Protective Effect Of H₂S Produced By Cse In Cerebral Vascular Endothelial Cells On Hypoxic Injury Of Hippocampal Neurons And The Mechanism Of Inhibiting RhoA-ROCK Signaling Pathway

Background:Stroke is an acute cerebrovascular disease with very high morbidity and mortality,and has become the second leading cause of disability and death.The main pathogenesis of cerebral apoplexy is ischemia-reperfusion,which can induce obvious oxidative damage to peripheral neurons.Hydrogen sulfide is a gas signaling molecule that plays an important role in stroke.Endogenous H₂S could be generated from Cys catalyzed by CBS and CSE.In addition,3-MST could combine with cysteine aminotransferase（CAT）to produce H₂S.CBS is mainly distributed in the central nervous system,and CSE is mainly exist in the cardiovascular system.3-MST was expressed in brain cells and vascular endothelial cells.Therefore,there are two pathways of H₂S produced by CSE（CSE-produced H₂S）and 3-MST-produced H₂S（3-MST-produced H₂S）in endothelial cells.The RhoA-ROCK signaling pathway is closely related to the pathological mechanism of cardiovascular and cerebrovascular diseases,and it is a new target of the cardiovascular and cerebrovascular system.RhoA is a small guanylate binding protein,and ROCK is a serine/threonine protein kinase,including two isoforms ROCK₁ and ROCK₂.Studies have shown that inhibition of RhoA-ROCK signaling pathway can protect brain neurons.In this study,we aimed to determine whether H₂S produced by CSE in endothelial cells has a protective effect on neuronal hypoxic injury and the interaction between H₂S produced by CSE in endothelial cells and RhoA-ROCK signaling pathway.Purpose:1.To investigate whether H₂S produced by CSE in endothelial cells has protective effect on OGD/R injury of hippocampal neurons.2.To investigate the effect of H₂S produced by CSE in endothelial cells on OGD/R injury in hippocampal neurons and the relationship with RhoA-ROCK signaling pathway.Methods:1 To investigate the effect of H₂S produced by CSE in cerebrovascular endothelial cells on OGD/R injury in primary rat hippocampal neurons,L-Cys was used as endogenous H₂S donor.Rat hippocampal nerve cells and cerebrovascular endothelial cells were primary cultured,and then the two cells were co-cultured.1.1 After co-culture,the two cells were divided into six groups:Control group,OGD/R group,OGD/R+Na HS(200μmol·L^-1)group,OGD/R+L-Cys(50,100,200μmol·L^-1)group.Na HS was used as a positive control to determine the appropriate dose of L-Cys.The activities of LDH,NSE and CCK8 were detected by kits,and the content of H₂S was tested by methylene blue method.1.2 After co-culture,the two cells were divided into five groups:Control group,OGD/R group,OGD/R+L-Cys(200μmol·L^-1)group,OGD/R+PPG group,OGD/R+L-Cys(200μmol·L^-1)+PPG group.The activities of LDH,NSE and CCK8 were detected by kits.H₂S content was determined by methylene blue method,and cell apoptosis was detected by Annexin-FITC/PI double staining kit.2 To investigate the effect of H₂S produced by CSE in cerebrovascular endothelial cells on OGD/R injury in primary hippocampal neurons and the relationship between H₂S and RhoA-ROCK signaling pathway.2.1 After co-culture,the cells were divided into five groups:Control group,OGD/R group,OGD/R+L-Cys(200μmol·L^-1)group,OGD/R+PPG group,OGD/R+L-Cys(200μmol·L^-1)+PPG group.The activity of RhoA and ROCK₂was detected by RhoA and ROCK₂ activity detection kits,and the protein expression of RhoA and ROCK₂ was tested by Western blot.2.2 Primary cultured cerebral vascular endothelial cells（EC）of CSE knockout and wild type mice and primary hippocampal neural cells（NC）of mice were divided into:（1）NC+EC^CSE-WT;（2）NC+EC^CSE-KO（AOAA,L-Asp,L-Cys were added to each group）.H₂S content was detected by methylene blue method.Western blot was used to detect the protein expressions of RhoA,ROCK₂ and their corresponding phosphorylated proteins.Results:1.1（1）Compared with the negative control group,the cell viability of the OGD/R group was significantly decreased,and the levels of LDH and NSE were significantly increased（P<0.01）.L-Cys(100,200μmol·L^-1)significantly attenuated the decrease of cell viability,LDH activity and NSE activity in OGD/R injured neurons.In addition,administration of positive control Na HS(200μmol·L^-1)also significantly attenuated the decrease of nerve cell activity,LDH activity and NSE activity caused by OGD/R injury.These results suggest that L-Cys may play a protective role in OGD/R injury by inhibiting the leakage of intracellular enzymes in hippocampal neurons.