Geniposide, a component of Gardenia jasminoides Ellis, inhibits NF-ĸB to attenuate LPS-induced injury in intestinal epithelial cells

Background: The nuclear factor-ĸB (NF-ĸB) transcriptional system is a major effector pathway involved in inammatory responses. Previous studies found that a Gardenia decoction (GD) inhibited the expression of NF-κB in a lipopolysaccharide (LPS)-stimulated mouse intestinal injury model. Herein, we hypothesized that geniposide (GE), a component of Gardenia jasminoides Ellis, also exerts anti-inammatory effects and inhibits NF-ĸB activity in LPS-induced intestinal epithelial cells (IEC-6). IEC-6 cells were stimulated with LPS, following which the effects of GE on NF-ĸB signaling in the IEC-6 cells were examined by western blotting to detect IĸB phosphorylation/degradation. The expression of NF-κB was determined by immunouorescence assay (IFA). Enzyme-linked immunosorbent assay (ELISA) was used to detect the inhibitory effect of GE on the release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) activated by LPS in IEC-6 cells. In addition, the migration ability of IEC-6 cells was observed by the scratch method. Results: These results showed that GE dose-dependently downregulated levels of the proinammatory cytokines TNF-α, IL-6 and IL-1β that had been upregulated by LPS and suppressed the phosphorylation of IĸB and NF-ĸB induced by LPS. Our ndings indicated that GE could reduce LPS-induced NF-ĸB signaling and proin ﬂ ammatory expression in IEC-6 cells and signicantly enhance the migration of IEC-6 cells. Moreover, GE inhibited the of NF-κB, nuclear transfer, and transcriptional activity in IEC-6 cells.


Background
The nuclear factor-ĸB (NF-ĸB) family is a group of nuclear protein factors that regulate the expression of a wide range of genes. These nuclear protein factors play an important role in regulating the transcription of genes related to in ammation; cell proliferation, differentiation and apoptosis; the immune response; and tumorigenesis. In most cells, NF-ĸB proteins can form homodimers or heterodimers that bind IĸBs in an inactive state in the cytoplasm. When stimulated by stimulants such as viruses, radiation, oxygen free radicals, bacterial lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α), the NF-ĸB-IĸBs complex is activated, NF-ĸB is released and transferred to the nucleus, and its binding at speci c sites promotes transcription of the corresponding genes, thus initiating and regulating the immune response, cell adhesion, differentiation, proliferation, apoptosis and in ammation.
Expression of the NF-ĸB promoter gene involves the activation of IKK, phosphorylation and ubiquitination of IĸB, cleavage of IĸB, migration of NF-ĸB to the nucleus, and binding of NF-ĸB to DNA. Overactivation of NF-ĸB can lead to a series of diseases, including asthma, rheumatoid arthritis, enteritis and so on.
Inhibiting signal transduction pathways with a core of NF-ĸB through drugs or gene transformation may be an effective way to prevent and treat some diseases. Some traditional Chinese medicine extracts, such as avonoids [1] and steroid-like compounds [2], can strongly block IKK activity, thus inhibiting the phosphorylation and degradation of IĸB and blocking the activity and protein-binding ability of NF-ĸB.
These compounds have advantages in the treatment of some in ammatory diseases.
In previous studies, we found that Gardenia decoction (GD) markedly inhibited in ammatory cytokine production and reduced the expression of NF-ĸB following LPS stimulation in vivo [3]. Geniposide (GE), the main active ingredient of Gardenia jasminoides, is a kind of iridoid terpenoid glucoside [4]. Modern studies have shown that GE has substantial effects on digestive system, cardiovascular system and central nervous system diseases. In addition, GE has certain anti-in ammatory and therapeutic effects on soft tissue injury. However, the mechanism through which GE mediates its anti-in ammatory effects is not completely understood.
In this study, we investigated the role of GE in protection against LPS using in vitro cultured IEC-6 cells.
Our results showed that GE exerts an anti-in ammatory effect and that GE blocks the activation of NF-ĸB signaling by targeting IĸB in IEC-6 cells in vitro.

