The Regulation Of H₂S On The Phosphorylation Of Ser188 Of RhoA And Its Protective Effect On Hypoxic/ischemic Brain Damage In Rats

BackgroundStroke is one of the most common causes of death and the main cause of persistent and acquired disability in the world.With population changes,the morbidity is further increasing,which brings a huge burden to people’s quality of life.Stroke is divided into hemorrhagic stroke and ischemic stroke,the latter is more common.The current treatment focuses on rapid reperfusion through intravenous thrombolysis and intravascular thrombectomy.However,reperfusion can also damage brain tissue.The available treatments for stroke are extremely limited.Research on prevention strategies is moving towards the forefront of stroke management.Some new developments have brought new hopes for neuroprotection.Ras homologous family member A（Rho A）is a widely distributed cytoplasmic protein belonging to the small GTPases family.Guanosine triphosphate hydrolase（Rho GTPases）constitutes a fairly universally expressed cytoplasmic molecular switch that controls the activity of a series of downstream effectors,Rho-dependent coiled-coil kinase（ROCK）is the most direct and main effector molecule of Rho A,and it has two subtypes:ROCK₁ and ROCK₂.Ischemic brain injury can induce Rho A activation and significant up-regulation of ROCK₂ expression.More and more evidences show that Rho A/ROCK pathway is involved in the pathological process of some diseases of the central nervous system.The phosphorylation of Rho A is one of the main ways to change its function and regulate its activity.Phosphorylation of Rho A at Ser188 can inhibit Rho A activation.Hydrogen sulfide（H₂S）is a ubiquitous second messenger molecule that acts as a neuromodulator.It has important functions in many mammalian organs and systems and participates in the physiological and pathological mechanisms of the brain.Endogenous H₂S synthesis enzymes mainly include cystathionine-γ-lyase（L-cystathionine-γ-lyase,CSE）,cystathionine-β-synthetase（l-cystathionine-β-synthetase,CBS）and 3-Mercaptopyruvate sulfurtransferase（3-mercaptopyruvate sulfurtransferase,3-MST）.Our previous studies have proved that hydrogen sulfide（H₂S）can promote the opening of KCa channels in rat cerebral vascular smooth muscle cells.H₂S produced by CSE has a protective effect on cerebral ischemia-reperfusion（I/R）injury in mice,but its effect The molecular mechanism is not clear.Earlier researchers reported that the brain endothelial/neurovascular unit（NVU）plays an important role.The autoregulation of cerebrovascular is very important to protect nerve cells from ischemic damage.The remodeling of ischemic brain tissue involves both neurons and microvascular cells.The interaction between the neurovascular units and the mechanism involved in the ischemic death of neurovascular units need to be understood in depth.Based on the above research background,we have done the following research:1.Construct and express Glutathione S-transferase（GST）tagged Rho A wild-type(GST-Rho A^wild)and Rho A Ser188 point mutation(GST-Rho A^S188A)proteins,and observe the effect of exogenous H₂S（Na HS）on its phosphorylation in vitro.2.To explore the target and mechanism of H₂S inhibiting the Rho A/ROCK pathway,and to study the effect of H₂S on promoting the phosphorylation of Rho A Ser188 on hippocampal neuronal hypoxia/reoxygenation injury and cerebral ischemia/reperfusion injury in rats.Purpose1.Observe the regulation of H₂S on the phosphorylation of Ser188 of Rho A;2.To explore the effect of H₂S on the Rho A Ser188 site on hippocampal neurons and its potential H/R injury protection mechanism;3.To explore the effect of H₂S at Rho A Ser188 site on the protection of ischemic brain injury in rats.Methods1.Construct Rho A^wild-p GEX-6p-1 and Rho A ^S188A-p