The Expression Study Of Th1/Th2Inflammatory Cytokines In Rat Treatment Model Of Ulcerative Colitis

Objective:Ulcerative colitis (UC) is a nonspecific inflammatory disorder primarily involving the mucosa and submucosa of the colon that shows clinical manifestations of diarrhea and mucous/bloody stool. Pathophysiological bases of this disease involve genetic factors, immune dysregulation, barrier dysfunction, and a loss of immune tolerance toward the enteric flora.A balance is maintained between pro-inflammatory and anti-inflammatory cytokines systems under physiological conditions, but it is impaired under pathological circumstances. The expression imbalance of CD4+T subtypes Th1/Th2cells and their regulatory cytokines IL-2, IFN-γ, IL-4, IL-10may be one of the main mechanisms of the pathogenesis of ulcerative colitis. The aim of the study is to investigate the potential protective role of Chinese medicine Jiechanging enema (Hunan Jiuzhitang Co., Ltd.) against dextran sodium sulfate (DSS)-induced acute colitis in rats.Methods:Forty SD rats were divided into normal group, colitis control group, sulfasalazine(SASP)-treated group, Jiechanging-treated group. Normal drinking water was fed to the normal group for10days. Colitis control and two treated groups were treated with4%(w/v) DSS(molecular weight5000) dissolved in drinking water for7days and fed with normal drinking water for an additional3days. From the fourth day of DSS administration, Sulfasalazine(SASP) and Jiechanging enema were administered respectively (10ml/kg) to the two colitis-treated groups once a day for7days, as0.9%saline enema to the normal group and the colitis control group were. Inhibitory effects of concomitant treatment with SASP and Jiechanging enema were assessed daily using a Disease Activity Index (DAI) and severity of histological changes. Cytokines IL-2, IFN-γ, IL-4, IL-10expression levels in blood serum and colon tissue were detected using ELISA and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) respectively.Results:1. General state of health and assessmet of DAI:Rats administered4%DSS developed severe colitis and suffered weight loss, pasty-to-liquid grossly bloody stool, even severe anemia, having significant differences compared with normal rats (P<0.01). DAI started increasing rapidly from day4to day7in the colitis control group, and even reached the summit in day7, whereas the DAI rised slowly by SASP and Jiechangning enema administration in the two treated group, and decreased quickly from day7to day10. Symptoms of the two treated groups were more attenuated than the colitis control group that showed improved mental state, weight gain, increased appetite, increased activity, and no bloody stools, sometimes soft (P<0.05). But there was no obvious difference in SASP-treated and Jiechangning-treated groups.2. Histological Evaluation:The acute phase of UC exhibits a number of histological features, which includes mucosal erosions, crypt shortening, edema, and infiltration of neutrophils in the mucosa and lamina propria. Compared to the normal group, the colitis control group exhibited marked erosion of the lamina propria mucosa, disappearance of glandular epithelium, inflammatory cell infiltration, and other related findings. In the SASP-treated group and the Jiechanging-treated group, pathological inflammation were less severe than in the colitis control group, and the histological disease scores were significantly lower compared to the colitis group (P<0.05). Compared to the SASP-treated group, the Jiechanging-treated group had slightly lower histological disease score, but there was no obvious difference between the two treated groups (P>0.05).3. Cytokines IL-2, IFN-γ, IL-4, IL-10protein relative expression level in blood serum:We observed that the control colitis group exhibited increased IFN-y and decreased IL-4, IL-10protein expression compared to the normal group (P<0.05). Both the SASP-treated and the Jiechangning-treated groups exhibited decreased IFN-y protein expression compared to the control colitis group (P<0.05), but its level was higher than the normal group (P>0.05). We found an increased IL-10protein expression in two treated groups and an increased IL-4protein expression only in Jiechangning-treated group, compared to the control colitis group (P<0.05). There was no significant difference of IL-2protein expression whether the normal group or the control colitis group and the two treated group (P>0.05). There was also no obvious difference between the two treated groups (P>0.05).4. Cytokines IL-2, IFN-γ, IL-4, IL-10mRNA expression level in colon tissue:We observed that the control colitis group exhibited increased IFN-γ, IL-4and decreased IL-10mRNA expression compared to the normal group (P<0.05). The SASP-treated and the Jiechangning-treated groups exhibited increased IL-10protein expression compared to the control colitis group (P<0.05), but its level was lower than the normal group (P>0.05). We found a decreased IFN-γ protein expression in two treated groups, but only the Jiechangning-treated group had a significant difference compare to the control colitis group (P<0.05). There was no significant difference of IL-2mRNA expression whether the normal group or the control colitis group and the two treated group (P>0.05). There was also no obvious difference between the two treated groups(P>0.05).Conclusions:The expression of Th1/Th2inflammatory cytokines IFN-γ, IL-4, IL-10was significantly changed in rat DSS UC model, suggesting that DSS destroyed the balance of Th1/Th2expression in the colon. Jiechangning enema could ameliorate dextran sulfate sodium-induced acute experimental colitis in rats through decreasing IFN-y level and increasing IL-10level both in blood serum and colon tissue so as to regulate the Thl/Th2balance and improve immune function.