Purified MCP (purity ≥90%) was obtained from Chenguang Biotech Group Co., Ltd. Methyl thiazole blue (MTT), trypan blue, RPMI-1640 medium, DMSO, trimethylol aminomethane, PBS and chloroform were obtained from Beijing Solarbio Science & Technology Co., Ltd. PrimerScript™ RT reagent Kit and TB Green™ Premix Ex Taq™ II (Tli RNase H Plus) kits were purchased from Takara Biotechnology Co., Ltd.

In total, 60 Specific pathogen-free BALB/c male mice (weighing 20±2 g; 6-8-week-old) were brought from the Academy of Life Sciences, Jinzhou Medical University (Jinzhou, China). Housed at a constant temperature of 25±2˚C and relative humidity of 50-70%, the mice were fed with food and water, and were permitted to feed themselves in a standard environment with a 12-h light/dark cycle. They were subjected to adaptive feeding for 1 week. These experiments were approved by the Ethics Committee of Jinzhou Medical University [Production License SCXY (Liao) 2019-0003; experimental animal ethics approval no. 2019014].

A total of six groups of animals were prepared by randomly choosing 10 healthy mice for each group, namely the normal (NG), the model (MG), the positive (PG), the MCP low-dose (MLG), the MCP medium-dose (MMG) and the MCP high-dose (MHG) groups.

The animal protocols in the present study were identical to those performed in previous studies (9-19). Briefly, each group of mice was given an intragastric administration via gavage once a day for 21 days according to the following experimental design: From days 1 to 5, the mice all groups except for NG were intraperitoneally injected with 80 mg/kg cyclophosphamide (CY; by Jiangsu Hengrui Pharmaceutical Co., Ltd.), whereas NG and MG mice were given distilled water by intraperitoneal injection. From day 1, PG mice were given 200 mg/kg levamisole whereas the MLG, MMG and MHG mice were given MCP at different doses (100, 200 and 300 mg/kg, respectively; 0.1 ml/10 g per day). During the feeding process, the activity of the mice was observed daily. The body weight of the mice was also measured every day and the doses of drugs administered were adjusted according to changes in body weight.

In the procedure used to determine the DTH, a sensitizer, such as DNFB, is injected into the abdominal cavity of mice before the ears of the mice are insulted with DNFB. Subsequently, the degree of swelling of the ears is measured. A total of five mice from each of the six different groups were treated for 7 days as follows: Before administration on the first day, the abdominal regions of mice in each group, except for those in the NG group, were depilated and 1% DNFB (Shanghai Jizhi Biochemical Technology Co., Ltd.)/acetone/olive oil solution was applied to the depilated region for sensitization. The next day, intensive application of the same solution was performed again On day 6 of the sensitization test, 20 µl DNFB/acetone/olive oil solution was used to sensitize the mice behind and in front of the right ear. After 24 h, the mice were sacrificed by dislocation of the cervical vertebrae before two ears of each mouse were removed using a puncher and weighed. DTH was expressed as the weight difference between the right and left ears.

After the mice had been sacrificed by cervical dislocation, their thymus and spleen were also dissected and rinsed in 0.9% normal saline. The surfaces of the thymus and spleen were dried with absorbent paper, after which their weights were measured using an analytical balance. The thymus index (TI) and spleen index (SI) were calculated (9) according to the following equations: TI=mean weight of thymus in the group/mean body weight in the group x10; SI=mean weight of spleen in the group/mean body weight in the group x10.

The spleens and thymuses of the mice were dissected, washed with normal saline, fixed with neutral formalin fixative before paraffin sections were prepared. Tissues were then dehydrated, made transparent and wax-soaked. Before sealing, slices were embedded, made sticky and then baked, dewaxed, immersed in alcohol, dyed and dehydrated. Pathological sections were prepared and structural changes in each tissue were observed as previously reported (10). Specific details were as follows: Tissues (≤3 mm) were fixed in 10% neutral formalin for 24 h, embedded in paraffin, and sliced at 3-5 µm. Subsequently, the sections were dewaxed in xylene, rehydrated using a series of ethanol (100, 95 and 80% for 10 min each), followed by washing in tap water and distilled water (for 1 min each). Staining was performed with hematoxylin for 4 min followed by washing with tap water for 2 min, differentiation with 1% hydrochloric acid ethanol for 20 sec, washing with tap water for 2 min, incubation with 6.1% diluted ammonia water for 30 sec, within with tap water 2 min and distilled water for 1 min. Eosin staining was subsequently performed for 90 sec, followed by dehydration with 80, 95 and 100% ethanol. The sections were washed with xylene and sealed with neutral gum. After staining, the pathological changes of the tissue were observed under a light microscope.

