Introduction
Ulmus macrocarpa Hance is a deciduous tree, mainly distributed in humid areas and endemic to the Far East Asia [10]. Extracts from the dried bark of U. macrocarpa are known to act against edema, mastitis, gastric cancer and inflammation in traditional oriental medicine [22]. Moreover, the bark extracts of U. macrocarpa has been used for food industry in Korea. However, a number of scientific researches about the extracts of U. macrocarpa are not so much. So far, extracts of U. macrocarpa have an efficacy of anti-oxidative activity, anti-inflammatory activity, anti-platelet activity and anti-protozoal efficacy [22]. Ingredients of bark or root extract has not defined all of them. A few compounds such as catechin and coumarin in the roots of U. macrocarpa were reported [11]. Although there have been studies on the anti-inflammatory effect and anti-oxidant activity of U. macrocarpa [17], effects about cell survival of a stem cortex extract of U. macrocarpa has not been explored. Plant extract studies have a benefit as a swift screening method of active compound from useful nature products like development of cell-based tonic materials [7, 16]. In our previous research for anti-immuno-senescence, U. macrocarpa water extract (UMWE) prolonged the splenocyte life span in general cell culture condition. It allowed us to investigate signal factors on cell longevity. The signalling pathways which are targets of phytochemicals include phosphatidylinositol-3 kinase (PI3K) [19], and mitogen-activated protein kinase (MAPK) [18]. MAPKs are members of distinct signalling cascades in the cell and serve as focal points in response to a variety of extracellular stimuli [2, 14]. In present study, we investigated the survival rate of splenocytes with UMWE comparable to normal splenocyte and the activation of signal mediators in splenocytes.
Materials and Methods
Splenocyte preparation
Male Balb/c mice were purchased from Samtako, Inc. (Osan, Korea). Mice used in all experiments were 12 weeks old. These mice were housed in a specific pathogen-free facility with appropriate temperature and humidity, and allowed free access to food and water. The mice for this study (DEU-R2013-002) were approved by the Institutional Animal Care and Use Committee at Dong-eui University. The spleens from mice were minced and further processed for splenocytes with the RBC lysis. The RBC lysis buffer was purchased from the biolegend Inc. (CA, USA) and the splenocyte was cultured in RPMI 1640 media. The splenocytes were measured using hemacytometer.
Preparation of U. macrocarpa water extract
U. macrocarpa Hance was purchased from Dae Han Herbal Medicine Inc. (Busan, Korea) and UMWE were prepared at Bio Port Korea (Busan, Korea). Briefly, 3 kg dried material was added into 10 L water, and extracts obtained by heating for 3 h at 80℃ in a water bath and repeating twice. The extracts were filtered and concentrated under reduced pressure at below 45℃ using a rotary vacuum evaporator (Eyela, Japan). The concentrates were dried using a freeze-dryer at -80℃, and then water extracts of about 320 g were obtained.
Hoechst33342 staining
The splenocytes (1×106) isolated from 12 week balb/c mouse were seeded into 24-well plates, and then treated with vehicle, UMWE (100 μg/ml) for 24 hr, 48 hr, 72 hr and 96 hr. Then, cells were stained with 2 μl of 20 μg/ml Hoechst 33342 (Sigma) for 30 min in the dark. After washing, cells were diluted to seed onto cytospin slide glass, then observed by fluorescence microscopy using appropriate filters for blue fluorescence (ESSEN BioScience. Inc., USA). Images of the cells were captured and then processed using Adobe Photoshop software version 7.0 (Adobe Systems, Inc., San Jose, CA, USA).
Western blotting
Balb/c splenocytes (1×107cell/ml) were treated with or without 100 μg/ml UMWE. After incubation, total cell extracts were lysed with an ice-cold lysis buffer consisting of 10 mM Tris-HCl (pH 7.4), 5 mM NaF, 1 mM Na3VO4, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA). Proteins in total cell extracts were separated with 10% SDS-PAGE and transferred to nitrocellulose transfer membranes (Whatman GmbH, Dassel, Germany). The membranes were blocked with 5% skim milk in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.05% Tween 20 (TBST) for 1 hr, washed, and then incubated with primary antibody (diluted 1/1,000 in 5% skim milk in TBST) overnight at 4℃. The membranes were then incubated for 1 hr with HRP-conjugated anti-mouse IgG or anti-rabbit IgG antibody (diluted 1/2,000 in 5% skim milk in TBST) and immunoreactive bands were visualized by an ECL method (Amersham, Little Chalfont, UK). β-actin was used as internal control to normalize gel loading.
Measurement of cytokine levels and immunoglobulins
Splenocytes (2×107 cell/ml) were seed into 96 well plate with or without 100 μg/ml UMWE for 48 h and 96 h. Levels of total interleukin (IL)-2, IL-4, IL-12, and interferon- γ (IFN-γ) in cell culture media were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences Pharmingen, CA, USA). The cytokines were measured according to the manufacturer’s instructions, and the concentrations of these cytokines were recalculated from the standard curves, respectively. Immunoglobulin levels were also measured using commercially available ELISA kits (Immunology Consultants Laboratory, Inc., OR, USA).
