Quantitative Profiling of Oxylipin Reveals the Mechanism of Pien-Tze-Huang on Alcoholic Liver Disease

Alcoholic liver disease (ALD) is a liver disease caused by long-term alcohol consumption. ROS-mediated oxidative stress is the leading cause of ALD. Pien-Tze-Huang (PZH), a traditional formula, is famous in China. This study was designed to evaluate the effects and explore the potential mechanisms of PZH in ALD. Forty mice were randomly divided into five groups: control group (normal diet + vehicle), model group (ethanol diet + vehicle), PZH-L group (ethanol diet + PZH (0.125 g/kg)), PZH-M group (ethanol diet + PZH (0.25 g/kg)), and PZH-H group (ethanol diet + PZH (0.5 g/kg)). The mice were sacrificed, and their liver and blood samples were preserved. Liver steatosis, triglyceride (TG), total cholesterol, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were assayed. Malondialdehyde (MDA), glutathione peroxidase (GSH-PX), and total superoxide dismutase were identified using commercial kits. Oxylipins were profiled, and the data were analyzed. The AMPK/ACC/CPT1A pathway was identified using real-time polymerase chain reaction and western blotting. The PZH-H intervention significantly alleviated hepatic steatosis and injury and reduced the levels of liver TG and serum ALT and AST. In addition, MDA levels were markedly reduced, and GSH-PX activity significantly increased after PZH-H intervention. Finally, PZH-H increased the levels of 17-HETE, 15-HEPE, 9-HOTrE, 13-HOTrE, and 5,6-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid, and reduced PGE2 levels. PZH-H intervention also promoted the phosphorylation of AMPK and ACC, and the expression of CPT1A. In conclusion, PZH reduced oxidative stress and alleviated hepatic steatosis and injury. The mechanism was correlated with the oxylipin metabolites/AMPK/ACC/CPT1A axis.


Introduction
Alcoholic liver disease (ALD) is a liver disease caused by long-term alcohol consumption. It is one of the most common types of chronic liver diseases globally and is currently one of the major chronic liver diseases in China [1][2][3]. Heavy drinkers (ethanol consumption of ≥40 g/day for men and ≥20 g/day for women over 5 years; or ethanol consumption of >80 g/day and binge drinking within 2 weeks) develop fatty liver, and about 20%-40% of them develop more severe ALD [1,4]. According to the International Classification of Diseases (ICD-10), ALD is classified as alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, alcoholic cirrhosis, and alcoholic liver failure. Studies have found that the prevalence of ALD in China is 4.5%, which is similar to that in European and American countries, among which the prevalence of ALD in people aged 40-49 years is the highest, accounting for more than 10% [5]. At present, the effective ways to reduce or terminate alcoholic liver injury are nutritional support and abstinence from alcohol, but some patients still need to undergo combined treatment with drug intervention. e guidelines for the prevention and treatment in 2018 included glucocorticoid, metadoxine, S-adenosylmethionine, polyene phosphatidylcholine, glycyrrhizic acid preparation, and silymarin. Although these drugs show some improvement in patients with ALD, strict and extensive sample data supporting effective treatment and improvement of ALD are still lacking in clinical trials.
Alcohol consumption can induce the generation of reactive oxygen species (ROS), which cause oxidative stress. ROS can change the structure and function of the protein by binding to them and finally generate host antigens that can induce immune responses [6,7]. ROS can also lead to lipid peroxidation and generate lipid peroxidation products, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). Studies have found that these products can bind to DNA bases and induce apoptosis and autophagy in cells [8][9][10].
erefore, ROS-mediated oxidative stress is the leading cause of ALD.

Experimental Design and Administration of PZH.
Mice were randomly divided into five groups: control group (normal diet + vehicle), model group (ethanol diet + vehicle), PZH-L group (ethanol diet + PZH (0.125 g/kg)), PZH-M group (ethanol diet + PZH (0.25 g/kg)), and PZH-H group (ethanol diet + PZH (0.5 g/kg)). Mice fed an ethanol diet were fed an ethanol Lieber-DeCarli diet (Trophic Animal Feed High-tech Co. Ltd, China) containing 5% (v/v) ethanol for 10 days. Mice with a normal diet were pair-fed with an equicaloric normal Lieber-DeCarli diet for 10 days. PZH was exclusively produced and certified by Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd., and the fingerprint spectrum was determined by HPLC-MS in a previous study [22]. PZH-treated mice were gavaged with PZH aqueous solution simultaneously as they were fed an ethanol diet for 10 days. On day 11, mice fed an ethanol diet were gavaged with a single dose of 31.5% (v/v) ethanol (20 μL/g body weight), and mice fed a normal diet were gavaged with equicaloric 45% (w/v) maltose dextrin (20 μL/g body weight). All mice were euthanized after 9 hours, and blood and liver samples were collected for further study.

