| Object:To investigate the therapeutic effects of Iridoid glycosides extract from Lamiophlomis rotate (IGLR) for inflammatory bowel disease (IBD) induced by Trinitrobenzene sulfonic acid (TNBS) and Dextran sulfate sodium (DSS) in rats, respectively, and the mechanism of the anti-inflammation effect of IGLR using the techniques of immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA).Methods:1. Groups assigning:90 Wistar rats were randomly divided into 9 groups (n=10 per group):the control group (C), TNBS-induced model group (M1), the treated groups (T1l, T1m, T1h) for TNBS-induced models with different doses of IGLR (300mg/kg,150mg/kg,75 mg/kg), salazosulfapyridine treated groups (S1), DSS-induced model group (M2), and the treated groups for DSS-induced models with SASP (S2) or 300mg/kg IGLR (T2).2. The inducement of IBD models:TNBS-50% ethanol solution was directly fill into colons through anuses of rats to build the TNBS-induced IBD model in rats; 6% DSS solution was given by drinking for 8 days to build the DSS-induced IBD model in rats.3. Administration and observation:the administration was started from the day after that model-building was completed. All the control groups were administered by i.g. with the equal volume of distilled water; the activities of the body and the appearance of feces were observed, and the occult blood was detected during the experiment.4. Detection:All rats were sacrificed after 7-day-administration and the samples of colon tissue and blood serum were taken out. HE stained tissue samples were observed under microscope. The IHC was utilized to determine the expression of MPO and EPO in colon tissues. ELISA method was used to determine the serum levels of INF-γ, IL-4, IL-17 and IL-10. Western blot and RT-PCR were adopted, respectively, to determine the protein and mRNA expression of T-bet, GATA-3, ROR-γt, and Foxp3.Result:1. General status:the rats in group Mi and Til were in poor status with the characteristics of abdominal distension, astriction, and gentle tendency of DAI curve. Compared with Mi group, the general status and defecation function of rats in groups S1, T1m and T1h started to recover gradually from d2 to d4 after the beginning of experiment, with a significant declining of DAI tendency curve. The general status of the rats in M2 group were also in poor status, with plenty loose and bright blood stools, weight loss, and slightly decline of DAI tendency curve. Compared with M2 group, the bloody stools in rats of S2 and T2 groups stopped on d2-d3 from the beginning of experiment. On d6-d7, the stool form was changed to solid spindle pellets, the occult blood in stool was turned to negative, and the DAI tendency curve was significantly declined. Moreover, the body weights were gradually gained while not in normal level.2. The morphological and histological alterations of the colon tissue:the morphological observation of the samples of M1 group showed there were a wide range of necrosis on colon wall, which were completely hardened and devitalized. The histological scores indicated there was a significant difference compared with C group (P<0.05). A moderate alleviation was observed in samples of Til group, while a significant decrease of ulcer area was observed with small piece-or point-ulcer in the samples of Si, Tih, and Tim groups, and the scores of these groups display a significant difference compared to Mi group(P<0.05). The colon walls in M2 group had congested and swollen severely, with obvious blood streak and hemorrhagic foci. The scores between M2 and C had significant difference (P<0.05). However, little hemorrhagic spot was observed in samples from S2 and T2. Compared with M2 group, the score of S2 and T2 groups was decreased significantly (P<0.05).After HE staining, ulcers were observed deep into muscular layers in samples from Mi under microscope. Compared with samples of M1, those from S1, T1l, T1m, and Tih had obvious attenuation. In samples from M2, cell swelling and necrosis was observed in mucosal layers, but no further damage in muscular layers. In samples from T2 and S2, the repair and revitalization of gland and goblet cells were observed. Compared with C group, the histological scores were significantly different in M1, M2 groups (P<0.05). Compared with M1 and M2 groups, the score in all treatment groups was decreased significantly (P<0.05).3. The expression of MPO and EPO by IHC detection:the IHC scores of MPO in Mi, M2 were increased compared with group C (P<0.05) and the IHC scores of MPO in S1, Tih, S2, and T2 were significantly decreased compared with groups of Mi and M2 (P<0.05). The IHC scores of EPO in Mi, M2 were significantly increased compared with group C (P<0.05) and the IHC scores of EPO in S1, T1m, T1h, S2 and T2 were significantly decreased compared with groups of M1 and M2 (P<0.05).4. The detection of protein level in blood serum by ELISA:the results indicated that, compared with group C, the levels of INF-y and IL-17 in M1 and M2 were significantly increased (P<0.05) while the IL-10 level was significantly decreased (P<0.05). Compared with M1 and M2 groups, the INF-y level was significantly decreased (P<0.05) while the IL-10 level was significantly increased (P<0.05). The IL-17 level was significantly decreased in groups of Si, Tim, Tih, S2, and T2 compared with groups M1 and M2 (P<0.05), while the IL-4 level was decreased only in M2 group (P<0.05). No significant alteration was observed in TNBS-induced groups, however, significant growth (P<0.05) was observed in the two treated groups of DSS-induced models (S2 and T2).5. Detection for protein level and mRNA expression in the ulcer tissue of colons by Western Blot and RT-PCR:the results indicated the expressions of T-bet and ROR-yt protein and mRNA in Mi and M2 groups were significantly increased compared with group C (P<0.05). After the treatment by IGLR and SASP, the expressions of T-bet and ROR-yt protein and mRNA in T2, S2, Tim were significantly decreased compared with group Mi and M2 groups (P<0.05). The expressions of GATA-3 protein and mRNA in Mi group failed to show any significant changes compared with group C, while exhibited a significant increase in M2 group(P<0.05), and a significant decrease in T2 and S2 groups (P<0.05). Compared with C group, the expressions of foxp3 protein and mRNA in Mi and M2 groups were significantly decreased, while after the treatment, mats in samples of all treated groups were significantly increased (P<0.05).Conclusions:1. TNBS-induced IBD model in rat were primarily characterized with abdominal distension, astriction, and weight loss. The HE stained samples were observed that the ulcers deep into muscular layer. Its immunopathological alterations suggested that the effects may be mediated by Th1 and Th17 cytokines. In general, this model was similar with the human Crohn’ disease. DSS-induced IBD model in rat were primarily characterized as diarrhea, loose stool, and hematochezia. The colon ulcers located on the mucosal layers and were mainly manifested as edema and haemorrhagia. In HE stained samples, crypt defects and goblet cell damages were observed. The immunopathological alteration of this model may be mediated by the interactions of Th1, Th2, and Th17 cytokines.2. IGLR had the pharmaceutical effects on colon ulcer in both the TNBS-and DSS-induced IBD models in rats, and restored the impairments in colon tissues by the inflammation at a certain degree.3. The alleviation effect of IGLR for the IBD rat models may be mediated through the regulation for the functions of Th1, Th2 and Th17 cytokines and the expression of their relevant transcription factors, including T-bet, GATA-3, and ROR-yt, respectively, and finally inhibit the inflammation.4. IGLR can promote the expression of Treg cytokines and their transcription factors such as foxp3 to exert its anti-inflammation function, and performed the treating effects for the IBD models. |
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