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Journal of Ethnopharmacology

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PG2, a botanically derived drug extracted from Astragalus membranaceus,promotes proliferation and immunosuppression of umbilical cord-derivedmesenchymal stem cells

Yu-Hua Chaoa,b,c, Kang-Hsi Wud,e, Chiao-Wen Linf,g, Shun-Fa Yangc,h, Wan-Ru Chaob,c,i,Ching-Tien Pengd,j, Han-Ping Wuk,l,⁎

a Department of Pediatrics, Chung Shan Medical University Hospital, Taichung, Taiwanb School of Medicine, Chung Shan Medical University, Taichung, Taiwanc Institute of Medicine, Chung Shan Medical University, Taichung, Taiwand Division of Pediatric Hematology-Oncology, Children's Hospital, China Medical University, Taichung, Taiwane School of Post-baccalaureate Chinese Medicine, China Medical University, Taichung, Taiwanf Institute of Oral Sciences, Chung Shan Medical University, Taichung, Taiwang Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwanh Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwani Department of Pathology, Chung Shan Medical University Hospital, Taichung, Taiwanj Department of Biotechnology and Bioinformatics, Asia University, Taichung, Taiwank Division of Pediatric General Medicine, Department of Pediatrics, Chang Gung Memorial Hospital, Taoyuan, Taiwanl College of Medicine, Chang Gung University, Taoyuan, Taiwan

A R T I C L E I N F O

Keywords:Astragalus polysaccharidesMesenchymal stem cellsPG2ProliferationImmunosuppression

A B S T R A C T

Ethnopharmacological relevance: Astragalus membranaceus is used to manage the deficiency of vital energyin traditional Chinese medicine and confirmed to have many biological functions. Mesenchymal stem cells(MSCs) possess immunosuppressive effects, and are widely used for regenerative medicine and immunedisorders.Aims of study: This study investigated the effects of Astragalus polysaccharides (APS) on umbilical cord-derived MSCs (UCMSCs), including morphology, surface marker expression, proliferation, differentiation, andin-vitro and in-vivo immunosuppressive capacities.Materials and methods: MSCs isolated from umbilical cords were used. PG2 injection, a botanically deriveddrug containing a mixture of APS, was added into the culture medium to prepare PG2-treated UCMSCs. Themorphology, surface marker expression, proliferation, and differentiation of UCMSCs were determined. The in-vitro immunosuppressive effects of UCMSCs were examined by peripheral blood mononuclear cell (PBMC)proliferation assay. The in-vivo effects were evaluated by circulatory inflammation-associated cytokine levels inmice with septic peritonitis induced by cecal ligation and puncture (CLP) operation.Results: Compared with control UCMSCs, UCMSCs had higher population doublings when exposed to PG2-containing medium (P = 0.003). The reduction rates of PBMC proliferation after phytohemagglutininstimulation increased significantly when UCMSCs were treated with PG2 (P = 0.004). The serum levels ofinflammation-associated cytokines, including TNF-α, IL-6, MCP-1, IFN-γ, and IL-1β, were significantly lower at6 h after CLP in the mice receiving PG2-treated UCMSCs.Conclusions: Our results demonstrated that PG2 can enhance UCMSC proliferation and their in-vitro and in-vivo immunosuppressive effects. Consequently, UCMSCs can be obtained in earlier passages to avoidsenescence, and sufficient cells can be acquired faster for clinical use. With stronger immunosuppressiveeffects, UCMSCs may treat immune disorders more effectively. Further studies are warranted.

http://dx.doi.org/10.1016/j.jep.2017.06.018Received 15 September 2016; Received in revised form 25 April 2017; Accepted 14 June 2017

⁎ Correspondence to: Division of Pediatric General Medicine, Department of Pediatrics, Chang Gung Memorial Hospital, No. 5, Fu-Hsin Street, Kweishan, Taoyuan, Taiwan.E-mail address: arthur1226@gmail.com (H.-P. Wu).

Journal of Ethnopharmacology 207 (2017) 184–191

Available online 23 June 20170378-8741/ © 2017 Elsevier B.V. All rights reserved.

