Estradiol Exerts Anti-inflammatory Effects By Restraining Phosphorylation Of I?B? On Synovicytes Of Osteoarthritis Via ER?

BackgroundOsteoarthritis(OA)is a kind of chronic disabling disease,the clinical manifestations of which are mainly joint pain,activity decreased obviously,joint rigidity and a decrease in activity.The pathological characteristics of osteoarthritis are articular cartilage degenerative,secondary bone/subchondral bone destruction and articular synovial hyperplasia.The course of OA involving the whole joints,including cartilage,subchondral bone,synovium and ligaments.Based on the current available results,OA associated risk factors including age,obesity,genetic,mechanical stress,gender,immune,etc,but its specific pathogenesis is still not very clear.Therefore,to explore the key molecules participated in important pathologic change and signaling pathways during the process of OA pathogenesis is crucial for prevention and treatment.Under normal circumstances,synovium is a thin film consisted of fibroblast-like synoviocytes(FLS)and macrophages.But in the process of OA pathogenesis,the synovium inflammatory change closely associated with osteoarthritis disease development.Meanwhile,the epidemiologic studys have show the correlation between estrogen and osteoarthritis,and estrogen-replacement therapy could delay the occurrence and development of OA,improve the clinical symptoms of patients.The nuclear-factor kappa B inhibitor alpha(I?B?)was choosen as a pointcut to investigate the correlation between estrogen and NF-?B pathway.Then,we coulid find how estradiol works in OA synoviocytes.Objective1.To obtain normal synovial tissue,isolate and cultivate synoviocyte,then identify the purity of sample cells.2.To verify whether synoviocytes express ER?.3.To verify whether estradiol plays an anti-inflammatory role in synoviocytes induced by inflammation factors.4.To investigate the correlation between the anti-inflammation of estradiol and ER?.5.To investigate the regulatory effect of estradiol on I?B? and NF-?B pathway in synoviocytes.Methods1.Obtained the normal knee joint synovial tissue,tissue explant adherent methods were used to harvest human FLS.Flow cytometry were used to identify the purity of the cell samples.2.Immunofluorescent staining and immunohistochemical method were used to observe the expression of ER? in human knee joint synovial tissue and synoviocytes.3.The synovicytes were divided into 4 groups: control group,group pretreated with IL-1?,group pretreated with IL-1? and estrogen,group pretreated with IL-1?,estradiol and specific antagonist of ER?-tetrahydrocannabinol(THC).Then fluorogenic quantitative PCR and western blot were used 36 hours later to observe the mRNA and protein levels of IL-6 in each group of synovicytes.4.Fluorogenic quantitative PCR and western blot were used to observe the mRNA and protein levels of I?B? and p-I?B? in each group of synovicytes.Result1.Flow cytometry showed that cellular purity met the basic stands of subsequent experiments.2.The result of cell immunofluorescence staining and immune histochemical showed that ER? widely expressed in FLS.3.Fluorogenic quantitative PCR and western blot showed that the expression of IL-6 mRNA and protein elevates in IL-1? treated cells compared to control group;mRNA and protein levels of IL-6 were inhibited in the cells pretreated with IL-1? and estradiol compared to IL-1? treated cells.MRNA and protein levels of IL-6 elevated in the cells treated with THC,estradiol and IL-1?,compared to IL-1? and estradiol treated cells.4.I?B? protein level declined while p-I?B? protein level and I?B? mRNA level elevated in IL-1? treated cells compared to in control group;I?B? protein level elevated while p-I?B? protein level and I?B? mRNA level declined in the cells pretreated with IL-1? and estradiol compared to IL-1? treated cells;estradiol and IL-1?,I?B? protein level declined while p-I?B? protein level and I?B? mRNA level elevated In the cells treated with THC compared to IL-1? and estradiol treated cells.Conclusion1.Macrophagocyte could be removed through the exchange of the cell medium and trypsin digestion.Thus FLS be purified.2.ER? widely widely expressed in the FLS.3.In the cells treated with IL-1?,IL-6 mRNA and protein level elevates compared to control group,which verified IL-1? could simulate the affect of inflammation.The rest of the three groups of cells verified estradiol can inhibit synoviocyte release of inflammatory mediators in the inflammatory conditions,thus protect synoviocyte.4.Estradiol could inhibit the phosphorylation of I?B?,and play a crucial role of anti-inflammation in synoviocyte.