Thymol mitigates lipopolysaccharide-induced endometritis by regulating the TLR4- and ROS-mediated NF-κB signaling pathways

The purpose of this study was to investigate the effects of thymol on lipopolysaccharide (LPS)-induced inflammatory responses and to clarify the potential mechanism of these effects. LPS-induced mouse endometritis was used to confirm the anti-inflammatory action of thymol in vivo. RAW264.7 cells were used to examine the molecular mechanism and targets of thymol in vitro. In vivo, thymol markedly alleviated LPS-induced pathological injury, myeloperoxidase (MPO) activity, and the production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in mice. Further studies were performed to examine the expression of the Toll-like receptor 4 (TLR4) -mediated nuclear factor-κB (NF-κB) pathway. These results showed that the expression of the TLR4-mediated NF-κB pathway was inhibited by thymol treatment. In vitro, we observed that thymol dose-dependently inhibited the expression of TNF-α, IL-1β, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW264.7 cells. Moreover, the results obtained from immunofluorescence assays also indicated that thymol dose-dependently suppressed LPS-induced reactive oxygen species (ROS) production. Silencing of TLR4 inhibited NF-κB pathway activation. Furthermore, H2O2 treatment increased the phosphorylation of p65 and IκBα, which were decreased when treated with N-acetyl cysteine or thymol. In conclusion, the anti-inflammatory effects of thymol are associated with activation of the TLR4 or ROS signaling pathways, contributing to NF-κB activation, thereby alleviating LPS-induced oxidative and inflammatory responses.


INTRODUCTION
Endometritis, defined as inflammation of the uterus, is a prevalent disease in high-producing dairy cows and consequently results in economic losses [1,2]. The endometrium is the first line of defense against invading pathogenic microorganisms, which cause histological lesions and inflammation of the endometrium [3]. E. coli is the major pathogenic bacteria in uterine infection, which produces the endotoxin lipopolysaccharide (LPS) [4]. LPS, an essential constituent of the outer membrane of Gram-negative bacteria, is one of the most efficient stimulators in the immune system [5]. It has been reported that LPS can induce TLR4 signaling pathway activation, which subsequently leads to an overexpression of inflammatory cytokines [6]. TLRs are important factors in the innate immune response and quickly activate multiple pathways upon recognition of microbial pathogens [7]. Many studies have demonstrated that when the expression of TLR4 is stimulated by LPS, it induces nuclear factor (NF)-κB pathway activation [8,9]. NF-κB is important for inflammatory responses and mediating the secretion of cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), to aggravate inflammatory damage [10,11].
Previous studies have shown that reactive oxygen species (ROS) serve as second messengers and participate in a large number of signal transduction pathways, including those of NF-κB [12,13]. Pro-inflammatory cytokines, such as IL-1β and TNF-α, promote ROS Research Paper generation and deteriorate the inflammatory response [14]. Moreover, COX-2 and iNOS also play a predominant role in LPS-stimulated inflammation [15]. Jung et al. have reported suppression of LPS-induced ROS excessive secretion via obstruction of the expression of iNOS and COX-2 [16]. Therefore, this pathway may be used as the theoretical basis for a potential drug for the treatment of inflammatory diseases.
Thymol (2-isopropyl-5-methylphenol, Figure 1A), a phenol derivative, is a main component of the essential oil of thyme and is widely used as a fragrance agent in a variety of household products [17]. It has wide-ranging pharmacological actions, such as anti-diabetic, antiinflammatory, and antioxidant effects [18][19][20]. Although some studies on the antioxidant effects of thymol have been reported [21,22], no research has shown the effect of thymol on LPS-induced endometritis in mice. In addition, macrophages stimulate and secrete cytokines during the inflammation cycle and then regulate the innate inflammatory system [23]. Thus, in the present study, mice with LPS-induced endometritis in vivo and RAW264.7 macrophages in vitro were used to examine the antiinflammatory molecular mechanism of thymol.

