Human acute monocytic leukemia cells THP-1 were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences (Shanghai, China). TRIzol^® RNA extraction reagent (Invitrogen), 1st Strand cDNA Synthesis kit gDNA Purge (Novoprotein; cat. no. E042-01B), SYBR qPCR SuperMix Plus (Novoprotein; cat. no. E096-01A) and reverse transcription RT-PCR primers (Invitrogen) were from Thermo Fisher Scientific, Inc. The primers for qPCR were commissioned to be synthesized by Genewiz, Inc. Primary antibodies, including Krüppel-like factor 4 (KLF4; cat. no. R1308-1), insulin receptor (INSR; cat. no. ET1705-22), IRS1 (cat. no. ER2001-35), Gal-3 (cat. no. ER1803-82), NF-κB (cat. no. ER0815), phosphorylated (p)-NF-κB p65 (S529) antibody (cat. no. ET1604-27), TLR4 (cat. no. ER1706-43) and GAPDH (cat. no. ER1706-83), were purchased from Hangzhou Hua'an Biotechnology Co., Ltd. The p-IRS1 (Ser1101) primary antibody (cat. no. 2385) was from Cell Signaling Technology, Inc. The following secondary antibodies were used: Horseradish peroxidase-conjugated goat anti-rabbit IgG (cat. no. HA1001; Hangzhou HuaAn Biotechnology Co., Ltd.) and FITC-conjugated goat anti-rabbit antibody, (cat. no. HA1004; Hangzhou HuaAn Biotechnology Co., Ltd.). DAPI was purchased from Beyotime Institute of Biotechnology, and the (3R,4R,5S,6R)6-(hydroxymethyl)- 3-[(7-nitrobenzo(c) (1,2,5) oxadiazol-4-yl) amino] tetrahydro-2H pyran-2,4,5-triol (2-NBDG) glucose uptake/transport fluorescent probe (cat. no. MX4511-1MG) was from Shanghai Mao Kang Biological Technology, Co., Ltd. The TLR4 protein inhibitor (TAK 242; cat. no. 243984-11-4) was also obtained from Merck KGaA. All protocols and the use of human peripheral blood were approved by the Affiliated Hospital of Medical School of Ningbo University (Ningbo, China; approval no. KY20190102). A total of 10 men (40-50 years old; including 5 healthy individuals and 5 patients with type 2 diabetes) and 10 women (40-50 years old; including 5 healthy individuals and 5 patients with type 2 diabetes) were admitted to the Affiliated Hospital of Ningbo University from January 2018 to December 2018 to detect the expression level of Gal-3 protein in the peripheral blood of patients. All subjects provided oral informed consent.

THP-1 cells were grown in a 6-well plate (5x10⁶ cells/well) and grown in an incubator at 37˚C and 5% CO₂ in RPMI-1640 (cat. no. ZQ-230) medium containing 10% FBS (cat. no. ZQ500; Zhejiang Ruyao Biotechnology Co., Ltd.). Cells between passage four to eight were selected for the experiments based on the cell growth state and cell morphology. A total of 1x10⁶ cells/well were inoculated in a 6-well plate and grown in an incubator at 37˚C and 5% CO₂ in RPMI-1640 medium containing 10% FBS. The differentiated macrophages were identified after treatment with 200 ng/ml 12-O-Tetradecanoyl-phorbol 13-Acetate (PMA; cat. no. P6741; Beijing Solarbio Science & Technology Co., Ltd.) for 3 days.

