The Effect And Mechanism Of TANIIA On Osteoarthritis Based On YAP/NF-κb Signaling Pathway

Objective: To explore the role and mechanism of TANIIA in osteoarthritis based on YAP/NF-κB signaling pathway.(1)Bioinformatics analysis was used to construct the theoretical basis for TANIA treatment of osteoarthritis,and osteoarthritis cartilage samples were used to verified the theory.(2)The effects of TANIIA on catabolism,inflammation,apoptosis and YAP/NF-κB signaling pathway of osteoarthoid chondrocytes were investigated in vitro.(3)In vivo experiments were conducted to investigate the effects of TANIIA on cartilage decomposition synthesis,inflammation and apoptosis in osteoarthritis mice,and the role of YAP in degenerative cartilage.(4)The effect of TANIIA on abnormal bone remodeling and angiogenesis of subchondral bone and the role of YAP in subchondral bone were investigated in mice with osteoarthritis.Methods:(1)1.The action targets of TANIA-OA were retrieved through TCMSP database,and the latest literature about TANIA was searched to supplement the targets not yet recorded in the database,and the protein target information was standardized through Uniprot database.2.Search for targets related to osteoarthritis through Genecards,OMIM and TDD databases,and searching for targets of clinical drugs for the treatment of osteoarthritis through Drugbank database as supplements.3.Venn plots of TANIIA-OA targets were prepared to obtain the action targets of TANIIA in the treatment of Osteoarthritis,and PPI protein interaction network was made by String database and Cytoscape software.4.Go and KEGG analysis of TANIIA-OA targets was performed by Metascape database.5.After decalcification,embedding and section of osteoarthritis cartilage samples of different K-L grades,Safranin O and Fast Green staining and immunohistochemical staining were performed to evaluate the degree of degeneration of cartilage in each group,the expression of catabolic factors and the relationship between YAP and the degree of degeneration of osteoarthritis.(2)1.The ATDC5 cells were placed in ITS medium,and the induction status of chondrocytes was detected by Alcian blue staining and collagen q RT-PCR.Osteoarthritis like chondrocytes were constructed by IL-1β intervention.2.CCK-8 and flow cytometry were used to evaluate the effect of TANIIA on the activity of osteoarthritis like chondrocytes,and the optimal concentration range of TANIIA in the in vitro experiment was determined.The morphological differences between OA and normal chondrocytes,the expression level and location of YAP,and the effect of TANIIA on OA chondrocytes were observed by YAP/F-actin fluorescence double-standard staining and electron microscopy.3.The expressions of inflammatory response,apoptosis,YAP/NF-κB signaling pathway related factors in osteoarthritis chondrocytes were detected by q RT-PCR and WB,as well as the effects of TANIIA on the expression of the above indexes.(3)1.The environment of abnormal stress on the joint surface caused by osteoarthritis was simulated by amputating the anterior ligament of mice,and then an in vivo model of osteoarthritis was constructed.2.In the preliminary experiment,the effect of TANIIA on degenerative cartilage in osteoarthritis mice was evaluated by HE and Safranin O and Fast Green staining,and the optimal dose of TANIIA was determined.3.In the formal experiment,the functional status of the affected limbs of osteoarthritis mice and the effect of TANIIA on them were evaluated by mouse foot imprinting test.The changes of articular surface transparent/calcified cartilage,proteoglycan loss,cartilage integrity,and the protective effect of TANIA on osteoarthritis cartilage in mice were evaluated by HE and Safranin O and Fast Green staining.4.By immunohistochemical staining,the expression of catabolic/anabolic factors on the surface of osteoarthritis cartilage and the influence of TANIIA on the expression levels of the above indexes were evaluated.5.The apoptosis rate of chondrocytes and the expression level of apoptosis-related factors mediated by YAP were evaluated by immunofluorescence technique,as well as the effect of TANIIA on the apoptosis of YAP and chondrocytes in vivo.(4)1.The microstructural changes of subchondral bone were preliminarily evaluated by Micro CT 3D reconstruction.The CTan software was used to analyze the bone volume fraction,bone plate thickness and trabecular factor of subchondral bone in each group,and to evaluate the influence of TANIIA on the microstructure of subchondral bone.2.Osteoclasts,osteoblasts and YAP in the subchondral bone of OA mice at 0,30 and 60 days after operation were stained to evaluate the dynamic changes of bone metabolism in the subchondral bone of OA mice.3.Osteoclast and osteoblast progenitor cells were labeled through TRAP and Osterix to evaluate the influence of TANIIA on their differentiation and formation at 30 days after surgery.4.The effect of TANIIA on the RANK/RANKL/OPG pathway related to osteoclast formation was evaluated by immunofluorescence staining.5.Micro CT angiography and CTan software were used to evaluate the subchondral bone angiogenesis in osteoarthritis.CD31/Endomucin immunodouble staining was used to evaluate the effect of TANIIA on the angiogenesis of subchondral bone,including H-type vessels.6.The effects of TANIIA on VEGF and HIF-1α mediated angiogenesis related factors were evaluated by immunohistochemical staining.Results:(1)1.Through TCMSP database,the OD value of TANIIA was 48.89%,the DL value was 0.40,and there were 51 related targets.Through Genecards,OMIM,TDD,DRUGbank,Pharmac GKB databases and the latest