In vivo study: Effect of thymol on LPS-induced histopathological changes
The severity of LPS-inducedendometritis was determined by histological analysis using H&E staining. Uterus morphology was observed in each group ( Figure  2A). Compared with the control group ( Figure 2F), the LPS group showed a more severely damaged uterus, with extensive inflammatory cell infiltration ( Figure 2B). However, the inflammatory cell infiltration was reduced, and the structure of the uterus was comparatively complete in the thymol groups ( Figure 2C-E). Effect of thymol on MPO activity and the production of inflammatory mediators MPO, an early marker in the prediction of inflammatory diseases, reflects the level of inflammation and oxidative stress [24]. As shown in Figure 3A, after administration with LPS, the MPO activity was greatly increased in the LPS group. However, the MPO activity was reduced in a dose-dependent manner by pretreatment with thymol at concentrations of 10, 20 and 40 mg/kg.
The expression of the pro-inflammatory mediators IL-1β and TNF-α were detected using a qPCR assay of the LPS-induced endometritis treatment. The results showed that the levels of TNF-α and IL-1β were significantly increased after LPS treatment, whereas treatment with thymol dose-dependently reduced the expression of these cytokines ( Figure 3B).

Effect of thymol on the expression of TLR4
To detect whether thymol could inhibit the inflammatory response by suppressing TLR4 expression, western blotting was performed to determine TLR4 expression. As shown in Figure 4A, these results showed that the expression of TLR4 was inhibited by thymol in LPS-induced endometritis.

Effect of thymol on NF-κB pathway activation
It is well known that the NF-κB signaling pathway plays a vital role in the production of inflammatory cytokines. To examine whether the suppression of the inflammatory response by thymol occurs via inhibition of the NF-κB pathway activation, western blotting was performed. The results indicated that thymol markedly inhibited the phosphorylation of p65 and IκBα proteins ( Figure 4B). To confirm these results, immunofluorescence assays were performed to determine if p65 translocated to the nucleus. As shown in Figure 5, the expression of p65 was significantly decreased in the nucleus with the administration of thymol.

In vitro study: Effect of thymol on cell viability
The potential cytotoxicity of thymol on RAW264.7 cells was determined using the MTT assay. These results showed that cell viability was not affected by thymol administration ( Figure 6A).

Effect of thymol on cytokines levels
The expression of the cytokines TNF-α and IL-1β in RAW264.7 cells was determined using ELISA. These results indicated that the expression of TNF-α and IL-1β was significantly increased in the LPS group. However, treatment with thymol dose-dependently decreased the levels of TNF-α and IL-1β ( Figure 6B). Moreover, iNOS and COX-2 are often used as inflammatory markers. We detected the expression of the cytokines iNOS and COX-2 in RAW264.7 cells using a qPCR assay. As shown in Figure 6C, LPS treatment significantly increased the expression of iNOS and COX-2. However, the expression of iNOS and COX-2 were dose-dependently inhibited by pretreatment with thymol.

Effect of thymol on ROS production
ROS, as signaling molecules involved in various processes, play a pivotal role in the inflammatory regulation [25]. Thus, we measured the generation of ROS to evaluate the antioxidant effect of thymol. As shown in Figure 7, these results showed that thymol dosedependently suppressed LPS-induced ROS production in RAW264.7 cells.

Effects of thymol on TLR4-mediated NF-κB pathway activation
Since TLR4 plays a critical role in the regulation of the LPS-induced inflammatory pathway, the expression of TLR4 in LPS-stimulated RAW264.7 cells was detected by western blot. These results suggested that the expression of TLR4 was remarkably increased after being treated with LPS, which was dose-dependently down-regulated by thymol ( Figure 8A).
It is well known that NF-κB participates in the regulation of inflammation. To examine the antiinflammatory molecular mechanism of thymol, activation of the NF-κB pathway was assessed. As shown in Figure  8B, compared with the LPS group, the phosphorylation of the p65 and IκBα proteins was inhibited by thymol administration.

