Influence of Rosiglitazone on the Expression of PPARγ, NF-κB, and TNF-α in Rat Model of Ulcerative Colitis

Aim. To observe the disease activity index (DAI) and the colonic mucosa damage index (CMDI), detect the colonic mucosal expression of PPARγ, NF-κB, and TNF-α in rats with ulcerative colitis (UC), and to investigate the protective role of rosiglitazone in UC. Methods. Sprague-Dawley (SD) rats were divided into three groups: a control group, a rosiglitazone treatment group, and a UC model group. Rats were sacrificed on days 7, 14, 21, or 35 following administration of treatment after enema and DAI, CMDI and colonic expression of PPARγ, NF-κB, and TNF-α were assessed. Results. In the UC model group, DAI, CDMI and the colonic expression of NF-κB and TNF-α increased significantly compared to the control group at all timepoints, but PPARγ decreased significantly. Furthermore, in the rosiglitazone treatment group, DAI and CMDI decreased significantly on the 14-day, 21-day, and 35-day timepoints compared to the UC model group; the colonic expression of NF-κB and TNF-α decreased compared to UC model group at all timepoints, but the PPARγ expression increased significantly. Conclusions. Rosiglitazone can alleviate colonic mucosal inflammation and have the protective role on UC by upregulating PPARγ expression and downregulating NF-κB and TNF-α expression.


Introduction
UC is a chronic inflammatory condition of the intestinum crassum whose etiology remains unknown. Cytokines are crucial components of the inflammatory pathways that take place during the active and chronic phases of UC.
NF-κB is a key regulator of inducible expression of many genes that are involved in immune and inflammatory responses in the intestinal tract [1]. NF-κB can activate anti-apoptotic genes and repress the apoptosis of some inflammatory cells, thereby elongating and worsening tissue inflammatory injury [2]. TNF-α is an important proinflammatory cytokine which is involved in systemic inflammation, and stimulates acute phase reactions [3]. Alterations in the regulation of TNF-α, especially TNF-α overexpression, have been implicated in a variety of symptoms associated with UC. Inflammation, anorexia, and weight loss are all associated with increased levels of TNF-α expression [4].
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of transcription factors. The antidiabetic drugs thiazolidinediones (TZDs) are ligands for the γ subtype of PPARs. High levels of PPARγ protein are expressed by the colonic epithelium. Recent evidence suggests a possible anti-inflammatory effect of PPARγ ligands, including TZDs, particularly in the colon [5]. Treatment with PPARγ ligands has been demonstrated to attenuate inflammatory cytokine production, inflammatory cell proliferation [6].
We conducted rats model of UC induced by the mixture solution of 2,4,6-trinitrobenzenesulfonic acid solution (TNBS) and ethanol through enema, and after these rats were treated with rosiglitazone, a kind of TZDs, by gastric lavage, the colonic mucosal expression of PPARγ, NF-κB, and TNF-α were detected to demonstrate the protective role of rosiglitazone to rats with UC.

Animal Treatment.
A total of 72 SD rats were randomly divided into three groups: a control group, a rosiglitazone treatment group, and a UC model group, each group had 24 rats. The SD rat model of UC was established by administering the mixture solution of TNBS (100 mg/kg) and 50% ethanol (0.25 mL) by enema. The control group was subject to enema and gastric lavage with normal saline. For the rosiglitazone treatment group, TNBS was administered by enema and rosiglitazone was administered by gastric lavage (8 mg/kg once a day). The UC model group was subjected to TNBS enema and the gastric lavage was saline control. Rats from all groups were sacrificed on days 7, 14, 21, and 35, 6 rats per group were sacrificed each day after the enema and colonic tissue, 2.0-10.0 cm from the anus, was collected for HE staining, RT-PCR and immunohistochemistry analysis. Rats were kept in a normally controlled breeding room with standard laboratory food and water for one week before the experiments. The rats were maintained in accordance with internationally accepted principles for laboratory animal use.
Rats were sacrificed at the timepoints indicated and the entire colon was excised from the cecum to the anus and opened longitudinally. Macroscopic damage was evaluated using a validated (CMDI) scoring system with slight modifications [8]. The numerical rating score was as follows: 0, no inflammation; 1, local hyperemia and edema without ulcers or erosions on mucosal surface; 2, ulceration without hyperemia; 3, ulceration and adhesions at one site; 4, two or more sites of inflammation and ulceration extending >1 cm; 5, ulceration extending more than 2 cm.

