Penthorum Chinense Pursh Protects Liver From Alcohol-induced Steatosis in Zebrash by Mechanisms Including Inhibition of Oxidative Stress and Increase in Autophagy

Abstract

2. PCP modulated AMPK-Dependent steatosis in zebra sh by inhibiting the synthesis of fatty acids and promoting the oxidation of fatty acids 3. PCP mitigated alcohol hepatosteatosis by up-regulating the antioxidant pathway of Keap1/Nrf2 and down-regulating the autophagy pathway of mTOR/ PI3K/Akt signaling pathways 4. ATP-activated P2X7R were involved in regulating Nrf2-mediated oxidative stress and mTORmediated autophagy in zebra sh with alcohol hepatosteatosis Background Alcoholic liver disease (ALD) caused by heavy alcohol is currently highly prevalent worldwide and its liver damage has been well characterized [1], but there is no effective curative therapy in the clinical, so an indepth study of its mechanisms is necessary [2]. Lipid deposition in liver is the rst stage of ALD, and then reactive oxygen species (ROS) is produced during the effects of ethanol metabolism, which causing the occurrence of oxidative-stress and autophagy [3]. However, the pathogenesis of ALD is not fully understood.
As an ATP-gated ion channel, P2 × 7 receptor (P2 × 7R) is present in an increasing number of different cell types, especially expressed in a variety of liver cells, including immune cells, HSC and liver cells [4]. P2 × 7R accounts for a fairly vital role in health and disease, which can be activated by extracellular ATP to induce a variety of downstream events, including lipid metabolism, in ammatory molecule release, oxidative stress, cell proliferation and death, and autophagy [5]. Its receptor affects lipid metabolism [6][7][8][9] by activating the dangerous molecule ATP derived from liver cells caused by alcohol [10]. Recent studies have found that P2 × 7R mediates the activation of NLRP3 in amemome and lipid deposition in hepatocytes, P2 × 7R blocker alleviates alcohol-induced steatosis and intestinal ora changes via MEK1/2-ERK1/2 signaling [11]. Moreover, P2 × 7R is a vital target in hepatitis-induced oxidative stressmediated autophagy, but the role of P2 × 7R in alcoholic liver remains to be further studied.
In order to further understand the pathogenesis of ALD, the development of rational targeted therapies is key to the clinical treatment or prevention of it. Penthorum chinense Push (PCP) (Ganhuangcao in Chinese), as a health food and folk medicine, is a plant from the family of Penthoraceae, which is traditionally used for hepatoprotection and hepatic diseases treatment, including alcoholic liver damage [12,13]. Recent pharmacological studies have shown PCP protects hepatocytes from ethanolinduced liver injury with anti-hepatic steatosis [14], antiaging [15], antiapoptotic [16], anti-in ammatory [17], and anti-oxidative stress [18]. While research on autophagy of PCP is minimal. We speculate that PCP may mediate alcohol-induced oxidative stress and autophagy in the liver. Therefore, further studies are needed.
Diseases such as neonatal cholestasis, cholangitis, polycystic liver disease, alcohol liver disease have been studied on zebra sh [19]. Thus, the present study was done to evaluate whether PCP could attenuate alcohol hepatosteatosis in zebra sh by regulating oxidative stress and autophagy related signaling pathway, which provides a new target for studying the mechanisms of alcohol hepatosteatosis. larvae at 3 dpf were randomly assigned to 5 groups in a 6-well plate (30 larvae per well): larvae were maintained in ltered sh water as a control group while the model group was exposed to 350 mM ethanol (2% EtOH) for 32 h at 28.5 °C [20]. In PCP group, larvae were exposed to 48 h different doses PCP pre-treatment (100 µg/mL, 50 µg/ mL, 25 µg/mL) followed by 350 mM ethanol (2% EtOH) incubated in 28.5 °C for 32 h. ATP was given to further activate P2 × 7R, and the experiment was divided into six groups, the control and model groups are treated in the same way as before and 1 mM ATP for another 30 min at 28.5 °C [21]. In PCP group, larvae were exposed to 48 h different doses PCP pre-treatment (100 µg/mL, 50 µg/ mL, 25 µg/mL) followed by 350 mM ethanol (2% EtOH) for 32 h, and 1 mM ATP incubated in 28.5 °C for another 30 min. Afterwards, larvae were collected for detection.

