Study On The Protective Role And Mechanism Of 3-MST-produced H₂S In Cerebrovascular Endothelial Cells Against Hypoxic-ischemic Injury

BackgroundGlobally,approximately 5.5 million people die from ischemic or hemorrhagic strokes each year.Stroke is the second most common cause of death after ischemic heart disease.It is characterized by high prevalence,high mortality,high recurrence rate,and high disability rate.At present,thrombolytic drugs are considered to be the key clinical treatment of acute stroke.However,timely restoration of blood supply in the ischemic area will lead to more severe structure and functional damage of brain tissue,also known as ischemia/reperfusion?I/R?injury.Endothelial cells?ECs?are the first barrier to blood vessels.Impaired endothelial function aggravates ischemic brain injury.Mitochondria are highly active organelles in cells.Mitochondrial dysfunction of endothelial cells has recently been considered as a new mechanism for secondary cell death after I/R injury.Cerebrovascular ECs and mitochondria are very sensitive to ischemia and hypoxia,and improving mitochondrial function has been recently proved to be a potential intervention to improve cardiovascular and cerebrovascular diseases.3-mercaptopyruvatesulfurtransferase?3-MST?,which is mainly localized to the mitochondria,is one of the endogenous H₂S synthetases and is the main source of H₂S production in the brain.A variety of functions of 3-MST/H₂S has been reported,including angiogenesis,vasodilation,antioxidant stress,and promoting bioenergy,but little is known about its role in cerebral ischemic injury.The Rho kinase signaling pathway is an important target of the cardiovascular and cerebrovascular system,and ROCK is the most important and direct effector molecule of Rho A.There have been many studies on the relationship between Rho A/ROCK pathway and function of ECs.Besides,activation of Rho A/ROCK pathway may impair mitochondrial dynamics and energetics.Therefore,we hypothesized that 3-MST/H₂S produced by ECs mitochondria could protect the cerebral ischemia and reperfusion injury via inhibiting the Rho A/ROCK signaling pathway,and designed in vitro and in vivo study to explore the potential mechanisms.Purpose:1.To explore the protective effects of 3-MST-produced H₂S on rat cerebral vascular ECs and mitochondria against OGD/R-induced injury together with the potential mechanisms related to Rho A/ROCK pathway.2.To investigate the protective effect of 3-MST-produced H₂S on endothelial injury and the potential mechanisms related to Rho A/ROCK pathway in CSE-/-mice subjected to ischemia/reperfusion injury.3.Endothelial damage as well as changes in 3-MST and H₂S level in the vascular specimens of the distal middle cerebral artery of human were studied.Method:1.An in vitro oxygen and glucose deprivation/reperfusion?OGD/R?model of primary cerebral vascular endothelial cells was established.Different concentrations of3-mercaptopyruvate?3-MP?and L-aspartic acid?L-Asp?were added into the culture medium during OGD/R,succinate dehydrogenase activity was evaluated by MTT assay.1.1 ECs was divided into the following five groups:?1?Control group;?2?OGD/R group;?3?L-Asp?10?M?group;?4?3-MP?90 n M?group;?5?3-MP+L-Asp group.1.2 The methylene blue method and Western blot method were used to detect changes in H₂S content and 3-MST protein expression levels in ECs,respectively.Changes in the levels of reactive oxygen species?ROS?,total antioxidant capacity?T-AOC?,and adenosine triphosphate?ATP?were used to assess the celluar injury.1.3 The subcelluar location of 3-MST and Rho A,ROCK₁,and ROCK₂ in primary EC was detected by immunofluorescence.Besides,purified mitochondria were isolated by differential centrifugation,and Western blotting was used to further verify the mitochondrial localization of 3-MST and Rho A,ROCK₁,and ROCK₂.1.4 The fluorescent dye 7-azido-4-methylcoumarin?Az MC?was used to detect changes in mitochondrial H₂S content,and Western blotting was used to detect changes in mitochondrial 3-MST and ATP5D protein expression levels.Transmission Electron Microscope?TEM?was used to observe the changes in mitochondrial ultrastructure.Mitochondrial reactive oxygen levels were detected using Mito SOX fluorescent probe.Changes in mitochondrial ATP synthase activity was assessed by enzyme-linked immunosorbent assay?ELISA?method.Changes in mitochondrial ATP content was detected by chemiluminescence method,and mitochondrial membrane potential was detected using JC-1 dye.1.5 Rho A activity together with ROCK₁ and ROCK₂ expression levels in celluar lysates and mitochondria were detected by ELISA and Western blotting,respectively.2.The focal middle cerebral artery occlusion/reperfusion?MCAO/R?model was established using suture embolism and removal method.At the end of the experiment,neurological score was finished,and brain infarct volume was detected by2,3,5-triphenyltetrazolium chloride?TTC?staining.2.1 Laser speckle flow imaging?LSFG?technology was used to detect cerebral blood flow in each group in mice.The time points were the following:baseline,post occlussion,and 10 min,1h and 22 h after reperfusion.2.2 At the end of the experiment,hematoxylin-eosin?HE?staining was carried out to detect the morphological changes of endothelium in the middle cerebral artery.Changes in the levels of H₂S as well as Rho A,ROCK₁ and ROCK₂ protein in middle arterial vessels were detected by methylene blue staining and immunohistochemistry,respectively.3.The distal middle cerebral artery of human brain were collected from patients with massive cerebral infarction?MCI?and deep benign tumor.Morphological changes of cerebral vascular endothelium was observed by HE staining and TEM.Changes in H₂S content were detected by methylene blue method.Changes in 3-MST protein levels in vascular tissue were detected by immunofluorescence?IF?and Western blotting.Results:1.Compared with the control group,the succinate dehydrogenase activity was significantly reduced in OGD?4 h?