Accepted Manuscript

Tamarix hohenackeri Bunge exerts anti-inflammatory effects on

lipopolysaccharide-activated microglia in vitro

Ru Chen , Yanqiu Yang , Jikai Xu , Yingni Pan ,

Wenqiang Zhang , Yachao Xing , Hui Ni , Yu Sun , Yue Hou ,
Ning Li

PII: S0944-7113(17)30209-X
DOI: 10.1016/j.phymed.2017.12.035
Reference: PHYMED 52337

To appear in: Phytomedicine

Received date: 9 August 2016

Revised date: 11 December 2017
Accepted date: 28 December 2017

Please cite this article as: Ru Chen , Yanqiu Yang , Jikai Xu , Yingni Pan , Wenqiang Zhang ,
Yachao Xing , Hui Ni , Yu Sun , Yue Hou , Ning Li , Tamarix hohenackeri Bunge exerts anti-
inflammatory effects on lipopolysaccharide-activated microglia in vitro, Phytomedicine (2017), doi:
10.1016/j.phymed.2017.12.035

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 ACCEPTED MANUSCRIPT

Tamarix hohenackeri Bunge exerts anti-inflammatory effects on

lipopolysaccharide-activated microglia in vitro

Ru Chena,1, Yanqiu Yang a,1, Jikai Xua, Yingni Panb, Wenqiang Zhanga,Yachao Xingb, Hui Nic, Yu Sunc, Yue Houa,*,
Ning Lib,*

a
College of Life and Health Sciences, Northeastern University, Shenyang, China

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b
School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design

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and Discovery, Shenyang Pharmaceutical University, Ministry of Education, Shenyang, China

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c
XinJiang Institute of Chinese Materia Medica and Ethnodrug, Urumqi, China

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These authors contributed equally to this work
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*Corresponding authors:
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College of Life and Health Sciences, Northeastern University, Shenyang 110004, China.

E-mail address: houyue@mail.neu.edu.cn (Y. Hou);

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School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang

110016, China,
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E-mail address: liningsypharm@163.com (N. Li).

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ABSTRACT
Background: Tamarix species are well known as the main host plants of Herba

Cistanches, a valuable Traditional Chinese Medicine. They are also traditional

medicinal plants themselves and are used to treat spleen problems, leucoderma and

ocular conditions.

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Purpose: The aim of the present study was to investigate the anti-inflammatory effect of

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Tamarix hohenackeri Bunge.

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Methods: In the present study, BV-2 microglial cells were used and stimulated with

lipopolysaccharide (LPS). Cell viability was tested using the MTT assay. The release of

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nitric oxide (NO) was determined using the Griess assay. The mRNA level of inducible

nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), interleukin (IL)-1β and
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IL-6 were investigated by quantitative real-time PCR (qRT–PCR). The protein levels of
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phosphorylated of IκBα, ERK and MEK, as well as the cytoplasmic and nuclear NF-B

p65 were tested by Western blot analysis. The translocation of the NF-B p65 subunit
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from the cytosol to the nucleus was investigated by immunofluorescence staining.

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Results: Ethyl acetate (EtOAc) extract of Tamarix hohenackeri Bunge significantly

inhibited the release of NO. Phytochemical research was performed to produce 13 main
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constituents. Among them, compounds 6, 7, 10 and 13 were identified to be the

effective components with anti-inflammatory activity. These compounds significantly

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inhibited the production of NO by LPS-activated BV-2 microglial cells. qRT–PCR

showed that compounds 6 and 7 significantly suppressed the LPS-induced transcription

of genes encoding pro-inflammatory mediators, including iNOS, TNF-α, IL-1β and IL-6.

Western blot analysis showed that compound 7 inhibited the LPS-induced

phosphorylation of IB and antagonized the LPS-induced reduction of cytoplasmic

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NF-B p65 and the increase of nuclear NF-B p65. Immunofluorescence staining

showed that nuclear translocation of NF-B p65 was suppressed by compound 7.

Western blot analysis showed that compound 7 inhibited the LPS-induced

phosphorylation of ERK and MEK.

Conclusion: The present study revealed, for the first time, the effective

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anti-inflammatory agents from T. Hohenackeri. Compound 7 exerted potent

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anti-inflammatory effects and its underlying mechanism may be associated with its

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capacity to inhibit NF-B signaling pathway and the MEK/ERK activation in activated

microglia. The compound may be potential candidate therapeutic agent for

neurodegenerative diseases.
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Keywords: Tamarix hohenackeri Bunge; Anti-inflammatory; Neurodegenerative diseases;

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Microglia; LPS
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Abbreviations: MAPK, Mitogen-activated protein kinase; NF-B, nuclear factor B; LPS,
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lipopolysaccharide; NO, nitric oxide; iNOS, inducible nitric oxide synthase; TNF-α,

tumor necrosis factor α; IL, interleukin; qRT–PCR, quantitative real-time PCR; EtOAc,
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ethyl acetate; ACE, angiotensin-converting enzyme; HPLC, High-performance liquid

chromatography; FBS, Fetal bovine serum; DMEM, Dulbecco’s Modified Eagel

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Medium; PDTC, Pyrrolidinedithiocarbamate; MINO, Minocycline; EtOH, ethanol; PE,

petroleum ether; n-BuOH, n-butanol; DMSO, dimethyl sulfoxide; PBS,

phosphate-buffered saline; GAPDH, glyceraldehyde phosphate dehydrogenase; BCA,

bicinchoninic acid; ANOVA, analysis of variance; LSD, least significant difference;

CNS, central nervous system.

