Immune Regulatory Effect And Therapeutic Mechanism Of DPSCs In Intervertebral Disc Degeneration And Rheumatoid Arthritis

Intervertebral disc degeneration(IVDD)and rheumatoid arthritis(RA)are widespread chronic bone and joint diseases.Both IVDD and RA have high morbidity and recurrence rates as well as low healing rates.These diseases have posed enormous threats to patients' life qualities,and added burden to the country and society.Autoimmune reaction plays a certain role in their pathogenesis.When immune tolerance is jeopardized,self-antigen released from tissues could be recognized by the immune system which activates immune cascade,causing immunopathological damage subsequently.Mesenchymal stem cells,which have great therapeutic potential and wide-range applications,are the most commonly used types of cells in stem cell therapy.As an important component of mesenchymal stem cells,dental pulp stem cells(DPSCs)have strong self-renewal ability,outstanding immune regulation capacity,and excellent chondrogenesis and osteogenesis properties.Our previous studies have proved the inflammation-regulatory effects and tissue regeneration function of DPSCs in treating chronic periodontal disease.Because of the fact that autoimmune response is an indispensable factor in the periodontitis pathogenesis,therefore,DPSCs may possess the ability to treat diseases caused by autoimmune reactions.Therefore,we evaluate the therapeutic effect of DPSCs on IVDD and RA animal models.Besides,by isolating patient primary nucleus pulposus(NP)cells and synovial cells,we elucidate the underlying mechanism of DPSCs to treat these diseases.This research is mainly divided into the following four parts.Part I:Objective To evaluate the changes in the intervertebral disc structure and the production of extracellular matrix of IVDD rat model after DPSCs treatment.Methods NP piercing rat model was treated with DPSCs administrated in situ.Micro-CT was used to evaluate the pathological changes of IVDD.H&E,Masson,Safranin Fast Green,and immunohistochemical staining were used to evaluate morphological and structural changes.The Masuda scoring system was used to provide quantitative analysis.Results Micro-CT proved that the height of the intervertebral disc was restored after DPSCs treatment with recovered imaging performance.Histopathological analysis confirmed that the tissue structure of intervertebral disc recovered,as the border of fibrous annulus(AF)and NP was clearer,the number of NP cells was increased,hypertrophy of cartilage endplate(EP)and atrophy of NP was relieved,and the extracellular matrix was increased.Conclusion DPSCs promote the restoration of intervertebral disc structure and the production of extracellular matrix in IVDD animal models.Part II:Objective To analyze the differential expressed genes(DEGs)and altered signal pathways of primary NP cells after DPSCs supernatant treatment.Methods Transcriptome sequencing was carried out after primary isolated NP cells were treated with DPSCs supernatant treatment.Bio-information analysis of the sequencing results was carried out to identify DEGs and enriched signal pathways.Western blotting and immunofluorescence staining were used to verify the sequencing results and analyze the phenotypic changes of NP cells.Results Transcriptome sequencing results proved that after the DPSCs supernatant treatment,inflammation-related genes(such as IL6,CCL7,IL1RL1 and TNFAIP6)were significantly down-regulated,and NP cell function-related genes(such as PIK3R1,ITGA10,IGFBP2 and LEP)were significantly up-regulated.GO analysis indicated that the secretion and binding process of inflammatory factors were weakened,and extracellular matrix secretion was enhanced.Pathway enrichment analysis suggested that up-regulated genes were mainly enriched in pathways related to extracellular matrix secretion(such as elastic fiber,collagen production-related signal pathways,etc.),and down-regulated genes were mainly enriched in inflammation-related signaling pathways(such as Jak-STAT signaling pathway,TNF signaling pathway,NF-?B signaling pathway,etc.).Western blotting results confirmed that after DPSCs supernatant treatment,NP cells produced less IL-6,more Col II,and less apoptosis-related protein Cleaved-Caspase-3.Akt signaling pathway was activated while NF-?B and STAT3 signaling pathways were deactivated.Immunofluorescence staining proved that after DPSCs supernatant treatment,the filamentous cytoskeleton structure(formed of F-actin)was protected.Less degradation,less apoptosis and more intact cell function were seen in these cells.Conclusion DPSCs supernatant treatment inhibits the activation of inflammation-related signal pathways in NP cells,reduces the release of inflammatory factors,and protects