Penthorum chinense Pursh alleviates ethanol-induced hepatic oxidative impairment in zebrash via AMPK / p62 / Nrf2 / mTOR pathway

Ethnopharmacological relevance: In China, Penthorum chinense Pursh (PCP) is renowned for its effectiveness in “promoting blood circulation” and “removing blood stasis”. It can “relieve the liver” and its application in the eld of liver protection, including viral hepatitis, alcoholic liver, liver brosis, has been known for hundreds of years. Aim of the study: Oxidative stress is widely believed to exert a key role in the pathophysiology of alcoholic liver disease (ALD). Therefore, antioxidant therapy reects a reasonable strategy for the prevention and treatment of ALD. Hence, this study aimed to elucidate the mechanism of PCP in ethanol-induced liver injury. Methods: Treatment of liver-specic transgenic zebrash larvae (lfabp: EGFP) at three days post-fertilization (3 dpf) with different concentrations of PCP (100, 50, 25 μg / mL) for 48 h was followed by soaking in 350 mmol / L ethanol for 32 h. Liver function and fat accumulation were identied by phenotypic indicators and biochemical kits. The related proteins and gene expression were further estimated by western blotting and quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). Finally, high performance liquid chromatography (HPLC) was adopted to analyze the chemical composition of PCP extract. Results: Firstly, PCP mediated alleviation of ethanol-induced steatosis and reduction of aspartate aminotransferase (AST), alanine transaminase (ALT), total cholesterol (TC) and triglyceride (TG) related indexes were evident. Dose-dependent decrease of intracellular reactive oxygen species (ROS) production, the activity of malondialdehyde (MDA) and increased the activity of glutathione (GSH), Superoxide dismutase (SOD) and catalase (CAT) in zebrash substantiated the role of PCP in relieving oxidative stress. Furthermore, PCP induced downregulation of sequestosome (p62 Atg13


Introduction
Alcohol liver disease (ALD) caused by heavy ethanol consumption is worldwide problem and the resultant liver damage has been well characterized (Asrani, Devarbhavi, Eaton, & Kamath, 2019;Rehm et al., 2009).
The World Health Organization (WHO) estimated that 2.3 billion people were active drinkers in 2016, about 3 million people die from ethanol each year, some of them from ALD (Mitra, De, & Chowdhury, 2020). The commonly used drug glucocorticoids have obvious side effects and are not suitable for longterm treatment, and many contraindications make its indication relatively limited (Lucey, Mathurin, & Morgan, 2009). In addition to abstinence, there is no effective clinical measures to cure ALD (You & Arteel, 2019). Thus, collective efforts are urgently needed to stem the rising tide of ALD on healthcare resources (Cholankeril et al., 2021).
Oxidative stress is widely believed to exert a key role in the pathophysiology of ALD. Therefore, antioxidant therapy re ects a reasonable strategy for the prevention and treatment of ALD (Michalak, Lach, & Cichoż-Lach, 2021). Nrf2 is a transcription factor that regulates the redox state of cells and is a promising intervention target for the prevention of ALD (N. Zhao, Guo, Xie, & Zeng, 2018). Nrf2 can mediate adaptive responses to oxidative stresses by degrading cytoplasmic Keap1-Nrf2 (Ohtsuji et al., 2008). Meanwhile, Nrf2 inhibits adipogenesis, supports β-oxidation of fatty acids and increases NADPH regeneration and purine biosynthesis (Hayes & Dinkova-Kostova, 2014). Recent study found that Nrf2 activation prevented acute ethanol-induced oxidative stress and accumulation of free fatty acids in liver (N. Zhao et al., 2018).
Autophagy is an essential survival pathway that ameliorates oxidative damage caused by ROS (Menk et al., 2018). Studies have shown that selective renewal of p62 impaired by autophagy leads to severe liver injury. In addition, there is increasing evidence of a functional association between dysfunctional autophagy and activation of the Nrf2 pathway (Komatsu et al., 2010). Furthermore, study has found that phosphorylated AMPK inhibits mTOR, thereby activating autophagy (Lu et al., 2021). Thus, the AMPK / p62 / Nrf2 / mTOR axis may play an important role in protecting ethanol-induced liver injury.
Recently researchers are increasingly turning their attention to multi-component, multi-targeted natural drugs, especially health food. Penthorum chinense Pursh (PCP) (Penthoraceae, Ganhuangcao in Chinese) is renowned for its effectiveness in "promoting blood circulation" and "removing blood stasis". It was rstly recorded in Jiuhuang Materia medica in Ming dynasty. Similar descriptions can be found in the National compilation of Chinese herbal medicine (A. Wang et al., 2020). It can relieve the liver and its application in the eld of liver protection, including viral hepatitis, alcoholic liver, liver brosis, has been known for hundreds of years (A. Wang et al., 2020;X. Zhao et al., 2020). Previous studies have revealed that PCP extract con rmed the presence of various polyphenols with potential antioxidant effects in vivo and in vitro (L. He et al., 2019;Y. Sun et al., 2021;Tao et al., 2021;X. Zhao et al., 2020), respectively. And PCP have achieved good clinical effect in the treatment of liver disease (X. Sun et al., 2020;A. Wang et al., 2020). These ndings indicated that PCP has great potential to relieve oxidative stress and prevent liver injury.
Thus, the aim of this study was done to investigate the protective effect of PCP on ethanol-induced hepatic oxidative impairment in zebra sh. Further study explored the underlying mechanism involving in AMPK / p62 / Nrf2 / mTOR signaling pathways, providing a new target in identifying the molecular mechanisms of ethanol hepatic steatosis.

