Research ArticleExperimental Studies

Ethanol Extract of Hedyotis diffusa Willd Affects Immune Responses in Normal Balb/c Mice In Vivo

YU-JUI KUO, JING-PIN LIN, YUNG-TING HSIAO, GUAN-LING CHOU, YU-HSIANG TSAI, SU-YIN CHIANG, JAUNG-GENG LIN and JING-GUNG CHUNG
In Vivo July 2015, 29 (4) 453-460;

Abstract

Numerous clinical anticancer drugs are obtained from natural plants and Hedyotis diffusa Willd (EEHDW) has been used as a major component in Traditional Chinese medicine formulas since a long time. Ethanol extracts of EEHDW have been shown to possess various biological activities including anticancer function in vitro. Our earlier studies have shown that EEHDW affects immune responses in WEHI-3-generated leukemia mice, but EEHDW has not been reported to affect immune responses in a normal mouse model. Herein, we investigated whether EEHDW could affect immune responses on normal murine cells in vivo. Normal BALB/c mice were orally treated with or without EEHDW at 0, 16, 32, and 64 mg/kg or 32 mg/kg by i.p. for 3 weeks, then were weighed, and blood, liver and spleen samples were collected for further experiments. Results indicated that EEHDW did not significantly affect body and liver weight but significantly increased the spleen weight by i.p. treatment when compared to control groups. Flow cytometric assays indicated that EEHDW promoted CD11b levels at 16, 32 and 64 mg/kg oral treatment, CD19 levels at 16, 32, 64 mg/kg oral treatment and i.p. treatment, and Mac-3 levels at 16, 32 and 64 mg/kg oral treatment, however, it did not significantly affect the levels of CD3. Oral treatment with 16 and 32 mg/kg of EEHDW significantly decreased macrophage phagocytosis from PBMC; 32 mg/kg of EEHDW by i.p. treatment significantly increased phagocytosis activity of macrophages obtain from the peritoneal cavity. EEHDW at 32 mg/kg by i.p. treatment led to an increase of NK cell activities compared to oil control groups. EEHDW at 32 mg/kg of EEHDW by i.p. treatment increased B- and T-cell proliferation. Based on these observations, EEHDW seems to have promoted immune responses in this murine model.

Leukemia is derived from the unregulated proliferation of immature blood cells, such as mutant hematopoietic stem cells (1). Currently, the major treatment for patients with leukemia is chemotherapy; however, it has a relatively low efficacy and high toxicity for the patient (2). Therefore, numerous studies have focused on natural products for treatment of human cancer including leukemia.

When an antigen enters the human body, several types of white blood cells are activated against it through interactions with each other (3). Major histocompatibility complex-1 (MHC-1), also named as human leukocyte antigen-1 (HLA-1), plays a central role in both adaptive and innate immunity (4). It has been reported that certain natural products can promote immune responses (5). We have reported that some crude extracts of natural plants can increase immune responses in normal or leukemia mice models (6-8). Herein, we further investigated crude extracts of natural plants to examine the effects of immune responses in vivo.

Traditional Chinese medicine (TCM) has been used to treat patients in Chinese population and keeps attracting interest as an alternative therapy for several diseases, due to minimal side-effects (9-11). Hedyotis Diffusa Willd, a traditional Chinese folk medicine widely distributed in northeast Asia, has been used for heat-clearing, detoxification, promotion of blood circulation and removal of blood stasis (12). Some of TCM formula prescriptions contain Hedyotis Diffusa Willd as a significant component to treat various types of cancers including breast and colon cancer (12-14). It has been reported that Hedyotis Diffusa Willd induces apoptosis of human colon carcinoma cells, inhibits tumor angiogenesis in vitro (15, 16) and inhibits colorectal cancer growth in vivo via inhibition of STAT3 signaling pathway (17). Furthermore, Hedyotis diffusa Willd inhibits colorectal cancer growth in vivo via inhibition of SHH-mediated tumor angiogenesis (18). We found that ethanol extract of Hedyotis diffusa Willd (EEHDW) upregulates G0/G1 phase arrest and induces apoptosis in human leukemia HL-60 cells by modulating caspase cascade signaling and altering the expression of associated genes (19).

Figure 1.
Figure 1.
Figure 1.

EEHDW effects on body, liver and spleen weights of normal BALB/c mice. Mice were randomly divided into 6 groups, Group I was treated with normal diet. Group II was treated with olive oil. Group III was treated with 16 mg/kg of EEHDW. Group IV was treated with 32 mg/kg of EEHDW. Group V was treated with 64 mg/kg of EEHDW. Group VI was treated with 32 mg/kg of EEHDW by i.p. All animals were treated for 27 days. Representative pictures of animal (A), liver and spleen (B), body (C), liver (D) and spleen (E) weights are shown. Total body weights were measured every 2 days.

