TM was obtained by submerged fermentation (16) and administered directly to experimental mice.

A total of 72 six-week BALB/c mice (18–22 g; 1:1 male:female; SCXK (JI)-2014-0003; Changchun Yisi Experimental Animal Technology Co., Ltd., Changchun, China) were housed at 20°C with a 12 h light/dark cycle and fed autoclaved standard chow and water ad libitum.

The experimental protocol was approved by the Animal Ethics Committee of Jilin University (Guangdong, China). A total of 60 mice were intraperitoneally injected with 80 mg/kg CTX for 3 days. Following confirmation of immunosuppression, the CTX-injected mice were separated randomly into 5 groups (n=12), and treated with saline (CTX mice) and 0.3, 1.0 and 2.0 g/kg TM (TM + CTX mice) orally once a day for 2 weeks. The positive control mice were intraperitoneally injected with 0.16 mg/kg thymosin α1 (Tα1 + CTX group) twice a week for 2 weeks. Another 12 mice were treated with normal saline during the whole experimental process and served as control (CTRL). All mice were weighed at day 0, 3, 10 and 17, and sacrificed at the end of the experiment by injection with 200 mg/kg pentobarbital. The spleen and thymus were extracted and weighed, and the organ index calculated.

Following 2 week's treatment, mice were sensitized with 0.2 ml of 2% (v/v) sheep red blood cells (SRBC; Yuanye Bio-Technology Co., Ltd., Shanghai, China) for 4 days via intraperitoneal injection, and then the thickness of hind paw was measured using a Vernier caliper. Previously sensitized mice were again challenged with 20 µl 20% (v/v) SRBC which was injected into the measured hind paw. The thickness of the hind paw was recorded at 24 and 48 h. The swelling of the hind paw was calculated as the different thickness prior to and following 20% SRBC injection.

Spleen cell suspension (1×10⁶; 100 µl) from each mouse, was produced as in a previous study (17), was seeded into a 96-well cell culture plate with 1×10⁵ of YAC-1 cells (cat. no. TCM28; Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) (effector-to-target ratio 10:1). For a control group, 100 µl RPMI-1640 medium was used in place of the spleen cell suspension. The cells were incubated under a humidified atmosphere containing 5/95% CO₂/air at 37°C for 4 h. Following centrifugation at 150 × g for 10 min at room temperature, the lactate dehydrogenase concentration in medium was detected using an in vitro Toxicology Assay kit (cat. no. TOX1; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's protocols. The killing rate of NK cells vs. YAC-1 cells was calculated as:

Where OD is optical density; OD_e+t is the average OD₄₉₀ of six wells for NK and YAC-1 cells; OD_e is the average OD₄₉₀ of six wells for NK effector control; and OD_t is the average OD₄₉₀ of six wells for YAC-1 target control (18).

Spleen cells (1×10⁶/100 µl) from each mouse were seeded into a 96-well plate and mixed with 10 µl RPMI1640 (control group) or 10 µl 200 µg/ml concanavalin (ConA; Yuanye Bio-Technology Co., Ltd.), and then incubated under a humidified atmosphere containing 5%/95% CO₂/air at 37°C for 48 h. As in a previous study (19), 3- (4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma-Aldrich; Merck KGaA) analysis was performed to detect cell viability. The stimulation index (SI) of T lymphocyte transformation was calculated as: SI=OD_test/OD_control.

Where OD_test is the average OD₄₉₀ of three wells for T lymphocytes and ConA and OD_control is the average OD₄₉₀ of three wells for T lymphocytes only.

Following the last treatment, blood was sampled from the caudal vein and the serum levels of immunoglobulin (Ig)G (cat. no. 40157) and IgA (cat. no. 40156), cytokines interleukin (IL)-2 (cat. no. 42903), IL-6 (cat. no. 42899), IL-10 (cat. no. 42912) and 12 (cat. no. 42910), interferon (IFN)-α (cat. no. 43102) and IFN-γ (cat. no. 42918), tumor necrosis factor (TNF)-α (cat. no. 42868)] and NF-κB (cat. no. 43059) were investigated via ELISA using commercial kits (Yuanye Bio-Technology Co., Ltd) according to the manufacturer's protocol.