Pathological Observation.
Paraffin-embedded and formalin-fixed colon samples were cut into 4-μm thick sections for HE staining and pathological observation.

PPARγ, NF-κB, and TNF-α mRNA Expression
. mRNA was extracted from colonic tissue samples using Trizol according to the manufacturer's protocols (Invitrogen) and reverse transcription polymerase chain reaction (RT-PCR) was done according to the instructions of the Takara RNA PCR kit3.0 (AMV). An equal amount of cDNA from each sample was amplified using primers specific to each gene (Table 1). DNA amplification was done using a thermocycler under the following conditions: for PPARγ, 35 cycles of denaturation at 94 • C for 30 s, annealing at 60 • C for 60 s, and extension at 72 • C for 60 s; for NF-κB p65, 35 cycles of denaturation at 94 • C for 30 s, annealing at 59 • C for 30 s, and extension at 72 • C for 60 s; for TNF-α, 35 cycles of denaturation at 94 • C for 45 s, annealing at 54 • C for 30 s, and extension at 72 • C for 60 s; for β-actin, 35 cycles of denaturation at 94 • C for 30 s, annealing at 62 • C for 30 s, and extension at 72 • C for 60 s. RT-PCR products were measured by photodensitometry using a gel image analysis system after agarose gel electrophoresis and ethidium bromide staining.

Measurement of PPARγ, NF-κB, and TNF-α Protein
Expression. Immunohistochemistry was done according to the Max Vision kit protocol. Image analysis software (Imagepro plus 6.0) was used to measure the light density of positive control cells in which the cytoplasm was tan-yellow or brown after 3,3'-diaminobenzidine (DAB) staining. For each section, the positive integrated optical density (IOD) and total area of five representative visual fields without overlap were observed under high-power microscope (×400). The ratio of IOD and total area represents the mean value of   optical density, with a higher ratio indicating a higher level of protein expression.

Statistical Analysis.
Data showed a normal distribution and are expressed as means ± standard deviation. The responses of different experimental groups were analyzed using one-way ANOVA. Spearman correlation analysis was used to study the relationship between PPARγ, NF-κB, and TNF-α. P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 19.0 for windows.

Results of DAI and CMDI.
The DAI and CMDI of the UC model group were significantly higher than that of the control group (P < 0.05) at all timepoints. The DAI and CMDI of the rosiglitazone treatment group were reduced significantly on days 14, 21, and 35 than that of the UC model group (P < 0.05; Table 2). Figure 1, the colonic mucosa structure was intact in the control group. Mucosa and submucosa defects could be seen with infiltrations of inflammatory neutrophils and lymphocytes in the lamina propria of the model group on day 14. The degree of mucosal inflammation relieved in the rosiglitazone treatment group on day 14. Regenerated epithelia covered the surface of ulcer and the mucosa tended to be complete. The base of ulcer was filled with tissues and scars.

Results of RT-PCR.
In the control group, expression of PPARγ mRNA in the colonic mucosa was significantly higher compared to the UC model group (P < 0.05), but the NF-κB and TNF-α expression was significantly less (P < 0.05). In the rosiglitazone treatment group, PPARγ mRNA expression increased significantly than that of the model group on days 7, 14, 21, and 35 (P < 0.05), but the NF-κB and TNF-α expression reduced significantly (P < 0.05) ( Table 3).

Results of Immunohistochemistry.
In the control group, expression of PPARγ protein in the colonic mucosa was significantly higher compared to the UC model group (P < 0.05), but the NF-κB and TNF-α expression was significantly less (P < 0.05). In the rosiglitazone treatment group, PPARγ protein expression increased significantly than that of the model group on days 7, 14, 21, and 35 (P < 0.05), but the NF-κB and TNF-α expression reduced significantly (P < 0.05) ( Table 4; Figures 2, 3 and 4).