We also found that EEHDW affects immune responses in leukemia mice in vivo (20). However, there exists no available information to show the effect of EEHDW on immune responses in normal mice. Therefore, in the present study, we investigated how EEHDW affects immune responses of normal BALB/c mice in vivo.

Materials and Methods

Materials and reagents. Tissue culture plastic wares were obtained from BD Bioscience (San Jose, CA, USA). Isolated cells were maintained in RPMI-1640 medium with fetal bovine serum (FBS), L-glutamine and penicillin-streptomycin (Gibco, Life Technologies, Carlsbad, CA, USA). EEHDW was kindly provided by Dr. Yu-Jui, Kuo (Department of Chinese Medicine, China Medical University, Taichung, Taiwan) and was dissolved in ddH2O and kept at −20°C in a 50 ml tube covered with aluminum paper to avoid light before use.

Male BALB/c mice. Sixty male BALB/c mice at an age of 4 weeks, weighing 22-25 g were obtained from the National Laboratory Animal Center (Taipei, Taiwan). All animals were housed in stainless steel mesh-bottomed cages that contained wooden chips and kept under specified pathogen-free conditions in the animal center of China Medical University (Taichung, Taiwan). Housing of the animals was standardized by a 12 h: 12 h dark-light cycle (dark phase: 9.00 a.m.-9.00 p.m.) at a room temperature of 22±2°C. Humidity was set at 50%. All mice were fed with clean water and normal nutrient food, under standard conditions based on international and institutional ethical guidelines (Affidavit of Approval of Animal Use Protocol) that have been reviewed and approved by the Institutional animal Care and Use Committee (IACUC) of China Medical University (Taichung, Taiwan), as described previously (20).

In vivo Treatment of animals with EEHDW. Sixty BALB/c mice were randomized into 6 groups (n=10). Group I mice received normal diet as control. Group II was treated with olive oil by intra-gastric administration. Group III was treated with 16 mg/kg of EEHDW by intra-gastric administration. Group IV was treated with 32 mg/kg of EEHDW by intra-gastric administration. Group V was treated with 64 mg/kg of EEHDW by intra-gastric administration. Group VI was treated with 32 mg/kg of EEHDW by intraperitoneal (i.p.). All animals were treated for 27 days. After 27 days all mice were weighed and sacrificed by euthanasia with CO2, as described previously (20).

Immunofluorescence staining of cell surface markers. After the treatment, all mice were individually weighed, and then blood, liver and spleen were individually collected. Splenocytes were isolated from each spleen of each mouse to measure cytotoxic activity of Natural killer (NK) cells as described previously (21). Leukocyte cell markers were measured from each blood sample from each mouse as described previously (21). Briefly, 1 ml blood sample was lysed with 1×Pharm Lyse™ lysing buffer (BD Biosciences Pharmingen Inc., San Diego, CA, USA) to destroy the red blood cells and was then centrifuged at 1500 × g for 15 min at 4°C to collect white blood cells. Isolated leukocytes were stained by PE-labeled anti-mouse CD3 and CD19, FITC-labeled anti-mouse CD11b and Mac-3 antibodies (BD Biosciences Pharmingen Inc., San Diego, CA, USA) for 30 min and were then stained with secondary antibody, before being analyzed by flow cytometry (Becton-Dickinson, San Jose, CA, USA), as previously described (20).

Measurements of macrophage phagocytosis. After treatment, the macrophages from each mouse of each group were isolated from PBMC and peritoneum as described previously (21, 22). Isolated cells were placed in 96-well plates. 50 μl of Escherichia coli-FITC were added, based on the PHAGOTEST® kit manufacturer's instructions (Orpegen, Heidelberg, Germany). Cells were then analyzed by flow cytometry. The percentage of phagocytosis was further quantified by CellQuest software (BD Biosciences Pharmingen Inc., San Diego, CA, USA) as described previously (20, 21).

Measurements of NK cell cytotoxic activity. Isolated splenocytes (1×105 cells/well) were placed in 15-ml tubes containing 1 ml of RPMI-1640 medium. YAC-1 cells (2.5×107 cells) were placed in serum-free RPMI 1640 medium and the PKH-67/Dil.C buffer (Sigma-Aldrich Corp., St Louis, MO) was added to the cells and then mixed thoroughly for 2 min at 25°C. PBS (2 ml) was added for 1 min, and then 4 ml of medium were added to the well. Cells were incubated for 10 min and then centrifuged at 1,200 rpm for 2 min at 25°C. NK cell cytotoxic activity was assayed by flow cytometry as described elsewhere (20, 21).

