A Study On The Inhibitory Effect Of Xenobiotics On The Chondrogenic Differentiation Of Human WJ-MSCs And Its Related Mechanisms

Osteoarthritis(OA)is a common disease in the elder people,which is a chronic joint disease with articular cartilage degeneration as the main pathological features,and OA is one of the main causes of pain and disability in the elderly population.The main component of articular cartilage matrix is proteoglycan(PG),which is the material basis of cartilage strength,tissue elasticity,and joint lubrication,and could maintain the homeostasis of cartilage.PG consists of a few glycosaminoglycan(GAG)chains attached to a core protein,and then,these components form a spatial network structure by cross-linking collagen fibers.Matrix degeneration or reduction is the main index to evaluate the quality of cartilage.Traditionally,OA is considered as the result of mechanical factors and age-related degeneration,however,large sample epidemiological studies found that the susceptibility to hand and hip joint OA after adultly is related to birth weight and the susceptibility is higher in the population with intrauterine growth retardation(IUGR)than normal.It indicates that OA might have a developmental origin.However,the etiology of developmental origin OA is complicated,and the related mechanism of pathophysiology is unknown,which makes the early diagnosis and prevention difficult in the clinic.Wharton's jelly-derived mesenchymal stem cells(WJ-MSCs)is a kind of relatively primitive stem cells,with a wide range of sources,and kept the mark of adverse effects on fetal development caused by adverse intrauterine environment,and it could be used for early warning of fetal diseases.WJ-MSCs could be induced to differentiate into chondrocytes,and in the process of differentiation,insulin-like growth factor 1(IGF1)pathway and transforming growth factor beta(TGF?)signaling pathway play a key role,the activation of the pathways could promote the expression of the target genes like the key genes of cartilage alpha 1 gene of type ? collagen(Col2?1)and aggrecan.Our previous study found that prenatal xenobiotic exposure(PXE)could lead to IUGR and fetal exposure to high concentrations of maternal glucocorticoid(GC),and furthermore,inhibit the development of cartilage which could lead to the increase of susceptibility to OA in the adult.However,do the WJ-MSCs from the IUGR newborn poorly differentiate to chondrocytes?After differentiation,are the cells susceptible to OA?How does xenobiotic affect chondrogenic differentiation?How is the role of the TGF-beta signaling pathway in the process of chondrogenic differentiation?All these questions are still unclear.Based on these questions,in this study,we firstly isolated WJ-MSCs from normal neonates,and established mature technology of isolating and culturing WJ-MSCs,and then different xenobiotic of different concentration was employed to treat WJ-MSCs to explore the effect of xenobiotic on the expression of cartilage phenotype affecting gene and its upstream pathways in WJ-MSCs;after that,normal and IUGR original WJ-MSCs were isolated,and we detected the expression of cartilage phenotype-related genes and the upstream pathway in the proliferation and chondrogenic differentiation model of WJ-MSCs,and furtherly,interleukin 1 beta(IL-1?)was employed to make OA model,and we tried to study the mark of effect on WJ-MSCs caused by adverse intrauterine environment and its effect on chondrogenic differentiation;on this basis,different concentrations of cortisol was employed to chondrogenic differentiation of normal newborn original WJ-MSCs,and we detected the expression of cartilage phenotype-related genes and the upstream pathway in the proliferation and chondrogenic differentiation model of WJ-MSCs,and furtherly,IL-1?was employed to make OA model,and we tried to study the effect of cortisol overexposure on chondrogenic differentiation of WJ-MSCs.On this basis,we explored the possibility of TGF-beta receptor(TGF?Rs)as a marker for early warning of developmental OA.The clarification of the scientific questions,could help deepen our understanding of the pathogenesis of fetal originated OA,and clear the role of TGF?Rs in cartilage matrix synthesis,and furtherly this study tried to clarify its possibility to be considered as fetal originated OA markers for early warning,and lay a theoretical and experimental basis for early warning of fetus originated OA.Part oneThe inhibition effect and mechanism of exogenous substances on chondrogenic related genes expression in human WJ-MSCs.Objective Our previous study found hypochondroplasia and adult osteoarthritis(OA)susceptibility in intrauterine growth retardation(IUGR)model caused by prenatal xenobiotic exposure in rats,and we also found that overexposure of maternal glucocorticoid(GC)is the common phenomenon.Human Wharton's jelly-derived mesenchymal stem cells(WJ-MSCs)are relatively primitive stem cells that could provide information about the relationship between birth weight and the long-term effects of adult diseases.Therefore,in this study,we established the proliferation model of WJ-MSCs,and explored the effect of a variety of xenobiotic(including caffeine,nicotine,and alcohol)on cartilage differentiation marker gene(Col2?1 and aggrecan)and tried to explore the related mechanism by studying GC barrier and TGF-beta signaling pathway related key genes.Methods Isolation and identification of the primary WJ-MSCs.Take the fourth-generation cells as our object of study,adding commonly