracted in our laboratory (seed oil of
   sea-buckthorn was purchased from Qinghai, China, Qinghua Bozhong
   Biotechnology Co., Ltd.) by the following processes: (i) extraction
   crude sterol from seabuckthorn seed oil by saponification; (ii) repeat
   recrystallization with anhydrous ethanol until SBS was purified. SBS
   has no hepatotoxicity, which has been validated in this study and other
   studies [[56]19]. CCl[4] (batch number: 56-23-5) and Bifendate (batch
   number: D25O10G100857) were purchased from Qinghai, China, Rhine
   Biotechnology Co., Ltd. The cyclooxygenase-2 (COX-2) kit (batch number:
   uifhwf41eb) and prostaglandin E2 (PGE2) kit (batch number: kex55l2533)
   were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China).
   The superoxide dismutase (SOD) detection kit (batch number: 20191123),
   glutathione peroxidase (GSH-PX) kit (batch number: 20191118),
   gamma-glutamyl transpeptidase (γ–GT) kit (batch number: 20191112),
   catalase (CAT) kit (batch number: 20191120), total antioxidant capacity
   (T-AOC) kit (batch number: 20191116), total protein (TP) kit (batch
   number: 20191115), and malondialdehyde (MDA) kit (batch number:
   20191119) were purchased from Nanjing Jiancheng Bioengineering
   Institute. Methanol, acetonitrile, ethanol, acetic acid, ammonium
   methyl acetate, chloroform, and methyl tert-butyl ether were supplied
   by Merck at HPLC grade, and the standard materials were supplied by
   BioBioPha. The standard materials were dissolved in dimethyl sulfoxide
   (DMSO) or methanol, and were stored at −20 °C. They were diluted with
   70% methanol before mass spectrometry in gradient concentrations.

2.2. Preparations of the Acute Liver Injury Model

   Sixty male SD rats were randomly divided into six groups (10 rats in
   each group): a blank control group (BG, oral administration of saline),
   a model group (MG, oral administration of saline), a bifendate control
   group (DDB, oral administration of bifendate 200 mg/kg), and low-dose
   (LD), medium-dose (MD), and high-dose (HD) SBS-treated groups (oral
   administration of 100, 200, 400 SBS mg/kg, respectively). All materials
   were treated for 7 days. After 2 h from the last oral administration,
   50% CCl[4] solved in olive oil (2 mL/kg) were administered orally to
   all animal groups except BG. The BG group was given the same volume of
   saline orally.

2.3. Pathological Examination of Liver Tissue

   Three rats in each group were randomly selected and anesthetized by
   intraperitoneal injection of 20% urethane. After laparotomy, samples
   were collected from the edge of the left lobe of the liver and placed
   in 3% dialdehyde. Ultrathin sections were obtained through the
   processes of fixing, dehydrating, and permeation embedding. Before
   observing these sections using the transmission electron microscope, we
   used the uranium acetate and lead citrate to stain them successively.
   In the same three rats, samples collected at the edge of the right lobe
   of the liver were placed and fixed in 10% neutral formaldehyde. After
   dehydration, pruning, embedding, sectioning, staining, sealing, and HE
   staining were performed, the histopathological changes of the rats’
   livers in each group were observed under a light microscope.

2.4. Measurement of Biochemical Index in Liver Tissue Homogenate

   Liver tissues were mixed with saline at a ratio of 1:9, and then the
   mixture was homogenized to make the liver tissue homogenate. After
   removing the supernatant, the levels of SOD, GSH-Px, Cat, γ-GT, T-AOC,
   total protein, MDA, COX-2, and PGE[2] were measured in the liver tissue
   homogenate according to the instructions of the test kit.

2.5. Sample Preparation

2.5.1. Extraction of Hydrophilic Compounds

   A quantity of 50 mg of sample was taken and homogenized with 1 mL of
   ice-cold methanol/water (70%, v/v). Cold steel balls were added to the
   mixture and homogenized at 30 Hz for 3 min. After stirring the mixture
   for 1 min, it was centrifuged in 12,000 rpm at 4 °C for 10 min. The
   collected supernatant was later used for LC-MS/MS analysis.

2.5.2. Extraction of Hydrophobic Compounds

   A quantity of 50 mg of sample was taken and homogenized with 1 mL of
   mixture (including methanol, MTBE, and internal standard mixture) and
   cold steel balls were added. After the steel balls were removed, the
   mixture was stirred for 2 min. Subsequently, 500 μL of water was added
   and the mixture was stirred for 1 min, and then it was centrifuged in
   12,000 rpm at 4 °C for 10 min. Following this, 500 μL of supernatant
   was concentrated and dried. The powder was dissolved in 100 μL of
   mobile phase B, and stored at −80 °C. Finally, the dissolving solution
   was taken into the sample bottle for LC-MS/MS analysis.

2.6. Analysis by UPLC

   The samples were analyzed using the LC-ESI-MS/MS system (UPLC,
   Shim-pack UFLC SHIMADZU CBM A system, [57]https://www.shimadzu.com/
   (accessed on 21 February 2020); MS, QTRAP^® System,
   [58]https://sciex.com/ (accessed on 21 February 2020)).

   The UPLC conditions of hydrophilic compounds were as follows. A Waters
   ACQUITY UPLC HSS T3 C18 (1.8 µm, 2.1 mm × 100 mm) column was used. The
   column temperature was 40 °C. The flow rate was 0.4 mL/min, and the
   injection volume was 2 μL. The mobile phase was water (0.04% acetic
   acid): acetonitrile (0.04% acetic acid). The gradient condition was as
   follows: 95:5 v/v at 0 min, 5:95 v/v at 11.0 min, 5:95 v/v at 12.0 min,
   95:5 v/v at 12.1 min, 95:5 v/v at 14.0 min.

