For convenient interpretation of the data discussed in the subsequent sections, the current paragraph highlights key features of the method section. The acute model is defined by rapid onset of severe clinical signs (sometimes interchangeably referred to as 'symptoms' in the rest of the text) that may be healed when the disease-causing agent (DSS) is withdrawn. The induction period in the acute model is shorter than in the chronic model and a higher DSS concentration is used. In the chronic model, induction of symptoms is slower but longer lasting with possible flare-up and remission cycles. DSS is administered in alternate cycles at lower concentrations for the chronic model (see Table and methods). The optimal concentration of PEO (75 mg/kg) for assessing its in vivo efficacy was determined in a pilot study using a dose response administration of PEO in DSS-treated animals (data not shown). Mesalamine/5-ASA, a standard first-line therapy for mild-to-moderate UC, administered at a recommended 50 mg/kg [22
], was used as a reference standard for in vivo models. In all instances, statistical significances of treatments of PEO and 5-ASA groups were compared to DSS group (Figs , , , Table ). The results are presented in the order of physiological, histopathological and molecular observations. In the sections to follow, the 'water-group' and the 'DSS-group' refer to the healthy and the disease controls respectively (see Table for details).
Disease activity index (DAI) (Fig ) combined the following 5 variables (clinical signs/symptoms): change in body weight, stool consistency, occult blood in stool, rear end bleeding and inflammation (visible), and rectal protrusion. DAI was used to compare the treatment response to PEO and 5-ASA with that of the DSS group following the scale described in Table [modified from [5
]]. Rectal prolapse, one of the variables for DAI (Table ) was observed only in the chronic colitis animals (Fig ) occurring with partial prolapse of the rectal lining. Therefore, the DAI shown for the acute model (Fig ) does not reflect this variable. Also, UC is an intermittent disease with periods of exacerbated symptoms and periods that are relatively symptoms free [11
]. Due to the longer experimental duration of the chronic model (Table ), the relapse-remission pattern among members of individual groups was observed (Fig ). In the acute model, both PEO and 5-ASA attenuated the general signs of UC after 5, 10 and 15 days of respective treatments (Fig ) with PEO performing notably better (PEO p
< 0.01, 5-ASA p
< 0.05) and comparing closely with the water (healthy) group (Fig ). In the chronic model, alleviation of clinical signs was not observed until after 10 days of treatment (Fig ). The PEO-treated group showed significant amelioration (p
< 0.01) of symptoms on day 12 and continued to experience further relief until the end of the experiment while the 5-ASA group did not show any significant remission (Fig ). In addition, the PEO- treated group experienced an attenuated spike in symptoms starting from the second day of treatment (p
< 0.01), suggesting that PEO may also prevent the relapsing nature of chronic colitis (Fig ).
Colon characteristics were analyzed next. Colon lengths were measured prior to flushing. The healthy water group typically had longer, non-thickened colons. In the diseased animals, the colons were often shortened (Fig ), likely due to loss in crypt structure and thickened due to edema formation, similar to previously reported studies with this model [24
]. The differences between the healthy and the DSS-receiving groups in terms of colon lengths were less pronounced in the acute study (data not shown) as compared with the chronic study (Fig ), likely due to shorter induction period (5 days vs 36 days). Therefore, relief from colon shortening-effects was not observed in the acute model. In the chronic study, all of the DSS-treated groups (Table ) had markedly shorter average colon lengths as compared with the healthy water-treated group (Fig ). The PEO-treated group exhibited pronounced attenuation of colon shortening (p
< 0.05, Fig ) as was observed terminally.