MARK

1. Introduction

Astragalus membranaceus (Fisch.) Bunge, also called Huangqi, isone of the most popular health-promoting herbs in traditional Chinesemedicine (http://www.theplantlist.org/tpl1.1/record/ild-32156). It ishistorically used to manage the deficiency of qi (vital energy)(McKenna et al., 2002). Astragalus polysaccharides (APS), the majorbioactive components of Astragalus membranaceus, were confirmedto have a variety of important biological functions, includingimmunomodulation, antioxidant and anti-neoplastic properties, anti-inflammation, hepato-protection, hematopoietic promotion, neuro-protection, etc. (Jin et al., 2014).

Mesenchymal stem cells (MSCs), which were first isolated frombone marrow by Friedenstein et al. (Friedenstein et al., 1966), areconsidered a promising platform for cell-based therapy. However,harvesting MSCs from bone marrow involves an invasive and painfulprocedure. Umbilical cords are rich in MSCs which can be easilyisolated and cultured (Kim et al., 2004; Wang et al., 2004; Secco et al.,2008). Our previous studies demonstrated that umbilical cord-derivedMSCs (UCMSCs) could proliferate faster and better than bone marrow-derived MSCs (BMMSCs) (Wu et al., 2011; Chan et al., 2012),suggesting that umbilical cords may be considered a good source ofMSCs for clinical use. Over the last decade, clinical application of in-vitro expanded MSCs has been evolving rapidly for a variety of diseases(Chao et al., 2012; Wu et al., 2013; Trounson and McDonald, 2015).Given their immunosuppressive properties, MSCs are being investi-gated in the management of diseases associated with aberrant immunereactions (Nauta and Fibbe, 2007). In our previous studies, we foundthat UCMSCs had greater immunosuppressive effects than BMMSCsand could be used to treat patients with severe acute graft-versus-hostdiseases effectively and safely (Wu et al., 2011; Chan et al., 2012).

In-vitro passaging leads to a gradual decrease in the proliferativepotential of MSCs. During culture, the occurrence of MSC senescence,such as the decrease of immunosuppressive capacity, also has a greatimpact on the outcomes in their clinical utility (Galipeau, 2013).Astragalus membranaceus is a traditional herb commonly-used toreinforce vital energy, but whether the proliferative and immunosup-pressive capacities of MSCs can be enhanced by Astragalus membra-naceus has not been reported. Here, we aimed to investigate theinfluence of PG2 (a mixture of APS extracted from the roots ofAstragalus membranaceus) on the characteristics of MSCs, includingmorphology, surface marker expression, differentiation, proliferation,and in-vitro and in-vivo immunosuppressive capacities.

2. Materials and methods

2.1. Chemicals and reagents

PG2 injection was purchased from PhytoHealth Corporation (Taipei,Taiwan) and reconstituted with normal saline before use. Culture medium(Dulbecco's modified Eagle medium; DMEM), fetal bovine serum (FBS),and antibiotic-antimycotic were from Gibco (Gaithersburg, MD). Exceptinsulin which was from Novo Nordisk A/S (Bagsværd, Denmark), allchemicals and reagents for osteogenic and adipogenic induction wereobtained from Sigma (St Louis, MO). Reagents for immunophenotypicanalysis and cytometric bead array immunoassay were purchased fromBD Biosciences (San Jose, CA). Reagents used in peripheral bloodmononuclear cell (PBMC) proliferation assay, including RPMI-1640 andphytohemagglutinin (PHA), were from Sigma-Aldrich (St Louis, MO).And cell proliferation reagent WST-1 was from Roche Diagnostics GmbH(Mannheim, Germany).

