Effects Of 3-MST-produced H₂S On Tension And Hypoxic Injury Of Endothelial Cells Of Rat Cerebral Artery

Cerebrovascular disease is a common clinical disease.Ischemic cerebrovascular disease is more common and is the main public health problem in the world.However,the pathophysiological mechanism of its development is still not very clear.The severity of cerebral ischemia is determined by the time the tissue is restricted by blood flow and becomes more severe after a longer ischemic time.This can lead to many complications such as tissue necrosis,apoptosis,inflammation,metabolism and mitochondrial dysfunction,and even organ failure.Hydrogen sulfide,a gas transmitter that diffuses through cell membranes,acts to regulate various signaling pathways that regulate cell survival.Endogenous hydrogen sulfide,as an important gas signal molecule,participates in vascular regulation like NO.It is mainly catalyzed by cysteine（l-cystathionine-?-synthetase,CBS）and cysteine-γ-lyase（CSE）.Hydrogen sulfide is produced by cysteine,L-Cys),and 3-mercaptopyruvate sulfur transferase（3-MST）in combination with cysteine aminotransferase.Our group has previously studied the effects of hydrogen sulfide produced by CSE on cerebral blood vessels.It is proved that H₂S produced by CSE can relax cerebral blood vessels and improve learning and memory dysfunction in mice caused by brain injury.3-MST is also an important H₂S-producing enzyme,but to date,little is known about the regulation of 3-MST and its hydrogen sulfide.Therefore,this paper studies the hydrogen sulfide produced by 3-MST and its effect on endothelial cells damaged by hypoxia.Purpose:（1）To study the relaxation effect of H₂S produced by 3-MST on rat BAs（2）To study the protective effect of H₂S produced by 3-MST on cerebral vascular BAs endothelial cells Methods:（1）Four-channel vascular tension tester detects changes in rat BAs tension;（2）Primary culture,identification of rat cerebral vascular endothelial cells and establishment of an endothelial cell hypoxia model;（3）MTT assay for cell viability and LDH lactate dehydrogenase assay;（4）Methylene blue method for detecting hydrogen sulfide content;（5）Fluorescence detection of active oxygen;（6）The effect of H₂S produced by 3-MST on the expression of 3-MST protein in rat vascular endothelial cells was detected by Western-Blot.Results:（1）3-MST inhibitor ASP(1×10^-5.51×10^-3 mol/L)has a tendency to cause vasoconstriction,but the difference is not statistically significant;compared with the control group,ASP pre-contracted U₄₆₆₁₉ The vascular ring has no significant effect;（2）3-MST substrate 3MP can relax U₄₆₆₁₉ contracted blood vessels;its agonist SIN-1can relax rat cerebral basilar artery in a concentration-dependent manner,compared with endothelial intact vascular ring,endothelium vascular ring pair The sensitivity of SIN-1 is reduced;ASP can attenuate the relaxation of SIN-1 on blood vessels.3MP can enhance the vasodilating effect of SIN-1.（3）Compared with the Control group,the ASP(10^-3mol/L)group had no significant effect on the H₂S content;while the concentration of H₂S was significantly increased after 3MP,the content of H₂S in the SIN-1 group also increased significantly.Compared with SIN-1 alone,ASP can reduce the increased hydrogen sulfide of SIN-1,while 3MP can further increase the hydrogen sulfide content.（4）After hypoxia,ASP can reduce the survival rate of endothelial cells and increase the LDH in the supernatant;3MP and SIN-1 alone can increase the survival rate of endothelial cells and reduce the LDH in the supernatant;SIN-1 The combination of3MP can significantly increase the survival rate of endothelial cells,and at the same time,the LDH in the supernatant is significantly decreased.（5）After hypoxia,the content of hydrogen sulfide in endothelial cells decreased significantly;ASP can inhibit the release of hydrogen sulfide,3MP and SIN-1 can promote the release of hydrogen sulfide;3MP+SIN-1 can significantly increase the release of hydrogen sulfide.（6）After hypoxia,3MP and ASP had no significant effect on the level of reactive oxygen species.SIN-1 and 3MP+SIN-1 could reduce the level of reactive oxygen species,and NaHS could decrease the level of reactive oxygen species.（7）For endothelial cells after hypoxia,the protein expression level of 3-MST decreased,ASP decreased the expression of 3-MST;SIN-1 and 3MP together could increase the expression of 3-MST.Conclusions:（1）H₂S produced by 3-MST has a relaxing effect on rat basilar artery.（2）The H₂S produced by 3-MST has protective effects on hypoxic cerebrovascular endothelial cells.