| Rheumatoid arthritis(RA)is an autoimmune disease.Fibroblast-like synoviocytes(FLS)are known to be the key effector cells in RA.Activated RA FLS are abnormally proliferative and highly aggressive similar to malignant tumor cells,and its imbalance of proliferation and apoptosis is the main cause of synovial hyperplasia and synovial inflammation.Therefore,FLS is a key target cell for RA prevention and treatment,and the intrinsic molecular processes that regulate the abnormal biological properties of RA FLS are essential for finding innovative drugs against RA.hedgehog(Hh)is a common pathway in vivo,and one of its branches,Sonic hedgehog(Shh),is most widely expressed in humans.A large body of literature shows that the Shh signaling pathway is aberrantly activated in RA synovial tissues and isolated cultured RA FLS;the activated Shh pathway significantly promotes the proliferation and metastasis of RA FLS,while the Smo inhibitor cyclopamine inhibits the Shh pathway and reduces the proliferative capacity of RA FLS,suggesting that the Shh pathway plays a close role in the pathogenesis of RA by regulating the biological properties of RA FLS aggressiveness.pathogenic role in RA pathogenesis.Numerous studies have shown that a hypoxic environment similar to that of tumor cells exists in RA.the main cause of RA hypoxia is the abnormal proliferation and differentiation of FLS.Hypoxia inducible factor-1α(HIF-1α)is a master regulator of cellular hypoxic response.Therefore,interventions targeting HIF-1α are of wide application.The group has synthesized and discovered a series of arylformamide derivatives,among which the most active compound is AMSP-30 m,which has been shown to affect the biological behavior of various tumor cells.In this study,the therapeutic effects and mechanism of action of AMSP-30 m,a HIF-1α inhibitor,on RA were investigated through in vitro and in vivo experiments:The group has synthesized and discovered a series of aryl carboxamide derivatives,among which the most active compound is AMSP-30 m,which has been shown to affect the biological behavior of various tumor cells.In this study,the therapeutic effects and mechanism of action of AMSP-30 m,a HIF-1α inhibitor,on RA were investigated through in vivo and in vitro experiments.1.Mechanisms by which AMSP-30 m regulates the biological behavior of RA FLS through inhibition of Shh signaling pathwayObjective: The aim of this study was to observe the effects of AMSP-30 m on proliferation,apoptosis,migration,invasion and inflammation of RA FLS(MH7A cells)under hypoxic stimulation.Methods: MH7 A cells were stimulated by hypoxia(1% O2)to simulate the hypoxic microenvironment of RA.MTT was performed to detect the cell viability level,ELISA to detect the level of pro-inflammatory factors(IL-1β,IL-6 and TNF-α),flow cytometry to detect cell cycle distribution and apoptosis,and wound healing and Transwell assays to detect cell migration and invasion.Ghost pen loop peptide staining was performed to detect cytoskeletal reorganization,and Western blot(WB)to detect the expression levels of pathway-related proteins.Results: AMSP-30 m inhibited cell proliferation by inhibiting c-Myc and Cyclin D1 and blocking the cell cycle in the G2/M phase of MH7 A cells.In addition,AMSP-30 m promoted apoptosis by activating the mitochondrial apoptotic pathway,as evidenced by Annexin V-PE/7-AAD staining showing an elevated apoptotic index in synovial cells and increased expression of Cleaved caspase-3 and Cleaved PARP.The reduction of proinflammatory factors as well as the results of wound healing assay and Transwell assay indicated that AMSP-30 m had strong anti-inflammatory and invasion inhibitory effects.AMSP-30 m reduced the protein levels of HIF-1α and Shh signaling pathwayrelated proteins Shh,Smo and Gil-1 in hypoxia-stimulated MH7 A cells,while Shh pathway activator(SAG)and inhibitor(cyclopamine)reversed and enhanced this result.Conclusion: The HIF-1α inhibitor AMSP-30 m regulates the abnormal biological behavior of RA FLS by blocking the Shh signaling pathway.2.Study on the therapeutic effects and some mechanisms of AMSP-30 m in rats with adjuvant arthritisObjective: This study was conducted to clarify the therapeutic effect of AMSP-30 m on adjuvant arthritis(AIA)rats and to explore its possible mechanisms.Methods: A rat model of AIA was constructed,and AMSP-30 m was injected intraperitoneally after successful modeling to observe its therapeutic and antiinflammatory effects.The effects of AMSP-30 m on proliferation and apoptosis in AIA rats were detected by immunohistochemistry(IHC)of Ki67 and TUNEL kit,VEGF content and vascular density were detected by IHC of CD31 and VEGF,and pathwayrelated protein levels were detected by WB.Results: AMSP-30 m,while inhibiting HIF-1α expression,also had potent anti-arthritic and anti-inflammatory effects in AIA rats,as evidenced by reduced foot swelling,arthritis index,histopathology scores,and production of IL-1β,IL-6,and TNF-α in serum and synovial tissues.AMSP-30 m decreased synovial Ki67 expression and increased TUNEL positive index,suggesting an anti-proliferative and pro-apoptotic effect on AIA synovial cells,which was associated with reduced Bcl-2 protein levels and increased Bax,Cleaved caspase-3 protein levels.In addition,AMSP-30 m had an anti-angiogenic effect in AIA synovial membranes,as evidenced by a decrease in synovial VEGF expression and a decrease in the number of blood vessels.In addition,AMSP-30 m inhibited the activation of the synovial Shh pathway,as evidenced by a decrease in pathway-related proteins such as Shh,Smo,Gil-1,Cyclin D1,and c-Myc.Conclusion: HIF-1α inhibitor AMSP-30 m may promote synovial apoptosis,reduce synovial angiogenesis and inhibit Shh pathway to have strong anti-arthritic effects in AIA rats... |
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