   The UPLC conditions of hydrophobic compounds were as follows. A Thermo
   C30 (2.6 μm, 2.1 mm × 100 mm) column was used. The mobile phase was a
   mixed solvent gradient system with the following contents:
   A—acetonitrile/water (60/40 V, 0.04% acetic acid, 5 mmol/L ammonium
   formate); and B—acetonitrile/isopropanol (10/90 V, 0.04% acetic acid, 5
   mmol/L ammonium formate). The gradient condition was as follows: A/B
   (80:20 v/v) at 0 min, 50:50 v/v at 3.0 min, 35:65 v/v at 5 min, 25:75
   v/v at 9 min, 10:90 v/v at 15.5 min. The flow rate was 0.35 mL/min. The
   column temperature was 45 °C, and the injection volume was 2 μL. The
   effluent was alternatively connected to an ESI-triple quadrupole-linear
   ion trap (QTRAP)-MS.

2.7. Analysis by ESI-Q TRAP-MS/MS

2.7.1. ESI-Q TRAP-MS/MS of Hydrophilic Compounds

   LIT and triple quadrupole (QQQ) scans were acquired on a triple
   quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP^® LC-MS/MS
   System, equipped with an ESI Turbo Ion-Spray interface, operating in
   positive and negative ion mode and controlled by Analyst 1.6.3 software
   (Sciex). The ESI source operation parameters were as follows: the
   source temperature was 500 °C, the ion spray voltage (IS) was 5500 V
   (positive) and −4500 V (negative), the ion source gas I (GSI), gas II
   (GSII), and curtain gas (CUR) were set at 55, 60, and 25.0 psi,
   respectively, and the collision gas (CAD) was high. Instrument tuning
   and mass calibration were performed with 10 and 100 μmol/L
   polypropylene glycol solutions in QQQ and LIT modes, respectively. A
   specific set of MRM transitions was monitored for each period according
   to the metabolites eluted within this period.

2.7.2. ESI-Q TRAP-MS/MS of Hydrophobic Compounds

   LIT and triple quadrupole (QQQ) scans were acquired on a triple
   quadrupole-linear ion trap mass spectrometer (Q TRAP), API QTRAP
   LC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface,
   operating in a positive ion mode and controlled by Analyst 1.6 software
   (AB Sciex). The ESI source operation parameters were as follows: the
   ion source, turbo spray, and source temperature were 550 °C, the ion
   spray voltage (IS) was 5500 V, the ion source gas I (GSI), gas
   II(GSII), and curtain gas (CUR) were set at 55, 60, and 25.0 psi,
   respectively, and the collision gas (CAD) was high. Instrument tuning
   and mass calibration were performed with 10 and 100 μmol/L
   polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ
   scans were acquired as MRM experiments with collision gas (nitrogen)
   set to 5 psi. DP and CE for individual MRM transitions were conducted
   with further DP and CE optimization. A specific set of MRM transitions
   was monitored for each period according to the metabolites eluted
   within this period.

2.8. Transcriptome Analysis

2.8.1. RNA Detection

   There were three steps: (i) Agarose gel electrophoresis: the integrity
   of RNA was analyzed and it was determined whether there was DNA
   pollution; (ii) Qubit 2.0 fluorometer: high-precision measurement of
   RNA concentration was performed; (iii) Agilent 2100 biological
   analyzer: accurate detection of RNA integrity was performed.

2.8.2. Library Setup

   There are two ways to obtain mRNA: one is to enrich the mRNA with
   poly-A tail by oligo (DT) magnetic beads using most of the mRNA of
   eukaryotes with poly-A tails. The other is to remove ribosomal RNA from
   total RNA to obtain mRNA, and then add the fragmentation buffer. The
   first strand of cDNA was synthesized by random hexamers. The second
   strand cDNA was synthesized by adding buffer, dNTPs (dUTP, dATP, dGTP,
   and DCTP), and DNA polymerase I. The double-stranded cDNA was purified
   by AMPure XP beads. Then, end repair, addition of the A-tail, and the
   connecting of sequencing adaptors were performed on the purified
   double-stranded cDNA. Next, AMPure XP beads were used for fragment size
   selection. Finally, the final cDNA library was obtained by performing
   PCR enrichment.

Library Quality Check

     * (i)
       Qubit2.0 was used for preliminary quantification, and Agilent 2100
       was used to detect the insert size of the library. The next
       experiment could only be carried out after the insert size met
       expectations;
     * (ii)
       The Q-PCR method accurately quantified the effective concentration
       of the library (the effective concentration of the library was >2
       nM). Then, the library check was successfully performed.

Sequencing on the Machine

   After the library was qualified, different libraries were pooled
   according to the target offline data volume and sequenced using the
   Illumina hiseq platform.

2.9. Statistical Analysis

   SPSS 26.0 statistical software was used to analyze the data, expressed
   as mean ± standard deviation, and ANOVA was used for data analysis,
   followed by Bonferroni post hoc tests, with p < 0.05 determining
   statistical significance. After the LC-MS/MS analysis of metabolomic
   data, the mass spectrometry data were processed by Software Analyst
   1.6.3. The genomic data filtered off-line data to obtain evident data.
   After performing comparisons with the designated reference genome,
   mapped data were obtained that were used for structural level analysis,
   such as alternative splicing analysis, new gene discovery, and gene
   structure optimization. According to the expression levels of genes in
   different samples or different groups, differential expression
   analysis, differential expression gene function annotation, function
   enrichment, and other expression levels were analyzed.

