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A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages
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A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages Lu Jin , Michael Schmiech , Menna El Gaafary , Xinlei Zhang , Tatiana Syrovets , Thomas Simmet PII: DOI: Reference:
S0944-7113(20)30014-3 https://doi.org/10.1016/j.phymed.2020.153181 PHYMED 153181
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
31 October 2019 24 January 2020 5 February 2020
Please cite this article as: Lu Jin , Michael Schmiech , Menna El Gaafary , Xinlei Zhang , Tatiana Syrovets , Thomas Simmet , A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages, Phytomedicine (2020), doi: https://doi.org/10.1016/j.phymed.2020.153181
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A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages Lu Jina, Michael Schmiecha, Menna El Gaafarya,b, Xinlei Zhang c, Tatiana Syrovetsa, Thomas Simmeta* a
Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University,
89081 Ulm, Germany b
Department of Pharmacognosy, College of Pharmacy, Cairo University, Cairo, 11562 Egypt
c
Department of Medicinal Chemistry, School of Pharmacy, Fourth Military Medical University,
710032 Xi’an, Shaanxi, P.R. China *Corresponding author: Thomas Simmet, Institute of Pharmacology of Natural Products and Clinical Pharmacology, Helmholtzstraße 20, 89081 Ulm, Germany Tel.: (+49) 731 500 65600; fax: (+49) 731 500 65602 E-mail address: [email protected]
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Abstract Background: Eleutherococcus senticosus or Siberian ginseng is a medicinal plant containing adaptogenic substances believed to regulate immune responses. Both, the root and stem bark are commonly used in traditional medicines. Purpose: The purpose of the present study is to chemically characterize E. senticosus root and bark extracts and to compare their effects on functions of human primary macrophages. Study design and methods: HPLC-DAD-MS analysis was used to characterize chemical constituents of alcoholic extracts from E. senticosus root and bark. The data obtained and available databases were combined for network pharmacology analysis. Involvement of predicted pathways was further functionally confirmed by using monocyte-derived human macrophages and endotoxin-free E. senticosus root and bark extracts. Results: Chemical analysis showed that the root extract contained more syringin, caffeic acid, and isofraxidin than the bark extract. At variance, bark extract contained more sesamin and oleanolic acid. Coniferyl aldehyde and afzelin were below the limit of quantification in both extracts. Network pharmacology analysis indicated that constituents of E. senticosus might affect the immune cell phenotype and signaling pathways involved in cell metabolism and cytoskeleton regulation. Indeed, both extracts promoted actin polymerization, migration, and phagocytosis of E. coli by macrophages pointing to macrophage polarization towards the M2 phenotype. In addition, treatment with E. senticosus root and bark extracts decreased phosphorylation of Akt on Ser473 and significantly reduced expression of the hemoglobin scavenger receptor CD163 by macrophages. Neither extract affected expression of CD11b, CD80, or CD64 by macrophages. In addition, macrophages treated with the bark extract, but not with the root extract, exhibited activated p38 MAPK and NF-κB and released increased, but still moderate, amounts of proinflammatory TNF- and IL-6, anti-inflammatory IL-10, and chemotactic CCL1, which all together point to a M2b-like macrophage polarization. Differently, the root extract increased the IL-4-induced expression of anti-inflammatory CD200R. These changes in monocytes are in agreement with an increased M2a macrophage polarization. Conclusion: The ability of E. senticosus root and bark extracts to promote polarization of human macrophages towards anti-inflammatory M2a and M2b phenotypes, respectively, might underlay the immunoregulatory activities and point to potential wound healing promoting effects of this medicinal plant. Keywords Alternatively activated macrophages, Eleutherococcus senticosus; Adaptogen; Network pharmacology; Wound healing; Cytoskeleton. 2
Abbreviations APC, allophycocyanin; BSA, bovine serum albumin; DAD, diode-array detector; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; HPLC-MS, high-performance liquid chromatography-mass spectrometry; IL, interleukin; LOD, limit of detection; LOQ, limit of quantification; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; CSF, macrophage colony-stimulating factor (colony stimulating factor 1, CSF1); MF, median florescence intensity; MFI, median florescence index (normalized to isotype control); mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa B; SEAP, secreted embryonic alkaline phosphatase; TNF-, tumor necrosis factor ; XTT, 2,3-bis-(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide.
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Introduction Eleutherococcus senticosus (Rupr. and Maxim.), commonly called Siberian ginseng, belongs to a small family of plants containing adaptogenic substances. They are believed to increase resistance to various exogenous stressors, and they might contribute to normalization of immune responses (Panossian and Wikman, 2009). In Chinese and Asian medicine in general, adaptogens including Panax ginseng and Eleutherococcus senticosus are more widely accepted compared to Western medicine as they might better suit the whole-body treatment approach adopted by Asian therapies. However, our understanding of the molecular mechanisms of adaptogens is still very limited. Most experimental data obtained with extracts from plants with adaptogenic activity originate from studies with either extracts or ingredients of Panax ginseng (Panossian and Wikman, 2009). Only few reports address biological activities of E. senticosus extracts. Some claim effects of E. senticosus extracts or components thereof on the Raw264.7 murine macrophage cell line or mouse peritoneal macrophages (Fei et al., 2014; Jung et al., 2007; Lin et al., 2008; Lin et al., 2007; Soo Kim et al., 2012). However, receptor and cytokine expression, and specific features, such as polarization into distinct phenotypes are grossly different in human and murine macrophages as well as in monocytic leukemia cell lines and primary cells (Murray, 2017; Loos et al., 2014; Lunov et al., 2011). In addition, the effects reported are often contradictory, which may be due to different extraction procedures leading to differences in the chemical composition of these extracts. Understanding functional cellular mechanisms of E. senticosus and other adaptogens might advance their rational application in distinct disease states. Macrophages express an array of receptors enabling them to monitor continuously their microenvironment and to maintain tissue homeostasis. Depending on their activation state, they may reprogram their phenotype to respond quickly to disturbed homeostasis (Murray, 2017). Reacting to damage- and pathogen-associated molecular patterns, macrophages undergo classical activation resulting in formation of the proinflammatory M1 macrophage phenotype, which exhibits increased microbicidal and tumoricidal activities. In response to non-pathogen related stimuli, macrophages undergo alternative activation and adopt the anti-inflammatory alternative M2 phenotype. Unlike M1 macrophages, M2 macrophages comprise a spectrum of different subtypes, such as M2a, M2b, and M2c. Polarization to each of these subtypes is induced by specialized stimuli (Mantovani et al., 2004; Murray, 2017). Obviously, modulation of macrophage polarization might offer therapeutic opportunities in patients suffering from cancer or chronic inflammatory diseases. 4
To investigate the effects of E. senticosus-derived adaptogens on macrophage polarization and thereby function, we have chemically characterized extracts from root and stem bark of E. senticosus and monitored their effects on the polarization profile of primary human macrophages.