After the mice were sacrificed by dislocation of the cervical vertebrae, they were soaked in 75% ethanol at room temperature for 3 min for disinfection. A total of 5 ml pre-cooled PBS was then injected into the abdominal cavities before they were gently massaged for ~2 min. After allowing the abdomen to rest for 5 min, the peritoneal fluid was extracted, centrifuged at ~670.8 x g and 4˚C for 5 min before the supernatant was discarded. The pellet was subsequently diluted in erythrocyte lysate (Red Blood Cell Lysis Buffer; Beijing Solarbio Science & Technology Co., Ltd.), allowed to stand and then centrifuged at ~670.8 x g and 4˚C for 5 min, before the supernatant was again discarded. The cells were suspended in RPMI-1640 complete culture medium [with 10% FBS (Zhejiang Tianhang Biotechnology Co., Ltd.)] for counting before adjusting to the required concentration of 5x10⁶ cells/ml. Subsequently, the cells were incubated in an incubator at 37˚C with 5% CO₂ for 18 h until complete adherence had occurred. The supernatant was subsequently removed and discarded, where non-adherent cells were removed. Fresh RPMI-1640 complete medium was added to obtain the purified peritoneal macrophages (11,12).

After the mice had been sacrificed by cervical dislocation, the mice were clamped with tweezers and soaked in 75% alcohol for 1-2 min for disinfection purposes. The spleens were extracted on a sterile clean bench, placed into a Petri dish with PBS for 1-2 min at 4˚C, transferred to RPMI-1640 medium and ground through a 200-mesh screen with the end of a syringe pull rod. The filtrate was then sucked into a centrifuge tube before being centrifuged at ~377.32 x g and 4˚C for 5 min, after which the supernatant was discarded and 3 ml erythrocyte lysis solution (Red Blood Cell Lysis Buffer) was added. The mixture was allowed to stand for 5 min at 4˚C, after which it was re-centrifuged at ~377.32 x g at 4˚C for 5 min and the supernatant was discarded again. This step was repeated until the erythrocytes were completely lysed. The precipitated cells were resuspended in RPMI-1640 complete culture medium (with 10% FBS) and filtered using a 70-µm cell strainer into another centrifuge tube. After trypan blue staining, cell viability was adjusted to >95% and RPMI-1640 complete culture medium was added until the cell density reached 5x10⁶ cells/ml, thereby forming the lymphocyte suspension (13). After the preparation of lymphocyte suspension, the cells were cultured for 48 h and were observed to be in good condition.

Lymphocyte suspension (200 µl; ~100-300 cells) was inoculated into a 96-well cell culture plate, cultured at 37˚C with 5% CO₂ for 48 h and subsequently 20 µl MTT (5 mg/ml) solution was added in each well. After 4 h (37˚C) of continuous culture, the supernatant was discarded and 150 µl DMSO was added to each well. Finally, the absorbance at 570 nm (Abs₅₇₀) was measured using a microplate reader and the proliferation rate of the mouse spleen lymphocytes was calculated (14,15) according to the following equation: Spleen lymphocyte proliferation rate (%)=(Abs₅₇₀ experimental group)/(Abs₅₇₀ control group) x100.

Mice peritoneal macrophages were collected before the cell concentration was adjusted to 2x10⁶ cells/ml in RPMI-1640 complete medium to ensure that the concentration of the macrophage suspension from each mouse was consistent. The macrophage suspension was then added into 96-well plates and the plates were cultured in an incubator at 37˚C with 5% CO₂ for 3 h. After observing the adherent cells under a microscope, the culture medium was removed and the cells were washed twice with PBS pre-heated to 37˚C to remove the nonadherent cells. Subsequently, 200 µl 0.1% neutral red dye (cat. no. G1310; Beijing Solarbio Science & Technology Co., Ltd.) was added to each well and the culture was allowed to continue for 3 h (13-15). After washing the cells three times with preheated PBS to remove excess neutral red, 100 µl cell lysis solution [comprising absolute ethanol/glacial acetic acid, 1:1 (v/v)] was added to each well followed by mixing. The plates were incubated overnight at 4˚C before absorbance at 540 nm was read using a microplate reader.

