Endogenous H₂S Protects Brain From Ischemic Injury Via Inhibition Of The RhoA-ROCK Pathway And It-mediated Vasodilation Of Total Flavones Of Rhododendra

Stroke remains a global health burden,with approximately 13.7 million people suffering from stroke and its complications,such as decreased motor function and neurobehavioral changes.Stroke can be classified as ischemic or hemorrhagic,the latter being less common.An ischemic stroke is caused by a blood clot that forms in the arteries of the brain and can lead to cerebral infarction and neurological deficits.Although reperfusion increases blood supply to the ischemic area,it promotes oxidative stress and inflammation,leading to further neuronal cell death.This phenomenon is called ischemia/reperfusion injury（Ischemia/Reperfusion,I/R）.It is well documented that the inflammatory response and the oxidative stress that occurs after ischemia are regulated by RhoA（Ras homologous gene family,member A）.Activation of RhoA and its downstream effector ROCK（Rho-dependent coiled-coil kinase）induces phosphorylation of LIM kinase（LIMK）and actin depolymerizing factor cofilin（CFL）,thereby exacerbating neuroinflammation and cytotoxicity.For brain I/R injury,the change in cerebral blood vessel diameter is crucial because it can supply the blood flow and oxygen required by the brain tissue through the contraction and relaxation of vascular smooth muscle.In blood vessels,hydrogen sulfide（H₂S）can regulate vascular tension,inhibit proliferation,and have a bidirectional effect on apoptosis and autophagy of vascular smooth muscle cells（VSMCs）.It is an important gas signaling molecule in the body.It is mainly composed of cystathionine-γ-lyase（CSE）,cystathionineβ-synthetase（CBS）and3-mercaptopyruvate thiotransferase（3-MST）catalyzed generation.In addition,the development of many vascular remodeling-related diseases,including hypertension,atherosclerosis,and pulmonary hypertension,has been shown to be associated with impaired H₂S production.But does endogenous H₂S act through the RhoA-ROCK signaling pathway?There have been no detailed reports in this regard.With the continuous research on traditional Chinese medicine,the role of traditional Chinese medicine is found to be more and more extensive.Studies have shown that Total flavones of Rhododendra（TFR）have the effects of protecting brain damage and dilating blood vessels,but the mechanism of its vasodilating effect is not clear.Therefore,this topic established a model of cerebral ischemia-reperfusion to observe the protective effect of H₂S on brain injury in vivo and its relationship with the RhoA-ROCK signaling pathway.The role of endogenous H₂S in basilar artery vasodilation and its relationship with RhoA-ROCK signaling pathway were observed in vitro,and the relationship between TFR vasodilation and endogenous H₂S was also discussed.Aims1.To study the relationship between H₂S produced by CSE in increasing cerebral blood flow and resisting ischemic brain injury and the RhoA-ROCK signaling pathway.2.To study the relationship between endothelial-derived H₂S-mediated cerebral vasodilation and RhoA-ROCK signaling pathway.3.To study the relationship between TFR vasodilation and H₂S pathway.Method1.The mouse cerebral ischemia-reperfusion model was established by bilateral common carotid artery ligation.The changes of cerebral blood flow were monitored by laser speckle flowmeter before ischemia,after ischemia and within 20 minutes of reperfusion.Experimental grouping:WT mice were divided into sham operation group,model group,L-Cys group,Na HS group,PPG group,and PPG and L-Cys combination group,knockout mice were divided into sham operation group,model group,L-Cys group.2.To observe the effects of L-Cys and Na HS on the pathological changes of the hippocampus of brain tissue after cerebral ischemia-reperfusion by pathological section staining.3.The methylene blue method was used to determine the content of hydrogen sulfide in serum after cerebral ischemia-reperfusion injury.4.Detection of serum lactate dehydrogenase content after cerebral ischemia-reperfusion injury using a kit.5.The content of neuron-specific enolase,the activities of RhoA and ROCK₂ in brain tissue after cerebral ischemia-reperfusion injury were detected by enzyme-linked immunosorbent assay.6.Western Blot was used to determine the expression of ROCK₂ protein in brain tissue after cerebral ischemia-reperfusion injury.7.To observe the relaxation effect of rat cerebral basilar artery on ACh and TFR under different inhibitor pretreatment by isolated vascular ring experiment.Results1.The results of cerebral blood flow detection showed that in WT mice,compared with the sham-operated group,the blood flow of the model group decreased significantly within 20 minutes after cerebral I/R.Compared with the model group,the cerebral blood flow of the mice in the substrate L-Cys group and Na HS group recovered significantly at5 and 10 minutes of reperfusion,respectively,and basically returned to the level of the sham-operated group at 20 minutes.The CSE inhibitor PPG had no obvious effect on cerebral blood flow,but could significantly attenuate the recovery effect of L-Cys on blood flow.In ROCK₂ knockout mice,compared with the model group,the mice in the L-Cys group did not return to pre-ischemic levels within 20 min after reperfusion.The results suggest that L-Cys can promote the recovery of cerebral blood flow in mice with brain I/R injury through CSE,which is closely related to the presence of ROCK₂.2.Observation of pathological morphological changes in