Study On The Mechanisms Of SOCE Regulating Intestinal Submucosal Microvasculature Vasorelaxation And Its Role In Colitis

Part Ⅰ Mechanisms of CPA-induced relaxation of intestinal submucosal microvasculature and its role in colitisObjective:To explore the mechanisms of CPA-induced relaxation of intestinal submucosal microvasculature and its role in colitis.Methods:The subjects were human intestinal submucosal microvasculature and the second-level mesenteric arteries from 18-22g,6-8weeks C57BL/6 male healthy mice and colitis mice.Changes of vessel tension were recorded using the Danish DMT520A microvascular tension system and Powerlab analytical system in vitro.Firstly,to observe the relaxing effect of CPA on human intestinal submucosal microvasculature.To explore the role of NO and PGI₂in the process of CPA-induced relaxation of human intestinal submucosal microvasculature by application of e NOS inhibitor L-NNA and endothelial COX inhibitor INDO.Because human tissues are not easy to obtain and our research team found that the second-level micro-arteries of the mice mesentery,which are close to the human intestinal mucosal microvessels,can mimic the functional characteristics of the former through preliminary experiments.It has long been used as a representative model of resistance vessels.Therefore,we replaced the human intestinal submucosal microvasculature by mice mesenteric micro-arteries in the subsequent experiments.By comparing CPA-induced vasorelaxation between endothelium-intact and endothelium-denuded mesenteric arteries,it is clear whether the relaxation effect of CPA depends on endothelial cells.To explore the role of NO and PGI₂in the process of CPA-induced relaxation of mesenteric arteries by application of e NOS inhibitor L-NNA and endothelial COX inhibitor INDO on blood vessels.To investigate whether the potassium channels are involved in CPA-induced relaxation of mesenteric arteries by application of high potassium.K_Cainhibitor TEA and activator SKA-31 were used to investigate whether endothelium-dependent hyperpolarization plays an important role in CPA-induced relaxation of the mesenteric arteries.To investigate whether Na⁺-K⁺ATPase（NKA）is involved in CPA-induced relaxation by application of NKA inhibitors ouabain and K⁺free solution.To investigate whether Na⁺-Ca²⁺ATPase（NCX）is involved in CPA-induced relaxation by application of NCX inhibitor SN-6.Application of SOCE inhibitors SKF96365、FFA and Orai inhibitor GSK-7975A to investigate if CPA-induced relaxation relied on SOCE.In addition,application of single cell Ca²⁺real-time measurement technique to detect the change of Ca²⁺signal after CPA acts on HUVEC under the conditions of normal PSS solution and 0 Ca²⁺solution respectively.Finally,investigate CPA-induced relaxation in DSS-induced colitis mice with or without L-NNA plus INDO.Results:CPA induced vasorelaxation in concentration-dependent manners when human intestinal submucosal microvasculature were pre-contracted with NE,with endothelium-intact Rmax of 86.24±3.28%.L-NNA and INDO have no effect on CPA-induced relaxation of human intestinal submucosal microvasculature.CPA induced vasorelaxation in concentration-dependent and endothelium-dependent manners when mice mesenteric arteries were pre-contracted with NE,with endothelium-intact Rmax of 91.46±5.09%and endothelium-denuded Rmax of 18.29±2.33%.L-NNA and INDO have no effect on CPA-induced relaxation of mesenteric arteries.High potassium contraction significantly inhibited CPA-induced relaxation of mesenteric arteries.A large portion of CPA-induced vasorelaxation was significantly attenuated by TEA;but potentiated by SKA-31.Ouabain、K⁺free solution and SN-6significantly inhibited CPA-induced relaxation of mesenteric arteries.In addition,CPA-induced relaxation was significantly inhibited by SKF96365、FFA and GSK-7975A.Moreover,CPA can increase HUVEC Ca²⁺signal through intracellular calcium release and external calcium influx,and the Ca²⁺signal of CPA-induced external calcium influx is inhibited by GSK-7975A.Finally,CPA-induced vasorelaxation through EDH was impaired in colitis mice.Conclusion:CPA mediated EDH by activation SOCE/Ca²⁺signal in vascular endothelial cells,which can promote the blood supply of the intestinal mucosa and its repair after injury by relaxing human intestinal submucosal microvasculature in concentration-dependent manners.The SOCE/Ca²⁺/EDH is impaired in colitis and can be used as a potential target for the prevention and treatment of colitis.Part Ⅱ To explore the molecular mechanisms of SOCE in regulating intestinal submucosal microvascular relaxation and its role in colitisObjective: Further to explore the molecular mechanisms of SOCE in regulating intestinal submucosal microvascular relaxation and its role in colitis.Methods: The subjects