2.2. Isolation of MSCs from umbilical cords

The study was approved by the institutional review board of ChungShan Medical University Hospital (CSMUH No: CS2-15091). With

written informed consents from parents, umbilical cords were obtainedfrom full-term infants immediately after birth. MSCs were isolated andcultured as per our previous studies (Chao et al., 2014; Wu et al.,2016). Shortly after, cord blood vessels were carefully removed toretain Wharton's jelly. Wharton's jelly was digested in 1 mg/mlcollagenase and then placed in high-glucose DMEM supplementedwith 10% FBS and antibiotic-antimycotic. Cells were incubated at 37 °Cwith 5% CO2 in a humidified atmosphere. After 48 h, the medium withsuspension of non-adhered cells was discarded, and fresh medium wasadded. Thereafter, the medium was changed twice a week. Whenreaching 80–90% confluence, cultured cells were detached withtrypsin-EDTA (Gibco, Carlsbad, CA) and replated for subculture.Cultured MSCs of passage 5 were used for further studies.

2.3. PG2, a preparation of APS

PG2 injection (purchased from PhytoHealth Corporation, Taiwan),a botanically derived drug, contains a mixture of APS extracted,isolated, and purified from the roots of Astragalus membranaceus(Chen et al., 2012; Kuo et al., 2015). The detailed information aboutPG2 injection can be found in the supplementary documents, and alsoat http://www.phytohealth.com.tw/en/product/8/product_detail.With high and consistent quality, the preparation of the rawmaterials, intermediates, and final products were in compliance withGMP requirements. The molecular weights of PG2 ranged between 20,000 and 60,000 Da, and the dominant polysaccharides were α-1,4-linked glucans with varying degrees of branching at the 6 positions ofthe backbone residues. Other polysaccharides and glycoproteins in PG2were arabinogalactans, rhamnogalacturonans, and arabinogalactanproteins (Kuo et al., 2015). Each vial of 500 mg of PG2 was preparedin sterile powder, and reconstituted with 10 ml of normal saline byshaking thoroughly until completely dissolved.

2.4. Preparation of PG2-treated UCMSCs and assessment ofproliferative capacity

To prepare PG2-treated UCMSCs, PG2 was added into the culturemedium to a final PG2 concentration of 200 mg/l. Control UCMSCswere grown in the regular medium without PG2 (i.e. DMEM supple-mented with 10% FBS and antibiotic-antimycotic). Cultured UCMSCsof 1 × 106 cells were plated in 10-cm dishes and incubated at 37 °C for72 h. Cells were then detached with trypsin-EDTA and collected. Thepopulation doubling was calculated using the following equation:population doubling = log2 (the number of cells at harvest/the numberof seeded cells).

2.5. Immunophenotypic analysis

MSCs were detached from culture dishes, and then washed and re-suspended in phosphate-buffered saline (PBS; Gibco, Gaithersburg,MD). After fixing and blocking, cells were immunolabeled withfluorescein isothiocyanate or phycoerythrin-conjugated mouse antihu-man antibodies specific to CD34, CD45, CD14, CD29, CD44, CD73,CD90, CD105, HLA-A, HLA-B, HLA-C or HLA-DR following themanufacturer's instruction. Nonspecific mouse IgG served as theisotype control. Data were analyzed by flow cytometry (FACSCalibur;BD Biosciences, San Jose, CA) with CellQuest software.

2.6. Assessment of osteogenic and adipogenic potential

To assess the differentiation potential, MSCs were plated in 60-mmdishes. Differentiation of MSCs was induced and evaluated as ourprevious studies (Chao et al., 2010, 2014; Wu et al., 2016). To induceosteogenesis, MSCs were grown in DMEM with 10% FBS, 10 mM β-glycerophosphate, 0.1 μM dexamethasone, and 0.2 mM ascorbic acid.On day 14, osteogenic differentiation was demonstrated by alkaline

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phosphatase (ALP) activity. For further quantification, 2 ml of 0.05 NNaOH in ethanol was added to each dish, and the extraction wasmeasured by spectrophotometry (Ultrospec 1100 pro; AmershamBiosciences) at 550 nm.

To promote adipogenesis, MSCs were incubated in DMEM with10% FBS, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine,0.1 mM indomethacin, and 10 μg/ml insulin. On day 14, adipogenicdifferentiation was demonstrated by intracellular accumulation of lipiddroplets stainable with oil red O. The dye content was eluted by ethanoland quantified spectrophotometrically.