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Introduction
Tamarix L., commonly known as Tamarisk, is a genus of the family Tamaricaceae.

The genus contains about 20 species which are characterized by their tolerance of dry

and salt-rich conditions. In China, the plants mainly grow in desert regions and the

saline-alkali fields of the north-west, especially in Xinjiang Province (Zhang et al.

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2003). Tamarix species are well known as the main host plants of Herba Cistanches, a

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valuable Traditional Chinese Medicine which enhances kidney function. In recent years,

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the quantity demanded has increased greatly because of the wide use of Herba

Cistanches for production of healthy food and drug. Now, Cistanches has become into

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an endangered plant in China. In order to protect wild plant source, Tamarix

hohenackeri Bunge has been extensively cultivated in Xinjiang Province for the large
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scale production of Herba Cistanches. As well as acting as host plants, Tamarix species
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are also traditional medicinal plants themselves, and are used to treat spleen problems,

leucoderma and ocular conditions (Sharma and Parmar 1998). They have astringent and
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diuretic properties, and are also used as an aperitif and as a stimulant of perspiration
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(van Oudenhoven et al. 2010). A wide variety of biologically important activities are

associated with Tamarix plant extracts. These include anti-inflammatory (Barakat and
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Nada 1996), anti-oxidant (Abouzid et al. 2009), hepatoprotective (Assiri 2006;

Sehrawat and Sultana 2006; Abouzid and Sleem 2011), anti-bacteria (Saïdana et al.
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2008) and anti-neoplastic (Wang et al. 2009) effects, as well as inhibition of

α-glucosidase (Chang et al. 2011). It has been demonstrated that certain Tamarix species

contain high levels of flavonoids, phenylpropanoids, organic acids, triterpenes, steroids,

tannins and lignans (Zhang and Tu 2008; Huang and Liang 2007). We have also

reported the effects of T. hohenackeri constituents on angiotensin‑converting enzyme

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(ACE) inhibition and inhibition of platelet aggregation (Xing et al. 2014).

Neuroinflammation serves as a crucial mechanism in protecting the brain from

neuronal damage and infectious agents (Li et al. 2014; Lee et al. 2012). However,

severe neuroinflammatory processes can lead to the neuronal injuries observed in a

number of neurodegenerative diseases (Li et al. 2014; Lee et al. 2012; Kanno et al.

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2012). In response to inflammatory stimulation, microglial cells, the macrophages in the

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brain, secrete pro-inflammatory molecules, such as interleukin 6 (IL-6), tumor necrosis

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factor α (TNF-α) and nitric oxide (NO), which can contribute to neuronal injuries,

particularly in neurodegenerative disorders (Barger and Harmon 1997; Saleppico et al.

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1996; Wang et al. 2014). Thus, hyperactive microglial cells are a potential target for

therapies to treat neurodegenerative disorders. To date, there are no effective methods

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for preventing or treating neurodegeneration.

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In our previous work, T. hohenackeri extract attracted our attention because of its

inhibitory effect on the production of NO in LPS-stimulated microglia. Here, we

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identified the potential neuroinflammation inhibitors from T. hohenackeri and

considered their plausible mechanisms of action.

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Materials and methods

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General
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Silica gel (SiO2: 200-300 mesh, Qingdao Marine Chemical Company, China),

Sephadex LH-20 (Pharmacia, Co., Switzerland), Macroporous resin HPD-100

(Cangzhou Bonchem Co., Ltd., China), and polyamide (Taizhou City Luqiao Sijia

Biochemical Plastic Factory, China) were used for column chromatography.

Semi-preparative HPLC was performed on a YMC ODS C-18 column (250 mm × 10

mm, 10 μm, ODS-A, Co., Ltd. Japan) using a SHIMADZU SPD-10A UV detector.
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Silica gel GF254 was used for TLC (SiO2: 200-300 mesh, Qingdao Marine Chemical

Company, China). The chromatograms were visualized under UV light (at 254 and 365

nm) before and after applying the sulfuric acid-ethanol (10%) spray, ferric

chloride-ethanol spray and bromocresol green-ethanol-sodium hydroxide spray. Fetal

bovine serum (FBS) was from Tianjinhaoyang Biological Manufactory Co., Ltd (Tianjin,

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China). Dulbecco’s Modified Eagel Medium (DMEM) was purchased from Gibco BRL

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(Grand Island, NY, USA). Lipopolysaccharide (LPS) was obtained from Sigma (St.

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Louis, MO, USA). (3-[4,5- Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

(MTT) was purchased from Beyotime Biotechnology (Beijing, China). Trizol reagent

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was from Invitrogen Co. (Carlsbad, CA, USA). Go ScriptTM Reverse Transcription

System and Go Taq qPCR Master Mix were obtained from Promega (Madison, WI,
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USA). Nuclear and Cytoplasmic Protein Extraction Kit was purchased from Beyotime
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(Beyotime, Shanghai, China). The antibodies against phospho-MEK1/2,

phospho-ERK1/2, phospho-IκBα, NF-B p65 were purchased from Cell Signaling

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Technology, Inc. (Danvers, MA, USA). Pyrrolidinedithiocarbamate (PDTC) and

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Minocycline (MINO) were from Sigma (St. Louis, MO, USA). The other reagents used

in this study were all analytical grade or higher and were purchased from Shandong
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Yuwang Co., Ltd. Chemical Engineering Branch and Tianjin Damao Chemical Reagent

Co.
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Plant material
The aerial parts of T. hohenackeri Bunge were collected from the Hetian District of

Xinjiang Province, China, in June 2011. The identity of the plant material was

confirmed by Prof. Xiaoguang Jia. (Xinjiang Institute of Chinese Materia Medica and

Ethnodrug) and a voucher specimen (No.20110613) was deposited in the School of

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Traditional Chinese Materia Medica at Shenyang Pharmaceutical University.