Biochemical Analysis.
e blood was centrifuged for 20 min at 4000 rpm at 4°C, and serum was collected for analysis. Liver tissue was homogenized in propanone/ethanol (1 : 1) and incubated overnight at 4°C. Homogenates were centrifuged for 10 min at 4000 rpm at 4°C, and the supernatant was collected for testing. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, liver triglyceride (TG), and total cholesterol (TC) levels were measured using an automatic biochemistry analyzer (TBA-40FR, Toshiba, Japan).

Histological Analysis.
e left lobe of the liver was cut in half. One half was fixed in 10% buffered formalin, and the other was frozen in liquid nitrogen. e fixed liver sections were dehydrated, embedded, and sectioned into 4 μm sections for staining with hematoxylin and eosin (H&E). e frozen liver sections were embedded and sectioned into 10 μm thick sections using a frozen microtome (Leica, Germany) for staining with Oil Red O, according to previous studies [25,26].

Measurement of MDA, GSH-PX, and T-SOD Levels in the
Liver. Liver tissue was homogenized and centrifuged for 10 min at 4000 rpm at 4°C, and the supernatant was collected. e levels of MDA, glutathione peroxidase (GSH-PX), and total superoxide dismutase (T-SOD) in liver tissue were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Oxylipins Data Analysis.
e raw data generated by UPLC-MS/MS were extracted, integrated, identified, and quantitatively analyzed for each metabolite using AB Sciex's Analyst software (V1.6.3, AB Sciex, Boston, MA, USA). Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and variable importance of projection (VIP) were performed to analyze the data. e formula used for calculating the Z-score was Z-scores � (x−mean)/SD. e Euclidean distance of Z-scores was clustered with the method in the heatmap. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched using Metaboanalyst 4.0.

Real-Time Polymerase Chain Reaction (RT-PCR).
Total RNA was isolated from liver tissue using RNAiso Reagent (Takara, Japan) and reverse-transcribed using PrimeScript ™ RT Master Mix (Takara, Japan). RT-PCR was performed using TB Green ® Premix Ex Taq ™ according to the manufacturer's instructions. Target gene expression was analyzed using the 2 -ΔΔCt method by normalization to β-actin. e primers used are listed in Table 1.

Statistical
Analysis. Data are presented as mean-± standard deviation (x ± s). Statistical significance was analyzed using one-way ANOVA in SPSS 24.0. Qualitative data among the three groups were assessed using one-way ANOVA, the Kruskal-Wallis test, or the chi-square test based on data distribution. Differences between the two groups were analyzed using Student's t-test. e values were considered statistically significant at P < 0.05.

PZH-H Alleviated Hepatic Steatosis in ALD-Mice.
Hepatic steatosis was significantly higher in the model group than in the control group, and the PZH-M and PZH-H interventions significantly improved hepatic steatosis (Figures 1(a) and 1(b)). In addition, liver TG and TC levels were markedly increased in the model group compared with those in the control group. e PZH-H intervention significantly reduced liver TG levels, but liver TC was increased in the PZH-H group compared with that in the model group (Figures 1(c) and 1(d)). Furthermore, serum ALT and AST levels were markedly increased in the model group compared with those in the control group. e PZH-H intervention markedly reduced ALT and AST levels, and the PZH-M intervention also markedly reduced ALT levels (Figures 1(e) and 1(f )). ese results indicated that PZH-M and PZH-H alleviated hepatic steatosis and injury in ALD mice, and the effect of PZH-H was better than that of PZH-M.

Effect of PZH on MDA, GSH-PX, and SOD.
Oxidative stress is one of the main pathogeneses of ALD; MDA, GSH-PX, and T-SOD are the primary biomarkers of oxidative stress.
us, we tested the levels of MDA, GSH-PX, and T-SOD in the livers of ALD-mice. Compared with the control group, the level of MDA in the model group was significantly increased, while the activities of GSH-PX and T-SOD were significantly decreased. Compared with the model group, the level of MDA in the three PZH groups was significantly decreased (Figure 2(a)), and the activity of GSH-PX in the PZH-H group was significantly increased ( Figure 2(b)). Although T-SOD activity in the PZH-H group was not different, its activity was significantly increased in the PZH-M group (Figure 2(c)). ese results indicate that PZH-H could improve oxidative stress.

Analysis of Oxylipin Metabolites among ree Groups.
Our results showed that PZH could alleviate hepatic steatosis, liver injury, and oxidative stress, and the effect of PZH-H was better than that of the PZH-L and PZH-M groups.
us, we chose the PZH-H group to explore the mechanism of oxidative stress improvement. Previous studies have proved that oxylipins play an important role in oxidative stress [13,15]. erefore, the profiling of oxylipins was performed and analyzed. e results indicated that the proportion of DHA was the highest, followed by that of LA (Figure 3(a)). DHA, LA, and EPA levels were significantly different among the three groups ( Figure 3(b)). Fifty-eight oxylipin metabolites were detected in the liver tissue of the mice (Figure 3    . Data were presented as means ± SD. # P < 0.05, ## P < 0.01, and ### P < 0.01 in the model group vs the control group; * P < 0.05 and * * P < 0.01 in the PZH groups vs the model group.  Table 2).