Effect of thymol on the TLR4-dependent or TLR4-independent pathways
To further confirm whether the effect of thymol on the NF-κB pathway is TLR4-dependent or TLR4-independent, specific interference RNA (TLR4-si) was used to silence TLR4 expression. When TLR4 was silenced, phosphorylation of NF-κB, p65 and IκBα were detected in RAW264.7 cells that had been treated with LPS or H 2 O 2 . These results showed that LPS induced excessive expression of TLR4, which was subsequently reduced by TLR4-si, and the LPSinduced phosphorylation of p65 and IκBα was also decreased by TLR4-si and thymol (40 μg/mL) ( Figure 9A, 9B). ROS are involved in the regulation of NF-κB pathway activation and therefore elicit a wide spectrum of responses [26]. H 2 O 2 is a strong oxidant and is often used as a representative ROS in modeling and inducing oxidative stress [27]. Thus, we also determined the effect of ROS on NF-κB activation in H 2 O 2 -stimulated, TLR4-si treated RAW264.7 cells by western blot. Interestingly, these results suggested that H 2 O 2 treatment increased the phosphorylation of p65 and IκBα; however, phosphorylation of these proteins was decreased by pre-treatment with NCA or thymol (40 μg/mL) ( Figure 10A). Moreover, further studies were performed to determine the translocation of NF-κB p65 in TLR4-si RAW264.7 cells that had been challenged with LPS using immunofluorescence assays (as shown in Figure 10B). In addition, LPS increased the expression of NF-κB downstream cytokines, IL-1β and TNF-α, which was dose-dependently alleviated by thymol ( Figure 10C). The above results indicate that thymol inhibits NF-κB pathway activation in a TLR4-dependent or TLR4independent manner.

DISCUSSION
ROS play essential roles in protection from the invasion of microbial pathogens and are thought to be responsible for tissue damage, inflammation and cell signaling pathways [28,29]. A recent study suggested that neutralization of ROS or inhibition of the redox pathway could relieve inflammation [30]. LPS, a bacterial cell wall component, is known to induce the production of several inflammatory cytokines, tissue edema, and injury [31]. As one of the most important immune cells, macrophage activation is a hallmark of inflammation and produces ROS, which trigger signal transduction pathways [32]. Although there have been many reports that have extensively described the anti-inflammatory function of thymol, a Chinese herbal medicine [19,33], a detailed analysis of its molecular targets has not been completed until now. In the present study, an LPS-induced endometritis mouse model was first used to evaluate the anti-inflammatory effects of thymol in vivo, and its complex mechanism was uncovered in LPS-stimulated macrophages in vitro.
Thus far, researchers have paid more attention to traditional Chinese medicine due to its ability to lower toxicity and its increased efficacy in the treatment of inflammatory diseases [6,34]. The results of in vivo experiments have indicated that thymol alleviates inflammatory injury and decreases MPO activity in LPSinduced mouse endometritis.
Inflammation is beneficial for the host defense against infection; however, excessive inflammatory response leads to injury [35]. Secretion of the cytokines IL-1β and TNF-α increases dramatically during the process of inflammatory pathological development [6,24]. IL-1β, the master regulator of inflammation, is well     to induce inflammation by activating a cascade of molecular pathways [36]. In addition, TNF-α, iNOS and COX-2 were also markedly increased in LPS-stimulated inflammatory disease [34]. In vitro studies revealed that thymol inhibited the expression of TNF-α, IL-1β, iNOS and COX-2 in LPS-stimulated RAW264.7 cells. Moreover, some reports have indicated that these proinflammatory cytokines are mainly activated by the NF-κB signaling pathway, which is also mediated by ROS [12,25]. NF-κB, a nuclear transcription factor, is a regulator  [37]. Upon stimulation, NF-κB moves to the nucleus and induces the expression of various genes, including IκBα [38]. In the present study, thymol administration inhibited the phosphorylation of IκBα and p65 in LPS-induced mouse endometritis and RAW264.7 cells.
TLR4, a Type I transmembrane receptor, is activated by LPS and plays an integral role in the innate immune system [6]. It is well known that LPS activates the TLR4-mediated signaling pathway and leads to the activation of NF-κB. Previous studies have reported that the activation of NF-κB is regulated by ROS in LPS-induced inflammation [39]. Therefore, we sought to investigate whether the anti-inflammatory mechanism of thymol inhibited the activation of NF-κB via TLR4-independent or TLR4-dependent pathways. In the present study, we found that LPS increased the expression of TLR4, which was reduced by thymol treatment. When TLR4 was silenced, phosphorylation of p65 and IκBα were decreased by TLR4-si and thymol (40 μg/mL) in LPS-induced RAW264.7 cells. H 2 O 2 is a strong oxidant and induces excessive ROS production; therefore, it contributes to signaling cascades, such as the NF-κB pathway [40]. Moreover, N-acetyl cysteine (NAC) is an efficient antioxidant and is often used as a positive control [32]. We observed that phosphorylation of p65 and IκBα was greatly increased in the H 2 O 2 treatment group, and phosphorylation of these proteins was decreased by NCA or thymol (40 μg/mL). Taken together, these results indicate that thymol inhibited the activation of NF-κB via TLR4-mediated or ROSregulated signaling pathways.
In conclusion, the current study clearly demonstrates that thymol can effectively inhibit the expression of pro-inflammatory cytokines in LPS-induced mouse endometritis. The promising anti-inflammatory effect of thymol on LPS-stimulated RAW264.7 cell is relevant to TLR4-mediated or ROS-dependent NF-κB signaling pathways, thereby suppressing LPS-induced inflammatory responses.

