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(7P,8S)-9-acetyl-dehydrodiconiferyl alcohol inhibits inflammation and migration in lipopolysaccharide-stimulated macrophages.

ABSTRACT

Background: (7R, 8S)-9-Acetyl-dehydrodiconiferyl alcohol (ADDA), a novel lignan compound isolated from Clematis armandii Franch (Ranunculaceae) stems, has been found to exert potential anti-inflammatory activities in vitro.

Purpose: To investigate the pharmacological effects and molecular mechanisms of ADDA on lipopolysaccharide (LPS)-induced activation and migration of macrophages.

Study design/methods: Macrophages were stimulated with LPS in the presence or absence of ADDA. Expression of inflammatory mediators, including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitric oxide (NO) were measured by Western blot and commercial NO detection kit. Cellular viability and chemotactic properties of macrophages were investigated using MTT and transwell migration assays. The activation and expression of mitogen activated protein kinases, nuclear factor-[kappa] B (NF-[kappa] B), protein kinase B (Akt), Src, and focal adhesion kinase (FAK) were analyzed by Western blot.

Results: Non-toxic concentrations (12.5-50 [micro]M) of ADDA concentration-dependently inhibited expression/ release of inflammatory mediators (COX-2, iNOS, and NO), suppressed Akt and c-jun N-terminal kinase 1/2 (JNK) phosphorylation, and NF-[kappa] B activation in LPS-stimulated macrophages. In addition, ADDA blocked LPS-mediated macrophage migration and this was associated with inhibition of LPS-induced Src and FAK phosphorylation as well as Src expression in a concentration dependent manner. Notably, the inhibitory effects of ADDA on iNOS, NO, and Src could be mimicked by a Src inhibitor PP2 or an iNOS inhibitor L-NMMA.

Conclusion: Our results suggested that ADDA attenuated LPS-induced inflammatory responses in macrophages and cell migration, at least in part, through inhibition of NF-[kappa] B activation and modulation of iNOS/Src/FAK axis.

Keywords:

(7R,8S)-9-Acetyl-dehydrodiconiferyl alcohol

Lipopolysaccharide

Raw264.7

Inflammation

Migration

Src

Introduction

Macrophages, present in almost all tissues constitute the first line of innate immune defense to pathogens and diverse external stimuli. Under physiological conditions, as a part of host defense response to infection, macrophages sense, move to the site of infection or injury, and engulf microorganisms, foreign particles, and apoptotic bodies (Friedl 2004; Gordon 2003). Under pathological stimuli, macrophages activate their effector functions and excessive macrophage activation is associated with the expression/release of several inflammatory mediators, importantly, the proinflammatory enzymes inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), nitric oxide (NO), prostaglandins (PGs) (Jiang et al. 2014). Furthermore, overproduction of NO and cytokines/chemokines by activated macrophages contribute to recruitment and activation of inflammatory cells (Maa et al. 2008), which results in a variety of pathological disorders such as sepsis and atherosclerosis. Lipopolysaccharide (LPS), a major constituent of the Gram-negative bacterial endotoxin, plays a pivotal role in the initiation of inflammation via toll-like receptor 4 (Dalpke and Fleeg 2002). LPS stimulates toll-like receptor 4 to ignite common downstream signaling pathways, i.e. activation of mitogen activated protein kinases (MAPK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and nuclear factor-[kappa] B (NF-[kappa] B) (Kyriakis and Avruch 2012), leading to expression of proinflammatory mediators (Israf et al. 2010; Liu et al. 2013b) and the resultant inflammatory processes and tissue injury.

Cellular Src is the prototype of Src family kinases (SFKs) of highly conserved proteins, including Blk, Fgr, Fyn, Pick, Lck, Lyn, and Yes (Miguelez et al. 2014). SFKs have been implicated in a spectrum of signaling pathways and cellular events. In macrophages, the majority of the myeloid-specific SFK members (i.e. Fgr, Hck and Lyn) are constitutively expressed and almost unaltered in response to inflammatory stimuli such as LPS (Maa et al. 2008). In contrast, Src is barely detectable in resting macrophages and is greatly up-regulated by LPS, leading to focal adhesion kinase (FAK) activation and cell motility (Chen et al. 2012; Leu et al. 2006). FAK, one of the Src substrates, plays a critical role in macrophage adhesion and motility (Owen et al. 2007). Mounting evidence indicates that LPS induces Src expression and subsequently FAK auto-phosphorylation at Tyr 397, triggering macrophage migration, which is iNOS-dependent (Fernandez-Arche et al. 2010; Leu and Maa 2002). Hence, targeting Src/FAK axis may effectively prevent LPS-triggered macrophages locomotion.