2.7. PBMC proliferation assay

For use as responder cells, PBMCs were cultured in RPMI-1640 as200 μl at 5 × 105 cells/ml per well in 96-well plates (Chan et al., 2012).100 μl of 5 µg/ml PHA was added to each well as a stimulator.Suppressive effects of MSCs (PG2 treated or not) on PBMC prolifera-tion were assessed. The ratio of PBMC:MSC was 10:1. Following co-culture for 3 days, 10 μl of cell proliferation reagent WST-1 was addedto each well and absorbance was measured with a microplate reader(Molecular Devices Corporation, Sunnydale, CA) at 550 nm. To furtherevaluate the influence of PG2 on PBMC proliferation, a series of PG2concentrations in the medium was used, including 50, 100, and200 mg/l. After incubation for 3 days, WST test was performed aspreviously described.

2.8. Evaluation of inflammation-associated cytokine levels in micewith septic peritonitis induced by cecal ligation and puncture (CLP)operation

CLP operation was performed to induce septic peritonitis in mice(Hubbard et al., 2005). Six-week-old male C57BL/6 mice werepurchased from BioLASCO Taiwan Co. Before surgery, the mice wereanesthetized by intramuscular injections of 75 mg/kg ketamine and5 mg/kg xylazine. After laparotomy, the distal one half of the cecumwas ligated with a 4-0 silk tie. A single through-and-through perfora-tion was made in the ligated segment with a 21-gauge needle, and a 1-mm column of fecal material was extruded through the puncture site.The cecum was then replaced into the abdomen and the abdominalincision was closed. Immediately after operation, one million MSCs(PG2 treated or not) in 0.5 ml PBS were administered via intraper-itoneal injection. Mice in the sham group underwent the sameoperative procedure but without ligation and needle perforation ofthe cecum; they received PBS in a volume of 0.5 ml with no cells afteroperation.

All mice were sacrificed at 6 h after operation, and blood wasobtained by cardiac puncture immediately after death. To determine

circulating cytokine levels, serum was separated by centrifugation at10,000g for 10 min at 4 °C. The concentrations of tumor necrosisfactor-α (TNF-α), interleukin (IL)-6, monocyte chemotactic protein(MCP)-1, interferon-γ (IFN-γ), and IL-1β were measured by cytometricbead array immunoassay (BD CBA Mouse Soluble Protein Flex SetSystem; BD Biosciences, San Jose, CA), according to the manufac-turer's instruction. Shortly after, a mixture of capture bead reagent(50 μl) was added to a serum sample (50 μl) and incubated for 1 h atroom temperature. 50 μl of the mixed PE detection reagent was thenadded and further incubated for 1 h. After washing, the samples wereanalyzed using flow cytometry (FACSCanto; BD Biosciences, San Jose,CA) with FCAP Array™ software.

2.9. Statistical analysis

Data analysis was performed using SPSS 17.0 for Windows.Students paired t-test and t-test were used to compare the character-istics of UCMSCs with and without PG2 treatment. The influence ofPG2 on PBMC proliferation was tested by one-way ANOVA, andGames-Howell test for post hoc analysis. A value of P < 0.05 wasconsidered to be statistically significant.

3. Results

3.1. Identification of UCMSCs

First, the basic properties of MSCs, including in-vitro morphology,surface marker expression, and differentiation potential (Dominiciet al., 2006), were used as indicators to identify MSCs isolated fromumbilical cords. In-vitro, UCMSCs adhered to culture plates andshowed a spindle-shaped fibroblastic morphology. They expressedCD29, CD44, CD73, CD90, CD105, HLA-A, HLA-B, and HLA-C, andwere negative for CD34, CD45, CD14, and HLA-DR. Under inductionconditions, UCMSCs were capable of achieving osteogenic and adipo-genic differentiation. These findings were in accordance with thecriteria of the International Society for Cellular Therapy (Dominiciet al., 2006).