Extraction and isolation

The ethanol (EtOH) extract, petroleum ether (PE) extract, ethyl acetate (EtOAc)

extract and n-butanol (n-BuOH) extract were obtained as previously reported [14].

Thirteen compounds were isolated from the EtOAc extract according to previously

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published approaches [14]. The purity of the identified compounds 1-13 were

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determined by HPLC analysis before the bioassay described in this paper, compounds 1

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(98.6%), 2 (98.9%), 3 (99.2%), 4 (98.3%), 5 (98.6%), 6 (99.1%), 7 (98.4%), 8 (98.7%),

9 (98.9%), 10 (99.0%), 11 (98.5%), 12 (98.6%), 13 (98.3%).

Qualitative analysis
chromatographic preparations
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the main constituents from
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The qualitative analysis was performed on a C18 column (250 mm x 4.6 mm, 5 μm,
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Dikma) at 16 ◦C. The flow rate was 0.8 ml/min. The mobile phase was a mixture of

methanol (A) and 0.5% acetic acid–water (B). For the mobile phase, the gradient
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program was as follows: 0–40 min, 20–80% A-B; and 40–60 min, 80–80% A-B. The

wavelength was 254 nm, and the injection volume was 10 μl . Individual stock solutions
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of the 4 reference compounds (compounds 6, 7, 10 and 13) used for the qualitative
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analysis were prepared by accurately weighing them, then dissolving them in methanol.

A solution containing a mixture of all 4 of the reference compounds was also prepared.
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All of solutions were centrifuged at 14,000 rpm, then passed through a 0.2-μm filter

membrane prior to injection.

Cell culture
The mouse microglial cell line BV-2 cells were cultured at 37 °C in a humidified

incubator with 5% CO2. The culture medium was DMEM supplemented with 10% FBS,

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2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Sample treatment protocols

Tamarix hohenackeri Bunge extracts or the isolated compounds were initially

dissolved in dimethyl sulfoxide (DMSO). Dilutions were made with phosphate-buffered

saline (PBS). The maximum concentration of DMSO in the culture media was 0.1% and

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was not toxic to the cells. The MTT reduction assay and nitrite assay were performed

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after 24 h of LPS treatment. To detect the mRNA levels of the proinflammatory factors

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by qRT-PCR, the microglial cells were pretreated with one of the compounds for 2 h

and then exposed to LPS for 4 h. For Western blot experiments to detect the protein

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levels of phosphorylated IκBα, ERK and MEK as well as cytoplasmic and nuclear
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NF-B p65, BV-2 microglia were pre-incubated with certain compound for 2 h and then

exposed to LPS for 30 min. Immunofluorescence staining was performed to investigate

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nuclear translocation of NF-B p65, BV-2 microglia were pre-incubated with compound

7 for 2 h and then exposed to LPS for 30 min.

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Cell viability measurements

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Cell viability was assessed using the MTT assay (Li et al. 2014; Hou et al. 2006).

BV-2 cells were seeded into 96-well plates. After treatment with the tested compounds
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and LPS (100 ng/ml) for 24 h, the cells were incubated with MTT (0.25 mg/ml) for 4 h

at 37 °C. Then, the supernatant was removed and the formazan crystals were dissolved
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by adding DMSO. The plates were then analyzed in a plate-reader at 490 nm (Bio-Tek,

USA) to determine the percentage of surviving cells.

Nitrite assay
NO synthase activity was determined by measuring the accumulation of nitrite

(NO2−) in the cell supernatant using the Griess reaction in culture supernatants (Li et al.

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2014; Hou et al. 2006). The cells were plated into 96-well microtiter plates, then

incubated with Tamarix hohenackeri Bunge extracts or the isolated compounds in the

presence of LPS (100 ng/ml) for 24 h. Fifty microliters of Griess reagent was mixed

with of 50 μl culture supernatant at room temperature. Fifteen minutes later, the

absorbance was determined at 540 nm using a plate reader (Bio-Tek, USA).

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Quantitative real-time PCR (qRT–PCR)

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The total RNA from the BV-2 microglial cells was extracted with Trizol according

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to the manufacturer's instructions and converted to cDNAs. qRT-PCR assays were

performed with the CFX ConnectTM real-time PCR detection system (Bio-Rad) using

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the GoTaq one-step real-time PCR kit with SYBR green. Primers sequences are shown

in Table 1. Gene expression values were normalized to those of GAPDH.

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Western blot analysis

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Western blot analysis was performed as previously described (Wang et al. 2014;

Meng et al. 2008). BV-2 cells were pretreated with certain compound for 2 h, followed
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by LPS for 30 min. For total protein extraction, cells were washed with ice-cold PBS
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and lysed with lysis buffer as previously described. For cytoplasmic and nuclear protein

extraction, the protein was extracted according to Nuclear and Cytoplasmic Protein
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Extraction Kit manufacturer’s instructions. The protein content was determined by BCA

assay. Cell lysates were electrophoresed using 12% SDS-PAGE and the gel was then
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transferred to a membrane. After soaking in blocking buffer, the membrane was

incubated overnight with primary antibodies, followed by horseradish

peroxidase-conjugated secondary antibodies.

