Hydrogen Sulfide Attenuates Endothelial Cell Hypoxia-Reoxygenation Injury Through Upregulation Of ACE2

Background:The morbidity and mortality rates of myocardial infarction（MI）remain high internationally.Currently,the main clinical treatment is percutaneous coronary intervention（PCI）,which can restore blood flow to the epicardial coronary arteries,thus reducing the mortality of patients.However,there is still a significant proportion of patients in whom myocardial injury worsens after restoration of perfusion,which is called"Myocardial ischemia-reperfusion injury（MIRI）".Angiotensin converting enzyme2（ACE2）,the main active substance in renin angiotensin system（RAS）,hydrolyzes angiotensin II（Ang II）to produce angiotensin（1-7）[Ang（1-7）],and then Ang（1-7）acts on Mas receptor and subsequently produces vasodilatory,anti-oxidative stress,and anti-inflammatory effects through various signal transduction pathways.Hydrogen sulfide（H₂S）plays an important role in cell protection,inflammation,vascular function,nervous system,tissue repair and healing,apoptosis and cell cycle,mitochondrial function and energy metabolism.Previous studies by our group have confirmed that exogenous H₂S can reduce H/R injury in endothelial cells,but whether this is achieved by regulating ACE2 has not been reported.Therefore,the purpose of this experiment is to further investigate the protective mechanism of H₂S on H/R endothelial cells and provide new ideas for the prevention and treatment of MIRI in clinical practice.Objective:To investigate the mechanism of the effect of hydrogen sulfide on hypoxia-reoxygenation injury in endothelial cells and its correlation with ACE2.Methods:1.To investigate the mechanism by which H2S attenuates hypoxia-reoxygenation injury in HUVECs,firstly,a model of endothelial cell hypoxia-reoxygenation injury was established,and cells were treated with different concentrations of Na HS and a blank control was set up,followed by the detection of cell viability and cell injury level using CCK-8 and LDH kits,respectively,to determine the optimal Na HS treatment concentration;secondly,cells were treated with Na HS and/or PPG,followed by the detection of vasodilator level,inflammation level and reactive oxygen species using NO kit,ELISA kit and DHE fluorescence Probe reagent assays were used to detect vasodilator levels,inflammation levels and reactive oxygen species levels,respectively.2.To investigate whether hypoxia-reoxygenation affects the protein expression level of ACE2 in HUVECs,the cells were randomly divided into blank control and hypoxia-reoxygenation groups,and the levels of Ang II and Ang（1-7）were detected by ELISA kits,and the protein expression level of ACE2 was detected by Western blot.3.To investigate the effect of H2S on ACE2 protein expression level in HUVECs,we investigated the effect of Na HS concentration on ACE2 protein expression level in HUVECs,the effect of treatment time of Na HS on ACE2protein expression level in HUVECs,the effect of Na HS concentration on ACE2protein expression level in hypoxic-reoxygenated HUVECs and H2S on ACE2protein expression level in hypoxic-reoxygenated HUVECs,ELISA kits were used to detect the levels of Ang II and Ang（1-7）in the supernatants of different groups of cell cultures,and Western blot was used to detect the protein expression levels of ACE2 in different groups.4.To investigate the role of ACE2 in H2S attenuating hypoxia-reoxygenation injury in HUVECs,we used Na HS and/or DIZE and/or MLN-4760 to treat the cells,followed by NO kit to detect the levels of NO in cell culture supernatant,ELISA kit to detect the levels of ET-1,IL-1β,IL-6,TNF-αAng II and Ang（1-7）in cell culture supernatant,DHE fluorescent probe reagent to detect the level of ROS in HUVECs,and Western blot to detect the protein expression level of ACE2 in HUVECs.Results:1.（1）CCK-8 and LDH results showed that cell viability was significantly weakened（P<0.01）and LDH activity was significantly increased（P<0.01）in the H/R group compared with the blank control group;cell viability was significantly enhanced（P<0.05）and LDH activity was significantly decreased（P<0.05）in the 100μM,200μM and 400μM groups compared with the H/R group.