The lymphocyte suspension was inoculated into 24-well plates and 2 ml lymphocyte suspension (~1x10⁴ cells) was used for each well. After 48 h of culture at 37˚C with 5% CO₂, the cells were centrifuged at ~377.32 x g and 4˚C for 10 min and the supernatant was collected. The concentrations of IFN-γ (cat. no. ml002277), IL-6 (cat. no. ml063159) and IL-12 (cat. no. ml037868;) secreted by the lymphocytes in the supernatant were measured using ELISA. These procedures were performed following the protocols of the mouse cytokine kit (all from Shanghai Enzyme-linked Biotechnology Co. Ltd.). The absorbance values were measured at a wavelength of 450 nm using a microplate reader and a standard calibration curve was generated to calculate the concentrations of the secreted compounds in pg/ml (16).

The lymphocyte suspension was inoculated on a 24-well cell culture plate and cultured at 37˚C with 5% CO₂ for 48 h. Subsequently, the cells were collected, centrifuged at 377.32 x g at 4˚C for 5 min and the supernatant was discarded, before washing the cells twice with PBS and TRIzol^® (Thermo Fisher Scientific, Inc.) was added to completely lyse the cells. Total RNA was extracted according to the protocols of the TRIzol kit and the ratio between the optical density measured at 260 and 280 nm was used to assess the RNA purity (17).

Following precisely the protocols of the PrimerScript™ RT reagent Kit with gDNA Eraser (cat. no. RR047A; Takara Bio, Inc.), RNA was reverse-transcribed into cDNA using the temperature protocols of 37˚C for 15 min and 85˚C for 5 sec. qPCR was subsequently performed using the TB Green™ Premix Ex Taq™ II kit, using the following thermocycling conditions: Initial denaturation at 95˚C for 30 sec, followed by 40 cycles of 95˚C for 5 sec and 60˚C for 30 sec. The primer pairs used for qPCR (Table I) were designed the mRNA levels were quantified using the 2^-ΔΔCq method and normalized to the internal reference gene β-actin (18).

Using a 24-well cell culture plate, 1 ml lymphocyte suspension (~2x10⁴ cells) was added into each well incubated at 37˚C for 48 h. Total RNA of lymphocytes was extracted using the TRIzol method according to the manufacturer's protocol. A protein/nucleic acid analyzer (NanoDrop One; Thermo Fisher Scientific, Inc.) was used to detect the RNA concentration and purity, before the total RNA concentration was adjusted to 400-600 ng/µl range. Reverse transcription was performed according to the instructions of PrimeScript™ RT Reagent Kit with gDNA Eraser (cat. no. RR047A; Takara Bio, Inc.), which was 37˚C for 15 min followed by 85˚C 5 sec. The products of qPCR (described in the previous paragraph) were used for 2% agarose gel electrophoresis. The following reagents were used: DL 1000DNA Marker Takara Japan (cat. no. 3591Q; Takara Bio, Inc.); gel stain 10,000X (Beijing TransGen Biotech Co., Ltd.).

Statistical analysis was performed using SPSS 21.0 software (IBM Corp.) and the experimental data are shown as the mean ± standard deviation. Statistical comparisons were performed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Compared with those in the NG mice, the thymus and spleen indices of mice in the MG group were significantly decreased (P<0.05; Table II), suggesting that the immune function of the mice was significantly inhibited. Compared with those MG mice, the TI was significantly increased in the PG, MHG, MMG and MLG groups (P<0.05; Table II), whereas the SI was increased significantly in the PG, MMG and MHG groups (P<0.05; Table II). The SI of mice in MLG was also significantly increased compared with that in the MG group (P<0.05). These data suggest that the immune organ indices of the immunosuppressed mice increased in response to MCP in a dose-dependent manner, although it remained lower compared with that of the NG mice.

The spleen tissue of MG mice was found to be markedly smaller compared with that of NG mice, with lower density, uneven distribution of lymphocytes and unclear boundaries between the red and the white medulla (Fig. 1B). Lymphocytes in the spleen tissue of mice in the MHG and PG groups were densely but unevenly distributed, with deep staining (Fig. 1C and F). The distribution of lymphocytes in the red medulla region of the spleen tissue from the MLG and MMG mice were loose with no evident damage found in the spleen tissues of mice in either group (Fig. 1D and E). These observations suggest that MCP can promote the development of spleen in mice with CY-induced hypoimmunity, thereby improving the immunity of mice (Fig. 1).

The number of lymphocytes in the thymus tissue of MG mice was decreased compared with that of NG mice, where the boundary between the medulla and cortex was not clear (Fig. 2B). In the PG, MMG and MHG mice, the cortical lymphocytes were dense, evenly distributed and deeply stained, where medullary lymphocytes were loosely distributed, and the blood vessels and reticular cells were clear (Fig. 2C, E and F). These observations suggest that MCP can enhance the development of lymphocytes. The lymphocyte distribution in the tissues of MLG mice was loose and uneven, which indicated that MCP was able to repair injuries sustained to the thymus of immunocompromised mice (Fig. 2D).

