| Osteoarthritis(OA)is a joint degenerative disease,the main pathological manifestations are joint strain,deformity and articular cartilage damage.Articular cartilage mainly composed of chondrocytes and extracellular matrix and does not contain blood vessels and nerves,threrfore it is difficult to repair itself after being damaged.Because of this,the treatment of OA cartilage damage is a difficult problem facing the clinic,traditional surgical cartilage repair methods include subchondral bone drilling,microfractures,and artificial joint replacement.These methods are limited in application,such as easy to cause harm to patients and the efficacy is not satisfactory.In recent years,with the development of cell transplantation treatment technology,the therapy of cartilage injury has seen new hope.Mesenchymal stem cell(MSC)is a kind of pluripotent stem cell with multidirectional differentiation and high self-renewal ability.Due to its low immunogenicity,there is no immune rejection after transplantation,and it has multidirectional differentiation and self-renewal,immune regulation and inflammation suppression capabilities.For these advantages,MSC become the seed cell for the treatment of OA cartilage damage and other related diseases.The aryl hydrocarbon receptor(AHR)is a ligand-activated nuclear transcription factor and exists in many tissues and cells of the human body.AHR participates in many important biological processes such as the differentiation,immune regulation or growth of many cells,what’s more,it also participates in the development of the body.Studies have shown that AHR exists in MSC,and after binding to ligands,it regulates the multi-differentiation potential of MSC and inhibits the adipogenic and osteogenic differentiation capabilities of MSC,but whether it affects the chondrogenic differentiation capability of MSC is still unknown.At present,a number of preclinical experiments and clinical trials have confirmed the safety and efficacy of MSC in the treatment of OA,and indicated that the role of MSC in cartilage repair is mainly through multidimensional differentiation potential and paracrine function.However,studies have shown that the therapeutic effect of MSC will be affected by the microenvironment in which it is located.The microenvironment in the joint cavity of patients with OA is complicated,which is not conducive to the therapeutic effect of MSC.According to reports,synovial fluid component of OA patients is abnormal and with the elevated levels of tryptophan and its metabolite kynurenine(KYN,a natural ligand of AHR).The abnormal synovial fluid whether could activate the expression of AHR in MSC,affect the chondrogenic differentiation potential of MSC and affect the efficacy has not been reported yet.For this purpose,we collected samples of synovial fluid from patients with OA and rheumatoid arthritis(RA).Human umbilical cord-derived mesenchymal stem cells(UC-MSC)were cultured with different concentrations of synovial fluid to study the activation of AHR and the change of chondrogenic differentiation potential in UC-MSC.KYN,the natural ligand of AHR,was used to stimulate UC-MSC in vitro to detect the effect of AHR activation on chondrogenic differentiation potential.Then,UC-MSC was transfected with lentivirus to construct a stable cell line(shAHR-UC-MSC)with low expression of AHR and then stimulated by KYN to detect the changes in the chondrogenic differentiation potential of UC-MSC after AHR knockdown.Meanwhile,OA rat model was established to study the therapeutic effect of shAHR-UC-MSC on OA rat model,so as to provide a new therapeutic method and experimental basis for the clinical treatment of OA.Objective: To study the effect of synovial fluid of OA and RA patients on the activation of AHR and on potential of chondrogenic differentiation in UC-MSC,and to explore the role of AHR in UC-MSC chondrogenic differentiation,as well as the effect of AHR on UC-MSC in the treatment of cartilage injury in OA rat model,so as to provide new therapeutic measures and experimental basis for the clinical treatment of cartilage injury diseases such as OA.Methods: UC-MSC was stimulated by synovial fluid from OA and RA patients at different concentrations for chondrogenic differentiation.AHR activation and expression of chondrogenic markers were detected by Real-time quantative PCR(qPCR),Western blot and immunofluorescence,and chondrocyte formation was detected by Alcian blue staining.UC-MSC was stimulated with different doses of KYN for chondrogenic differentiation,and the activation of AHR and the expression of chondrogenic differentiation markers were detected by qPCR,Western blot and immunofluorescence.UC-MSC was transfected with lentivirus,and the stable low expression of AHR cell line shAHR-UC-MSC and negative control shNC-UC-MSC were obtained for chondrogenic differentiation.The changes of chondrogenic differentiation ability were detected by