（2）Compared with the H/R group,the cell culture supernatant in the Na HS+H/R group showed a significant increase in NO content（P<0.05）,a significant decrease in ET-1 content（P<0.01）,a significant decrease in ROS fluorescence intensity（P<0.01）,and a significant decrease in IL-1β,IL-6 and TNF-αcontent（P<0.05）.In contrast,the PPG+H/R group had significantly reduced NO content（P<0.05）,significantly increased ET-1 content（P<0.05）,significantly enhanced ROS fluorescence intensity（P<0.01）,and significantly increased IL-1β,IL-6,and TNF-αcontent（P<0.05）.And the re-addition of Na HS to the PPG+H/R group reversed this series of phenomena.The above results suggest that H₂S attenuates endothelial cell H/R injury and its protective effects are related to the correction of the imbalance of vasodilatory factors and the inhibition of oxidative stress and inflammatory responses.2.Compared with the Control group,the Ang II content in the culture supernatant of the H/R group was significantly increased（P<0.01）,but the Ang（1-7）content was significantly decreased（P<0.01）,while the protein expression level of ACE2 was significantly decreased（P<0.01）.The results indicate that H/R could reduce the protein expression of ACE2 in HUVECs,which in turn altered the levels of Ang II and Ang（1-7）.3.（1）In a certain Na HS concentration range（200-400μM）,Ang II content in culture supernatant was significantly reduced（P<0.01）,while Ang（1-7）content was significantly increased（P<0.01）,and ACE2 protein expression levels were significantly increased in the 200μM and 400μM groups（P<0.05）.（2）Treatment with 200μM Na HS for a range of time（6-12h）resulted in a significant decrease in Ang II content in culture supernatant（P<0.05）,along with a significant increase in Ang（1-7）content（P<0.01）and a significant increase in ACE2 protein expression level（P<0.05）.（3）In the H/R condition in a certain Na HS concentration range（200-400μM）,Ang II content in culture supernatant was significantly reduced（P<0.01）,while Ang（1-7）content was significantly increased（P<0.01）,and ACE2 protein expression level was significantly increased in the 200μM group（P<0.05）.（4）Compared with the H/R group,the Na HS+H/R group showed a significant decrease in Ang II content（P<0.01）and a significant increase in Ang（1-7）content（P<0.01）,as well as a significant increase in ACE2 protein expression level（P<0.05）.In contrast,the PPG+H/R group showed a significant increase in Ang II content（P<0.05）,Ang（1-7）content（P<0.01）and ACE2 protein expression levels were significantly reduced（P<0.05）.And the re-addition of Na HS to the PPG+H/R group reversed this series of phenomena.The above results indicate that H₂S can affect the expression of ACE2in endothelial cells in different states,and thus alter the content of Ang II and Ang（1-7）.4.（1）Compared with the H/R group,the DIZE+H/R and Na HS+H/R groups showed a significant decrease in Ang II content（P<0.01）,a significant increase in Ang（1-7）content（P<0.01）,and a significant increase in ACE2 protein expression level（P<0.01）,while the MLN-4760+H/R group showed a significant increase in Ang II content in the culture supernatant（P<0.01）,Ang（1-7）content was significantly decreased（P<0.01）,and ACE2 protein expression level was not significantly changed in the MLN-4760+H/R group.Compared with the Na HS+H/R group,the culture supernatant of the Na HS+DIZE+H/R group showed a significant decrease in Ang II content（P<0.01）,a significant increase in Ang（1-7）content（P<0.01）,and a significant increase in ACE2 protein expression level（P<0.01）,while the culture supernatant of the Na HS+MLN-4760+H/R group showed a Ang II content was significantly increased（P<0.01）,Ang（1-7）content was significantly decreased（P<0.01）,and ACE2 protein expression level was not significantly changed.（2）Compared with the H/R group,the DIZE+H/R and Na HS+H/R groups showed a significant increase in NO content（P<0.01）,a significant decrease in ET-1 content（P<0.01）,a significant decrease in ROS fluorescence intensity（P<0.05）,and a significant decrease in IL-1β,IL-6 and TNF-αcontent（P<0.05）,while the MLN-4760+H/R group showed a significant decrease in NO content（P<0.01）,a significant increase in ET-1 content（P<0.05）,a significant increase in ROS fluorescence intensity（P<0.01）,and a significant increase in IL-1β,IL-6 and TNF-αcontent（P<0.01）.Compared with the Na HS+H/R group,the Na HS+DIZE+H/R group showed a significant increase in NO content（P<0.01）,a significant decrease in ET-1 content（P<0.01）,a significant decrease in ROS fluorescence intensity（P<0.05）,and a significant decrease in IL-1β,IL-6,and TNF-αcontent（P<0.05）,whereas the Na HS+MLN-4760+H/R group showed a significant increase in NO content（P<0.01）,a significant decrease in ET-1 content（P<0.05）,a significant decrease in ROS fluorescence intensity（P<0.05）,and a significant decrease in IL-1β,IL-6,and TNF-αcontent（P<0.05）.The above results suggest that the corrective vasodilator imbalance,antioxidant stress and anti-inflammatory effects of H₂S are dependent on ACE2 and that H₂S attenuates endothelial cell H/R injury by upregulating ACE2.Conclusion:1.H₂S attenuates endothelial cell hypoxia-reoxygenation injury and its protective effects are associated with correction of the imbalance of vasodilatory factors and inhibition of oxidative stress and inflammatory responses.2.The inhibitory effect of H₂S on hypoxia-reoxygenation injury in endothelial cells is achieved by upregulating the protein expression of ACE2.