Untargeted Metabolomic Analysis Of Anterior Cruciate Ligament Remnant Based On Time From Injury To Surgery

PurposeWith the method of clinical study and untargeted metabolomic analysis,we aimed to screen the differential metabolites of anterior cruciate ligament(ACL)remnant between the acute and non-acute stage,explore the potential biomarkers and their possible functions,and provide evidence for early ACL reconstruction with remnant preservation.MethodsThe subjects were patients with ACL injuries admitted by our institute for arthroscopic surgery.Pre-operatively,the injured knee of the patients was examined by International Knee Documentation Committee(IKDC)score,Lysholm score,Tegner score and visual analogue scale(VAS)score.The side to side difference(SSD)was measured with KT2000 arthrometer.The ACL remnants were collected during the surgery.The samples were divided into two groups: experimental group with injury time ≤ 3 weeks and control group with injury time > 3 weeks.After sample preparation and processing,spectrum data were obtained.Firstly,the metabolites of level 1 and 2were annotated and classified,to analyze metabolites in a wholistic perspective.The principal component analysis(PCA)and orthogonal partial least squares-discriminant analysis(OPLS-DA)were carried out to screen the differential metabolites in positive and negative ionic modes.In addition,the biomarkers were identified with machine learning of least absolute shrinkage and selection operator(LASSO)and Boruta algorithms.Subsequently,gene set enrichment analysis(GSEA)was utilized to investigate the metabolic pathways of the biomarkers.Meanwhile,the correlation analysis between biomarkers and clinical data was performed.ResultsWith 39 subjects recruited,the study included 19 cases in the experimental group and 20 cases control group.In total,1032 metabolites were identified through annotation and classification,with lipids most numerous.84 differential metabolites were screened,including 66 up-regulated metabolites and 18 down-regulated metabolites.There were 24 Lasso-feature metabolites and 14 Boruta-feature metabolites.A total of 8 shared metabolites of LASSO and Boruta algorithms were identified as biomarkers,including 5,6,7,8-tetrahydromethanopteri(H4MPT),NP-020871,(E)-4-octenoic acid,cyanidin 3-O-(6-O-p-coumaroyl)glucoside-5-O-glucoside(shisonin),testosterone,5’-deoxyadenosine(5’-dAdo),6-thioguanosine monophosphate(6-TGMP)and methyl dodecanoate.In addition,the GSEA revealed the metabolic pathways of 4 biomarkers.6-TGMP were enriched to AMPK signaling pathway.5’-dAdo involved in the biosynthesis of branched-chain amino acids(BCAAs),digestion and absorption of carbohydrate,along with the biosynthesis of steroid and steroid hormone.The enriched metabolic pathway of H4 MPT is biosynthesis of unsaturated fatty acids.Shisonin involved in the amino sugar and nucleotide sugar metabolism along with the biosynthesis of unsaturated fatty acids.Finally,we found that the expression of H4 MPT,shisonin,NP-020871,6-TGMP,5’-dAdo and(E)-4-octenoic acid had a significant negative correlation with IKDC score and Lysholm score.The expression of testosterone had a significant positive correlation with IKDC score.The expression of 5’-dAdo had a significant positive correlation with VAS score.ConclusionsWith the method of untargeted metabolomic,this study identified biomarkers differentiating ACL remnant of acute and non-acute stage.Among these biomarkers,5’-dAdo,6-TGMP,methyl dodecanoate and the related metabolic pathways indicated the healing potential of ACL remnant in acute stage.The neuro-restoration of ACL remnant of acute stage could be demonstrated by biomarker named shisonin.The enrichedAMPK signaling pathway of 6-TGMP served as indicator that ACL remnant of acute stage could prevent cartilage damage.This study provided metabolomic evidence for early ACL reconstruction with remnant preservation.