the hippocampus of the brain by pathological section staining The experimental results showed that in WT mice,the neurons in the hippocampus of the sham-operated group were larger and round,with obvious nucleoli.The cells are arranged in multiple layers,neat and dense.However,hippocampal neurons or pyramidal cells in the model group were triangular or irregular in shape,with shrunken cells with invisible nucleoli and disordered arrangements.Compared with the model group,the Na HS group and the L-Cys group could significantly improve the pathological damage,most of the nerve cells returned to normal,only a few nerve cells shrank,the neuron necrosis was improved,and the number of normal cells increased.The injury did not improve in the PPG group,but PPG reduced the protective effect of L-Cys.In ROCK₂ knockout mice,L-Cys did not improve the damage in the pathological area compared with the model group.The results showed that L-Cys could protect the I/R injury of hippocampal neurons in mouse brain tissue through CSE,and this effect was closely related to ROCK₂.3.By measuring the content of H₂S and the activity of LDH,the results showed that in WT mice,compared with the sham operation group,the content of H₂S in the serum of the model group was significantly decreased,while the activity of LDH was significantly increased.Compared with the model group,the content of H₂S in the serum of the Na HS group and the L-Cys group was significantly increased,and the LDH activity was significantly decreased.PPG had no significant effect on serum H₂S content and LDH activity,but could significantly attenuate the effect of L-Cys on increasing serum H₂S content and reducing LDH activity.In ROCK₂ knockout mice,compared with the model group,the serum H₂S content in the L-Cys group did not increase,and the LDH activity did not change.It is indicated that L-Cys can inhibit the decrease of H₂S and the increase of LDH in the serum of mice induced by brain I/R injury by producing H₂S through CSE,and this effect is closely related to ROCK₂.4.By measuring the activities of NSE,RhoA and ROCK₂,the results showed that in WT mice,compared with the sham-operated group,the NSE content in the brain tissue of the model group was significantly decreased,and the activities of RhoA and ROCK₂were significantly increased.Compared with the model group,the content of NSE in the Na HS group and the L-Cys group was significantly increased,and the activities of RhoA and ROCK₂ were decreased.PPG had no effect on the activities of NSE,RhoA and ROCK₂ in brain tissue,but could significantly attenuate the effect of L-Cys on the I/R injury-induced reduction of NSE content and the increase of RhoA and ROCK₂ activities.In ROCK₂ knockout mice,the activities of NSE,RhoA and ROCK₂ in the brain tissue of the L-Cys group were not significantly different from those of the model group.The results showed that L-Cys could reduce NSE and increase the activities of RhoA and ROCK₂ in brain tissue caused by I/R injury in mice by producing H₂S from CSE,and this effect was closely related to ROCK₂.5.Western Blot method to determine the expression of ROCK₂ protein The experimental results showed that in WT mice,compared with the sham operation group,the expression of ROCK₂ protein in the brain tissue of the model group was significantly increased,and Na HS and L-Cys could significantly reduce the expression of ROCK₂protein.Elevation of ROCK₂ protein expression induced by mouse brain I/R.PPG had no significant effect on the expression of ROCK₂ protein,but could significantly attenuate the effect of L-Cys on the increase of ROCK₂ protein expression induced by I/R injury.The results showed that L-Cys could inhibit the increase of ROCK₂ protein expression in mouse brain tissue induced by I/R injury through the production of H₂S by CSE.6.The results of the isolated vascular ring experiment showed that ACh in the range of1×10^-7 mol·L^-1～1×10^-5 mol·L^-1 could significantly and concentration-dependently relax the basilar artery of the rat brain,and could be significantly attenuated by the CSE inhibitor PPG(1×10^-4 mol·L^-1)pretreatment,but not affected by the 3-MST inhibitor Asp(1×10^-3 mol·L^-1)pretreatment,and PPG and Asp themselves did not change the diameter of blood vessels within a certain concentration range.The RhoA inhibitor CCG-1423 and the ROCK₂ inhibitor KD-025 can relax the blood vessels pre-constricted by KCl to different degrees within a certain concentration range,and CCG-1423(1×10^-6 mol·L^-1)and KD-025(1×10^-7 mol·L^-1)pretreatment can significantly attenuate the ACh-induced relaxation of the cerebral basilar artery in rats.CCG-1423 and KD-025 combined with PPG pretreatment can further attenuate the ACh-induced vasodilation.Relaxation effect,but pretreatment with Asp did not further attenuate the relaxation effect of ACh.TFR can dilate the cerebral basilar artery in rats,up to 70%.The CSE enzyme inhibitor PPG pretreatment can significantly reduce the relaxation effect of TFR,while the 3-MST inhibitor Asp does not significantly reduce the relaxation effect of TFR.Combined use of PPG and Asp can further reduce the relaxation effect of TFR.Conclusion1.H₂S produced by CSE has a protective effect on brain I/R injury in mice,and its effect may be related to inhibiting RhoA-ROCK signaling pathway and increasing cerebral blood flow.2.Endothelial-derived H₂S vasodilation is related to the inhibition of RhoA-ROCK signaling pathway.3.The role of TFR in relaxing cerebral blood vessels is related to the activation of endogenous H₂S production.