Mechanism Of Atractylenolide Ⅲ Regulates Trim63-Myh11 To Promote BMSC Chondrogenic Differentiation

Objective:Articular cartilage defects are common in the field of orthopedics clinically,which usually caused by trauma or other diseases,such as chondritis,osteoarthritis,and osteonecrosis.Due to the self-repair ability of articular cartilage limitation,articular cartilage defect has always been a therapeutic challenge in clinic.Bone marrow mesenchymal stem cells(Bone Mesenchymal Stem cell,BMSC)are recognized as ideal seed cells for repairing articular cartilage defects.Therefore,efficient promotion of chondrogenic differentiation of BMSCs is the key to repairing articular cartilage defects.Therefore,more and better efficient promotion of chondrogenic differentiation of BMSC is the key point to repair cartilage tissue.In our previous study,we found that Atractylenolide Ⅲ,one of active ingredient of Chinese medicine Atractylodes macrocephala,can promote the BMSC chondrogenic differentiation with upregulating the expression of cartilage marker genes like Sox9,Collagen Ⅱ and Aggrecan.However,the mechanism of action is unclear and deserves further research.Indepth study revealed that Atractylenolide Ⅲ could upregulate the total ubiquitination level and the expression of E3 ligase Trim63.Based on this,our study proposes the scientific hypothesis that Atractylenolide Ⅲ may promote BMSC chondrogenesis by regulating ubiquitin E3 ligase Trim63 and affect post-translational modification.In this study,we explored the role of E3 ligase Trim63 on BMSC chondrogenic differentiation and the molecular mechanism of Atractylenolide Ⅲ regulation by establishing a targeted differentiation chondrosphere model of BMSC and a rat articular cartilage defect model.Methods:1.To explore the effect of Atractylenolide Ⅲ on BMSC chondrogenesis,a chondrosphere model was constructed by BMSC differentiation for this research.(1)Classical induction medium was used for constructing a BMSC chondrosphere for 7 days,and the expression of cartilage marker genes were detected by western bolt.(2)Cytotoxicity produced by different concentration of Atractylenolide Ⅲ,which was detected by CCK8 to determine the appropriate concentration for culturing BMSC.The concentration range of Atractylolide Ⅲacting on BMSC chondrogenesis was screened by western blot.(3)Alcian blue staining was used to observe the morphological changes of Atractylolide Ⅲ promoting BMSCs chondrogenesis.2.The ubiquitin E3 ligase Trim63 was discovered respectively from transcriptomics and translational omics by analyzing the previous omics data of our research group.And further study explored the role of Atractylolide Ⅲ in promoting chondrogenic differentiation of BMSC by regulating E3 ligase Trim63.(1)Autodock was used to calculate the affinity of small molecular binding with the protein.(2)Within the appropriate range concentration of Atractylolide Ⅲ promoting chondrogenic differentiation of BMSC,the effect of Atractylolide Ⅲ on Trim63 was detected by western blot.(3)Atractylolide Ⅲ stimulated BMSC chondrogenesis and the change of total ubiquitination level was detected by western blot.(4)To confirm the region of Atractylolide Ⅲ regulating Trim63 promoter,a dualluciferase reporter gene for the Trim63 promoter was constructed.(5)RNA interference was used for knocking out Trim63 in BMSC,and RT-qPCR was used to detect whether the transcription of Atractylolide Ⅲ promoting chondrogenic differentiation of BMSC was dependent on the expression of Trim63.3.The regulation mechanism of E3 ligase Trim63 on the chondrogenic differentiation of BMSC in vivo was researched by using the BMSC chondrosphere model and the rat articular cartilage defect model.(1)The total ubiquitination level of BMSC chondrogenesis was detected by western blot.(2)The protein level and transcription level of Trim63 in BMSC chondrosphere model was detected by RT-qPCR and western blot.(3)The phenotype changes of BMSC chondrosphere were observed,and the expression of cartilage marker genes and total ubiquitination levels were detected by RT-qPCR and western blot after interfering Trim63.(4)Proteins that can bind to Trim63 were enriched by co-immunoprecipitation(COIP),predicted by LC-MS.The interaction between Trim63 and target gene was verified by western blot.