the survival and function of NP cells.Part III:Objective To evaluate the clinical manifestations,pathological and radiological changes of DPSCs-HGF or DPSCs-Null treated collagen-induced arthritis(CIA)mouse model.Methods Adenovirus was used to overexpress HGF molecule,which has been reported to have immune regulation and immune tolerance reconstruction ability,in DPSCs to construct DPSCs-HGF cells.DPSCs-HGF and DPSCs-Null were intravenously injected into CIA mice.The clinical manifestations scoring was carried out.To evaluate histopathological changes of synovitis,joints were stained with H&E and scored according to Kreen criteria.Micro-CT was carried out to evaluate bone destruction.Results The clinical scores of each group showed that joint swelling and skin rashes in DPSCs-Null treated mice were relatively mild,the clinical scores of DPSCs-HGF treated mice were low in the early period,yet rose rapidly in the late period.Histopathological results showed that DPSCs-Null treatment could effectively reduce synovitis.The DPSCs-HGF treatment group had more infiltrated inflammatory cells in joint cavity,thicker joint synovial lining layers,and more intense synovial cell density at end point.Micro-CT proved that after DPSCs-Null or DPSCs-HGF treatment,the bone destruction alleviated with no statistical differences between the two treatments.Conclusion DPSCs-Null treatment can recede clinical manifestations of CIA mice and can protect joint synovitis;DPSCs-HGF treatment is beneficial to CIA mice in the early period but disease relapses in the late period.Part IV:Objective To clarify the mechanism for disease relapse in the late period of DPSCs-HGF treatment.Methods Flow cytometry was carried out to analyze the subtypes of peripheral blood immune cells of CIA mice at different time points after treatment.The levels of inflammatory factors in the serum of CIA mice at different time points after treatment were detected by using Multi-factor detection system.Immunohistochemical staining was used to analyze the expression of IL-6 molecules in joints after treatment.Patients' primary synovial cells were separated and treated with HGF and/or pathway inhibitors.Clone formation assays,Western blotting and cell cycle staining experiments were carried out to analyze NP cell proliferation,apoptosis,cell cycle and other biological phenotypes.Results The immunological analysis of CIA mice proved that in DPSCs-HGF treatment group,elevated levels of Treg cells appeared relatively earlier,but maintained for a shorter time;peripheral blood Th1/Th2 ratio increased;serum and joints IL-6molecules increased rapidly in the late period of treatment.While in DPSCs-Null treatment group,the elevated levels of Treg cells appeared later but more intense,and maintained for a longer time;peripheral blood Th1/Th2 ratio decreased;serum and joint IL-6 remained low.In vitro experiments of synovial cells proved that HGF promoted synovial cells to secret IL-6 in a time and dose-dependent manner;DPSCs-HGF culture supernatant or exogenous supplementation of HGF molecules activated c-Met receptor and downstream Akt signal pathway,induced the expression of Survivin molecules,and enhanced the clonogenic ability and apoptosis resistance of synovial cells.Inhibitors of c-Met and Akt activation or Survivin molecules caused G2/M phase arrest in synovial cells,CDK1 and Cyclin B1 molecules expression were reduced,and HGF induced enhancement of clonogenic ability and apoptosis resistance were effectively reversed.Conclusion DPSCs-HGF treatment can induce Treg cells,reduce IL-6 cytokine secretion,and control arthritis clinical manifestations in the early period;however,in the late period,HGF activates downstream Akt signaling pathway by binding to c-Met receptor,induces the expression of Survivin protein,promotes synovial proliferation and apoptosis resistance,and accelerates the deterioration of arthritis.Based on the above-mentioned results,this research clarifies the following aspects.(1)DPSCs inhibit the activation of NP cell's inflammation-related signal pathways,reduce the release of inflammatory factors,protect the survival and function of NP cells,and promote the recovery of intervertebral disc structure,all of which empower potential therapeutic effects of DPSCs on the IVDD.(2)DPSCs have therapeutic effect on RA by increasing the immunosuppressive Treg cells,reducing pro-inflammatory Th1 cells and IL-6 molecules,and controlling synovial inflammation.(3)In the early period,HGF controls arthritis by suppressing the autoimmune system;while in the late period,HGF promotes arthritis by binding to c-Met receptors on synovial cell membrane and activating downstream Akt signaling pathway to enhance cell proliferation and apoptosis resistance.