A total of 2x10⁶ cells/well were inoculated in a 24-well plate and grown in an incubator at 37˚C and 5% CO₂ in RPMI-1640 medium containing 10% FBS for 2 h. Cultured macrophages were washed with PBS and fixed with methanol at 4˚C for 20 min. After fixation, all subsequent steps were carried out at room temperature. Samples were washed 1X PBS and stained with hematoxylin for 3 min and eosin for 5 sec. After dehydration, xylene was added and neutral gum was used for sealing. Cultured THP-1 cells that did not receive PMA induction were centrifuged at 800 x g room at temperature for 5 min, resuspend 100 µl in 1X PBS and then dropped onto a slide. After air drying, the cells were fixed with methanol at 4˚C for 20 min, followed by HE staining using the same method as described above. Prior to HE staining, both groups of cells were photographed under a phase contrast microscope to observe their morphology (magnification, x100 and x400). In addition, THP-1 cells were cultured in a 24-well plate with the inoculation density of 2x10⁶ cells per well, and used for microsphere phagocytosis test. A drop of polystyrene microspheres (cat. no. P107780; Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to 2.5 ml PBS, mixed thoroughly and then 5 µl diluted beads were added to the PMA-treated THP-1 cells. The cells were incubated at 37˚C for 50 min and then washed twice with 1x PBS. The phagocytosed microspheres were counted under an inverted microscope (magnification, x100). Phagocytosis rate (%)=(the number of phagocytic pellets/the number of cells in one field).

Macrophages in good growth condition and in the logarithmic growth phase were treated with 0, 200, 400 or 600 µM PA (cat. no. P0500; Merck KGaA) for 24 h. Subsequently, the cells were tested for glucose uptake ability according to the glucose uptake assay protocol described below. The cell supernatant was collected via centrifugation at 800 x g for 20 min at 4˚C and the inflammatory factors, including interleukin (IL)-1β (cat. no. SEKH-0002), IL-6 (cat. no. SEKH-0013) and TNF-α (cat. no. SEKS-0003), were detected by ELISA (all from Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocol.

For the glucose uptake assay, 1x10⁵ macrophages were used following treatment with PA (0, 200, 400 or 600 µM), inoculated (1x10⁵ cells/well) in a 6-well plate, cultured at 37˚C for 48 h, washed three times with PBS and incubated with 100 µM 2-NBDG at 37˚C for 20 min. The cells were washed three times with precooled PBS, digested with 0.25% trypsin, collected via centrifugation at 800 x g for 5 min at 4˚C and examined using an Attune NxT flow cytometer (Thermo Fisher Scientific, Inc.) with FlowJo 10 software (FlowJo LLC).

Macrophages were collected in the logarithmic growth phase and cell viability was detected using the MTT method. A total of 100 µl macrophages were added to each well, and the density of the cells was adjusted to 5x10³ macrophages/well (the edge holes were filled with sterile PBS). The cells were incubated at 37˚C and 5% CO₂ until the macrophage monolayer covered the bottom of the well (96-well flat-bottom plate). After the adherent growth of macrophages, cells were treated with 100 µl drug-containing complete medium (PA, 0, 200, 400 and 600 µM) in each well. After incubation at 37˚C for 48 h, 20 µl 5% MTT was added to each well for 4 h. The culture medium in the well was removed, 150 µl DMSO was added. The absorbance was measured at an optical density of 490 nm using a microplate reader (MultiskanGO; Thermo Fisher Scientific, Inc.).

The macrophages (1x10⁶) were removed from the CO₂ incubator after 24 h treatment with PA and washed twice with precooled PBS. The remaining PBS was aspirated, the cells were lysed with RIPA lysis buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) on ice and then the macrophage lysate was pipetted to destroy the DNA. A total of 3 µl lysate was used for BCA quantification. Further, 4X protein loading buffer was added to the remaining samples, incubated at 95˚C for 10 min to denature and stored at -20˚C until further analysis. Proteins (40 µg) were separated by 10% SDS-PAGE, transferred to a PVDF membrane at a constant current of 200 mA for 2 h and blocked with 5% skimmed milk for 1 h at room temperature. The membranes were incubated with the primary antibody (rabbit anti p-IRS1/IRS1/INSR/KLF4/Gal-3; all, 1:1,000) overnight at 4˚C. The internal reference antibody GAPDH was added and incubated for 1 h at room temperature (cut according to marker size). The membrane was washed three times with 1X TBST (0.15% Tween-20), then incubated with the secondary antibody at room temperature for 2 h, followed by three washes with TBST. Proteins were visualized using ECL solution (cat. no. PE0010; Beijing Solarbio Science & Technology Co., Ltd.). The ChemiDoc-It Imaging System (UVP, LLC) was used for observation and ImageJ 8.0 (National Institutes of Health) was used for quantification. When assessing the protein levels of the downstream TLR4 pathway, four groups were constructed: The control group; the PA group, which received induction with 400 µm PA; the TAK-242 group, in which macrophages were treated with 1 µM TAK-242; and the TAK-242 + PA group, where macrophages were treated with 400 µM PA and 1 µM TAK-242. After administration, macrophages were cultured for 24 h under 5% CO₂ and 37˚C. Western blot analysis was performed at the end of treatment using the same procedures as described above.