Assessment of liver phenotype
After treatment, Zebra sh larvae were subjected to a series of pre-treatments including washed with fresh medium and subsequently anesthetized with tricaine, then xed in CMC-Na, and adjusted to the lateral position. Then, zebra sh larvae were photographed under Leica M165Fic uorescence microscope (Leica Microsystems, Germany). Finally, the uorescence integral optical density of zebra sh livers were quanti ed using Image Pro Plus 6.0 software (Media Cybernetics, USA) was applied to quantify.

Assessment of tissue biochemical indicators
After treatment, zebra sh larvae were collected and broken using an ultrasonic cell disruption system at 4 °C Triglyceride (TG) and total cholesterol (TC), alamine aminotransferase (ALT), aspartate aminotransferase (AST), γ-GT were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). After treatments, 30 larvae were cleaned by precooled PBS for three times, then homogenized and the supernatants were aspirated according to the instructions.

Whole-Mount Oil Red O Staining
Oil red O staining was used to determine hepatic lipid deposition. After treatment, zebra sh larvae were xed overnight with 4% PFA overnight. The rest of the procedure is routine [22]. Finally, the oil red O positive staining of zebra sh livers were photographed and related parameters was measured.

Assessment of ROS accumulation
2′,7′-dichlorodihydro uorescein diacetate (DCF-DA) was used as uorescence probes to investigate intracellular production of ROS. Then operated according to instructions, the accumulation of ROS was measured.

Assessment of oxidative stress factors
Indicators of oxidative stress and oxidation resistance were measured. After treatment, zebra sh larvae were collected and broken using an ultrasonic cell disruption system at 4 °C. Superoxide dismutase (SOD), malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT) assay kit was obtained from Elabsciense Bio-technology Co., Ltd. (Shanghai, China). After treatments, 30 larvae were cleaned by precooled PBS for three times, 270 µL Slurry medium was added and homogenized, then the supernatants were aspirated according to the instructions.

Assessment the expression of related proteins via western blot analysis
At 4 °C, RIPA lysis buffer containing PMSF and a protease inhibitor cocktail was used to extract zebra sh protein. Additional phosphatase inhibitors are required to extract of phosphorylated protein, then 50 µg protein samples of p-AMPK, AMPK, PI3K, p-PI3K and p-ACC, ACC, p-mTOR, mTOR were determined using wester blotanalysis, the membranes were detected on a Tanon 5200 automatic chemiluminescence imaging analysis system (Shanghai, China) and quanti ed by Image-Pro Plus (version 6.0).

Assessment the expression of related genes via quantitative RT-qPCR
Trizol reagent was used to extract total RNA from zebra sh larvae and dissolved it in RNase-free water at 4 °C. Ct values were obtained (reaction conditions: 95 °C 10 min, 95 °C 15 s, 60 °C 30 s (40 cycles)) and the relative gene mRNA expression was determined on ABI7500 qPCR system and calculated using the 2 −ΔΔCt method. The gene primer sequences used for RT-qPCR were listed in supplementary table 1.

Statistical analyses
All values were expressed as means ± SD. One-way analysis of variance (ANOVA) and Student's-test were used to assess the differences between the groups. The statistical analyses and graphs were generated by GraphPad Prism 6.0 (GraphPad, San Diego, CA, USA). Results were considered to be statistically signi cant when p < 0.05.

