| Rheumatoid arthritis (RA) is a kind of systemic autoimmune disease with chronic, progressive, and invasive arthritis as its main manifestation. The patients' condition will deteriorate, leading to joint deformity and losing function eventually if they are not be treated regularly. Owing to the high rate of mutilation, RA seriously affects the health and the quality of life of the patients, and causes huge economic burden to patients'families and society.Many studies have shown that food is closely related to the incidence of RA. Now the RA animal model induced by subcutaneous injection of type II collagen is the best animal model of RA. The subcutaneous injection of type II collagen to rat can successfully induce the occurence of RA, but the induction of subcutaneous injection is incompatible with the pattern of human feeding, because it ignored the importance of gastrointestinal tract in the incident of RA, so a very importan gap appears in the research of pathogenesis of RA:the mechanism how the food antigen pass intestinal epithelium mucous membrane barrier into lamina propria of intestinal mucosa in the process of human feeding, and cause a series of immune responses of gut associated immune cells, and desrupt the inherent and adaptive immune responses of the intestinal mucosal immune tissues, and break immune tolerance by molecular mimicry and bystander effect, leading to the occurence of RA ultimately are remain unclear.ObjectivesThe objectives of this study are to establish rat model of RA induced by oralling bovine type II collagen and make sure the relevance of oralling food antigen between RA; On the basis of the animal model, we will preliminarily explore the role of intestinal permeability in the pathogenesis of RA induced by food antigen and provide theoretical basis for clinical prevention and treatment of RA.MethodsThe Wistar rats were selected as experimental animals in this study. The bovine typeⅡcollagen was extract from bovine cartilage. The function of rats'intestinal mucosal barrier was damaged with aspirin first in the experiment, then the rats were fed with larger doses of bovine typeⅡcollagen to induce the occurrence of rheumatoid arthritis. The 4-5 week-old's male Wistar rats were allocated into four groups randomly: control group(physiological saline; physiological saline), group M1 (physiological saline; physiological saline+CCl4+LPS), group M2 (5mg/kg aspirin; bovine type II collagen+CCl4+LPS), and group M3 (10mg/kg aspirin; bovine type II collagen+CCl4+LPS). During the period of aspirin feeding, the rats fed with a certain amount of lactulose and mannitol every seven days, then the rats'24h urinary were collected with metabolic cage, and lactulose and mannitol ratio(L/M) were detected and calculated by high performance liquid chromatography (HPLC), finally the comparisions were done between group M2,group M3 and control groups for evaluating the changes of intestinal permeability indirectly. After modeling, the serum, jejunum tissue and ankle joint tissue were obtained from rats. During the modeling, the diameter of the right hind ankle was measured and recorded with vernier caliper once two weeks, for a total of five times, and the articular index (AI) was assessed; Double antibody ELISA method was used to detected the level of special anti-bovine-type-II-collagen antibody, and the level of TNF-a, IFN-r and IL-4; The the jejunum tissues and the ankle joint tissues were put into fixative, made into paraffin sections, and HE stained; The pathological changes of the structure and the infiltration situation of lymphocytic were observed under light microscope; The method of immunohistochemistry was used to locate the bovine type II collagen in jejunum, in order to observe whether it can pass intestinal epithelium mucous membrane barrier into lamina propria of intestinal mucosa; The method of immunohistochemistry was used to locate the bovine type II collagen on the joint synovial, in order to observe its deposition on joint synovial. The reliability of model-establishing method in this experiment was comprehensively evaluated through the above indicators.ResultsAfter the modeling, varying degrees of ankle swelling were observed in rats of group M2 and group M3; The level of blood serum anti-CII antibody of group M2 and group M3 had a significant rise compared with cotrol group (P＜0.05), the level of blood serum TNF-a and IFN-r of group M2 and group M3 had a significant rise compared with cotrol group (P<0.05), and the level of blood serum IL-4 of group M2 and group M3 had a significant decline compared with cotrol group (P＜0.05); Pathological changes in jejunum of group M2 and group M3 rats were significant. The changes included that the intestinal mucosa was damaged severely, submucosal neutrophils infiltrated obviously, intestinal villi were dropsical, and intestinal epithelial cells losed widespreadly. It showed that undigested or incompletely digested macromolecules bovine type II collagen can pass intestinal epithelium mucous membrane barrier into lamina propria of intestinal mucosa with immunofluorescence. Synovial hyperplasia of histological specimens of the ankle joint in group M2 and group M3 was observed, the destruction of local bone resorption was obvious, surface collagen fiber of cartilage was released and broken, cartilage cells were degenerated and necrosed, and the surface of cartilage was regged. The study found that bovine type II collagen was deposited on the joints synovial of experimental group rats with immunofluorescence. The study also found that the concentration of mannitol in urine of group M2 and group M3 rats was declined significantly(P<0.05), the concentration of lactulose increased(P<0.05), L/M also increased(P<0.05) with double-sugar tests, which indicated that intestinal permeability of rats in group M2 and group M3 was increased.ConclusionsThe occurence of rheumatoid arthritis in rats can be induced successfully by oralling bovine type II collagen, under the condition of intestinal mucous membrane damaged. |
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