3.2. No alteration in morphology, surface marker expression, anddifferentiation potential after PG2 treatment

The influence of PG2 on the basic properties of UCMSCs, includingmorphology, surface marker expression, and differentiation potential,were evaluated. Both UCMSCs with and without PG2 treatment shareda similar spindle-shaped morphology in-vitro (Fig. 1) and a consistentimmunophenotypic profile which was positive for CD29, CD44, CD73,CD90, CD105, HLA-A, HLA-B, and HLA-C, and negative for CD34,

Fig. 1. Morphology. In-vitro culture, both UCMSCs with and without PG2 treatment had a similar spindle-shaped fibroblastic morphology (×100).

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Fig. 2. Differentiation potential. Under specific induction conditions, both UCMSCs with and without PG2 treatment could differentiate into osteocytes (ALP activity, ×100) andadipocytes (Oil red O stain, ×100). Further quantification by spectrophotometry revealed that PG2 treatment did not alter the capacities of UCMSCs for osteogenesis (P = 0.865) andadipogenesis (P = 0.798). n = 10. All procedures were performed in triplicate. Students paired t-test was used for statistical analysis.

Table 1Influence of PG2 treatment on UCMSCs.

UCMSCs PG2-treated UCMSCs P value

Proliferative potentialPopulation doublings 1.648 ± 0.414 1.976 ± 0.603 0.003*

Differentiation potentialOsteogenesis (alkaline phosphatase activity) 0.240 ± 0.131 0.238 ± 0.138 0.865Adipogenesis (oil red O stain) 0.796 ± 0.258 0.784 ± 0.186 0.798

Suppressive effects of UCMSCs on PBMC proliferation after PHA stimulationIncrease of PBMC proliferation 0.067 ± 0.017 0.035 ± 0.018 0.002*

Reduction rate of PBMC proliferation 0.454 ± 0.112 0.716 ± 0.159 0.004*

PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; UCMSCs, umbilical cord-derived mesenchymal stem cells.Data are presented as mean ± standard deviation.

* P < 0.05.

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CD45, CD14, and HLA-DR. Both were able to achieve osteogenic andadipogenic differentiation under specific induction conditions, asdemonstrated by ALP activity and Oil red O stain, respectively(Fig. 2). As shown in Table 1 and Fig. 2, further quantification byspectrophotometry revealed that PG2 treatment did not alter thecapacities of UCMSCs for osteogenesis and adipogenesis (P = 0.865and 0.798, respectively).

3.3. Enhancement of UCMSC proliferation after PG2 treatment

The impact of PG2 on proliferative potential of UCMSCs wasassessed. When exposed to PG2-containing medium, UCMSCs hadsignificantly higher population doublings compared with controlUCMSCs (P = 0.003), as shown in Table 1 and Fig. 3. An augmentationof greater than 10% could be found in eight of the ten, and greater than30% in three. Our results indicated that PG2 had positive effects on theproliferation of UCMSCs.

3.4. Augmentation of suppression on PBMC proliferation fromUCMSCs after PG2 treatment

We evaluated the suppressive effects of UCMSCs on PBMC pro-liferation, and compared the difference between UCMSCs with andwithout PG2 treatment (Table 1 and Fig. 4A). Consistent with ourprevious report (Chan et al., 2012), the increase of PBMC proliferationafter PHA stimulation was significantly inhibited when PBMCs co-cultured with UCMSCs (P < 0.001). When UCMSCs were treated withPG2 before co-culture, the inhibition effects were amplified signifi-cantly (P = 0.002). As shown in Fig. 4B, the reduction rates of PBMC

proliferation after PHA stimulation increased significantly whenUCMSCs were treated with PG2 (P = 0.004). Our results indicatedthat the suppressive effects of UCMSCs on PBMC proliferation could beaugmented by PG2 treatment.

The impact of PG2 on PBMC proliferation was further assessed.PBMC proliferation increased after PHA stimulation, whether PG2 wasadded into the medium or not (Fig. 5A). Compared with the regularmedium (i.e. no PG2 within the medium), adding PG2 into the mediumof 50, 100, or 200 mg/l did not significantly alter the increase of PBMCproliferation (P = 0.975). Our results implied that PG2 alone did notexhibit suppressive effects on PBMC proliferation (Fig. 5B).