After 24 h of treatment with PA, the treated macrophages were collected (1x10⁶) and total RNA was extracted by the TRIzol^® method. An ultramicro-spectrophotometer (NanoDrop One/OneC; Thermo Fisher Scientific, Inc.) was used to detect nucleic acid concentration, and the integrity of the extracted mRNA was verified by agarose gel electrophoresis. After incubating samples at 25˚C for 5 min, the 1st Strand cDNA Synthesis kit (cat. no. E047-01A; Novoprotein) was applied and used according to the manufacturer's protocol, incubating samples at 42˚C for 60 min for RT. The reaction was terminated by heating at 70˚C for 5 min, and the mRNA was reverse transcribed into cDNA. qPCR was conducted using SYBR qPCR SuperMix Plus for analysis, along with the 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction mixture contained 20 ng cDNA, 1.5 mmol of each primer and 2.5 mM MgCl₂, and the thermocycling conditions were as follows: Initial denaturation at 95˚C for 2 min; followed by 40 cycles of denaturation at 95˚C for 10 sec, annealing at 60˚C for 10 sec and extension at 72˚C for 30 sec. The relative expression of Gal-3 was calculated using the 2^-ΔΔCq method (19). Primer 3 (Premier Biosoft International.) was used to design primer pairs; the sequences were as follows: Gal-3, forward 5'-CGGAGCCAGCCAACGAG-3' and reverse, 5'-AACGCATCATGGAGCGAAAA-3'; GAPDH forward, 5'-GCACCGTCAAGGCTGAGAAC-3' and reverse, 5'-TGGTGAAGACGCCAGTGGA-3'.

THP-1 cells (1x10⁶) were inoculated on a 6-well plate with a cover glass slide placed on the plate. After inducing macrophages with PMA, the cells were then slip-fed and cultured at 37˚C and 5% CO₂ for 24 h until a 70% fusion degree was reached. The next day, 400 µM PA and 37 nM GB1107 (Shanghai ZrBiorise Biotechnology Co., Ltd.; cat. no. 1978336-61-6) were added, and set up the experimental group with the above two reagents, the standard medium was used for the negative control. The groups were divided into four: The control group; the PA group, where macrophages were treated with 400 µM PA; the GB1107 group, where macrophages were treated with 37 nM GB1107; and the PA+GB1107 group, where macrophages were treated with 400 µM PA and 37 nM GB1107. Cells were incubated for 8 h at 37˚C with 5% CO₂. The cells were then fixed with 4% paraformaldehyde for 20 min at room temperature and incubated with rabbit antibody-3 antibodies (1:100) at room temperature for 2 h. Cells were then incubated with the FITC-labeled secondary antibody (goat anti rabbit; 1:500). The nuclei were stained and sealed with anti-fluorescence quenching solution (including DAPI; cat. no. P0131; Beyotime Institute of Biotechnology) was added. A Leica DM500 fluorescence microscope was used to observe and obtain images (Leica Microsystems GmbH). Image-Pro Plus 6.0 (Media Cybernetics, Inc.) software was used for the quantification of results.

The names of all proteins were put into the string website to predict the relationship between proteins (https://string-db.org/).

All experiments were repeated three times separately, and the data are expressed as the mean ± standard deviation, differences between two groups were measured by unpaired Student's t-test using SPSS 22.0 software (IBM Corp.). One-way ANOVA was used to analyze differences between multiple groups, followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The adherent macrophages induced by PMA were observed directly under a light microscope and phase contrast microscope after HE staining (Fig. 1A and B). The adherent cells exhibited clear boundaries and blunt round protrusions. The nuclei were large and irregularly shaped at one end of the cell. THP-1 cells without PMA induction demonstrated a round karyotype and less cytoplasm. In addition, the macrophages phagocytosed the microspheres, indicating that the PMA-induced THP-1 cells exhibited phagocytic ability (Fig. 1C), which was statistically significant compared with the number of phagocytosed microspheres in the uninduced THP-1 cells (P<0.05; Fig. 1D). The results indiacted that PMA could induce macrophage differentiation successfully.