Clematis armandii Franch. (Ranunculaceae) (Caulis clematidis armandii, or "Chuan-Mu-Tong" in Chinese), a flowering climbing plant, is frequently found in southwestern China, especially in Si-Chuan (Szechwan) Province (Xiong et al. 2014). Clematis armandii has long been used for the treatment of inflammation conditions, such as rheumatism and urinary tract infection (Chawla et al. 2012). Previously, we reported that (7R, 8S)-9-acetyldehydrodiconiferyl alcohol (ADDA, Fig. 1A), a novel lignan isolated and identified from the dried stems of Clematis armandii, had anti-inflammatory and cytoprotective effects in activated microglial cells in vitro (Xiong et al. 2014). The current study was conducted to investigate the pharmacological effects and underlying mechanism of ADDA on inflammatory responses in LPS-stimulated murine RAW264.7 macrophages.

Materials and methods

Reagents and antibodies

Dulbecco's modified Eagle's medium (DMEM) medium and fetal bovine serum (FBS) were from GIBCO-BRL (USA). 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT), 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), [N.sup.G]-monomethyl-L-arginine (L-NMMA), and LPS (E. coli 055:B5) were obtained from Sigma (St. Louis, MO, USA). Antibodies against [beta]-actin, iNOS, Hck, and COX-2 were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against Lamin A/C, NF-[kappa] B p65, FAK, and Src were from Epitomics (Burlingame, CA). Antibodies against total- and phosphor (p)- c-Jun N-terminal kinase 1/2 (JNK) ([Thr.sup.183] and [Tyr.sup.185]), total- and p- p38 ([Thr.sup.180] and [Tyr.sup.182]), total- and p- extracellular signal-regulated kinase 1/2 (ERK) ([Thr.sup.202]/[Tyr.sup.204]), total- and p- Akt, p-Src ([Tyr.sup.416]), p-FAK ([Tyr.sup.397]) and p-p65 ([Ser.sup.563]) were purchased from Cell Signaling Technology (Danvers, MA).

Isolation and purity of ADDA

The dried stem of Clematis armandii (10 kg) was pulverized and extracted with 95% EtOH at room temperature to give a brown crude extract (500g), which was suspended in [H.sub.2] O and then extracted with C[H.sub.2] [Cl.sub.2]. After removal of the solvent under reduced pressure, the C[H.sub.2] [Cl.sub.2] extract (200 g) was chromatographed over a silica gel column with a gradient elution of C[H.sub.2][Cl.sub.2]-MeOH (90:1 to 0:1) to afford seven fractions (Fr. 1-Fr. 7). Fr. 4 (15.0 g) was subjected to silica gel CC with PE-acetone (3:1 to 0:1) to yield five subfractions (Fr. 4.1-Fr. 4.5). Fr. 4.2 and Fr. 4.3 were purified by semi-preparative HPLC [Waters e2695 system with a Waters 2998 Photodiode Array Detector (PAD) and a Waters 2424 ELSD; a SunFire ODS column (5|im, 250 x 10 mm) was utilized] eluted with MeCN-[H.sub.2] O (65:35) at 3.0ml/min to furnish (7R, 8S)-9-acetyl-dehydrodiconiferyl alcohol (1, 5.7 mg, [t.sub.R] = 15.0 min). The purity (>98.5%) of this compound was deduced based on the HPLC profile and its proton NMR spectrum (supplemental can be found in the online version at http://dx.doi.Org/10.1016/j.jep.2014.03.036) (Xiong et al. 2014). In present study, ADDA was dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO was less than 0.1%.

Cell culture and treatment

The murine macrophage cell line, RAW264.7 (American Type Culture Collection), was cultured and propagated in DMEM medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 [micro]g/ml streptomycin (GIBCO) at 37[degrees]C in humidified atmosphere of 5% C[O.sub.2].

Cell viability assay

Cell viability was evaluated by MTT assay as described previously (Liu et al. 2013b).

