The Role And Mechanism Of Hyperglycemia Activated S1P-S1PR3 Signaling Pathway In Hepatic Ischemia-reperfusion Injury

Part one: The effects of hyperglycemia on S1P/S1 PRS system,and further study the effect of activated S1P-S1PR3 signaling pathway on hepatic ischemia-reperfusion injury in vivo Objective To study the effect of hyperglycemia on the S1P/S1PRS(1-3)system,and further explore the effect and mechanism of this signaling pathway on hyperglycemia-related IRI.Methods we enrolled diabetic patients with hepatic benign disease who had liver resection and used streptozotocin induced hyperglycemia mice.We established a 90-min ischemic 70% liver and 6,24-hour reperfusion model on diabetic mice and normal mice.A sham-operated group as a control(shame group).We examined the expression of S1P/S1 PRS system in each group by Quantitative RT-PCR(qRT-PCR),Western blot and ELISA.According to the results,we established a 90-min ischemic 70% liver and 6 reperfusion model again but injected S1PR3 inhibitor CAY-10444(1 mg/kg)intraperitoneally before reperfusion.We tested the ALT,AST,inflammatory genes and infiltration of inflammatory cells in different groups.We also examined molecules marker and pathways related to macrophage polarization by using qRT-PCR,Western blot and immunofluorescence.Result S1 P levels were higher in liver tissues from patients with diabetes mellitus and mice with streptozotocin-induced diabetes.S1PR3,but not S1PR1 and S1PR2,was activated in liver tissues and KCs under hyperglycemic conditions.S1PR3 antagonist CAY-10444 attenuated hyperglycemia-related liver IRI based on hepatic biochemistry,histology,and inflammatory responses.Diabetic liver expressed higher levels of M1 markers but lower levels of M2 markers at baseline and post-IR.Dual-immunofluorescence staining showed that hyperglycemia promoted M1(CD68/CD86)differentiation and inhibited M2(CD68/CD206)differentiation.Importantly,CAY10444 reversed hyperglycemia-modulated M1/M2 polarization.Conclusion Hyperglycemia specifically triggers S1P/S1PR3 signaling and exacerbates liver IRI by regulating M1/M2 polarization.Part two: The effect of hyperglycemia on S1P-S1PR3 signaling pathway in macrophages,and further explore the role and mechanism of S1P-S1PR3 pathway aggravating inflammatory response in vitro Objective To confirm that hyperglycemia activates the S1P-S1PR3 axis and further investigates the effect of this pathway on the regulation of M1/M2 polarization in macrophages in vitro.Methods Mouse bone marrow-derived macrophages(BMDMs)were extracted and cultured with high glucose(30 mM)and low glucose(5 mM)respectively.They were set as high glucose(HG)group and low glucose(LG)group.Then they were stimulated with LPS(1 ug/ml),and were set as HG+LPS group and LG+LPS group.At the same time,the siRNA interference technique was used to inhibit the expression of S1PR3 by S1PR3-siRNA and then stimulated with LPS,which set as HG+LPS /S1PR3 group.The NC-siRNA group was used as a control and set as the HG+LPS /NC group.The cell supernatant and cell protein of the different group were collected at 0h,2h,6h,12 h and 24 h after stimulation.We examined the expression of S1P-S1PR3 pathway and molecules marker as related to macrophage polarization by using qRT-PCR Western blot and ELISA.Result In vitro,high glucose(HG)concentrations also triggered S1P-S1PR3 signaling,promoting M1 polarization and inhibiting M2 polarization.After LPS stimulation,the S1P/S1PR3 was activated in HG+LPS group.Compared with the LG+LPS group,the secretion of pro-inflammatory factors such as TNF-α and IL-6 was significantly increased in the HG+LPS group.In contrast,the S1PR3 knockdown group under high glucose after LPS stimulation significantly reduced the secretion of pro-inflammatory factors such as TNF-α and IL-6,but increased anti-inflammatory factors such as IL-10.At the same time,WB results also showed that STAT1 activation was significantly(phosphorylation)decreased in the S1PR3 knockdown group,but activation of STAT3 and STAT6 was increased in S1PR3 knockdown group.Conclusion High glucose in vitro also activates the S1P-S1PR3 axis of macrophages.The S1P-S1PR3 axis aggravates the macrophage inflammatory response by promoting M1 type polarization of macrophages and inhibiting M2 polarization in vitro.