Active Components from sea buckthorn (Hippophae rhamnoides L

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Bioactive Constituents, Metabolites, and Functions

Active Components from sea buckthorn (Hippophae rhamnoides L.) regulate hepatic stellate cell activation and liver fibrogenesis Guokun Zhang, Yifei Liu, and Ping Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05306 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 7, 2018

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Journal of Agricultural and Food Chemistry

Active Components from sea buckthorn (Hippophae rhamnoides L.) regulate hepatic stellate cell activation and liver fibrogenesis Guokun Zhang,† Yifei Liu, § and Ping Liu*# †Institute

of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, Jilin, 130112, China

#College

of Forestry, Shenyang Agricultural University, Shenyang, Liaoning, 110866, China

§Liaoning

Academy of Forestry Science, Shenyang, Liaoning 110032, China

*Correspondence information: Ping Liu; Tel: +86 13709816862; Fax:+86 02486903334: E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Sea buckthorn (Hippophae rhamnoides L.) is a berry-bearing with multiple nutritional

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properties. In this study, 46 compounds were isolated from sea buckthorn berries.

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Preliminary data showed that the components, C13, C15 and C32 exhibited profound

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inhibitory effect on the activation of hepatic stellate cells (HSCs) induced by

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transforming growth factor-β (TGF-β), and decreased the levels of inflammatory

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factors. Furthermore, these compounds over-regulated the proteins of DNA damage

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signaling pathway and alpha-smooth muscle actin (α-SMA). Moreover, active

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components of sea buckthorn berries (ACSB) treatment attenuated fibrosis

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development in rats after BDL, reducing liver injury and inflammation, and reviving

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liver function in a dose-dependent manner. Moreover, ACSB down-regulated the

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expression of α-SMA, as while over-regulated the DNA damage signaling pathway

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and the related genes. These suggest that ACSB inhibit DNA repair of HSCs, make

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them in a damaged state, inhibit the expression of TGF-β, and induce apoptosis.

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KEYWORDS: sea buckthorn, liver fibrosis, HSCs, DNA damage

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INTRODUCTION

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Liver fibrosis is a serious health disrupter that can develop into liver cirrhosis and

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cancer.1 Hepatic fibrosis is due to the excessive accumulation of extracellular matrix

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(ECM), which destroys the structure of normal liver.1, 2 The ECM has the capacity to

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activate quiescent hepatic stellate cells (HSCs), these activated HSCs increase

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proliferation, acquire contractility and pro-inflammatory properties, and express

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α-smooth muscle actin (α-SMA) which is a myogenic marker, to become the major

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Col1a1-producing cells.3 DNA damage is closely related to liver injury.4, 5 Without

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proper function of DNA damage response, It may cause abnormal proliferation of

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cells (e.g. HSCs), resulting in cell transformation and carcinogenesis. DNA damage

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response is mediated by multiple signal transduction processes in which the

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ATR-Chk1 and ATM-Chk2 pathways activate single-stranded DNA breaks (SSB) and

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DNA double-strand breaks (DSB), respectively.6 The balance of DNA damage

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responses is critical in liver; they can also regulate other biological phenomena such

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as apoptosis or cellular senescence.6, 7

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There is no effective therapy for liver fibrosis, some small molecules from plant

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have entered the eyes of researchers; they may become a new vision to reverse liver

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fibrosis. Sea buckthorn (Hippophae rhamnoides L.), a spiny deciduous shrub of the

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Elaeagnaceae family, has recently gained worldwide attention due to its nutritional

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and medicinal potential. Sea buckthorn is naturally distributed in Europe and Asia,

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and has been planted all over the world in recent years.8 Sea buckthorn berries are rich

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in nutrients and medicinal potential, which have been traditionally used to treat liver 3



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injuries, cardiovascular problems and gastric disorders in oriental medicinal system.8, 9

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There have been some preliminary reports on the role of sea buckthorn against liver

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injury,9 but the research on its mechanism and the specific efficacy components have

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not been perfected. This study was aimed to identify and clarify the chemical

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constituents isolated from sea buckthorn through chromatographic fractionation and

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bioactivity assays in vitro; and evaluate its anti-liver fibrotic effect and explore its

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mechanism in vivo.

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MATERIALS AND METHODS

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Materials. Sea buckthorn (Hippophae rhamnoides L.) berries were collected from Aohan banner, Inner Mongolia, China.

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Extraction and Isolation. The steps for extracting active compounds from sea

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buckthorn are as follows, the 20 kg of berries were extracted with 75% EtOH (bp

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78.1 °C) under reflux at 80 °C. The ethanol solution was rotationally vaporized, the

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residual aqueous solution was subsequently extracted using H2O, petroleum ether (PE,

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260 g), ethyl acetate (EtOAc, 260 g), and n-butanol (n BuOH, 135 g). The extraction

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and isolation scheme was reference to Zhong et al.10

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Cell Culture. Rat hepatic stellate cell (HSC) was purchased from Beijing Ding

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Guo Biotechnology Co., Ltd. (Beijing China). HSCs were routinely maintained in

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DMEM (Gibco, New York, USA) added on 10% FBS (Gibco, New York, USA) and

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antibiotics (100 U/ml streptomycin and penicillin) in a humidified atmosphere

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containing 5% CO2 at 37 °C.