(5)To confirm the ubiquitination modification,ubiquitin mutants with different lysine residues were constructed.(6)Co-transfecting ubiquitin,Trim63 and target gene,antiFlag tag was used for CO-IP,which was detected by vertical electrophoresis.Quality inspection were verified by Coomassie blue,and ubiquitin modification sites of target gene were predicted by LC-MS/MS.(7)After transfecting ubiquitin mutants into BMSC,induction medium were used for chondrophere model.The capability and effectiveness of chondrogenesis of BMSC were observed,and the expression of cartilage marker genes would be detected.(8)In order to verify the role of Trim63 in repairing articular cartilage defects in vivo,the rat cartilage defect model was constructed.In order to verify the role of Trim63 in repairing cartilage defects,a rat cartilage defect model was constructed.The BMSC were infected with adeno-associated virus to overexpress Trim63,and then transplanted into animals to repair articular cartilage defects.Defect repair was evaluated by HE staining,Alcian blue staining,and immunofluorescence of cartilage marker gene.Results:1.Atractylenolide Ⅲ promotes BMSC chondrogenesis(1)Constructing BMSC chondrosphere model:After centrifuging,the P4 generation BMSC were cultured with classic chondrogenic medium within 15ml centrifuge tube for 7 days.Obviously,a chondrocytes pellet formed.(2)The results of CCK8 experiments show that the range of Atractylenolide Ⅲ working concentrations acts between 0.1 μM and 160μM,which has no obvious toxic and side effects on BMSC.Subsequently,Atractylenolide Ⅲ was used to induce the culture of BMSC into chondrosphere,and the protein levels expression of cartilage marker genes Sox9,Co12A and Aggrecan were detected by western blot.The experimental results showed that compared with the induction group,Atractylenolide Ⅲ could significantly upregulate the cartilage marker genes with the working concentration between 1 μM and 20μM.(3)Alcian blue staining were observed the morphological changes of the spheres and proteoglycan content after inducing by Atractylenolide Ⅲ.The results showed that there was no significant difference in proteoglycan content among the groups.Compared with the induction group,the volume of BMSC chondrosphere induced by Atractylenolide Ⅲ was greater.The density of cell to cell in BMSC chondrosphere was higher when the working concentration was 1 μM.2.Atractylenolide Ⅲ regulates the transcription of ubiquitin E3 ligase Trim63 promoting BMSC chondrogenesis.(1)The small molecule-protein docking result showed the affinity score of AtractylenolideⅢ and Trim63 was-6.7,which indicated the strong possibility of binding between them.(2)Atractylenolide Ⅲ induced BMSC chondrogenesis,the results revealed that ubiquitination levels were significantly upregulated in the Atractylenolide Ⅲ induced group of concentration within 1 μM to20μM compared with the induced group.(3)Analyzing and screening the differentially expressed genes of transcriptomics and translational omics on BMSC chondrogenesis.The results showed that the expression of the ubiquitin E3 ligase Trim63 was significantly increased in the transcriptional and translational levels,which have been further verified by RT-qPCR and western blot.In the range of Atractylenolide Ⅲ concentration within 1μM to20μM,the expression of Trim63 was upregulated.(4)Trim63 was knocked out by RNA interference technology,and the expression of cartilage marker genes was detected by western blot.The results showed that cartilage marker genes and total ubiquitin levels were significantly down-regulated.The BMSC chondrosphere culture results determined that BMSC could not be induced into a complete cartilage sphere after knocking out Trim63.