LPS (L-2880:B5) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rhein (purity >98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). ELISA kits used to detect the inflammatory cytokines IL-1β, IL-6, IL-8, IL-10, and TNF-α were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The antibodies directed against IL-1β, IL-6, IL-8, IL-10, TNF-α, TLR-4, and β-actin, as well as the 3,3′-diaminobenzidine (DAB) reagent were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). The TLR4 inhibitor TAK-242 was purchased from EMD Millipore (Billerica, MA, USA). SDS-PAGE and polyvinylidene fluoride (PVDF) membranes were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The Enhanced Chemiluminescence (ECL) Advanced Western Blotting Detection kit was purchased from GE Healthcare Life Sciences (Chalfont, UK). All other reagents used in the present study were purchased from Sigma-Aldrich. Anti-NF-κB p65 (phospho S536; cat.no. ab28856) and total anti-NF-κB p65 antibody (cat. no. ab16502) were purchased from Abcam Trading (Shanghai) Company Ltd. (Shanghai, China).

Four-week-old male BALB/c mice, weighing 18–22 g, were provided by Shanghai Laboratory Animals Center Co., Ltd. (Shanghai, China). The mice were maintained in controlled conditions at 25±1°C, a relative humidity of 45% with 12 h light/dark cycles, and were given ad libitum access to food and water. The mice were randomly assigned to five experimental groups, consisting of a normal control group, an LPS-stimulated model group, a rhein group, a TLR4 inhibitor group and a rhein + TLR4 inhibitor TAK-242 group (n=10, each group). The mice in each group were orally administered 100 mg/kg rhein (14), and 0.3 mg/kg TAK-242 (15) for 1 h prior to being injected intraperitoneally with 20 mg/kg LPS (16) for a further 18 h. The mice were subsequently anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital (Merck KGaA, Darmstadt, Germany), prior to having their blood samples taken from the retinal vein. The mice were sacrificed under anesthesia with ketamine at 65 mg/kg and xylazine at 6.5 mg/kg⁻¹, i.p. (Jiancheng Bioengineering Institute) and 1 cm from the rectum and 1 cm from the cecum were harvested in order to evaluate the changes in intestinal injury. The length of the colons were measured and recorded. The present study was approved by the Institutional Animal Care and Use Committee of Zhengzhou People's Hospital, (Zhengzhou, China).

The extent of intestinal mucosal injury was graded following inspection under a microscope (Olympus IX71; Olympus, Tokyo, Japan), according to the standard classification described by Chiu et al (17). Intestinal injury was graded as follows: 0, normal mucosal villi; 1, the gap in the epithelium increased, and capillary congestion at the tip of villi was observed; 2, the gap in the epithelium was expanded with moderate separation of the epithelium and lamina propria; 3, severe separation of the epithelium and lamina propria with damage to the villus tip; 4, villi were damaged and the capillaries exposed in the lamina propria, an increase in the cellular components of the lamina propria was also observed; and 5, the intrinsic layer was damaged with incomplete bleeding and ulcers.

Sections of 4 μm formalin-fixed, paraffin-embedded colon tissue was cut for immunohistochemistry. Following deparaffinization in xylene, the tissue sections were hydrated using graded ethanol (100, 95 and 70%). The sections were subsequently washed three times in deionized H₂O for 1 min at room temperature, prior to being incubated in 3% hydrogen peroxide for 10 min, in order to block the endogenous peroxidase activity. Each section was then sequentially washed with phosphate-buffered saline (PBS) for 5 min prior to being blocked with 5% horse serum (Gibco-Brl, Gaithersburg, MD, USA). The sections were then incubated overnight at 4°C with 1:100 dilution of primary rabbit anti-TLR4 antibody (cat. no. sc-16240; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Following 3–5 min washes with PBS, the sections were treated with biotin-conjugated goat anti-rabbit secondary antibody for 2 h at 37°C. The sections were then treated with an avidin biotin enzyme reagent (Wuhan Boster Biological Technology, Ltd.) for 30 min, prior to being washed three times with PBS. The sections were subsequently stained with DAB and DAB enhancer, prior to staining with hematoxylin for 5 sec. Dehydration was performed using ethanol and xylene. Finally, the sections were covered with a glass coverslip and observed under an Olympus IX71 microscope.

