Inhibition Of RhoA And ROCK Targets In Vasodilation Of Rats By H₂S And Its Follow-up Mechanism

Cerebrovascular disease is a common clinical disease and the main cause of deaths around the world,of which ischemic cerebrovascular disease is more common.However,the specific pathophysiology of its occurrence and development is still not very clear.Hydrogen sulfide?H₂S?is the third gas signal molecule found in the body after nitric oxide?NO?and carbon monoxide?CO?.Like NO,endogenous H₂S can also be produced and released by vascular endothelial cells,regulating vascular tone and organ blood flow.Rho A-ROCK signaling pathway participates in the contraction of smooth muscle cells in the vascular bed,plays an important role in the regulation of vascular tone,and is a new target of the cardiovascular system.Both small G protein Rho A and ROCKs play an important role in cardiovascular pathogenesis,and ROCK can also be expressed on vascular smooth muscle.ROCKs is the main effector of small guanosine binding protein Rho A.At the same time,some studies show that rocks are expressed in vascular endothelial cells and smooth muscle cells,ROCK has two subtypes,ROCK₁ and ROCK₂.They have some homology in amino acid sequence and kinase domain.At rest,Rho A is located in the cytoplasm and can be transferred to the cell membrane after activation.Subsequently activate the downstream ROCK,phosphorylate MBS of MLCP,and inactivate MLCP.Phosphorylated p-MLC cannot be dephosphorylated,and ROCK also directly phosphorylates MLC,increasing p-MLC levels,enhancing smooth muscle contractility,and eventually causing arterial spasm reactions,thereby causing a series of cardiovascular diseases.We have previously studied the role of H₂S-Rho A-ROCK signaling pathway in regulating the tension of the basilar artery in mice by CSE knockout model.The results show that H₂S may induce the vasodilation by inhibiting Rho A-ROCK signaling pathway.However,the exact target of Rho A ROCK inhibition is not clear.That is whether H₂S inhibits Rho A,ROCK,and whether it is selective for its subtype.At the same time,the effects on the expression of p-MLC and intracellular free Ca²⁺were also studied.Therefore,we studied the inhibition of Rho A and ROCK targets and its follow-up mechanism in the vasodilation of rats by H₂S.Purpose:1. To study the relationship between Rho A and ROCK in cerebrovascular function of H₂S relaxation rats.2.To study the relationship between the effect of H₂S relaxation rat cerebral vascular smooth muscle cells and Rho A and ROCK.3.To study the effect of H₂S inhibition of Rho A and ROCK on p-MLC protein expression and intracellular free Ca²⁺.Methods:1. Using si RNA in vivo interference technology to establish a transfection model to reduce the expression of ROCK₁and ROCK₂ proteins in cerebral blood vessels.2. In vitro microvascular pressure diameter measurement was used to detect the dilation of rat basilar artery.3. Primary culture and identification of rat cerebral vascular smooth muscle cells.4. Computer image analysis system to measure the relaxation amplitude of smooth muscle cells5. The Elisa kit detects ROCK₁ and ROCK₂ activity.6. Calcium ion imaging system for measuring calcium ion fluorescence intensity in smooth muscle cells7. Western-Blot was used to detect the effect of H₂S on the phosphorylation expression of ROCK downstream protein myosin light chain.Results:1.In the range of 1×10^-6 to 1×10^-3 mol/L,Na HS induced concentration-dependent relaxation of rat brain basal in a concentration-dependent manner,but this relaxation can be induced by 1×10^-5 mol/L Rho A inhibitor C3 Transferase pretreatment was significantly weakened.ROCK₁-si RNA or ROCK₂-si RNA transfection in vivo can significantly attenuate ROCK₁ or ROCK₂ expression in rat basilar artery.In rat brain basilar artery with knockdown of ROCK₁ or ROCK₂ expression,Na HS-induced vasodilation is significantly reduced.2.Compared with the control group,Na HS?100?mol/L?induces relaxation of rat basal artery smooth muscle cells after reaching a certain concentration.However,this relaxation can be attenuated by 1?g/ml C3 transferase pretreatment.Pretreatment with Y27632 at 10?mol/l also attenuated the diastolic effect of Na HS.At the same time,under the condition of 30 mmol/L KCl preshrinking,C3 transferase pretreatment group was larger than Y27632 pretreatment group in reducing the diastolic amplitude of Na HS to rat basilar artery smooth muscle cells.3.Compared with the control group,exogenous H₂S?Na HS:100?mol/L?can significantly reduce the activity of ROCK₁ or ROCK₂.After treatment with C3 transferase alone,it can also reduce the activity of ROCK₁ or ROCK₂.Compared with the Na HS group,pretreatment with C3 transferase and then adding Na HS will reduce the activity of ROCK₁ and ROCK₂.4.Compared with the control group,exogenous H₂S?Na HS:100?mol/L?caused a significant decrease in p-MLC protein expression in rat basilar artery smooth muscle cells.Pretreatment with C3 transferase or ROCK inhibitor Y27632 can significantly inhibit Na HS-induced decrease in p-MLC protein expression.5.Compared with the control group,exogenous H₂S?Na HS:100?mol/L?can significantly attenuate the fluorescence intensity of Ca²⁺in rat brain base artery smooth muscle cells.Compared with the Na HS group,the pretreatment with C3 transferase or Y27632 can make Na HS reduce the degree of decrease in the Ca²⁺ fluorescence intensity in the rat basilar artery smooth muscle cells.Conclusions:1.H₂S acts on Rho A,ROCK₁ and ROCK₂ to cause Rho A-ROCK signaling pathway inhibition play the role of dilation of rat cerebrovascular.2.H₂S leads to the inhibition of Rho A-ROCK signaling pathway by acting on Rho A and ROCK,thus exerting the effect of relaxing rat cerebral vascular smooth muscle cells.3.Inhibition of Rho A and rock can reduce the effect of H₂S on protein expression and intracellular Ca²⁺content of p-MLC.