3. Results

3.1. Effects of SBS on SOD, MDA, and GSH-P[X] in Rats with CCl[4]-Induced
Liver Injury

   As shown in [59]Figure 1, compared with group BG, the levels of SOD and
   GSH-Px in the MG group decreased significantly, while the MDA content
   showed a considerable increase (p < 0.01). Compared to the MG group,
   the MD and HD groups exhibited an increase in the SOD level with
   statistically significant differences (p < 0.01). The level of SOD in
   the MD and HD groups was close to that in group BG. The values of MDA
   content in the LD, MD, and HD groups were all significantly decreased
   (p < 0.05 or p < 0.01), and the BG group showed a similar MDA content
   value to group HD. In addition, there was an increase in the level of
   GSH-Px in the LD, MD, and HD groups, with statistically significant
   differences (p < 0.01). Furthermore, the level of GSH-Px in the HD
   group was close to that in group BG. These results indicated that SBS
   has the effects of anti-lipid peroxidation and reducing lipid
   peroxidation products in the defense system of the body.

Figure 1.

   [60]Figure 1
   [61]Open in a new tab

   Effects of SBS on SOD, MDA, and GSH-Px in liver tissue. Data are
   presented as mean ± S.E.M. (n = 10). Significant differences compared
   with the BG group were designated as # p < 0.01 and ## p < 0.05, with
   the MG group as * p < 0.05 and ** p < 0.01, and with the DDB groups as
   ▲ p < 0.05.

3.2. Effects of SBS on CAT and T-AOC in Rats with CCl[4]-Induced Liver Injury

   [62]Figure 2 demonstrates that the CAT and T-AOC levels in group MG
   were considerably lower than those in the BG (p < 0.01) group. Compared
   to the MG group, the levels of CAT in the LD, MD, and HD groups were
   significantly higher (p < 0.01). The increase in T-AOC level in the HD
   group showed a statistical significance (p < 0.01) compared with the MG
   group. These results suggested that SBS has the effect of anti-lipid
   peroxidation.

Figure 2.

   [63]Figure 2
   [64]Open in a new tab

   Effects of SBS on CAT and T-AOC in liver tissue. Data are presented as
   mean ± S.E.M. (n = 10). Significant differences compared with the BG
   group were designated as ## p < 0.05, with the MG group as ** p < 0.01,
   and with the DDB groups as ▲ p < 0.05.

3.3. Effects of SBS on γ-GT, TP, PGE[2], and COX-2 in Rats with
CCl[4]-Induced Liver Injury

   As shown in [65]Figure 3 and [66]Figure 4, the increased levels of
   γ-GT, PGE2, and COX-2, and the reduction in TP in the MG group were
   significant (p < 0.01) in comparison to the BG group. The level of γ-GT
   in HD was significantly lower than that in the MG group (p < 0.01). The
   comparison to group MG showed that the PGE[2] levels in the MD and HD
   groups were significantly reduced (p < 0.01). The levels of PGE[2] in
   the MD and HD groups were close to that in the BG group. Similar
   observations were also found by comparing the COX-2 levels in the
   SBS-treated group and the MG group, and a significant decrease (p <
   0.01) was observed in the HD group. In addition, a close level of COX-2
   in the HD group and BG group was found. As for the TP level, there was
   a considerable increase (p < 0.05 or p < 0.01) in the LD, MD, and HD
   groups, and two groups (MD and HD) shared a similar level of TP with
   the BG group. These results provided evidence that SBS can lower the
   level of inflammation and alleviate the liver injury.

Figure 3.

   [67]Figure 3
   [68]Open in a new tab

   Effects of SBS on COX-2 and PGE2 in liver tissue. Data are presented as
   mean ± S.E.M. (n = 10). Significant differences compared with the BG
   group were designated as ## p < 0.05, with the MG group as ** p < 0.01,
   and with the DDB groups as ▲ p < 0.05.

Figure 4.

   [69]Figure 4
   [70]Open in a new tab

   Effects of SBS on γ-GT and TP in liver tissue. Data are presented as
   mean ± S.E.M. (n = 10). Significant differences compared with the BG
   group were designated as # p < 0.01 and ## p < 0.05, with the MG group
   as * p < 0.05 and ** p < 0.01, and with the DDB groups as ▲ p < 0.05.

3.4. Electron Microscopic Observations of Liver Tissue

   An important observation could be obtained based on [71]Figure 5, where
   the structure of liver tissues of group BG was obvious and normal.
   However, the MG group showed a fuzzy liver structure and obvious damage
   (e.g., necrotic liver cells, a large number of swollen mitochondria,
   many lipid droplets in the cytoplasm, and a large number of the
   expanded endoplasmic reticulum) was found. In the DDB group, the liver
   structure was partially clear, but some mitochondria were still
   slightly swollen and autophagic, and some liver cells were suspected to
   be necrotic. In addition, a small number of lipid droplets were formed.
   The liver structure of the LD and MD groups was clear, but there were
   still some rough expansions of endoplasmic reticulum and a small number
   of lipid droplets, as well as a few necrotic hepatocytes. The HD group
   had a relatively evident liver structure with abundant mitochondria in
   the cytoplasm, and the mitochondrial structure was basically obvious
   and clear. It was also found that necrotic hepatocytes and lipid
   droplets were greatly reduced.

Figure 5.

   [72]Figure 5
   [73]Open in a new tab

   Electron microscopic observation.

   These results of the electron microscopic observations showed that
   liver injury in the MG group was obvious in comparison to the BG group.
   The LD, MD, and HD groups showed different degrees of improvement in
   terms of liver damage expansion, among which the HD group showed the
   most obvious effect.