The colon tissue samples were harvested from the BALB/c mice of each group, prior to being frozen and stored at −80°C until further use. The colon tissue samples were homogenized in ice-cold radioimmunoprecipitation lysing buffer (Sigma-Aldrich) and were centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was subsequently collected in order to examine protein expression levels using western blot analysis. Protein concentrations were determined using a bicinchoninic acid protein assay reagent kit (Pierce Biotechnology, Inc., Rockford, IL USA). Equal amounts of supernatant protein (40 μg) were separated by 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were blocked with 5% skimmed dry milk in tris-buffered saline with 0.05% Tween^® 20 for 1 h, and were then co-incubated overnight at 4°C with the following primary antibodies IL-1β (1:1,000; cat. no. sc-7884), IL-6 (1:1,000; cat. no. sc-1265), IL-8 (1:1,000; cat. no. sc-8427), IL-10 (1:1,000; cat. no. sc-1783), TNF-α (1:500; cat. no. sc-65440), TLR-4 (1:500; cat. no. BA1717), Phospho-NF-kB p65 (1:500), total NF-kB p65 (1:500) and β-actin (1:400; cat. no. BM0005). The membranes were subsequently washed with PBS supplemented with 0.05% Tween^® 20 and incubated for 2 h at room temperature with horseradish peroxidase-bound secondary antibodies (1:1,000; Santa Cruz Biotechnology, Inc.). β-actin was used as a loading control. Finally, the proteins were visualized using ECL reagents, and the intensity of the immunoreactive bands was quantified and normalized to the respective β-actin content using Image Pro Plus (IPP) software (Media Cybernetics, Inc., Rockville, MD, USA).

Following sacrifice, blood samples of the mice were collected in heparinized tubes and centrifuged at 10,000 × g for 10 min in order to obtain the plasma. The colon tissue samples of the mice in each group were subsequently harvested, weighed, and homogenized in iced saline. The samples were then centrifuged at 10,000 × g for 10 min in order to obtain the supernatant. The levels of inflammatory cytokines IL-1β, IL-6, IL-8, IL-10, and TNF-α present in the supernatants were assayed by ELISA, according to the manufacturer's instructions. The optical densities of the samples were detected using a microplate reader (ELx800; BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 450 nm.

The data from at least three experiments were presented as the mean ± standard deviation. The statistical significance of the differences between the experimental groups was analyzed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance and post hoc Tukey tests were used to analyze the data. For all experiments, P<0.05 was considered to indicate a statistically significant difference.

Following stimulation with 20 mg/kg LPS, the length of the murine colon was markedly shortened, as compared with that of the normal mice (P<0.01). However, following treatment with 100 mg/kg rhein, the length of the colon was significantly increased. Notably, treatment with either 0.3 mg/kg TLR4 inhibitor TAK-242 alone or together with rhein, also increased the length of the damaged colon (P<0.05 and P<0.01, respectively). However, no statistically significant difference was observed between the mice treated with rhein alone, or the mice co-treated with TAK-242 (Table I).

LPS, the main endotoxin present during sepsis in the case of severe systemic inflammatory syndrome, has been shown to induce colon damage. As demonstrated in Table I, significant damage was observed in the LPS-induced mice, as compared with the normal mice. However, this damage was notably attenuated following treatment with rhein in the presence or absence of TAK-242 (P<0.01). These results support the hypothesis that rhein is able to attenuate LPS-induced colon injury.

An LPS-induced inflammatory response was associated with damage to the mouse colon. ELISA was performed in order to detect the expression levels of the following inflammatory cytokines: IL-1β, IL-6, IL-8, IL-10, and TNF-α, in both the plasma and colon tissue samples. As shown in Table II, exposure to LPS markedly increased the concentration of the inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α, whereas it markedly decreased the concentration of IL-10 in the plasma and colon tissue samples, as compared with the control (P<0.01). Notably, the rhein-treated animals exhibited reduced expression levels of IL-1β, IL-6, IL-8, and TNF-α, whereas they exhibited increased expression levels of IL-10, as compared with the LPS model group (P<0.05, P<0.01, respectively). Furthermore, treatment with TLR4 inhibitor TAK-242 significantly attenuated the LPS-induced inflammatory response, increasing IL-10 expression levels and decreasing IL-1β, IL-6, IL-8, TNF-α expression levels. These results were concordant with those of the western blot analysis (Fig. 2). These results suggest that the mechanism underlying the protective effects of rhein during LPS-induced intestinal toxicity is similar to that of TAK-242.

In order to evaluate the effects of rhein on TLR4, immunohistochemistry and western blot analyses were performed to determine the expression levels of TLR4 in the colon. The results from the immunohistochemical imaging of the sections demonstrated the localization of TLR4 to the colon in the LPS-treated mice, which in turn resulted in increased expression levels of TLR-4 (Fig. 3A–D). However, the expression levels of TLR4 were significantly reduced following treatment with rhein, both in the presence and absence of TAK-242 (P<0.01). Notably, the expression levels of TLR4 in the rhein or TAK-242-treated mice were close to normal. In addition, there was no statistically significant difference between the expression levels of TLR4 in the mice co-treated with rhein and TAK-242, as compared with the mice treated with rhein alone.

To further investigate the effects of the rhein signaling pathway on LPS-induced intestinal toxicity during sepsis, the expression levels of both phosphorylated and total NF-κB were analyzed. Following treatment with LPS, the expression levels of the phosphorylated NF-κB were notably increased, as compared with the control. This upregulation was reduced following treatment with 100 mg/kg rhein (P<0.05). However, the expression levels of total NF-κB were not significantly different in any of the four groups, as compared with the control group. Notably, LPS-mediated NF-κB phosphorylation was inhibited by treatment with TAK-242 (Fig. 4).