Materials and methods General experimental procedures XTT cell proliferation assay was from Roche (Filderstadt, Germany). Macrophage colonystimulating factor (M-CSF, CSF1) and interleukin 4 (IL-4) were from Miltenyi Biotec (Bergisch Gladbach, Germany). Medium and antibiotics were from Gibco (Carlsbad, CA), FCS and Biocoll separation solution was from Biochrom (Berlin, Germany). Lipopolysaccharide (LPS) O55:B5 from E. coli and fibrinogen were purchased from Sigma. Dylight650-phalloidin and antibodies used for Western immunoblotting were obtained from Cell Signaling Technology (Danvers, MA). APC-labeled antibodies to CD64, CD163, CD11b, and respective IgG controls were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Anti-CD200R-FITC and the respective IgG control were from Bio-Rad (Hercules, CA). Anti-CD80 was from BD (Franklin Lakes, NJ), Alexa Fluor 647-conjugated secondary antibody was from Jackson ImmunoResearch (Dianova, Hamburg, Germany). pHrodo™ Green E. coli were from Molecular Probes (Eugene, OR). Polymyxin B sulfate was from Enzo Life Sciences (Farmingdale, NY). Fractionation and analytical characterization of E. senticosus extracts E. senticosus ethanolic root extract Eleu curarina® (batch #1601) was from Harras Pharma Curarina (Munich, Germany). Eleu curarina extract (25 ml) was lyophilized to yield 1.0856 g of dry extract, which was further dissolved in DMSO. E. senticosus bark extract was a gift from Dr. Myung Hwan Park (Ambo Institute, Daejeon, South Korea). Briefly, 100 g of dried and chopped stem bark of E. senticosus were twice extracted with 600 ml of 70% methanol by reflux for 3 h. The filtrate was concentrated to dry powder (9.2 g) and used to prepare stock solutions in DMSO, which were further diluted in medium prior to experiments. Final DMSO concentration was 0.1%. Absence of endotoxin in E. senticosus extracts was confirmed using the EndoLISA assay, LOD = 0.05 EU/ml (Hyglos, Bernried/Starnberger See, Germany) according to the manufacturer’s instructions. Fingerprint characterization of E. senticosus root and bark extracts was carried out by highperformance liquid chromatography coupled with photodiode array detection and mass spectrometry (HPLC-DAD-MS). Syringin, isofraxidin, sesamin (all from AKos, Steinen, 5
Germany) and caffeic acid, afzelin, coniferyl aldehyde, oleanolic acid (all from Sigma) were used as references compounds for quantification. For separation, a Dr. Maisch ReproSil-Pur Basic C18-HD, 3 μm, 125 x 3 mm column was used. The flow rate was set to 600 μL/min and the injection volume was 5 μL. The mobile phase consisted of eluent A, MilliQ water, and eluent B, methanol, both acidified with 0.2 % acetic acid. The elution gradient was: 0 - 15 min, 10 95% B; 15 - 23 min 95% B; 23 - 24 min, 95 - 10% B; 24 - 30 min, 10% B. The UV/Vis detection was carried out at 210 nm, 254 nm, and 280 nm. MS analysis was performed in the negative electrospray ionization mode and the selected ion monitoring detection mode. Quantification of the compounds was performed using the external calibration method based on the corresponding selected ion chromatograms or UV/Vis chromatograms. The quantification method was validated in terms of linearity, the limit of detection (LOD), and limit of quantification (LOQ). Macrophages Macrophages were obtained by differentiation of human peripheral blood monocytes with 15 ng/ml M-CSF for 6 days (Li et al., 2007). Macrophages were treated with indicated concentrations of E. senticosus root or bark extracts or vehicle control (0.1% DMSO). LPS (100 ng/ml), LPS with IFN- (20 ng/ml), fibrinogen (50 ng/ml), or IL-4 (20 ng/ml) were used as positive controls. Network pharmacology analysis Caffeic acid, chlorogenic acid, coniferyl aldehyde, eleutheroside A, eleutheroside B1, (isofraxidin 7-0-glucoside), eleutheroside B4 [(-)-sesamin], eleutheroside B (syringin), eleutheroside E, hyperin, isofraxidin, and oleanolic acid were used as reference compounds for the root extract, whereas afzelin, chlorogenic acid, isofraxidin-7-O-β-D-glucoside, friedelin, liriodendrin, eleutheroside B (syringin), eleutheroside E were used for the bark extract (Li et al., 2016; Nishibe et al., 1990; Slacanin et al., 1991). Their targets were predicted by ChemMapper (Gong et al., 2013) using ChEMBL database. The similarity score threshold of potential targets was set to 1.2 and the top-scored 30 targets of each compound were selected for further analysis. After removing duplicates, 110 proteins highly relevant for macrophage activation and function (Jones, 2000; Lawrence and Natoli, 2011; Xue et al., 2014) (DrugBank and Therapeutic Target Database) were further used for the network construction. The network was constructed by connecting identified nodes with protein-protein interaction information from String, HPRD, HIPPE, and Reactome databases. The records were filtered according to 6
reliability and only interactions proved experimentally or highly ranked (for String, > 400) were preserved. The initial network was consolidated by igraph in R 3.5.3, filtered by NetworkAnalyzer, and visualized by Cytoscape 3.7.1. The enrichment analysis and biological annotations were performed by ClueGo (Bindea et al., 2009). Analysis of macrophage polarization and function Cell viability was analyzed by XTT assay. Absorbance was measured with a Tecan Infinite M1000PRO reader (Tecan, Männedorf, Switzerland) at 450 nm with a 630 nm reference filter. Data were normalized to vehicle control. Cell surface marker expression was analyzed after 24 h treatment flow cytometrically by using a FACSVerse (BD Biosciences) and quantified by FlowJo (Treestar, OR). Median fluorescent values of respective IgG controls were subtracted from the sample values. Cytokine and chemokine release was analyzed in supernatants of macrophages treated for 24 h by using corresponding ELISAs (R&D Systems, Minneapolis, MN). Actin polymerization was analyzed in macrophages grown in ibidi µ-glass-bottom plates stimulated for 40 min with either extract or fibrinogen control for 40 min and stained with DyLight 650-phalloidin. Cells were acquired under a Nikon eclipse Ti microscope using 20x objective and NIS-elements software (Nikon Corporation, Tokyo, Japan). The fluorescence of samples was quantified using a Tecan Infinite M1000PRO and normalized to fluorescence of the control sample. For analysis of migration, a cell-free zone in macrophage monolayer was introduced and cells were treated with either extract or LPS control and monitored for 18 h by Nikon eclipse Ti microscope with a 10x objective. The area of the cell-free zone was calculated with MRI Wound Healing Tool (ImageJ, NIH). For analysis of phagocytic activity, macrophages treated with either extract or LPS for 24 h were incubated with 15 µg/ml E. coli particles pHrodo for 2 h followed by flow cytometric analysis. Phosphorylation of Akt and p38 MAPK were analyzed by Western immunoblotting in whole cell extracts of cells treated for 1 h (Fuchs et al., 2016). NF-κB activation was analyzed in macrophage J774 reporter cells (InvivoGen, San Diego, CA) by assessing the activity of secreted embryonic alkaline phosphatase (SEAP) after 24 h.
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Statistics Results are expressed as mean ± SEM of at least three independent macrophage isolations. For two-group comparisons, results were analyzed with the two-tailed Student’s t-test. Multigroup analyses were performed with ANOVA followed by the Tukey or Dunnett posthoc test using SigmaPlot. Significance levels are *p < 0.05, **p < 0.01, ***p < 0.001.
Results E. senticosus root and bark extracts contain different compounds and both extracts are not toxic to human macrophages HPLC-MS analysis (Fig. 1) showed that the root and bark extracts contain different amounts of reference compounds with the root extract containing more syringin, caffeic acid, and isofraxidin than the bark extract. By contrast, the bark extract contained more sesamin and oleanolic acid. Coniferyl aldehyde and afzelin were present in very low concentrations and were below the LOQ in both extracts (Fig 1B). Neither extract affected the viability of human macrophages after 24 h of treatment (Fig. S1). Network pharmacology-based analysis revealed biological processes that might be targeted by E. senticosus root and bark extracts The results revealed that compounds, which we have identified in the E. senticosus extracts and those reported in the literature (Li et al., 2016; Nishibe et al., 1990; Slacanin et al., 1991), might affect immune cell functions, cell metabolism, actin cytoskeleton reorganization, cell migration regulation, and Fc receptor-mediated phagocytosis (Fig. 2A). Enrichment analysis showed that the majority of possible protein targets of E. senticosus are located in the cytoplasm functioning as a hub for binding of regulatory molecules, such as ATP. Besides, the respective protein targets play essential roles in responses to cytokines and other organic substances, the regulation of immune responses, the response to stress, and in cell motility. In addition, MAPK, PI3K/Akt/mTOR, and Toll-like receptor 4/NF-κB signaling pathways were predicted as possible targets of compounds present in E. senticosus (Fig. 2B). E. senticosus root and bark extracts promote actin polymerization, migration, and E. coli phagocytosis by macrophages Actin cytoskeleton regulation was identified in network analysis as a possible target of E. senticosus. Indeed, E. senticosus root and bark extracts significantly enhanced actin polymerization in macrophages, although to a lesser extent than fibrinogen (Fig. 3A). Reorganization of actin from soluble to polymerized filamentous F-actin within macrophages 8
is important for cell migration and their phagocytic activity. Both extracts induced migration of macrophages leading to the reduction of a cell-free zone (Fig. 3B). Also, the phagocytosis of E. coli by macrophages was increased after they had been incubated for 24 h with either root or bark extract of E. senticosus (Fig. 3C). Despite the differences in the extracts’ compositions, both extracts exhibited similar potency in activating macrophage actin polymerization, migration, and phagocytosis. E. senticosus root and bark extracts reduce Akt activation and CD163 expression in human macrophages E. senticosus root and bark extracts did not affect the expression of the macrophage markers CD64, CD80, and CD11b (Fig. 4A). However, both extracts reduced concentration-dependently the cell-surface expression of the hemoglobin scavenger receptor CD163 (Fig. 4B). It has previously been shown that activation of the mTORC2/Akt axis increases the CD163 expression on macrophages (Shrivastava et al., 2019). In addition, the network analysis indicated possible effects of E. senticosus constituents on the mTOR/Akt signaling pathway. Hence, phosphorylation of Akt on Ser473 was further analyzed. The results demonstrate that E. senticosus root and bark extracts equally decrease the phosphorylation level of Akt S473 (Fig. 4C) indicating that the E. senticosus extracts might reduce expression of CD163 through downregulation of Akt activation. The E. senticosus bark but not the root extract induces p38 MAPK kinase activation and cytokine and chemokine secretion by human macrophages Extract of E. senticosus bark significantly increased the release of proinflammatory TNF-α and anti-inflammatory IL-10 by macrophages, whereas non-stimulated or macrophages stimulated with E. senticosus root extract show non-detectable amounts of TNF- and low amounts of IL10 (Fig. 5A). LPS is a very strong activator of TNF- by macrophages, which, in turn, activates production of anti-inflammatory IL-10 (Wanidworanun and Strober, 1993). E. senticosus extracts used in our study contained no detectable LPS as analyzed by EndoLISA assay (LOD = 0.05 EU/ml). To confirm that the E. senticosus bark extract-induced TNF- release is not due to any residual LPS contamination, we stimulated cells in the presence of polymyxin B, which binds to LPS thereby blocking LPS-induced effects. As expected, polymyxin B inhibited the LPS-induced TNF- and IL-10 release, but had no effect on the cytokine release induced by the E. senticosus bark extract (Fig. 5A, right hand panel). The extract of E. senticosus bark likewise significantly increased secretion of chemotactic CCL1 and proinflammatory IL-6 (Fig. 5B). 9
Release of proinflammatory mediators is tightly regulated in macrophages. Activation of MAPK signaling and of NF-κB is necessary to trigger transcription of proinflammatory genes (Carter et al., 1999; Lawrence and Natoli, 2011; Syrovets et al., 2001). E. senticosus bark extract did not affect the phosphorylation pf ERK1/2 and JNK MAPK, but it increased phosphorylation of p38 MAPK (Fig. 5C). Similarly, the bark extract induced activation of NF-κB in a mouse macrophage reporter cell line (Fig. 5D). Of note, the observed p38 and NF-κB activation was much less pronounced than that induced by the strong proinflammatory activator LPS. E. senticosus root and bark extracts differentially regulate the IL-4-induced CD200R expression by human macrophages Neither E. senticosus root nor bark extract affected the basal CD200R expression on macrophages (Fig. 6A, left hand panel). By contrast, treatment with IL-4 induced strong expression of CD220R by macrophages (Fig. 6A, right hand panel). Interestingly, the root extract increased the IL-4 induced CD200R expression, but the bark extract decreased the IL-4 induced CD200R expression (Fig 6B).
