Study On Abnormal Osteogenesis Mechanism Of AS Based On IPSCs Technology And Intervention Mechanism Of Total Glucosides Of Paeony

Objective Ankylosing spondylitis(AS)is an autoimmune disease characterized by abnormal osteogenesis and inflammation.It is mainly axial joint disease.With the progress of the disease,the normal structure and function of the joints are gradually lost,and in severe cases the spine movement function is completely lost.In this study,iPSCs of normal humans and AS disease models were established and induced to differentiate into MSCs.The differences in osteogenic differentiation between normal humans and AS iPS-MSCs were observed,and the possible molecular mechanism was explored.To explore the role and mechanism of total glucosides of paeony on AS iPS-MSCs osteogenesis,total glucosides of paeony was used to interfere with AS iPS-MSCs osteogenic differentiation.This will provide theoretical basis for traditional Chinese medicine treatment of AS.Methods Part ?:After obtained urine cells from urine of healthy volunteers and AS patients,pCXLE-hOCT3/4-shp53-F,pCXLE-hSK.pCXLE-hUL,pCXLE-EGFP,pCXWB-EBNA1 plasmids were transferred into urine cells.The iPSCs clones were generated after co-cultivation on MEF.The iPSCs clones were picked for expansion.Their morphology,expression of endogenous and exogenous genes,surface markers,and pluripotent differentiation potential were identifiedPart ?:Induced iPSCs to MSCs by direct adherence method,and obtained fibroblast-like cells with consistent morphology through multiple passages.The acquired cells differentiated into osteogenesis,adipogenesis,and chondrogenesis.The ability of three lineage differentiation were identified.To verify whether the induced cells meet the diagnostic criteria of MSCs,the expression of surface markers were anlysised by flow cytometry.Part ?:Normal iPS-MSCs and AS iPS-MSCs are induced to osteogenesis.The osteogenesis indicators,autophagy indicators and ?-catenin gene and protein expression were observed.Autophagy inhibitor(3-MA)and Wnt/p-catenin agonist(SKL2001)were used to intervent cells,and the changes of osteogenesis indicators,autophagy indicators and ?-catenin gene and protein expression were observedPart ?:Intervention of AS iPS-MSCs with total glucosides of paeony was to observe the gene and protein expression of osteogenesis indicators,autophagy indicators and ?-catenin.Then added the autophagy-promoting drug intervention,the gene and protein expression changes of osteogenesis,autophagy indicators and ?-catenin were observed after intervention.Results Part ?:The iPSCs produced on MEF were similar in shape to ESCs:the cells were round,tightly arranged,and present as colonies with clear edges around the MEF cells.RT-PCR results of normal iPSCs and AS iPSCs showed that endogenous KLF,MYC,NANOG,OCT4,and SOX2 genes were increased in both cells.The genes of the exogenous plasmids of pCXLE-hOCT3/4-shp53-F,pCXLE-hSK,pCXLE-hUL and pCXLE-EGFP were not expressed in those cells.This indicated that the transferred genes did not integrate with their own genome.The immunofluorescence of normal iPSCs and AS iPSCs cells showed that they expressed totipotent specilic proteins OCT4 and NANOG,and also expressed ESCs-specific surface antigens TRA-1-60 and SSEA4.ALP staining of normal iPSCs and AS iPSCs showed high ALP expression,which suggested that both cells were undifferentiated and had strong proliferation potential.Normal iPSCs and AS iPSCs can autonomously form embryoid bodies in vitro.Normal iPSCs and AS iPSCs in vivo teratoma pathological section showed endoderm,mesoderm and ectoderm structure.The chromosome karyotype analysis showed that the number and structure of iPSCs and AS iPSCs were normal.and no deletions occurred.Part ?:The iPSCs showed a consistent fibroblast-like morphology after induction in MSC induction medium.Alizarin red staining and ALP staining were positive after iPS-MSCs osteogenesis induction,indicating that iPS-MSCs had calcium nodule formation and alkaline phosphatase expression after induction.Oil droplets were visible in oil red O stained in cells after adipogenesis.The culture of cartilage aggregates showed the formation of cartilage aggregates.Pathological sections of Alicin blue staining showed type ? collagen formation in the aggregates.In order to identify the surface markers of iPS-MSCs,this study used flow cytometry to detect the expression of cell surface molecules.Among them,CD73,CD90,and CD105 were positively expressed on the surface of induced cells,while CD14,CD34,and CD45 were negative expression,which met the criteria of identifying MSCs surface markers Part ?:At 24h,48h and 72h,CCK8 tests of normal iPS-MSCs and AS iPS-MSCs showed no significant difference.After 14 days induced to osteogenesis,Alizarin red staining,ALP staining color,Alizarin red amount,and ALP activity level of AS iPS-MSCs were higher than normal iPS-MSCs.The expression levels of MAP1LC3B(LC3),OCN,COL1A1,CTNNB1(?