| Background and aimInflammatory bowel disease (IBD) is a kind of non-specificity chronic inflammation, which contains Crohn’s Disease (CD) and Ulcerative Colitis (UC). The exact causes of diseases are not very clear, but the environment, heredity, infection, immunity are all associated with them. Although there are lots of ways to treat IBD, based on anti-inflammation and immunity-depression, the relapse and side effects of drugs always perplex us, like allergy and granulocytopenia. It may come as a shock to many that most drugs are effective in only50%of patients, and in IBD, a remission rate of20%to30%with a response rate of50%to60%is often considered highly acceptable.As one of the extracts of garlic, allicin is a safety drug and has few desirable adverse effects. It has effects like anti-inflammation, anti-oxidant, anti-atherosclerosis, apoptosis stimulation, and anti-tumor. In a meta-analyse of colorectal and stomach cancer, the random-effects relative risk (RR) estimate of colorectal cancer and garlic consumption, excluding garlic supplements, was0.69. For stomach cancer, the random-effects RR estimate was0.53. Allicin is a strong inducer of apoptosis in tumor cells and can cause different expressions of some proteins like Annexin I, Galectin, VDAC-1, etc. Sulfurocompound from garlic inhibited colon cancer cells (SW620and HCT-116) growth through induction of apoptotic cell death by modulating of NF-κB. Allicin may have anti-inflammatory function through NF-κB pathway because it is the key point of IBD.It is an indespensible way to choose and establish a suitable animal model to study a drug. Chemical inflammatory is a widely used and stable way to imitate IBD. TNBS enema, a classic way, can induce chronic colitis which imitate human CD, according to immune type. This process can maintain a long time. In order to prove the anti-inflammatory effects of allicin in vivo, we tested the inflammatory index after interfering with different drugs on TNBS-induced rats.To study the mechanisms of allicin on anti-inflammation, we chose human colon adenocarcinoma cell line, Caco-2, which present mature characters of intestinal epithelial structure and function. It is an ideal cell line to research the expression of proteins and functions of intestinal epithelial. It can imitate inflammatory status after stimulated by IL-1β. Interfered with allicin, we evaluated the anti-inflammatory effects of allicin on cells by examine the levels of cytokines in the culture medium and the quantity of NF-κB p65in the nuclear. We still tested the phosphorylation of p38, ERK and JNK pathways.Based on the background and theory above, we studied the effects of allicin on IBD in vivo and ex vivo and the relationship between it and MAPK pathways.1.Established TNBS-induced IBD model on rats.2.Observed the effects of mesalazine, sulfaslazine and allicin alone or in combinations. 3.Tested the change of MAPK pathways after the treatment of allicin on IL-1β-induced Caco-2cells.Methods1. Experiments were conducted in male Wistar rats weighted180～220g.80rats were divided into8groups (10rats per group):control group, TNBS group, allicin prevention group (30mg/kg), allicin gavage group (30mg/kg), mesalazine group (30mg/kg), sulfaslazine group (100mg/kg), allicin combined with mesalazine group and allicin combined with sulfaslazine group (dose as above). Colitis was induced by rectal administration of50mg/kg of2,4,6-trinitrobenzene sulfonic acid in50%ethanol using a vinyl catheter positioned8cm from the anus. Control rats underwent identical procedures but were instilled with50%ethanol. Allicin prevention group was gavaged with allicin daily for14days before the enema. After the administration, control group, TNBS group and allicin prevention group were gavaged with physiological saline solution daily. Others were gavaged with drugs depending on the groups. All rats were killed14days after the first TNBS administration. Body weights were recorded every other day. Serum and colons were harvested after death. Calculate histologic scores and exam the level of TNF-α, IL-1β, IL-4and IL-10.2. Induced Caco-2cells with various concentrations of allicin and IL-1β for different time. MTT test was presented to confirm the appropriate concentration and time. Cells were treated for12h,24h or48h with various concentration of IL-1β, alone or in combination with allicin. Culture medium and nuclear protein were collected for the examination of IL-8and NF-κB p65. Total protein was still needed for the test of MAPK pathways.3. Statistics:The results were reported as means±S. One-way ANOVA was used for parametric test. Kruskal-Wallis H test was used for non-parametric test. P<0.05was considered statistically significant. Results1.The weight of rats:The weight was lower in TNBS group than control group2days after TNBS was used. It was higher in all therapeutic groups than TNBS group. But there was no significant differences between allicin group and sulfaslazine combined with allicin group and mesalazine combined with allicin group. At the end of the experiment, there was still no significant differences between allicin group and mesalazine group and mesalazine combined with allicin group.2. The histological score was lower in TNBS group than control group. The score in mesalazine combined with allicin group increased significantly compared to other therapeutic groups.3. Compared to the control group, the levels of TNF-α and IL-1β were rised significantly in TNBS group. It was lower in allicin group, but still higher than mesalazine combined with allicin group. The tendency was opposite in IL-4. There was no significant different among TNBS group and other therapeutic groups.4. Caco-2cells were treated with various concentrations of allicin and IL-1β for12h,24h or48h. In IL-1β-induced Caco-2cells, the IL-8level in culture medium was significant increased, and the quantity of NF-κB in nuclear was upgraded as the same, depending on the rise of concentrations. Allicin can not affect the production of IL-8, but it restrained the transfer of NF-κB.5.1ng/ml IL-1β stimulated p38, ERK, JNK pathways. Pretreated with allicin can depress this phenomenon except ERK pathway. Cells treated with allicin alone can not stimulate p38, JNK pathways. So, allicin could restrain the inflammation through depress p38and JNK pathways.Conclusions1. Allicin could relieve the inflammation induced by TNBS on rats, and better than prevention. There is no significant difference among allicin group, sulfaslazine group and mesalazine group. But the inflammation can be significantly reduced by using of allicin combined with mesalazine.2. IL-1β can increase the expression of IL-8in the serum of Caco-2cells with dose-dependent effects. Pretreated with allicin for2hours before IL-1β induction does not change it compared with IL-1β alone, while that does not happened on cells with allicin alone as well.3. In the nuclear of IL-1β-induced Caco-2cells, the expression of NF-κB p65increases as the elevation of IL-1β. Pretreated with allicin for2hours before IL-1β induction can lead to the decrease of it compared with IL-1β alone, which was not happened on cells with allicin alone. This effect can be enhanced by increase the concentration of allicin.4. Allicin depresses p38, JNK and NF-κB pathways, which could be the cause of the change of cytokines. |
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