Discussion Root or stem bark are the main parts of E. senticosus used medicinally as sources of tonifying and immunostimulatory preparations (Huang et al., 2011; Kalke, 2014). Extracts from both parts of the medicinal plant might exhibit effects on macrophages. The anti-inflammatory activity is one of the best-studied effects of E. senticosus extracts (Panossian and Wikman, 2009). However, most studies analyzed effects of E. senticosus on LPS-stimulated M1 macrophages using murine cells. Thus, an extract from whole plant decreased LPS-induced iNOS and COX-2 expression by inhibiting JNK and Akt activation in RAW264.7 macrophages (Jung et al., 2007), a root extract of E. senticosus decreased IL-6 and TNF-α secretion in LPSinduced acute lung injury mouse model by inhibiting NF-κB signaling pathway (Fei et al., 2014), and a stem bark extract inhibited nitric oxide production in LPS-stimulated murine RAW264.7 macrophages (Lin et al., 2007). In human whole blood, the root extract inhibited the release of granulocyte colony-stimulating factor, IL-6, and IL-13 at low concentrations, but increased the same cytokine production at higher concentrations (Schmolz et al., 2001). Still, a comparative analysis of E. senticosus root and bark extract effects on human primary macrophages has not been performed yet. In addition, effects of E. senticosus extracts were analyzed under inflammatory conditions, in cells activated with LPS. Different from previous studies, we show that under physiological conditions, in the absence of LPS, E. senticosus affects quiescent M0 macrophages as well, polarizing them towards anti-inflammatory M2 subtypes. 10
Both E. senticosus extracts used in our study have complex chemical compositions. A computational analysis allowed us to spot targeted pathways and proteins based on the chemical constituents of the extracts. Thus, network pharmacology analysis pointed to modulation of macrophage phenotype, migration, and actin cytoskeleton regulation, as well as MAPK, Akt, and NF-κB pathway activation as possible intracellular targets of E. senticosus extracts. Our study shows that both E. senticosus extracts induce actin polymerization, increase macrophage migration and phagocytosis, which are characteristic features of M2 macrophages (Van Ginderachter et al., 2006). Such changes in macrophage phenotypes indicate that E. senticosus might exert its anti-inflammatory activity through promotion of macrophage polarization towards the anti-inflammatory M2 subtype. Interestingly, the root and bark extracts obviously have different effects on M2 macrophage subtype polarization. Whereas the root extract inhibited an important proinflammatory signaling, that is NF-κB, the bark extract slightly increased the NF-κB and MAPK p38 activation and proinflammatory cytokine release by human macrophages. Of note, the proinflammatory cytokine release and the NF-κB and p38 activation induced by the E. senticosus bark extract were much less pronounced than those induced by LPS, a M1-polarization stimulus, findings that are are consistent with macrophage polarization into a M2b phenotype (Mantovani et al., 2004). The root extract also increased the expression of CD200R in the presence of IL-4, a M2 polarization stimulus. In addition to increased phagocytic activity after root extract treatment, this points to increased polarization of human macrophages towards the M2a phenotype (Fuchs et al., 2016; Jaguin et al., 2013; Van Ginderachter et al., 2006). Changes exerted by E. senticosus extracts in human macrophages might be important for wound healing. Thus, in the inflammatory phase of wound healing, the presence of M2b macrophages may provide a clean environment by eliminating infectious bacteria and apoptotic neutrophils at the wound site. While, in the proliferation and maturation phase, the presence of M2a macrophages will limit inflammatory response and participate in immunoregulation (Krzyszczyk et al., 2018). Interestingly, E. senticosus extracts are used in skin care products (Khan and Abourashed, 2010) and have been shown to promote wound healing after UV or pressure capping assaults (Bone and Mills, 2013). The increased CD163 expression is associated with transplant-related complications including acute graft-versus-host disease (Wolff et al., 2018). In addition, CD163 expression on macrophages is required for their tumor promoting activity (Shiraishi et al., 2018). Hence, inhibition of CD163 expression by both E. senticosus extracts, might point to its beneficial 11
effect in treatment of transplant-related diseases and tumors. These data are supported by experimental evidence of antitumor activity of E. senticosus in animal models and also by reports on improved survival of cancer patients in Russia (Bone and Mills, 2013; Khan and Abourashed, 2010). Changes exerted by E. senticosus extracts in human macrophages although significant are not high. Also under physiological conditions, borders between the two macrophage populations are fluent, and in vivo many intermediate forms with various roles in different diseases have been described (Mantovani et al., 2004). This study provides a scientific basis of potentially immunomodulatory activities of prescribed E. senticosus preparations.
Conclusion Target prediction, protein-protein interaction, and network analysis were used to predict biological processes affected by E. senticosus root and bark extracts. Experimental analysis confirmed that both extracts promoted actin polymerization, migration, and E. coli phagocytosis by human macrophages indicating increased M2 macrophage polarization. HPLC-MS analysis identified differences in the chemical composition of E. senticosus root and bark extracts, which might explain their differential effects on p38 MAPK and NF-κB activation, proinflammatory cytokine release, and CD200R expression, which are consistent with the induction of M2b-like polarization by the E. senticosus bark extract and M2a-like polarization by the E. senticosus root extract. These effects on macrophages might explain immunoregulatory effects of E. senticosus-containing medications. Compliance with ethical standards The collection of human blood, isolation of peripheral blood mononuclear cells, and macrophage differentiation thereof were approved by the University’s Ethics Committee (reference number 177/18). The volunteers provided written informed consent to participate in the study. Acknowledgements This work was supported by State Ministry of Baden-Württemberg for Sciences, Research and Arts through the Academic Center for Complementary and Integrative Medicine (AZKIM), High-Performance Computing Program (bwHPC) of Baden-Württemberg and Chinese Scholarships Council (No. 201708080165). We are grateful to Dr. Myung Hwan Park, Ambo Institute, Daejeon, South Korea, for the gift of the bark extract of E. senticosus. Conflict of interest 12
The authors have stated that there is no conflict of interest associated with the publication and no financial support, which could have influenced the outcome.
References Bindea, G., Mlecnik, B., Hackl, H., Charoentong, P., Tosolini, M., Kirilovsky, A., Fridman, W.H., Pages, F., Trajanoski, Z., Galon, J., 2009. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 25, 1091-1093. Bone, K., Mills, S., 2013. Principles and practice of phytotherapy. Elsevier Health Sciences. Carter, A.B., Knudtson, K.L., Monick, M.M., Hunninghake, G.W., 1999. The p38 mitogenactivated protein kinase is required for NF-κB-dependent gene expression. The role of TATAbinding protein (TBP). J Biol Chem 274, 30858-30863. Fei, X.J., Zhu, L.L., Xia, L.M., Peng, W.B., Wang, Q., 2014. Acanthopanax senticosus attenuates inflammation in lipopolysaccharide-induced acute lung injury by inhibiting the NFκB pathway. Genet Mol Res 13, 10537-10544. Fuchs, A.K., Syrovets, T., Haas, K.A., Loos, C., Musyanovych, A., Mailander, V., Landfester, K., Simmet, T., 2016. Carboxyl- and amino-functionalized polystyrene nanoparticles differentially affect the polarization profile of M1 and M2 macrophage subsets. Biomaterials 85, 78-87. Gong, J., Cai, C., Liu, X., Ku, X., Jiang, H., Gao, D., Li, H., 2013. ChemMapper: a versatile web server for exploring pharmacology and chemical structure association based on molecular 3D similarity method. Bioinformatics 29, 1827-1829. Huang, L., Zhao, H., Huang, B., Zheng, C., Peng, W., Qin, L., 2011. Acanthopanax senticosus: review of botany, chemistry and pharmacology. Pharmazie 66, 83-97. Jaguin, M., Houlbert, N., Fardel, O., Lecureur, V., 2013. Polarization profiles of human M-CSFgenerated macrophages and comparison of M1-markers in classically activated macrophages from GM-CSF and M-CSF origin. Cell Immunol 281, 51-61. Jones, G.E., 2000. Cellular signaling in macrophage migration and chemotaxis. J Leukoc Biol 68, 593-602. Jung, C.H., Jung, H., Shin, Y.C., Park, J.H., Jun, C.Y., Kim, H.M., Yim, H.S., Shin, M.G., Bae, H.S., Kim, S.H., Ko, S.G., 2007. Eleutherococcus senticosus extract attenuates LPS-induced iNOS expression through the inhibition of Akt and JNK pathways in murine macrophage. J Ethnopharmacol 113, 183-187. Kalke, D., 2014. Assessment report on Eleutherococcus senticosus (Rupr. et Maxim.) Maxim., radix. European Medicines Agency, https://www.ema.europa.eu/en/medicines/herbal/eleutherococci-radix, pp. 1-51. Khan I. A., Abourashed E. A., 2010. Leung’s encyclopedia of common natural ingredients, 3rd ed. John Wiley & Sons, Hoboken, New Jersey. 13