used culture medium prepared in the laboratory for IUGR models with caffeine(final concentrations of 0,0.1,1,10,100?M),nicotine(final concentrations of 0,0.1,1,10,100?M)and ethanol(final concentration respectively.0,15,30,60mM).Continuous treatment for 5 days,and replace fresh culture medium every other day.The control group was given the same volume routine culture medium.MTS was employed to detect the cell vitality,real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of key genes in GC activation system,TGF-beta signal pathway and expression of cartilage phenotype gene.Results WJ-MSCs in the 4th generation was confirmed to be purified by flow cytometry.Various xenobiotics of different concentrations treated WJ-MSCs for 5 days,MTS showed that the various xenobiotics used in this experiment doesn't change significantly the vitality of WJ-MSCs compared with the control group.RT-qPCR results suggest that various xenobiotics can significantly reduce the cartilage phenotype of gene alpha 1 chain of collagen type ?(Col2?1)and aggrecan expression(P<0.01);after treated by the highest concentration of each xenobiotics,the key genes expression of TGF-beta signaling pathway TGF?R?/?were lower(P<0.01);the expression of early growth response protein 1(Egr-1)was increased(P<0.01),while the expression of 11 beta-hydroxysteroid dehydrogenase types 2(11 beta-HSD-2)was decreased(P<0.01).Conclusion In this study,we established a stable isolation and in vitro culture system of human WJ-MSCs.For the first time,we confirmed that xenobiotics(including caffeine,nicotine,and alcohol)inhibit the expression of chondrogenic marker genes(Col2al and aggrecan)on the proliferation model of WJ-MSCs,and the related mechanism might be linked to TGF-beta signaling pathway inhibition mediated by GC barrier.Part twoThe chondrogenic differentiation potential of IUGR original human WJ-MSCs is lower and the related mechanism.Objective Epidemiological studies have shown that birth weight is associated with osteoarthritis in adults.The first part of the study found that a variety of xenobiotics(including caffeine,nicotine,ethanol)could inhibit the chondrogenic differentiation marker gene(Col2?1 and aggrecan),injure the glucocorticoid(GC)barrier and inhibit the expression of the key gene of TGF-beta signaling pathways on WJ-MSCs proliferation model.All these indicate that the chondrogenic differentiation potential of WJ-MSCs from IUGR newborn that suffered bad intrauterine environment might be lower.In this study,we established the WJ-MSCs proliferation model and chondrogenic 3D differentiation model,and we detect the expression of key genes of GC barrier and chondrogenic genes,to identify the chondrogenic differentiation potential of WJ-MSCs from IUGR newborns.Methods Using the established method in the first part,we isolated primary WJ-MSCs from normal neonates and IUGR neonates,and proliferation and 3D chondrogenesis model established at the fourth generation of WJ-MSCs.On the proliferation model,after culturing with a routine medium for 5 days,we detected the expression of cartilage phenotype genes,key genes of TGF-beta signaling pathway and GC barrier related genes in normal and IUGR WJ-MSCs.Sodium alginate was employed as a scaffold to establish a 3D chondrogenic model,and the model was treated with the same differentiation medium for 21 days.Sodium alginate microspheres were taken to fix and make safranin O and alcian blue special staining analysis;part microspheres were dissolved to extract RNA,and we detected the expression of and the chondrocytes phenotype genes and key genes of TGF-beta the signal pathway with RT-qPCR;the rest were given IL-1? for 24h to make OA model,part of these microspheres after OA modeling were carried out make safranin O and alcian blue special staining analysis,residual microspheres were dissolved to extract RNA,and we detected the expression of and the chondrocytes phenotype genes and matrix degradation related genes with RT-qPCR.Results On the proliferation model,compared with the normal group,cartilage phenotype gene Col2?1 and aggrecan in IUGR group is lower(P<0.01);the expression of key genes of TGF-beta signaling pathway were lower(P<0.05,P<0.01);the expression of Egr-1 higher(P<0.01),while the expression of 11?-HSD2 is lower(P<0.01),which suggests that WJ-MSCs from IUGR newborns caused by intrauterine exposure factors has produced GC barrier damage mark,and TGF-beta signaling pathway dysfunction.On the differentiation model,matrix synthesis in IUGR group is less than normal group,as safranin O and alcian blue special staining analysis showed cell number is less,hypochromasia and the expression of aggrecan,and Col2?1 is lower,also in the upstream TGF-beta signaling pathway,the mRNA expression of TGF?Rs is lower than normal group;after OA modeling by IL-1?,chondrocytes in IUGR group are more susceptible to OA,compared with the normal group,as safranin O and alcian blue special staining analysis showed hypochromasia and the expression of aggrecan,and Col2al is further reduced lower,while MMP3,13 and ADAMTS5 expression were increased,suggesting that matrix degradation was enhanced.Conclusion This is the first study to confirm that the chondrogenic differentiation of WJ-MSC from IUGR newborns is poor on proliferation and 3D chondrogenesis model,and after OA modeling by IL-1?,chondrocytes in IUGR group are more susceptible to OA.The mechanism might be related to intrauterine GC barrier injury and TGF? pathway inhibition during the fetal period,and the