NO assay

Brief, RAW246.7 cells were plated in 12 well cell culture plates. After incubated with various concentrations (12.5 [micro]M, 25 [micro]M, 50 [micro]M) of ADDA, then stimulated with or without LPS (1 [micro]g/ml), NO production in the medium was assessed by measuring nitrite/nitrate using the NO detection kit (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer's instructions. Optical density at 540 nm was measured and calculated using a standard curve obtained by series concentrations of sodium nitrite.

Lysate preparation and western blot analysis

Total lysis of the cells was carried out with RIPA buffers as described before (Pan et al. 2013). The cytoplasmic and nuclear fractions of cells were extracted using nuclear and cytoplasmic extraction reagents (ThermoFisher Scientific, Shanghai, China) according to the manufacturer's instructions. The protein concentration was determined by protein assay kit (ThermoFisher Scientific). Western blot was performed as previously described (Liu et al. 2010; Liu et al. 2013a). In brief, equal amounts of protein were separated in SDS-polyacrylamide gels and transferred to a nitrocellulose membrane (GE healthcare). The membrane was probed with primary antibody overnight at 4[degrees]C. Immune complexes were detected by enhanced chemiluminescence (Millipore) with horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG as the secondary antibody (ThermoFisher Scientific). Signal intensity was quantified using the software Alpha Imager (Alpha Innotech Corp, San Leandro, CA). The results were expressed as fold changes by normalizing the data to the control values.

Cell migration assay

The migration of RAW264.7 cells was determined by using a modified Boyden chamber. RAW264.7 cells were pretreated with ADDA (25 [micro]M) or Src inhibitor PP2 (10 [micro]M) for 4h, then the cells were re-suspended and a total of 2 x [10.sup.5] cells in 300 [micro]l of DMEM medium with 1% fetal calf serum were seeded in the upper chamber with 8-[micro]m pore size membrane. The lower wells of the chamber were filled with DMEM medium FBS free in the presence or absence of LPS, the chamber was then incubated at 37[degrees]C for 24 h to initiate migration. Non-migrated cells were wiped off with a cotton swab and then the filter was fixed and stained with viola crystalline, and counted. The number of migrated cells in five random microscopy fields per well were counted at 400 x magnification.

Immunofluorescence

The immunofluorescence of interest protein exposed to LPS was determined as described before (Pan et al. 2011). The RAW264.7 cells were grown in 8-well culture slides (BD Biosciences). Cells were fixed in 4% paraformaldehyde for 30 min at room temperature. Immunostained using mouse anti-Src antibody (dilution in 1:100) and Alexa Fluor 488 conjugated goat anti-mouse IgG (1:1000; ThermoFisher Scientific), and counterstained for nuclei with DAP1. Immunofluorescence was visualized using a confocal laser scanning microscope (Carl Zeiss Inc.). The results were based on three independent analyses.

Statistical analysis

Results are expressed as mean [+ or -] standard error of the mean (SEM). All values for each treatment group have been normalized to the unstimulated cells. All data analysis was performed with the use of GraphPad Prism 5 software. Differences between mean values of multiple groups were analyzed by one-way analysis of variance with Tukey's test for post hoc comparisons. Statistical significance was considered at p < 0.05.

Results

ADDA inhibited LPS-mediated inflammatory mediators in RAW 264.7 macrophages

To investigate potential anti-inflammatory activities of ADDA, we firstly examined its effects on LPS-induced expression of iNOS, COX-2 and production of NO in RAW264.7 cells. LPS stimulation resulted in a significant increase in iNOS (597.3%) and COX-2 (769.0%) expression (Fig. 1B and C). The increase in iNOS and COX-2 was remarkably inhibited by pretreatment with ADDA (12.5-50 [micro]M) for 4 h in a concentration dependent manner. The maximal inhibitory effects of ADDA were observed at the concentration of 50 [micro]M (65.3% and 30% on iNOS and COX-2, respectively). Since iNOS is mainly responsible for NO production during inflammatory conditions, we examined the effect of ADDA on NO production. Similarly, ADDA concentration-dependently suppressed LPS-induced NO production (16.5%, 42.2%, and 47.7%, respectively) (Fig. 1D). To evaluate potential cytotoxicity of ADDA on RAW264.7 cells, cell viability after treatment with ADDA at 12.5-200 [micro]M was evaluated by MTT assay. The data showed that ADDA at this concentration range did not affect cell survival (Fig. 1E). Together, these data indicated that ADDA attenuated LPS-induced iNOS and COX-2 expression and NO production in RAW264.7 macrophages.