Immunofluorescence staining
Immunofluorescence staining was used to test the nuclear localization of NF-B

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p65 as previously reported (Hou et al. 2017). Briefly, the cells were pretreated with

compound 7 (100 μM) for 2 h, followed by LPS for 30 min. Then, the cells were fixed,

permeabilized, blocked and incubated with NF-B p65 primary antibody. After

incubation with fluorescein (FITC)-conjugated anti-rabbit lgG, the cells were stained

with DAPI and photographed.

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Statistical analysis

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Statistical procedures were performed using SPSS 17.0 software for Windows

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(SPSS Inc., Chicago, IL, USA). Data were analyzed by ANOVA (one-way analysis of

variance). All results are expressed as the mean ± SEM. For comparison of groups, post

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hoc analyses were carried out with Fisher’s least significant difference (LSD) test or

Dunnett’s T3 test. The differences were considered significant at P < 0.05.

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Results and discussion

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Microglia are the resident immune cells in the CNS. They are responsible for

immune surveillance and for defending the brain against injury or disease (Ransohoff
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and Perry 2010; Lue et al. 2010). When subjected to abnormal stimulation, they readily
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become activated and initiate the release of various proinflammatory mediators,

including NO, TNF-α, IL-1β and IL-6, which are associated with a number of
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neurodegenerative diseases (Lu et al. 2010; González-Scarano and Baltuch 1999; Wang

et al. 2014). Therefore, inhibiting the activation of microglial cells to diminish the level
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of proinflammatory mediators is regarded as an important therapeutic strategy for

neuroinflammatory diseases (Wang et al. 2014). This is the first report demonstrating

that Tamarix hohenackeri Bunge exerts anti-inflammatory effects on LPS-activated

microglia, which may be associated with inhibition of NF-B signaling pathways and

the activation of MEK/ ERK.

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Effects of Tamarix hohenackeri Bunge extracts on LPS-induced

NO production
The anti-inflammatory activities of the extracts were assayed in LPS-stimulated

BV-2 microglia by monitoring the production of NO. The results showed that the four

Tamarix hohenackeri Bunge extracts (EtOH, PE, EtOAc and n-BuOH) significantly

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inhibited NO production (Fig. 1). The IC50 values of the extracts were 19.5 μg/ml for

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EtOH, 11.3 μg/ml for PE, 6.0 μg/ml for EtOAc and 22.1 μg/ml for BuOH. The EtOAc

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extract was the most effective.

Effects of constituents isolated from the EtOAc extract on

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LPS-induced NO production and iNOS mRNA expression
The anti-inflammatory properties of the compounds isolated from the EtOAc
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extract were further investigated in LPS-stimulated BV-2 microglial cells by monitoring

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production of NO. At the same time, the cytotoxic effects of the compounds on BV-2

microglial cells were also tested using the MTT assay because it is difficult to separate
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their anti-inflammatory activities from their toxicity. As shown in Fig. 2, compounds 2,

4, 5, 9 and 12 were toxic to the cells at a concentration of 100 μM, which may affect
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their inhibitory effects on LPS-induced NO release. Taking the anti-inflammatory and

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cytotoxic activities into consideration, a reasonable conclusion may be drawn.

Compounds 6, 7, 10 and 13 can greatly inhibit the production of NO without showing

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cytotoxicity. Using the UPLC-DAD method, the main effective constituents (6, 7, 10

and 13) were characterized from the total extract (Fig. 4A) and the bioactive EtOAc

extract (Fig. 4B) by comparing with the retention time of the standards (6, 7, 10 and 13)

(Fig. 4C). By comparing the bioassay results (Fig. 2 and Table 2) and the structures of

the compounds (Fig. 3), the activities of the different structures could be discussed

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briefly. First, the substitution of methoxyl group at C-7 may greatly reduce the activities

in 3, 4, 7-trihydroxy-4′-methoxyflavonoid (4). The same effect was observed at

C-3′methoxyl in 3, 4, 7, 3′-tetrahydroxy-4′-methoxyflavonoid (6). Second, the methyl

esterification of the carboxyl group in the phenolic acids (compounds 10 and 12) could

enhance the anti-inflammatory activity. Moreover, the structures with the 3, 4,

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5-trihydroxyl benzoic acid (10) exhibited better effects than those with 3, 4- dihydroxyl

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benzoic acid (11).

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NO is synthesized by the enzyme NOS from L-arginine in various cells and tissues

(Mayer et al. 1989). NOS has several different isoforms, and the isoform present in

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microglia is inducible NOS (iNOS), which can continuously release NO when activated

(Lijia et al. 2012). Therefore, the effects of the four active compounds (6, 7, 10 and 13)
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on LPS-induced iNOS mRNA expression were investigated by qRT–PCR. One-way

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ANOVA analysis showed significant differences between the groups ([F(6,14) =

355.722，P < 0.001], Fig. 5). By Fisher’s LSD test, the LPS group showed increased
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iNOS mRNA expression compared to the control group, while compounds 6, 7, 10 and

13 (100 μM) significantly inhibited iNOS mRNA expression in LPS-stimulated BV-2

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cells (P < 0.001), indicating a direct basis for their ability to reduce NO production.
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Effects of active compounds (6, 7, 10 and 13) on LPS-induced

mRNA expression of pro-inflammatory mediators
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TNF-, IL-1β and IL-6 are the major pro-inflammatory cytokines produced by

activated microglial cells during inflammation (Dai et al. 2011). Therefore, the effects

of the four active compounds (6, 7, 10 and 13) on the LPS-induced mRNA expression

of these cytokines were also investigated by qRT–PCR. For TNF-, the one-way

ANOVA analysis showed significant differences between the groups ([F(6,14) =

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261.255，P < 0.001], Fig. 5). Fisher’s LSD test showed that the LPS group had increased