3.5. Light Microscopic Observations of Liver Tissue

   [74]Figure 6 and [75]Table 1 show light microscopic observations of
   liver tissue. Glisson’s capsule of group BG was intact, and the
   morphology of hepatocytes was normal. Compared with group BG, fatty
   degeneration and feathery degeneration of hepatocytes were found, and
   the hepatocytes showed multiple punctate necrosis with obvious injury
   in the MG group. Comparisons between the MG and DDB groups indicated
   that Glisson’s capsule was intact and the degree of hepatocyte necrosis
   was significantly reduced in the DDB group. Additionally, Glisson’s
   capsule of two groups (LD and MD) was slightly damaged, and only a few
   hepatocytes were necrotic, as well as showing a small amount of fatty
   degeneration and feathery degeneration. In particular, the HD group
   showed an intact Glisson’s capsule, and the degree of liver cell lesion
   was greatly reduced.

Figure 6.

   [76]Figure 6
   [77]Open in a new tab

   Light microscopic observation.

Table 1.

   Pathological results.
   Group Results
   BG (−)
   MG Feathery degeneration of hepatocyte (++)
   Fatty degeneration of hepatocytes (++)
   Hepatocyte necrosis (++)
   Inflammatory cell infiltration (+)
   DDB A small amount of feathery degeneration of hepatocytes (+)
   A small amount of fatty degeneration of hepatocytes (+)
   Hepatocyte necrosis (+)
   LD Feathery degeneration of hepatocytes (++)
   Fatty degeneration of hepatocytes (+)
   Hepatocyte necrosis (+)
   Inflammatory cell infiltration (+)
   MD Feathery degeneration of hepatocytes (++)
   Fatty degeneration of hepatocytes (+)
   Hepatocyte necrosis (++)
   Inflammatory cell infiltration (+)
   HD A small amount of feathery degeneration of hepatocytes (+)
   A small amount of fatty degeneration of hepatocytes (+)
   Hepatocyte necrosis (+)
   [78]Open in a new tab

   Observations from light microscopy demonstrated that the degree of
   pathological change in the MG group was significantly aggravated
   compared to the BG group. Through further analysis, it was concluded
   that in comparison to group MG, the degrees of pathological change in
   the LD, MD, and HD groups were all alleviated, and the alleviation in
   group HD was the most obvious.

3.6. Identification and Analysis of Metabolites

3.6.1. Discriminant Analysis of Orthogonal Partial Least Squares Method

   Based on the self-built Metware Database (MWDB), qualitative analysis
   was performed according to the retention time (RT), ion-pair
   information, and secondary spectrum data. The hierarchical clustering
   map ([79]Figure 7) and the Partial least squares discriminant analysis
   (PLS-DA) shown in [80]Figure 8 were used to analyze the metabolic
   pattern of each group. It was found that the global metabolic statuses
   were significantly different between group MG and group BG, indicating
   that CCl4 caused an obvious metabolic disorder in the liver. In
   addition, the global metabolic statuses of the LD, MD, and HD groups
   were highly different from that of the MG group. Meanwhile, the
   metabolic trend of the HD group was close to that of group BG. These
   results indicated that SBS can effectively regulate the metabolic
   disorders induced by CCl4.

Figure 7.

   [81]Figure 7
   [82]Open in a new tab

   Heat map. The x-axis represents groups, and the y-axis represents
   differentially expressed genes. Data (peak areas) were normalized
   between −5 and 5 (green—the lowest level; red—the highest level).

Figure 8.

   [83]Figure 8
   [84]Open in a new tab

   OPLS-DA S-plot. S-plot from OPLS-DA. Covariance and correlation were
   plotted on the x- and y-axis, respectively. The variables located far
   from the origin represent influence with high reliability in the model.

3.6.2. Determination of Relevant Metabolite

   The S-plot ([85]Figure 8) and variable importance in projection (VIP)
   were used to identify potential metabolites. The altered metabolites
   with statistical significance were screened using S-plot, and they were
   located in the upper right quadrant or in the lower left quadrant
   (i.e., situated away from the origin). The metabolites that changed
   significantly were selected according to the fold change (more than 1.5
   or less than 0.67), the VIP score (more than 1.0), and FDR < 0.05. The
   t-test and ANOVA were employed to test the significant difference of
   metabolites between the groups. Referring to literature reports and
   searching in the online database (HMDB), 17 metabolites (shown in
   [86]Table 2) related to the inhibitory effect of SBS on CCl[4]-induced
   liver damage were selected. The criteria for selecting the metabolite
   was based on the selection of the common differential metabolites that
   appeared in each group, which were related to acute liver injury. In
   the MG group, the levels of L-malic acid, 7Z, 10Z, 13Z, 16Z,
   19Z-docosapentaenoic acid, creatine, n-acetyl-l-alanine,
   N-acetylaspartate, Trigonelline, 4-guanidinobutyric acid,
   N-amidino-L-aspartate, CE (16:1), CE(18:2), PE (16:1/16:0), DG
   (16:0/20:2/0:0), TG (14:0/18:0/18:2), TG (14:0/18:0/20:4), and TG
   (16:0/16:1/22:5) were significantly increased, and levels of
   n-glycyl-l-leucine and FFA (6:0) were obviously reduced compared to the
   BG group. The above-mentioned metabolites were significantly modulated
   in SBS groups with different doses in comparison to the MG group. As
   the metabolic trend of the HD group was close to that of group BG,
   which means that there was no significant difference in the metabolites
   mentioned above, HD vs. BG is not shown in [87]Table 2. These results
   suggested that SBS can regulate metabolic disorders through the key
   metabolites analyzed above.