GEX-6p-1 prokaryotic plasmids,induce expression and purification of GST-Rho A^wild and GST-Rho A^S188A proteins,and perform in vitro phosphorylation assays.2.Recombinant Rho A^wild-p EGFP-N1 and Rho A^S188A-p EGFP-N1 eukaryotic plasmids were constructed,and rat primary hippocampal neural cells（HNCs）were cultured and identified,and they were electrotransfected into HNCs,after adding different concentrations of Na HS,the expression levels of Rho A Ser188 phosphorylated protein and its membrane protein and ROCK₂ protein were determined by western blot,and the activity of Rho A and ROCK₂ was determined by enzyme-linked immunoassay（ELISA）.3.Whole-cell patch clamp records the changes of Na HS on the large-conductance calcium-activated potassium channel（BKCa）channel current of neurons transfected with the recombinant plasmid.4.Establish a hypoxia-reoxygenation injury model（Hypoxia-Reoxygenation,H/R）,and use a cell counting kit（CCK-8）to detect changes in nerve cell viability after transfection of recombinant plasmids,lactate dehydrogenase（LDH）,and neurospecific enolase（NSE）Release changes to assess damage.5.Culture and identification of primary rat brain vascular endothelial cells（ECs）,primary endothelial cells of CSE^-/-mice and 3-MST^-/-rats are co-cultured with nerve cells,and acetylcholine（ACh）is added to stimulate the release of endothelial cells H₂S,the expression levels of Rho A Ser188 phosphorylated protein and its membrane protein and ROCK₂ protein,the activity of Rho A and ROCK₂,and the content of H₂S released by the two endothelial cells were measured.6.The Rho A^wild-p EGFP-N1 and Rho A^S188A-p EGFP-N1 eukaryotic plasmids were transfected into HNCs and co-cultured with CSE^-/-ECs.Acetylcholine（ACh）was added to stimulate endothelial cells to release H₂S and cause H/R damage.Measure the expression levels of Rho A Ser188 phosphorylated protein and its membrane protein and ROCK₂ protein,measure the activity of Rho A and ROCK₂,and the changes of CCK8,LDH,and NSE.7.Hoechst 33258 staining,observation of nerve cell nuclear damage under a fluorescence microscope,Annexin V/PI staining to detect apoptotic cells by flow cytometry.8.Ca²⁺imaging system fluorescence microscope（Olympus IX73）detects the content of Ca²⁺in nerve cells and obtains fluorescence images of intracellular Ca²⁺signals.9.The brain was stereotactically transfected with Rho A^wild-p EGFP-N1 and Rho A^S188A-p EGFP-N1 eukaryotic plasmids into rat hippocampus.The expression of Rho A^wild and Rho A^S188A protein was detected by western blot,and the transfection result was observed under a fluorescence microscope on frozen sections.10.Bilateral common carotid artery ligation with 2VO is used to establish a cerebral ischemia-reperfusion model（I/R）in successfully transfected rats.Laser speckle imaging（LSI）is used to detect changes in blood flow and observe whether the model is successful or not.After 24 hours of ischemia-reperfusion,hematoxylin-eosin（HE）staining was used to observe the damage of hippocampus,TUNEL to detect the apoptosis of hippocampal tissues,western blot to detect the expression levels of Rho A Ser188 phosphorylated protein and ROCK₂ protein,and to determine the activity of Rho A and ROCK₂,And changes in LDH and NSE.Results1.Results of autoradiography showed that H₂S donor Na HS（100μmol/L）significantly promoted the phosphorylation of GST-Rho A^wild protein in the presence of PKG1,there was no phosphorylation of GST-Rho A^S188A protein.These results indicated that Na HS could promote phosphorylation of Rho A,and Ser188 is required for the promoted phosphorylation.2.Exogenous H₂S（Na HS）and Endothelial-derived CSE-produced H₂S promoted phosphorylation of Rho A in the neurons transfected with empty or GFP-Rho A^wildor GFP-Rho A^S188Aplasmids as well as phosphorylation of GFP-Rho