Methods
Reagents GE (purity > 98% SG8100) was purchased from Solarbio Co. (Beijing, China). LPS (Escherichia coli 055: B5) was obtained from Sigma Chemical Co. (St. Louis, MO, USA). G. jasminoides Ellis extract was obtained according to a previous method [3] with slight modi cations. In the previous experiment, we determined the composition and content of G. jasminoides Ellis extract by LC/MS Analysis. Brie y, the mixture was ltered through a 100-mesh metal sieve. The ltrate (crude decoction) was sterilized using a and 100 U•mL − 1 penicillin) and 10 µg•mL − 1 bovine insulin. Cells were maintained at 37°C in a 5% CO 2 atmosphere. After 24 h, the cells were harvested, plated on 96-well plates at a density of 5,000 cells/well and then treated with LPS, GE and GD at different concentrations. After 24 h of incubation, 20 µL of MTT (5 mg/mL) was added to each well, and the cells were incubated for 4 h at 37 °C. The medium was replaced with 100 mL of dimethyl sulfoxide (DMSO), and the cells were incubated for 5 min while shaking. Cellular viability was determined by measuring the optical density (OD) at 490 nm with a spectrophotometer.
Cell treatment IEC-6 cells were cultured in 96-well plates at a density of 5 × 10 3 cells/well. The cells were divided into a normal control group, LPS model group, GD treatment group (treated with different concentrations of GD) and GE treatment group (treated with different concentrations of GE). After pretreatment with GD and GE for 24 h, LPS was added to the cell culture for 12 h. Cells were then collected for subsequent testing.

Cell migration assays
The IEC-6 cells were seeded into 12-well cell culture plates for 24 h at 37 °C in a humidi ed atmosphere containing 5% CO2, at a concentration of 5 × 10 3 cells/mL, and cultured in a medium containing 10% FBS to produce a nearly con uent cell monolayer. A linear wound was subsequently generated in the monolayer using a cutting knife in the monolayer, a scratch was made and the cell scraped off the other side. Any cellular debris was removed by washing the wells with PBS. Place the dish in a tissue culture incubator at 37 °C for 12 h. After incubation, the dish was placed under a phase-contrast microscope, the photographed region was aligned, and images were acquired. The acquired images of each sample were further quantitatively analyzed with computational software. The number of cells across each scratch was then determined using Image-Pro Plus software.

Statistical analysis
Data are expressed as the means ± SDs and were analyzed by one-way ANOVA. The mean separation was assessed by Tukey's multiple comparison tests (GraphPad Prism version 5 software). Differences with P < 0.05 were considered statistically signi cant.

MTT assays
The chemical structure of GE is shown in Fig. 1A [3]. The results of MTT assays showed that the activity of IEC-6 cells treated with GD and GE alone was not signi cantly different with than of IEC-6 cells in the normal control group (P > 0.05) (Fig. 1B, C); however, 25 µg/mL LPS signi cantly inhibited the activity of IEC-6 cells (P < 0.01) (Fig. 1D). Therefore, LPS with concentration of 10 µg/mL was selected to prepare IEC-6 cells induce model.

GE promoted the migration of IEC-6 cell in vitro
The effect of GE on cell migration after injury is shown in Fig. 2. Treatment with GD (100 µg/mL) or GE (100 µM) remarkably promoted cell migration compared to that in the normal control group (P < 0.01, Fig. 2A, B). The LPS-induced injury of IEC-6 cells signi cantly inhibited cell migration, and the migration rate in the LPS group at 12 h decreased to 64% of that of the control group (Fig. 2).

GE inhibited in ammatory cytokine production in IEC-6 cells stimulated by LPS
We measured the effect of GD and GE on levels of the in ammatory cytokines TNF-α, IL-6 and IL-1β using ELISAs. A signi cant increase in the levels of TNF-α, IL-6 and IL-1β in IEC-6 cells induced by LPS treatment was detected compared with their levels in the control group (P < 0.01). Compared with LPS model group, the expression of in ammatory cytokines in GD group was signi cantly lower (Fig. 3A). Furthermore a signi cant dose-dependent decrease in in ammatory cytokine production in the GE treatment group compared with the LPS model group (Fig. 3B) (P < 0.01).
GE inhibited the NF-ĸB / IĸB-α proteins pathway IEC-6 cells As can be seen from Fig. 4, Compared with the normal control group, LPS signi cantly activated the phosphorylation of p-NF-κB p65 and p-IκB-α protein (P < 0.01), while GD (Fig. 4A) inhibited the phosphorylation of p-NF-κB p65 and p-IκB-α protein induced by LPS. The effect of GE on p-NF-κB p65 and p-IκB-α protein was consistent with the trend of GD (Fig. 4B).

