The Effect Of Prolactin On The Expressions Of MMP-9 And IL-1β Of The Adjuvant Arthritis

IntroductionRheumatoid arthritis (RA) is a chronic inflammatory joint disease accompanying aberrant T lymphocyte function. The major pathological changes in RA lesions include dyregulated proliferation of synovial cells, intensive lymphocyte infiltration, neoangiogenesis, and cartilage destruction in the affected joints. The majority of these events primary depend on excessive synovial cells function,maily through the secretion of proinflammatory cytokines such as TNF - α, IL,IFN and tissue destructive proteolytic enzymes such as matrix metalloprotein-ases ( MMPs).Prolactin (PRL) originally identified as a pituitary hormone with lactation modulating activity has immunoregulatory potential. The relationship between PRL and autoimmune diseases has attracted many researchers. Clinical observations and experimental animal studies have already implicated that PRL plays a immportant role in the pathogenesis of autoimmune diseases such as systemical lipus erythematosus, autoimmune thyroid diseases and autoimmune Addison's diseases etc. Numerous reports described the abnormal plasma level of PRL in RA, The plasma PRL levels elevate and show a significant correlation with RA.The functinary mechanisms of PRL are very complicated. It can effect multiple process of RA including the regulation of the immune system, the interaction with the inflammatory medium and the regulation of the cytokins,etc. But the precise mechanisms are not completely understood. To determine the exactrole of PRL in the pathogenesis of RA and supply experimental basis for clinical treatment of RA, we examine the expression of MMP-9 and IL-1β of synovium and cartilage of adjuvant arthritis at both hypoprolactinemic and hyperprolactine-mic levels.Materials and methods1. Experimental animals; forty male Wistar rats,6 -8 weeks old, weighing 160 ~ 180grams.2. Experimental reagents; Freud's compelet adjuvant, 17β-estradiol, di-ethylstilbestrol, bromocriptine (SIGMA) , goat anti rat MMP-9 antibody, goat an-ti rat IL-1β antibody, ECL kit, ( Zhongshan Company, Beijin) , gelatine, NC (Huamei Company, Shanghai) .3. Experimental intruments:stir apparatus,low temperature centrifugal machine, electrophoresis apparatus,transmark apparatus .freeze microtome, Luzex-F image analysis system.4. Methods; The forty male Wistar rats were divided into the following groups:I normal rats, II adjuvant arthritis rats, II diethylstilbestrol- induced hy-perprolactinemic adjuvant arthritis rats, IV bromcryptine-induced hypoprolactinemic adjuvant arthritis rats. The animals were killed 21 days after the adjuvant injections. The synovium was removed and then frozen immediately until further analysis. The whole joints were dissected and subsequently fixed in phosphate formalin for a week and decalcified in 10% EDTA for three weeks. The content of PRL in the serum was detected by radio - immunoassay methods. Gelatine zy-mography was used to analyze the expression levels of MMP-9 and Western blot was used to measure the expression of MMP-9 and IL-1β of the synovium. Im-munohistochemistry was used to determine the expression of MMP-9 and IL-1 β of the synovium and cartilage.Data from above methods were expressed as mean ± standard deviation (SD). The differences between groups were determined using unpaired Student T-test. A value P <0.05 was considered significant.Results1. The result of radioimmunoassay methods:The level of PRL in the serum of the AA rats treated with estrogen increased significantly compared to that of the arthritis controls (25 ±7. 5vsl33. 0 ±42.0,n=8,p<0.01). The level of PRL in the serum of the AA rats treated with BRC decreased significantly compared to that of the arthritis controls (25 ± 7.5 vsl33.0± 42.0,n= 8,p<0.01).2. The result of gelatinase zymography methods;There were significant difference of the enzyme activity: H > II > I > W ( I vsII : 23814.33 ±8439.729 vs61870.33 ± 21867.86,n =8,p <0.01; E vs 1:61870.33 ±21867.86 vs 98134.00 ± 26642.43 ,n=8,p<0.05; II vsIV: 61870.33 ±21867.86 vs 39098.33 ± 10498.05,n =8,p <0.05; I vs IV: 23814.33 ±8439.729 vs 39098.33 ± 10498.05,n =8,p <0.05).3. The result of Western blot methods:Western blot analysis revealed that the molecular weight of MMP-9 and IL-1B were respectively 92kDa and 17 kDa. There were significant differences of MMP-9 and IL-1B expression levels: I > II > I > IV.4. Immunohistochemical analysis methods;Immunohistochemical analysis clearly showed positive staining in synovial cell and inflammatory cell chondrocyte. There are significant difference of MMP-9 and IL-1B expression in the four groups of AA.DiscussionThere were have been numerous reports about the relationship between PRL and RA. Clinical observations and experimental animal studies have already implicated the dysfunction of the secretion of PRL and the plasma PRL levels showed a significant correlation with RA. PRL could affect multiple processes of RA including; the regulation of the immune system, the interaction with the inflammatory medium and the regulation of the cytokins, etc. Recently, Brenna,et al proposed that there is some association between HAL-DR4 and PRL gene, both located close together on the short arm of chromosome 6. The increased risk of RA associated with breastfeeding (hyperprolactinemic status) may be related to HAL-DR4. But the precise mechanisms of PRL were not completely understood.To determine the exact role of PRL in the pathogenesis of RA and supply experimental basis for clinical treatment of RA, we examine the expression of MMP-9 and IL-113 of adjuvant arthritis at both hyperprolactinemic levels induced by implanting subcutaneous diethylstilbestrol(DES) and hypoprolactinemic levels induced by injection of bromocriptine ( BRC). Our result indicated that the production of MMP-9 and IL-ip were augmented at hyperprolactinemic levels and inhibited at hypoprolactinemic levels. Our findings suggested that PRL may play its role by regulating the secretion of proinflammatory cytokines such as IL-1 p and tissue destructive proteolytic enzymes such as MMP-9 in RA. It has been verified that expressions of inflammatory cytokine and tissue destructive proteolytic enzymes by synovial cells could be stimulate and inhibited when treated with PRL and BRC seperatelly in vitro. Our conclusions were in good agreement with their reports.Recent research suggested that infiltrating T lymphocytes and fibroblast-like synovial cells could produce PRL and express PRL receptor (PRLR) in RA. The combination of PRL and PRLR could activate MAPKs or JAK/STAT signal transduction pathway, so the PRL/ PRLR pathway might be an important way for PRL to produce a marked effect. It has been reported that the inflammatory cytokines have direct effects on the secretion of MMP-9, the changes of MMP-9 may be stimulated by PRL directly or be stimulated by IL-1 (3 indirectly.In conclusion, this study demonstrated firstly that the level of PRL in RA rats could influence the expression of inflammatory cytokine IL-1 (3 and tissue destructive proteolytic enzyme MMP-9. The production of MMP-9 and IL-1 {5 were stimulated at hyperprolactinemic levels and inhibited at hypoprolactinemic levels. Whether the changes of MMP-9 expression was stimulated by PRL directly or by IL-1 (3 indirectly need further research.