The Role Of S-sulfhydration Of RhoA Kinase In The Neuroprotection Of Exogenous And Endothelial Hydrogen Sulfide Against Hypoxic Injury

Background:Cerebral ischemia-reperfusion injury is one of the main diseases with high disability rate and high fatality rate in clinical practice.Hydrogen sulfide（H₂S）is an considerable gas signal numerator in the airframe.It Involves a lot of physiology and pathology processes,and has the influences of anti-neuronal cell apoptosis,anti-inflammatory response,and anti-oxidative stress.The latest research shows that H₂S can modify the cysteine residues of the target protein with S-sulfhydration,which can change the spatial conformation of the goal protein,so as to realize the target of regulating the vigor and effect of the protein.RhoA and its below acting molecule Rho related coiled coil protein kinase（ROCK）are concerning to cell growing,mobile,death and gene utterance,and are of good meaning in life events.Researchs have found that the Rho/ROCK₂ semaphore route is involved in cerebral brain ischemic damage.ROCK is a member of the serine and threonine albumen enzyme clan.There are two genre of ROCK1 and ROCK2.ROCK2 is mainly expressed in the brain,heart and muscle.Studies have shown that ROCK₂activation promotes ischemic neuronal damage,while inhibiting ROCK₂activity or reducing ROCK₂ protein expression can reduce neuronal damage after ischemia-reperfusion.Given that the cysteine-rich zine finger-like motifdomain（CRD）in the molecular structure of ROCK₂ is rich in cysteine residues,This study intends to investigate the ROCK₂ S-sulfhydration mechanism of the protective effect of exogenous and endothelial H₂S on rat nerve cels from H/R harm,for the purpose of supply new belief and means for the therapy of related illness caused by brain ischemic damage.Purpose:1.To study the effect of H₂S on the S-sulfhydration modification of ROCK₂ in nerve cels.2.To study the relationship between exogenous and endothelial-derived H₂S protection of nerve cels from H/R injury and S-sulfhydration modification.Method:1.To study the effect of exogenous H₂S on the S-sulfhydration and activity of ROCK₂in rat hippocampal neurons,Na HS was used as the exogenous H₂S donor.1.1 Primary cultured rat hippocampal nerve cels were divided into four groups after immunofluorescence identification,（1）Control group;（2）Na HS(50μmol·L^-1)group;（3）Na HS(100μmol·L^-1)group;（4）Na HS(200μmol·L^-1)group.1.2 The Modified biotin switch method was used to purify the S-sulfhydration protein in the nerve cels of the co-culture system,and the Western blot method was used to detect the expression of ROCK₂protein and S-sulfhydration modified ROCK₂（SSH-ROCK₂）protein,and the kit detects the activity of ROCK₂ and S-sulfhydration ROCK₂.2.To study the effect of endothelial-derived H₂S on ROCK₂ S-sulfhydration and activity of rat hippocampal nerve cels,primary culture of CSE gene knockout and wild-type mouse brain vascular endothelial cels（EC）,rat hippocampal nerve cels（NC）,After identification,the two kinds of cells were co-cultured using Transwell co-culture system.Add acetylcholine（ACh）,2-methoxy-N-acetoacetylaniline（o-Acetoacetaniside,AOAA）and L-aspartic acid（L-Asp）.2.1 The two kinds of cells were divided into two groups after co-culture,（1）NC+EC^CSE-WTgroup;（2）NC+EC^CSE-KO group.