LPS from Escherichia coli and aloin (purity≥97%; Fig. 1A) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) and Aladdin Industrial Corporation (Shanghai, China), respectively. The aloin was dissolved in dimethyl sulfoxide and diluted with sterile PBS. DAPI was obtained from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Anti-phosphorylated (phosphor)-STAT3 (Tyr705, sc7993) and phospho-IκB (B-9, sc8404) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against phospho-p38 MAPK (p-p38 MAPK; Thr180/Tyr182, 4511S), phospho-ERK (Thr202/Tyr204, 4376S), phospho-JNK (Thr183/Tyr185, 4668S), p38 MAPK (8690S), ERK (4695S), JNK (9258S), phospho-JAK1 (Tyr1034/1035, 3331S), phospho-JAK2 (Y1007/1008, 3771S), phospho-STAT1 (Tyr701,9167S), JAK1 (3332S), JAK2 (3230S), STAT1 (14994S), STAT3 (12640S), COX-2 (4842S), iNOS (13120), GAPDH (5174S) and β-actin (4970S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The anti-phospho-IκB (IKK; S176/177, ab194528) antibody was purchased from Abcam (Cambridge, UK). Secondary antibodies coupled to IRDye 800 fluorophore used in the western blot analysis (926-3221 and 926-32210) were obtained from LI-COR Biosciences (Lincoln, NE, USA). The Alexa Fluor^® 555 goat anti-rabbit IgG secondary antibody used in the confocal microscopy experiment was obtained from Invitrogen (Z25305; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Murine macrophage RAW264.7 cells were purchased from Kunming Cell Bank of Type Culture Collection, Chinese Academy of Sciences (Kunming, China) and cultured in high glucose Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc.), 100 µg/m streptomycin and 100 U/ml penicillin at 37°C in 5% CO₂. The cells were passaged every 2 days.

Cell viability was detected using a Cell Counting Kit-8 (CCK-8; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's protocol. Briefly, RAW264.7 cells were treated at 37°C with different concentrations of aloin (20, 40, 80, 100, 200 and 400 µg/ml) for 24 h and then incubated at 37°C with 10 µl CCK-8 working solution for 2 h. The absorbance was detected using a Multiskan™ GO spectrophotometer (Thermo Fisher Scientific, Inc.) at 450 nm. All experiments were repeated in triplicate, and the data are presented as mean ± standard deviation (SD).

Following a 2 h pre-treatmentat 37°C with different aloin concentrations (100, 150 and 200 µg/ml), RAW264.7 cells were seeded at a density of 1 ×10⁶/well in 12-well cell culture plates and stimulated with LPS (100 ng/ml) for 16 h at 37°C. The levels of TNF-α, IL-1β and IL-6 in the cell culture supernatants were detected using TNF-α (P06804), IL-1β (P10749) or IL-6 (P08505) ELISA kits (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer's protocol. The experiments were repeated in triplicate for all aloin concentrations. The results are presented as mean ± SD.

RAW264.7 cells were pre-treated at 37°C with aloin (100, 150 and 200 µg/ml) for 2 h followed by a 16 h LPS treatment. NO production in the cell culture medium was determined using a Total Nitric Oxide Assay kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol. The absorbance at 540 nm was measured using a Multiskan™ GO spectrophotometer (Thermo Fisher Scientific, Inc.). Each experiment was repeated in triplicate for all aloin concentrations.

RAW264.7 cells were seeded at a density of 2 ×10⁵/well in 12-well cell culture plates. Following aloin (100,150 and 200 µg/ml) pre-treatment at 37°C, the cells were stimulated with LPS for 30 min, and intracellular total ROS was detected using a Reactive Oxygen Species Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, following treatment, the cell culture medium was removed, and dichloro-dihydro-fluorescein diacetate (DCFH-DA) was added to a final concentration of 10 µM. Then, the cells were incubated in a CO₂ incubator for 20 min at 37°C and washed 3 times with PBS to completely remove the DCFH-DA from the cells. ROS production was observed by inverted fluorescence microscopy (magnification, x 100; Olympus Corporation, Tokyo, Japan) and quantified using ImageJ software version 1.46 (National Institutes of Health, Bethesda, MD, USA). The experiments were repeated in triplicate.