ADDA suppressed LPS-activated JNK and Akt signaling

MAPK (JNK, p38, and ERK) and PI3K/Akt signaling were involved in LPS-induced inflammatory signaling pathways (Liu et al. 2013b). We examined if ADDA exerted anti-inflammatory activities by modulating these signaling pathways. MAPK and PI3K/Akt activation was examined by Western blot using specific phosphorylated antibodies. As shown in Fig. 2, LPS stimulation time-dependently induced the phosphorylation of JNK, ERK, p38, and Akt. A peak level of phosphorylated MAPK (JNK, ERK, and p38 by 1.8, 2.8 and 3.3 times, respectively) and Akt (by 2 times) were observed after 30 min of LPS stimulation. ADDA at 25 [micro]M, time-dependently blocked LPS-induced phosphorylation of JNK (Fig. 2A) and Akt (Fig. 2D), but not that of ERK (Fig. 2B) and p38 (Fig. 2C). Subsequently, cells stimulated with LPS for 30 min were chosen to study the concentration-related inhibitory effects of ADDA on JNK, p38, ERK, and Akt phosphorylation. Similarly, pre-treatment with ADDA for 4h, at 12.5-50 [micro]M, significantly inhibited LPS-induced phosphorylation of JNK (an inhibition by 3.9%, 26.4%, and 42.1%, respectively) and Akt (an inhibition by 14.7%, 33.7%, and 54.7%, respectively) in a concentration dependent manner (Fig. 3A and D), whereas it had little effect on ERK or p38 (Fig. 3B and C). Collectively, the results suggested that inhibition of JNK and Akt signaling by ADDA contributed to its anti-inflammatory activities in LPS-stimulated RAW264.7 cells.

ADDA blocked LPS-mediated NF-[kappa] B activation

NF-[kappa] B is an important transcriptional factor involved in the regulation of inflammatory mediators. Therefore, we explored whether ADDA regulated LPS-mediated NF-[kappa] B activity. Phosphorylation of p65 at serine 536 appeared to play a critical role in NF-[kappa] B DNA binding activity (Liu et al. 2012). As shown in Fig. 4A, LPS time-dependently induced the phosphorylation of NF-[kappa] B p65 subunit and the peak level of phosphorylated NF-[kappa] B p65 was observed at 30 min. ADDA (25 [micro]M) time-dependently blocked LPS-induced phosphorylation of NF-[kappa] B p65 subunit. In addition, LPS markedly induced nuclear translocation of NF-[kappa] B p65 subunit (208.5%), which was concentration-dependently attenuated by pretreatment with ADDA (18.4%, 37.4%, and 43.4%, respectively) (Fig. 4B). These findings suggested that ADDA down-regulated LPS-mediated NF-[kappa] B activation, thereby inhibiting expression of inflammatory mediators in RAW264.7 cells.

ADDA inhibited LPS-induced macrophage migration

LPS was documented to induce cell mobilization in macrophages (Maa et al. 2010). Next, we examined the migratory potential of RAW264.7 cells treated without or with ADDA (25 [micro]M) or PP2 (10 [micro]M) prior to LPS exposure. As shown in Fig. 5, LPS markedly induced RAW264.7 cells migration. ADDA (25 [micro]M) or PP2 (10 [micro]M) suppressed LPS-induced RAW264.7 cell migration. These results indicated that ADDA-mediated signaling have functional consequences related to macrophage motility.

ADDA suppressed Src expression and Src/FAK axis activation in LPS-stimulated RAW264.7 cells

Src has been demonstrated to be induced, activated and involved in LPS-mediated macrophage mobilization (Maa et al. 2011; Shimojo et al. 2015). Subsequently, the effect of ADDA on Src expression and activation in LPS-exposed macrophages was examined. As illustrated in Fig. 6A, LPS stimulation for 45 min markedly induced Src activation (202.5%), which was concentration-dependently (1.23%, 24.4%, and 37.0%, respectively) attenuated by pretreatment with ADDA at 12.5-50 [micro]M. FAK was a substrate of Src and its activation played a critical role in macrophage mobilization (Owen et al. 2007). Consistent with a previous study (Maa et al. 2010), LPS stimulation for 24 h markedly induced Src expression (297.3%) and FAK phosphorylation at Tyr 397 (204.0%), which were also suppressed by ADDA in a concentration dependent manner. In contrast, the expression of other myeloid-specific SFKs (i.e., Hck) was almost unaltered before and after LPS exposure (Fig. 6B and C). Taken together, our results suggested inhibition of macrophages migration by ADDA might be attributable to attenuation of Src expression and Src/FAK axis activation in LPS-exposed macrophages.