Table 2.

   Effects of SBS on metabolic in liver tissues of rats with acute liver
   injury induced by CCl[4] (X ± s, n = 6).
   NO. Metabolites RT MG/BG LD/MG MD/MG HD/MG DDB/MG Pathway
   1 L-Malic Acid 0.88 ↑ * ↓/- ↓ * ↓ * ↓/- Citrate cycle
   (TCA cycle)
   2 7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenoic acid 11.3 ↑ * ↓ * ↓ * ↓ * ↓/-
   Biosynthesis of unsaturated fatty acids
   3 creatine 0.78 ↑ * ↓/- ↓ * ↓ * ↓ * Arginine-proline metabolism
   4 n-acetyl-l-alanine 1.3 ↑ * ↓/- ↓ * ↓ * ↓ * -
   5 N-Acetylaspartate 0.69 ↑ * ↓/- ↓ * ↓ * ↓ * Alanine, aspartate and
   glutamate metabolism
   6 Trigonelline 0.77 ↑ * ↓/- ↓ * ↓ * ↓/- Nicotinate and nicotinamide
   metabolism
   7 4-guanidinobutyric acid 0.85 ↑ * ↓ * ↓ * ↓ * ↓ * Arginine-proline
   metabolism
   8 N-Amidino-L-Aspartate 0.96 ↑ * ↓ * ↓ * ↓ * ↓ * -
   9 n-glycyl-l-leucine 1.8 ↓ * ↑/- ↑ * ↑ * ↑/- -
   10 FFA(6:0) 0.73 ↓ * ↑ * ↑ * ↑ * ↑ * Fat digestion and absorption
   11 CE(16:1) 13.12 ↑ * ↓ * ↓ * ↓ * ↓ * -
   12 CE(18:2) 13.21 ↑ * ↓ * ↓ * ↓ * ↓ * -
   13 PE(16:1/16:0) 6.25 ↑ * ↓ * ↓ * ↓ * ↓ * Glycerophospholipid
   metabolism
   14 DG(16:0/20:2/0:0) 9.18 ↑ * ↓/- ↓ * ↓ * ↓/- Glycerolipid metabolism
   15 TG(14:0/18:0/18:2) 11.85 ↑ * ↓/- ↓ * ↓ * ↓ * Glycerolipid metabolism
   16 TG(14:0/18:0/20:4) 11.81 ↑ * ↓/- ↓ * ↓ * ↓ * Glycerolipid metabolism
   17 TG(16:0/16:1/22:5) 11.41 ↑ * ↓/- ↓ * ↓ * ↓ * Glycerolipid metabolism
   [88]Open in a new tab

   * p < 0.05, there was a significant difference between two groups. /-,
   there was no significant difference between two groups.

3.6.3. Metabolic Pathway Enrichment Analysis

   The Enrichment Analysis was conducted based on all the metabolites. We
   set all the substances with KEGG annotation as the background, without
   screening. The KEGG Enrichment formulation is as follows:
   [MATH:
   <mrow><mrow><mi>P</mi><mo>=</mo><mn>1</mn><mo>−</mo><munderover><mstyle
   mathsize="140%"
   displaystyle="true"><mo>∑</mo></mstyle><mrow><mi>i</mi><mo>=</mo><mn>0<
   /mn></mrow><mrow><mi>m</mi><mo>−</mo><mn>1</mn></mrow></munderover><mfr
   ac><mrow><mrow><mo>(</mo><mrow><mtable><mtr><mtd><mi>M</mi></mtd></mtr>
   <mtr><mtd><mi>i</mi></mtd></mtr></mtable></mrow><mo>)</mo></mrow><mrow>
   <mo>(</mo><mrow><mtable><mtr><mtd><mrow><mi>N</mi><mo>−</mo><mi>M</mi><
   /mrow></mtd></mtr><mtr><mtd><mrow><mi>n</mi><mo>−</mo><mi>i</mi></mrow>
   </mtd></mtr></mtable></mrow><mo>)</mo></mrow></mrow><mrow><mrow><mo>(</
   mo><mrow><mtable><mtr><mtd><mi>N</mi></mtd></mtr><mtr><mtd><mi>n</mi></
   mtd></mtr></mtable></mrow><mo>)</mo></mrow></mrow></mfrac></mrow></mrow
   > :MATH]

   where N denotes the number of metabolites with KEGG annotation in the
   entire list of metabolites, and n is the number of differential
   metabolites in N metabolites. In addition, M represents the number of
   metabolites of a KEGG pathway in N, and m is defined as the
   differential metabolite number of a KEGG pathway in M metabolites. The
   enrichment analysis process was carried out using our script, and the
   ggplot2 was used to draw the plots. The rich factor means m/M.

   [89]Figure 9 shows the metabolic pathway enrichment analysis. Using
   MetaboAnalyst 3.5, the pathways affected by SBS were analyzed. The dots
   in [90]Figure 9 reveal that: (i) the larger the dot size, the greater
   the number differential metabolites that were enriched in the pathway;
   (ii) the redder the dot, the more significant the enrichment of the
   pathway. As shown in [91]Table 2 and [92]Figure 9, the metabolism of
   citrate cycle (TCA cycle), biosynthesis of unsaturated fatty acids,
   arginine-proline metabolism, alanine, aspartate and glutamate
   metabolism, niacin and niacinamide metabolism, fat digestion,
   absorption and glycerophospholipid metabolism, and glycerolipid
   metabolism were the most affected metabolic pathways when SBS regulated
   the disordered metabolic levels induced by liver injury. These results
   indicated that SBS affects the metabolism of liver tissue by regulating
   the above metabolic pathways, thus reducing the degree of liver injury.

