Obacunone (C₂₆H₃₀O₇, MW: 454.516, CAS: 751-03-1, HPLC purity ≥ 98%) was obtained from Meilun Biological Technology Co., Ltd. (Dalian, China). RAW264.7 mouse macrophage cells and NCM460 human colonic epithelial cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640, and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, United States). DEPC water, LPS, formalin, paraformaldehyde, and ethanol were from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) and Tween 20 were obtained from Sangon Biotech Company (Shanghai, China). Antibodies against p-p65 (#SC-33039), p-IκBα (#SC-8404), and actin (#SC-47778) were from Santa Cruz Biotechnology (CA, United States). The anti-TLR4 (ab13556) and inducible nitric oxide (iNOS, ab129372) antibody were purchased from Abcam (Cambridge, MA, United States), while the anti-ZO-1 (A11417) and anti-occludin (A2601) antibodies were purchased from ABclonal Technology (Wuhan, China). All the other antibodies were from Cell Signaling Technology (Danvers, MA, United States), as follows: COX-2 (#12282P), IFN-γ (#3159), TNF-α (#3707S), and MyD88 (#42835). All reagents for quantitative polymerase chain reaction (qPCR) and the Reverse Transcriptase Kit were from Takara Biotechnology (Shiga, Japan). The Cell Counting Kit 8 (CCK-8) was from Meilun Biological Technology Co., Ltd. All other reagents were obtained from Thermo Fisher Scientific (Waltham, MA, United States).

Healthy C57BL/6 mice (8 weeks of age, 20–22 g) were purchased from the Shanghai Laboratory Animal Center. All mice were maintained in specific pathogen-free facility and kept under controlled conditions at a humidity of 60–70% and stationary temperature of 23–25°C with a 12 h light/dark cycle and with access to autoclaved food and drinking water. This study was carried out in accordance with the principles of the declaration recommendations of the Animal Experimentation Ethics Committee at Shanghai University of Traditional Chinese Medicine (Animal license key: SYXK2014-0008).

All mice were randomly divided into the following six groups (n = 10 mice per group): vehicle control group, obacunone-only group (100 mg/kg), dextran sulfate sodium (DSS) group, and three obacunone-treated groups (low dosage, middle dose, and high dose, respectively). The dosages of obacunone were 25, 50, and 100 mg/kg/day per body weight, respectively. Acute experimental colitis was induced in mice by administration of 3.5% DSS (MW: 36,000–50,000 Da, MP Biomedicals, Irvine, CA, United States) for 7 days as previously described (Yue et al., 2018). Obacunone was dissolved in 0.5% methylcellulose and administered by oral gavage once a day, starting from 2 days before DSS treatment and continuing to the end of the experiment. Body weight and bloody diarrhea were recorded daily. Mice were euthanized under anesthesia after the last oral gavage. The entire colon was removed and the total length was measured. After that, the distal colon tissues were collected for hematoxylin–eosin (H&E) staining. And the images were taken by the Olympus DP20 optical digital microscope camera (Tokyo, Japan). Histological injury was assessed by a combined score of inflammatory cell infiltration (score 0–3) and epithelial damage (score 0–3) using a double-blind method as described previously (Luo et al., 2017).

Murine peritoneal macrophage RAW264.7 cells were cultured in DMEM supplemented with 10% FBS under 5% CO₂ at 37°C. Cells were treated with different concentrations of obacunone (0–100 μM) for 24 h. NCM460 human colonic epithelial cells were cultured in RPMI-1640 supplemented with 10% FBS under 5% CO₂ at 37°C. NCM460 cells were treated with different concentrations of obacunone (0–100 μM) for 2 h followed by coincubation with tumor necrosis factor (TNF-α) (20 ng/mL) for an additional 22 h. A CCK-8 assay was then performed to measure cell viability. The absorption values were measured at 540 nm using a microplate reader.

RAW264.7 cells were incubated with different concentrations of obacunone (0–100 μM) for 2 h before being stimulated by LPS (1 μg/mL) for an additional 22 h. The supernatant was then collected and a Griess assay was performed to measure the relative NO secretion levels in each group. Finally, the absorbance of each well was measured at 450 nm using a microplate reader as previously described (Luo et al., 2017).