Chemical characteristics of PCP extract
Previous studies showed that avonoids were the main chemical constituents of PCP, which possess strong antioxidant activities [18]. The extract of PCP was analyzed by HPLC to determine its main chemical constituents, which was consistent with the previous studies that PCP extract contained a lot of avonoids ( Fig. 1). Six peaks were identi ed as gallic acid, rutin, quercetin, luteolin, apigenin and kaempferol. The contents of the six compounds were quanti ed using corresponding chemical standards. Speci cally, the contents of gallic acid, rutin, quercetin, luteolin, apigenin and kaempferol in PCP were 1.2025, 0.8244, 0.4967, 0.0924, 0.026, 0.0970 mg/g, respectively.
PCP improved the liver function and attenuated hepatic accumulation Liver uorescence of Transgenic (fabp10: EGFP) zebra sh will be reduced after modeling ( Fig. 2A), compared with the control group. We found that the liver uorescence integral optical density (IOD) decreased signi cantly after modeling (Fig. 2B) and caused the remarkable increase of ALT, AST and γ-GT levels (Fig. 3A) (P < 0.01), in addition, the rate of AST/ALT > 2 (Fig. 3A). PCP pretreatment strongly increased IOD in a dose-dependent manner, as well as decreased ALT, AST accumulation (P < 0.01), however only 100 µg/mL PCP pretreatment decreased γ-GT accumulation (P < 0.05), and signi cantly improved liver function (P < 0.01). Ethanol treatment caused a signi cant accumulation of TG, TC concentrations, TG accumulation dominated (Fig. 3B). PCP pretreatment strongly reduced TG accumulation (P < 0.01) compared to those in the ethanol group in a dose-dependent manner, however only 100 µg/mL PCP pretreatment signi cantly reduced TC accumulation (P < 0.01). These results are consistent with whole-mount oil red O staining (Fig. 4), which demonstrated that PCP reduced liver lipid deposition.

PCP enhanced the level of oxidative stress-related factors
Drinking alcohol leads to the release of large amounts of intracellular ROS, as seen in Fig. 5A, a bright and strong uorescent image was observed in the model group. PCP dose-dependently decreased intracellular ROS production in zebra sh (Fig. 5B). Compared with control group, the MDA levels of the ethanol-treated groups were signi cantly elevated (Fig. 6). Conversely, the levels of SOD, CAT and GSH markedly declined (Fig. 6). However, PCP pretreatment reduced the activity of MDA elevation and increased the activity of GSH and SOD in a dose-dependent manner, however only 100 µg/mL PCP pretreatment signi cantly improved the activity of CAT (P < 0.01). Thus, PCP alleviates alcohol hepatosteatosis by inhibiting oxidative stress.
PCP alleviated lipid deposition, oxidative stress and enhanced autophagy mRNA expression of PCP in fatty acid synthesis and β-oxidation To investigate the effect of PCP on fatty acid synthesis, as compared with control group, the upstream genes SIRT1, LKB1 and AMPK were decreased signi cantly in model group and reversed by PCP (Fig. 7A). RT-qPCR for the expression of four lipid metabolism-related genes, SREBP1, CHREBP, FAS, and ACC1. The results (Fig. 7C) showed that, mRNA SREBP1, CHREBP and FAS were increased signi cantly in model group and reversed by 100 µg/ml PCP in compared with control group (P < 0.001), and SREBP1 and FAS were decreased by 50 µg/ml PCP (P < 0.001). However, there was no signi cant of ACC1 expression. Moreover, β-oxidation related genes including PPARα decreased, PPARγ and CPT1 showed signi cantly increased in model group which can be reversed by 100 µg/ml PCP (Fig. 7C) (P < 0.001) and 50 µg/ml PCP (P < 0.01), but no difference between the low doses compared with the model group (p > 0.05).

mRNA expression of PCP on anti-oxidant relevant signaling pathway
To further study the mechanism. Anti-oxidant effect of PCP was investigated with three related genes expression of keap1, Nrf2, HO-1 were examined by RT-qPCR. The results showed in Fig. 7E, indicated that, the levels of three increased genes in model group were signi cant in a dose-dependent decreased after PCP administration (P < 0.001).
mRNA expression of PCP on autophagy relevant signaling pathway Subsequently, RT-qPCR for detection of the autophagy effect of PCP was investigated with ve mTOR signaling pathways related genes expression of mTOR, Atg13 and Beclin 1 and Upstream targets PI3K and Akt, and as shown in Fig. 6F, the expression of PI3K, Akt and mTOR increased, while Atg13 and Beclin 1 decreased signi cantly in model group (P < 0.001). Moreover, our data showed that the stimulation effect of alcohol hepatosteatosis in zebra sh was signi cantly reversed by high-concentrations of PCP (P < 0.001), moreover PI3K, Akt, mTOR decreased in a concentration dependent manner after PCP administration.