Chemicals and Reagents
The dried PCP, obtained from Chengdu HeHuaChi Chinese Herbal Medicine Market Co. (Sichuan, China) were extracted for three times by decoction, 1 h each time. After the solution is ltered, it is evaporated under reduced pressure to obtain a dry powder 17 % (g / g) and stored at -20°C for further use.
Oil red O dyeing solution was collected from Shanghai Solarbio Bioscience and Technology Co., Ltd (Shanghai, China). DCF-DA was purchased from Yeasen Bio-technology Co., Ltd. (Shanghai, China).

Experimental animals and drug administration
Transgenic (lfabp: EGFP) zebra sh were obtained from China Zebra sh Resource Center (Wuhan, China), and raised at 28.5 ± 1.0°C on a 14 h light /10 h dark cycle. Transgenic (lfabp: EGFP) zebra sh 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 ethanol treatment group was exposed to 350 mM ethanol for 32 h at 28.5°C (Howarth & Passeri, 2011). In PCP group, larvae were exposed to different concentrations PCP pre-treatment (100 µg / mL, 50 µg / mL, 25 µg / mL) for 48 h followed by 350 mM ethanol incubated in 28.5°C for 32 h (Cruz & Leite, 2013). 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 larvae were quanti ed using Image Pro Plus 6.0 software (Media Cybernetics, USA) was applied to quantify.

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 (Yu & Gong, 2020). 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. After the treatments, the zebra sh were moved to a six-well plate (15 larvae per well) and treated with the DCF-DA (0.05 µM) solution. After 1 h incubation in dark, the zebra sh larvae were photographed under a Leica M165Fic uorescence microscope and 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 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.

Nucleus protein extraction
The extract was isolated using the Minute TM Hyston/DNA-Binding Protein Extraction Kit according to the manufacturer's protocol. In short, zebra sh larvae were digested with trypsin, then they were collected by low-speed centrifugation at 500 x g for 3 min, washed once with pre-cooled PBS, and transferred to a 1.5 mL centrifuge tube, centrifuged at 500 x g for 1 min, and the supernatant was discarded Suspend the precipitate in 100 µL cytoplasmic extraction reagent I, swirl for 15 seconds, incubate on ice for 10 min, mix 4, Centrifuge at 16000 x g for 5 min. Transfer the supernatant (the supernatant is the cytoplasmic component) to a new precooled 1.5 mL centrifuge tube and resuspend the precipitate with 0.5 mL precooled PBS. Centrifuge at 8000 x g for 3-5 min to clean the precipitate to reduce cytoplasmic protein contamination 15 seconds, incubate on ice for 1 minute and then repeat the shock for 15 seconds, incubate on ice for 1 minute for 4 times and quickly transfer the nucleus extract into the pre-cooled centrifuge tube sleeve, 16000 x g, centrifuge for 30 seconds and discard the centrifuge tube string, according to the manufacturer's instructions and heated at 95°C for 5 min.

Assessment the expression of related proteins via western blot analysis
Samples were then separated in denaturing PAGE (Novex, 10 % or 15 % bis-Tris gel) using amount of protein (50 µg) was loading per lane at 100 V for 1 h. Following electrophoresis, samples were then transferred onto a PVDF membrane (Millipore, Billerica, MA, USA) by electro-blotting at 120 V for 1 h, which were subsequently blocked in 5 % skimmed milk and probed with anti-p62, Keap1, Nrf2, p-AMPK, AMPK and p-mTOR, mTOR antibody solution (diluted 1:500). As internal reference, 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).