The nuclear and cytoplasmic proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction kit (P0028; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The extraction of total intracellular protein was as follows: Cells were pretreated with aloin (100, 150, 200 µg/ml) for 2 h, then stimulated with 100 ng/ml LPS for different times (30 min, 4 or 16 h) at 37°C. Pre-treated RAW264.7 cells were washed twice with ice-cold PBS and lysed in ice-cold cell lysis buffer (P0013; Beyotime Institute of Biotechnology) including 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃VO₄ and leupeptin. Following lysis on ice for 30 min, the lysates were centrifuged (14,300 × g) for 15 min at 4°C, and the protein samples were quantified using a BCA/Bradford assay. Then, equal amounts (50 µg) of protein were denatured in SDS and electrophoresed on a 12% SDS-PAGE prior to transferring to a nitrocellulose membrane (Pall Corporation, Port Washington, NY, USA). The membrane was blocked in 5% skimmed milk dissolved in TBST for 1 h at room temperature. Following washing with TBS and 0.1% Tween^®-20 3 times, the membrane was incubated overnight at 4°C with primary antibodies diluted 1:500 with TBST, followed by incubation with IRDye 800-conjugated IgG secondary antibodies (1:5,000; LI-COR Biosciences) at room temperature for 1 h. The antigen-antibody complex was visualised using an Odyssey^® infrared imaging system (LI-COR Biosciences). ImageJ version 1.46 software (National Institutes of Health) was used for the densitometry analysis.

Intracellular total RNA was extracted from RAW264.7 cells using TRIzol^® reagent (Life Technologies; Thermo Fisher Scientific, Inc.), and a RevertAid™ First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) was used to synthesise cDNA. The PCR primers were: 5′-GGGTCTTGTTCACTCCACGG-3′ (iNOS forward), and 5′-GCTCAGAACAGCACAAGGGG-3′ (iNOS reverse); 5′-GGAGAGTGTTTCCTCGTCCC-3′ (GAPDH forward), and 5′-ACTGTGCCGTTGAATTTGCC-3′ (GAPDH reverse). The thermocycling conditions were as follows: 94°C for 3 min, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min and 72°C for 1 min with a final extension step at 72°C for 10 min. The PCR products were detected by agarose gel (1.5%) electrophoresis and visualized with GoldView (Service Biological Technology Co., Ltd., Wuhan, China). The image was captured by the Gel Doc™ EZ imager (Bio-Rad Laboratories, Inc., Hercules, CA, USA). ImageJ version 1.46 software (National Institutes of Health) was used for densitometry analysis.

RAW264.7 cells were seeded in a small confocal laser dish at 500 cells/well. RAW264.7 cells were pre-treated with 200 µg/ml aloin for 2 h, and then stimulated with LPS for 4 h at 37°C. Following treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilised with 0.2% Triton X-100, blocked with 3% bovine serum albumin in PBS for 1 h at room temperature and incubated with STAT1 and STAT3 primary antibodies diluted 1:100 with PBS overnight at 4°C. Following rinsing with PBS, the cells were incubated with a goat anti-rabbit IgG Alexa Fluor^® 555 conjugated fluorescent secondary antibody diluted 1:200 with PBS for 1 h in the dark at room temperature. Finally, the cells were stained at room temperature with 0.1 µg/ml DAPI for 3 min in the dark. Images were captured using a TCS SP8 confocal microscope (magnification, ×200; Leica Microsystems GmbH, Wetzlar, Germany).

All data were presented as mean ± standard deviation. Statistical analyses were performed using Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). The results were analyzed by one-way analysis of variance followed by a post hoc Tukey's test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The present study investigated aloin cytotoxicity using a CCK-8 cell viability assay. Aloin did not affect cell viability, even at a high dose of 200 µg/ml (Fig. 1B). As iNOS and COX-2 are considered to be associated with LPS stimulation (7), the effects of aloin on LPS-induced iNOS and COX-2 expression were investigated. RAW264.7 cells were pre-treated with different doses of aloin for 2 h, then treated with LPS for 16 h, and the iNOS and COX-2 levels were determined by western blot analysis. Aloin treatment suppressed iNOS expression dose-dependently but did not affect COX-2 expression (Fig. 2A). According to these results, aloin concentrations of 100, 150 and 200 µg/ml were selected for subsequent experiments. RT-PCR and western blot analyses were used to detect iNOS expression levels in RAW264.7 cells pre-treated with different aloin doses (100, 150 and 200 µg/ml) for 2 h and then LPS for 6 or 16 h. Aloin markedly attenuated iNOS expression at mRNA and protein levels in a dose-dependent manner (Fig. 2B and C).

To investigate the anti-inflammatory effect of aloin, the levels of pro-inflammatory cytokines and mediators, including TNF-α, IL-1β, IL-6 and NO in LPS-stimulated RAW264.7 cells were first determined. The obvious increasement of the levels of TNF-α, IL-1β, IL-6 and NO resulting from the LPS stimulation were inhibited by aloin in a dose-dependent manner (Fig. 3A–D). Collectively, these results suggest that aloin inhibited pro-inflammatory cytokine and mediator release, exhibiting an anti-inflammatory effect.