（ACh,AOAA,L-Asp were added to each group）The modified biotin conversion method was used to purify the S-sulfhydration protein in the nerve cels of the co-culture system.The Western blot method was used to detect the expression of ROCK₂ protein and S-sulfhydration modified ROCK₂ protein.The kit was used to detect the activity of ROCK₂ and S-sulfhydration modified ROCK₂,and the cell H₂S content was detected by methylene blue method.3.The effect of S-sulfhydration modification on the protection of H/R injury of rat hippocampal neurons by Na HS3.1 Primary cultured rat hippocampal nerve cells,constructed a hypoxia/reoxygenation（H/R）model and divided into seven groups,namely:（1）Control group;（2）H/R group;（3）Na HS(50μmol·L^-1)group;（4）Na HS(100μmol·L^-1)group;（5）Na HS(200μmol·L^-1)group;（6）inhibitor DTT(50μmol·L^-1)group;（7）Na HS(200μmol·L^-1)+DTT group.3.2 After 4 hours of hypoxia/12 hours of reoxygenation,the cell viability was tested by CCK-8 kit,and the activity of lactate dehydrogenase（LDH）and neuron-specific enolase（NSE）in the culture supernatant was detected by the kit.Flow cytometry was used to detect cell apoptosis.4.To study the effect of S-sulfhydration modification on the protection of H/R injury of hippocampal neurons by endothelial H₂S4.1 After constructing the cellular H/R model,the experiment was divided into five groups:（1）Control group;（2）H/R group;（3）NC+EC^CSE-WT group;（4）NC+EC^CSE-KO group;（5）Inhibitor DTT(50μmol·L^-1)group;（6）NC+EC^CSE-WT+DTT group.（All groups have joined ACh,AOAA,L-Asp）4.2 After 4 hours of hypoxia/12 hours of reoxygenation,the cell viability was tested by CCK-8 kit,and the activity of lactate dehydrogenase（LDH）and neuron-specific enolase（NSE）in the culture supernatant was detected by the kit.Flow cytometry was used to detect cell apoptosis.Results:1.The effect of exogenous H₂S on ROCK₂ S-sulfhydration and activity of rat hippocampal neurons1.1 Compared with the NS control group,Na HS(100,200μmol·L^-1)can significantly reduce the expression of ROCK₂ protein in rat hippocampal neurons（P<0.01）;Na HS(200μmol·L^-1)can significantly reduce the hippocampus ROCK₂ activity in nerve cels（compared with NS control group,P<0.01）.After biotinylation treatment,each group of S-sulfhydration modified proteins was obtained,and the ratio of the amount of S-sulfhydration modified ROCK₂ protein and total ROCK₂ protein was used to quantitatively analyze the influence of Na HS on S-sulfhydration modified ROCK₂ protein,compared with the NS control group,Na HS(100,200μmol·L^-1)can significantly increase the content of SSH-ROCK₂ protein in hippocampal neurons（P<0.01）.The total ROCK₂ protein in the control group and the S-sulfhydration ROCK₂ protein activity in the Na HS-treated group were measured.For the sake of comparison,the total ROCK₂ protein content in the total protein in the control group and the S-sulfhydration protein content in the Na HS-treated group were determined by Western blot method.After adjusting the total ROCK₂ protein content in the control sample to be consistent with the S-sulfhydration ROCK₂ protein content in the Na HS-treated group,the ROCK₂ protein activity was determined.The results showed that the SSH-ROCK₂ activity in the Na HS(200μmol·L^-1)treatment group was significantly lower than the ROCK₂ activity in the control group,suggesting that ROCK₂ S-sulfhydration modification can significantly reduce its activity.2.The effect of endothelial-derived H₂S on ROCK₂ S-sulfhydration and activity of rat hippocampal neuronsCompared with the co-culture group of nerve cels and wild-type mouse endothelial cells(NC+EC^CSE-WT),the co-culture group of nerve cels and CSE knockout mouse endothelial cels(NC+EC^CSE-KO)can significantly reduce the expression of ROCK₂ protein in rat hippocampal nerve cels（P<0.01）;The NC+EC^CSE-KO co-culture group can significantly increase the ROCK₂ activity and H₂S content in hippocampal neurons(compared with the NC+EC^CSE-WT co-culture group,P<0.01);after biotinylation,the S-sulfhydration modification of each group can be obtained Compared with NC+EC^CSE-WT,NC+EC^CSE-KOcan significantly reduce the content of SSH-ROCK₂ protein in primary cultured rat hippocampal neurons（P<0.01）;The total ROCK₂ protein in the control group and the SSH-ROCK₂ protein activity in the NC+EC^CSE-WT group were determined.For the sake of comparison,the total ROCK₂ protein