Multi-omics Analysis Of The Pathogenesis Of Primary Frozen Shoulder

ObjectivePrimary frozen shoulder(PFS)is one of the most common shoulder disorders,which affects 2%-5%population worldwide.The main clinical symptoms of the disease are shoulder pain and limitation of motion.According to statistics,PFS is the most common form of arthrofibrosis in the upper and lower extremities and requires the most surgical interventional treatment in all kinds of arthrofibrosis,consuming a large amount of medical resources each year.Conservative treatment(cortisol injection,physiotherapy,release under anesthesia,etc.)is the main treatment for PFS,but quite a lot patients left shoulder pain and limited activity symptoms after treatment,which brings great inconvenience to daily life.The specific pathogenesis of PFS is still unclear.Multiple factors,such as inflammation,immune reaction and fibrosis reaction,participate in the occurrence and progression of the disease,leading to the poor effect of conservative treatment in some patients.Therefore,it is important to explore the pathogenesis of PFS and to find treatments that target the causes of the disease.With the development of endoscopic techniques,arthroscopic release for PFS is becoming more and more widely used.Surgical release can quickly restore the original range of motion and relieve pain in a short period of time.However,the consensus on the timing of surgery is unclear,and the biomarkers that reflect the severity of the disease and determine the timing of surgery are still lacking.Hence,the present study conducted a multi-omics analysis of tissue and blood samples from PFS patients and corresponding control patients,in order to explore the specific mechanism of PFS pathogenesis and screen related biomarkers.MethodsSubjects meeting the relevant inclusion criteria were recruited for this study and divided into a severe group(patients with PFS),a mild group(patients with rotator cuff injury combined with shoulder stiffness)and a control group(patients with non-joint stiffness such as shoulder dislocation)according to the range of motion of the shoulder joint.Tissue and blood samples from three groups of patients were collected for transcriptomic,metabolomic and modificationomic analyses.Combined multi-omics analysis was used to explore the pathogenesis of PFS at the three levels of gene expression,metabolite and post-translational modification,and potential biomarkers were screened.ResultsA total of 30 subjects were recruited in this study,including 13 patients in the severe group,12 patients in the mild group,and 5 patients in the control group,and an additional10 blood samples were taken from healthy volunteers.Through metabonomics analysis,we found that lipid metabolism and amino acid metabolism were significantly dysregulated in tissue samples of the severe group,and the citrullination related pathway was strongly associated with the progression of PFS.Metabolomic analysis of plasma samples screened for two phospholipid molecules(SOPC,PC16:0)that could be used as biomarkers to distinguish between mild and severe frozen shoulder.In transcriptomic analysis,muscle development and muscle contraction related pathways were found to be significantly activated in the severe group.Metabolomic combined with transcriptomic analysis revealed that arginine-citrullination related pathways were significantly upregulated in the severe group,and immunohistochemical staining verified that two enzymes catalyzing citrulline production:Nitric Oxide Synthase 1(NOS1)and Peptidyl Arginine Deiminase Ⅱ(PADI2)were highly expressed in the severe group.Immunoinfiltration analysis of transcriptome data showed that T cells,B lymphocytes,monocytes,neutrophils and CD8~+T cells infiltrated significantly in the tissue samples of the severe group,suggesting that the combination of protein citrullination and immune response affected progression of frozen shoulder.Modification omics analysis confirmed the significant up-regulation level of protein citrullination in the severe group,which was verified by immunofluorescence staining.The outcome of enrichment analysis of differentially modified sites was consistent with transcriptomics,and the muscle development and muscle contraction related pathways were significantly activated in the severe group.The combined analysis of the modification omics and the transcriptomics showed that three proteins(MYH6,TTN,ITIH4)were up-regulated in the severe group tissues at the gene expression level and the citrullination level.The analysis of the protein interaction network showed that the interaction of these three citcitlinated proteins was closely related to the development of frozen shoulder.ConclusionsLipid metabolism and amino acid metabolism were significantly dysregulated in severe frozen shoulder tissues,and muscle development as well as muscle contraction related pathways were highly activated.Two phospholipids(SOPC,PC16:0)can be used as potential biomarkers to differentiate between mild and severe frozen shoulder.The level of protein citrullination that catalyzed by PADI2 was significantly up-regulated in the severe group,which was closely related to immune response.Three proteins(MYH6,TTN,ITIH4)interact with each other may influence the development and progression of PFS.Future studies are needed to explore the relationship between citrullination,immune response and disease phenotype.