Study On The Mechanism Of Icaritin Against Osteoarthritis Based On Metabolomics Combined With Transcriptomics

Background and Purpose:The development of Osteoarthritis（OA）is associated with an increase in ageing cells in the joint tissue and the degradation of cartilage,possibly due to mitochondrial dysfunction and oxidative stress.Therefore,the reduction of oxidative stress injury to chondrocytes is often regarded as an effective treatment for OA.Epimedium,a Traditional Chinese herb,is commonly used in basic research and clinical treatment of OA.The unique flavonoids of which Icariin are designated as an index component in the quality evaluation of Epimedium herbal medicines in the Chinese Pharmacopoeia,and have various biological activities such as scavenging of reactive oxygen species（ROS）.In vivo,Icariin exerts its therapeutic effects through its metabolite Icaritin,however,the therapeutic advantages and therapeutic mechanisms of Icaritin in the treatment of OA are still unclear.Therefore,in this study,a comparative evaluation study of Icariin and other characteristic flavonoid components of Epimedium herb was first conducted through in vitro cellular assays.On the premise of confirming the superiority of Icaritin,a combined metabolomics and transcriptomics approach was further employed to elucidate the protective mechanism of Icaritin on chondrocytes.This study provides a scientific basis for the treatment of OA with Icaritin.Methods:1.Using H₂O₂ to treat chondrocytes in order to establish a chondrocyte model of OA,the CCK-8 method was used to screen the flavonoid compounds in Epimedium for their chemical composition effective against OA.2.Vitro experiments were used to study the mechanism of Icaritin in the treatment of OA,to detect the levels of ROS,CAT,SOD,GSH and NO,and Western-Blot to detect the expression of MMP3 and MMP13,and to explore their therapeutic mechanism initially.3.The chondrocyte samples were assayed using UPLC-Q-ESI-Orbitrap in the metabolomic analysis,metabolite identification was performed by database comparison,and pathway analysis was performed using a multivariate statistical analysis of differential metabolites between the control,OA and Icaritin groups to reveal the potential mechanism of action of Icaritin in the treatment of OA at the metabolic level.In the transcriptomic analysis,total RNA samples of cells were first extracted and detected by Illumina to identify differentially expressed genes based on the expression of m RNA.Then,differential genes co-adjusted in the control,OA and Icaritin groups were screened for GO functional enrichment and KEGG pathway analysis.Finally,joint analysis of metabolomic and transcriptomic results,combining differential metabolites along with differential genes,was performed to further clarify the mechanism of action of Icaritin in protecting OA chondrocytes.Results:1.The protective effects of the flavonoids of Icariin on OA chondrocyte pairs were verified.The results were best for the protection of OA chondrocytes with 10mM Icariin and Icaritin 10mM.2.The results of the assay of intracellular ROS,CAT,SOD,GSH and NO in chondrocytes showed that the protective effect of Icaritin was superior to that of Icariin.The Western-Blot results showed that Icaritin was able to reduce intracellular MMP3and MMP13 expression.Network pharmacology predicts that the mechanism of OA treatment by both drugs may be through counteracting oxygen stress and anti-inflammation and promoting cellular matrix and collagen synthesis to achieve therapeutic effects.3.Metabolomic results showed that 49 metabolites（19 up-regulated and 30 down-regulated）were differentially expressed in the control and OA groups;108 metabolites（15 up-regulated and 93 down-regulated）were differentially expressed in the OA and Icaritin groups when compared.These differential metabolites were strongly associated with the development of OA.L-glutamine was the most significantly differentially expressed metabolite,while altered expression levels of L-aspartate,L-arginine and L-citrulline were also observed.The pathways involved in these metabolites were mainly involved in the metabolism of D-glutamine and D-glutamate,nitrogen metabolism and arginine biosynthesis.Transcriptomics results showed that Icaritin was able to significantly reduce the expression levels of various cytokines（IL-6,MMP13）and chemokines（CCL5,CCL2 and CXCL10）.4.Metabolomics combined with transcriptomics further showed that these metabolites and genes are mainly associated with energy metabolism,lipid metabolism,oxidative stress,and inflammatory pathways.Icaritin has powerful antioxidant effects,scavenging some of the ROS and restoring the balance of the intracellular redox system,while preventing further damage to cellular lipids from ROS.Conclusion:This study confirmed that Icaritin has strong antioxidant effects by scavenging ROS,reducing glutathione depletion by H₂O₂ and reducing the stress on the antioxidant system of cells.In addition,Icaritin also reduced intracellular lipid damage by ROS,decreased intracellular arachidonic acid content,and lowered the expression of MMP13and IL-6.In this study,we screened potential biomarkers and H₂O₂-induced oxidative stress pathways based on the analysis of integrated multi-omics data.This study provides an experimental basis for investigating the mechanism of action of Icaritin in the protection of OA chondrocytes at the molecular level.