Animal treatment and experimental groups
A total of 50 female BALB/c mice (8 weeks old, approximately 25 g in weight) were used in this study. The mice were obtained from Hubei Provincial Center for Experimental Animal Research (Wuhan, China). The mice were fed a standard diet and housed in a temperaturecontrolled room with a 12 h dark/light cycle for one week prior to the experiments. The present study was performed according to the guidelines of the Huazhong Agricultural University Animal Care Committee and the care and use of Laboratory Animals published by the US National Institutes of Health.
Mice were randomly divided into the following 5 groups with 10 mice in each group to establish the model of endometritis: a control group, thymol (10, 20, and 40 mg/kg) + LPS groups, and an LPS group. Thymol was solubilized by dimethyl sulfoxide (the DMSO concentration in the solution was not greater than 0.1%) to obtain final concentrations of 10, 20, and 40 mg/kg. The method for inducing the endometritis model in mice was performed as previously described [34]. Briefly, each side of the mouse uterus was perfused with 50 μL of LPS (1 mg/mL) to induce endometritis. After 24 h, the blank group received normal saline. The thymol groups received an intraperitoneal injection of differing concentrations of thymol (10, 20, and 40 mg/kg) three times (once every six hours). Next, the mice were sacrificed by CO 2 inhalation, and the uterus tissues were collected and stored at -80°C.

Histopathological assessment
Uterus tissues were isolated, and approximately one centimeter-sized tissue was fixed in10% formalin for subsequent histopathological experiments. The tissues were dehydrated, paraffin embedded, and then cut into 5-μm-thick sections for hematoxylin and eosin (H&E) staining. Finally, the sections were observed using an optical microscope (Olympus, Japan).

Myeloperoxidase (MPO) analysis
The MPO activity was measured according to the manufacturer's instructions. Briefly, uterine tissue, weighing approximately 100 mg, was fixed with phosphate buffered saline (PBS, weight/volume ratio 1:19) and homogenized. The supernatants were analyzed using the MPO kit (Jiancheng biotechnology, China) and detected at an absorbance value of 460 nm.
Cell culture and treatment RAW264.7 cells were purchased from the American Type Culture Collection (Manassas, USA)

Cytokine analysis
The uterine tissues were homogenized in pre-chilled PBS, centrifuged and the supernatants were collected. RAW264.7 cells were seeded onto a 6-well-plate, and the cells were treated as indicated. The tissue and cell supernatants were harvested to detect the expression of IL-1β and TNF-α using ELISA kits (Bio-Swamp, China) according to the manufacturer's instructions. The absorbance value was read at 450 nm using a microplate reader (Thermo, USA).