GE inhibited the LPS-induced translocation of NF-κB in IEC-6 cells
As shown in Fig. 5, NF-κB was obviously retained in the cytoplasm of IEC-6 cells in the normal control group. The translocation of NF-κB to the nucleus was increased, and the immunostaining signal in the cytoplasm after LPS stimulation was weaker than that in the normal control group (Fig. 5). However, treatment with GE (100 µm) prevented the effects of LPS on nuclear NF-κB translocation. Discussion GE, a bioactive iridoid glucoside derived from G. jasminoides, has anti-in ammatory effects [5]. In sepsis model mice, GE directly bound LPS and neutralized LPS in vitro, and signi cantly protecting against sepsis [6]. Gardenia, a plant belonging to the Rubiaceae family, contains various iridoid glycosides, which are unstable, nonvolatile, thermally labile, polar natural compounds [7]. The fruit of G. jasminoides Ellis (gardenia fruit) is an Asian folk medicine that has been widely used for centuries for the treatment of in ammation, jaundice, headache [8], edema, fever, hepatic disorders [9] and hypertension [10]. The iridoid compounds GE and genipin were found to be the anti-in ammatory components of gardenia fruit.
Both GE and genipin have anti-in ammatory activities, as shown via their inhibition of exudate and nitric oxide (NO) production in a carrageenan-induced rat paw edema model. Genipin suppressed both NO production and cyclooxygenase expression at an early stage and exhibited stronger anti-in ammatory activity than GE [11]. Genipin-mediated inhibition of NO production has been associated with the inhibition of NF-κB activation [12]. However, the material basis of the anti-in ammatory activity gardenia fruit is not completely clear.
In this study, we investigated the impact of GE on LPS signaling in IEC-6 cells and elucidated its mechanism of action.
Activation of the NF-κB signaling pathway can induce expression of the proin ammatory cytokines IL-6 [13], TNF-α, and IL-1β [14], and these proin ammatory factors can further activate NF-κB, thus aggravating in ammation [15]. IL-6, TNF-α and IL-1β are important cytokines secreted by cells used to evaluate the in ammatory response. The secretion of IL-6, TNF-α and IL-1β is signi cantly increased in in ammatory cells [16,17]. Much evidence indicates that LPS-induced in ammation is closely related to increased secretion of the in ammatory factors IL-6, TNF-α [18] and IL-1β [19]. Therefore, we detected the levels of IL-6, TNF-α and IL-1β in IEC-6 cells treated with LPS to observe the effects of LPS-induced in ammation.
In this study, LPS induction was used to establish a model of in ammation in IEC-6 cells. LPS treatment increased IL-6, TNF-α and IL-1β secretion by IEC-6 cells and thus upregulated in ammation in IEC-6 cells. We found that GE at a certain concentration could downregulated the expression levels of IL-6, TNF-α and L-1β in LPS-damaged IEC-6 cells. Therefore, we speculated that GE at this concentration can effectively regulate the expression levels of IL-6, IL-1β and TNF-α through the IκB/NF-κB signaling pathway to further control the occurrence of in ammatory reactions.
NF-κB is an important transcription factor that regulates the in ammatory response. At reset, NF-κB binds its inhibitor, IκB, and has no biological activity [20]. When cells are stimulated by LPS, IκB-mediated inhibition of NF-κB is relieved, and the NF-κB complex is dissociated and transferred to cells. After binding the corresponding site on speci c DNA, it can induce the transcription of related genes and promote the release of cytokines, thus generating a systemic in ammatory response [21]. The research shows that GD could reduce the level of in ammatory cytokines in the injury model and had antiin ammatory effects on tissues [3]. However, the treatment of LPS-induced IEC-6 cells with GE has not been investigated. Western blot analysis showed that the protein expression of p-IĸB-α was increased, and the distribution of p-NF-κB p65 was signi cantly increased in IEC-6 cells stimulated by LPS. GE prevented activation of the NF-κB pathway and decreased the nuclear transfer of p-NF-κB p65 in cells induced by LPS (Fig. 6).
Cell migration plays an important role in the repair of intestinal mucosal injury. IEC-6 cells have been widely used in the study of intestinal mucosal repair [22]. Scholars have conducted in-depth and systematic research on the physiological regulation [23], signaling pathways [24] and pathology [25]