ADDA-elicited Src reduction and Src/FAK axis inactivation was iNOS-dependent

iNOS/NO system was essential for LPS-mediated Src induction and macrophage migration (Maa et al. 2011). To determine if iNOS/NO is involved in ADDA-induced Src reduction, the cells were treated with a Src inhibitor PP2 or an iNOS inhibitor L-NMMA. As shown in Fig. 7A-C, LPS markedly induced NO production as well as iNOS and Src expression, which was significantly suppressed by ADDA (25 [micro]M) (55.5%, 70.4%, and 56.0%, respectively) and mimicked by PP2 (57.8%, 64.4%, and 57.1%, respectively) or L -NMMA (45.7%, 68.1%, and 51.9%, respectively) pretreatment. ADDA (25 [micro]M) alone didn't affect the NO production as well as iNOS and Src expression. Furthermore, the effect of ADDA on LPS-mediated Src expression was confirmed by immunofluorescence examination (Fig. 7D). Taken together, our data indicated that ADDA attenuated LPS-mediated Src induction and macrophage migration, which was iNOS-dependent.

Discussion

Activated macrophages play an important role in the progression of various inflammatory diseases. Correspondingly, suppression of over-activated macrophages represents a therapeutic strategy to prevent inflammatory related diseases. Herein, we reported that ADDA, a novel compound isolated from Clematis armandii, exerted anti-inflammatory effects in LPS-stimulated RAW264.7 macrophages: inhibition of production of various inflammatory mediators, modulation of macrophage migration, and blockade of NF-[kappa]B and Src/FAK pathways.

Clematis armandii has been shown to elicit numerous pharmacological effects. In our previous study, a total of 17 related compounds were isolated (Xiong et al. 2014) and the anti-neuroinflammatory bioassay showed that 4 compounds out of them displayed significant inhibitory effects on LPS-mediated NO production in BV2 microglia (Xiong et al. 2014). Among them, ADDA exhibited the most potent anti-inflammatory activity against LPS-stimulated NO production, with an [IC.sub.50] value of 9.3 [micro]M (Xiong et al. 2014). Activated macrophages release various inflammatory cytokines that exacerbate the inflammatory response and cause tissue injury (Fujiwara and Kobayashi 2005). Overproduction of NO generated by iNOS could stimulate macrophage activities to release high levels of inflammatory cytokines and induce macrophage migration (Chong and Sriskandan 2011; Park and Lee 2013). Upregulation of iNOS and COX-2 were involved in the pathogenesis of many chronic diseases. Therefore, inhibition of these inflammatory mediators might be a therapeutic strategy for treating inflammatory diseases. In the present study, our results clearly demonstrated that ADDA attenuated LPS-induced iNOS and COX-2 expression as well as NO production in a concentration dependent manner. In addition, ADDA significantly suppressed LPS-mediated migration of macrophages. Taken together, our results for the first time supported the traditional application of this herbal medicine on inflammation-related diseases (Chawla et al. 2012). It has been reported that NF-[kappa]B plays a crucial role in inflammation and it regulates inflammatory genes encoding iNOS and COX-2 (Liu et al. 2013b; Shembade et al. 2010). Thus, we speculated that ADDA might, at least partially, inhibit the effects of NF-[kappa]B signaling. As expected, our present results was in line with previous reports that LPS significantly induced NF-[kappa]B activation, as evidenced by an increased phosphorylation and nuclear translocation of NF-[kappa]B p65, which was concentration-dependently reverted by ADDA pretreatment. A growing body of evidence had demonstrated that a number of intracellular signaling pathways, including MAPK (ERK, p38, and JNK) and PI3K/Akt, were involved in NF-[kappa]B activation (Guma et al. 2011; Zhang et al. 2013; Zhao et al. 2012). We further investigated the role of ADDA on LPS-activated MAPK and PI3K/Akt signaling pathways. In our present study, ADDA concentration- and time-dependently inhibited LPS-mediated phosphorylation of Akt and JNK, but not p38 or ERK in RAW264.7 cells, suggesting that the inhibitory effect of ADDA on LPS-mediated NF-[kappa]B activation by was exerted, at least in part, through modulation of Akt and JNK signaling pathway. This finding suggested that ADDA, at least in LPS-stimulated RAW264.7 cells, exerted its anti-inflammatory activity by inhibition of Akt and JNK signaling and modulation NF-[kappa]B activation.

