| IntroductionRheumatoid arthritis ( RA) , an autoimmune disease, is disordered mainly in cell -based immunity. As a common systemic chronic inflammatory disease, renal involvement is an extra - articular manifestation. But in the early RA, it is often ignored or hardly associated with RA without obvious symptoms and signs. Renal impairment in RA is a progressive disease that may be one cause of death. More and more scientists have noticed it. Estrogen is a hormone modulating immune responses which can not only depress Thl - medicated cell - based immunity and but also stimulate Th2 - medicated humoral - based immunity. It may affect many steps in RA by regulating the immune system. Meanwhile, the effect of estrogen on RA has differences and more studies should be made to explore the mechanism of it. Recent studies on estrogen and its receptor(ER) implicate that there is ER B in the cellular membrane of renal cells and inflammatory cells. So estrogen likely influences the expressions of proteins and cytokines in the level of transcription and exerts its effect through them. In the present study, we quantified interleukin 18 and Matrix metalloproteinas 3 expression in the kidney of rats with RA to explore the mechanisms of estrogen on renal impairment in RA by means of immunohistochemistry and Western blot and provide the basis of the therapy of the disease.Materials and methodsExperimental animals;fifty female Wistar rats, 6-8 weeks old, weighing160 ±20 grams.Experimental reagent: Freud's complete adjuvant, E2 - 17(3 (SIGMA) , rabbit anti rat IL -18 antibody and MMP -3 antibody, immunoglobulin marked with HRP(Boshide Company, Wuhan) ,ECL kit, immunohistochemistry SP kit, NC (Zhongshan Company, Beijing) .Experimental instruments: stir apparatus, low temperature centrifugal machine , electrophoresis apparatus, transmark apparatus, Luze - F image analysis system.Methods: The fifty female wistar rats were devided into the following groups with random: I normal rats;H rats with adjuvant arthritis( AA);IK ovariecto-mized(OVX) AA rats;IV low - dose E2 - 17(3 - induced AA rats of OVX V high -dose E2 - 17(3 - induced A A rats of OVX. The animals were killed 21 days after the adjuvant injections. Some of kidneys were removed and then frozen immediately for Western blot to analyze. Others were done and kept for immunohistochemistry to analyze. The content of E2 in the serum was detected by radioimmunoassay. Western blot and immunohistochemistry were used to analyze the expression levels of IL - 18 and MMP -3.Data from above methods were expressed as mean standard deviation ( SD) . The differences between groups were determined by unpaired student t - test. P <0.05 was statistically significant.Result1. The result of radioimmunoassay: the level of E2 in the serum of the AA rats of OVX decreased significantly compared to that of the normal controls;The level of the AA rats treated with high - dose of E2 - 17(3 increased significantly compared to that of the group of AA rats of OVX;And the levels of the normal controls, the A A group and the low - dose of E2 - 17(3 have no obvious difference.2. The result of Western blot method: Western blot analysis revealed that the molecular weight of IL - 18 was 18KDa. There was significant difference of IL - 18 expression levels;M>H >IV>V> I. And the molecular weight ofMMP-3 was 57KDa. There was significant difference of MMP -3 expression levels:]! > II > IV > V > I .3. The result of immunohistochemiatry: Immunohistochemical analysis clearly showed positive staining in renal cell. There are significant difference of IL -18 and MMP -3 expression in the five groups.DiscussionThe expression of ER B decreased in the kidney of rats with RA;IL - 18 and MMP - 3 is overexpressed. They demonstrated that estrogen, IL - 18 and MMP -3 all are involved in the renal impairment in RA. In the present study, we found that the expression of IL -18 and MMP - 3 in the kidney of rats with RA decreased when induced E2 - 17(3. The result indicates that estrogen has an important role on the renal impairment in RA by downregulating the expression of IL-18 and MMP-3.We evidenced that the expression of IL - 18 and MMP -3 in the kidney of AA rats of OVX is significantly increased and is decreased when induced E2 -17(3;There is difference between the low - dose group and high - dose group. Given previous findings that estrogen have a direct role on inflammatory cyto-kines or through hormones - medicated, estrogen can suppress inflammatory response on the kidney of rats with RA. The mechanism of estrogen on the disease is complicated. It has been reported that there is ER B in the celluar membrane of kidney and in the early RA, the expression of IL -18, a proinflammatory cy-tokine, is increased firstly. And then IL-18 stimulates the proliferation of Thl cells and Thl immune responses. Meanwhile, IL-18 causes the changes in the expression of MMPs, IL - 1 and TNF - a. So estrogen may combine with ER B firstly and then medicate the downregulation of IL - 18 through respective sign paths, next downstream target gene including MMP -3.IL-18 can induce INF-"/ strongly, modulate immune system, stimulate Thl cells proliferation and Thl immune responses. Studies have found that the level of IL - 18 in serum of patients with RA is obviously increased and that of INF - 7 is decreased by other cytokines. They indicate that IL-18 have a posi-tive role on immune system in RA but the mechanism is unknown. MMP - 3 is one of degrading enzymes in kidney as an important part of MMPs and has a direct role on the expression of extracellular matrix. In RA MMP - 3 is activated by many enzymes and activates other MMPs. Since the immune system is disordered in RA , the interaction between estrogen, IL - 18, MMP -3 and other cy-tokines shouldnt be ignored and the relationships need to be studied.In summary, by analyzing IL - 18 and MMP -3 expression in the kidney of rats, we found that the levels of them are higher in RA compared with those of the normal group;and the levels are decreased by administration of adequate exogenous estrogen. This study explained the mechanism of renal impairment in RA. Farther studies on the effect of estrogen on the renal impairment in RA will provide new ways to prevention and clinical treatment of the disease.ConclusionAfter induction of adjuvant arthritis, the expression of IL - 18 and MMP -3 in the kidney was increased. The serum level of E2 in rats with adjuvant arthritis could regulate the expression of IL -18 and MMP -3 in the kidney.Our findings suggested that E2 might play its role in renal impairment in RA by downregulating the secretion of IL - 18 and MMP -3.
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