| Background:The formation of new blood vessels occurs throughout the course of rheumatoid arthritis(RA)and is involved in synovial malignant hyperplasia,synovitis and even bone destruction.In the RA inflammatory state,the metabolic balance in the vascular system composed of vascular endothelial cells(VECs)is broken,and activated VECs choose aerobic glycolysis as the main metabolic way in response to the surge of energy versus material demands.In contrast to oxidative phosphorylation,glycolysis supply not only meets the demands of biological functions such as proliferation,budding due to its rapid ATP supply,metabolic intermediates,but also serves as an important signal for intercellular communication.Metabolic reprogramming in the synovial inflammatory microenvironment dictates the important role of glycolytic metabolism in RA angiogenesis,and defining changes in the way endoglin is metabolized may provide an important direction for anti angiogenic therapeutic strategies for RA.Geniposide(GE),as the main active component of the traditional Chinese medicine Gardenia jasminoides Ellis,was found to have significant therapeutic effects on experimental arthritis,by inhibiting the inflammatory response and angiogenesis mediated by sphingosine kinases 1(Sph K1)and its downstream signals,whereas GE with multi hydroxyl active structures with Sph K1 binding site prediction suggested potential targets for GE action.But the mode of action of GE at the cellular level is not well defined.Therefore,this project used the disease-related changes in differential metabolites of glycometabolism to investigate the mechanisms by which RA glycolytic metabolism is involved in angiogenesis at the metabolic molecular level,and then provided new ideas for RA angiostatic therapy.Objective:To screen and verify whether the differential metabolites and rate limiting enzymes of the glycolytic pathway associated with RA disease are PFKFB3,to clarify that GE exerts its activity intracellularly,to target Sph K1 to regulate PFKFB3 mediated glycolytic metabolism in RA angiogenesis,to elucidate the pathogenesis of RA angiogenesis from the perspective of reprogramming of endothelial glucose metabolism,and to provide a basis for GE to exert an RA angiogenesis inhibitory therapy.Methods:1.Establishment of AA rats and TNF-α Induced inflammatory cell model of HUVECs,sample processing methods and analytical methods for constructing glycolytic pathway signatures the metabolomics analysis platform was developed in combination with UPLC-Q-Tof / MS and LC-MS / MS,and the blood of RA patients,the synovial tissue of AA rats,and the cell lysate of HUVECs was used to isolate and identify potential differential metabolites and to content the targeted metabolites.The screened rate limiting enzymes were functionally validated by WB and ELISA methods.2.The AA rat model and TNF-α were firstly observed by HE,IHC and Western blot Levels of glycolytic metabolism and expression of signaling proteins in the induced HUVECs model.Sph K1-si RNA knockdown of Sph K1 detection of the expression and glycolysis level of intracellular signaling proteins demonstrated that Sph K1 was involved in regulating the glycolysis pathway.Finally,signaling protein expression was analyzed by signal specific agonist and inhibitor intervention to clarify the up-and downstream relationship of Sph K1-PI3K-Akt-PFKFB3 signaling.Signaling protein expression total and phosphorylated protein levels of Sph K1,PI3 K,Akt,and PFKFB3,as well as the interaction between Co-immunoprecipitated reactive proteins,were determined by WB,respectively;The level of glycolytic metabolism was revealed by detection of intracellular glucose uptake,intracellular lactate accumulation,and flow and imaging analysis of the fluorescent glucose analog 2-NBDG.3.Detection of GE inside the cell of different FLSs and HUVECs by liquid-mass spectrometry;Synthetic fitc-ge the intracellular distribution and subcellular localization of fitc-ge were analyzed by laser confocal analysis,providing visual verification that GE functions inside the cell.Second,the intervention of GE and Sph K1 inhibitor PF543 at different concentrations in AA rats and TNF-α induced HUVECs,analyzed by WB and CO-IP for signal total and phosphorylated protein expression;The effects of GE on in vitro model angiogenesis were analyzed by Matrigel plug assay,VECs proliferation,migration and tube formation assay.Results:1.Metabolomic analysis of glycolytic metabolic levels in experimental arthritis model with Ge interventionMetabolomic studies of serum and synovial tissue from AA rats identified 11 and13 glycolytic metabolites that affect disease,respectively,and combined with quantitative results showed that the serum of RA patients had decreased glucose and increased lactate metabolites,while the content of glycolytic metabolites in the synovial tissue of AA rats and the cell model of TNF-α induced HUVECs were