3.5. Augmentation of immunosuppression on mice with septicperitonitis from UCMSCs after PG2 treatment

Fig. 6 illustrates the circulatory inflammation-associated cytokinelevels in the mice with CLP-induced septic peritonitis at 6 h afteroperation. Compared with the mice in the control group (i.e. micewithout any intervention) and sham group, CLP could induce inflam-matory reactions in the mice, as shown in an increase in the serumlevels of TNF-α, IL-6, MCP-1, IFN-γ, and IL-1β at 6 h after operation.These inflammation-associated cytokine levels were significantly lowerin the mice receiving PG2-treated UCMSCs than in those receivingcontrol UCMSCs after CLP, indicating the augmentation of immuno-suppressive capacity in UCMSCs by PG2 treatment.

4. Discussion

Clinical application of in-vitro expanded MSCs is evolving rapidlyfor a variety of diseases (Chao et al., 2012; Wu et al., 2013; Trounsonand McDonald, 2015). In clinical practice, it is an important concern toobtain sufficient cells with better functions in a shorter period of time.In the present study, we found that PG2 could enhance the proliferativeand in-vitro and in-vivo immunosuppressive capacities of UCMSCswithout alterations in their morphology, surface marker expression,and differentiation potential. To our knowledge, no other plant producthas been reported to have influenced the characteristic features ofMSC's proliferative and immunosuppressive capacities. This is the firstreport about the effects of APS on human MSCs, and our study suggeststhe useful role of PG2 in the preparation of MSCs for clinical use.

In traditional medicine, Astragalus membranaceus was historicallyused to manage the deficiency of vital energy (McKenna et al., 2002). Itis not only widely used as a Chinese medicinal herb in many Asiacountries, but also popularly used in Western countries as functionalfood and a dietary supplement. Despite its complicated constituents,modern phytochemistry has proven that APS is the main bioactiveingredient of Astragalus membranaceus (Jin et al., 2014). PG2, apurified extract of Astragalus membranaceus, is mostly composed ofAPS with consistent and high quality (Kuo et al., 2015). A double-blind,randomized controlled study demonstrated that PG2 could treatcancer-related fatigue effectively in patients with advanced cancer(Chen et al., 2012). In-vitro passaging and culturing could result inthe occurrence of MSC senescence, such as decreases in the prolif-erative and immunosuppressive capacities, and these characteristicchanges may influence the outcomes in their clinical use (Galipeau,2013). In the present study, we found that adding PG2 into the culturemedium could increase the proliferative and in-vitro and in-vivoimmunosuppressive capacities of UCMSCs, just like reinforcing vitalenergy into the MSCs.

To get sufficient cells for clinical use, in-vitro expansion of MSCs isessential. However, passaging during culture causes MSCs to graduallylose their progenitor properties (Banfi et al., 2000; Dominici et al.,2006). Therefore, it is important to acquire enough MSCs of earlierpassages in clinical applications. The present study demonstrated thatPG2-treated UCMSCs had higher population doublings. Therefore, thetime needed to acquire enough cells could be shorter, and younger cells

Fig. 3. Proliferative potential. When exposed to PG2-containing medium (PG2 concen-tration, 200 mg/l), UCMSCs had significantly higher population doublings comparedwith control UCMSCs (P = 0.003). n = 10. All procedures were performed in triplicate.Students paired t-test was used for statistical analysis.

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of earlier passages could be obtained. Our study suggested that addingPG2 into the culture medium can be useful in the preparation of MSCsfor clinical use.

MSCs interact with a variety of immune cells, and have beeninvestigated in the management of clinical diseases associated withaberrant immune responses (Nauta and Fibbe, 2007). We found thatUCMSCs are able to effectively treat patients with severe graft versushost disease, which is typical of immune-mediated host tissue damage(Wu et al., 2011). Our previous studies also demonstrated thebeneficial effects of MSCs on septic animals by exerting their immu-nosuppressive functions (Chao et al., 2014; Wu et al., 2016). Moreover,we found that UCMSCs had greater immunosuppressive effects thanBMMSCs (Wu et al., 2011; Chan et al., 2012). We speculated that usingMSCs with more potent immunosuppressive capacities could increasethe efficacy of disease control. In the present study, there was noadverse effect of PG2 treatment on PBMC proliferation which wascomparable to the findings of Zhuge et al. (2012). The increase ofPBMC proliferation was inhibited when PBMCs co-cultured withUCMSCs, and the inhibition effects were amplified when UCMSCswere treated with PG2. We speculated that PG2 could increase the