Figure 9.

   [93]Figure 9
   [94]Open in a new tab

   Pathway enrichment. Summary of pathway analysis of significantly
   reversed metabolites between rats in MG and SBS-treated rats. Rich
   factor and KEGG pathway were plotted on the x- and y-axis,
   respectively. The larger the dot, the greater the number of metabolites
   that were enriched in the pathway. The darker the dot color, the more
   significant the enrichment. (a) Nicotinate and nicotinamide metabolism.
   (b) Alanine, aspartate, and glutamate metabolism. (c) Citrate cycle
   (TCA cycle). (d) Arginine-proline metabolism. (e) Glycerophospholipid
   metabolism. (f) Glycerolipid metabolism.

3.7. Identification and Selection of Differential Genes

3.7.1. Differential Gene Volcano Map

   [95]Figure 10 shows the differentially expressed genes that were
   screened out under the condition of |log[2]FoldChange| = 1 and FDR <
   0.05. The overall distribution of differentially expressed genes can be
   seen in the volcano map. Compared to the BG group, there were 959 genes
   expressed differently in group MG, of which 379 genes were
   down-regulated and 580 genes were up-regulated. On the other hand,
   comparing the MG group with the three SBS groups, we found that: (i)
   229 genes were expressed differently in the LD group, with 65
   down-regulated genes and 164 up-regulated genes; (ii) 42 out of 75
   differentially expressed genes in the MD were down-regulated and the
   rest were up-regulated; (iii) among 404 differentially expressed genes
   in the HD group, there were 211 down-regulated genes and 193
   up-regulated genes. Furthermore, comparing HD with BG, there were only
   four differentially expressed genes, which were meaningless
   differences. These results suggested that SBS can regulate gene
   expression disorders in rats.

Figure 10.

   [96]Figure 10
   [97]Open in a new tab

   Volcano. Fold change in gene expression and significance level of the
   differential gene are plotted on the x- and y-axis, respectively.
   Up-regulated genes are represented by red dots, down-regulated genes
   are represented by green dots, and genes without differential
   expression are represented by blue dots.

3.7.2. Expression of Key Genes

   There were three overlapping differentially expressed genes among the
   six groups. These overlapping genes probably represented the related
   genes that alleviated liver injury using SBS. [98]Figure 11 indicates
   that SBS was capable of down-regulating the expression levels of CYP1A1
   and Noct, and up-regulating the expression of TUBB6 in the model group.
   The level of CYP1A1 in HD group was close to that of the BG group. As a
   result, SBS showed the ability to down-regulate the expression of NOCT,
   thereby reducing the hepatocyte steatosis induced by CCl[4]. In
   addition, SBS increased the TUBB6 expression, which may be related to
   the inhibition of inflammatory response by SBS. The reduction in CYP1A1
   overexpression in group MG indicated that SBS can inhibit lipid
   peroxidation and reduce lipid peroxidation products, thus mitigating
   the CCl[4]-induced liver damage.

Figure 11.

   [99]Figure 11
   [100]Open in a new tab

   Heat map. The x-axis represents groups, and the y-axis represents
   differentially expressed genes. Data (peak areas) were normalized
   between −4 and 4 (green—the lowest level; red—the highest level).

4. Discussion

   After being treated with CCl[4], liver cytochrome P-450 in rats is
   activated, and then the trichloromethyl free radical (·CCl[3]) is
   produced, which is covalently coupled with cell macromolecules, thus
   affecting cell functions. The produced CCl[3] attack the phospholipid
   molecules on the liver cell membrane, causing a lipid peroxidation
   reaction, as well as destroying the integrity of the structure and
   function of the liver cell membrane. Meanwhile, the levels of SOD, MDA,
   and other related lipid peroxidation indices in liver tissues are
   altered, ultimately leading to impaired liver function [[101]20].
   Therefore, liver cells are encouraged to release various enzymes into
   the blood, including ALT, AST, and AKP, which are sensitive indicators
   of liver cell damage [[102]21]. In addition to the above reactions,
   CCl[4] also causes inflammation in the body, resulting in changes in
   inflammatory cytokines in the blood (IL-6 and TNF-α) and in liver
   tissues (COX-2 and PGE[2]) [[103]22]. In terms of metabolomics, after
   CCl[4] is absorbed by the body, the level of cytochrome P450 increases,
   leading to a series of lipid peroxidation reactions, resulting in
   disorder of the TCA circulation, and other related pathways are also
   affected [[104]23]. The present work studied the effects of SBS on
   pathological changes of liver tissues and liver indices in rats with
   acute liver injury induced by CCl[4]. Our previous study reported that
   SBS can reduce ALT, AST, AKP, TNF-α, and IL-6 levels in the blood of
   rats.

   The current study found that SBS reduces CYP1A1 and, therefore, affects
   CCl[4]-induced lipid peroxidation. Furthermore, the levels of SOD, MDA,
   as well as other related lipid peroxidation indices in liver tissues
   are regulated. Then, the TCA cycle is modulated. Due to the fact that
   TCA cycle connects to other pathways, as shown in [105]Figure 12, these
   pathways are modulated. The experimental results ([106]Section 3.7.2)
   showed that SBS can also regulate Noct and TUBB6 in rats. By
   investigating existing studies, we found that ALT, AST, AKP, TNF-α, and
   IL-6, which were analyzed in our previous study, have connections to
   this present work in three aspects: (i) IL-6 is related to
   branched-chain amino acids (BCAA); (ii) ALT and AST are associated with
   alanine, aspartic acid, and glutamate metabolism; (iii) there are
   connections between TNF-α and TUBB6. Detailed explanations about these
   connections and corresponding conclusions will be given in the
   following sections.

