Multi-omics Screening Of Biomarkers Of Knee Osteoarthritis And Exploration Of ITIH1 And NQO2 As Potential Biological Targets For Early Diagnosis And Treatment

Objective: Through proteomics,modified proteomics and metabolomics,the pathogenesis of osteoarthritis(OA)was deeply explored,potential biomarkers were screened,potential early diagnosis targets ITIH1 and therapeutic targets NQO2 were discovered,and the feasibility of ITIH1 and NQO2 as biological targets of OA and their roles and influencing factors in pathogenesis were explored.Methods:1.Joint tissue samples from 54 female patients with primary OA who underwent total knee replacement in the Department of Orthopedics,General Hospital of Ningxia Medical University were collected.Multi-omics joint analysis of collected clinical specimens to screen biological targets and validation: some clinical specimens undergo proteomics,modified proteomics,metabolomics experiments,and some specimens undergo Parallel Reaction Monitoring(PRM)and protein immunoblotting(Wester).blotting,WB),immunohistochenistry(IHC)and other experiments to biologically verify the biological targets screened by multi-omics.2.Eighty female 8-week-old C57BL/6J mice were selected to construct a model of Destabilization of the medial meniscaliscus(DMM)for knee osteoarthritis,which were taken from the 2 weeks(2W)group and 8 weeks(8W)group after molding.Finger O-solid green staining and hematoxylin-eosin staining were used for OARSI grading to evaluate the knee joint lesions of mice,immunohistochemistry and immunofluorescence(IF)were used to detect relevant factors,and transmission electron microscope(TEM)was used to observe the fine structure changes of mouse chondrocytes.2.Results:1.Through bioinformatics analysis of clinical cartilage tissue samples,we found that 52 proteins were expressed differently in Medial cartilage tissue(MCT)and lateral cartilage tissue(LCT)of OA,and PRM experiments confirmed the inclusion of the protein quinone reductase 2(NQO2)The 29 peptides,including peptides,are consistent with the trend of mass spectrometry results.The low expression of NQO2 in the medial cartilage tissue of the OA tibial platform was confirmed by WB experiments.Animal experiments found that NQO2 was poorly expressed in the mouse DMM model group,and a large number of chondrocytes mitochondrial membrane crest were broken and disappeared in the mouse DMM model group,and OA damage was aggravated.2.Proteomic studies of clinical subchondral bone tissue samples found that 46 proteins were expressed differently in Medial subchondral bone(MSB)and lateral subchondral bone(LSB)tissues of OA.PRM experiments confirmed that 26 peptides,including α-trypsin inhibitor heavy chain(ITIH1)peptides,were consistent with the trend of proteomics mass spectrometry results.WB experiments confirmed that the vascular-associated factors CD31 and EMCN were highly expressed in the MSB group.In mouse experiments,CD31 and EMCN were highly expressed in the subchondral bone of the DMM group.ITIH1 is involved in the development of OA course in early subchondral bone.3.N-glycoproteomic in clinical subchondral bone tissue samples found that 295N-glycosylation sites were significantly different from LSBs in OA MSBs.Bioinformatics analysis revealed that N-glycosylation involves multiple signaling pathways.PRM confirmed the difference in N-glycosylation sites in 7 of these peptides.Protein N-glycosylation modification is one of the mechanisms of OA subchondral bone pathogenesis.4.Through clinical subchondral bone tissue sample acetylated proteomics,50 acetylatation sites were found to be expressed differently in MSB and LSB of OA,and protein acetylation modification was involved in the pathogenic mechanism of OA subchondral bone.5.Metabolomics of clinical subchondral bone tissue samples found that there were 64 metabolites with differential expression between MSB and LSB of OA.Of these,31 can serve as potential biomarkers that are key metabolites in the pathogenesis of OA.Multi-omics joint analysis found that the main biological pathways involved in the pathogenesis of OA are: HIF-1,CAMP,NF-kappa B,Fat digestion and absorption,PPAR,Neurotrophiny,Adipocytokine and other signaling pathways.These are key biological pathways in the pathogenesis of OA.Conclusion: The potential early diagnostic target ITIH1 and therapeutic target NQO2 were screened through multi-omics analysis.The acetylation and N-glycosylation modification of subchondral bone proteins are involved in the pathogenesis of osteoarthritis,resulting in cell death and changes in the level of inflammatory factors in subchondral osteocytes,which ultimately affect the course of osteoarthritis.The main biological pathways involved in the pathogenesis of primary KOA are: HIF-1,CAMP,NF-kappa B,Fat digestion and absorption,PPAR,Neurotrophiny,Adipocytokine,etc.These are key biological pathways in the pathogenesis of OA.