TNF- mRNA expression compared to the control group and that compounds 6 and 7

(100 μM) significantly inhibited TNF- mRNA expression in LPS-treated BV-2

microglia (P < 0.001). For IL-1β, the one-way ANOVA analysis showed significant

differences between the groups ([F(6,14) = 786.582，P < 0.001], Fig. 5). Dunnett’s T3

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test also revealed that the LPS group showed increased IL-1β mRNA expression

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compared to the control group and that compounds 6, 7 and 13 (100 μM) significantly

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inhibited the LPS-induced IL-1β mRNA expression in BV-2 microglial cells

(compounds 6 and 7: P < 0.01, compound 13: P < 0.05). For IL-6, the one-way ANOVA

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analysis showed significant differences between the groups ([F(6,14) = 130.338，P <

0.001], Fig. 5). Dunnett’s T3 test also revealed that the LPS group showed significantly
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higher IL-6 mRNA expression than the control group and that compounds 6, 7 and 13
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(100 μM) significantly inhibited IL-6 mRNA expression in LPS-activated BV-2 cells (P

< 0.01). Together, these results showed that compounds 6 and 7 exerted more potent
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anti-inflammatory effects. Compound 6 and 7 have almost similar structure, except for
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the substitution of methoxyl group at C-4′. As reported, the effect of quercetin

(compound 7) on the pro-inflammatory mediators was consistent with our results. For
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example, quercetin markedly reduced the mRNA or protein levels of inflammatory

mediators, including IL-6, IL-1β and TNF-α in microglia (Bournival et al. 2012; Yang et
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al. 2017). Similarly, quercetin also inhibited the protein expressions of TNF-α, IL-1β

and IL-6 induced by LPS in RAW 264.7 macrophages (Endale et al. 2013).

Effects of active compound(s) on LPS-induced NF-B signaling

pathway and MEK/ERK activation

Accumulating evidence has revealed that the NF-B signaling pathway is involved

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in regulating inflammatory processes (Wang et al. 2014; Meng et al. 2008） The NF-B

protein is generally found in the cytoplasm of unstimulated cells, where it binds to its

inhibitor, IB. LPS can induce the activation of NF-B in microglial cells by

phosphorylating IκBα (Wang et al. 2014; Meng et al. 2008). Therefore, the effects of

active compounds 6, 7, 10 and 13 on LPS-induced IκBα phosphorylation were tested.

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One-way ANOVA analysis showed significant differences among the groups ([F(6,14) =

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14.934，P < 0.001], Fig. 6). Fisher’s LSD test also revealed that LPS significantly

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increased IκBα phosphorylation (P < 0.001) and that compounds 6, 7 and 10 at the

concentration of 100 μM significantly inhibited its phosphorylation (compounds 6 and 7:

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P < 0.001, compound 10: P < 0.01). Among them, compound 7 exhibited the strongest

effect. As mentioned above, compound 6 and 7 were identified as effective components

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which exhibited better activity in inhibiting LPS-induced mRNA expressions of

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pro-inflammatory mediators. In addition, compound 6 and 7 have almost similar

structure. Take all these into consideration, the following study was focused on
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compound 7. Its effect on NF-B signaling pathway was further investigated by testing
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the protein level of NF-B p65. For cytoplasmic NF-B p65 protein, one-way ANOVA

analysis showed significant differences among the groups ([F(5,12) = 33.342，P <
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0.001], Fig. 7A). Fisher’s LSD test also revealed that LPS significantly reduced

cytoplasmic NF-B p65 (P < 0.001) and compound 7 (100 μM: P < 0.001) significantly
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inhibited the reduction of cytoplasmic NF-B p65. For nuclear NF-B p65 protein,

one-way ANOVA analysis showed significant differences among the groups ([F(5,12) =

80.738，P < 0.001], Fig. 7B). Fisher’s LSD test also revealed that LPS significantly

increased nuclear NF-B p65 (P < 0.001) and compounds 7 (10 M, 30M and 100 μM:

P < 0.001) significantly inhibited the increase of nuclear NF-B p65. The results were

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further confirmed by the observation in the immunofluorescence staining that treatment

of compound 7 prevented the translocation of NF-B p65 to the nucleus induced by

LPS (Fig. 7C).

MAPKs (mitogen-activated protein kinases) regulate numerous cellular processes,

including inflammatory responses (Wang et al. 2014; Zhao et al. 2011). LPS-induced

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microglial activation and pro-inflammatory cytokine expression is regulated by the

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ERK/MAPK signaling pathway (Wang et al. 2014; Zhao et al. 2011). We therefore used

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Western blot to investigate whether ERK was involved in the mechanism of action of

compound 7. For phosphorylation of ERK, one-way ANOVA analysis showed

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significant differences among the groups ([F(5,12) = 36.227，P < 0.001], Fig. 8A). The
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Fisher’s LSD test also revealed that LPS significantly increased ERK phosphorylation

(P < 0.001) and that compound 7 significantly inhibited its phosphorylation (10 and 30
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μM: P < 0.01, 100 μM: P < 0.001). Since the phosphorylation of ERK is regulated by

the activation of MEK (ERK kinase) (Caunt and Keyse, 2013), the phosphorylation of
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MEK was then detected. For phosphorylation of MEK, one-way ANOVA analysis
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showed significant differences among the groups ([F(5,12) = 27.109, P < 0.001], Fig.

