S1PR2/RhoA/ROCK1 Pathway Promotes IBD By Inducing Intestinal Vascular Endothelial Barrier Damage And M1 Macrophage Polarization

Objectives: Vascular and immune dysfunctions are thought to be related to the pathogenesis of inflammatory bowel disease(IBD),but behind this,the exact mechanism of mucosal vascular endothelial barrier dysfunction and macrophage phenotypic transition is not fully understood.Here,we explored the role of sphingosine 1-phosphate receptor 2(S1PR2)and its downstream G protein RhoA/Rhokinase 1(ROCK1)signaling pathway in the intestinal endothelial barrier damage and M1 macrophage polarization in IBD.We first clarified the expression of S1PR2 in IBD patients,animal and cell models of IBD,and then explored the regulatory effect of S1PR2 and downstream RhoA/ROCK1 on the intestinal vascular endothelial barrier and M1 polarization of macrophages in IBD to clarify its specific mechanism further.Methods:(1)According to the Consensus opinion on the diagnosis and Treatment of Inflammatory bowel disease Chinese Medical Association in 2018,the pathological colon tissues of 10 IBD patients were collected,and normal colon tissues were used as control.The expression of S1PR2 in intestinal mucosal vascular endothelial cells and macrophages of IBD patients was detected by immunofluorescence double staining.(2)DSS was used to induce colitis in mice,and the mice were sacrificed after 7 days of feeding.Body weight,stool status,colon length,colon pathology,and other indicators were recorded.Immunofluorescence was used to detect the expression of S1PR2 in vascular endothelium and macrophages.Then the mice were treated with JTE-013,an S1PR2 antagonist.Evans vascular permeability test was used to evaluate the intestinal vascular endothelial barrier function,and immunofluorescence was used to evaluate the M1 polarization of intestinal macrophages to observe the effect of S1PR2 inhibition on the intestinal vascular endothelial barrier and M1 polarization of macrophages in mice with DSS-induced colitis.At the same time,LPS was used to induce inflammatory stimulation of vascular endothelial cells and macrophages in vitro.S1PR2 siRNA was constructed and transfected using Lipo RNAi MAX and Lipo 3000.S1PR2 was inhibited by siRNA or JTE-013.The barrier function of endothelial cells was detected by monolayer permeation assay.The effect of S1PR2 inhibition on LPS-induced vascular endothelial barrier damage and M1 polarization of macrophages was observed at the cellular level.(3)At the cell level,the cells were divided into a control group,an LPS group,and an LPS+JTE-013 group.ELISA or Western blot was used to detect the activity or expression of downstream RhoA and ROCK1 in the two types of cells.The cells were then treated with RCOK1 antagonist Y-27632.The morphological changes of the endoplasmic reticulum of vascular endothelial cells were detected by transmission electron microscope.Western blot was used to detect the expression of Endoplasmic reticulum stress(ER stress)markers Bip and CHOP in endothelial cells.Immunofluorescence detected the expression of intercellular junction protein VE-cadherin,and endothelial barrier function was detected.Finally,TUDCA,an endoplasmic reticulum stress inhibitor,was used to observe the effect of TUDCA on LPS-induced barrier dysfunction of endothelial cells.To clarify the mechanism of S1PR2 regulating intestinal vascular endothelial cell barrier dysfunction in IBD through the ROCK1 signaling pathway.On the other hand,the expression of glycolytic key enzyme PFKFB3 in macrophages was detected by Western blot.Flow cytometry and Real-time PCR were used to detect the changes in macrophage M1 polarization markers CD86,MCP-1,iNOS,and TNF-α.Next,the glycolysis inhibitor 2-DG was used to detect the effect on the M1 polarization of macrophages.Thus,the mechanism of the S1PR2/ROCK1 signaling pathway regulating M1 polarization of intestinal macrophages in IBD was elucidated.Results:(1)The expression of S1PR2 was significantly increased in intestinal vascular endothelial cells and macrophages of IBD patients.(2)The expression of S1PR2 in intestinal vascular endothelial cells and macrophages was significantly increased in DSS-induced colitis mice,and S1PR2 inhibition could reverse DSS-induced colon injury in mice and reverse the damage of vascular endothelial barrier and M1 polarization of macrophages in vitro and in vivo.(3)LPS can up-regulate the expression of S1PR2 in HUVECs,and S1PR2 inhibition can reverse the LPS-induced elevation of RhoA and RCOK1,impaired barrier function,decreased expression of VE-cadherin and increased endoplasmic reticulum stress in vascular endothelial cells.Inhibition of ROCK1 or ER stress reversed LPS-induced impairment of endothelial barrier function and decreased VE-cadherin expression.(4)LPS up-regulated the expression of S1PR2 in macrophages,and inhibition of S1PR2 reversed the increase of RhoA,RCOK1,PFKFB3 and M1 polarization of macrophages induced by LPS.Inhibition of ROCK1 or glycolysis reversed the M1 polarization of macrophages induced by LPS.Conclusion:(1)S1PR2 mediates endothelial endoplasmic reticulum stress through RhoA/ROCK1 signaling pathway to affect VE-cadherin expression,thereby regulating intestinal vascular endothelial barrier dysfunction in IBD.(2)S1PR2 mediates macrophage glycolysis through the RhoA/ROCK1 signaling pathway to regulate M1 polarization of intestinal macrophages in IBD.