To Study The Expression Of SOX5、SOX6in RA And The Effect Of Celastrol On SOX5、SOX6/RANKL Pathway In CIA Mouse

Objective：To study the expression of SOX5、SOX6in RA and the effect of Celastrol onSOX5、SOX6/RANKL pathway in CIA mouseMethods：1.Clinical information and analyses40RA patients’ mRNA of PBMC were measured by PCR analysis for relativeexpression of SOX family. Collecting blood serum and synovial fluid of RA/OA/HCpatients, SOX5/SOX6secretary protein levels were measured by ELISA.Collectingsynovial tissues of RA/OA/HC patients, SOX5/SOX6protein expression levels andlocalization were measured by immunohistochemistry.2. Vitro study12RA patients’ mRNA of PBMC were effected by TNF-α(50ng/ml) for6、24and48hs, then the mRNA were measured by PCR analysis for relative expression ofRANKL、SOX5and SOX6.Generation and activity of healthy pers’ PBMC effected by Cel were measuredby CCK-8, then using Cel of different concentration to effect healthy pers’ PBMC for48hs, the mRNA were measured by PCR analysis for relative expression of RANKL、SOX5and SOX6.Generation and activity of MH7A effected by Cel were measured by CCK-8,then using safe concentration Cel or IL-1β（20ng/ml）effect MH7A for24hs, themRNA were measured by PCR analysis for relative expression ofRANKL/SOX5/SOX6/IL-6/TNF-α and IL-8.3. Vivo study CIA mouse model was establishment to observe therapeutic action of Cel forCIA and the effection and the mechanism of RA bone erosion.To be specific：1.Toobserve inflammation and bone erosion by immunopathogenesis.2.To observeprotein expression of RANKL/SOX5/SOX6in mouse joints Blank/CIA/CIA+Celgroups by immunopathogenesis.3.Via REAL TIME-PCR，to observe the relativeexpression of RANKL/SOX5/SOX6/IL-1β/IL-6/TNF-αand IL-8，to extracte mousejoints RNA and measuring by，to confirm inflammation inhibition again and toapproach the effect and the molecule mechanism of Cel for CIA bone erosion.Results：1.Clinical information and analysesIn RA patients’ mRNA of PBMC, SOX5and SOX6express more than the otherin SOX family. SOX5and SOX6in RA patients’ blood serum and synovial fluid,express more than OA and HC groups, and express both on synovial macrophage-likecells and synovioblasts.2.Vitro studyThe Cel doesn’t effect the generation and activity of MH7A when itsconcentration is less then2μM. The expressions of RANKL、OPG、IL-6、TNF-αandIL-8in MH7A were significantly increased in MH7A upon stimulation with IL-1β(p<0.05). Celastrol could inhibit the expressions of RANKL、OPG、IL-6、TNF-αandIL-8in a dose-dependent manner and notably elevated the ratio of OPG/RANKL axis.3.Vivo studyThe pathematology scores of CIA model demonstrated that Cel could inhibitarthrositis of CIA mouse compared between CIA and CIA+Cel group（p<0.01）；HEstaining and the pathematology scores of joints confirm the result again；Immunohistochemical staining of ankle-joints and knee joints demonstrated that Celcould inhibit the protein expression of SOX5and SOX6（p<0.001）；The REALTIME-PCR results demonstrated that Cel could inhibit the relative expression ofRANKL、SOX5and SOX6in gene level compared with CIA group（p<0.05），to increase OPG expression at the same time，notably elevated the ratio of OPG/RANKLaxis.Conclusion：1.The transcription factor SOX5and SOX6may have important function in RA.2.Cel could inhibite the expression of RANKL、inflammatory factor and chemotaticfactor inRA FLS.3.Cel could inhibite inflammation and bone erosion in CIA mouse, the possiblemechanism is via transcription factor SOX5and SOX6to adjust the expression ofRANKL, and to elevate the expression of OPG, in order to elevated the ratio ofOPG/RANKL axis.