The Study On The Correlation Between Estrogen And Pathomechanisms Of Osteoarthritis

Purposes: OA is the most prevalent disease in middle-aged and older,a heavy burden to society, and the mechanisms of which is still not known. With the aging of the human society, OA increase rapidly. To the medical workers, especially health care workers, it is necessary to further study the pathogenesis of OA. Epidemiological studies have found that in post-menopause women the incidence of OA increase quickly, so whether post-menopausal or not is a watershed for women suffering from OA, suggesting that changes in estrogen levels may play an important role in the pathogenesis of OA. OA mainly manifested as pathological degradation of articular cartilage, and synovial membrane and subchondral bone change. As the only cell type in the mature cartilage tissue, increased chondrocyte apoptosis is considered the core of the pathogenesis of OA. NO and Fas pathways are two main channels of chondrocyte apoptosis. Others including Bcl-2, Bax, Caspase-3 and other apoptosis regulatory proteins also play important roles. Our research focuses on the relationship between changes in the level of estrogen and chondrocyte apoptosis and the pathological changes, we have assumed: First, if the level of estrogen decrease , chondrocyte apoptosis may increase through Fas and NO passways, leading to articular cartilage degradation, and may aggravate the pathological changes in the course of OA, a series of apoptosis regulatory proteins may play important roles. Second, estrogen bind to the estrogen receptors in chondrocyte surface, regulating the expression of cellular proteins, which may play a protective effect on cartilage cells. Our experiment is set up to verify whether the above assumptions, as well as the mechanisms.Methods: We designed the following tests: First, bilateral ovariectomy in female rat as ovariectomized model, after 6 weeks, 10weeks, the the knees from different rats, 5μm sections after decalcification, TUNEL staining for apoptosis rates, HE staining and toluidine blue staining for Mankin score to observe the pathological changes in articular cartilage, the cartilage slices were observed by immunohistochemical expression for apoptosis-related proteins, to understand the links between the level of estrogen and chondrocyte apoptosis and cartilage pathological changes, as well as apoptosis-related proteins; Second, Hulth method to establishe OA model in female rats, simultaneously bilateral ovariectomied to establisht the OVX models.After 6w, 10w we take the rat knees, and make TUNEL staining for the rate of chondrocyte apoptosis, HE staining and toluidine blue staining for Mankin score ,immunohistochemical staining for the expression of iNOS ,to verify if the level of estrogen decrease after OVX will reduce chondrocyte apoptosis and the original impact of the pathological changes of OA, and reveale if NO change in articular cartilage may be the impact of OA-like changes; Third, take the rabbit knee joint chondrocytes in primary culture cells, using the sodium nitroprusside(SNP), a nitric oxide donor to the cell culture system to establish the in vitro model of chondrocyte apoptosis .To observe if precultured with 10-3 ~ 10-9mM estrogen for 2 hours will change chondrocyte apoptosis rates and to reveal its possible mechanisms.Results: We found that: I: In ovariectomized rats ,articular cartilage fibrosis and cystic changes,can be seen with HE staining , toluidine blue staining of articular cartilage can even see the loss of dye staining shallow. Mankin scores were significantly higher than sham-operated group, and estrogen replacement therapy can reduce the Mankin score, improve the changes of articular cartilage. TUNEL detection see OVX increase the rate of chondrocyte apoptosis, which may play an important role in the OA pathological changes. Immuno- histochemical staining showed that OVX groups iNOS, Fas, Bax, Caspase-3- positive cells increase and Bcl-2-positive cells decrease, that means NO and Fas apoptosis pathways may be involved in OA. Apoptosis regulatory proteins Bcl-2, Bax, Caspase-3 may play important roles too; II: OVX can significantly aggravate OA changes of the articular cartilage in female rat models, the Mankin score was significantly higher than non-OVX rats, iNOS staining show that OVX increase iNOS protein expression. TUNEL staining also verify chondrocyte apoptosis increased, perhaps due to lower estrogen levels, which exacerbates the changes of articular cartilage; NO level increase in the cartilage tissue may be one reason. Third, 2mmol / L SNP can induce in vitro apoptosis of rabbit chondrocytes, and 10-3 ~ 10-9mM estrogen can inhibit this effect. That estrogen may bind to ERs ,regulate apoptosis-related proteins such as Bcl-2, Bax, Caspase-3 expression and reduce NO-induced chondrocyte apoptosis.Conclusions: Bilateral ovariectomize in female rats can successfully establish OA model.We have found that reduce the level of estrogen will induce chondrocyte apoptosis, and cartilage degradation in animals, resulting in OA-like changes, and further studies confirmed chondrocytes apoptosis through NO and Fas ways , a variety of apoptosis and apoptosis-related proteins such Bcl-2,Bax,Caspase-3 play a regulatory role. We have found that reducing the level of estrogen will increase the original OA animals change, and further studied the possibility of occurrence of the process mechanisms. Chondrocytes cultured in the system by precultured with different concentrations of estrogen, we found estrogen can prevent the SNP-induced chondrocyte apoptosis, which may play an protective effect on chondrocytes in OA pathogenesis.From our experiments, we speculated that estrogen levels after ovariectomy reduced may lower the protective effect from attacks outside or may be induced by increased apoptosis of articular cartilage, chondrocyte apoptosis is exacerbated by increased intra-articular changes in physiology, the results may lead to the degradation of articular cartilage and thus give rise to OA lesions. The application of exogenous estrogen to chondrocytes may reduce apoptosis and cartilage degeneration, inhibit the development of OA pathology. Therefore, estrogen application in post-menpause women should be a routine treatment for the prevention and treatment of OA.Of course, in our tests, we also found that application of exogenous estrogen, can only reduce apoptosis in cartilage and ease the development of OA pathology and can not preven OA completely, so we think that there should be other mechanisms play important roles, it is necessary to continue more researches.