content in the total protein in the control group and the NC+EC^CSE-WT co-culture group were determined by Western blot.After adjusting the content of thiolated protein in the control group,the total ROCK₂ protein content in the control group was adjusted to be consistent with the thiolated ROCK₂ protein content in the NC+EC^CSE-WTco-culture group,and then the ROCK₂ protein activity was determined.The results showed that the activity of SSH-ROCK₂ in the cels of the NC+EC^CSE-WT group was significantly lower than the activity of ROCK₂ in the cells of the control group,suggesting that ROCK₂S-sulfhydration modification can significantly reduce its activity.3.The effect of S-sulfhydration modification on the H/R injury of Na HS in protecting rat hippocampal neuronsCompared with the normal control group,the cell viability of the H/R model group was significantly reduced,the LDH and NSE activities in the cell supernatant were significantly increased,and the apoptosis rate was increased（P<0.01）.The administration of exogenous H₂S donor Na HS(100,200μmol·L^-1)can significantly increase the decrease in cell viability caused by H/R damage and can significantly reduce the leakage of LDH and NSE from nerve cels caused by H/R,and significantly reduce the increase of apoptosis rate caused by H/R injury（P<0.01,compared with model group）;Na HS(50μmol·L^-1)can also significantly reduce the NSE activity in the culture supernatant of nerve cels after H/R.The S-sulfhydration modification blocker DTT alone has no significant effect on the reduction of H/R injury-induced rat hippocampal neuronal cell viability,the increase of LDH and NSE activities in the cell supernatant,and the apoptosis rate,but the combined application of DTT significantly block the inhibitory effect of Na HS(200μmol·L^-1)on the reduction of cell viability induced by H/R injury and the increase of LDH and NSE activities in the supernatant,and significantly reduce the effect of Na HS(200μmol·L^-1)on H/R Inhibition of the increased apoptosis rate of hippocampal neurons induced by injury in rats(compared with 200μmol·L^-1 Na HS group,P<0.01).4.To study the effect of S-sulfhydration modification on the protection of H/R injury of hippocampal neurons by endothelial H₂SCompared with the control group,the cell viability of the H/R model group was significantly reduced,the LDH and NSE activities in the supernatant were significantly enhanced,and the apoptosis rate was increased（P<0.01）;And endothelial-derived H₂S donors,namely the co-culture group of nerve cels and wild-type mouse endothelial cels(NC+EC^CSE-WT)and the co-culture group of nerve cels and CSE knockout mouse endothelial cels(NC+EC^CSE-KO)can significantly increase the decrease in cell viability caused by H/R damage,reduce the leakage of LDH and NSE in the cell supernatant caused by H/R damage,and significantly reduce the increase in apoptosis rate caused by H/R damage（P<0.01,Compared with the model group）;The S-sulfhydration modification blocker DTT alone has no significant effect on the decrease of H/R injury-induced rat hippocampal neuronal cell viability,the increase of LDH and NSE activity,and the apoptosis rate,but DTT significantly blocks the combined application The inhibitory effect of the NC+EC^CSE-WT group on the decrease of cell viability induced by H/R injury,the increase of LDH and NSE activity,and the increase of the apoptosis rate of rat hippocampal neurons(compared with the NC+EC^CSE-WT group,P<0.01).Conclusion:1.Exogenous and endothelial-derived H₂S can modify ROCK₂ in rat hippocampal neurons by S-sulfhydration.2.Exogenous and endothelial-derived H₂S has a significant protective effect on H/R injury of rat hippocampal neurons.The mechanism is related to the decrease of ROCK₂ activity and the decrease of ROCK₂ protein expression caused by S-sulfhydration.