Histology of the bowel sections of the experimental animals revealed a number of cellular changes in the colons as described in Table [11
]. However, the population size available for histopathology study (Fig ) was smaller (n = 5) as compared to the observations for gross colon length (n = 10) (Fig ). This is because half of the 10 colons were used for biopsy samples (discussed later, Fig ) and half were available for histopathological evaluations. The PEO-treated group had the lowest average pathological score (p
= 0.07), closely followed by the 5-ASA group (p
= 0.08) (Fig ). Qualitative differences were widespread among the bowels of both the PEO (Fig ) and 5-ASA (Fig ) groups relative to the DSS group (Fig ). In the bowels of the DSS treated animals, moderate to extensive infiltration of submucosa ('SM', Fig ) and superficial muscularis ('ME', Fig ) by a mixed population of inflammatory cells (shown in white triangles with red border, Fig ), such as lymphocytes, plasma cells, and macrophages (all mononuclear type), was observed. In some samples the infiltration extended transmurally through the muscularis and into the serosa (data not shown). The epithelium in small patches had frequently lost the majority of their goblet cells (shown in yellow triangles with black borders, Fig ) or ulcerated away from mucosa with intense infiltration of polymorphonuclear neutrophylic leukocytes (PMNs) beneath ulcerated areas, in contrast to the representative healthy colon section (Fig ). Rectal parts of the colons were generally characterized with the worst pathology including extensive infiltration, the presence of hyperplastic squamous epithelium and an increased mitotic index (data not shown). In the bowels of PEO- and 5-ASA-treated groups (Figs ) some signs of ulceration, partially inflamed submucosa, and low inflammatory cell infiltration were also observed, but to a much lesser extent and infrequently, in contrast to the DSS group (Fig ). Additionally, crypt structures with intact goblet cells were frequently visible (Figs ).
In order to address the biochemical basis of histological alterations, the cytokine response in the gut was evaluated by measuring the release of the pro-inflammatory cytokine, Interleukin 1β (IL1β) from colon tissue. A significant reduction in the average production of IL1β in the proximal (p < 0.01) and distal (p < 0.05) colon biopsies was observed among PEO-treated animals as compared with the DSS group. There was also a marked reduction in cytokine production in the mid colon for the PEO group (p = 0.06) (Fig ). The average IL1β production was reduced by 5-ASA in the middle and distal colons.
In the present study, histopathological analyses showed infiltration by various inflammatory cells including macrophages in the diseased samples and a reduction of such infiltration in the PEO treated group. Subsequently, we performed a gene array analysis (Table ) using the same in vitro macrophage based system to elucidate additional immune signaling genes that could be potential targets of PEO activity. Twenty one out of 84 genes (25%) induced by LPS, were suppressed three-fold or more by 10 μM PEO as compared to LPS activation alone (Table ). LPS is a known agonist of toll-like receptor 4 signaling that plays a critical role in colitis [25
]. Two genes that were down-regulated by LPS and up-regulated by PEITC were Htr2b (5-hydroxytryptamine, NM_008311) and Zap70 (zeta chain associated protein, NM_009539). LPS activation (or suppression) was separately compared to non activated levels of gene expressions (data not shown). The 21 down regulated genes represent key transcription factors and inflammatory mediators, at least 7 of which including IL6 were previously unknown to be affected by PEITC/PEO treatment (Table ). There is one very recent report [26
], however, showing in vitro down regulation of some oncogenes by PEITC in response to IL6 elicitation but effects of PEITC on IL6 expression was not reported. Out of these 7 responding genes, concentration-dependent changes in IL6, CXCL10 and STAT1 (signal transducer and activator of transcription1) expressions have been confirmed using real time RT-PCR (Fig ) and Western Blot analyses respectively (Fig ). These three genes were selected for further examination since their activation has been reported for human ulcerative colitis [27
]. At 10-15 μM, a decrease in mRNA and protein levels was observed for all these three genes (p
< 0.01) (Figs , ).
IL-6 signaling is mediated by STAT3 and various inflammatory diseases including UC are associated with STAT3 activation [26
]. But since activity of PEITC on STAT3 activation is known [26
], our subsequent investigation focused on effects of PEO on STAT1 activation and expression. Concentration dependent suppression of cytoplasmic STAT1 protein (Fig ) and mRNA (data not shown) levels were observed. Additionally, CXCL10, a STAT1 responsive gene [30
] was also suppressed by PEO in activated macrophages (Fig ). We previously reported suppression of another STAT1 responsive gene, iNOS by PEO [19
]. Therefore, we wanted to study the effects of PEO on activated (phosphorylated at Tyr701
) STAT1 (pSTAT1). Since within seconds after phosphorylation STAT1 translocates into the nucleus for binding to the DNA of its target genes [31
], we determined the levels of pSTAT1 in the nuclear fractions of the PEO treated cells. A concentration dependent attenuation of pSTAT1 was observed (Fig ). Densitometric analysis revealed a 2.5-, 4-, and 5-fold (p
< 0.05, with respect to LPS control) reduction in pSTAT levels by one, 5 and 10 μM PEO. At 15 μM PEO treatment, pSTAT was almost absent (p
< 0.001) as was also observed in non-stimulated cells (Fig ).