Statistical analyses
All statistics were evaluated by t-test or Mann-Whitney U test (two groups) and one-way ANOVA or Kruskal-Wallis test followed by pairwise comparisons (three or more groups) depending on whether the data were normally distributed. The statistical analyses and graphs were generated by GraphPad Prism 6.0 (GraphPad, San Diego, CA, USA). All values were expressed as means ± SD. Results were considered to be statistically signi cant when p < 0.05.

Results
PCP decreased ethanol-induced liver function injury and fat accumulation Liver area of zebra sh (lfabp: EGFP) was reduced after ethanol treatment ( Figure 1A), compared with the control group. We found that the liver uorescence integral optical density (IOD) decreased signi cantly (P < 0.001) after ethanol treatment ( Figure 1B) and caused the remarkable increasing of ALT, AST levels (P < 0.001) ( Figure 1E, F). While PCP pretreatment strongly increased IOD in a concentration-dependent manner (P < 0.001), as well as decreased ALT, AST accumulation (P < 0.001) and signi cantly improved liver function. Ethanol treatment caused a signi cant accumulation of TG, TC concentrations (P < 0.001), PCP pretreatment strongly reduced TG, TC accumulation in a concentration-dependent manner (P < 0.01), and TG accumulation dominated ( Figure 1G, H). These results were consistent with whole-mount oil red O staining ( Figure 1C, D), which demonstrated that PCP reduced liver lipid deposition.

PCP reduced oxidative stress in ethanol-induced liver injury
Drinking ethanol leads to the release of large amounts of intracellular ROS, as seen in Figure 2A, a bright and strong uorescent image was observed in the ethanol treatment group. PCP concentrationdependently decreased intracellular ROS production in zebra sh (P < 0.001) ( Figure 2B). Compared with control group, the MDA levels of the ethanol-treated groups were signi cantly elevated (P < 0.001) ( Figure  2C). Conversely, the levels of SOD, CAT and GSH markedly declined (P < 0.001). While PCP pretreatment reversed the process in a concentration-dependent manner ( Figure 2D, E, F). However, the CAT activity was signi cantly increased only at 100 µg/mL PCP pretreatment (P < 0.001). Thus, PCP alleviated ethanol hepatosteatosis by inhibiting oxidative stress.

PCP decreased Keap1 expression and promoted Nrf2 transferring into nucleus via p62
To further explore the underlying mechanism. Western blot experiments were conducted to detect the expression of related proteins, as shown in Figure 3. In ethanol treatment group, the protein expression of p62 was upregulated (P < 0.01), while a decreased protein expression of p62 was observed ( Figure 3A) when PCP pretreated with zebra sh in a concentration dependent manner (P < 0.001). And There is increasing evidence of a functional association between dysfunctional autophagy and Nrf2 pathway activation. Based on this, the Keap1 and Nrf2 expression ( Figure 3B, C) were detected in our study. We found PCP could downregulate the Keap1 protein expression (P < 0.001) and promote Nrf2 transferring into nucleus (P < 0.01). These data suggested that PCP alleviated ethanol-induced liver injury via activating p62 / Keap1 / Nrf2 pathway.
PCP alleviated ethanol-induced liver injury via activating AMPK / mTOR pathway Next, effort was made to investigate that whether PCP could activate AMPK / mTOR pathway to induce autophagy. As showed in gure 3D, E, PCP was no signi cant effect on total proteins expression including AMPK and mTOR, whereas it could enhance the phosphorylation of AMPK (p-AMPK) (P < 0.01),and decrease p-mTOR in a concentration dependent manner (P < 0.001), thus enhancing autophagy. In addition, a decrease of p-AMPK and an increase of p-mTOR expression was showed in ethanol treatment group (P < 0.001). These data suggested that the possible mechanisms of PCP alleviated ethanol-induced liver injury were primarily through phosphorylation of AMPK / mTOR pathways.

Chemical characteristics of PCP extract
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 various polyphenols ( Figure 5). Five peaks were identi ed as gallic acid, rutin, quercetin, luteolin, and apigenin. The contents of the ve compounds were quanti ed using corresponding chemical standards. Speci cally, the contents of gallic acid, rutin, quercetin, luteolin, and apigenin in PCP were 1.2025, 0.8244, 0.4967, 0.026, 0.0970 mg / g, respectively.