Collected spleen samples were homogenized with RIPA buffer containing protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride (all from Sigma-Aldrich; Merck KGaA). Following determination of the total protein concentration, 30 µg protein was separated with 10% SDS-PAGE and then transferred onto a nitrocellulose membrane (0.45 µm, Bio Basic, Inc., Markham, ON, Canada). The transferred membrane was blocked through being incubated with 5% bull serum albumin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 3 h at room temperature, and then incubated with primary antibodies phosphorylated (P)-inhibitor of NF-κB kinase (IKK) α/β (ab195907; Abcam, Cambridge, UK), total (T)-IKKα/β (ab178870; Abcam) and glyceraldehyde-3-phosphate dehydrogenase (ab8245; Abcam) all at 1:1,000 dilution at 4°C overnight. The membranes were then treated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (sc-3836; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) diluted 1:2,000 (IH-0011; Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). Bands were visualized with an ECL detector (GE Healthcare Life Sciences, Chalfont, UK) and quantified by ImageJ software version 1.48 (National Institutes of Health, Bethesda, MD, USA).

All values were expressed as the mean ± standard error. A one-way analysis of variance was used to detect statistical significance followed by Dunn's test using SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Suppressed growth rate, low organ indexes of thymus and spleen, and a high degree of hind paw swelling were observed in CTX mice (P<0.01; Table I). Comparatively, Tα1 and TM improved gain in body weight, enhanced thymus and spleen indexes, and suppressed the swelling of the hind paw (P<0.05; Table I).

The cytotoxicity of NK cells, the first defense against tumor cells, was investigated. Compared with CTX mice, TM enhanced the activity of NK cells ≤14.2% (P<0.05; Fig. 1A); meanwhile, Tα1 enhanced the activity of NK cells to 14.8% (P<0.05; Fig. 1A).

The lymphocyte proliferative response is responsible for immune function. Compared with CTX mice, TM and Tα1 enhanced the stimulation index by ~20% following 2 weeks of treatment (P<0.05; Fig. 1B).

Ig is responsible for identification and neutralization of foreign substances (20). CTX injection significantly suppressed IgG and IgA levels, which had been successfully enhanced by 2 weeks of TM treatment (P<0.05; Fig. 2). Unlike TM, Tα1 upregulated only IgA levels in CTX mice (P<0.05; Fig. 2A).

IL serves important roles in immune function by mediating the activation and proliferation of immune cells (21). In CTX mice, low levels of IL-2 and IL-12 and high levels of IL-6 and IL-10 were noted (P<0.05; Fig. 3). TM reversed these abnormal changes (P<0.05, Fig. 3). Unlike TM, Tα1 demonstrated no significant effect on IL-10 level (P<0.05, Fig. 3C).

IFN is associated with immunostimulatory and immunomodulatory effects. CTX resulted in a significant reduction on serum levels of IFN-α and IFN-γ, and an increase of TNF-α level (P<0.05; Fig. 4A-C). Tα1 reversed these abnormal changes in immunosuppressed mice with the exception of IFN-α (P<0.05, Fig. 4B and C). Compared with CTX mice, TM enhanced by 55.4 and 20.9% respectively serum levels of IFN-α (P<0.001, Fig. 4A) and IFN-γ (P<0.01, Fig. 4B), and reduced TNF-α level by 15.9% (P<0.05, Fig. 4C).

Due to the central function served by NF-κB during inflammation development, the effects of TM on the NF-κB signaling pathway were investigated. TM significantly reduced the activities of NF-κB in serum compared with CTX mice (P<0.05; Fig. 5A). Furthermore, TM strongly reduced the activations of NF-κB and IKKα/β in the spleens of CTX-induced immunosuppressed mice (P<0.05; Fig. 5B). Similarly, Tα1 demonstrated the same regulatory effects as TM (P<0.05, Fig. 5B).