Mechanism And Gene Therapy Of Nrf2-CHI3L1 On Traumatic Osteoarthritis

OBJECTIVE:The aim of this study is to investigate the mechanism of the nuclear factor erythroid 2-related factor 2(Nrf2)-chondroitin gibberellin 3-like-1(CHI3L1)axis in regulating traumatic osteoarthritis(PTOA)and to develop an inflammation-responsive hydrogel system for achieving long-lasting and controlled Nrf2 gene therapy and to validate its efficacy in vivo and in vitro.METHODS:A mouse knee PTOA model was constructed and Nrf2 overexpression lentiviral vector was injected intra-articularly,and the cartilage condition was assessed by HE,Masson,and red solid green staining and OARSI score.Protein expression in synovial tissue was assessed by western blotting,immunohistochemical staining and ELISA.An overexpression Nrf2 plasmid was constructed to transfect mouse fibroblast-like synovial cells,which were treated with LPS to induce inflammation.Nrf2 and CHI3L1 expression,reactive oxygen species levels,and inflammatory factor levels(TNF-α,IL-1β,IL-6)were measured 24 hours later.The vector polymers were synthesized,and their structures were verified by nuclear magnetic resonance(NMR).After evaluating plasmid compounding ability of polymers,nanocarriers were prepared by compounding carrier polymers with plasmids,and the morphology,particle size and potential of nanocarriers were characterized.The gene delivery function of nanocarriers were evaluated by flow cytometry,fluorescence microscopy,and real-time fluorescence quantitative PCR(qPCR).The effects of Nrf2 overexpression on reactive oxygen levels,inflammatory factor levels and catabolic enzymes in synovial cells were assessed by qPCR,immunofluorescence staining,and Elisa.Changes in the expression levels of Nrf2 and CHI3L1 were detected by western blotting.Inflammation-responsive hydrogels were synthesized,and their morphology,rheological properties and inflammatory response functions were characterized.The hydrogels were injected intra-articularly into the joint cavity and their distribution and degradation were assessed by in vivo imaging.A mouse PTOA model was constructed and treated with inflammation-responsive hydrogel.Synovial inflammation and biosafety were assessed by HE staining,cartilage damage was assessed by toluidine blue staining and safranine-green staining,bone destruction was assessed by Micro-CT,Nrf2 and catabolic enzyme expression were assessed by immunohistochemical staining.RESULTS:In vivo,overexpression of Nrf2 reduced persistent cartilage destruction and significantly decreased expression of matrix metalloproteinase 13(MMP13)in mice with PTOA.CHI3L1 expression and TNF-α,IL-1β and IL-6 levels were reduced in Nrf2 overexpressing mice compared to controls.Findings in vitro were consistent with in vivo,where synovial cells treated with LPS exhibited dose-dependent increases in ROS,TNF-α,IL-1β,IL-6,Nrf2 and CHI3L1 levels,and their increases were inhibited by Nrf2 overexpression.The NMR results indicated that the vector polymers were successfully synthesized and could be efficiently loaded with plasmids to synthesize nano vectors.Nano vectors enabled gene delivery and efficient overexpression of Nrf2.Nano vector treatment inhibited LPS-induced expression of reactive oxygen species,inflammatory factors and catabolic enzymes in synovial cells.Mechanistic studies showed that Nrf2 overexpression inhibited CHI3L1 production.The inflammation-responsive hydrogel was formed by cross-linking nanocarriers with polyvinyl alcohol.It can achieve inflammation-responsive nanocarrier release and long-lasting Nrf2 overexpression.The inflammation-responsive hydrogel treatment effectively inhibited synovial inflammation,cartilage damage and bone destruction in a PTOA model.CONCLUSION:Our data suggest that Nrf2 negatively regulates CHI3L1.Nrf2-CHI3L1 axis can modulates the progression of PTOA.The inflammation-responsive hydrogel constructed in this study can achieve long-lasting controlled overexpression of Nrf2 and showed good efficacy in vivo and in vitro.