The degree of DTH (Table III) in the MG mice was significantly decreased compared with that in the NG mice (P<0.05). The DTH in PG mice and those administered with MCP at concentrations of 200 or 300 mg/kg (MMG and MHG) was significantly increased compared with that in MG mice (P<0.05). The difference in DTH resulting from treatment with MCP at a concentration of 100 mg/kg (MLG) was smaller compared with that in the MMG and MHG groups, but remained significant compared with that in the MG group (P<0.05).

The effect of MCP on the proliferation of splenic lymphocytes show that CY treatment in MG mice significantly reduced lymphocyte proliferation compared with that in NG mice (P<0.05; Table III). The proliferation of splenic lymphocytes in the MCP treatment groups (MLG, MMG and MHG) appeared to be dose-dependent (Table III). The proliferation of splenic lymphocytes in the PG, MMG and MHG mice was significantly increased compared with that in the MG mice (P<0.05; Table III). These data suggest that MCP can effectively promote the proliferation of spleen lymphocytes in immunosuppressive mice.

As shown in Table III, the phagocytic activity of peritoneal macrophages in MG mice was significantly decreased compared with that in NG mice (P<0.05), suggesting that CY treatment reduced the activity of phagocytotic macrophages in mice. The phagocytic activity of peritoneal macrophages in the PG, MMG and MHG mice was significantly higher compared with that in the MG mice (P<0.05; the activity of phagocytotic macrophages). By contrast, cell phagocytosis of mice in the MLG group was also markedly enhanced compared with that of MG mice, although these changes were found to be not significant.

The effects of MCP on the levels of the cytokines IFN-γ, IL-6 and IL-12 in immunosuppressed mice are shown in Table IV. In MG mice, the levels of the helper T (Th) cell type 1 cytokines IL-12 and IFN-γ and the Th2 cytokine IL-6 were significantly lower compared with those in the NG mice (P<0.05). Compared with those in the MG mice, the levels of IL-12 and IFN-γ in the PG, MLG, MMG and MHG mice were significantly higher (P<0.05; Table IV). The levels of IL-6 in MMG and MHG mice were significantly increased (P<0.05), whereas those in MLG mice was also significantly increased but to a lesser extent (P<0.05; Table IV), compared with those in the MG group. As the MCP dose increased, the levels of secreted IFN-γ, IL-6 and IL-12 were also increased.

The present study next examined the mRNA expression levels of IFN-γ, IL-6 and IL-12 after MCP was administered at different concentrations (Fig. 3). The mRNA expression levels of IFN-γ, IL-6 and IL-12 in the spleen lymphocytes of MG mice were significantly lower compared with those in the spleen lymphocytes of NG mice (P<0.05). Fig. 4 also shows that the expression levels of the cytokines IFN-γ, IL-6 and IL-12 in the spleens of MG mice were markedly lower compared with those in the NG group. After treatment with different concentrations of MCP, the mRNA expression levels of IFN-γ, IL-6 and IL-12 were observed to increase by varying degrees. The mRNA expression levels of IFN-γ, IL-6 and IL-12 in the PG and MCP dosage groups were significantly higher compared with those in MG mice (P<0.05; Fig. 3). In addition, the mRNA expression levels of IFN-γ, IL-6 and IL-12 showed a certain dose-effect relationship with the dose of MCP used. Fig. 5 shows the results of the cell viability test measured with lymphocytes and macrophages at 570 nm. Although the results obtained with the MHG and PG mice were comparable, they did not fully return to normal levels. Finally, the cell viability of CY-immunosuppressed mice was found to be increased in the MLG mice. Compared with that in the NG mice, the lymphocyte viability of mice in the MG group was significantly decreased (P<0.05; Table II). Compared with the MG mice, the lymphocyte viability was significantly increased in the PG, MHG, MMG and MLG groups (P<0.05; Table II). These data suggested that the lymphocyte viability of the immunosuppressed mice increased in response to MCP in a dose-dependent manner, although it remained lower compared with that of the NG mice (Fig. 5A). Compared with that in the NG group, the macrophage viability of mice in the MG group was significantly decreased (P<0.05; Table II). Compared with the MG mice, the macrophage viability was significantly increased in the PG, MHG, MMG and MLG groups (P<0.05; Table II). These data suggested that the macrophage viability of the immunosuppressed mice increased in response to MCP in a dose-dependent manner (Fig. 5B).