（2）Compared with the negative control group,the concentration of H₂S in neurons of OGD/R group was significantly decreased,but after L-Cys(100,200μmol·L^-1)was added,the concentration of H₂S in neurons of OGD/R group was significantly increased（P<0.01 or P<0.05）;In addition,the positive control Na HS(200μmol·L^-1)and L-Cys(100,200μmol·L^-1)had similar effects.The results suggested that the concentration of H₂S in the supernatant of hippocampal neurons after OGD/R injury could be increased by adding L-Cys to cerebral vascular endothelial cells.1.2（1）After pretreatment with CSE inhibitor(PPG 100μmol·L^-1)for 30 minutes,compared with L-Cys group,the cell viability of neurons was significantly decreased（P<0.01）,LDH activity significantly increased（P<0.01）,increased NSE activity（P<0.01）,while H₂S production significantly decreased（P<0.01）.These results suggest that CSE inhibitor PPG pretreatment can inhibit L-Cys protection against OGD/R injury and increase of H₂S production in primary rat hippocampal neurons.（2）Compared with the control group,the fluorescence intensity of OGD/R group was significantly increased（P<0.01）;Compared with OGD/R group,the fluorescence intensity of L-Cys group was significantly decreased（P<0.01）;Compared with L-Cys group,the fluorescence intensity was increased in L-Cys+PPG group（P<0.01）.The results showed that the treatment of cerebral vascular endothelial cells with L-Cys could reduce the apoptosis of hippocampal neurons induced by OGD/R injury,while the pretreatment with CSE inhibitor PPG could inhibit the protective effect of L-Cys on the apoptosis of primary rat hippocampal neurons induced by OGD/R injury.2.1 Compared with the normal control group,the protein expression and activity of RhoA and ROCK₂ were significantly increased in the OGD/R model group（P<0.01）;Compared with the OGD/R group,CSE substrate L-Cys(200μmol·L^-1)after OGD/R injury could reduce the protein expression of RhoA and ROCK₂（P<0.01）,and decreased the activities of RhoA and ROCK₂.CSE inhibitor PPG alone did not affect the protein expression of RhoA and ROCK₂and the activity of RhoA and ROCK₂.Compared with L-Cys group,the protein expressions of RhoA and ROCK₂ were significantly increased in CSE inhibitor PPG and L-Cys group（P<0.05）,and the activities of RhoA and ROCK₂ increased.The results showed that L-Cys could inhibit the up-regulation of RhoA and ROCK₂ protein expression and the increase of RhoA and ROCK₂ activity in hippocampal neurons caused by OGD/R injury.CSE inhibitor PPG pretreatment can inhibit L-Cys protection of OGD/R injury in primary rat hippocampal neurons caused by the decrease of RhoA and ROCK₂ protein expression and activity.2.2 Compared with the NC+EC^CSE-WT group,the H2S content in the supernatant of nerve cells in the NC+EC^CSE-KO group decreased,the protein expressions of RhoA and ROCK₂ increased significantly,and the protein expressions of p-RhoA/total RhoA and p-ROCK₂/total ROCK₂decreased significantly.The results showed that H₂S produced by CSE in cerebrovascular endothelial cells could reduce the protein expression of RhoA and ROCK₂,and promote the phosphorylation of RhoA and ROCK₂ in nerve cells.Conclusions:1.H₂S produced by CSE in primary rat cerebral vascular endothelial cells can protect primary rat hippocampal nerve cells from OGD/R injury.2.H₂S produced by CSE in cerebrovascular endothelial cells can inhibit the RhoA-ROCK signaling pathway by promoting the phosphorylation of RhoA and ROCK₂ proteins,and play a protective role in OGD/R injury of hippocampal nerve cells.