(5)In order to prove that Atractylolide Ⅲ promotes the high-efficiency transcription of Trim63 in the process of BMSC chondrogenesis by activating the enzymatic activity of Trim63 promoter,Trim63 promoter region plasmids were constructed which would be verified by dual luciferase reporter gene system.The results showed that the relative fluorescence value of the 2000bp region of the Trim63 promoter increased significantly with the1 μM to 10μM concentration of Atractylolide Ⅲ.Then,the Trim63 promoter regions were detected segmentally,and the results showed that the relative fluorescence value of Atractylolide Ⅲ acting on the 500bp to 1000bp region of the Trim63 promoter increased significantly.3.Mechanism of ubiquitin E3 ligase Trim63 regulating BMSC chondrogenic differentiation(1)In the progress of BMSC Chondrogenesis,the total ubiquitination level and the expression of the ubiquitin E3 ligase Trim63 increased.To explore the mechanism of Trim63 regulating chondrogenic differentiation of BMSC,CO-IP and LC-MS were used for discovering the protein bounded with Trim63.The results showed that during the chondrogenic differentiation of BMSC,Trim63 enriched a large number of cytoskeletal proteins,and myosin11(Myh11)was screened in three biological replicate groups.Subsequently,CO-IP and western blotting experiments revealed that Trim63 increased binding to Myh11 in BMSC chondrogenic differentiation.(2)In order to realize the expression of Myh11,the western bolt experiment results showed that Myh11 protein expression was significant downregulated in BMSC chondrogenic differentiation.Overexpression of Myhll in BMSCs,and the changes of chondrosphere morphology and the cartilage marker genes would be observed and detected.The results showed that compared with the induction group,the expression of cartilage marker genes in the overexpressed Myh11 induction group was significantly down-regulated,and complete chondrosphere could not be formed.(3)To verified whether the Myhll ubiquitination level depending on the expression of Trim63,co-transfected ubiquitin and Myh11 with Trim63,and then CO-IP experiment were performed.The result showed that compared with group of ubiquitin and Myhll,the ubiquitination level of ubiquitin and Myh11 with Trim63 group were definitely increased.Mutations of different Ubiquitin lysine residue K6R,K11R,K27R,K29R,K33R,K48R,K63R were constructed and co-transfected with Myh11 and Trim63 in order to qualified the ubiquitin sites.The results showed that the ubiquitination level of K27R group was prominently decreased.(4)Focusing on Trim63-mediated Myhll ubiquitin modification sites,LC-MS/MS was performed.CO-IP samples were tested after quality inspecting by Coomassie blue.The detection results showed that K1520,C382 and T490 may be the potential sites which were mediated Myhll by Trim63 modification.K1520R,C382R and T490R mutants were constructed and then confirmed by CO-IP and western blot.The results showed that the ubiquitination level of C382R group was remarkable decreased.(5)To clarify the role of Myh11 ubiquitin site C382 during BMSC chondrogenesis,C382R mutant was transfected into BMSC.The results showed that BMSC chondrosphere could not be formed in C382R induction group comparing with induction group.4.Overexpressing Trim63 repairs the rat articular cartilage defects.Successfully constructed the articular cartilage defect models in rat to verify the repairing effect of ubiquitin E3 ligase Trim63 on articular cartilage.BMSCs were infected with adenoassociated virus negative control or overexpressing Trim63 and cultured with chondrocytes induction for 7 days,then transplanted into rat articular cartilage defects.The animals were fed freely for 6 to 8 weeks and subsequently sacrificed.The results of HE staining showed that the repair of the articular defects surface in the Trim63 overexpression group was relatively smooth.The results of Alcian blue staining showed that compared with the NC group and the Matrix group,the proteoglycan staining of the repair surface of the articular defects in the overexpression Trim63 group was more obvious.Immunofluorescence results showed that the cartilage marker gene Co12A performed distinguished fluorescent expression in the repairing site of articular defects in the Trim63 overexpression group.Conclusions:1.Atractylenolide Ⅲ,an active ingredient of the traditional Chinese medicine Atractylodes macrocephala,can target binding to ubiquitin E3 ligase enzyme Trim63,and regulate the total ubiquitination level,playing a significant role in the progress of BMSC chondrogenesis.2.During BMSC chondrogenesis,the ubiquitin E3 ligase Trim63 promotes the ubiquitination of the K27-linked cysteine residue C382 of Myh11.3.The ubiquitin E3 ligase Trim63 can promotes BMSC chondrogenesis and repairs the rat articular cartilage defect.