Krzyszczyk, P., Schloss, R., Palmer, A., Berthiaume, F., 2018. The role of macrophages in acute and chronic wound healing and interventions to promote pro-wound healing phenotypes. Front Physiol 9, 419. Lawrence, T., Natoli, G., 2011. Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nat Rev Immunol 11, 750-761. Li, Q., Laumonnier, Y., Syrovets, T., Simmet, T., 2007. Plasmin triggers cytokine induction in human monocyte-derived macrophages. Arterioscler Thromb Vasc Biol 27, 1383-1389. Li, T., Ferns, K., Yan, Z.Q., Yin, S.Y., Kou, J.J., Li, D., Zeng, Z., Yin, L., Wang, X., Bao, H.X., Zhou, Y.J., Li, Q.H., Zhao, Z.Y., Liu, H., Liu, S.L., 2016. Acanthopanax senticosus: Photochemistry and anticancer potential. Am J Chin Med 44, 1543-1558. Lin, Q.-Y., Jin, L.-J., Cao, Z.-H., Xu, Y.-P., 2008. Inhibition of inducible nitric oxide synthase by Acanthopanax senticosus extract in RAW264.7 macrophages. J Ethnopharm 118, 231-236. Lin, Q.Y., Jin, L.J., Ma, Y.S., Shi, M., Xu, Y.P., 2007. Acanthopanax senticosus inhibits nitric oxide production in murine macrophages in vitro and in vivo. Phytother Res 21, 879-883. Loos, C., Syrovets, T., Musyanovych, A., Mailänder, V., Landfester, K., Simmet, T., 2014. Amino-functionalized nanoparticles as inhibitors of mTOR and inducers of cell cycle arrest in leukemia cells. Biomaterials 35, 1944-1953. Lunov, O., Syrovets, T., Loos, C., Beil, J., Delacher, M., Tron, K., Nienhaus, G.U., Musyanovych, A., Mailander, V., Landfester, K., Simmet, T., 2011. Differential uptake of functionalized polystyrene nanoparticles by human macrophages and a monocytic cell line. ACS Nano 5, 1657-1669. Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A., Locati, M., 2004. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25, 677686. Murray, P.J., 2017. Macrophage polarization. Annu Rev Physiol 79, 541-566. Nishibe, S., Kinoshita, H., Takeda, H., Okano, G., 1990. Phenolic-compounds from stem bark of Acanthopanax senticosus and their pharmacological effect in chronic swimming stressed rats. Chem Pharm Bull (Tokyo) 38, 1763-1765. Panossian, A., Wikman, G., 2009. Evidence-based efficacy of adaptogens in fatigue, and molecular mechanisms related to their stress-protective activity. Curr Clin Pharmacol 4, 198219. Schmolz, M.W., Sacher, F., Aicher, B., 2001. The synthesis of Rantes, G-CSF, IL-4, IL-5, IL-6, IL-12 and IL-13 in human whole-blood cultures is modulated by an extract from Eleutherococcus senticosus L. roots. Phytother Res 15, 268-270. Shiraishi, D., Fujiwara, Y., Horlad, H., Saito, Y., Iriki, T., Tsuboki, J., Cheng, P., Nakagata, N., Mizuta, H., Bekki, H., Nakashima, Y., Oda, Y., Takeya, M., Komohara, Y., 2018. CD163 is required for protumoral activation of macrophages in human and murine sarcoma. Cancer Res 78, 3255-3266. Shrivastava, R., Asif, M., Singh, V., Dubey, P., Malik, S.A., Lone, M.U.D., Tewari, B.N., Baghel, 14
K.S., Pal, S., Nagar, G.K., Chattopadhyay, N., Bhadauria, S., 2019. M2 polarization of macrophages by Oncostatin M in hypoxic tumor microenvironment is mediated by mTORC2 and promotes tumor growth and metastasis. Cytokine 118, 130-143. Slacanin, I., Marston, A., Hostettmann, K., Guedon, D., Abbe, P., 1991. The isolation of Eleutherococcus senticosus constituents by centrifugal partition chromatography and their quantitative determination by high performance liquid chromatography. Phytochem Anal 2, 137-142. Soo Kim, H., Young Park, S., Kyoung Kim, E., Yeon Ryu, E., Hun Kim, Y., Park, G., Joon Lee, S., 2012. Acanthopanax senticosus has a heme oxygenase-1 signaling-dependent effect on Porphyromonas gingivalis lipopolysaccharide-stimulated macrophages. J Ethnopharm 142, 819-828. Syrovets, T., Jendrach, M., Rohwedder, A., Schule, A., Simmet, T., 2001. Plasmin-induced expression of cytokines and tissue factor in human monocytes involves AP-1 and IKKβmediated NF-κB activation. Blood 97, 3941-3950. Van Ginderachter, J.A., Movahedi, K., Hassanzadeh Ghassabeh, G., Meerschaut, S., Beschin, A., Raes, G., De Baetselier, P., 2006. Classical and alternative activation of mononuclear phagocytes: picking the best of both worlds for tumor promotion. Immunobiology 211, 487501. Wanidworanun, C., Strober, W., 1993. Predominant role of tumor necrosis factor-α in human monocyte IL-10 synthesis. J Immunol 151, 6853-6861. Wolff, D., Greinix, H., Lee, S.J., Gooley, T., Paczesny, S., Pavletic, S., Hakim, F., Malard, F., Jagasia, M., Lawitschka, A., Hansen, J.A., Pulanic, D., Holler, E., Dickinson, A., Weissinger, E., Edinger, M., Sarantopoulos, S., Schultz, K.R., 2018. Biomarkers in chronic graft-versushost disease: quo vadis? Bone Marrow Transplant 53, 832-837. Xue, J., Schmidt, S.V., Sander, J., Draffehn, A., Krebs, W., Quester, I., De Nardo, D., Gohel, T.D., Emde, M., Schmidleithner, L., Ganesan, H., Nino-Castro, A., Mallmann, M.R., Labzin, L., Theis, H., Kraut, M., Beyer, M., Latz, E., Freeman, T.C., Ulas, T., Schultze, J.L., 2014. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 40, 274-288.
Figure legends Fig. 1. Chemical characterization of E. senticosus root and bark extracts. (A) HPLC-DAD fingerprints of root and bark extracts at 210 nm. (B) Quantification of isolated compounds by HPLC-DAD-MS, mean, n = 3. (C) Chemical structures of compounds from E. senticosus root and bark extracts. Fig. 2. Network pharmacology-based analysis of possible E. senticosus targets in macrophages. (A) Network of intracellular pathways targeted by compounds from E. senticosus. Yellow (root) or orange (bark) nodes show the reference compounds present in high amounts. The node in the middle (cyan) depicts targeted networks. Three green nodes on the right show pathways that 15
might be affected by E. senticosus treatment. (B) Proteins predicted to be targeted by E. senticosus compounds arranged into networks by String database using Gene ontology and KEGG datasets. Fig. 3. E. senticosus root and bark extracts increase actin polymerization, migration, and phagocytosis by macrophages. (A) Macrophages were treated for 40 min and actin polymerization (F-actin) was visualized by phalloidin staining followed by microscopy and fluorimetry. Left hand panels, representative photomicrographs are shown. (B) Macrophages were treated as indicated and allowed to migrate for 18 h. Wound Healing Tool in ImageJ was used to quantify the cell-free wound area. (C) Cells were treated for 24 h and phagocytosis of fluorescent E. coli was analyzed by flow cytometry. All data are mean ± SEM, n = 3-5, *p < 0.05, **p < 0.01. Fig 4. E. senticosus root and bark extracts decrease Akt activation and CD163 expression by macrophages. (A) E. senticosus root and bark extracts do not affect expression of CD11b, CD80, and CD64 markers. (B) E. senticosus root and bark extracts (24 h treatment) reduce CD163 expression as measured by flow cytometry. Treatment with LPS (100 ng/ml)/IFN- (20 ng/ml) and IL-4 (20 ng/ml) for 24 h – positive controls. Representative histograms are shown. (C). Treatment with E. senticosus root and bark extracts (both, 30 µg/ml) for 1 h reduces basal Akt activation by phosphorylation (Ser473) in macrophages. LPS (100 ng/ml) – positive control. All data are mean ± SEM, n = 3-6, *p < 0.05, **p < 0.01, ***p < 0.001. Fig. 5. Extract from E. senticosus bark, but not from the root, induces moderate p38 MAPK and NF-κB activation and cytokine release by human macrophages. (A) Cells were treated with extracts, LPS (100 ng/ml), or IL-4 (20 ng/ml) for 24 h and TNF- and IL-10 were analyzed in supernatants by ELISA. Polymyxin B (10 µg/ml) significantly reduced the LPS-induced cytokine release, but had no effect on the cytokine release triggered by E. senticosus bark extract confirming absence of LPS contamination. (B) Cells were treated as in (A) and CCL-1 and IL6 secretion were analyzed by corresponding ELISAs. LPS (100 ng/ml) – positive control. (C) E. senticosus bark, but not root extract, activates p38 MAPK. Cells were treated for 1 h and analyzed by Western immunoblotting. (D) E. senticosus bark, but not root extract, activates NFκB. Macrophage J774 NF-κB reporter cells were treated for 24 h and secreted SEAP enzyme quantified. All data are mean ± SEM, n = 3-6, *p < 0.05, **p < 0.01, ***p < 0.001. Fig. 6. E. senticosus root and bark extracts differentially regulate the IL-4-induced CD200R expression in macrophages. (A) Extracts alone do not affect expression of CD200R. Cells were treated with extracts, LPS (100 ng/ml), or IL-4 (20 ng/ml) for 24 h and analyzed by flow 16
