7428 MOLECULAR MEDICINE REPORTS 17: 7428-7434, 2018 Tumor necrosis factor-α-induced protein 8-like-2 is involved in the activation of macrophages by Astragalus polysaccharides in vitro JIE ZHU1, YUANYUAN ZHANG2, FANGHUA FAN1, GUOYOU WU1, ZHEN XIAO1 and HUANQIN ZHOU1 1Department of Clinical Laboratory, Zhejiang Hospital, Hangzhou, Zhejiang 310013; 2Department of Pulmonology, The Children's Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang 310052, P.R. China Received April 6, 2017; Accepted September 11, 2017 DOI: 10.3892/mmr.2018.8730 Abstract. In previous years, studies have shown that Introduction Astragalus polysaccharides (APS) can improve cellular immu- nity and humoral immune function, which has become a focus Macrophages are important immune cells, which are involved of investigations. Tumor necrosis factor-α-induced protein in the stimulation of the innate immune system, including the 8-like 2 (TIPE2) is a negative regulator of immune reactions. release of inflammatory cytokines and inflammatory molecules, However, the effect and underlying mechanisms of TIPE2 on including tumor necrosis-factor (TNF)-α, interleukin (IL)-1β, the APS-induced immune response remains to be fully eluci- IL-6 and nitric oxide (NO) (1,2). These cytokines and inflam- dated. The present study aimed to examine the role of TIPE2 matory molecules are often regulated by the mitogen-activated and its underlying mechanisms in the APS-induced immune protein kinase (MAPK) signaling pathway (3,4). response. The production of nitric oxide (NO) was detected TNF-α-induced protein-8 like 2 (TNFAIP8L2, also known in macrophages in vitro following APS stimulation. In addi- as TIPE2), is a novel member of the TNFAIP8 (TIPE) family. tion, the present study interfered with the expression of TIPE2 TIPE2 was originally identified as a negative regulator of in macrophages, and examined the production of cytokines, immune homeostasis, being expressed mainly in lymphoid NO and components of the mitogen-activate protein kinase tissues, inflammatory tissues and immune cells, including (MAPK) signaling pathway following APS stimulation. The macrophages (5). results showed that APS was able to activate macrophages by Astragalus, a well-known Chinese traditional medicine inducing the production of interleukin (IL)-1β, tumor necrosis and edible food, has multiple effects in pharmacological factor (TNF)-α, IL-6 and NO. Furthermore, RAW264.7 cells and biological processes. One of its bioactive components is were stimulated with APS when TIPE2 was silenced, and Astragalus polysaccharides (APS), which can improve cellular it was found that the production of TNF-α, IL-6, IL-1β and and humoral immunity, and regulate certain cytokines (6-8). NO were upregulated, and the signaling pathway of MAPK Although it is well known that APS is an immune regulator, was activated. Taken together, these results demonstrated that few studies have reported on the molecular mechanism under- TIPE2 had an important negative effect on the APS-induced lying the effect of APS on the immune system. MAPKs are a production of inflammatory cytokines and NO via the MAPK family of serine/threonine protein kinases, which are respon- signaling pathway. sible for the majority of cellular responses to cytokines and are crucial for regulating the production of inflammation media- tors. A small number of natural extracts have been shown to inhibit the expression of inflammatory genes by regulating the phosphorylation of MAPK pathways (3-4,9). The present study aimed to determine what effect TIPE2 has on activating macrophages induced by APS in vitro. Materials and methods Correspondence to: Miss Huanqin Zhou, Department of Clinical Laboratory, Zhejiang Hospital, 12 Linying Road, Hangzhou, Drugs. APS (cat. no. 151001B) for clinical application with Zhejiang 310013, P.R. China E-mail: [email protected] an endotoxin content of <0.1 Eu/mg was purchased from Pharmagenesis, Inc. (Palo Alto, CA, USA); it is a polysaccharide Abbreviations: APS, Astragalus polysaccharides; TIPE2, tumor with a molecular weight of 20,000-60,000, and the polysac- necrosis factor-α-induced protein 8-like-2; NO, nitric oxide; MAPK, charides consist of α-1, 4 (1,6) glucan, arabinose-galactose mitogen-activated protein kinase polysaccharides, hamnose-galacturonic acid polysaccharides and arabinose-galactose protein polysaccharides. Key words: Astragalus polysaccharides, macrophages, activation, downregulation, tumor necrosis factor-α-induced protein 8-like-2 Cell line and culture. Murine macrophage RAW264.7 cells (Institute of Biochemistry and Cell Biology, Shanghai, China) ZHU et al: APS-INDUCED ACTIVATION OF MACROPHAGES 7429 were grown in RPMI-1640 medium (Invitrogen; Thermo extracted from cells using TRIzol (Takara Bio, Inc., Otsu, Fisher Scientific, Inc., Waltham, MA, USA) with penicillin Japan) according to the manufacturer's protocol. Single-strand 100 IU/ml, streptomycin 100 IU/ml and 