8B). The Fisher’s LSD test also revealed that LPS significantly increased MEK
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phosphorylation (P < 0.001) and that compounds 7 significantly inhibited its

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phosphorylation (30 μM: P < 0.05, 100 μM: P < 0.001).

Conclusions
The present study identified, for the first time, the effective anti-inflammatory

agents from T. Hohenackeri. Flavanoids and phenol acids were characterized as

bioactive components representative for the observed effect. It is suggested that

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compound 7 has potent anti-inflammatory effects, which may be associated with its

capacity to inhibit the phosphorylation of MEK, ERK and IκBα as well as the nuclear

translocation of NF-B p65 in LPS-activated microglial cells. It is a natural bioactive

product with low toxicity and exhibit anti-inflammatory activity in vitro. Further in vivo

study merits being performed to verify its activities by using neuroinflammation animal

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model.

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Conflicts of interest

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There are no conflicts of interest to declare.

Acknowledgments
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This research is supported by the National Natural Science Foundation of China
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(81473330, U1603125, U1403102, 81673323, 81473108), Program for Liaoning

Excellent Talents in University (LR2015022), the Fundamental Research Funds for the
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Central Universities of China (N162004003, N130120001), Research Project for Key

laboratory of Liaoning Educational Committee (LZ2015067), Natural Science

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Foundation of Liaoning Province (2015020732), Shenyang science and technology

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research project (F15-199-1-26).

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Figure legends

Fig. 1. Anti-inflammatory activity of Tamarix hohenackeri Bunge in BV-2

microglial cells. BV-2 microglial cells were treated with the Tamarix hohenackeri

Bunge extracts in the presence of LPS (100 ng/ml) for 24 h at the indicated

concentrations. The culture supernatants were isolated and analyzed for nitrite

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production. The data are expressed as the means ± SEM of three independent

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experiments performed in triplicates; ###P < 0.001 compared with the control group, *P

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< 0.05, **P < 0.01 and ***P < 0.001 compared with the LPS group. Minocycline was

used as positive control. (EtOH: ethanol, PE: petroleum ether, EtOAc: ethyl acetate,

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n-BuOH: n-butanol, NO: nitric oxide, LPS: lipopolysaccharide)
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Fig. 2. Anti-inflammatory activity of the isolated compounds from the Tamarix

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hohenackeri Bunge EtOAc extract in BV-2 microglial cells. BV-2 microglial cells

were treated with the isolated compounds in the presence of LPS (100 ng/ml) for 24 h at
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the indicated concentrations. The culture supernatants were isolated and analyzed for
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nitrite production. The cell viability was determined using the MTT assay. The data are

expressed as the means ± SEM of three independent experiments performed in

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triplicates. Minocycline was used as positive control. ##P < 0.01, ###P < 0.001

compared with the control group, *P < 0.05, **P < 0.01 and ***P < 0.001 compared
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with the LPS group.

Fig. 3. Structures of the identified characteristic constituents from the

anti-inflammatory T. Hohenackeri extract.

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Fig. 4. HPLC chromatograms of the total ethanol extract, EtOAc extract and the

effective constituents. A: HPLC chromatogram of the total ethanol T. Hohenackeri

extract. B: HPLC chromatogram of the T. Hohenackeri EtOAc extract. C: HPLC

chromatogram of the effective compounds (6, 7, 10, and 13).

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Fig. 5. Effects of the effective compounds (6, 7, 10, and 13) on the LPS-induced

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mRNA levels of the pro-inflammatory mediators in BV-2 microglial cells. BV-2

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microglial cells were pretreated with each compound (100 μM) for 2 h and then

stimulated with LPS (100 ng/ml) for 4 h. The mRNA levels of iNOS, TNF-, IL-1 and
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IL-6 were measured by qRT-PCR. The data are expressed as the means ± SEM of three

independent experiments performed in triplicates. Minocycline (30 μM) was used as

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positive control. ##P < 0.01, ###P < 0.001 compared with the control group, *P < 0.05,
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**P < 0.01, ***P < 0.001 compared with the LPS group.
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Fig. 6. Effects of the effective compounds (6, 7, 10, and 13) on LPS-induced IκBα
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phosphorylation in BV-2 microglial cells. BV-2 microglial cells were pretreated with

the compounds 6, 7, 10, and 13 or PD98059 (20 μM) for 2 h and then stimulated with
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LPS for 30 min. Cell lysates were prepared to detect IκBα phosphorylation by Western

blot. The data are expressed as the means ± SEM of three independent experiments

performed in triplicates; ###P < 0.001 compared with the control group, **P < 0.01,

***P < 0.001 compared with the LPS group.

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Fig. 7. Effect of compound 7 on LPS-induced NF-B p65 protein expression in

BV-2 microglial cells. BV-2 microglial cells were pretreated with the compound 7 (10

M, 30 M, 100 μM) or MINO (30 M) for 2 h and then stimulated with LPS for 30

min. Cell lysates were prepared to detect cytoplasmic NF-B p65 (A) and nuclear

NF-B p65 protein (B) by Western blot. Immunofluorescence staining was used to test

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the translocation of NF-κB p65 (C). The data are expressed as the means ± SEM of

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three independent experiments performed in triplicates; ###P < 0.001 compared with

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the control group, **P < 0.01, ***P < 0.001 compared with the LPS group.