reduction of TGF?Rs positive cells might be also an important mechanism.Part threeThe epigenetic regulation mechanism of cortisol on the chondrogenesis inhibition of human WJ-MSCs and susceptibility to osteoarthritis.Objective Our previous study found that overexposure to maternal GC is a common phenomenon in the rat model of IUGR caused by prenatal xenobiotics exposure(PXE).TGF-beta signal pathway is one of the key pathways in chondrogenic differentiation and cartilage development.In the first part of this study,we have confirmed that TGF-beta signaling mediates cartilage dysplasia caused by xenobiotics exposure(including caffeine,nicotine,and alcohol),and in the second part,we confirmed poorly chondrogenic differentiation of WJ-MSCs from IUGR newborns,the differentiated cells are susceptible to OA and TGF-beta signaling pathway mediated the related mechanism.In this study,we aimed to confirm the effects of different concentrations of cortisol on the differentiation of WJ-MSCs and the susceptibility to osteoarthritis in WJ-MSCs from normal newborns;and also,we will try to explore the possibility of TGF?Rs as a target for early prediction of fetal OA.Methods The fourth generation of WJ-MSCs with good growth condition was chosen to establish chondrogenic three-dimensional differentiation model of alginate microspheres,besides routine differentiative culture medium,the cells were treated with 150,300,600 and 1200nM cortisol.In the proliferation model,MTS was carried out to explore cortisol effect on cell proliferation;in the differentiation model,WJ-MSCs were induced chondrogenic differentiation for 21 days with treatment of different concentrations of cortisol,and after differentiation,microspheres morphology changes were observed,and the expression of chondrocyte phenotype genes and key genes of TGF-beta signaling pathway were detected.Then the differentiated cells in all the groups were given IL-1? for 24h for OA modeling after that microsphere morphological changes were observed,and the expression of chondrocyte phenotype genes and genes related to matrix degradation.At the same time,we established of three-dimensional model of chondrogenic differentiation model for 7 days,and the model was treated with a final concentration of 0,300 and 1200 nM cortisol,adding GR antagonist,after 7-day differentiation,mRNA expression and protein of chondrocyte phenotypic genes and key genes of TGF-beta signaling pathway key genes were detected.At last,the final concentration of 0,300 and 1200 nM cortisol were given to WJ-MSCs for 48h,and induce WJ-MSCs chondrogenic differentiation,after that,cells were collected,and the acetylation of histone H3K9,H3K14,and H3K27 change of TGF?Rs of and chondrogenic gene Col2?1and aggrecan were detected by CHIP-PCR.Results After treatment of different concentrations of cortisol on WJ-MSCs for 5 days,the results of MTS showed that cell metabolism activity in each concentration of cortisol group was not significantly changed compared with the control group.After 21-day chondrogenic differentiation of WJ-MSCs treated with different concentrations of cortisol,safranin O staining and alcian blue special staining analysis showed:compared to physiological cortisol concentration group(150 and 300 nM),the matrix synthesis in pathological concentration(600 and 1200 nM)treatment group was inhibited,and after OA modeling induced by IL-1?,safranin O and alcian blue special staining analysis showed,cell number reduced to shallow,the expression of aggrecan and Col2al was decreased more significantly.After 21-day chondrogenic differentiation of WJ-MSCs treated with different concentrations of cortisol,the mRNA expression of Col2?1 and aggrecan in physiological concentrations group was increased while that in pathological concentration was decreased;at the same time,mRNA expression of the key genes of the upstream TGF-beta signal pathway,TGF?R? and TGF?R?was increased in physiological concentrations group,and decreased in pathological concentration group.After OA modeling,the expression of MMP3,13 and ADAMTS5 was increased in a concentration-dependent manner,suggesting that the matrix degradation was increased.After GR antagonist mifepristone 10nM alone or together with 0,3 00,600nM cortisol were given to the chondrogenic differentiation of human primary WJ-MSCs for 7 days,the result showed that mifepristone could partially reverse the effect of physiological and pathological concentrations of cortisol on the expression of TGF?R?/?,Col2?1 and aggrecan(P<0.05,P<0.01).after 48-hours chondrogenic differentiation of human primary WJ-MSCs treated with 0,300,600nM cortisol,CHIP-PCR found that H3K9 histone of TGF?R? in physiological concentration group was increased significantly while that in pathological concentrations was significantly decreased(P<0.05,P<0.01),and H3K9 histone of TGF?R? in pathological concentration group was significantly decreased(P<0.01).Conclusion This study demonstrated for the first time high concentrations of cortisol could directly cause poor chondrogenic differentiation of human primary WJ-MSCs and the susceptibility of the differentiated cells to osteoarthritis,and the mechanism might be related to the activation of glucocorticoid receptor(GR),that could induce TGF?Rs histone acetylation,thereby inhibiting TGF-beta signaling and cartilage the marker genes(Col2?1 and aggrecan)expression,causing poor chondrogenic differentiation.TGF?Rs could be used as a molecular target for early warning of OA susceptibility in neonates with IUGR.