cytometry. (B) E. senticosus root extracts increases, whereas bark extract decreases the IL-4induced CD200R. Macrophages were treated as in A. All data are mean ± SEM, n = 3, *p < 0.05, ***p < 0.001. Credit Author Statement Lu Jin: Investigation, Formal Analysis, Conceptualization, Visualization, Writing – Original Draft, Funding Aquisition Michael Schmiech: Methodology, Data Curation, Validation, Resources Menna El Gaafary: Methodology, Validation Xinlei Zhang: Methodology, Formal Analysis Tatiana Syrovets: Conceptualization, Supervision, Data Curation, Validation, Visualization, Writing - Review and Editing, Project Administration Thomas Simmet: Conceptualization, Supervision, Writing - Review and Editing, Project Administration, Funding Acqusition
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Graphical Abstract
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A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages Lu Jin , Michael Schmiech , Menna El Gaafary , Xinlei Zhang , Tatiana Syrovets , Thomas Simmet PII: DOI: Reference:
S0944-7113(20)30014-3 https://doi.org/10.1016/j.phymed.2020.153181 PHYMED 153181
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
31 October 2019 24 January 2020 5 February 2020
Please cite this article as: Lu Jin , Michael Schmiech , Menna El Gaafary , Xinlei Zhang , Tatiana Syrovets , Thomas Simmet , A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages, Phytomedicine (2020), doi: https://doi.org/10.1016/j.phymed.2020.153181
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A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages Lu Jina, Michael Schmiecha, Menna El Gaafarya,b, Xinlei Zhang c, Tatiana Syrovetsa, Thomas Simmeta* a
Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University,
89081 Ulm, Germany b
Department of Pharmacognosy, College of Pharmacy, Cairo University, Cairo, 11562 Egypt
c
Department of Medicinal Chemistry, School of Pharmacy, Fourth Military Medical University,
710032 Xi’an, Shaanxi, P.R. China *Corresponding author: Thomas Simmet, Institute of Pharmacology of Natural Products and Clinical Pharmacology, Helmholtzstraße 20, 89081 Ulm, Germany Tel.: (+49) 731 500 65600; fax: (+49) 731 500 65602 E-mail address: [email protected]
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Abstract Background: Eleutherococcus senticosus or Siberian ginseng is a medicinal plant containing adaptogenic substances believed to regulate immune responses. Both, the root and stem bark are commonly used in traditional medicines. Purpose: The purpose of the present study is to chemically characterize E. senticosus root and bark extracts and to compare their effects on functions of human primary macrophages. Study design and methods: HPLC-DAD-MS analysis was used to characterize chemical constituents of alcoholic extracts from E. senticosus root and bark. The data obtained and available databases were combined for network pharmacology analysis. Involvement of predicted pathways was further functionally confirmed by using monocyte-derived human macrophages and endotoxin-free E. senticosus root and bark extracts. Results: Chemical analysis showed that the root extract contained more syringin, caffeic acid, and isofraxidin than the bark extract. At variance, bark extract contained more sesamin and oleanolic acid. Coniferyl aldehyde and afzelin were below the limit of quantification in both extracts. Network pharmacology analysis indicated that constituents of E. senticosus might affect the immune cell phenotype and signaling pathways involved in cell metabolism and cytoskeleton regulation. Indeed, both extracts promoted actin polymerization, migration, and phagocytosis of E. coli by macrophages pointing to macrophage polarization towards the M2 phenotype. In addition, treatment with E. senticosus root and bark extracts decreased phosphorylation of Akt on Ser473 and significantly reduced expression of the hemoglobin scavenger receptor CD163 by macrophages. Neither extract affected expression of CD11b, CD80, or CD64 by macrophages. In addition, macrophages treated with the bark extract, but not with the root extract, exhibited activated p38 MAPK and NF-κB and released increased, but still moderate, amounts of proinflammatory TNF- and IL-6, anti-inflammatory IL-10, and chemotactic CCL1, which all together point to a M2b-like macrophage polarization. Differently, the root extract increased the IL-4-induced expression of anti-inflammatory CD200R. These changes in monocytes are in agreement with an increased M2a macrophage polarization. Conclusion: The ability of E. senticosus root and bark extracts to promote polarization of human macrophages towards anti-inflammatory M2a and M2b phenotypes, respectively, might underlay the immunoregulatory activities and point to potential wound healing promoting effects of this medicinal plant. Keywords Alternatively activated macrophages, Eleutherococcus senticosus; Adaptogen; Network pharmacology; Wound healing; Cytoskeleton. 2
Abbreviations APC, allophycocyanin; BSA, bovine serum albumin; DAD, diode-array detector; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; HPLC-MS, high-performance liquid chromatography-mass spectrometry; IL, interleukin; LOD, limit of detection; LOQ, limit of quantification; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; CSF, macrophage colony-stimulating factor (colony stimulating factor 1, CSF1); MF, median florescence intensity; MFI, median florescence index (normalized to isotype control); mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa B; SEAP, secreted embryonic alkaline phosphatase; TNF-, tumor necrosis factor ; XTT, 2,3-bis-(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide.
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Introduction Eleutherococcus senticosus (Rupr. and Maxim.), commonly called Siberian ginseng, belongs to a small family of plants containing adaptogenic substances. They are believed to increase resistance to various exogenous stressors, and they might contribute to normalization of immune responses (Panossian and Wikman, 2009). In Chinese and Asian medicine in general, adaptogens including Panax ginseng and Eleutherococcus senticosus are more widely accepted compared to Western medicine as they might better suit the whole-body treatment approach adopted by Asian therapies. However, our understanding of the molecular mechanisms of adaptogens is still very limited. Most experimental data obtained with extracts from plants with adaptogenic activity originate from studies with either extracts or ingredients of Panax ginseng (Panossian and Wikman, 2009). Only few reports address biological activities of E. senticosus extracts. Some claim effects of E. senticosus extracts or components thereof on the Raw264.7 murine macrophage cell line or mouse peritoneal macrophages (Fei et al., 2014; Jung et al., 2007; Lin et al., 2008; Lin et al., 2007; Soo Kim et al., 2012). However, receptor and cytokine expression, and specific features, such as polarization into distinct phenotypes are grossly different in human and murine macrophages as well as in monocytic leukemia cell lines and primary cells (Murray, 2017; Loos et al., 2014; Lunov et al., 2011). In addition, the effects reported are often contradictory, which may be due to different extraction procedures leading to differences in the chemical composition of these extracts. Understanding functional cellular mechanisms of E. senticosus and other adaptogens might advance their rational application in distinct disease states. Macrophages express an array of receptors enabling them to monitor continuously their microenvironment and to maintain tissue homeostasis. Depending on their activation state, they may reprogram their phenotype to respond quickly to disturbed homeostasis (Murray, 2017). Reacting to damage- and pathogen-associated molecular patterns, macrophages undergo classical activation resulting in formation of the proinflammatory M1 macrophage phenotype, which exhibits increased microbicidal and tumoricidal activities. In response to non-pathogen related stimuli, macrophages undergo alternative activation and adopt the anti-inflammatory alternative M2 phenotype. Unlike M1 macrophages, M2 macrophages comprise a spectrum of different subtypes, such as M2a, M2b, and M2c. Polarization to each of these subtypes is induced by specialized stimuli (Mantovani et al., 2004; Murray, 2017). Obviously, modulation of macrophage polarization might offer therapeutic opportunities in patients suffering from cancer or chronic inflammatory diseases. 4
To investigate the effects of E. senticosus-derived adaptogens on macrophage polarization and thereby function, we have chemically characterized extracts from root and stem bark of E. senticosus and monitored their effects on the polarization profile of primary human macrophages.