qPCR,Western blot,immunofluorescence and Alecine blue staining.In vivo experiment,the OA model of rats was established according to Hulth method,and the rats were injected with shNC-UC-MSC and shAHR-UC-MSC in the articular cavity.After four weeks of administration,and the rat’s knee joints were examined by X-rays and Kellgren-Lawrence Scoring;operating microscope to observe the femoral condyle and tibial plateau of the rat knee joint,and perform ICRS score;Safranin O fast green staining method to observe the pathological changes of the rat knee joint,and perform the modified Mankin score;immunohistochemical detection of the expression of Aggrecan and Collagen Ⅱ in the cartilage tissue of the knee joint of rats in the group.Results: 1.Identification of UC-MSC The cells grew well after resuscitation,with large and clear cell nuclei.The cells grew adherently after 6 hours of culture,and the concentration of cells reached more than 80% after 48 hours of culture.The results of flow cytometric determination of cell surface antigens showed that the expression rates of CD11 b,CD34 and CD45 were all lower than 5% and were negative,and the expression rates of CD73,CD90 and CD105 were all higher than 95% and were positive.The identification results of the differentiation potential of the three lines show that the cells can differentiate into adipocytes,bone cells and chondrocytes.2.The effect of synovial fluid in patients with OA on AHR activation and chondrogenic differentiation of UC-MSC The results of qPCR and Western blot showed that compared with the control group,the expression levels of AHR gene and protein of UC-MSC were significantly increased after OA and RA synovial fluid culture at different concentrations,and the expression levels of AHR downstream target genes CYP1A1 and CYP1B1 were also significantly higher.Increased,while the gene and protein expression of chondrogenic differentiation markers SOX-9,COL2A1 and Aggrecan decreased significantly.Immunofluorescence results showed that after UC-MSC cultured in OA synovial fluid,AHR was activated into the nucleus,the nuclear expression increased significantly,and the expression of SOX-9 and COL2A1 decreased.The results of CCK-8 showed that the cell viability of UC-MSC began to decrease after the third day of synovial fluid culture,and the decrease was more obvious on the fifth and seventh days.The results of alcian blue staining showed that compared with the control group,the number of chondrocytes differentiated from UC-MSCs cultured in OA synovial fluid was significantly reduced,and the blue staining effect was poor.3.The effect of KYN stimulation on the AHR activation and chondrogenic differentiation ability of UC-MSC The results of qPCR and Western blot showed that KYN stimulation can promote the m RNA expression of AHR and its downstream specific target genes CYP1A1 and CYP1B1,increase the protein expression of AHR,and inhibit the m RNA and protein expression of chondrogenic differentiation markers SOX-9,COL2A1 and Aggrecan.Immunofluorescence results showed that after KYN stimulation,AHR was activated into the nucleus,the nuclear expression was significantly up-regulated,and the expression of SOX-9 and COL2A1 decreased.The results of alcian blue staining showed that the number of UC-MSCs differentiated into chondrocytes after KYN stimulation was significantly reduced,and the content of proteoglycans was reduced.4.Lentivirus transfects UC-MSC to obtain shAHR-UC-MSC with stable and low expression of AHR The shNC and shAHR lentiviral vectors were purchased from Shanghai Jikai Gene Biology Co.,Ltd.,and UC-MSCs were transfected with shNC and shAHR lentiviral vectors respectively.Fluorescence microscope was used to observe the expression of GFP in UC-MSCs under different transfection conditions.The results showed that when the MOI of P group was 60,the GFP expression was over 80%,and the transfection effect was the best.The optimal transfection conditions for the P group when the MOI was 60 were used to transfect UC-MSCs,and the shAHR-UC-MSCs obtained were identified.Fluorescence microscope observation of GFP expression showed that the control group did not express GFP and shNC-UC.