2.2. MPO Activity and IL-1β Levels in Colon
The colonic inflammation induced by TNBS was characterized by increased levels of colonic MPO and IL-1β. When GSPE or SASP was administered to colitic rats, a significant reduction in colonic MPO activity and IL-1β levels were observed in comparison with TNBS control group (
Figure 2 and
Figure 3). Howere, there was a significant difference between the different GSPE on IL-1β levels, IL-1β of the medium dose of GSPE was the lowest.
Figure 1.
Effects of proanthocyanidins from grape seeds (GSPE) on microscopic of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced rat colitis (original magnification, ×40). n = 12–14 per group. (A). normal control group; (B). 50% Ethanol control group showing oedema of the mucosa; (C). TNBS control group showing complete ulceration of the mucosa, oedema and intense diffuse transmural inflammatory infiltrate; (D). SASP group (500 mg/kg); (E). GSPE-L (100 mg/kg); (F). GSPE-M (200 mg/kg) and (G). GSPE-L (400 mg/kg) showing focal denudation with the presence of granulation tissue near well-preserved mucosa which, in turn, shows a loss of goblet cells.
Figure 1.
Effects of proanthocyanidins from grape seeds (GSPE) on microscopic of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced rat colitis (original magnification, ×40). n = 12–14 per group. (A). normal control group; (B). 50% Ethanol control group showing oedema of the mucosa; (C). TNBS control group showing complete ulceration of the mucosa, oedema and intense diffuse transmural inflammatory infiltrate; (D). SASP group (500 mg/kg); (E). GSPE-L (100 mg/kg); (F). GSPE-M (200 mg/kg) and (G). GSPE-L (400 mg/kg) showing focal denudation with the presence of granulation tissue near well-preserved mucosa which, in turn, shows a loss of goblet cells.
Figure 2.
Colonic MPO activity after treatment with three doses of GSPE in rats with TNBS-induced colitis. Data were means ± SD, n = 12–14 per group. ** Significant at P < 0.01 vs. normal control group. # P < 0.05 and ## P < 0.01 vs. TNBS control group (One-way ANOVA test). MPO: Myeloperoxidase; GSPE: Proanthocyanidins from grape seeds at GSPE-L 100 mg/kg, GSPE-M 200 mg/kg, or GSPE-H 400 mg/kg; TNBS:2,4,6-trinitrobenzenesulfonic acid; SASP: Sulfasalazine.
Figure 2.
Colonic MPO activity after treatment with three doses of GSPE in rats with TNBS-induced colitis. Data were means ± SD, n = 12–14 per group. ** Significant at P < 0.01 vs. normal control group. # P < 0.05 and ## P < 0.01 vs. TNBS control group (One-way ANOVA test). MPO: Myeloperoxidase; GSPE: Proanthocyanidins from grape seeds at GSPE-L 100 mg/kg, GSPE-M 200 mg/kg, or GSPE-H 400 mg/kg; TNBS:2,4,6-trinitrobenzenesulfonic acid; SASP: Sulfasalazine.
Figure 3.
Colonic IL-1β levels after treatment with three doses of GSPE in rats with TNBS-induced colitis. Data were means ± SD, n = 12–14 per group. ** Significant at P < 0.01 vs. normal control group. ## P < 0.01 vs. TNBS control group. †† P < 0.01 vs. GSPE-M group (One-way ANOVA test). IL-1β: Interleukin-1β; GSPE: Proanthocyanidins from grape seeds at GSPE-L 100 mg/kg, GSPE-M 200 mg/kg, or GSPE-H 400 mg/kg; TNBS: 2,4,6-trinitrobenzenesulfonic acid; SASP, Sulfasalazine.
Figure 3.
Colonic IL-1β levels after treatment with three doses of GSPE in rats with TNBS-induced colitis. Data were means ± SD, n = 12–14 per group. ** Significant at P < 0.01 vs. normal control group. ## P < 0.01 vs. TNBS control group. †† P < 0.01 vs. GSPE-M group (One-way ANOVA test). IL-1β: Interleukin-1β; GSPE: Proanthocyanidins from grape seeds at GSPE-L 100 mg/kg, GSPE-M 200 mg/kg, or GSPE-H 400 mg/kg; TNBS: 2,4,6-trinitrobenzenesulfonic acid; SASP, Sulfasalazine.