Materials and methods General experimental procedures XTT cell proliferation assay was from Roche (Filderstadt, Germany). Macrophage colonystimulating factor (M-CSF, CSF1) and interleukin 4 (IL-4) were from Miltenyi Biotec (Bergisch Gladbach, Germany). Medium and antibiotics were from Gibco (Carlsbad, CA), FCS and Biocoll separation solution was from Biochrom (Berlin, Germany). Lipopolysaccharide (LPS) O55:B5 from E. coli and fibrinogen were purchased from Sigma. Dylight650-phalloidin and antibodies used for Western immunoblotting were obtained from Cell Signaling Technology (Danvers, MA). APC-labeled antibodies to CD64, CD163, CD11b, and respective IgG controls were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Anti-CD200R-FITC and the respective IgG control were from Bio-Rad (Hercules, CA). Anti-CD80 was from BD (Franklin Lakes, NJ), Alexa Fluor 647-conjugated secondary antibody was from Jackson ImmunoResearch (Dianova, Hamburg, Germany). pHrodo™ Green E. coli were from Molecular Probes (Eugene, OR). Polymyxin B sulfate was from Enzo Life Sciences (Farmingdale, NY). Fractionation and analytical characterization of E. senticosus extracts E. senticosus ethanolic root extract Eleu curarina® (batch #1601) was from Harras Pharma Curarina (Munich, Germany). Eleu curarina extract (25 ml) was lyophilized to yield 1.0856 g of dry extract, which was further dissolved in DMSO. E. senticosus bark extract was a gift from Dr. Myung Hwan Park (Ambo Institute, Daejeon, South Korea). Briefly, 100 g of dried and chopped stem bark of E. senticosus were twice extracted with 600 ml of 70% methanol by reflux for 3 h. The filtrate was concentrated to dry powder (9.2 g) and used to prepare stock solutions in DMSO, which were further diluted in medium prior to experiments. Final DMSO concentration was 0.1%. Absence of endotoxin in E. senticosus extracts was confirmed using the EndoLISA assay, LOD = 0.05 EU/ml (Hyglos, Bernried/Starnberger See, Germany) according to the manufacturer’s instructions. Fingerprint characterization of E. senticosus root and bark extracts was carried out by highperformance liquid chromatography coupled with photodiode array detection and mass spectrometry (HPLC-DAD-MS). Syringin, isofraxidin, sesamin (all from AKos, Steinen, 5
Germany) and caffeic acid, afzelin, coniferyl aldehyde, oleanolic acid (all from Sigma) were used as references compounds for quantification. For separation, a Dr. Maisch ReproSil-Pur Basic C18-HD, 3 μm, 125 x 3 mm column was used. The flow rate was set to 600 μL/min and the injection volume was 5 μL. The mobile phase consisted of eluent A, MilliQ water, and eluent B, methanol, both acidified with 0.2 % acetic acid. The elution gradient was: 0 - 15 min, 10 95% B; 15 - 23 min 95% B; 23 - 24 min, 95 - 10% B; 24 - 30 min, 10% B. The UV/Vis detection was carried out at 210 nm, 254 nm, and 280 nm. MS analysis was performed in the negative electrospray ionization mode and the selected ion monitoring detection mode. Quantification of the compounds was performed using the external calibration method based on the corresponding selected ion chromatograms or UV/Vis chromatograms. The quantification method was validated in terms of linearity, the limit of detection (LOD), and limit of quantification (LOQ). Macrophages Macrophages were obtained by differentiation of human peripheral blood monocytes with 15 ng/ml M-CSF for 6 days (Li et al., 2007). Macrophages were treated with indicated concentrations of E. senticosus root or bark extracts or vehicle control (0.1% DMSO). LPS (100 ng/ml), LPS with IFN- (20 ng/ml), fibrinogen (50 ng/ml), or IL-4 (20 ng/ml) were used as positive controls. Network pharmacology analysis Caffeic acid, chlorogenic acid, coniferyl aldehyde, eleutheroside A, eleutheroside B1, (isofraxidin 7-0-glucoside), eleutheroside B4 [(-)-sesamin], eleutheroside B (syringin), eleutheroside E, hyperin, isofraxidin, and oleanolic acid were used as reference compounds for the root extract, whereas afzelin, chlorogenic acid, isofraxidin-7-O-β-D-glucoside, friedelin, liriodendrin, eleutheroside B (syringin), eleutheroside E were used for the bark extract (Li et al., 2016; Nishibe et al., 1990; Slacanin et al., 1991). Their targets were predicted by ChemMapper (Gong et al., 2013) using ChEMBL database. The similarity score threshold of potential targets was set to 1.2 and the top-scored 30 targets of each compound were selected for further analysis. After removing duplicates, 110 proteins highly relevant for macrophage activation and function (Jones, 2000; Lawrence and Natoli, 2011; Xue et al., 2014) (DrugBank and Therapeutic Target Database) were further used for the network construction. The network was constructed by connecting identified nodes with protein-protein interaction information from String, HPRD, HIPPE, and Reactome databases. The records were filtered according to 6
reliability and only interactions proved experimentally or highly ranked (for String, > 400) were preserved. The initial network was consolidated by igraph in R 3.5.3, filtered by NetworkAnalyzer, and visualized by Cytoscape 3.7.1. The enrichment analysis and biological annotations were performed by ClueGo (Bindea et al., 2009). Analysis of macrophage polarization and function Cell viability was analyzed by XTT assay. Absorbance was measured with a Tecan Infinite M1000PRO reader (Tecan, Männedorf, Switzerland) at 450 nm with a 630 nm reference filter. Data were normalized to vehicle control. Cell surface marker expression was analyzed after 24 h treatment flow cytometrically by using a FACSVerse (BD Biosciences) and quantified by FlowJo (Treestar, OR). Median fluorescent values of respective IgG controls were subtracted from the sample values. Cytokine and chemokine release was analyzed in supernatants of macrophages treated for 24 h by using corresponding ELISAs (R&D Systems, Minneapolis, MN). Actin polymerization was analyzed in macrophages grown in ibidi µ-glass-bottom plates stimulated for 40 min with either extract or fibrinogen control for 40 min and stained with DyLight 650-phalloidin. Cells were acquired under a Nikon eclipse Ti microscope using 20x objective and NIS-elements software (Nikon Corporation, Tokyo, Japan). The fluorescence of samples was quantified using a Tecan Infinite M1000PRO and normalized to fluorescence of the control sample. For analysis of migration, a cell-free zone in macrophage monolayer was introduced and cells were treated with either extract or LPS control and monitored for 18 h by Nikon eclipse Ti microscope with a 10x objective. The area of the cell-free zone was calculated with MRI Wound Healing Tool (ImageJ, NIH). For analysis of phagocytic activity, macrophages treated with either extract or LPS for 24 h were incubated with 15 µg/ml E. coli particles pHrodo for 2 h followed by flow cytometric analysis. Phosphorylation of Akt and p38 MAPK were analyzed by Western immunoblotting in whole cell extracts of cells treated for 1 h (Fuchs et al., 2016). NF-κB activation was analyzed in macrophage J774 reporter cells (InvivoGen, San Diego, CA) by assessing the activity of secreted embryonic alkaline phosphatase (SEAP) after 24 h.
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Statistics Results are expressed as mean ± SEM of at least three independent macrophage isolations. For two-group comparisons, results were analyzed with the two-tailed Student’s t-test. Multigroup analyses were performed with ANOVA followed by the Tukey or Dunnett posthoc test using SigmaPlot. Significance levels are *p < 0.05, **p < 0.01, ***p < 0.001.
Results E. senticosus root and bark extracts contain different compounds and both extracts are not toxic to human macrophages HPLC-MS analysis (Fig. 1) showed that the root and bark extracts contain different amounts of reference compounds with the root extract containing more syringin, caffeic acid, and isofraxidin than the bark extract. By contrast, the bark extract contained more sesamin and oleanolic acid. Coniferyl aldehyde and afzelin were present in very low concentrations and were below the LOQ in both extracts (Fig 1B). Neither extract affected the viability of human macrophages after 24 h of treatment (Fig. S1). Network pharmacology-based analysis revealed biological processes that might be targeted by E. senticosus root and bark extracts The results revealed that compounds, which we have identified in the E. senticosus extracts and those reported in the literature (Li et al., 2016; Nishibe et al., 1990; Slacanin et al., 1991), might affect immune cell functions, cell metabolism, actin cytoskeleton reorganization, cell migration regulation, and Fc receptor-mediated phagocytosis (Fig. 2A). Enrichment analysis showed that the majority of possible protein targets of E. senticosus are located in the cytoplasm functioning as a hub for binding of regulatory molecules, such as ATP. Besides, the respective protein targets play essential roles in responses to cytokines and other organic substances, the regulation of immune responses, the response to stress, and in cell motility. In addition, MAPK, PI3K/Akt/mTOR, and Toll-like receptor 4/NF-κB signaling pathways were predicted as possible targets of compounds present in E. senticosus (Fig. 2B). E. senticosus root and bark extracts promote actin polymerization, migration, and E. coli phagocytosis by macrophages Actin cytoskeleton regulation was identified in network analysis as a possible target of E. senticosus. Indeed, E. senticosus root and bark extracts significantly enhanced actin polymerization in macrophages, although to a lesser extent than fibrinogen (Fig. 3A). Reorganization of actin from soluble to polymerized filamentous F-actin within macrophages 8
is important for cell migration and their phagocytic activity. Both extracts induced migration of macrophages leading to the reduction of a cell-free zone (Fig. 3B). Also, the phagocytosis of E. coli by macrophages was increased after they had been incubated for 24 h with either root or bark extract of E. senticosus (Fig. 3C). Despite the differences in the extracts’ compositions, both extracts exhibited similar potency in activating macrophage actin polymerization, migration, and phagocytosis. E. senticosus root and bark extracts reduce Akt activation and CD163 expression in human macrophages E. senticosus root and bark extracts did not affect the expression of the macrophage markers CD64, CD80, and CD11b (Fig. 4A). However, both extracts reduced concentration-dependently the cell-surface expression of the hemoglobin scavenger receptor CD163 (Fig. 4B). It has previously been shown that activation of the mTORC2/Akt axis increases the CD163 expression on macrophages (Shrivastava et al., 2019). In addition, the network analysis indicated possible effects of E. senticosus constituents on the mTOR/Akt signaling pathway. Hence, phosphorylation of Akt on Ser473 was further analyzed. The results demonstrate that E. senticosus root and bark extracts equally decrease the phosphorylation level of Akt S473 (Fig. 4C) indicating that the E. senticosus extracts might reduce expression of CD163 through downregulation of Akt activation. The E. senticosus bark but not the root extract induces p38 MAPK kinase activation and cytokine and chemokine secretion by human macrophages Extract of E. senticosus bark significantly increased the release of proinflammatory TNF-α and anti-inflammatory IL-10 by macrophages, whereas non-stimulated or macrophages stimulated with E. senticosus root extract show non-detectable amounts of TNF- and low amounts of IL10 (Fig. 5A). LPS is a very strong activator of TNF- by macrophages, which, in turn, activates production of anti-inflammatory IL-10 (Wanidworanun and Strober, 1993). E. senticosus extracts used in our study contained no detectable LPS as analyzed by EndoLISA assay (LOD = 0.05 EU/ml). To confirm that the E. senticosus bark extract-induced TNF- release is not due to any residual LPS contamination, we stimulated cells in the presence of polymyxin B, which binds to LPS thereby blocking LPS-induced effects. As expected, polymyxin B inhibited the LPS-induced TNF- and IL-10 release, but had no effect on the cytokine release induced by the E. senticosus bark extract (Fig. 5A, right hand panel). The extract of E. senticosus bark likewise significantly increased secretion of chemotactic CCL1 and proinflammatory IL-6 (Fig. 5B). 9
Release of proinflammatory mediators is tightly regulated in macrophages. Activation of MAPK signaling and of NF-κB is necessary to trigger transcription of proinflammatory genes (Carter et al., 1999; Lawrence and Natoli, 2011; Syrovets et al., 2001). E. senticosus bark extract did not affect the phosphorylation pf ERK1/2 and JNK MAPK, but it increased phosphorylation of p38 MAPK (Fig. 5C). Similarly, the bark extract induced activation of NF-κB in a mouse macrophage reporter cell line (Fig. 5D). Of note, the observed p38 and NF-κB activation was much less pronounced than that induced by the strong proinflammatory activator LPS. E. senticosus root and bark extracts differentially regulate the IL-4-induced CD200R expression by human macrophages Neither E. senticosus root nor bark extract affected the basal CD200R expression on macrophages (Fig. 6A, left hand panel). By contrast, treatment with IL-4 induced strong expression of CD220R by macrophages (Fig. 6A, right hand panel). Interestingly, the root extract increased the IL-4 induced CD200R expression, but the bark extract decreased the IL-4 induced CD200R expression (Fig 6B).
