A Study On The Intervention Of Geniposide On The Sphingolipid Metabolism Disorder Of Fibroblast-like Synoviocytes In Rheumatoid Arthritis In Hypoxic Microenvironment

1.ObjectiveEstablish a metabolomics analysis platform using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS)and ultra-high liquid chromatography tandem triple quadrupole mass spectrometry(UHPLC-MS/MS)to study Changes of sphingolipid(SPL)metabolites in rheumatoid arthritis Fibroblast-like synoviocyte(MH7A),rat serum and synovium.The aim is to explore the mechanism of Geniposide(GE)in the treatment of rheumatoid arthritis(RA)from the perspective of sphingolipid metabolism.2.MethodsSD rats were randomly divided into control group,Adjuvant arthritis(AA)model group and GE administration group.After continuous administration for two weeks,the changes of arthritis index and arthritis index(AI)in each group were observed;MH7A cells were randomly divided into normoxic control group,normoxic administration group(10 μm),hypoxia control group and hypoxia administration group(GE(10 μm)).The hypoxia group was cultured in anoxic chamber,and the SPL extract of MH7 A was collected.The pathological conditions of synovial tissue were detected by sections,and serum and synovial tissue samples were collected.The UPLC-Q-TOF/MS analysis method of the three biological samples was established to obtain the original data.Multivariate statistical analysis was performed by combining the Progenesis QI software,matching the online database,self-built database and secondary mass spectrometry information in MSE mode,and screening and identification of the differential SPL metabolites.To reveal the changes of SPL metabolism in the pathological process of RA.The specific metabolites of sphingolipids were quantitatively analyzed by UHPLC-MS/MS.3.Results3.1 The AI value and paw swelling degree of AA rats were significantly higher than those of the control group,accompanied by inflammatory infiltration in synovial histological sections.GE(60 mg/kg)could effectively relieve the degree of secondary foot swelling in AA rats,significantly reduce AI index,and reverse the pathological changes of synovial tissue,suggesting that GE has anti-inflammatory and immunomodulatory effect on AA rats.3.2 Rat serum and synovial sphingolipidomics analysis: In synovial samples,compared with the control group,the levels of two SPL metabolites,Lyso SM(d18:0)and Cer P(d18:1/22:0),were up-regulated in the AA group,GE administration can effectively correct its metabolic level;In serum samples,compared with the control group,the AA group had up-regulated levels of 3 SPL metabolites,Lyso SM(d18:0),Araliacerebroside and SM(d18:1/18:0).SM(d18:1/23:0),SM(d18:1/22:0)and SM(d18:1/24:1(15Z))3SPL metabolite levels are down-regulated,GE administration can effectively correct SPL Metabolites tend to be normal.3.3 MH7 A sphingolipidomics analysis: Compared with the normoxic control group,the7 SPL metabolites in the hypoxia group had metabolic disorders.After the intervention of GE administration(10 μM)under hypoxia and normoxia,Lyso SM(d18:0),Palmitoyl sphingomyelin,Araliacerebroside,SM(d18:1/18:0),Cer(d18:0/14:0)and Cer(d18:0/16:0).The levels of 6 metabolites were down-regulated.3.4 Based on UPLC-Q-TOF/MS high-resolution mass spectrometry,the SPL metabolites in the sample were scanned to identify the differential SPL metabolites,and the key metabolite SM(d18:1/18:0)was selected using UHPLC-MS/MS uses the MRM pattern to identify the parent ion(731.6075)and the product ion(184.0739)to establish the ion pair 731.6075-184.0739 and perform quantification.The lower limit of quantification is: MH7 A sample 10 pg/L,serum sample 100 pg/L;intraday repeatability:MH7A sample RSD<4.3%,rat serum sample RSD<5.2%.Recovery rate: The recovery rate of the three concentration levels of MH7 A sample is in the range of 87.0-105.9%,and the recovery rate of serum sample is in the range of 86.2%-112.5%,which meets the methodological requirements.4.ConclusionBased on UPLC-Q-TOF/MS technology and sphingolipidomics methods,8differential sphingolipid metabolites related to the occurrence of AA were identified(synovium: 2 differential sphingolipid metabolites;serum: 7 differential sphingolipid metabolites(One is the same)),GE corrects 7 different sphingolipid metabolites.Reveal the mechanism of GE from the perspective of sphingolipid metabolism.It reveals that there are significant changes in the levels of seven SPL metabolites in MH7 A under hypoxia,and GE intervention can adjust their levels.Provide a solid foundation for studying the pathological mechanism of RA and the mechanism of GE intervention in RA.