Correlation of PPARγ, NF-κB, and TNF-α Protein Expression in Rosiglitazone Treatment Group. Correlation studies
showed that the expression of PPARγ protein was significantly negatively correlated with that of NF-κB protein. The correlating factor was −0.881 (P < 0.05). The NF-κB protein expression was significantly positively correlated with that of TNF-α. The correlating factor was 0.943 (P < 0.05).

Discussion
UC is a chronic inflammatory condition of the intestinum crassum that etiology remains unknown. Cytokines are crucial components of the inflammatory pathways that take  Figure 3: Colonic mucosal NF-κB protein expression in the control group, UC model group, and rosiglitazone treated group on days 7, 14, 21, 35. In the control group, expression of NF-κB protein in the colonic mucosa was significantly less compared to the UC model group (P < 0.05). In the rosiglitazone treatment group, NF-κB protein expression reduced significantly than that of the model group on days 7, 14, 21, and 35 (P < 0.05).
PPARγ is predominantly detected in adipose tissue, intestine, and macrophages. A growing body of evidence suggests that PPARγ play a role in inhibiting inflammation. Impaired expression of PPARγ in colonic epithelial cells in UC and increased expression in hypertrophic mesenteric adipose tissue in Crohn's disease (CD) have been reported [9]. Decreased gene expression of PPARγ was found from rectal biopsies in patients with active UC and its expression was negatively correlated with the severity of endoscopic disease activity [10].
Our results demonstrated that the colonic mucosal expression of PPARγ in the control group was significantly higher compared to the UC model group, either on the mRNA level or protein level. The evidence suggests that PPARγ plays an important protective role in the process of UC. The molecular mechanisms by which PPARγ regulates inflammatory response are not fully understood. PPARγ has been proposed as a key inhibitor of colitis through attenuation of NF-κB activity [9]. Ricote et al. demonstrated that PPARγ inhibits iNOS and MMP-9 expression by antagonizing the AP-1, signal transducer, and activator of transcription (STAT) and NF-κB pathways [11]. In a recent paper, it was shown that, similar to that reported for PPARα, PPARγ inhibits NF-κB-driven transcription by physically interacting with both p65 and p50 [12].
The antidiabetic drugs thiazolidinedione (TZDs) are ligands for the γ subtype of PPARs. Previous in vitro, in vivo, and uncontrolled human studies have suggested a potential anti-inflammatory effect of PPARγ ligands including TZDs in the colon [5]. Pedersen and Brynskov showed that rosiglitazone (a kind of TZDs) enemas could improve impaired PPARγ activity in inflamed colonic epithelium and have beneficial clinical effect in patients with active ulcerative colitis [13]. In our study, the results showed that the DAI and CMDI of the rosiglitazone treatment group reduced significantly on days 14, 21, and 35 compared to the UC model group, and meanwhile, PPARγ expression increased significantly on days 7, 14, 21, and 35, but the NF-κB and TNF-α expression reduced significantly. We verified that rosiglitazone can alleviate the UC symptoms as well as mucosal inflammation and ulceration through improve the colonic mucosal expression of PPARγ.
Furthermore, we used Spearman correlation analysis to study the relationship between PPARγ, NF-κB, and TNF-α protein expression in rosiglitazone treatment group. Correlation studies showed that the PPARγ expression was negatively correlated with NF-κB. The NF-κB protein expression was significantly positively correlated with that of TNF-α. The result might imply that impaired activity or decreased expression of PPARγ in UC colonic mucosa was deprived of the suppressive capability of NF-κB, which led to the high expression of NF-κB transfer from cytoplasmic form to nuclei and then upregulate the expression of proinflammatory factors such as TNF-α.
In conclusion, rosiglitazone can alleviate colonic mucosal inflammatory response and has the protective role on UC by upregulating PPARγ colonic expression and downregulating NF-κB and TNF-α expression.