Immune activity analysis

A total of 60 Female Balb/c mice (specific-pathogen free grade,18–24 g, 5 weeks) were purchased from the Laboratory Animal Center of Lanzhou University and kept in room temperature at 24 ± 2°C and relative humidity of 60 ± 5% under an automatic 12 h light/12 h dark cycle. Six groups (10 mice in each group) were used in experiments: group 1 (normal saline control, PS): animals were treated with saline (1–30 d); group 2 (cyclophosphamide, CTX): animals were administrated with cyclophosphamide (1–10 d) (80 mg kg^–1⋅bw) and saline (11–30 d); group 3 (levamisole hydrochloride, LH): animals were administrated with cyclophosphamide (1–10 d) and levamisole hydrochloride (11–30 d); group 4 (High-SLMPs-1-1 doses, SLMPs-H): animals were administrated with cyclophosphamide (1–10 d) and SLMPs (11–30 d, 800 mg kg^–1⋅bw); group 5 (Mid-SLMPs-1-1 doses, SLMPs-M): animals were administrated with cyclophosphamide (1–10 d) and SLMPs (11–30 d, 400 mg kg^–1⋅bw); group 6 (Low-SLMPs-1-1 doses, SLMPs-L): animals were administrated with cyclophosphamide (1–10 d) and SLMPs (11–30 d, 400 mg kg^–1⋅bw); At the end of the treatment, mice were sacrificed within 24 h, and their spleen and thymus were dissected under sterile conditions (26).

The whole blood samples of mice in each group was obtained by taking eyeball under sterile conditions and the serum was separated by centrifugation (8,000 rpm, 5 min) at 4°C for 10 min. The levels of IgG, IFN-γ, IL-4, and TNF-α in mice serum were determined using a ELISA kits (Solarbio Biological Reagent Co., Ltd., Beijing, China) by the manufacturer’s instructions. The color intensity was read using absorbance (A₄₅₀) by a tunable microplate reader (Fisher FC, Thermo Co., United States), and the concentration of different cytokine were calculated according to a standard curve (27).

The spleen and thymus tissues were fixed in 10% PFA for at 37°C for 24 h, washed by flowing water for 24 h, dehydrated in a graded series of ethanol, soaked in xylene for 5 min, embedded in paraffin, and sectioned at 5 μm using a Leica RM2255 a microtome (Leica Biosystems Inc., Germany). Then the paraffin sections were stained with hematoxylin and eosin (HE) method, and observed under an Olympus Simon-01 microscope (Olympus Optical Co., Japan) (28).

RT-qPCR analysis was employed in detection of the mRNA expression of TLR4 and NF-κB in mice spleens extracted from each group (29). The total RNA of mice spleens in each group was extracted using a Trizol reagent Kit (TransGen Biotech Co., China). The concentration and purity of RNA were determined by ultraviolet spectrophotometry at 260 and 280 nm, aliquoted and stored at –80°C for future use. RNA was reverse-transcribed to cDNA using a PrimeScript™ RT reagent kit with cDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s introduction. RT-qPCR was performed to quantify mRNA expression by a CFX96 Real-time PCR System (Bio-Rad, Hercules, United States) with SYBR Green Real-time Master Mix (Toyobo, Japan). The PCR program was: 95°C, 10 min; 95°C, 15 s; 60°C, 30 s, 40 cycles; melting curve analysis 65→95°C to detect the fluorescence signal every 0.5°C –cycle, and the reaction system was 2.0 μL cDNA, 1.5 μL 2.5 μM primers, 7.5 μL 2 × RT-qPCR Mix, 4 μL ddH₂O, a total of 15 μL. The used primers are presented in Table 1.

The primer sequence for RT-qPCR.

A protein extraction Kit (Solarbio, China) was used to isolated the total protein of the spleen tissue in each group mice, and a BCA protein quantitative Kit (Solarbio, China) was used to measure protein concentration. Then the protein (20 μg) was separated by 10% SDS-PAGE gel, transferred onto a 0.2 μm polyvinylidene difluoride (PVDF) membrane through a trans-blot Turbo transfer system (Bio-Rad, Hercules, United States) for 10 min at 25 V. The membrane was blocked in 0.02 mol/L PBS buffer (containing 5% skim milk powder (w/v) and 0.05% Tween-20, pH 7.5) at room temperature for 1 h, then incubated in a primary antibody solution at 4°C for 24 h. Thereafter, the membrane washed by TBST, incubated with secondary antibody at room temperature for 1 h. The protein bands were observed by using the ECL Western Blotting Analysis System (Bio-Rad, Hercules, United States) on an Image Quant LAS 4000 mini imager (GE, Life Science, United States) (30).