Macrophages were treated with 0, 200, 400 or 600 µM PA, and 2-NBDG was used to detect the sugar uptake ability of macrophages. The results showed that the glucose uptake was significantly lower in the PA-treated groups compared with uptake in the control group (P<0.01; Fig. 2A). In addition, the protein expression levels of p-INSR, INSR, and IRS1 were detected by western blotting. The results demonstrated that the expression levels of INSR were downregulated following PA treatment, and p-IRS1 was significantly decreased (P<0.05) (Fig. 2B and C), indicating that PA induced insulin resistance in macrophages. At the same time, the relative protein expression level of KLF4 was significantly decreased in PA-treated cells compared with the control group (P<0.01; Fig. 2D and E). After treatment with different PA concentrations, the levels of inflammatory cytokines IL-6, IL-1β and TNF-α in the cells increased significantly compared with untreated control cells (P<0.01; Fig. 2F-H, respectively).

Western blotting analysis of the peripheral blood showed that Gal-3 was significantly upregulated in T2DM patients compared with healthy individuals (P<0.05; Fig. 3A and C). Moreover, compared with the control group, different concentrations of PA significantly increased the expression of Gal-3 in treated macrophages (P<0.01; Fig. 3B and D). Therefore, it was speculated that Gal-3 may be involved in the regulation of insulin resistance by macrophages. Fig. 3E suggested that 100, 400 or 600 µM PA significantly increased Gal-3 expression when compared with the control group. The MTT viability assay was performed on macrophages treated with different concentrations of PA. When the PA treatment concentration was 400 µM, cell viability was not significantly affected compared with that in the control group (P>0.05; Fig. 3F). However, at 600 µM, cell viability decreased significantly compared with that in the control group (P<0.01). Taking into account the effects of different concentrations of PA on the protein and gene expression levels and cell viability of GAL-3 in macrophages, 400 µM PA was selected as the appropriate treatment concentration for further experiments.

Following PA treatment, macrophages were treated with the Gal-3 inhibitor GB1107 (37 nM), and the results showed that GB1107 could significantly slow down the weakening effect of PA on the glucose uptake capacity of macrophages (P<0.01; Fig. 4A), thereby alleviating the PA-induced insulin resistance of macrophages. In addition, after adding GB1107, the relative protein expression level of Gal-3 in PA-treated macrophages was significantly reduced, but the protein of Gal-3 was not affected (20) (P<0.01; Fig. 4B and C). Western blotting analysis and immunofluorescence demonstrated that compared with the control, Gal-3 protein was significantly down regulated in the GB1107 group (Fig. 4D), whereas Gal-3 protein expression in the PA + GB1107 group returned to the same level as in the control group. These data indicated that PA may mediate the occurrence of macrophage insulin resistance by affecting the expression of Gal-3.

The expression levels of TLR4 protein and the downstream proteins and p-NF-κB, as well as Gal-3 were detected by western blot analysis (Fig. 5A). The results showed that compared with the control group, the expression of TLR4 in the PA group was significantly upregulated (P<0.01), and the expression of the protein KLF4 decreased (Fig. 5B). In addition, compared with the control group, the p-NF-κB significantly increased (P<0.01) and the expression of Gal-3 was significantly upregulated (P<0.01) in the PA group The macrophages were cultured for 24 h at 5% CO₂ and 37˚C after co-administration of PA (400 µM) and the TLR4 protein inhibitor (TAK-242; 1 µM), the expression levels of KLF4 and Gal-3 protein returned to control levels, and the p-NF-κB protein level was significantly inhibited (P<0.01). Bioinformatics analysis showed an interactive relationship between KLF4, Gal-3 and TLR4 proteins (Fig. 5C), The results suggested that PA may affect TLR4, p-NF-κB and Gal-3 by inhibiting the expression of KLF4, thus mediating the development of insulin resistance in macrophages.