Discussion Root or stem bark are the main parts of E. senticosus used medicinally as sources of tonifying and immunostimulatory preparations (Huang et al., 2011; Kalke, 2014). Extracts from both parts of the medicinal plant might exhibit effects on macrophages. The anti-inflammatory activity is one of the best-studied effects of E. senticosus extracts (Panossian and Wikman, 2009). However, most studies analyzed effects of E. senticosus on LPS-stimulated M1 macrophages using murine cells. Thus, an extract from whole plant decreased LPS-induced iNOS and COX-2 expression by inhibiting JNK and Akt activation in RAW264.7 macrophages (Jung et al., 2007), a root extract of E. senticosus decreased IL-6 and TNF-α secretion in LPSinduced acute lung injury mouse model by inhibiting NF-κB signaling pathway (Fei et al., 2014), and a stem bark extract inhibited nitric oxide production in LPS-stimulated murine RAW264.7 macrophages (Lin et al., 2007). In human whole blood, the root extract inhibited the release of granulocyte colony-stimulating factor, IL-6, and IL-13 at low concentrations, but increased the same cytokine production at higher concentrations (Schmolz et al., 2001). Still, a comparative analysis of E. senticosus root and bark extract effects on human primary macrophages has not been performed yet. In addition, effects of E. senticosus extracts were analyzed under inflammatory conditions, in cells activated with LPS. Different from previous studies, we show that under physiological conditions, in the absence of LPS, E. senticosus affects quiescent M0 macrophages as well, polarizing them towards anti-inflammatory M2 subtypes. 10
Both E. senticosus extracts used in our study have complex chemical compositions. A computational analysis allowed us to spot targeted pathways and proteins based on the chemical constituents of the extracts. Thus, network pharmacology analysis pointed to modulation of macrophage phenotype, migration, and actin cytoskeleton regulation, as well as MAPK, Akt, and NF-κB pathway activation as possible intracellular targets of E. senticosus extracts. Our study shows that both E. senticosus extracts induce actin polymerization, increase macrophage migration and phagocytosis, which are characteristic features of M2 macrophages (Van Ginderachter et al., 2006). Such changes in macrophage phenotypes indicate that E. senticosus might exert its anti-inflammatory activity through promotion of macrophage polarization towards the anti-inflammatory M2 subtype. Interestingly, the root and bark extracts obviously have different effects on M2 macrophage subtype polarization. Whereas the root extract inhibited an important proinflammatory signaling, that is NF-κB, the bark extract slightly increased the NF-κB and MAPK p38 activation and proinflammatory cytokine release by human macrophages. Of note, the proinflammatory cytokine release and the NF-κB and p38 activation induced by the E. senticosus bark extract were much less pronounced than those induced by LPS, a M1-polarization stimulus, findings that are are consistent with macrophage polarization into a M2b phenotype (Mantovani et al., 2004). The root extract also increased the expression of CD200R in the presence of IL-4, a M2 polarization stimulus. In addition to increased phagocytic activity after root extract treatment, this points to increased polarization of human macrophages towards the M2a phenotype (Fuchs et al., 2016; Jaguin et al., 2013; Van Ginderachter et al., 2006). Changes exerted by E. senticosus extracts in human macrophages might be important for wound healing. Thus, in the inflammatory phase of wound healing, the presence of M2b macrophages may provide a clean environment by eliminating infectious bacteria and apoptotic neutrophils at the wound site. While, in the proliferation and maturation phase, the presence of M2a macrophages will limit inflammatory response and participate in immunoregulation (Krzyszczyk et al., 2018). Interestingly, E. senticosus extracts are used in skin care products (Khan and Abourashed, 2010) and have been shown to promote wound healing after UV or pressure capping assaults (Bone and Mills, 2013). The increased CD163 expression is associated with transplant-related complications including acute graft-versus-host disease (Wolff et al., 2018). In addition, CD163 expression on macrophages is required for their tumor promoting activity (Shiraishi et al., 2018). Hence, inhibition of CD163 expression by both E. senticosus extracts, might point to its beneficial 11
effect in treatment of transplant-related diseases and tumors. These data are supported by experimental evidence of antitumor activity of E. senticosus in animal models and also by reports on improved survival of cancer patients in Russia (Bone and Mills, 2013; Khan and Abourashed, 2010). Changes exerted by E. senticosus extracts in human macrophages although significant are not high. Also under physiological conditions, borders between the two macrophage populations are fluent, and in vivo many intermediate forms with various roles in different diseases have been described (Mantovani et al., 2004). This study provides a scientific basis of potentially immunomodulatory activities of prescribed E. senticosus preparations.
Conclusion Target prediction, protein-protein interaction, and network analysis were used to predict biological processes affected by E. senticosus root and bark extracts. Experimental analysis confirmed that both extracts promoted actin polymerization, migration, and E. coli phagocytosis by human macrophages indicating increased M2 macrophage polarization. HPLC-MS analysis identified differences in the chemical composition of E. senticosus root and bark extracts, which might explain their differential effects on p38 MAPK and NF-κB activation, proinflammatory cytokine release, and CD200R expression, which are consistent with the induction of M2b-like polarization by the E. senticosus bark extract and M2a-like polarization by the E. senticosus root extract. These effects on macrophages might explain immunoregulatory effects of E. senticosus-containing medications. Compliance with ethical standards The collection of human blood, isolation of peripheral blood mononuclear cells, and macrophage differentiation thereof were approved by the University’s Ethics Committee (reference number 177/18). The volunteers provided written informed consent to participate in the study. Acknowledgements This work was supported by State Ministry of Baden-Württemberg for Sciences, Research and Arts through the Academic Center for Complementary and Integrative Medicine (AZKIM), High-Performance Computing Program (bwHPC) of Baden-Württemberg and Chinese Scholarships Council (No. 201708080165). We are grateful to Dr. Myung Hwan Park, Ambo Institute, Daejeon, South Korea, for the gift of the bark extract of E. senticosus. Conflict of interest 12
The authors have stated that there is no conflict of interest associated with the publication and no financial support, which could have influenced the outcome.
References Bindea, G., Mlecnik, B., Hackl, H., Charoentong, P., Tosolini, M., Kirilovsky, A., Fridman, W.H., Pages, F., Trajanoski, Z., Galon, J., 2009. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 25, 1091-1093. Bone, K., Mills, S., 2013. Principles and practice of phytotherapy. Elsevier Health Sciences. Carter, A.B., Knudtson, K.L., Monick, M.M., Hunninghake, G.W., 1999. The p38 mitogenactivated protein kinase is required for NF-κB-dependent gene expression. The role of TATAbinding protein (TBP). J Biol Chem 274, 30858-30863. Fei, X.J., Zhu, L.L., Xia, L.M., Peng, W.B., Wang, Q., 2014. Acanthopanax senticosus attenuates inflammation in lipopolysaccharide-induced acute lung injury by inhibiting the NFκB pathway. Genet Mol Res 13, 10537-10544. Fuchs, A.K., Syrovets, T., Haas, K.A., Loos, C., Musyanovych, A., Mailander, V., Landfester, K., Simmet, T., 2016. Carboxyl- and amino-functionalized polystyrene nanoparticles differentially affect the polarization profile of M1 and M2 macrophage subsets. Biomaterials 85, 78-87. Gong, J., Cai, C., Liu, X., Ku, X., Jiang, H., Gao, D., Li, H., 2013. ChemMapper: a versatile web server for exploring pharmacology and chemical structure association based on molecular 3D similarity method. Bioinformatics 29, 1827-1829. Huang, L., Zhao, H., Huang, B., Zheng, C., Peng, W., Qin, L., 2011. Acanthopanax senticosus: review of botany, chemistry and pharmacology. Pharmazie 66, 83-97. Jaguin, M., Houlbert, N., Fardel, O., Lecureur, V., 2013. Polarization profiles of human M-CSFgenerated macrophages and comparison of M1-markers in classically activated macrophages from GM-CSF and M-CSF origin. Cell Immunol 281, 51-61. Jones, G.E., 2000. Cellular signaling in macrophage migration and chemotaxis. J Leukoc Biol 68, 593-602. Jung, C.H., Jung, H., Shin, Y.C., Park, J.H., Jun, C.Y., Kim, H.M., Yim, H.S., Shin, M.G., Bae, H.S., Kim, S.H., Ko, S.G., 2007. Eleutherococcus senticosus extract attenuates LPS-induced iNOS expression through the inhibition of Akt and JNK pathways in murine macrophage. J Ethnopharmacol 113, 183-187. Kalke, D., 2014. Assessment report on Eleutherococcus senticosus (Rupr. et Maxim.) Maxim., radix. European Medicines Agency, https://www.ema.europa.eu/en/medicines/herbal/eleutherococci-radix, pp. 1-51. Khan I. A., Abourashed E. A., 2010. Leung’s encyclopedia of common natural ingredients, 3rd ed. John Wiley & Sons, Hoboken, New Jersey. 13