literature results,3176 targets related to osteoarthritis were identified.34 TANIA-OA targets,including YAP and NF-κB factors,were obtained by Venn diagram.2.GO analysis showed that the main biological processes involved in TANIIA treat osteoarthritis included lipopolysaccharide response,hormone response,aging,endogenous apoptosis signaling pathway,muscle tone response and glucocorticoid response.Its main molecular functions include binding to DNA transcription factors,polypeptides,amino compounds,ubiquitinprotein kinases and NF-κB.KEGG analysis showed that the pathways of TANIIA in the treatment of OA were mainly enriched in IL-17,apoptosis-related,TNF,osteoclast differentiation related and NF-κB signaling pathways.3.The results of Safranin O and Fast Green staining of osteoarthritis cartilage indicated that with the increase of K-L grade,the loss of articular cartilage proteoglycan increased gradually,and the thickness of cartilage decreased gradually.Meanwhile,the expression levels of YAP,COX-2 and Bax in cartilage were increased.(2)1.ATDC5 cells could be induced into chondrocytes with high COLII expression by ITS medium for 2 weeks,and osteoarthritis-like chondrocytes model could be constructed by the intervention of IL-1β.2.CCK-8 and flow cytometry results indicated that 1.25-5 μM TANIA had little effect on the activity of chondrocytes and inhibited the apoptosis of osteoarthritic chondrocytes.The results of cell fluorescence indicated that YAP expression increased in osteoarthritic chondrocytes and accumulated in the nucleus while the transformation of chondrocytes to hypertrophic phenotype.2.5μM TANIA can effectively inhibit the expression and nuclear translocation of YAP in osteoarthritis like chondrocytes,and reduce the occurrence of hypertrophic chondrocytes.The results of electron microscopy showed that the apoptotic bodies of osteoarthritic chondrocytes were significantly increased.q RT-PCR and WB results showed that TANIIA could inhibit the YAP/NF-κB(YAP,p-IκBα,p-p65)signaling pathway,and reduce the expression and activation of catabolism-,inflammatory response-(IL-6,TNF-α,COX-2)and apoptosisrelated factors(Bax,c-Casp3,c-Casp9)in osteoarthid-like chondrocytes.(3)1.In the preliminary experiment,the protective effect of TANIIA on articular cartilage of OA mice was evaluated by HE and Safranin O and Fast Green staining,and the optimal administration dose was determined.2.The results of HE staining in the formal experiment indicated that there was no significant change in hyaline/calcified cartilage 30 days after the operation,while hyaline cartilage decreased,calcified cartilage increased and subchondral bone erosed the base of cartilage appeared 60 days after the operation.Safranin O and Fast Green staining showed that only a small amount of proteoglycan was lost at 30 days postoperatively,but not only the proteoglycan was lost significantly at 60 days postoperatively,but the integrity of cartilage was destroyed.The intervention of TANIIA can effectively inhibit the degeneration of cartilage surface and protect the intact structure of cartilage.3.Immunohistochemical results showed that the expressions of anabolic factors(Aggrecan and Lubricin)were significantly reduced in osteoarthritis cartilage,while the expressions of YAP and catabolic factors(COX-2,MMP13)were significantly increased.However,TANIA could maintain the homeostasis of cartilage decomposition and synthesis and reduce cartilage injury.4.Immunofluorescence results indicated that the apoptosis rate of chondrocytes in OA mice was significantly increased,the level of anti-apoptotic protein Bcl-2 was decreased,and the expression of pro-apoptotic protein Bax was increased.TANIIA can inhibit the apoptosis of osteoarthritis chondrocytes by regulating apoptosis-related proteins.(4)1.Micro CT data analysis indicated that subchondral bone marrow cavity enlargement and bone loss were observed in OA mice 30 days after surgery,and abnormal subchondral bone mass increase and sclerotic bone formation were observed 60 days after opaeration.TANIIA can effectively maintain the microstructure of subchondral bone in OA mice.2.TANIIA can maintain the normal differentiation and formation level of osteoclasts/osteoblasts in subchondral bone by regulating YAP and RANK/RANKL/OPG pathways.3.Micro CT angiography indicated that the number and volume of total blood vessels,including H-type blood vessels,in subchondral bone of OA mice were significantly increased compared with those in Sham group.TANIIA can reduce the formation of abnormal blood vessels by inhibiting the expression of VEGF and HIF-1α.Conclusion:(1)TANIIA acts on osteoarthritis through34 targets including YAP and NF-κB factors,and reduces the levels of catabolism,inflammatory response and apoptosis of osteoarthritic chondrocytes by inhibiting YAP/NF-κB signaling pathway.(2)By regulating chondrocyte metabolism and apoptosis in osteoarthritis mice,TANIA maintained the normal structure and collagen level of articular cartilage,alleviated cartilage degeneration caused by abnormal stress,and delayed the progression of osteoarthritis.(3)By regulating YAP and RANK/RANKL/OPG mediated bone metabolism,TANIA can inhibit the formation of bone cysts and bone islands of subchondral bone caused by stress changes,and maintain the microstructure of subchondral bone.At the same time,inhibiting the expression of VEGF and HIF-1α while reduce the formation of H-type blood vessels in subchondral bone,restore the mechanism of bone-vascular physiological coupling,further stabilize the microstructure of subchondral bone,and delay the progression of osteoarthritis.