Effects Of Geniposide On S1P-S1PR1 And Its Downstream RhoA-F-actin-NF-κB Signaling Pathway In The Intervention Of Fibroblast-like Synoviocytes On The Biological Function Of Vascular Endothelial Cells

Part Ⅰ: Preparation of conditioned medium and its effect on the biological function of human umbilical vein endothelial cells(HUVEC)Objectives The purposes of this experiment were to culture MH7 A in vitro,establish a tumor necrosis factor-α(TNF-α)-induced MH7 A inflammatory injury model,and prepare conditioned medium from MH7 A and to determine the appropriate concentration of HUVEC induced by MH7 A conditioned medium,and to observe the effect of MH7 A conditioned medium on HUVEC proliferation and biological function and its mechanism.Methods: MH7 A was successfully cultured in vitro and promoted proliferation with TNF-α at concentrations of 2.5 ng/mL,5 ng/mL,10 ng/mL,20 ng/mL,and 40 ng/mL.The CCK-8 method detected the proliferative activity of MH7 A after TNF-αtreatment to determine a suitable modeling concentration.After modeling for 24 h,the MH7 A culture solution was extracted,and the extracted culture solution was subjected to quality evaluation from the aspects of clarity,sterility,pH,and the like.After successful culture of HUVEC in vitro,HUVEC was induced by different concentrations of MH7 A conditioned medium,and CCK-8 method was used to detect the proliferation of HUVEC in different groups to determine the appropriate concentration of HUVEC induced by MH7 A conditioned medium.The conditioned medium and the common medium were used to induce the experimental group and the control group for 24 h respectively.The changes of HUVEC proliferation and migration activity were observed by CCK-8 method,cell scratching and cell migration method.Angiotensin-1(Ang-1),Fibroblast growth factor(FGF)and Angiostatin(AS)secretion in HUVEC supernatant were detected by ELISA.Western-blot and RT-PCR were used to detect the protein expression level and the amount of RNA expression of S1PR1,RhoA,NF-κB p65,NF-κB p-p65 and F-actin in S1P-S1PR1 and its downstream RhoA-F-actin-NF-κB signal pathway in HUVEC.Results: TNF-α at concentrations of 5 ng/mL,10 ng/mL,and 20 ng/mL all inducedMH7 A proliferation,and 10 ng/mL TNF-α was the appropriate concentration for modeling.The extracted medium is a pink clear transparent solution,sterile,pH 7.28± 0.12,and the pH of the solution is suitable for most cell culture(7.2-7.4).MH7 A conditioned medium stocks,double and quadruple dilutions were able to induce HUVEC proliferation.In the subsequent experiments,MH7 A conditioned medium stock was used as stimulator.After induction by MH7 A conditioned medium,the proliferation and migration ability of HUVEC were significantly increased,and the difference was statistically significant(p<0.05).The proangiogenic factors Ang-1 and FGF secreted by HUVEC were significantly increased,and the angiogenic factor AS was significantly decreased,which promoted the promotion of angiogenesis.Western blot analysis showed that the protein expression levels of S1PR1,RhoA,F-actin,NF-κB p65 and NF-κB p-p65 increased.The mRNA expression levels of S1PR1,RhoA,F-actin and NF-κB p65 in HUVEC were significantly increased by RT-qPCR.The PCR results were consistent with the protein results.Conclusion: The conditioned medium from MH7 A in accordance with the requirements of the cell culture medium was successfully prepared.Conditioned medium from MH7 A can successfully induce HUVEC proliferation and migration.MH7 A conditioned medium has the effect of promoting angiogenesis of HUVEC,and its mechanism of action is achieved by activating S1P-S1PR1 and its downstream RhoA-F-actin-NF-κB signaling pathway.Part Ⅱ Therapeutic effect of Geniposide(GE)on the biological function of HUVEC induced by MH7 A conditioned mediumObjectives: The purposes of this study were to investigate the therapeutic effect and mechanism of conditioned medium-induced changes in the biological function of HUVEC after GE administration.Methods: HUVEC induced by MH7 A conditioned medium were cultured for 24 h in vitro.After adding PE solution at concentrations of 6.25 μM,12.5 μM,25 μM,50 μM,100 μM and 200 μM for 24 h,the proliferation activity of HUVEC was determined by CCK-8 method to determine the appropriate concentration of GE.The migration ability of HUVEC was observed by cell scratching and cell migration.The secretion of Ang-1,FGF and AS in HUVEC supernatant was detected by ELISA.The number and distribution of F-actin in HUVEC were observed by fluorescence staining.Western-blot and RT-PCR were used to detect the key proteins S1PR1 and Rho A in the S1P-S1PR1-Rho A-F-actin-NF-κB signaling pathway in HUVEC induced by MH7 A conditioned medium.NF-κB p65,NF-κB p-p65 and F-actin protein expression and RNA expression.Results: Different doses of GE could reduce the abnormal proliferative response of HUVEC to vary degrees,and the GE(25 μM,50 μM,100 μM)group had the most significant effect.Therefore,these three doses were used for subsequent experiments.After administration by GE,the increased proliferative activity and migration ability of HUVEC were significantly inhibited.The pro-angiogenic factor/angiogenic factor that lost its dynamic balance in HUVEC was improved,and the levels of pro-angiogenic factors Ang-1 and FGF were down-regulated,and the level of anti-angiogenic factor AS was up-regulated.The expressions of S1PR1,Rho A,NF-κB p65,NF-κB p-p65 and F-actin increased protein expression and RNA expression were down-regulated.Conclusion: GE can improve the induction of HUVEC by MH7 A conditioned medium,and its mechanism of action is achieved by inhibiting S1P-S1PR1 and downstream Rho A-F-actin-NF-κB signaling pathways.These new findings help us further to elucidate the mechanism of action of GE in inhibiting angiogenesis and cytoskeletal reorganization of rheumatoid arthritis.