Krzyszczyk, P., Schloss, R., Palmer, A., Berthiaume, F., 2018. The role of macrophages in acute and chronic wound healing and interventions to promote pro-wound healing phenotypes. Front Physiol 9, 419. Lawrence, T., Natoli, G., 2011. Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nat Rev Immunol 11, 750-761. Li, Q., Laumonnier, Y., Syrovets, T., Simmet, T., 2007. Plasmin triggers cytokine induction in human monocyte-derived macrophages. Arterioscler Thromb Vasc Biol 27, 1383-1389. Li, T., Ferns, K., Yan, Z.Q., Yin, S.Y., Kou, J.J., Li, D., Zeng, Z., Yin, L., Wang, X., Bao, H.X., Zhou, Y.J., Li, Q.H., Zhao, Z.Y., Liu, H., Liu, S.L., 2016. Acanthopanax senticosus: Photochemistry and anticancer potential. Am J Chin Med 44, 1543-1558. Lin, Q.-Y., Jin, L.-J., Cao, Z.-H., Xu, Y.-P., 2008. Inhibition of inducible nitric oxide synthase by Acanthopanax senticosus extract in RAW264.7 macrophages. J Ethnopharm 118, 231-236. Lin, Q.Y., Jin, L.J., Ma, Y.S., Shi, M., Xu, Y.P., 2007. Acanthopanax senticosus inhibits nitric oxide production in murine macrophages in vitro and in vivo. Phytother Res 21, 879-883. Loos, C., Syrovets, T., Musyanovych, A., Mailänder, V., Landfester, K., Simmet, T., 2014. Amino-functionalized nanoparticles as inhibitors of mTOR and inducers of cell cycle arrest in leukemia cells. Biomaterials 35, 1944-1953. Lunov, O., Syrovets, T., Loos, C., Beil, J., Delacher, M., Tron, K., Nienhaus, G.U., Musyanovych, A., Mailander, V., Landfester, K., Simmet, T., 2011. Differential uptake of functionalized polystyrene nanoparticles by human macrophages and a monocytic cell line. ACS Nano 5, 1657-1669. Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A., Locati, M., 2004. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25, 677686. Murray, P.J., 2017. Macrophage polarization. Annu Rev Physiol 79, 541-566. Nishibe, S., Kinoshita, H., Takeda, H., Okano, G., 1990. Phenolic-compounds from stem bark of Acanthopanax senticosus and their pharmacological effect in chronic swimming stressed rats. Chem Pharm Bull (Tokyo) 38, 1763-1765. Panossian, A., Wikman, G., 2009. Evidence-based efficacy of adaptogens in fatigue, and molecular mechanisms related to their stress-protective activity. Curr Clin Pharmacol 4, 198219. Schmolz, M.W., Sacher, F., Aicher, B., 2001. The synthesis of Rantes, G-CSF, IL-4, IL-5, IL-6, IL-12 and IL-13 in human whole-blood cultures is modulated by an extract from Eleutherococcus senticosus L. roots. Phytother Res 15, 268-270. Shiraishi, D., Fujiwara, Y., Horlad, H., Saito, Y., Iriki, T., Tsuboki, J., Cheng, P., Nakagata, N., Mizuta, H., Bekki, H., Nakashima, Y., Oda, Y., Takeya, M., Komohara, Y., 2018. CD163 is required for protumoral activation of macrophages in human and murine sarcoma. Cancer Res 78, 3255-3266. Shrivastava, R., Asif, M., Singh, V., Dubey, P., Malik, S.A., Lone, M.U.D., Tewari, B.N., Baghel, 14
K.S., Pal, S., Nagar, G.K., Chattopadhyay, N., Bhadauria, S., 2019. M2 polarization of macrophages by Oncostatin M in hypoxic tumor microenvironment is mediated by mTORC2 and promotes tumor growth and metastasis. Cytokine 118, 130-143. Slacanin, I., Marston, A., Hostettmann, K., Guedon, D., Abbe, P., 1991. The isolation of Eleutherococcus senticosus constituents by centrifugal partition chromatography and their quantitative determination by high performance liquid chromatography. Phytochem Anal 2, 137-142. Soo Kim, H., Young Park, S., Kyoung Kim, E., Yeon Ryu, E., Hun Kim, Y., Park, G., Joon Lee, S., 2012. Acanthopanax senticosus has a heme oxygenase-1 signaling-dependent effect on Porphyromonas gingivalis lipopolysaccharide-stimulated macrophages. J Ethnopharm 142, 819-828. Syrovets, T., Jendrach, M., Rohwedder, A., Schule, A., Simmet, T., 2001. Plasmin-induced expression of cytokines and tissue factor in human monocytes involves AP-1 and IKKβmediated NF-κB activation. Blood 97, 3941-3950. Van Ginderachter, J.A., Movahedi, K., Hassanzadeh Ghassabeh, G., Meerschaut, S., Beschin, A., Raes, G., De Baetselier, P., 2006. Classical and alternative activation of mononuclear phagocytes: picking the best of both worlds for tumor promotion. Immunobiology 211, 487501. Wanidworanun, C., Strober, W., 1993. Predominant role of tumor necrosis factor-α in human monocyte IL-10 synthesis. J Immunol 151, 6853-6861. Wolff, D., Greinix, H., Lee, S.J., Gooley, T., Paczesny, S., Pavletic, S., Hakim, F., Malard, F., Jagasia, M., Lawitschka, A., Hansen, J.A., Pulanic, D., Holler, E., Dickinson, A., Weissinger, E., Edinger, M., Sarantopoulos, S., Schultz, K.R., 2018. Biomarkers in chronic graft-versushost disease: quo vadis? Bone Marrow Transplant 53, 832-837. Xue, J., Schmidt, S.V., Sander, J., Draffehn, A., Krebs, W., Quester, I., De Nardo, D., Gohel, T.D., Emde, M., Schmidleithner, L., Ganesan, H., Nino-Castro, A., Mallmann, M.R., Labzin, L., Theis, H., Kraut, M., Beyer, M., Latz, E., Freeman, T.C., Ulas, T., Schultze, J.L., 2014. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 40, 274-288.
Figure legends Fig. 1. Chemical characterization of E. senticosus root and bark extracts. (A) HPLC-DAD fingerprints of root and bark extracts at 210 nm. (B) Quantification of isolated compounds by HPLC-DAD-MS, mean, n = 3. (C) Chemical structures of compounds from E. senticosus root and bark extracts. Fig. 2. Network pharmacology-based analysis of possible E. senticosus targets in macrophages. (A) Network of intracellular pathways targeted by compounds from E. senticosus. Yellow (root) or orange (bark) nodes show the reference compounds present in high amounts. The node in the middle (cyan) depicts targeted networks. Three green nodes on the right show pathways that 15
might be affected by E. senticosus treatment. (B) Proteins predicted to be targeted by E. senticosus compounds arranged into networks by String database using Gene ontology and KEGG datasets. Fig. 3. E. senticosus root and bark extracts increase actin polymerization, migration, and phagocytosis by macrophages. (A) Macrophages were treated for 40 min and actin polymerization (F-actin) was visualized by phalloidin staining followed by microscopy and fluorimetry. Left hand panels, representative photomicrographs are shown. (B) Macrophages were treated as indicated and allowed to migrate for 18 h. Wound Healing Tool in ImageJ was used to quantify the cell-free wound area. (C) Cells were treated for 24 h and phagocytosis of fluorescent E. coli was analyzed by flow cytometry. All data are mean ± SEM, n = 3-5, *p < 0.05, **p < 0.01. Fig 4. E. senticosus root and bark extracts decrease Akt activation and CD163 expression by macrophages. (A) E. senticosus root and bark extracts do not affect expression of CD11b, CD80, and CD64 markers. (B) E. senticosus root and bark extracts (24 h treatment) reduce CD163 expression as measured by flow cytometry. Treatment with LPS (100 ng/ml)/IFN- (20 ng/ml) and IL-4 (20 ng/ml) for 24 h – positive controls. Representative histograms are shown. (C). Treatment with E. senticosus root and bark extracts (both, 30 µg/ml) for 1 h reduces basal Akt activation by phosphorylation (Ser473) in macrophages. LPS (100 ng/ml) – positive control. All data are mean ± SEM, n = 3-6, *p < 0.05, **p < 0.01, ***p < 0.001. Fig. 5. Extract from E. senticosus bark, but not from the root, induces moderate p38 MAPK and NF-κB activation and cytokine release by human macrophages. (A) Cells were treated with extracts, LPS (100 ng/ml), or IL-4 (20 ng/ml) for 24 h and TNF- and IL-10 were analyzed in supernatants by ELISA. Polymyxin B (10 µg/ml) significantly reduced the LPS-induced cytokine release, but had no effect on the cytokine release triggered by E. senticosus bark extract confirming absence of LPS contamination. (B) Cells were treated as in (A) and CCL-1 and IL6 secretion were analyzed by corresponding ELISAs. LPS (100 ng/ml) – positive control. (C) E. senticosus bark, but not root extract, activates p38 MAPK. Cells were treated for 1 h and analyzed by Western immunoblotting. (D) E. senticosus bark, but not root extract, activates NFκB. Macrophage J774 NF-κB reporter cells were treated for 24 h and secreted SEAP enzyme quantified. All data are mean ± SEM, n = 3-6, *p < 0.05, **p < 0.01, ***p < 0.001. Fig. 6. E. senticosus root and bark extracts differentially regulate the IL-4-induced CD200R expression in macrophages. (A) Extracts alone do not affect expression of CD200R. Cells were treated with extracts, LPS (100 ng/ml), or IL-4 (20 ng/ml) for 24 h and analyzed by flow 16
cytometry. (B) E. senticosus root extracts increases, whereas bark extract decreases the IL-4induced CD200R. Macrophages were treated as in A. All data are mean ± SEM, n = 3, *p < 0.05, ***p < 0.001. Credit Author Statement Lu Jin: Investigation, Formal Analysis, Conceptualization, Visualization, Writing – Original Draft, Funding Aquisition Michael Schmiech: Methodology, Data Curation, Validation, Resources Menna El Gaafary: Methodology, Validation Xinlei Zhang: Methodology, Formal Analysis Tatiana Syrovets: Conceptualization, Supervision, Data Curation, Validation, Visualization, Writing - Review and Editing, Project Administration Thomas Simmet: Conceptualization, Supervision, Writing - Review and Editing, Project Administration, Funding Acqusition
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