Colon segments (∼1.5 cm near the anus) or cultured cells were homogenized or lysed in lysis buffer (Thermo Fisher Scientific, Mannheim, MA, United States) containing protease and phosphatase inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, GER). The lysate was centrifuged (4°C, 12,000 × g, 15 min) and the supernatant was collected. The procedure for immunoblotting was performed as previously described (Yue et al., 2018). In brief, proteins (30 μg) was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked in 5% (w/v) skim milk for 2 h at room temperature and immunoblotted with primary antibody. Then blots were washed and incubated with HRP-coupled secondary antibody at room temperature. Finally, the blots were observed by enhanced chemiluminescence (ECL) detection reagents. Protein expressions were analyzed by a GS-700 imaging densitometer (Bio-Rad, CA, United States). β-actin (Santa Cruz, CA, United States) was used as an internal control.

RAW264.7 cells were treated with different concentrations of obacunone (0–100 μM) for 2 h before being stimulated by LPS (1 μg/mL) for an additional 22 h. Total RNA was extracted from cultured cells using TRIzol reagent. Reverse transcription and qPCR were carried out using SYBR Premix ExTaq Mix in an ABI Prism 7900HT Sequence Detection System (Life Technologies, Carlsbad, CA, United States) as previously described (Dou et al., 2012). The following thermal cycler parameters were used: 1 cycle of 95°C for 30 s and 40 cycles of denaturation (95°C, 5 s) and combined annealing/extension (60°C, 30 s). Relative mRNA expression levels were calculated by the comparative Ct method, and the values were normalized as the ratio of the optimal density relative to β-actin. The sequences of the primers were listed in Table 1.

Mice feces were collected and stored at −80°C. Genomic DNA was extracted from the fecal samples using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, GA, United States), according to the manufacturer’s protocol. A NanoDrop 2000 UV-vis spectrophotometer (Thermo Fisher Scientific, DE, United States) was used to assess the concentration and quality of the DNA. The V3–V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) in a GeneAmp 9700 ABI thermocycler PCR system (Carlsbad, CA, United States). PCR reaction was conducted using the following cycling conditions: an initial denaturation at 95°C for 3 min, followed by 27 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 45 s, and a final extension at 72°C for 10 min. Sequencing was performed using the high-throughput Illumina MiSeq platform (Illumina, CA, United States) according to standard protocols (Majorbio, Shanghai, China). Raw fastq files were quality-filtered by Trimmomatic and merged by FLASH. All the results were based on sequenced reads and operational taxonomic unit (OTU) clustering was performed with a 97% similarity cutoff and a 70% confidence threshold.

Significance between groups was evaluated by one-way analysis of variance (ANOVA) using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, United States). All data were presented as the mean ± standard deviation (SD). p-values < 0.05 (two-sided) were considered significant (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001). All the 16S rDNA sequencing data were analyzed on the online Majorbio I-Sanger Cloud Platform¹.

Dextran sulfate sodium-induced colitic mice exhibited body weight loss, accompanied by diarrhea, and bloody stools, compared with vehicle- or obacunone only-treated mice. However, treatment with obacunone (50 or 100 mg/kg) significantly attenuated disease symptoms in DSS-treated mice, including weight loss, diarrhea, bloody stool, and colonic shortening (Figures 1A–D). Moreover, the mice in the obacunone (100 mg/kg)-only treatment group showed an almost identical disease hallmark to that of vehicle-treated mice, indicating that obacunone had no obvious toxic side effects.

Additionally, examination of pathological colon tissue sections showed that DSS treatment resulted in severe intestinal epithelial injury, including crypt loss, mucosal ulceration, muscle thickening, and neutrophil infiltration. Obacunone (25, 50, or 100 mg/kg)-treated mice presented reduced loss of mucosal architecture, fewer ulcerations, and less cellular infiltration (Figures 2A,B). Because obacunone treatment at 100 mg/kg produced the best phenotypes, 100 mg/kg treatment group was used in the subsequent experimental analyses.