2.3. The Expression of Colonic IκK and PIκBα
A significant increase of I
κK and PI
κBα expression in cytoplasm of the TNBS control group compared with the normal control group. Compared with those of the TNBS control group, the expression of I
κK and PI
κBα in the 3 GSPE treatment groups and SASP group were decreased (
Figure 4 and
Figure 5), but the decreased expression of PI
κBα in the SASP group did not reach significance in comparison with the TNBS control group (
Figure 5).
Figure 4.
Colonic IκK expression after treatment with three doses of GSPE in rats with TNBS-induced colitis. Data were means ± SD, n = 12–14 per group. ** Significant at P < 0.01 vs. normal control group. # P < 0.05 and ## P < 0.01 vs. TNBS control group (One-way ANOVA test). IκK: Inhibitor kappa B kinase; GSPE: Proanthocyanidins from grape seeds at GSPE-L 100 mg/kg, GSPE-M 200 mg/kg, or GSPE-H 400 mg/kg; TNBS: 2,4,6-trinitrobenzenesulfonic acid; SASP: Sulfasalazine.
Figure 4.
Colonic IκK expression after treatment with three doses of GSPE in rats with TNBS-induced colitis. Data were means ± SD, n = 12–14 per group. ** Significant at P < 0.01 vs. normal control group. # P < 0.05 and ## P < 0.01 vs. TNBS control group (One-way ANOVA test). IκK: Inhibitor kappa B kinase; GSPE: Proanthocyanidins from grape seeds at GSPE-L 100 mg/kg, GSPE-M 200 mg/kg, or GSPE-H 400 mg/kg; TNBS: 2,4,6-trinitrobenzenesulfonic acid; SASP: Sulfasalazine.
2.4. The Expression of Colonic NF-κB
NF-
κB is a heterodimer complex of p50 and p65 subunits and plays a central role in various inflammatory responses. On exposure to various inflammatory stimuli, NF-
κB is activated and translocates from the cytoplasma to the nucleus, where it then regulates the expression of various proinflammation cytokines such as IL-1, MPO. In our study, we measured NF-
κB p65 of nuclear extracts. Rats in the TNBS control group exhibited higher levels of NF-
κB p65 than those in the normal control group. However, the NF-
κB p65 levels were reduced by GSPE treated in a dose-dependent manner, compared with the TNBS control group, the NF-
κB p65 levels in the GSPE-L, GSPE-M and SASP groups were obviously down-regulated (
Figure 6).
Figure 5.
Colonic pIκBα expression after treatment with three doses of GSPE in rats with TNBS-induced colitis. Data were means ± SD, n = 12–14 per group. ** Significant at P < 0.01 vs. normal control group. # P < 0.05 and ## P < 0.01 vs. TNBS control group (One-way ANOVA test). pIκBα: Phospho-I kappaB-alpha; GSPE: Proanthocyanidins from grape seeds at GSPE-L 100 mg/kg, GSPE-M 200 mg/kg, or GSPE-H 400 mg/kg; TNBS: 2,4,6-trinitrobenzenesulfonic acid; SASP: Sulfasalazine.
Figure 5.
Colonic pIκBα expression after treatment with three doses of GSPE in rats with TNBS-induced colitis. Data were means ± SD, n = 12–14 per group. ** Significant at P < 0.01 vs. normal control group. # P < 0.05 and ## P < 0.01 vs. TNBS control group (One-way ANOVA test). pIκBα: Phospho-I kappaB-alpha; GSPE: Proanthocyanidins from grape seeds at GSPE-L 100 mg/kg, GSPE-M 200 mg/kg, or GSPE-H 400 mg/kg; TNBS: 2,4,6-trinitrobenzenesulfonic acid; SASP: Sulfasalazine.
Figure 6.
NF-κB p65 expression of nuclear extracts after treatment with three doses of GSPE in rats with TNBS-induced colitis. Data were means ± SD, n = 12–14 per group. ** Significant at P < 0.01 vs. normal control group. # P < 0.05 and ## P < 0.01 vs. TNBS control group (One-way ANOVA test). NF-κB: Nuclear factor-kappa B; GSPE: Proanthocyanidins from grape seeds at GSPE-L 100 mg/kg, GSPE-M 200 mg/kg, or GSPE-H 400 mg/kg; TNBS: 2,4,6-trinitrobenzenesulfonic acid; SASP: Sulfasalazine.
Figure 6.