Effect of PZH on AMPK-ACC-CPT1A Pathway.
Previous studies have indicated that oxylipin metabolites can regulate the AMPK signaling pathway [27][28][29][30]. e results showed that although the phosphorylation of AMPK was not significantly different in the model group compared with the control group, PZH-H intervention markedly promoted the phosphorylation of AMPK (Figure 7(a)). In addition, compared with the control group, the phosphorylation of ACC was significantly reduced in the model group, and PZH-H intervention markedly promoted the phosphorylation of ACC. However, the mRNA levels of ACC1 and ACC2 in the model group were significantly increased, the mRNA levels of ACC1 were significantly decreased, and ACC2 levels were significantly increased after PZH-H intervention (Figures 7(a)-7(c)). Compared with the control group, the mRNA and protein levels of CPT1A were significantly decreased in the model group, and PZH-H intervention significantly promoted CPT1A expression (Figures 7(a) and 7(d)). Although the phosphorylation of PPARα was not significantly different in the model group compared with the control group, the PZH-H intervention markedly promoted the phosphorylation of PPARα. Moreover, the expression of SREBP1 was not significantly different among the three groups (Figures 7(e)-7(g)).

Discussion
In the present study, we showed that PZH could improve hepatic steatosis and injury in ALD mice, consistent with previous studies. In addition, PZH reduced the level of MDA and increased the activity of GSH-PX, ultimately ameliorating oxidative stress, which is consistent with a previous study [31]. rough the analysis of oxylipin profiling, we found that PZH promoted the levels of 17-HETE, 15-HEPE, 5,6-dihydroxy-8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid, 9-HOTrE, and 13-HOTrE, and reduced PGE2 levels, further activating the AMPK pathway. Previous studies have also shown that PZH can reduce tumor necrosis factor-alpha and interleukin-1β secretion to ameliorate hepatic inflammation [31] and can ameliorate hepatic injury by inhibiting the PERK/eIF2α pathway [22]. In addition, PZH could also ameliorate hepatic fibrosis by suppressing the NF-kappa B pathway [21]. Panax notoginseng saponins and polysaccharides have hepatoprotective effects against alcoholic liver damage [32,33], but the mechanism is not precise. Our study is the first to demonstrate that PZH regulates the oxylipin-metabolites/AMPK pathway to reduce oxidative stress and improve hepatic steatosis and injury in ALD-mice. is study fully confirmed the mechanism of action of PZH on ALD through the quantitative profiling of oxylipin.

Evidence-Based Complementary and Alternative Medicine 5
Oxidative stress is one of the main pathogeneses of ALD. As a primary biomarker of oxidative stress, MDA was markedly increased in ALD and promoted the generation of etheno-DNA adducts to induce damage to the liver [9,10]. In the present study, MDA levels were also markedly increased in the ALD mice, and PZH intervention significantly reduced MDA levels. In addition, the activity of GSH-PX, an antioxidant enzyme, was significantly reduced in ALD-mice, consistent with a previous study [34], and PZH increased the activity. ese results indicated that PZH could attenuate oxidative stress.
e AMPK-ACC-CPT1A pathway is involved in the regulation of oxidative stress and ALD [43][44][45][46][47]. Previous     studies have proved that EPA could improve palmitateinduced endothelial dysfunction and lipotoxicity by activating AMPK pathway [28,48]. In addition, LA can reduce hepatic lipid metabolism [29,30], insulin resistance, and adiposity [49,50] via the AMPK pathway. PGE2 can negatively regulate the AMPK pathway [51,52]. In this study, we found that PZH increased the levels of EPA and LA and reduced PGE2 levels. In addition, PZH can activate AMPK and promote the phosphorylation of ACC, which reduces the ACC activity [53] and finally promotes the expression of CPT1A. Although studies have shown that PPARα and SREBP-1 could improve oxidative stress [54][55][56], PZH did not regulate their expression in our study, indicating that PZH did not play a regulatory role in ALD through PPARα and SREBP-1. However, in this study, we found that the levels of 9-HOTrE, 13-HOTrE, 5,6-dihydroxy-8Z, 11Z, 14Z, 17Z-eicosatetraenoic acid, 17-HETE, and 15-HEPE were increased in the liver of ALD-mice, and PZH further increased their levels. PGE2 levels were decreased in the liver of ALD-mice, and PZH further decreased this level. We speculated that oxylipin metabolites may need to reach certain concentrations in vivo and activate the AMPK/ACC/ CPT1A pathway to protect against hepatic injury. However, further studies are required to elucidate the underlying mechanism. Although our results showed that PZH could regulate oxylipin metabolites and speculated that Panax notoginseng may mainly enhance oxylipin metabolites, further studies are also needed.

Conclusions
In summary, PZH reduced oxidative stress and alleviated hepatic steatosis and injury. e mechanism was correlated with the oxylipin metabolites/AMPK/ACC/CPT1A pathway ( Figure 8).

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
is study (PZSHUTCM200703007) was approved by the Animal Experiment Ethics Committee of Shanghai University of Traditional Chinese Medicine.

Conflicts of Interest
e authors declare that they have no conflicts of interest.