Exploration Of The Pathogenesis Of Knee Osteoarthritis And Exploration Of The Therapeutic Effect And Mechanism Of Lignans Of Lignans On Knee Osteoarthritis

Objective:Explore the potential pathogenesis and therapeutic targets of Knee Osteoarthtitis(KOA),and evaluate the postoperative improvement of Tolal Knee Arthroplasty(TKA).Confirm the therapeutic effect of lignans of schisandra chinensis on KOA and explore its possible mechanism on KOA.Methods:(1)The GEO database was used to analyze the transcriptome data of cartilage tissues from non-KOA and KOA patients,analyze the differential genes between the two groups,and conduct KEGG signal enrichment for the differential genes and GSEA enrichment analysis for the whole gene expression profile.(2)The knee joint imaging function of EOS system was used to take pictures of KOA patients who received TKA(observation group)and of young patients with unilateral meniscus or cruciate ligament injury(control group),and the parameters were compared and analyzed.The VAS score and the levels of Collagen Ⅰ,IL-1β,IL-6 and MMP13 in serum of the control group and observation group were measured before and 4 months after TKA.(3)The potential targets and signaling pathways lignans of schisandra chinensis in the treatment of KOA were analyzed based on network pharmacology strategy;A compound-target and proteinprotein interaction(PPI)network was constructed to analyze the potential core targets of lignans in KOA.The potential core targets of lignans in KOA were used to conduct the GO and KEGG enrichment analysis.Molecular docking was performed between lignans and their potential core targets in KOA.(4)In vitro SW1353 cell model induced by IL-1β and in vivo mouse KOA model induced by papain injection into knee joint were replicated,and the cell viability was measured with lignans treatment,and the swelling degree of toes of the mice was evaluated.PCR and/or western blot were used to detect the gene and protein expression levels of inflammatory factors such as IL-1β,IL-6,IL-17,Crp and potential targets such as SRC,EGFR,MAPK14 in SW1353 cells or in right knee cartilage of mice.Results:(1)GSE169077 dataset series was selected as the analysis object,5 cases were nonKOA patients and 6 cases were KOA patients.Differential gene analysis showed that 164 genes were significantly up-regulated and 61 genes were significantly down-regulated in the knee cartilage tissue of KOA patients under two-fold differential conditions.Expressions of genes represented by different types of collagen and matrix degradation related genes such as MMP13 were significantly increased in articular cartilage tissues of KOA patients.Lipid transport represented by ABCA1,CEBPB and APOD,and metabolity-related genes represented by PCK1 and HILPDA were significantly decreased in articular cartilage tissues of KOA patients.KEGG signal enrichment analysis showed that the differential genes were enriched to 205 signals under the two-fold difference condition,and 17 of them had a statistical significance.The top five signaling pathways with the most statistical significance were ECM-receptor interaction,Protein Digestion and Absorption,PI3K-Akt signaling Pathway,Ribosome and Human Papillomavirus infection.GSEA enrichment analysis of the whole gene profile showed that the genes were significantly enriched to 15 signaling pathways,among which ECM-receptor interaction decreased most significantly in KOA patients,while Ribosome increased most significantly in KOA patients.(2)Nine parameters(HKA,mLDFA,AMA,JLCA,mLDTA,TT,TC,TMM,LDT-GSA)obtained from the coronal plane of knee and four parameters(PDFA,PPTA,ADTA,ADT-GSA)obtained from the vertical plane of knee by sterEOS software were compared between the control group and observation group.TC was significantly increased and ADT-GSA significantly decreased in KOA patients with either varus or valgus.The LDT-GSA parameters distinguished the difference of skeletal parameters between varus and valgus patients in KOA,and the differences were significant when compared to control group.The presence or absence of pain symptoms affects the imaging perspective of KOA patients before TKA.TKA significantly corrected most of abnormal parameters in patients with KOA.Compared with the control group,the levels of Collagen I,IL-1β,IL-6 and MMP13 in serum of KOA patients were significantly increased before surgery,while the levels of these factors were significantly decreased after TKA treatment.(3)Network pharmacology analysis results showed that TCMSP and SWISS databases predicted 105 potential targets of 6 lignans,OMIM and Genecard databases identified 2612 KOA disease targets.Combining potential targets with disease targets,53 potential action targets were identified for lignan compounds in KOA.The PPI network confirmed that the degree values of mitogenactivated protein kinase 1(MAPK1),epidermal growth factor receptor(EGFR)and mammalian target of rapamycin(mTOR)were significantly higher than other targets,suggesting that they are potential key targets for the treatment of KOA by lignans.Topological MCC algorithm identified 15 core genes of potential targets of lignans in KOA.The results of KEGG analysis showed that SRC,EGFR and MAPK14 were the key targets for the treatment of KOA.The molecular docking results of the lignans with 15 core genes showed that schisandrae phenols and schisandrae b could bind to the protein structures of most genes at low energy,while schisandrae phenols and schisandrae b could stably bind to SRC.(4)The six lignin compounds with concentrations of less than 50μM had no effect on the activity of SW1353 cells.The six lignin compounds with concentrations of 12.5,25 and 50μM up-regulated the decreased Collagen Ⅱ level in IL-1βincubated SW1353 cells,and inhibited the increased gene and protein expression of Collagen 13 in IL-1β-incubated SW1353 cells.In the KOA mouse model,schisandrae chinensis,schisanhenol and schisandrae b significantly improved the symptom of joint swelling in KOA mice,and inhibited the mRNA expression of pro-inflammatory cytokines IL-1β,IL-6,IL-17 and Crp in the cartilage of KOA mice.At the same time,they significantly up-regulated the decreased CollagenⅡ gene expression and down-regulated the increased MMP13 gene expression in the cartilage of KOA mice.Western blot results of SW1353 cells showed that different lignin compounds inhibited the increased protein expression levels of SRC,EGFR and MAPK14 in model cells.PCR results in mice showed that schisandrae chinensis,schisanhenol and schisandrae b significantly inhibited the mRNA levels of SRC,EGFR and MAPK14 in the cartilage of model mice.Conclusion:MMP mediated matrix degradation and inflammation are the as the core mechanisms of KOA.Related molecules in EEM-receptor interaction and Ribosome signaling pathway are potential targets for diagnosis and treatment of KOA.Pain affects the postoperative recovery of TKA in KOA,and the EOS system is conducive to improve the postoperative evaluation of TKA.SRC,EGFR and MAPK are key targets for the treatment of KOA by lignin compounds in schisandrae chinensis.Lignin compounds inhibit SRC,EGFR and MAPK14 gene expression to reduce matrix degradation and play to extract therapeutical effect on KOA.