Figure 12.

   [107]Figure 12
   [108]Open in a new tab

   Metabolite pathway. Summary diagram of metabolic pathway relationships.
   The up-regulated substances are represented in red font, and the
   down-regulated substances are represented in green font. Dashed lines
   represent indirect relationships between substances, and solid lines
   represent direct relationships between substances.

   A large number of studies have shown that the liver has a strong
   antioxidant defense mechanism [[109]24], and there is a certain
   relationship between lipid oxidation and liver diseases [[110]25]. SOD
   helps to remove free radicals, inhibit lipid peroxidation, and reduce
   lipid peroxidation products in the defense system of the body
   [[111]26]. GSH-Px is an important type of enzyme that catalyzes
   hydrogen peroxide (H[2]O[2]), which can scavenge lipid peroxidase
   caused by reactive oxygen species (ROS), thus protecting the structural
   and functional integrity of cell membranes [[112]27]. It should be
   mentioned that the main function of CAT is to remove lipid
   hydroperoxide and H[2]O[2]. After SOD disproportionates oxygen free
   radicals into H[2]O[2], CAT decomposes them into water, thereby
   removing active oxygen in the body [[113]28]. T-AOC is an indicator
   that reflects the state of the body’s antioxidant defense system. The
   higher activity of T-AOC and SOD in the body means a faster removal of
   free radicals [[114]29]. The content of lipid peroxidation product
   malondialdehyde (MDA) reflects the degree of tissue peroxidation
   damage, and the damage of cells can also be indirectly reflected
   [[115]30]. Furthermore, CCl[4] increases the levels of PGE[2], COX-2,
   and γ-GT, and decreases the TP level. The content of prostaglandins is
   closely related to the occurrence and development of inflammation
   [[116]31]. In particular, PGE[2] plays the main role in the
   inflammatory process and is an important mediator of the inflammatory
   response [[117]32]. As a kind of inducible enzyme, COX-2 also
   participates in liver inflammatory injury. When cells are stimulated by
   pathogenic factors, the synthesis and release of COX-2 will be induced
   [[118]33]. Moreover, γ-GT plays a critical role in the clinical
   diagnosis of liver injury diseases, which is also one of the most
   important indicators for evaluating liver functions [[119]34]. In
   addition, the TP level reflects the synthetic ability of the liver
   [[120]35]. In the present study, SBS was found to significantly
   increase the levels of antioxidant enzymes (SOD, GSH-Px, and CAT), and
   the activity of T-AOC ([121]Section 3.2). In addition, the levels of
   γ-GT and PGE[2], and the content of MDA, were reduced ([122]Section
   3.1). These results suggest that SBS has the effect of antagonizing
   acute liver injury, and the mechanism is probably related to the
   anti-lipid peroxidation and inhibition of the inflammatory reaction.

   The TCA cycle is reported as the final common oxidation pathway of fat,
   carbohydrates, and amino acids, and is also the most important central
   pathway that connects almost all metabolic pathways of individuals
   [[123]36]. In addition, the TCA cycle is closely related to some liver
   diseases. The accumulation of lipids and loss of insulin lead to two
   changes in the livers of mice: (i) an increase in oxidation and (ii)
   the replenishment pathways of the TCA cycle [[124]37]. Slowing down the
   TCA cycle can reduce the production of reactive oxygen species (ROS)
   and protect the liver [[125]38]. In this work, as shown in [126]Table 2
   and [127]Figure 12, SBS reduces the level of malic acid in the MG and
   alleviates the disorder of the TCA cycle.

   Arginine is known as a semi-essential amino acid, which has many
   biological functions in the human body [[128]39]. It is also an
   intermediate in the urea cycle, the substrate for protein synthesis,
   and acts as the precursor of nitric oxide (NO), proline, glutamic acid,
   polyamines, and creatine [[129]40]. In this study, as shown in
   [130]Table 2 and [131]Figure 12, after treatment with CCl[4], there is
   an abnormal increase in the level of arginine caused by creatine, but
   the SBS treatment reduces the creatine level in liver tissues. It
   indicates that SBS has the ability to mediate the dysfunction of
   creatine or any arginine intermediate products that could be converted
   into creatine, which means that SBS can effectively reduce liver damage
   in rats.

   Branched-chain amino acids (BCAA), including leucine, isoleucine, and
   valine, play important roles in the liver, such as inducing
   mitochondrial biogenesis, inhibiting the production of reactive oxygen
   species (ROS), and stimulating both the production of hepatocyte growth
   factors and the synthesis of albumin and glycogen. BCAA has also been
   found to participate in hepatocyte apoptosis and improve insulin
   resistance [[132]41]. In addition, branched-chain amino acids have been
   shown to: (i) effectively reverse CCl[4]-induced liver injury by
   down-regulating the TGF-β1 and Smad3/Smad7 signaling pathways; (ii)
   reduce the pro-fibrotic mediators TGF-β1 and IL-6; and (iii) improve
   glucose metabolism and insulin resistance [[133]42]. Leucine is a kind
   of BCAA, which stimulates hepatic stellate cells (HSCs) to produce the
   hepatocyte growth factor for promoting protein synthesis. The
   simulation means that leucine also has a positive effect on patients
   with chronic liver disease [[134]43]. In the present study (shown in
   [135]Table 2 and [136]Figure 12), compared to group BG, the leucine
   level in group MG decreased, while the level of leucine increased
   significantly when using SBS. In our previous study, SBS showed the
   effect of decreasing the level of IL-6. This result is consistent with
   the conclusion of this study, i.e., SBS can alleviate the liver injury
   in rats by increasing the level of BCAA and decreasing the level of
   IL-6.