Bacterial 16S rRNA gene sequencing was used to evaluate the effect of obacunone on DSS-induced changes in gut microbiota composition. Principal component analysis (PCA) revealed that each group was clustered separately (Figures 3A,B). The Shannon index was used to characterize the overall microbial diversity. The results showed that microbial diversity was significantly decreased in DSS-treated mice, whereas obacunone treatment mitigated these changes (Figure 3A), although the difference was not significant. The major intestinal bacteria at the phylum level included Firmicutes, Bacteroidetes, Proteobacteria, Deferribacteres, Cyanobacteria, and Verrucomicrobia (Figure 3C). Moreover, DSS treatment decreased the relative abundance of Bacteroidetes and enriched the abundance of Proteobacteria compared with that of the vehicle-treated group; however, obacunone treatment mitigated the DSS-induced phylum-level changes Supplementary Table 1. At the genus level, the DSS-treated group exhibited proportional decreases in the abundances of Lachnospiraceae-NK4A136-group, Rikenellaceae-RC9-gut-group, and (Eubacterium)-fissicatena-group, but these changes were attenuated by obacunone administration (Figures 4A,B). Furthermore, the abundance of some pathogenic bacteria such as Escherichia–Shigella and Enterococcus, was significantly increased in DSS-treated group, whereas the abundance of Escherichia–Shigella and Enterococcus was decreased as a result of obacunone treatment (Figures 4A,B and Supplementary Table 2). Collectively, our results revealed that DSS treatment disturbed gut microbiota homeostasis, and this gut microbiota imbalance could be reversed by obacunone treatment.

To assess the integrity of the intestinal barrier in colitic mice, we determined the expression levels of the tight junction proteins, TJP1 and occludin, in colonic tissue of DSS-induced colitic mice. The results showed that the expression levels of TJP1 and occludin were markedly downregulated in DSS-treated mice (Figures 5A,B). However, obacunone treatment significantly rescued the reduced expression of these barrier proteins, indicating that obacunone might exert a protective effect against DSS-induced disruption of intestinal epithelial barrier integrity.

To investigate the mechanisms underlying obacunone-associated attenuation in DSS-induced intestinal inflammation, we evaluated the effect of obacunone on the protein expression levels of inflammatory mediators. Significant increase in the expression levels of nitric oxide synthase, cyclooxygenase 2 (COX-2), interferon gamma (IFN-γ), and TNF-α was observed in the colon of DSS-induced colitic mice (Figures 6A,B). However, obacunone administration greatly reduced the expression levels of these inflammatory mediators in the colons of colitic mice. Moreover, the protein expression levels of TLR4, myeloid differentiation primary response gene 88 (MyD88), p-p65 (a subunit of NF-κB), and p-IκBα (an inhibitor of NF-κB) in colonic tissue was significantly increased after DSS treatment (Figures 6C,D). However, all these abnormally increased expression levels were markedly downregulated in colitic mice with obacunone treatment. Combined, these results indicated that obacunone exerted a protective effect against DSS-induced colitis through attenuation of TLR4/NF-κB signaling cascades.

RAW264.7 mouse macrophages were used to further evaluate the anti-inflammatory effects of obacunone. The initial cytotoxicity evaluation suggested that obacunone was almost non-cytotoxic at dosages up to 100 μM (Figure 7A). Additionally, we found that obacunone markedly suppressed LPS-stimulated NO production in RAW264.7 cells in a concentration-dependent manner (0–100 μM) (Figure 7B). Meanwhile, the protein expression levels of the proinflammatory mediators iNOS and COX-2 were significantly increased in RAW264.7 cells after exposure to LPS. However, obacunone concentration-dependently decreased the LPS-induced increase in the levels of iNOS and COX-2 (Figures 7C,D). We then quantified the mRNA expression levels of pro-inflammatory mediators. The results showed that the mRNA levels of IL-1α, IL-1β, IL-16, COX-2, and iNOS were markedly upregulated in LPS-stimulated macrophages; however, obacunone treatment attenuated the LPS-induced increase in the mRNA levels of these proinflammatory mediators (Figure 7E).

The human colonic epithelial cell line NCM460 is commonly used to investigate the mechanisms of inflammatory damage and repair in intestinal epithelial barrier (Tsoi et al., 2017). We found that obacunone markedly increased NCM460 cell viability in a concentration-dependent manner (Figure 8A). Further, our study showed that TNF-α treatment downregulated the expression levels of the tight junction proteins TJP1 and occludin; however, these decreased expression levels were significantly reversed by obacunone treatment (Figure 8B).