Determination of ROS in RAW264.7 cells
ROS production in RAW264.7 cells was measured using the oxidative conversion of cell permeable 2′, 7′-dichlorofluorescein diacetate (DCFH-DA, Beyotime, China) to fluorescent dichlorofluorescein (DCF). Cells were seeded at a density of 1×10 5 cells mL -1 into 6-well plates. Next, they were incubated with control media or 1 μg/mL LPS in the presence or absence of thymol (10, 20, and 40 μg/mL) for 3 h. The cells were incubated with DCFH-DA for 30 min at 37°C and then washed three times with PBS to remove extracellular DCFH-DA. The DCF fluorescence was observed using a fluorescence microscope (Leica, Germany).

Quantitative PCR assay
Total RNA was extracted from RAW264.7 cells with TRIzol (Invitrogen, USA). Next, cDNA was synthesized using a reverse transcription kit (Takara, Japan) and primers, which are shown in Table 1 . The PCR reaction (40 cycles) was performed using a SYBR qPCR Mix (Roche, Swiss). The housekeeping gene GAPDH served as an internal standard. The relative quantification of the target gene expression levels was calculated using the 2 -ΔΔCt comparative approach.

Western blotting analysis
The total proteins of the uterine tissue and RAW264.7 cells were extracted with a RIPA lysis solution containing a phosphatase repressor. The concentration of proteins was determined using a BCA kit. Next, samples with equal amounts of protein were fractionated on a 10% SDS polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane. The membrane was incubated in blocking buffer (5% skim milk) and subsequently treated with primary antibody (1:1000 dilution) at 4°C overnight and then washed three times with PBS for 30 min. Next, the membranes were incubated with secondary antibody for 1 h at room temperature, and the protein expression levels were determined using the ECLPlus western blot Detection System. β-actin served as an internal standard.

TLR4-siRNA transfection
Negative control siRNA mice and TLR4-siRNA mice were obtained from Ribo (Guangzhou, China). For TLR4-siRNA transfection, RAW264.7 cells were seeded onto 6-well plates and allowed to reach approximately 70% confluency. Transfection was performed in Opti-MEM with NC-siRNA or TLR4-siRNA using Lipofectamine™ 2000 (Invitrogen, USA) for 6 h, and the medium was replaced with fresh RPMI-1640. Next, the treatment group was pre-treated with thymol (40 μg/ mL) for 1 h, and then LPS (1 μg/mL) was added for 3 h. For the LPS group, cells were treated with LPS for 3 h. H 2 O 2 (1 mM) or N-acetyl cysteine (NAC, 15 mM) was considered the negative or positive ROS control, respectively, in order to compare similar processes as described in a previous study [41], and the cells were cleaved for further analysis.

Immunofluorescence staining
Uterine tissues were fixed in 10% buffered formaldehyde for 24 h and then embedded in paraffin. Tissue sections were permeabilized with PBS containing 0.3% Triton X-100 (Sigma, USA) and 10% BSA. RAW264.7 cells (1×10 5 cells mL -1 ) were seeded onto a twelve-well-plate. After the cells were treated as indicated, immunofluorescence staining was performed. Sections of tissue and cells were incubated with special antibody for p-p65 (1:100) overnight at 4°C and then incubated with a Cy3 secondary antibody (1:200) in the dark for 2 h at 25°C. Next, p-p65 protein was mounted using a mounting medium supplemented with 4,6-diamidino-2-phenylindole (DAPI, Beyotime, China) for nuclear counterstaining and observed using fluorescence microscopy (Olympus, Japan). www.impactjournals.com/oncotarget

Statistical analyses
Statistical data were represented as the mean ± S.E.M. Data were assessed using Student's t-test or oneway analysis of variance (ANOVA). p-value ≤ 0.05 were considered a statistically significant difference.