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Fig. 8. Effect of compound 7 on LPS-induced MEK/ERK activation in BV-2

microglial cells. BV-2 microglial cells were pretreated with compound 7 (10 M, 30
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M, 100 μM) or MINO (30 M) for 2 h and then stimulated with LPS for 30 min. Cell
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lysates were prepared to detect the phosphorylation of ERK (A) and MEK (B) by

Western blot. The data are expressed as the means ± SEM of three independent
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experiments performed in triplicates; ###P < 0.001 compared with the control group, *P
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< 0.05, **P < 0.01, ***P < 0.001 compared with the LPS group.
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Table 1

Primer sequences used in the qRT-PCR assay

Gene Forward primer Reverse primer

iNOS 5′-GGCAAACCCAAGGTCTACGTT-3′ 5′-GAGCACGCTGAGTACCTCATTG-3′

TNF-α 5′-CCCTCACACTCAGATCATCTTCT-3′ 5′-GCTACGACGTGGGCTACAG-3′

IL-1β 5′-TGACGGACCCCAAAAGATGA-3′ 5′-TCTCCACAGCCACAATGAGT-3′

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IL-6 5′-TAGTCCTTCCTACCCCAATTTCC-3′ 5′-TTGGTCCTTAGCCACTCCTTC-3′

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GAPDH 5′-AGGTCGGTGTGAACGGATTTG-3′ 5′-TGTAGACCATGTAGTTGAGGTCA-3′

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Table 2

Effects of the isolated compounds from T. Hohenackeri on NO production by LPS-activated

microglia cells (IC50 ± SEM)

Sample name IC50 Sample name IC50

Compound 1 ﹥100 Compound 8 ﹥100
a a
Compound 2 27.28 ± 1.27 Compound 9 25.91 ± 1.30
Compound 3 ﹥100 Compound 10 21.99 ± 1.79
a
Compound 4 5.83 ± 0.95 Compound 11 ﹥100

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a a
Compound 5 72.29 ± 2.05 Compound 12 4.68 ± 1.23

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Compound 6 29.38 ± 1.15 Compound 13 8.14 ± 1.26
Compound 7 13.55 ± 1.21 Minocycline 32.46 ± 3.13

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a
Compounds 2, 4, 5, 9, 12 and minocycline showed cytotoxicity at 100 µM.

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Fig. 1

140 140
(-)LPS (-)LPS
(+)LPS (+)LPS
120 120
### ###
NO (% of LPS control)

100

NO (% of LPS control)
* 100

***
80 80

60 ***
60

***
40 40
***

20

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20

0
0 0 0.1 1 10 50 0
0 0 0.1 1 10 50

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EtOH extract (μg/mL)
PE extract (μg/mL)

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140 140
(-)LPS (-)LPS
(+)LPS (+)LPS
120 120

### ###
NO (% of LPS control)

100
NO (% of LPS control)

100

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80 80 *

60 60

** **
40 40
**
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20 20

0 0
0 0 0.1 1 10 50 0 0 0.1 1 10 50

EtOAc extract (μg/mL) n-BuOH extract (μg/mL)

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Fig. 2

140 140
(-)LPS (-)LPS
(+)LPS (+)LPS
120 120
###
NO (% of LPS control)

100 100

Cell Viability (%)

**
80 80

60 60

40 40

20 20

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0 0
0 0 0.1 1 10 100 0 0 0.1 1 10 100

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Compound 1 (μM) Compound 1 (μM)

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140 140
(-)LPS (-)LPS
(+)LPS (+)LPS
120 120

###
NO (% of LPS control)

100 100
Cell Viability (%)

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80 80

60 60

40 40
***
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20 20
***

0 0
0 0 0.1 1 10 100 0 0 0.1 1 10 100

Compound 2 (μM) Compound 2 (μM)

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140
140
(-)LPS
(-)LPS
(+)LPS
(+)LPS
120
120
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###
100
NO (% of LPS control)

100
Cell Viability (%)

**
80
80
***
60
60
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40
40

20
20
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0
0 0 0 0.1 1 10 100
0 0 0.1 1 10 100
Compound 3 (μM)
Compound 3 (μM)
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140
140 (-)LPS
(-)LPS (+)LPS
(+)LPS
120
120

###
100
NO (% of LPS control)

100
Cell Viability (%)

** *
80 ***
80
*
60
60

40
40

*** 20
20

0
0 0 0 0.1 1 10 100
0 0 0.1 1 10 100
Compound 4 (μM)
Compound 4 (μM)

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140 140
(-)LPS (-)LPS
(+)LPS (+)LPS
120 120
##
NO (% of LPS control)

100 100

Cell Viability (%)

80 80
***

60 **
60

40 40

20
20

0
0 0 0.1 1 10 100 0
0 0 0.1 1 10 100
Compound 5 (μM)

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Compound 5 (μM)

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140 140
(-)LPS
(-)LPS
(+)LPS
(+)LPS
120 120

##

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100
NO (% of LPS control)

100 Cell Viability (%)

80 80

60 60

40

20

0
**

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20

0
0 0 0.1 1 10 100
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0 0 0.1 1 10 100

Compound 6 (μM) Compound 6 (μM)

140
140
(-)LPS
(-)LPS
(+)LPS
(+)LPS
120
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120
##
NO (% of LPS control)

100
100
Cell Viability (%)