Statistical analysis
For statistical analysis, three independent experiments were carried out. The p-values were determined through one-way ANOVA with less than 0.01 considered to be statistically significant. The p-values are represented as an asterisk (*) or #.
Results and Discussions
UMWE improved the survival rate of splenocytes in general cell culture condition
Tumor cells or self-renewal cells survive in vitro cell culture condition. Those cells have proliferative ability within in vitro culture condition using general cell culture media. However, natural cell apoptosis occur in cell culture media in case of general primary cell. In other words, splenocytes isolated from spleen in complete cell culture media also start to die out. In our previous research, we found that UMWE was able to prolong cell life span via Incucyte analysis using general cell culture media. Thus, in order to confirm delayed natural cell apoptosis of UMWE, live cells were stained with Hoechst dye at various time intervals. The viability of splenocytes with 100 μg /ml UMWE was observed under fluoroscence microscope for 24 hr (Fig. 1A), 48 hr (Fig. 1B), 72 hr (Fig. 1C), and 96 hr (Fig. 1D) by Hoechst 33342 stain. Splenocyte numbers were gradually decreased by time dependent manner in natural cell culture condition and UMWE-treated culture condition. However, the number of splenocytes was enhanced by the UMWE at a dose of 100 μg/ml for 24 hr, 48 hr, 72 hr and 96 hr compared to that of control at each time (Fig. 1). Furthermore, UMWE-treated cells showed a healthy morphology via phase contrast image and a longer-lasting survival rate than the group with no treatment (Fig. 1). This result suggested that active compounds related to cell viability were contained in the UMWE and led us to perform further study on prolonged splenocyte.
Fig. 1.UMWE augment survival rate in live splenocyte staining by Hoechst 33342. Splenocytes were treated with media (negative control), 100 μg/ml UMWE for 24 hr, 48 hr, 72 hr and 96 hr. The cells were fixed, stained with Hoechst 33342 and observed under phase contrast and fluorescent microscope at 40X. Representative images of cells after treatment for 24 hr, 48 hr, 72 hr and 96 hr. Live cell morphology was confirmed by merge between phase contrast and Hoechst 33342 image.
UMWE activated various signalling pathways such as PI3K, ERK, and Bcl-2 to improve the cell survival
Cell survival requires the activity inhibition of natural apoptosis. To confirm the UMWE and signal mechanism for inhibition of time-mediated apoptotic cell death in general cell culture condition, the expression levels of survival and apoptotic marker proteins were estimated by Western blot analysis. The activation of PI3K is important to improve the cell survival and prevent the apoptosis [1]. As shown in Fig. 3, PI3K phosphorylation was slightly elevated at 48 hr and 96 hr in UMWE-treated cells. The MAPKs are an essential part of signal transduction machinery involved in the gene expression associated with the regulation of cell survival [9]. The ERK signalling pathway, also known as the p42/p44 MAPK pathway, is a major determinant of cell survival. Thus, to estimate the level of ERK1/2 phosphorylation of UMWE, cells were stimulated with 100 μg/ml UMWE. 100ug /ml UMWE was shown to cause increases in the level of ERK1/2 phosphorylation used at 48 hr and 96 hr (Fig. 2). ERK1/2 is usually associated with pro-survival signalling [21], the up-regulation of the anti-apoptotic protein Bcl-2, and non-transcriptional inhibition of Bcl-xL/Bcl-2-associated death promoter [23]. To determine the mechanism involved, we examined the effect of the UMWE on Bcl-2 anti-apoptotic signaling molecule. Bcl-2 protein level in splenocytes with UMWE compared to the pro-survival protein Bcl-2 under resting splenocytes were elevated at 48 hr and 96 hr (Fig. 2). Thus, UMWE via MAPK-related signalling may regulate the prevention of apoptosis and promotion of cell survival.
Fig. 2.Effect of UMWE on cell survival signaling pathway. Splenocytes (2×107 cell/ml) were cultured with 100 μg/ml UMWE for 42 hr and 96 hr and whole cell lysates were prepared. The phospho- or total protein levels of PI3K, ERK and Bcl-2 were analyzed by immunoblotting analysis. β-actin was used as internal control to normalize gel loading.
Fig. 3.Effect of UMWE on induction of pro-apoptosis related proteins. Splenocytes (2×107 cell/ml) treated with 100 μg/ml for 42 hr and 96 hr were introduced to prepare whole cell lysates and total and active form levels of caspase 3and ICAD. β-actin were then detected by immunoblotting analysis.