PCP improved the liver function and attenuate hepatic accumulation
To further explore the underlying mechanism. Western blot experiments were conducted to detect the expression of related proteins, as shown in Fig. 8. In our study, there were no signi cant change in total proteins expression including AMPK, ACC and mTOR, mainly through phosphorylation, as the protein expression of p-AMPK/AMPK, p-ACC/ACC, and p-mTOR/mTOR were signi cantly decreased in model group (P < 0.001), which were signi cantly reversed by high-concentrations of PCP (P < 0.01), moreover, p-AMPK/AMPK and p-mTOR/mTOR increased in a dose-dependent manner after PCP administration and the results were consistent with the mRNA expression. Although, in the PCR results, we saw no signi cant change in mRNA expression of ACC, while the protein level PCP played its role mainly through phosphorylation of ACC. These data suggested that the possible mechanisms of PCP were primarily through phosphorylation of AMPK and mTOR targets.
ATP-activated P2 × 7R were involved in regulating oxidative stress and autophagy.
To further explore whether P2 × 7R mediated the possible mechanism of PCP inhibiting the oxidation and autophagy pathway, RT-qPCR for mRNA levels of P2 × 7R, AMPK, Keap1, Nrf2 oxidation-related, mTOR, PI3K, and Akt autophagy-related genes. Compared with the ethanol treatment group, the mRNA expression of P2 × 7R in the ethanol plus ATP group was signi cantly increased ( Fig. 9A-1), then PCP signi cantly decreased its expression compared with both control and ethanol group in a dose-dependent manner. In the ethanol plus ATP group, activated P2 × 7R further increased the expression of Keap1 (Fig. 9A-3) related to oxidative stress, and signi cantly reduced the expression of AMPK and Nrf2 (Fig. 9A-2,4), moreover, compared with the ethanol plus ATP treatment group, mTOR, PI3K, Akt decreased, however PCP treatment signi cantly reversed these in a dose-dependent. More interestingly, mRNA expression of PI3K and Akt signi cantly decreased (P < 0.001) compared with ethanol plus ATP group (Fig. 9B).