-catenin)genes in AS iPS-MSCs were higher than those in normal iPS-MSCs,and SQSTM1(P62)gene expression was lower than normal iPS-MSCs.The expression of OCN,COL1A1,non-phosphorylated ?-catenin protein and LC3 ?/? protein ratio of AS iPS-MSCs were higher than normal iPS-MSCs,and the expression level of P62 protein was lower than normal iPS-MSCs.After intervent with autophagy inhibitor 3-MA,Alizarin Red staining color,Alizarin Red mass,ALP staining color,ALP activity of normal iPS-MSCs and AS iPS-MSCs weakened.MAP1LC3B(LC3),OCN,COL1A1 gene expression levels and OCN,COL protein expression,LC3 ?/? protein ratio decreased,while SQSTM1(P62)gene expression and P62 protein expression level increased,there was no significant difference between normal iPS-MSCs and AS iPS-MSCs,which indicated the increase of autophagy levels in AS iPS-MSCs promoted their osteogenesis.We also detected that the expression level of non-phosphorylated ?-catenin protein and CTNNB1 gene also decreased,suggesting that autophagy is likely to regulate the expression of CTNNB1 gene and levies of ?-catenin activation.In order to further confirm whether autophagy induced the increase in osteogenesis of AS iPS-MSCs by inducing P-catenin activation,we intervened with SKL2001,an agonist of the Wnt/?-catenin signaling pathway,and found that after 14 days of osteogenesis induction,SKL2001 promoted osteogenic differentiation of AS iPS-MSCs with autophagy inhibition.Alizarin red staining,Alizarin red mass,ALP staining and ALP activity levels were higher than AS iPS-MSCs with autophagy inhibition.The expression levels of OCN,COLIA1,and CTNNB1 genes and the expression of OCN,COL,and non-phosphorylated?-catenin proteins were also increased,while there was no difference in the MAP1LC3B(LC3),SQSTM1(P62),LC3 ?/? protein ratio,and P62 protein levels.After SKL2001 intervention,however the osteogenesis ability of AS iPS-MSCs could not be restored to the level of non-interfering SKL2001,which suggested that autophagy may promote the osteogenic differentiation of AS iPS-MSCs in part by promoting ?-catenin activation.Part ?:In order to evaluate the appropriate use concentration of total glucosides of paeony in the intervention of AS iPS-MSCs.This study used 62.5ug/mL,125ug/mL,250ug/mL,500ug/mL total glucosides of paeony to cultivate cells,and found that 250ug/mL,500ug/m L total glucosides of paeonia inhibited the proliferation of AS iPS-MSCs significantly,while 62.5 ug/mL and 125 ug/mL total glucosides of paeonia had less effect on proliferation.Therefore,62.5 ug/mL and 125 ug/mL total glucosides of paeony were selected to interfere with osteogenic differentiation of AS iPS-MSCs in this study.According to the effect of total glucosides of paeony on cell proliferation,the control group(without drug),62.5ug/mL,125ug/mL total glucosides of paeony were used to intervene the osteogenic differentiation of AS iPS-MSCs.After 14 days of intervention,125ug/mL total glucosides of paeony were significantly reduced in Alizarin red staining,Alizarin red mass,ALP staining,and ALP activity.The autophagy,osteogenesis,and ?-catenin detection revealed 125ug/mL total glucosides of-paeony in AS iPS-MSCs reduced the expression levels of MAP1 LC3B(LC3),OCN,COL1A1,CTNNB1 genes,and expression levels of LC3?/?,OCN,COL and non-phosphorylated ?-catenin protein;SQSTM1(P62)gene and P62 protein levels were higher than those of the control group.In order to observe whether the total glucosides of paeony affected osteogenesis through autophagy,we used an autophagy agnonist Rapamycin(RA)0.2 ?M intervention and divided into control group,125ug/mL total glucosides of paeony intervention group and RA+125ug/mL total glucosides of paeony intervention group.After 14d induction osteogenesis,it was observed that RA restored the osteogenesis ability of total glucosides of paeony to interfere with osteogenic differentiation of AS iPS-MSCs.The autophagy level,osteogenic genes and proteins were higher than those interfere with total glucosides of paeony,but the level of P62 gene and protein reduced.It indicated total glucosides of paeony can inhibit the osteogenic differentiation of AS iPS-MSCs by inhibiting autophagy.Conclusion In this study,urine cells derived from AS patients can be successfully induced to AS disease model iPSCs,and the iPSCs can be induced to MSCs.AS iPS-MSCs exhibit stronger osteogenesis than normal iPS-MSCs.AS iPS-MSCs have a higher autophagy level during osteogenesis,which induces an increase in activated ?-catenin.This indicates that AS iPS-MSCs have stronger osteogenic differentiation ability.The total glucosides of paeony can inhibit the autophagy level of AS iPS-MSCs during osteogenic differentiation and reduce the activation of ?-catenin,thereby reducing the osteogenic differentiation ability of AS iPS-MSCs.