2 mM/l-glutamine cDNA (100 ng) was generated from total RNA using reverse supplemented with 10% bovine serum albumin (PPA, Ferlach, transcriptase (Toyobo Co., Ltd., Osaka, Japan). RT-qPCR Austria). The cells were maintained subconfluent at 37˚C in analysis, using 5 µl SYBR-Green detection chemistry mix humidified air containing 5% CO2. The RAW264.7 cells were (Roche Diagnostics, Basel, Switzerland), was performed on a harvested by gentle scraping, washed in PBS, resuspended in 7500 Real-Time PCR system (Applied Biosystems; Thermo culture medium and plated. Non-adherent cells were removed Fisher Scientific, Inc.). For mouse TIPE2, 1 µl qPCR primers by repeated washing following incubation at 37˚C for 4 h. were: Forward, 5'-TCT CAG AAA CAT CCA AGG CC-3' and Each experimental unit comprised 5x105/ml cells. reverse, 5'-TTT GAG CTG AAG GAC TCC ATG-3'. For mouse β-actin, the qPCR primers were: Forward, 5'-AGT GTG ACG RNA interference. The TIPE2-specific small interfering TTG ACA TCC GT-3' and reverse, 5'-GCA GCT CAG TAA (si)RNA-517 (GenePharma Co., Ltd., Shanghai, China) CAG TCC GC‑3'. PCR amplification program was performed sequences were as follows: Sense 5'-GCA UCA GGC ACG UGU under the following two steps. Stage 1: 50˚C for 2 min, 95˚C UUG ATT-3' and antisense 5'-UCA AAC ACG UGC CUG AUG for 10 min (1 cycle). Stage 2: 95˚C for 15 sec, 60˚C for 35 sec CTT‑3'. The TIPE2‑specific siRNA‑166 (GenePharma Co., Ltd.) (39 cycles). Relative expression fold change was calculated sequences were as follows: Sense 5'-CCG UGG CGC AUC UCU by the 2∆∆Cq method, and β-actin was used as the endogenous CUU UAU TT-3' and antisense 5'-AUA AAG AGA UGC GCC reference gene to normalize the expression level of target gene. ACG GTT‑3'. The TIPE2‑specific siRNA‑351 (GenePharma Co., Ltd.) sequences were as follows: Sense 5'-GCU ACA CGA Western blot analysis. The cells were washed twice with cold UUU CGU CAG ATT-3' and antisense 5'-UCU GAC GAA AUC PBS and lysed with cell lysis buffer (Cell Signaling Technology, GUG UAG CTT-3'. The control siRNA sequences (GenePharma Inc., Boston, MA, USA) supplemented with protease inhibitor Co., Ltd.) were as follows: Sense 5'-UUC UCC GAA CGU GUC mixture (Beyotime Institute of Biotechnology, Shanghai, ACG UTT-3' and antisense 5'-ACG UGA CAC GUU CGG AGA China). The protein concentrations of the cell lysis extracts ATT-3'. For each well, 8.4 ng of siRNA duplexes was diluted were measured using a BCA assay (Pierce; Thermo Fisher into 100 µl of medium and mixed. INTERFERin (4 µl) was Scientific, Inc.) and equalized with the extraction reagent. added to the mixture, which was immediately homogenize by Protein extracts (30 µg) were loaded and subjected to vortexing for 10 sec. It was then incubated for 10 min at room separation on an 10% SDS-PAGE gel, following which they temperature to allow transfection complexes to form between were transferred onto a PVDF membrane. The membrane siRNA duplexes and INTERFERin. During complex forma- was blocked with 5% skim milk in TBST for 1 h at 25˚C and tion, the growth medium was removed and 0.5 ml of fresh then incubated with rabbit anti murine ERK (cat. no. 9102), pre-warmed complete medium added per well. Then, 100 µl of JNK (cat. no. 9252), P38 (cat. no. 8690), phosphorylated transfection mix was added to the cells and homogenized by ERK (cat. no. 9101), phosphorylated JNK (cat. no. 9251), and gently swirling the plate. The final volume was 600 µl, and the phosphorylated P38 antibody (cat. no. 9215; Cell Signaling siRNA concentration 20 nM. The plate was incubated at 37˚C Technology, Inc.) for 1 h at 25˚C. Following washing three and gene silencing measured after 48 h. times in TBST for 10 min each time, the membrane was incubated with HRP conjugated goat anti rabbit-IgG (cat. NO assay. The quantity of stable NO generated by activated no. 18-8816-33; Rockland, Gilbertsville, Philadelphia, PA, macrophages was determined using Griess reagent (Promega USA) for 1 h and the antibody‑specific protein was visualized Corp., Madison, WI, USA). The TIPE2 siRNA- or control by using an ECL kit (Biological Industries, Kibbutz Beit siRNA-transfected RAW264.7 cells (5x105/ml) were treated Haemek, Israel) in the enhanced chemiluminescence detection with different concentrations of APS (10, 100, 200 and 400 µg) system as previously described (10). The intensities of the and LPS (10 µg/ml) at room temperature for different dura- protein bands were analyzed by Gel-Pro Analyzer software. tions (4, 8, 16, 24, 32, 40 and 48 h). Subsequently, 50 µl of the All of the above antibodies were diluted to 1:1,000. cell culture supernatant was mixed with an equal volume of Griess reagent and incubated at room temperature for 15 min. Statistical analysis. The results are expressed as the The absorbance was measured at 550 nm using a microplate mean ± standard deviation of the indicated number of experi- reader, and a standard curve was plotted using a serial known ments.