A^wildin the GFP-Rho A^wild plasmids-transfected neurons,p-Rho A/Rho A ratio or p-GFP-Rho A^wild/GFP-Rho A^wildratio of increased markedly compared with those in the control group.But had no effect on phosphorylation of GFP-Rho A^S188A,p-GFP-Rho A^S188A/GFP-Rho A^S188A ratio in the GFP-Rho A^S188Aplasmid-transfected neuron did not significantly change in comparison with that in the control group.These results suggested that H₂S could induce Rho A phosphorylation in rat hippocampal neurons,and the phosphorylation was mediated by Rho A Ser188.3.H₂S significant decreases of the locations of Rho A and GFP-Rho A^wild in the plasma membrane,but increases of their location in the cytosol.However,had no obvious effect on location of GFP-Rho A^S188Ain the plasma membrane and the cytosol.These results indicated that the Ser188 of Rho A is required for the H₂S-caused translocation of Rho A from plasma membrane to cytosol in the neuron and suggested that H₂S could inhibit Rho A activity through Ser188.4.The increase of Na HS on the BKCa channel current of the nerve cells transfected with GFP-Rho A^S188A plasmid is significant lower than that of the empty plasmid and GFP-Rho A^wild plasmid group（30 m V-70 m V）.These results indicate that Na HS has an effect on the BKCa channel current.Increase and activation are regulated by Rho A Ser188.5.H₂S significantly inhibited the ROCK₂ protein expression and its activity in rat hippocampal neurons transfected with either empty plasmid or GFP-Rho A^wild plasmid.But in GFP-Rho A^S188A plasmid-transfacted neurons,this inhibition is reduced.These results indicated that Ser188 of Rho A is associated with the H₂S-induced inhibition of ROCK₂ protein expression and its activity in the neuron.6.In the empty plasmid or GFP-Rho A^wild plasmid-transfected neurons,H₂S significantly inhibited the decrease cell viability and the increased activities of LDH and NSE.However,those effects of H₂S were markedly reduced in GFP-Rho A^S188Aplasmid-transfected neurons,These results indicated that Rho A Ser188 mediated in protective effect of H₂S on H/R injury.7.H/R injury induced significant increases of apoptotic cells and intracellular free Ca²⁺fluorescence intensity in CSE^-/-EC-co-cultured neurons transfected with either empty plasmid or GFP-Rho A^wildplasmid or GFP-Rho A^S188Aplasmid.CSE^+/+EC could significantly decreased H/R injury-induced increase of apoptotic cells and intracellular free Ca²⁺fluorescence intensity in co-cultured neurons transfected with empty or GFP-Rho A^wild plasmids,but had no effect in co-cultured neurons transfected with GFP-Rho A^S188A plasmids.These results suggested that endothelial CSE-produced H₂S could protect the neuron from H/R injury via inhibiting increase of intracellular free Ca²⁺,and the effects were mediated by Rho A Ser188.8.In the rat hippocampus transfected with the empty plasmid or GFP-Rho A^wild plasmid plasmid,Na HS can promote the phosphorylation of Rho A,inhibit the activity of Rho A,and reduce the expression and activity of ROCK₂ at the same time,reduces the increase of LDH and NSE in serum induced by I/R injury,as well as the nerve cell damage and apoptosis in the hippocampus.However,these effects were significantly reduced in hippocampal tissues transfected with GFP-Rho A^S188A plasmid.These results indicate that Na HS reduces hippocampal I/R damage is related to Rho A Ser188.Conclusion1.Na HS can promote the phosphorylation of Rho A through Ser188 site.2.Na HS may increase the BKCa channel current in rat hippocampal neurons through Rho A Ser188.3.Exogenous and endogenous H₂S protects hippocampal neurons from H/R damage by promoting the phosphorylation of Rho A Ser188 in rat hippocampal neurons.4.Rho A Ser188 mediates the protective effect of H₂S on rat cerebral ischemia reperfusion injury.