-MSC and shAHR-UC-MSC groups express GFP strongly.The results of qPCR and Western blot showed that the m RNA level of AHR in the shAHR-UC-MSC group was significantly reduced,and the protein expression was significantly reduced.Flow cytometry results of shAHR-UC-MSC surface antigen showed that the expression of CD11 b,CD34 and CD45 was negative,and the expression of CD73,CD90 and CD105 was positive.5.The effect of KYN stimulation on the AHR activation and chondrogenic differentiation ability of shAHR-UC-MSC The qPCR results showed that compared with shNC-UC-MSC,shAHR-UC-MSC after KYN stimulation,the m RNA levels of AHR,CYP1A1 and CYP1B1 decreased significantly,and the m RNA expression of SOX-9,COL2A1 and Aggrecan increased.Western blot results are similar to qPCR results.Immunofluorescence staining showed that the AHR of shNC-UC-MSC was significantly activated into the nucleus after KYN stimulation,and the AHR of shAHR-UC-MSC was weakly activated and expressed less in the nucleus.Compared with shUC-MSC,the expression of SOX-9 and COL2A1 of shAHR-UC-MSC was significantly increased.The results of Alcian Blue staining showed that after KYN stimulation,compared with shNC-UC-MSC,shAHR-UC-MSC differentiated into more chondrocytes and the degree of blue staining was deeper.6.Therapeutic effect of shAHR-UC-MSC on OA rat model Both shNC-UC-MSC and shAHR-UC-MSC articular cavity injection can improve the condition of OA rats,but shAHR-UC-MSC has a better therapeutic effect,and there are statistical differences between the groups.The X-ray results of the rat knee joint showed that the knee joint of the normal group was intact and smooth,the joint space was clear,and the X-ray score was the lowest.The articular surface of the rats in the model group was severely damaged,the joint space was blurred and the osteophytes formed obvious,and the X-ray score was the highest.Compared with the model group,after shNC-UC-MSC and shAHR-UC-MSC treatment,the X-ray score of the knee joint of rats was reduced,and the X-ray score of the shAHR-UC-MSC treatment group was significantly lower than that of the shNC-UC-MSC treatment group.A general observation of the knee joints of the rats in each group showed that the surface of the knee joint of the normal group was intact and smooth,and the surface of the knee joint of the model group was severely damaged with thick synovial fluid attached,and the ICRS score was significantly reduced.There are still small defects on the knee joint surface of rats in the shNC-UC-MSC treatment group.The knee joint surface of the rats in the shAHR-UC-MSC treatment group is almost intact without osteophyte formation.The ICRS score is also significantly higher than that in the shNC-UC-MSC group,And there are statistical differences.The results of pathological Safranine O fast green staining of the rat knee joint showed that the normal group Safranine O staining became a strong positive staining.The cartilage surface of the knee joint in the OA group was damaged and the cartilage was eroded and thinned.Safranine O stained a lot.Missing.The shNC-UC-MSC treatment group showed less joint damage than the OA group,and the cartilage tissue thickness increased.In the shAHR-UC-MSC treatment group,the articular cartilage surface was intact,and the cartilage tissue thickness was similar to that of the normal group.Safranin O staining was strongly positive.The immunohistochemical results of rat knee joint tissues showed that compared with the model group,after shNC-UC-MSC and shAHR-UC-MSC treatment,the expressions of Aggrecan and Collagen Ⅱ increased,and the levels of both in the shAHR-UC-MSC group Both are higher than the shNC-UC-MSC group.Conclusions: 1.After culturing the synovial fluid of OA patients,the AHR in UC-MSC is activated,and the chondrogenic differentiation ability of UC-MSC is inhibited,suggesting that the synovial fluid of OA patients can activate AHR in UC-MSC and affect the chondrogenic differentiation of UC-MSC ability.2.KYN is the natural ligand of AHR.After stimulating UC-MSC,AHR is significantly activated,and the chondrogenic differentiation ability of UC-MSC is inhibited,suggesting that KYN-mediated AHR activation participates in the chondrogenic differentiation of UC-MSC.3.The lentivirus shNC and shAHR were transfected into UC-MSC,and the stable shNC-UC-MSC and shAHR-UC-MSC cell lines were successfully constructed.After KYN stimulated shNC-UC-MSC and shAHR-UC-MSC,AHR in shNC-UC-MSC was obviously activated and the chondrogenic differentiation ability was reduced.The degree of AHR activation in shAHR-UC-MSC is weak,and the chondrogenic differentiation ability is significantly stronger than shNC-UC-MSC.It is suggested that AHR regulates the chondrogenic differentiation of UC-MSC.AHR is activated after ligand stimulation and inhibits the chondrogenic differentiation potential of UC-MSC.Knockdown of AHR expression can inhibit AHR activation and weaken its inhibitory effect on UC-MSC chondrogenic differentiation.4.Injecting shNC-UC-MSC and shAHR-UC-MSC into the joint cavity can alleviate the condition of OA rats and repair damaged articular cartilage.shAHR-UC-MSC has a better therapeutic effect.It is suggested that the therapeutic effect of UC-MSC is related to the environment.The articular cavity microenvironment of OA rats may activate AHR in UC-MSC,inhibit its chondrogenic differentiation ability,and affect the therapeutic effect of UC-MSC;knock down AHR expression and inhibit Its activation eliminates the inhibitory effect of AHR activation on UC-MSC chondrogenic differentiation and can produce better therapeutic effects. |
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