UMWE activated various signalling pathways such as caspase 3 and ICAD
Among the numerous proteins and genes involved, members of the Bcl-2 and caspase play important roles in inhibiting apoptosis [8]. In view of the roles of caspase and Bcl-2 in the apoptotic pathway, we tried to examine the expression of the key caspase in UMWE-treated cells. It is well known that in caspase family, caspase-3 plays the central role. Western blot assay was used to detect protein level of the caspase-3. Splenocytes were cultured with UMWE (100 μg/ml) for 42 hr and 96 hr, then the cell lysate was used. In natural cell culture condition at 48 hr and 96 hr, the caspase-3 level was maintained to the control level. However, UMWE decreased caspase-3 level at 48 hr and 96 hr (Fig. 3). In fact, caspase-3 is responsible for ICAD cleavage, with this nuclease giving rise to the typical apoptotic nuclei [6]. Then, the expression of ICAD protein was further examined by Western blot. ICAD protein increased at 48 hr culturing time (Fig. 3). Together, these observations indicated that UMWE promoted anti-apoptosis in splenocyte involving caspase-3 reduced activation and weak cleavage of its substrate ICAD.
UMWE increased the expression level of hematopoietin cytokine family in cell culture supernatant
Throughout life, lymphocytes are maintained at fairly stable numbers by various homeostatic mechanisms. These mechanisms are mainly governed by cytokines. These signals do not induce proliferation but instead allow the cells to survive for prolonged periods in a quiescent state. Of several cytokine families, hematopoietin cytokine family plays a role to maintain lymphocyte homeostsis. To evaluate the effects of UMWE on soluble hematopoietin cytokine production (IL-2 and IL-4) in cell culture media, the cytokine levels were measured by ELISA. UMWE reduced the cytokines IL-2 at 96 hr (p<0.0001) but increased IL-4 at 96 hr compared with control in UMWE-treated-cells (Fig. 4). T cell proliferation and survival are regulated by cytokines [5]. IL-2, IL-7, and IL-15 are particularly critical for T cell homeostasis. IL-7 is important for T lymphopoiesis and promotes survival of naive, activated, and memory T cells [12], while IL-15 selectively promotes proliferative renewal of activated/memory CD8+ T cells [20]. The role of IL-2 is more complex. IL-2- develop T lympho-proliferation because IL-2 is required for survival of T regulatory cells that limit conventional T cell proliferation [13]. However, increased levels of IL-2 can also stimulate conventional T cells [4]. All of these cytokines bind to receptors that contain cytokine receptor common γ-chain (γc) 3 [15]. IL-4, another γ-chain-associated cytokine, has a direct activating effect that potently promotes proliferation and survival of T lymphocyte. IL-2 is Th1-type cytokine and IL-4 is Th2-type cytokine. It suggests that UMWE can extend splenocyte life span and facilitate Th2 immune response for humoral immunity.
Fig. 4.Effect of UMWE on the production of hematopoietin cytokine. After 42 hr and 96 hr, IL-2 and IL-4 cytokine levels in cell culture media were measured using ELISA assay. 100 μg/ml UMWE increased the expression level of IL-4 cytokines related to B and T cell immunity. The levels of IL-2 were slightly decreased by UMWE. P values are indicated by ***p<0.0001 compared to the control.
UMWE increased the expression level of other cytokine family IL-12 and IFN-γ in cell culture supernatant
We investigated whether UMWE could produce other cytokine family IL-12 and IFN-γ during cell cultivation. Increased IFN-γ levels were verified in the supernatant of UMWE-treated cells in all periods (48 hr and 96 hr). However, higher levels of IFN-γ were observed in late period (p<0.0001) (Fig. 5). Increased patterns in the production of IL-12 cytokine occurred as compared with control after 48 and 96 hr in UMWE-treated-cell cultures (Fig. 5). In the innate immune response, macrophages are activated by IFN-γ made by NK cells and in turn produce the cytokine IL-12 [3]. This binds to IL-12 receptors on the NK cells, including further secretion of IFN-γ and maintenance of macrophage activation. In an adaptive immune response, IL-12 secreted by activated macrophages acts on activated Th1 cells, including their differentiation into IFN-γ secreting Th1 cells, which interact with the macrophage to strengthen its activation. CD8 cytotoxic T cells are also responsive to the IL-12 produced by the macrophage and produce more IFN-γ. Thus, our results suggest that UMWE has a sufficient role to cause mutual activation of macrophages and effector lymphocytes in the innate and adaptive immune responses to intracellular stimulation.
Fig. 5.Effect of UMWE on the production of IL-12 and IFN-γ family. After 42 hr and 96 hr, IL-12 and IFN-γ cytokine levels in cell culture media were measured using ELISA assay. 100 μg/ml UMWE increased the expression level of IL-12 and IFN-γ cytokines. P values are indicated by ***p<0.0001 compared to the control.
In conclusion, our results suggest that the effect of UMWE on splenocytes possess its ability to increase cell longevity against loss of immune cells such as an immune-senescence condition and UMWE contains potent components that could be used to modulate immune cell homeostasis in the manner required by these therapies.