Measurements of T- and B-cell proliferation. Isolated splenocytes (1×105 cells/well) were placed in 96-well plates and 100 μl of RPMI-1640 medium was added to the well. Concanavalin A (Con A, 5 μg/ml) and lipopolysaccharide (LPS, 5 μg/ml) were separately added to the cells to stimulate for 3 days for T and B cell proliferation assay, respectively. All samples were assayed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) as previously described (20, 21).

Statistical analysis. All data were presented as the means±standard deviation (S.D.) of values of three independent experiments. Comparisons between control and EEHDW-treated groups were analyzed by the Student's t-test. A probability of 0.05 or less was considered statistically significant.

Results

EEHDW affected the body, liver and spleen weights of normal BALB/c mice. During experimental periods, mice, every 2 days, were individually weighed and monitored. Representative animal, liver and spleen pictures are presented in Figure 1A and B. The body, liver and spleen weights are shown in Figure 1C, D and E. These results indicated that EEHDW did not significantly affect body and liver weights (Figure 1C and D) but significantly increased the spleen weight from i.p. treatment compared to control mice, as shown in Figure 1E. All other doses of EEHDW-treated groups did not show any significant effects.

EEHDW affected cell markers of white blood cells from normal BALB/c mice. At the end of incubation, blood samples were collected from each mouse and then levels of cell markers CD3, CD11b, CD19 and Mac-3 were measured by flow cytometry. The results are shown in Figure 2A, B, C and D. Results indicated that EEHDW promoted CD11b (Figure 2B) levels at 16, 32 and 64 mg/kg oral treatment, CD19 (Figure 2C) levels at 16, 32 and 64 mg/kg oral treatment and 32 mg/kg i.p. treatment, and Mac-3 (Figure 2D) levels at 16, 32 and 64 mg/kg oral treatment, however, it did not significantly affect the levels of CD3 (Figure 2A).

EEHDW affected macrophage phagocytosis from PBMC and peritoneal cavity of normal BALB/c mice. At the end of treatment, cells were isolated from PBMC and the peritoneal cavity of each animal and then levels of phagocytosis were measured by flow cytometry. Results are shown in Figure 3. EEHDW treatment at all oral treatment groups did not significantly affect macrophage phagocytosis from peritoneal cavity. However, i.p. treatment with 32 mg/kg EEHDW significantly increased macrophage phagocytosis from the peritoneal cavity. EEHDW treatment at 16 and 32 mg/kg oral treatment groups significantly increased macrophage phagocytosis from PBMC.

EEHDW affected the cytotoxic activity of NK cells from normal BALB/c mice. In order to investigate how EEHDW affected the activities of NK cell activities, isolated splenocytes were incubated with YAC-1 target cells and were then assayed by flow cytometry. Results shown in Figure 4 indicated that i.p. EEHDW-treated cells exhibited a higher NK cell activity compared to the control groups.

EEHDW affected T- and B-cell proliferation from normal BALB/c mice. In order to investigate the effects of EEHDW on T- and B-cell proliferation, isolated splenocytes were pretreated with Con A or LPS and were then assayed. Results shown in Figure 5A and B indicate that EEHDW treatment decreased T-cell proliferation at oral treatment (Figure 5B). B cell proliferation was increased following 16 mg/kg of oral treatment and 32 mg/kg of EEHDW i.p. treatment; other doses did not have a significant effect (Figure 5A).

Discussion

It is well-documented that many clinically-used anticancer drugs are obtained from natural plants such as paclitaxel from Taxus brevifolia, vinblastine (from Catharanthus roseus), and camptothectin (from Camptotheca acuminata) (22). Researchers are constantly studying more extracts of natural plants. Since a long time, Traditional Medicine was being used as alternative and complementary approach for cancer therapy (23). EEHDW has been used as traditional Chinese herbal medicine since long ago and our earlier studies showed that EEHDW suppressed proliferation of A549, H1355, HL-60, WEHI-3, and B16F10 cells, as well as reduced cell viability in a concentration-dependent manner. We also found that EEHDW triggered an arrest of HL-60 cells at G0/G1 phase and induced the formation of a sub-G1 population (apoptotic cells) in Hl-60 cells (19). Although we have shown that EEHDW affects immune responses on WEHI-3-induced leukemia mice in vivo (19), there were no available and reliable data regarding the effects of EEHDW on normal human patients or a normal animal model in vivo. Thus, in the present study, we investigated the effects of EEHDW on immune responses in normal BALB/c mice in vivo.