both increased.GE intervention significantly alleviated the motor performance,joint symptoms and synovial pathology of AA rats,downregulated the content of glycolytic metabolites in the synovial tissues and HUVECs cells of AA rats and differently in the synovial tissues of addback AA rats.Combined with the results of rate limiting enzyme validation,GE significantly downregulated PFKFB3 expression and its catalytic product fructose 2,6-bisphosphate,but significantly affected PFK1 expression.The results indicated that glycolytic metabolism was abnormally elevated in experimental arthritis and that GE played a therapeutic role against experimental arthritis,possibly related to the inhibition of PFKFB3 mediated glycolytic metabolism.2.Mechanistic insight into Sph K1 regulation of PFKFB3 induced endoglycolytic metabolismConstruction of TNF-α induced HUVECs cells in vitro under aerobic or anaerobic conditions,intracellular glucose uptake and lactate accumulation were increased,indicating an aerobic glycolytic metabolic way.Analysis of target total and phosphorylated proteins in AA rats and HUVECs revealed that Sph K1,PI3K-Akt signaling,and PFKFB3 were hyperactivated.Transfection of HUVECs with Sph K1-si RNA revealed that the expression of the target proteins Sph K1,p-sphk1,p-PI3 K,p-Akt,PFKFB3,p-PFKFB3 were all inhibited,while PI3 K and Akt expression was unchanged;and the increased cellular glucose uptake and lactate accumulation indicated that the glycolysis level was inhibited.Co-immunoprecipitation results showed that there was a protein-protein interaction of Sph K1 with PI3 K and p-PI3 K.Signaling protein analysis after treatment with different tool drugs,including K6PC-5(Sph K1 agonist),PF543(Sph K1 inhibitor),LY294002(PI3K inhibitor),MK2206(Akt inhibitor),and PFK15(PFKFB3 inhibitor),showed that the expression and activation of PI3K-Akt protein were significantly decreased by the inhibition of Sph K1;after PI3K-Akt signaling inhibition had no effect on Sph K1 expression and activation,but up-regulated PFKFB3 protein expression and activation;the expression and activation of Sph K1 and PI3K-Akt were not significantly changed by PFKFB3 inhibition.Analysis of cellular glycolysis levels after intervention with different inhibitors revealed that intracellular glucose uptake and lactate accumulation levels were inhibited to different degrees.The results indicated that PFKFB3 was positively regulated by Sph K1-PI3K-Akt to induce glycolytic metabolic levels in HUVECs.3.Angiogenesis under glycolytic metabolic disorders and the role and mechanism of GEDifferent concentrations of GE intervention in AA rats and TNF-α induced HUVECs revealed that the amount of glucose,lactate and glucose 2,6-bisphosphate in the serum of GE addback AA rats and that PFKFB3 and p-PFKFB3 in synovial tissue were downregulated to different degrees;TNF-α induced decrease in glucose uptake and accumulation of lactate in HUVECs.WB and co-immunoprecipitation analysis of cell signaling proteins showed that GE inhibited protein-protein interactions of Sph K1 and PI3K,p-PI3 K,and total and phosphorylated Sph K1-PI3K-Akt signaling proteins to various degrees.Transfection of PFKFB3-si RNA into HUVECs inhibited cell proliferation,migration and tube formation,indicating that inhibition of glycolysis can effectively alleviate the level of angiogenesis in vitro.Evaluation of angiogenesis by GE in vitro and in vivo revealed that GE inhibited angiogenesis,structure and function of HUVECs in vivo,as well as altered biological functions of HUVECs proliferation,migration and tube formation induced by TNF-α in C57 BL / 6 mice.Mode of action analysis of GE showed that the presence of GE prototypes was found in both FLS and HUVECs cell lysates.Laser confocal analysis FITC-GE showed that FITC-GE could diffuse in the cytosol of FLS and HUVECs cells,after which it distributed in the organelles in the cytosol,which were preferentially aggregated in the endoplasmic reticulum compared with the mitochondria,possibly to the endoplasmic reticulum to exert physiological activities,and had a colocalization with the cell membrane shown by orange fluorescence.The results indicated that GE could enter cells to exert its effect and inhibit glycolysis mediated by Sph K1 and its downstream signaling to improve angiogenesis.Conclusions:1.Glycolytic metabolism is abnormally elevated in experimental arthritis,and GE plays a therapeutic role in experimental arthritis,possibly related to the inhibition of PFKFB3 mediated glycolytic metabolism.2.Sph K1 regulated the glycolytic metabolic level of HUVECs,which was associated with activating the downstream signal PI3K-Akt to induce PFKFB3 expression and activation.3.GE inhibited PFKFB3 activated by Sph K1-PI3K-Akt,which in turn downregulated the level of glycolytic metabolism and played a role in angiogenesis of therapeutic trial arthritis,and GE may enter cells to play the above roles. |