Discussion
Serum levels of ALT and AST are vital and sensitive biochemical signals of liver function, indicating early stages of ALD, whose abnormal elevation can cause liver cell damage and necrosis [23]. In addition, γ-GGT has been used as a marker of hepatic impairment due to alcohol consumption [24]. Consistent with our results, compared with the model group, different doses of PCP effectively reduced ALT, AST and γ-GT levels and improved liver function. Moreover, during the pathological development of ALD, the most common phenomenon is an imbalance between lipid synthesis and fatty acid oxidation. Hepatic steatosis as a reversible process that can exacerbate disease progression [25]. AMPK signaling plays a meaningful role in lipid homeostasis regulation, as a highly conserved sensor for low intracellular ATP levels, which is produced in large amounts in alcoholic fatty liver. P2 × 7R is a vital regulatory point for transmitting extracellular ATP signals that allow AMPK to be activated. It has been reported that dihydroquercetin mediates P2 × 7R amelioration of alcoholic hepatic steatosis [8]. In addition, AMPK phosphorylation regulates downstream ACC, where ACC is a key enzyme in fatty acid synthesis, catalyzing acetyl coenzyme A carboxylation to malonyl coenzyme A, and then regulating PPAR-α and CPT1 pathway promoting free fatty acid oxidation; PPARγ showed the opposite pharmacological effect. moreover, SREBP-1c is a fat synthesis protein (e.g., FAS), which is the primary regulator of triglyceride conversion and decreased lipogenesis, TG accumulation by upregulating AMPK signaling by SREBP-1c and by downregulating FAS.
Oxidative stress is a key point in the process of ALD [26]. Alcohol consumption leads to high ROS production, suppresses antioxidant defense systems, and leads to oxidative stress in the liver [27]. Lipid peroxidation products (e.g., MDA) indirectly re ects the extent of free radical damage to the liver, and it is also an indicator of hepatocyte recovery after PCP administration. In contrast, the antioxidant defense systems GSH, CAT and SOD, which directly re ect the antioxidant capacity of the liver, remove of lipid peroxides and protect hepatocytes from ROS damage, while PCP treatment signi cantly reduced ROS. MDA level was decreased and increased SOD, GSH and CAT level in our study. Moreover, P2 × 7R activation induces ROS production and antioxidative defense system imbalances [28], which is key factors in oxidative stress-driven cell fate [29], and the downstream AMPK prevents oxidative stress primarily by regulating glucose and lipid metabolism [30], which activates Nrf2. Nrf2 is the key to cellular resistance to ROS, and studies suggest that targeting Keap1/Nrf2 can effectively reduce oxidative stress [31], and related studies have also found that AMPK mediates oxidative stress by increasing intracellular levels of its co-substrate, NAD + , and that hyperphosphorylation of AMPK inhibits Nrf2 activation and nuclear translocation by modifying cysteine residues in Keap1, which in turn inhibits oxidative stress by inducing the expression of various antioxidant genes, such as HO-1 [32]. Consistent with our experimental results, we observed Keap1 was increased and AMPK, Nrf2 was decreased in the model group at the gene level, while Nrf2 was increased after administration of PCP indicating the inhibition of oxidative stress, and P2 × 7R was activated by ATP and was involved in Keap1/Nrf2mediated oxidative stress, which suggests that P2 × 7R may be involved in lipid metabolism via Keap1/Nrf2-mediated oxidative stress.
Recently, autophagy has become a protective mechanism against ALD, although there are currently divergent mechanisms in acute and chronic alcohol-induced alcoholic hepatitis [33][34][35]. Some studies found that P2 × 7R played an important role in ALD, signals from P2 × 7R and adipoR1 regulate the PI3K-Akt and/or AMPK-Foxo3A pathway to restore the mitochondrial autophagy (mitosis) inhibited by ethanol intoxication [36]. Moreover, as the upstream target of PI3K/Akt, mTOR plays a vital role in autophagy, which mediates autophagy may be a potential mechanism for treating liver injury [37]. In our results, at the genetic level we observed that the administration of mTOR was able to reverse the expression of mTOR compared to the model group and further veri ed from the protein level that it acted mainly through phosphorylation of the expression. In addition, this paper also observed that the classical autophagy signaling pathway mTOR/PI3K/Akt was altered after activation of the P2 × 7R by administration of ATP, and PI3K/Akt changed signi cantly.
Moreover, there is some crosstalk between oxidative stress and autophagy. They can work together to participate in lipid metabolism, P2 × 7R plays a key role in this process, suggesting that P2 × 7R is a vital mediator of alcoholic hepatitis, mediates the development of alcoholic hepatitis, and is a new target for alcoholic hepatitis treatment. PCP was rst used in an ALD zebra sh model to investigate the bene cial effects of alcohol via P2 × 7R in mediating lipid metabolism, oxidative stress and autophagy (Fig. 10). The mechanism of PCP in alcoholic liver was revealed more comprehensively, which provided a new idea for the treatment of alcoholic liver.

Declarations
Ethics approval and consent to participate

Consent for publication
All the authors read the nal manuscript and approved for publication.

Availability of data and materials
The datasets used in this study are available from the corresponding author upon reasonable request.  expressed as the mean ± SD. ###P < 0.01 compared with control group; ***P < 0.001, **P < 0.05 compared with model group Figure 5 (A) Fluorescence micrographs of ROS in zebra sh larvae. (B) The distribution and amounts of superoxide anions were quanti ed according to the uorescence intensity. Data expressed as the mean ± SD. ###P < 0.001 compared with control group; ***P < 0.001 compared with model group.

Figure 6
Different doses of PCP effect on serum biochemical indicators of liver function. The following six liver function markers in the serum were assayed: MDA, SOD, GSH, CAT. Data expressed as the mean ± SD.