NF-κB p65 expression of nuclear extracts after treatment with three doses of GSPE in rats with TNBS-induced colitis. Data were means ± SD, n = 12–14 per group. ** Significant at P < 0.01 vs. normal control group. # P < 0.05 and ## P < 0.01 vs. TNBS control group (One-way ANOVA test). NF-κB: Nuclear factor-kappa B; GSPE: Proanthocyanidins from grape seeds at GSPE-L 100 mg/kg, GSPE-M 200 mg/kg, or GSPE-H 400 mg/kg; TNBS: 2,4,6-trinitrobenzenesulfonic acid; SASP: Sulfasalazine.
2.5. Discussion
It was previously demonstrated that GSPE protects against TNBS-induced colonic damage in rats. The protective effect of GSPE is probably associated with reducing granulocyte infiltration and decreasing the production of proinflammatory cytokine IL-1β in the colon of rats, in addition to its antioxidant effects [
7].
NF-
κB is a crucial transcription factor which mediates transcriptional activation of many inflammatory genes. In the normal physiological state, NF-
κB exists in the cytoplasma as a heterodimer complex of p65/p50 subunits combined with an inhibitory protein, I
κB. Inflammatory stimulation provokes rapid degradation of I
κB, and subsequently the free NF-
κB molecule translocates into the nucleus, binds to the promoter regions of the target genes, and induces active transcription of inflammatory genes [
10,
11].
The precise molecular mechanisms responsible for the anti-inflammatory effects of GSPE remain unclear. Both IL-1β and TNF-α have been suggested to be important mediators involved in the initiation and perpetuation of colonic inflammation in IBD [
1,
12]. And NF-
κB activation is the most critical step for IL-1β and TNF-α gene transcription. Activation of NF-
κB may be a pivotal event in pro-inflammatory signal transduction [
10,
13]. Thus, we hypothesized that GSPE suppresses inflammatory responses that are possibly associated with the expression of NF-
κB. In order to elucidate the mechanisms, the expression of the NF-
κB and I
κB systems have been measured [
1].
In this study, we also determined IL-1β and MPO. The results further document that GSPE significantly reduces: (1) the degree of colonic injury; (2) neutrophil infiltration and oxidative damage; (3) the release of proinflammatory cytokine IL-1β; (4) NF-κB expression caused by TNBS in the colon. The different doses of GSPE showed significantly dose-dependent effects on IκK levels in TNBS-induced rat colitis. However, GSPE did not show dose-dependent effects on MPO, IL-1β, PIκBα and NF-κB levels. The cause may be that the low dose of GSPE or medium dose of GSPE shows maximum therapeutic effect, so the effect is not increase obviously along with the increase in other doses.
These results suggest that oral administration of GSPE effectively suppresses mucosal inflammation in the colon through the inhibition of NF-κB signal transduction pathways. Thus, it is possible to consider that GSPE suppresses IκK activation, and the inactivated IκK complex suppresses the phosphorylation-induced degradation of IκBα. Therefore the NF-κB signal can be abrogated by newly-synthesized IκBα which enters the nucleus, removes NF-κB dimers from DNA, and results in their exportin-mediated transport to the cytoplasm. Inhibition of NF-κB signal transduction pathways may be one of the major mechanisms underlying the prevention of the development of TNBS-induced colitis. Thus, the inhibitory effect of GSPE on the development of TNBS-induced colitis was associated with the blockade of NF-κB in the colon tissue.
Some studies show that IL-1β and hydroxyl radical can activate NF-
κB, and likewise, activated NF-
κB would be up-regulating proinflammatory target genes for cytokines production and oxidative damage [
14]. It was reported that IL-1β and oxidative damage are significantly increase as well as the expression of NF-
κB in colonic tissue with colonitis. This could have attracted more neutrophils on the site of inflammation, further increasing the generation of hydroxyl radical and the damage to surrounding tissues [
15,
16,
17]. It has been well demonstrated in our study that IL-1β elevation, oxidative damage and neutrophil infiltration are involved in the pathogenesis of colitis as they are present in the colon tissues and can be detected in the inflamed tissues. The decrease of the colonic inflammation, oxidative damage, IL-1β and NF-
κB demonstrated in current study expands possible mechanisms for the good effect of GSPE observed. The inhibition of the oxidative damage, IL-1β production and neutrophil infiltration by GSPE observed in current study is most likely attributed to the inhibitory effect on the activation of NF-
κB. In addition, the inactivated NF-
κB would be down-regulating IL-1β, neutrophil infiltration and oxidative damage.