immunosuppressive capacities of UCMSCs which may contribute to theincrease of the suppressive effects on PBMC proliferation. Moreover,the circulating levels of inflammation-associated cytokines were sig-nificantly lower in the mice receiving PG2-treated UCMSCs than inthose receiving control UCMSCs after CLP-induced septic peritonitis.Our studies demonstrated that PG2 treatment increased the immuno-suppressive capacities of UCMSCs, both in-vitro and in-vivo.

5. Conclusions

Our data provide the first evidence that PG2 can enhance theproliferative and immunosuppressive capacities of UCMSCs. As acommonly-used traditional medicine that is highly safe, Astragalusmembranaceus can be useful in modern cell-based therapy. Furtherstudies are warranted.

Author contributions

Yu-Hua Chao designed the study and wrote the manuscript. Kang-Hsi Wu, Chiao-Wen Lin, and Shun-Fa Yang carried out the experi-

Fig. 4. Suppressive effects of UCMSCs on PBMC proliferation. (A) When PBMCs co-cultured with UCMSCs, the increase of PBMC proliferation after PHA stimulation was inhibitedsignificantly (P < 0.001). When UCMSCs treated with PG2 before co-culture, the inhibition effects augmented significantly (P = 0.002). (B) When UCMSCs treated with PG2 before co-culture, the reduction rates of PBMC proliferation after PHA stimulation were significantly increased (P = 0.004). n = 10. All procedures were performed in triplicate. Data are presentedas mean ± SEM. Students paired t-test was used for statistical analysis.

Fig. 5. Impact of PG2 on PBMC proliferation. (A) PBMC proliferation increased after PHA stimulation, whether PG2 was added into the medium or not. (B) Compared with the regularmedium (i.e. no PG2 within the medium), adding PG2 into the medium of 50, 100, or 200 mg/l did not make significant alteration in the increase of PBMC proliferation (P = 0.975). n =10. All procedures were performed in triplicate. Data are presented as mean ± SEM. One-way ANOVA was used for statistical analysis.

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ments. Wan-Ru Chao and Ching-Tien Peng performed the statisticalanalysis. Han-Ping Wu conceived and designed the study.

Conflict of interests

The authors declare that they have no conflict of interests.

Acknowledgments

This study was supported by the Chung Shan Medical UniversityHospital (CSH-2016-C-013); the China Medical University Hospital(DMR-105-039); the Ministry of Science and Technology (MOST 105-2314-B-040-017); the Research Laboratory of Pediatrics, Children'sHospital, China Medical University; the Taiwan Ministry of Health andWelfare Clinical Trial Center (MOHW106-TDU-B-212-113004).

Appendix A. Supplementary material

Supplementary data associated with this article can be found in theonline version at doi:10.1016/j.jep.2017.06.018.

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Fig. 6. Immunosuppressive effects of UCMSCs on the mice with CLP-induced septic peritonitis. Serum levels of inflammation-associated cytokines, including TNF-α, IL-6, MCP-1, IFN-γ, and IL-1β, were measured by cytometric bead array immunoassay at 6 h after surgery. Compared with the mice in the control group (i.e. mice without any intervention) and shamgroup (i.e. mice receiving sham operation), the serum levels of TNF-α, IL-6, MCP-1, IFN-γ, and IL-1β were significantly increased in the mice receiving CLP, suggesting that CLP couldinduce inflammatory reactions in the mice. These inflammation-associated cytokine levels were significantly lower in the mice receiving PG2-treated UCMSCs than in those receivingcontrol UCMSCs after CLP, indicating the augmentation of immunosuppressive capacity in UCMSCs after PG2 treatment. Data are presented as mean ± SEM. Students t-test was usedfor statistical analysis. n = 6–7 mice/group.

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