It has been suggested that the JAK-STAT, MAPK and NF-κB signalling pathways are involved in LPS-triggered inflammatory responses (7,10,20). Therefore, to investigate the underlying molecular mechanism of aloin-based inhibition of LPS-induced inflammatory reactions, the effects of aloin on LPS-activated STAT and MAPK signalling pathways were determined. Firstly, the phosphorylation levels of MAPKs and STATs following LPS stimulation of RAW264.7 cells were assessed. LPS stimulation was performed for different time periods, and western blot analysis was used to detect MAPK and STAT activation. At 10 min of LPS stimulation, p38 MAPK, ERK and JNK were activated, peaking at ~30 min visually (Fig. 4A). To additionally explore the effects of aloin on MAPK phosphorylation induced by LPS, the cells were pre-treated with aloin for 2 h and LPS for 30 min. Activation was detected by western blot analysis. LPS-induced phosphorylation of p38 MAPK, ERK and JNK were not affected by aloin treatment (Fig. 4B). A previous study demonstrated that NF-κB became activated following the activation of its upstream kinase IKK and IκB regions (21,22). To explore the effects of aloin on the LPS-induced NF-κB signaling pathway, levels of IκB kinase (IKK) and IκB phosphorylation were determined by western blot analysis. The results of the present study demonstrated that aloin exhibited a minimal effect on the phosphorylation levels of upstream IKK and IκB (Fig. 4C). Therefore, the transcriptional activity of downstream transcription factor NF-κB was not examined. STAT1 and STAT3 phosphorylation increased at 0.5 h, peaked at 4 h and was sustained until 6 h following LPS stimulation as observed visually (Fig. 4D). The increased levels of STAT1 and STAT3 phosphorylation were markedly decreased by aloin in a dose-dependent manner (Fig. 4E). Collectively, these data suggested that aloin attenuated the LPS-triggered inflammatory response by suppressing STAT1 and STAT3 activation, but not MAPKs, IKK or IκB.

As STAT transcription factors have been determined to be activated by the JAKs (23), the effects of aloin on JAK signals were investigated. RAW264.7 cells were stimulated with LPS for different time intervals, and JAK1 and JAK2 phosphorylation was determined by western blot analysis. JAK1 and JAK2 phosphorylation increased at 5 min and peaked at ~15 min following LPS treatment (Fig. 5A). RAW264.7 cells were also incubated with aloin for 2 h and treated with LPS for 15 min to detect JAK1 and JAK2 activation by western blot analysis. Aloin pre-treatment suppressed LPS-induced JAK1 phosphorylation, whereas JAK2 phosphorylation was not affected (Fig. 5B). Collectively, these results indicated that aloin inhibited STAT1 and STAT3 phosphorylation, potentially via the inhibition of JAK1 in LPS-induced inflammatory responses.

Activated STATs undergo dimerisation and translocate into the nucleus to initiate transcription. Therefore, the present study investigated whether aloin may inhibit the nuclear translocation of phosphorylated STAT1 and STAT3. The cytoplasmic and nuclear proteins of RAW264.7 cells pre-treated with aloin for 2 h and stimulated with LPS for 4 h were extracted to determine cytoplasmic and nuclear STAT1 and STAT3 levels. LPS stimulation induced the nuclear translocation of STAT1 and STAT3 and aloin pre-treatment significantly inhibited their nuclear translocation (Fig. 6A). A confocal microscopy experiment was performed to detect the location of STAT1 and STAT3. In the control and aloin groups, STAT1 and STAT3 were primarily localised in the cytoplasm, indicated by red staining. In the LPS-treated cells STAT1 and STAT3 transferred into the nuclei, as indicated by blue staining patterns (Fig. 6B and C). However, the levels of nucleocytoplasmic translocation of STAT1 and STAT3 induced by LPS were markedly decreased by aloin. Collectively, the results indicated that aloin inhibited LPS-induced inflammatory responses by suppressing JAK1-STAT1/3 activation and the nuclear translocation of STAT1 and STAT3, at least partially.

ROS are crucial to LPS-induced inflammation in RAW264.7 cells through the activation of STAT transcription factors (23), and Simon et al (24) suggested that ROS production contributing to JAK-STATs activation. Furthermore, a previous study has revealed that aloin exhibits an antioxidan effect (25). Therefore, the present study investigated whether the anti-inflammatory effect of aloin was due in part to its inhibition of ROS accumulation. RAW264.7 cells were pre-treated with aloin for 2 h and stimulated with LPS for 30 min. A ROS detection kit was used to assess ROS accumulation. Aloin significantly decreased LPS-induced ROS production in a dose-dependent manner (Fig. 7). The data of the present study demonstrated that aloin may function as an antioxidan. The anti-inflammatory mechanism of aloin may involve the inhibition of ROS-mediated JAK1-STAT1/3 signalling pathway activation.