   Nicotinate is known as one of the 13 essential vitamins for the human
   body, which can be converted into nicotinamide [[137]44]. Nicotinamide
   is a component of coenzyme I and II, and participates in lipid
   metabolism, tissue respiratory oxidation processes, and the anaerobic
   decomposition of carbohydrates [[138]45]. Glycerin phospholipid is the
   most abundant phospholipid in the human body [[139]46], known as an
   important bile component and membrane surfactant. It is also involved
   in the recognition of protein signal transduction through the cell
   membrane [[140]47]. Alanine, aspartate, and glutamate metabolism,
   involved in the immune regulation system and in lymphocyte regeneration
   [[141]48], are related to ALT and AST. By analyzing the results shown
   in [142]Table 2 and [143]Figure 12, SBS was found to alleviate
   CCl[4]-induced liver injury by improving the biological pathways that
   were mentioned above.

   Cytochrome P4501A1 (CYP1A1) is known as a subtype of the cytochrome
   P450 family, which was found to be an important regulator of reactive
   oxygen generation [[144]49]. CYP1A1 is capable of inducing oxidative
   stress as a result of the over-production of reactive oxygen [[145]50].
   Some studies have shown that blocking CYP1A1 partially inhibits MDA
   deposition, indicating that the deletion of CYP1A1 helps to alleviate
   excessive deposition of lipid peroxide, and the concentration of ROS
   and SOD can be affected by the treatment of HepG2 cells with
   CYP1A1-siRNA [[146]51]. This means that CYP1A1 regulates lipid
   peroxidation by affecting the ROS and SOD levels [[147]52].
   Furthermore, overexpression of CYP1A1 was found to increase the level
   of MDA and reduce the concentration of SOD, further indicating the
   importance of CYP1A1 in lipid peroxidation [[148]53]. The present study
   shows that SBS reduces CYP1A1 overexpression in group MG (shown in
   [149]Figure 11 and [150]Figure 12). This means that SBS is capable of
   inhibiting lipid peroxidation and reducing lipid peroxidation products.
   These findings are consistent with the detection results of MAD and SOD
   in our experiments.

   TUBB6 belongs to one type of β-tubulin. Cell proliferation is closely
   related to the synthesis of β-tubulin. The positive and negative
   effects of regulatory factors on β-tubulin are likely to be able to
   regulate cell proliferation [[151]54]. Existing studies have shown that
   TNF-α not only inhibits cell proliferation, but also inhibits the
   expression of β-tubulin mRNA [[152]55]. TNF-α is capable of: (i)
   down-regulating the number of parathyroid hormone receptors, (ii)
   inhibiting the concentration of cAMP and cytosolic free Ca^++; and
   (iii) reducing the polymerized tubulin [[153]56]. Our previous study
   found that SBS plays important roles in effectively inhibiting the
   inflammatory response of hepatocytes mediated by TNF-α, protecting
   hepatocytes, and significantly reducing the inflammatory lesions and
   structural damage of hepatocytes, thereby evidently improving liver
   functions of rats with acute liver injury. In this study, SBS increased
   TUBB6 in the MG group ([154]Section 3.7.2), which was probably related
   to the decrease in the TNF-α level caused by SBS. The decreasing of the
   TNF-α level will reduce the inhibition of TUBB6. This mechanism
   alleviates the damage of hepatocytes caused by CCl[4].

   Nocturnin (NOCT) belongs to the superfamily of exonuclease,
   endonuclease, and phosphatase. Previous works have shown that NOCT null
   mice (Noct −/−) are resistant to high-fat-diet-induced obesity and
   hepatic steatosis. Further studies indicate that Noct −/− mice show a
   decrease in circulating triglyceride levels [[155]57]. In 3T3-L1 cell
   lines, NOCT overexpression enhances adipogenesis when cells are
   cultured in adipogenic medium, whereas the reduction in NOCT in 3T3-L1
   cells decreases their potential adipogenesis [[156]58]. These results
   suggest that NOCT is essential for the regulation of lipid transport.
   In the current study, SBS showed the ability to down-regulate the
   expression of NOCT, thereby reducing the hepatocyte steatosis induced
   by CCl[4].

   This study provides the experimental basis for the liver-protective
   effects and mechanisms of SBS. The significant protective effects of
   pre-treated SBS on CCl[4]-induced liver injury provide solid support
   for our further study on the post-treatment effect of SBS. Referring to
   previous works [[157]59], the pre-treated experiment is also a common
   way to study the protective effect of a substance that needs to be
   analyzed in rats. We believe that the present study is sufficient to
   draw a conclusion that SBS is endowed with protective effects against
   CCl[4]-induced liver injury in rats. We will consider the vitro and
   vivo experiments on the effect of SBS on CYP1A1 and Noct in future
   work.

5. Conclusions

   In conclusion, the present study found that SBS effectively alleviated
   the liver inflammation and hepatocyte pathological damage of rats with
   acute liver injury induced by CCl[4]. In addition, SBS was able to
   alleviate the metabolic disorders. Furthermore, the expression levels
   of CYP1A1, NOCT, and TUBB6 were regulated, indicating the effects of
   improving the antioxidant capacity of liver tissues, reducing the
   steatosis of hepatocytes, and inhibiting the inflammatory reaction of
   hepatocytes, respectively.

Acknowledgments