were human intestinal submucosal microvasculature and the second-level mesenteric arteries from 18-22 g,6-8 weeks C57BL/6 male healthy mice and colitis mice.Changes of vessel tension were recorded using the Danish DMT520 A microvascular tension system and Powerlab analytical system in vitro.Firstly,to observe the relaxing effect of endoplasmic reticulum calcium chelator TPEN on human intestinal submucosal microvasculature.To explore the role of NO and PGI2 in the process of TPEN-induced relaxation of human intestinal submucosal microvessels by application of e NOS inhibitor L-NNA and endothelial COX inhibitor INDO.To investigate whether the potassium channels are involved in TPEN-induced relaxation of human intestinal submucosal microvasculature by application of high potassium.The IKCa and SKCa selective blockers TRAM-34、apamin were used to investigate whether endothelium-dependent hyperpolarization plays an important role in TPEN-induced relaxation of the human intestinal submucosal microvessels.Secondly,because human tissues are not easy to obtain and our research team found that the second-level micro-arteries of the mice mesentery,which are close to the human intestinal mucosal microvessels,can mimic the functional characteristics of the former through preliminary experiments.It has long been used as a representative model of resistance vessels.Therefore,we replaced the human intestinal submucosal microvasculature by mice mesenteric micro-arteries in the subsequent experiments.By comparing TPEN-induced vasorelaxation between endothelium-intact and endothelium-denuded mesenteric arteries,it is clear whether the relaxation effect of TPEN depends on endothelial cells.To explore the role of NO and PGI2 in the process of TPEN-induced relaxation of mesenteric arteries by application of e NOS inhibitor L-NNA and endothelial COX inhibitor INDO on blood vessels.High potassium was applied to investigate whether the potassium channels are involved in TPEN-induced relaxation of mesenteric arteries.IKCa 、 SKCa selective inhibitors TRAM-34 、 apamin and activator SKA-31 were used to investigate whether endothelium-dependent hyperpolarization plays an important role in TPEN-induced relaxation of the mesenteric arteries.To investigate whether Na+-K+ ATPase（NKA）is involved in TPEN-induced relaxation by application of NKA inhibitor ouabain.To investigate whether Na+-Ca2+ ATPase（NCX）is involved in TPEN-induced relaxation by application of NCX inhibitor SN-6.Application of SOCE inhibitor SKF96365 to investigate if TPEN-induced relaxation relied on SOCE.In addition,application of Orai selective inhibitor GSK-7975 A,STIM selective inhibitor ML-9 and Brefeldin A（BFA）which inhibits vesicular translocation to investigate if TPEN-induced relaxation relied on STIM/Orai-mediated-SOCE.Finally,to investigate TPEN-induced relaxation in DSS-induced colitis mice.Results: TPEN induced vasorelaxation in concentration-dependent manners when human intestinal submucosal microvasculature were pre-contracted with NE,with endothelium-intact Rmax of 95.67 ± 0.91 %.High potassium contraction significantly inhibited TPEN-induced relaxation of human intestinal submucosal microvasculature.L-NNA and INDO have no effect on TPEN-induced relaxation of human intestinal submucosal microvasculature.Furthermore,a large portion of TPEN-induced vasorelaxation of human intestinal submucosal microvasculature was significantly attenuated by TRAM-34 and apamin.TPEN induced vasorelaxation in concentration-dependent and endothelium-dependent manners when mesenteric arteries were pre-contracted with NE,with mice endothelium-intact Rmax of 96.13 ±4.74 %,mice endothelium-denuded Rmax of 11.33 ± 2.91 %.L-NNA and INDO have no effect on TPEN-induced relaxation of mesenteric arteries.High potassium contraction significantly inhibited TPEN-induced relaxation of mesenteric arteries.A large portion of TPEN-induced vasorelaxation was significantly attenuated by TRAM-34 and apamin,but SKA-31 can in turn potentiate EDH-type arterial relaxation.Ouabain and SN-6 significantly inhibited TPEN-induced relaxation of mesenteric arteries.TPEN-induced relaxation was significantly inhibited by SKF96365 、 ML-9 、 GSK-7975 A and BFA.Finally,TPEN-induced vasorelaxation was partially impaired in colitis mice.Conclusion: TPEN chelates endoplasmic reticulum calcium,stimulates the STIM/Orai-mediated-SOCE and Ca2+ signals to mediate EDH in vascular endothelial cell,which can promote the blood supply of the intestinal mucosa and its repair after injury by relaxing human intestinal submucosal microvasculature in concentration-dependent manners.Although the SOCE/Ca2+/EDH is partially impaired in colitis,STIM/Orai may serve as a potential molecular target for the prevention and treatment of colitis,which is worthy of further study.