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Cell Viability Assay. HSCs were cultured using 96-well plates (1 × 104 cells/well) 4


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overnight, and then the individual compounds (0, 20, 40, 80, 160, 320 μM) were

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added in the cultured system with (10 ng/ml) or without transforming growth factor-β

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(TGF-β) (PeproTech, USA) for 48 h. Removed the liquid from wells, and then added

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100 μl MTT (Beyotime, Shnghai, China) reagents (5 μg/ml) to each well for

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incubation of 3 h at 37 °C. After that, the formazan crystals were dissolved with

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dimethyl sulfoxide, cell viability was determined via measuring the absorbance at 490

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nm. Note that, each of the 46 compounds was dissolved in dimethyl sulfoxide to give

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a 0.1 M stock solution.

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Cell Cycle Analysis. C13, C15, or C32 (40 μM) was added to the HSC cultured

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system and incubated for 24 hours, respectively. Briefly, HSCs were collected (5 ×

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106 cells/group), washed, and suspended in cold 75% ethanol over night at 4°C. Cells

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were further centrifuged, washed and stained with 50 μg/ml propidium iodide (PI) and

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50 μg/ml RNase-A (Sigma, USA) dissolved in 500μL PBS. The suspension was

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incubated for another 30 min. and analyzed using flow cytometry (FACS Calibur,

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Becton Dickinson, USA).

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Animal and Treatment. 8-week-old male Sprague Dawley (SD) rats were

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provided by Changsheng Co., Ltd. (Benxi, China). All of the experimental rats

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received humane care, and were approved by the Guide for the Care and Use of

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Laboratory Animals at Shenyang Agricultural University (No. SYA20170563R),

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Shenyang, China.

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The rats were randomly divided into groups. Rats were anesthetized with chloral

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hydrate. All of the surgical procedures were performed under aseptic conditions. Bile 5



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duct ligation (BDL) was performed in rats as described previously.11 Controls

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underwent a sham operation. Three groups of ligated rats were given daily intragastric

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administrations of 20 and 40 mg/kg active components of sea buckthorn berries

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(ACSB) for 4 weeks, respectively, and the positive control group rats was treated with

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40 mg/kg silymarin. Liver tissues were collected under general. The serum was

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collected and stored at -70 °C for the liver function assays of albumin (ALB), alkaline

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phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST)

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and total bilirubin (TBIL). The kits were purchased from Biosino Bio-Technology and

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Science Inc (Beijing, China). The livers were then divided into three portions: (1)

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immediately used for protein and RNA isolation; (2) preserved in 4%

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paraformaldehyde for histological examination.

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Histological examination. The liver sections imbedded in paraffin were cut (4 μm)

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and stained with Hematoxylin-eosin and Masson’s trichrome. The liver fibrosis stage

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was assessed by Ishak scale.12

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Immunohistochemical (IHC) was performed according to staining kit instructions

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(KeyGEN BioTECH, Nanjing, China). Paraffin sections of 4% paraformaldehyde

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fixed liver samples were rehydrated and subjected to antigen retrieval in 10 mM

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sodium citrate buffer (pH 6.0). The sections were incubated in 10% goat serum for 0.5

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h, followed by avidin-biotin block. And then the sections were incubated with α-SMA

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(Boster, BM0002, China, 1: 100 dilution) overnight at 4ºC. Next day, sections were

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incubated with the secondary antibodies for 10 minutes and color developed with

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DAB. Sections were then dehydrated and closure. 6


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RNA Isolation and quantitative PCR (qPCR). Total RNA was extracted from

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the liver tissues (20 mg) of the rats or HSCs (1 × 106) using with TRIzol Reagent

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(Thermofisher, USA) and processed according to the manufacturer. Next, 2 μg of

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RNA from each sample was retrotranscribed. For 20 μl of PCR, 50 ng of cDNA was

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mixed with primers and FastStart Universal SYBR Green Master (ROX) (Roche,

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Basel, Switzerland). QPCR was carried out using the ABI Prism 7900-HT sequence

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detection system as previously.7 The primers used for quantitative PCR are described

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as follows: tumor necrosis factor-α (TNF-α), forward

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5′-CCCTCACACTCAGATCATCTTCT-3′ and reverse

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5′-GCTACGACGTGGGCTACAG-3′; IL-1, forward

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5′-GCAACTGTTCCTGAACTCAACT-3′ and reverse

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5′-ATCTTTTGGGGTCCGTCAACT-3′; IL-6, forward

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5′-TAGTCCTTCCTACCCCAATTTCC-3′and reverse

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5′-TTGGTCCTTAGCCACTCCTTC-3′; β-Actin was used as internal control. QPCR

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data were analyzed using the 2-ΔΔCt method to calculate the relative level of each

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mRNA.