Mice were treated with or without EEHDW at various doses for 27 days and results indicated that EEHDW at all doses did not significantly affect body, liver and spleen weights when compared to untreated groups of mice (Figure 1). EEHDW did not induce any cytotoxic effects on normal animals. This is in agreement with our earlier studies, where EEHDW extract did not significantly affect body weight but significantly reduced the weights of spleen and liver in WEHI-3 leukemic mice (19).

Figure 2.
Figure 2.

EEHDW effects on the levels of cell markers in white blood cells from normal BALB/c mice. A: CD3; B: CD11b; C: CD19; D: Mac-3. Data are expressed as the mean±S.D. of three experiments (n=10). *p<0.05; significant difference between Group II and EEHDW-treated groups.

Results from Figure 2A, B, C and D indicate that EEHDW promoted CD19 (Figure 2B) levels at oral treatment group, CD11b (Figure 2C) levels at oral treatment group and i.p. treatment group, and Mac-3 (Figure 2D) levels at oral treatment group, however, it did not significantly affect the levels of CD3 (Figure 2A). These findings require further investigation. However, there is a slight difference with our earlier studies in WEHI-3 leukemia mice, where EEHDW had increased the percentage of CD11b cell surface marker (monocytes) and it reduced the percentage of CD3 (T-cell) and CD19 (B-cell) markers, but did not affect the level of Mac-3 (19).

Figure 3.
Figure 3.

EEHDW effects on macrophage phagocytosis from PBMC and peritoneal cavity of normal BALB/c mice. Blood samples were individually collected from EEHDW-treated and untreated animals and macrophages were isolated from PBMC and the peritoneum of each mouse. Isolated macrophages were assayed for phagocytosis by flow cytometry and quantified by CellQuest as described in Materials and Methods. *p<0.05; significant difference between Group II and EEHDW-treated groups.

Figure 4.
Figure 4.

EEHDW effects on the cytotoxic activity of NK cells in normal BALB/c mice. Isolated splenocytes (1×105 cells/well) from each animal were placed in 1 ml of RPMI-1640 medium in 96-well plates. Target YAC-1 cells (2.5×107 cells) and the PKH-67/Dil.C buffer was added to the cells for the measurement of the NK cell cytotoxic activity by flow cytometry, as described in Materials and Methods. *p<0.05; significant difference between Group II and EEHDW-treated groups.

Figure 5.
Figure 5.

EEHDW effects on T- and B-cell proliferation in normal BALB/c mice. Isolated B-cells were pre-treated with LPS and cell proliferation was analyzed by flow cytometry (A). T-cells were pre-treated with Con A and cell proliferation was analyzed by flow cytometry (B), as described in Materials and Methods. *p<0.05; significant difference between Group II and EEHDW -treated groups.

T-cells also play an important role against foreign antigen, such as T cytotoxic cells, and T helper cells can promote B-cells for producing antibody to attached antigen (24). Furthermore, macrophages can inhibit intracellular bacterial growth and can also destroy the infection in vivo, through phagocytosis (25). This is in agreement with our earlier studies, where EEHDW had no influence on phagocytosis by macrophages from peripheral blood mononuclear cells and the peritoneal cavity in leukemic mice (19).

To further confirm whether or not EEHDW affects the activities of NK cell activities, YAC-1 target cells were added to the isolated splenocytes and were then incubated and assayed by flow cytometry. Results indicated that EEHDW-treated cells had lower NK cell activities compared to control groups. Figures 5A and B showed that EEHDW treatment decreased T (Figure 5B) and B (Figure 5A) cell proliferation. However, our earlier studies indicated that isolated splenocytes from EEHDW extract-treated leukemic mice demonstrated an increase of T and B cell proliferation following i.p. treatment in vivo (19).

In conclusion, the present study indicated that EEHDW did not significantly change body, liver and spleen weights in normal BALB/c mice in vivo. Leukocyte population assay indicated that EEHDW promoted cellular population of CD19 (B cells), increased CD11b (monocytes) and Mac-3 (macrophages) but did not significantly affect CD3 (T-cells) population in normal mice. Furthermore, EEHDW did not increase macrophage phagocytosis and NK cell activity in BALB/c mice in vivo.

Acknowledgements

This work was supported by grant CMU102-ASIA-20 from China Medical University, Taichung, Taiwan.

  • Received December 2, 2014.
  • Revision received May 15, 2015.
  • Accepted May 22, 2015.

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Ethanol Extract of Hedyotis diffusa Willd Affects Immune Responses in Normal Balb/c Mice In Vivo
YU-JUI KUO, JING-PIN LIN, YUNG-TING HSIAO, GUAN-LING CHOU, YU-HSIANG TSAI, SU-YIN CHIANG, JAUNG-GENG LIN, JING-GUNG CHUNG
In Vivo Jul 2015, 29 (4) 453-460;
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