| Rheumatoid arthritis(RA)is a chronic autoimmune disease.Abnormally activated fibroblast-like synoviocytes(FLSs)can secrete a variety of inflammatory factors involved in arthritic reactions in RA joints.1-phosphate sphingosine(S1P),as an important inflammatory factor,can be combined with 1-phosphate sphingosine receptors(S1PRs)to couple with G proteins to participate in the inflammatory response through regulating downstream signaling pathway.S1PR1/2/3 played important roles in cell proliferation,angiogenesis,and endothelial barrier function,which is widely expressed in various cells.However,S1 P plays an important role in regulating inflammation by regulating the downstream signaling pathway mediated by Gαi/Gαs,which affects the expression level of cyclic adenosine monophosphate(c AMP).But whether S1P-S1PR1/2/3 has interfered with the biological function of FLSs through mediating Gαi/Gαs conversion has not been reported.Geniposide(GE),as an anti-inflammatory drug candidate,has good prospects for development.The previous study of the research group found that GE played a role in the treatment of RA by inhibiting S1P/S1PR1 and its downstream signaling pathways,restoring the dynamic balance of promoting/suppressing inflammatory factors,inhibiting abnormal proliferation and angiogenesis in FLSs.Based on the previous good experimental foundation and the research status of S1P/S1 PRs.This study intends to establish S1 P induced inflammatory injury model of synovial cell line MH7 A in patients with RA in vitro and to investigate whether GE inhibits S1PR1/2/3,regulates the expression level of coupled Gαi/Gαs,and restores the dynamic balance of Gαi/Gαs to inhibit FLSs abnormal proliferation.It provides important evidence for revealing that S1 PRs coupled Gα protein participates in the process of RA arthritis and the possible mechanism of GE.It also provides new ideas and experimental basis for the drug therapeutic target of RA operational components of traditional Chinese medicine.Objective: The purpose is to clarify the effect of S1P-S1PR1/2/3-Gαi/Gαs signaling pathway on the biological function of MH7 A,and to reveal the mechanism of S1PR1/2/3 regulating the occurrence of Gαi/Gαs conversion.Further,explore the effect of GE mediated S1PR1/2/3 regulation of Gαi/Gαs conversion on the biological function of MH7 A.Methods: The model of S1 P induced MH7 A inflammatory injury was established by cultured in vitro.To clarify the mechanism of S1 P regulating Gαi/Gαs conversion through S1PR1/2/3,to investigate the effects of tool drugs such as selective antagonists of S1PR1、S1PR2、S1PR3、Gαi or agonist of Gαs on the biological function of MH7 A were discussed.To further explores the effect of GE on the biological function of MH7 A and its mechanism of mediating Gαi/Gαs conversion by S1PR1/2/3.To observe the effects of GE(25,50,100 μmol/L),selective antagonists of S1PR1/3,S1PR2,Gαi,and agonist of Gαs intervention on the biological functions of MH7 A were detected.CCK-8,scratch experiment,transwell and flow cytometry were used to detect the proliferation activity,migration and invasion ability,and apoptosis rate of MH7 A.ELISA was used to detect the secretion levels of PGE2,IL-1β,IL-6,IL-8,TGF-β1 and c AMP in MH7 A.And immunofluorescence was used to detect the localization and coupling conditions of S1PR1/2/3 and Gαi/Gαs proteins in each group.RT-q PCR and Western blot methods were used to detect the expression of key genes and proteins in S1PR1/2/3 coupling Gαi/s conversion and its downstream signaling pathways.Results:1 Establishment of S1 P induced rheumatoid arthritis fibroblast synovial cell line MH7 A in vitro inflammation injury model The S1 P of 0.1-10 μmol/L can promote the proliferation of MH7 A at 12,24 and48 h.After S1 P stimulation,c AMP secretion level and c AMP m RNA expression level in MH7 A was significantly reduced.Particularly,5 μmol/L and 24 h more significant.The results of immunofluorescence and western blot showed that S1P(5 μmol/L) stimulated MH7 A significantly up-regulated the protein expression of S1PR1/2/3.Therefore,S1P(5 μmol/L)for 24 h was used as the optimal model condition for subsequent experiments.2 Effects of S1 P coupling Gαi/Gαs conversion on biological function of MH7 A through S1PR1/2/3The selective tools drugs of S1PR1 antagonist(W146),S1PR2 antagonist(JTE-013),S1PR3 antagonist(CAY10444),Gαi antagonist(PTX)and Gαs agonist(CTX),and other tool drugs,were used to interfere with the expression of S1PR1/2/3,Gαi/Gαs in S1 P induced MH7 A.And other groups were found to significantly inhibit MH7 A proliferation,migration and invasion ability,and promote its apoptosis,in addition to inhibiting S1PR2 only inhibits MH7 A invasion ability.Extracellular secretion levels of IL-1β and PGE2 in MH7 A in each group were significantly reduced,while the intracellular c AMP content and c AMP m RNA expression level were significantly increased.The other groups can down-regulate the expression of Gαi protein in MH7 A and up-regulate the expression of Gαs protein.Except for inhibiting S1PR2,Gαs protein in MH7 A is not up-regulated significantly.The results suggest that S1PR1/3 functions are similar,and later experiments will be studied together.3 Effects of geniposide mediated S1PR1/2/3 coupling Gαi/Gαs conversion on MH7 A biological function The S1 P induced MH7 A was intervened by using several drugs,such as GE(25,50,100 μmol/L),selective double antagonist of S1PR1/3(VPC 23019),S1PR2antagonist(JTE-013),Gαi antagonist(PTX)and Gαs agonist(CTX).The results showed that GE could inhibit S1PR1/3 and Gαi gene and protein expression levels,activate the Gαs gene and protein expression levels,mediate Gαi/Gαs conversion,and restore the dynamic balance of Gαi/Gαs,to perform regulatory roles in the downstream signaling pathways,including down-regulating the expressions of p-Erk1/2,proliferation marker protein Ki67 and anti-apoptotic protein Bcl-2,un-regulating the expressions of pro-apoptotic proteins caspase-3 and Bax,inhibiting the secretion levels of pro-inflammatory factors IL-1β,IL-6,IL-8 and inflammatory mediator PGE2,up-regulating the secretion of anti-inflammatory factors TGF-β1 and c AMP,and restoring the balance between pro-and anti-inflammatory factors,which would further inhibit the proliferation,migration and invasion,and promoting apoptosis of S1 P induced MH7 A.Additionally,GE could also inhibit the activation of the S1PR2-Gαi signaling pathway in MH7 A and the related inflammatory responses caused by the activation of S1PR2.Conclusion:S1P can activate the S1PR1/3 receptors in MH7 A cells,up-regulate the expression level of Gαi protein and down-regulate the expression level of Gαs protein,cause the dynamic balance of Gαi/Gαs,disrupt the balance of promoting/suppressing inflammatory factors,and promote the abnormal proliferation of MH7 A.By mainly inhibiting S1PR1/3,mediating Gαi-Gαs conversion and restoring the dynamic balance of Gαi/Gαs,GE can regulate the downstream signaling pathways,including down-regulating the expressions of proliferative proteins and anti-apoptotic proteins,up-regulating the expressions of pro-apoptotic proteins,restoring the balance of pro-/anti-inflammatory factors,as well as inhibiting the abnormal proliferation of MH7 A,thereby exerting anti-inflammatory and immune regulatory effects. |
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