-R?20 h?group?P<0.01?,and pretreatment of 3-MP?10,30,90 n M?induced concentration gradient increases of succinate dehydrogenase activity?P<0.01?.While pretreatment of L-Asp?1?M and above?further aggravated cellular damage after OGD/R?P<0.05;P<0.01?.The results of methylene blue assay showed that H₂S content of ECs decreased significantly after OGD/R injury,and pretreatment of L-Asp?10?M?further inhibited production of H₂S?P<0.01?.While pretreatment of 3-MP?90 n M?significantly increased cellular H₂S content,and this effect was significantly attenuated by L-Asp addition.Besides,contrary to the effect of L-Asp,3-MP pretreatment significantly reversed the decrease of 3-MST expression caused by OGD/R.2.Compared with the control group,OGD/R-induced injury of ECs included significantly increased ROS levels,decreased T-AOC levels,and decreased ATP content?P<0.01?.Results of fluorescent probe and flow cytometry methods confirmed that3-MP pretreatment significantly inhibited cellular ROS generation and increased T-AOC and ATP levels,while L-Asp pretreatment exhibited the opposite effect.3.Results of immunofluorescence and Western blotting showed that 3-MST was localized not only in cytoplasm but also in mitochondria of primary ECs.Compared with the control group,mitochondrial H₂S fluorescence was significantly decreased after OGD/R injury.Compared with the model group,L-Asp pretreatment further reduced mitochondrial H₂S fluorescence,while 3-MP pretreatment reversed the decreases in mitochondrial H₂S fluorescence.Immunoblotting of purified mitochondria showed that compared with the control group,3-MST expression of mitochondria decreased after OGD/R injury?P<0.01?;compared with the model group,3-MP pretreatment increased 3-MST expression levels?P<0.01?,this effect of 3-MP can be attenuated by L-Asp pretreatment.4.Compared with the control group,OGD/R injury lead to mitochondrial ultrastructural damage,increased mitochondrial ROS generation?P<0.01?,and decreased mitochondrial ATP synthase activity and ATP5D expression level?P<0.01?,resulting in significant decreases in ATP production and mitochondrial membrane potential.In contrast to the effect of L-Asp,3-MP pretreatment significantly reversed these changes in mitochondrial dysfunction.5.Compared with the control group,Rho A activity,ROCK₁ and ROCK₂ expression in mitochondria-excluded cell lysates and mitochondria were significantly increased after OGD/R injury,and L-Asp pretreatment increased Rho A activity,ROCK₁ and ROCK₂expression.Compared with the model group,pretreatment with 3-MP significantly inhibited Rho A activity,ROCK₁and ROCK₂ expression in each group?P<0.01?.6.After MCAO/R injury in CSE^-/-mice,results of TTC staining and neurological score suggested that the model was successfully established.LSFG results showed that brain CBF in the model group was significantly reduced?P<0.01?.Pretreatment of L-Asp exerted a significant inhibitory effect on brain CBF,while 3-MP pretreatment increased brain CBF after 22 h of reperfusion?P<0.05?.Compared with the sham group,HE staining showed that morphological structure of the endothelium in the middle cerebral artery from model group was significantly damaged,while the endothelial structure in the 3-MP pretreatment group was relatively complete and the cells were neatly arranged.7.After MCAO/R injury,the H₂S content in the middle arteries of CSE^-/-mice decreased significantly,contrary to the effect of L-Asp,3-MP significantly increased the H₂S content in vascular tissues.Immunohistochemical results showed that,after ischemia-reperfusion injury,the expression level of Rho A,ROCK₁,and ROCK₂ in the cerebral vascular endothelium of CSE^-/-mice was significantly increased.Contrary to L-Asp pretreatment,3-MP pretreatment significantly inhibited their expression.8.Compared with the non-ischemic group,the morphological structure of vascular endothelium and mitochondria in the middle cerebral artery of MCI patients was damaged,including disrupture of cell membrane,margin concentration of nuclear chromatin,mitochondrial swelling,rupture of ridge,and vacuolization.At the same time,cerebrovascular H₂S content as well as the expression level of 3-MST in MCI patients were also significantly down-regulated.Conclusion:1.Significant down-regulation of 3-MST-produced H₂S was detected in the cerebral vascular ECs subjected to OGD/R injury.2.3-MST-produced H₂S protects ECs against OGD/R-induced injury via preserving of mitochondrial function.3.3-MST-produced H₂S protects ECs against OGD/R injury via inhibition of the Rho A/ROCK signaling pathway in cell and mitochondria.4.3-MST-produced H₂S protects against cerebral ischemia/reperfusion injury through increasing cerebral blood flow.This was related to inhibition of the Rho A/ROCK signaling pathway in cerebrovascular endothelium.5.Results of human cerebrovascular specimens suggest that significant down-regulation of 3-MST and H₂S was found in patients with ischemic stroke.