**
80
80
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60
60

40
40
**

20
20
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0
0 0 0.1 1 10 100 0
0 0 0.1 1 10 100
Compound 7 (μM)
Compound 7 (μM)
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140 140
(-)LPS (-)LPS
(+)LPS (+)LPS
120 120
##
NO (% of LPS control)

100 100
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Cell Viability (%)

80 80

60 60

40 40

20 20

0 0
0 0 0.1 1 10 100 0 0 0.1 1 10 100

Compound 8 (μM) Compound 8 (μM)

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140
140 (-)LPS
(-)LPS (+)LPS
(+)LPS
120
120

###
100
NO (% of LPS control)

100

Cell Viability (%)

***
80
80

60
60

40
40
***
20
20

0
0 0 0 0.1 1 10 100
0

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0 0.1 1 10 100
Compound 9 (μM)
Compound 9 (μM)

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140
(-)LPS 140

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(+)LPS (-)LPS
(+)LPS
120
120
###
NO (% of LPS control)

100
100
Cell Viability (%)

***
80
80

60

40

20
***
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40
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20

0
0 0 0.1 1 10 100 0
0 0 0.1 1 10 100
Compound 10 (μM)
Compound 10 (μM)
M

140
140 (-)LPS
(-)LPS (+)LPS
(+)LPS
120
120
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##
100
NO (% of LPS control)

100
Cell Viability (%)

80
80

60
60
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40
40

20
20
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0
0 0 0 0.1 1 10 100
0 0 0.1 1 10 100
Compound 11 (μM)
Compound 11 (μM)
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140
140 (-)LPS
(-)LPS (+)LPS
(+)LPS
120
120

###
100
NO (% of LPS control)

100
Cell Viability (%)

* 80
80

** ***
60
60

40
40

***
20
20

0
0 0 0 0.1 1 10 100
0 0 0.1 1 10 100
Compound 12 (μM)
Compound 12 (μM)

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140
(-)LPS 140
(+)LPS (-)LPS
(+)LPS
120
120
###
NO (% of LPS control)

100
100
* *

Cell Viability (%)

80
80
**
60
60

40 ***
40

20
20

0
0 0 0.1 1 10 100 0
0

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0 0.1 1 10 100
Compound 13 (μM)
Compound 13 (μM)

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140 140
(-)LPS
(-)LPS

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(+)LPS
(+)LPS
120 120
Cell Viability (%)
###
100
NO (% of LPS control)

100
*

80 80

60

40
***

***
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40

20
***
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20

0
0 0 0 1 10 30 100
0 0 1 10 30 100
M i n o c y c l (μM)
ine
Minocycline (μM)
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Fig. 3

Tamarix hohenackeri Bunge

Anti-inflammatory effect assay by BV2 cells

Ethyl acetate extract

Triterpenes Flavonoids Phenolic acid

OCH3 OCH3
O COOH COOH
O H3CO O HO O HO COOH

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OH OH H3CO HO OH HO OH
OH O OH O OCH3 OH
HO

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1 3 4 8 9 10
COOH OCH3 OH
HO OCH3
OCH3 COOH COOCH3 COOH
HO HO O

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CO HO O
O
2 OH OH HO HO OH HO OCH3
OH O OH O OH OH OH
5 6 11 12 13
OH
OH
HO

OH O
O

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OH
Bioactive components
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Fig. 4

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Fig. 5

140 140
(-)LPS (-)LPS
(+)LPS (+)LPS
120 120
### ###
100 100
iNOS/GAPDH(%)

TNF-/GAPDH(%)
80 80
***
***
60 60
*** ***

40 *** 40
***
***
20 20

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***

0 0
0 0 #6 #7 #10 #13 MINO 0 0 #6 #7 #10 #13 MINO

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Compound (100 μM)
Compound (100 μM)

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140 140
(-)LPS (-)LPS
(+)LPS (+)LPS
120 120

## ##
100 100
IL-1/GAPDH(%)

IL-6/GAPDH(%)

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80 80 **
**
* **
60 60 **
*
40 40
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20 ** 20
**
0 0
0 0 #6 #7 #10 #13 MINO 0 0 #6 #7 #10 #13 MINO
Compound (100 μM) Compound (100 μM)
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Fig. 6

140
(-)LPS
(+)LPS
120 ###
Relative level of p-IB/-actin

100

80 **

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**
60

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40 ***

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***
20

0
0 0 #6 #7 #10 #13 PDTC

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Compound (100 μM)
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Fig. 7

140 A
(-)LPS
(+)LPS
Relative level of NF-B p65 (C)/-actin

120

***
100

80
**

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60

###

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40

20

CR
0
0 0 10 30 100 MINO

Compound 7 (μM)

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140 B
(-)LPS
Relative level of NF-B p65 (N)/Histone H3

(+)LPS
120
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###
100

*** ***
80
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*** ***
60

40
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20

0
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0 0 10 30 100 MINO

Compound 7 (μM)

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Fig. 8

140 A
(-)LPS
(+)LPS
120
Relative level of p-ERK/-actin

###
100

** **
80

T
60

IP
40 ***

20 ***

CR
0
0 0 10 30 100 MINO

Compound 7 (μM)

US
AN
M

140 B
ED

(-)LPS
(+)LPS
120
Relative level of p-MEK/-actin

###

100
PT

*
80

***
CE

60

40

***
AC

20

0
0 0 10 30 100 MINO

Compound 7 (μM)

39
 ACCEPTED MANUSCRIPT

Graphical Abstract

T
IP
CR
US
AN
M
ED
PT
CE
AC

40