Mounting evidence demonstrated that dysregulated macrophage migration played an important role in misdirected immune responses, leading to tissue damage and chronic inflammation (Zicha et al. 1998). In the present study, we first proved ADDA significantly suppressed LPS-mediated migration of RAW264.7 macrophages. Concurrent with the decreased motility, ADDA effectively inhibited LPS-elicited Src activation, critical in regulating macrophage movement (Maa et al. 2008; Shimojo et al. 2015; van Vliet et al. 2005), while the expression of another SFK family protein Hck was not altered. The critical role of Src in macrophage migration suggested that the inhibition of Src activation and induction by ADDA might contribute to prevention of LPS-mediated inflammation responses. The enhanced Src activity was associated with increased activities of FAK, which was a substrate of Src and a critical molecule in regulating cell spreading and migration (Maa et al. 2011; Parsons et al. 2000). In addition, we demonstrated that ADDA was able to suppress phosphorylation of FAK at Tyr 397 in LPS-stimulated macrophages. The phosphorylation of FAK at Tyr 397 facilitated the formation of the FAK-Src bipartite kinase complex, which is pivotal in cell motility (Parsons et al. 2000). Previous studies demonstrated that the induction of Src expression and its activity are iNOS-dependent and its induction formed a positive feedback loop with NF-[kappa]B in macrophages (Maa et al. 2008; Maa et al. 2011). Intriguingly, ADDA-mediated reduction of iNOS and Src was mimicked by a Src inhibitor PP2 or an iNOS inhibitor L-NMMA pretreatment, indicating reduction of iNOS by ADDA, at least in part, contributes to Src inhibition in LPS-stimulated macrophages.

Conclusion

Our data demonstrated that ADDA, a novel compound from Clematis armandii, inhibited production of proinflammatory mediators by suppressing JNK/NF-[kappa]B signaling in LPS-stimulated RAW264.7 cell. Additionally, ADDA impaired LPS-mediated Src/FAK axis activation and macrophages migration. Therefore, ADDA might be a promising candidate for treating various inflammatory diseases.

http://dx.doi.org/10.1016/j.phymed.2016.02.018

ARTICLE INFO

Article history:

Received 2 September 2015

Revised 18 February 2016

Accepted 18 February 2016

Abbreviations: ADDA, (7R,8S)-9-acetyl-dehydrodiconiferyl alcohol; COX-2, cyclooxygenase-2; FAK, focal adhesion kinase; iNOS, inducible nitric oxide synthase; L-NMMA, [N.sup.G]-monomethyl-l-arginine; LPS, lipopolysaccharide; JNK, c-jun N-terminal kinase 1/2; MAPK, mitogen activated protein kinases; NF-[kappa] B, nuclear factor-[kappa] B; NO, nitric oxide; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; SFKs, Src family kinases.

Conflict of interest

There are no conflicts of interest to declare.

Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (no. 81573420; 81470164), a key laboratory program of the Education Commission of Shanghai Municipality (no. ZDSYS14005) and the National Basic Research Program of China (973 Program, no. 2013CB530700).

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Li-Long Pan (a), Qiu-Yang Zhang (a), Xiao-Ling Luo (a), Juan Xiong (b), Peng Xu (a), Si-Yu Liu (a), Jin-Feng Hub (b), **, Xin-Hua Liu (a), *

(a) Shanghai Key Laboratory of Bioactive Small Molecules and Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China

(b) Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China

* Corresponding author. Tel.: +86 21 51980159.

** Corresponding author. Tel.: +86 21 51980172; fax: +86 21 51980172.

E-mail addresses: jfhu@fudan.edu.cn (J.-F. Hu), liuxinhua@fudan.edu.cn (X.-H. Liu).
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Author:Pan, Li-Long; Zhang, Qiu-Yang; Luo, Xiao-Ling; Xiong, Juan; Xu, Peng; Liu, Si-Yu; Hu, Jin-Feng; Liu,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:May 15, 2016
Words:5010
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