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Western Blotting Analysis. Proteins were extracted from liver tissues (100 mg)

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or HSCs with RAPI buffer (Beyotime, Nanjing, China). The protein contents were

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detected using BCA Protein Assay Kit (Solarbio, Beijing, China). 20.0 μg of extracted

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proteins were mixed with loading buffer, and then protein solutions were denatured at

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95 °C for 10 min prior to electrophoresis on SDS gel. The samples (40 μl) were

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separated by polyacrylamide SDS gel and electrophoretically transferred onto 7



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polyvinylidene fluoride membranes (Millipore, MA), and were blocked with TBST

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buffer containing 3.0 % Bovine Serum Albumin (BSA) for 1 h, incubated with the

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primary antibody at 4 °C overnight. The membranes were incubated with the

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secondary antibody for 1 h after washing three times. The levels of proteins were

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visualized using an ECL system (Alpha FluorChem®HD2, USA). The target protein

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bands were quantified by scanning densitometry using Image-J

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(https://imagej.nih.gov/ij/ ) and normalized to the signal intensity of α-tubulin.

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Antibodies used in western blot were shown as follows: α-SMA; ataxia telangiectasia

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mutated (ATM); checkpoint kinase 1 (Chk1); ATM and Rad3-related protein (ATR);

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checkpoint kinase 2 (Chk2); p53; α-SMA; cell division control protein 25 (CDC25);

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p53; α-tubulin.

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Statistical Analysis. Results were expressed as mean ± s.e.m. Statistically

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evaluated by ANOVA using GraphPad Prism 6.0. Differences were considered

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significant: P 95%, as determined by HPLC analysis..

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Screening and Evaluation of Inhibitory Activity Against Self-Activated HSCs.

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All of and compounds isolated from sea buckthorn were assayed for their inhibitory

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activities against self-activated HSCs. IC50 values were analyzed via MTT assay, and

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the results were shown in Table 1. The total extract compounds greatly inhibited the

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activation of HSCs, with IC50 values of 87.24±5.82 μM. At the same time,

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compounds 12-15, 32 and 33 showed potent anti-fibrosis activity with a relative order

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of efficacy of 15> 13 > 32 > 33 > 14 > 12, with IC50 values of 46.03, 57.18, 58.28,

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87.30, 87.90, and 93.59 μM, respectively. The strong inhibitory activity of

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compounds 13, 15 and 32 toward the activated HSCs could possibly be related to their

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different phytochemical properties, which are due to their reduction bonds (e.g.

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hydroxyls).

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Compounds 13, 15 and 32 Inhibit Cell Viability and Decrease Cytokine

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Released via TGF-β-Activated HSCs. Based on preliminary screening and

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evaluation data for growth inhibition, further studies were conducted on compounds

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13,15 and 32 (Figure 1). HSCs were treated with these compounds (one at a time) plus

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TGF-β (10 ng/ml) for 48 h, and the growth of the HSCs was measured. The results

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showed that compounds 13, 15 significantly decreased the cell viability of activated

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HSCs in a dose-dependent manner (Figure 1a). The levels of TNF-α, IL-1 and IL-6 9



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(Figure 1b-d), which are HSC activation-associated factors and inflammatory

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cytokines, were increased in TGF-β activated HSCs compared with control. However,

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TGF-β activated HSCs treated with compounds 13, 15, and 32 decreased the

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transcript levels of TNF-α, IL-1 and IL-6, while C32 having no effect on the level of

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IL-1.

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Compounds 13, 15, and 32 induce HSC cell cycle arrest. Smooth cycle of HSC

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cell cycle is an important cause of liver fibrosis. The results of the cell cycle

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distribution of HSCs treated with C13, C15, or C32 showed that, the percentage of

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G1-phase cells of TGF-β activated HSCs was decreased compared to control group

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(83.33% vs. 86.53%), while the percentage of G2-phase cells was increased (8.67% vs.

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5.47%); however, treatment of these cells with compounds 13, 15, and 32 increased

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the levels of G1-phase cells (89.22%, 88.29%, and 88.41%), while decreased the

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levels of G2-phase cells (2.78%, 3.71%, and 3.59%) (Figure 2). The cell cycle results

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suggested that C13, C15, and C32 effectively induced G1-phase and G2-phase cell

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cycle arrest of HSCs.

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Compounds 13, 15, and 32 Change the Expression Levels of DNA damage

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signaling gene. In liver fibrosis, the DNA damage signaling pathway is highly

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activated, preventing malignant transformation of damaged cells by inducing growth

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arrest and cell death, p53 (a receptor of DNA damage pathway) is likely involved in

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the protective role in livers damage.5, 19 As shown in Figure 3, the related proteins

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including ATM, ATR, Chk1, Chk2, CDC25, and p53 were decreased in TGF-β

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treated group compared to control group (P

Active Components from sea buckthorn (Hippophae rhamnoides L

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