Discussion
Excessive ethanol consumption is increasing every year globally, especially among young people, affecting 10-15 per cent of the population, posing a signi cant medical, social and economic burden (Carvalho, Heilig, Perez, Probst, & Rehm, 2019). Apart from abstinence, there is no effective cure. Therefore, preventing the occurrence of disease may be a more reliable way to treat the disease better.
PCP has been used to treat liver-related diseases with no side effects or toxicity observed in clinical use (A. Wang et al., 2020). Previous studies showed that polyphenols were the main chemical constituents of PCP, which possessed strong antioxidant activities (Hu & Wang, 2015). Our data indicated that PCP reduced ethanol-induced hepatic steatosis and oxidative stress in zebra sh and the related molecular mechanism was further discussed.
The liver is an important organ for ethanol metabolism, which can be damaged by by-products of ethanol decomposition, such as ROS. While excessive production of ROS can lead to oxidative stress, which can lead to liver damage (Yan, Nie, Luo, Chen, & He, 2021). Recently, the prevention and treatment of oxidative stress-driven liver diseases by medicinal plant extracts has attracted wide attention. PCP, acted as traditional Miao medicine, its hepatoprotective effect on inhibiting oxidative stress in vivo was reported in previous literatures. It has been reported that PCP had a signi cant protective effect on tert-butyl hydroperoxide (t-BHP) induced hepatocyte damage, resulting in resistance to the ROS induced mitochondrial oxidative stress (A. Wang et al., 2016). Excessive ROS production induced oxidative stress, which led to the damage of protein and DNA in cells as well as the production of lipid peroxides, such as MDA. In our research, MDA level obviously increased in ethanol treatment group, whereas PCP, acting as scavengers of ROS, markedly decreased this tendency. In response to oxidative stress, the zebra sh larvae need to elevate the activities of antioxidant enzymes and activate non-enzyme antioxidant system, such as SOD, CAT and GSH. In our results, PCP exerted strong antioxidant ability, thereby attenuating the oxidative stress induced by ethanol.
In addition, recent papers reported that PCP enhanced the oxidant defense systems via the activation of Nrf2 / HO-1 pathway against chronic ethanol-induced liver injury (Cao et al., 2015). Nrf2, as important nucleus transcription factor, is retained by binding to its inhibitor and inactivating Keap1 in the cytoplasm, which serves as a Nrf2 degradation adapter. When responsing to oxidative stress, Nrf2 translocates into nucleus and regulates the expression of antioxidant genes, such as HO-1, NQO-1, which become the key pathway for plant extracts and natural products to inhibit ethanol-induced oxidative stress. And our results showed the same trend.
Recently, autophagy has become a protective mechanism against ALD (Babuta & Furi, 2019). p62 is considered to be a speci c autophagy marker, and the increase of p62 protein level indicates that autophagy is inhibited and autophagy ux is blocked (Pankiv et al., 2007). Previous studies have shown that disruption of early autophagy pathways leads to the accumulation of ubiquitinated proteins and an increase in ROS levels and mitochondrial dysfunction (Zhu et al., 2021). Increasing evidence suggests a functional association between dysfunctional autophagy and activation of the Nrf2 pathway, which appears to occur through the physical interaction between autophagy connector p62 and Nrf2 inhibitor Keap1, thereby binding to and competing with Keap1, promoting Nrf2 release by Keap1 and increasing Nrf2 stability and transcriptional activity of induced antioxidant gene expression . Our results were consistent with recent studies, indicating that PCP can effectively change the autophagy ux in ethanol-induced oxidative impairment.
In addition, AMPK is an energy sensor that is an upstream molecule of autophagy (W. S. He, Wu, Ren, Yu, & Zhao, 2021). In the former publications, Monascin regulated AMPK-mediated ethanol-induced liver injury by regulating p62 and autophagy crosstalk with the Keap1 / Nrf2 pathway (Lai, Hsu, Pan, & Lee, 2021). Similarly, in our study, an increase of p-AMPK protein expression was observed by pretreatment with PCP, and then promoting p62-mediated autophagic degradation of Keap1. The above results suggested PCP could indeed active the expression of p-AMPK to exert a vital role in the PCP alleviating oxidative stress and autophagy impairment.
Moreover, AMPK is an upstream regulator of mTOR, which can activate mTOR phosphorylation and thus mediate autophagy signaling pathway (H. Wang et al., 2019). Interactions between the p62 and mTOR pathways have been reported, and it has been found that upregulation of p62 activates mTORC1 by directly acting on the mTOR regulatory protein Raptor, thereby inhibiting autophagy (Zhang, Bao, Cong, Fan, & Li, 2020). Combined with our experimental results, the expression of p62 and mTOR increased, blocking the autophagy ux in ethanol treatment group. While after pretreatment with PCP, phosphorylated AMPK inhibited p-mTOR, thereby activating autophagy. The above results suggested PCP could indeed active the expression of p-AMPK to inhibit p-mTOR, alleviating autophagy impairment.
Above all, the mechanism of PCP in protecting ethanol-induced hepatic oxidative impairment in zebra sh was revealed more comprehensively, and the AMPK / p62 / Nrf2 / mTOR axis may play an important role in the treatment of ethanol-induced liver injury.