The Endothelium-derived Cse-produced H₂S Mediates Total Flavones Of Rhododendra To Protect Brain From Cerebral Hypooxic-ischemic Injury Via Inhibition Of RhoA-ROCK Pathway In Neurons

The brain is an extremely sensitive organ to hypoxia.Ischemia can lead to a series of secondary reactions,brain tissue damage and brain function damage.The recovery of blood flow after ischemia will further aggravate the inflammatory reaction and then aggravate brain injury,also known as cerebral ischemia/reperfusion injury（I/R）.Therefore,it is particularly important to explore potential therapeutic drugs for cerebral ischemia reperfusion injury.We found that the total flavonoids of rhododendra（TFR）extracted from rhododendron plants has certain biological activities,and its main components include quercetin,hyperoside and other flavonoids,which have positive effects on cerebral ischemia reperfusion injury.Endogenous hydrogen sulfide（H₂S）is a gas signal molecule that can freely pass through the cell membrane.In blood vessels,it is mainly produced by L-cysteine catalyzed by CSE in endothelial cells.It has important effects such as anti-inflammatory,antioxidant stress,promoting angiogenesis and vasodilation.Rho A-ROCK pathway is a signal pathway related to cell growth,proliferation and differentiation in the body,and participates in the pathological mechanism of ischemic brain injury.Our previous studies have shown that TFR can inhibit Rho A-ROCK pathway by promoting H₂S production,but the exact mechanism is still unclear.Therefore,in this study,CSE gene knockout mice（CSE KO）and ROCK₂ gene semi-knockout mice（ROCK₂ HK）were used to explore the mechanism of TFR protecting cerebral ischemia-reperfusion injury by promoting CSE produced H₂S and inhibiting ROCK₂in vivo and in vitro.Purpose:1.To explore the effect of H₂S produced by vascular endothelium-derived CSE on the protection of TFR against cerebral ischemia-reperfusion injury in mice,and its relationship with Rho A-ROCK pathway.2.To observe the protective effects of ROCK₂ heterozygous knockout and ROCK inhibitor KD025 on cerebral ischemia-reperfusion injury in mice.3.To study the protective effect of H₂S produced by CSE in endothelial cells on hypoxic injury of nerve cells,and its relationship with Rho A-ROCK pathway.Methods:1.Middle cerebral artery occlusion（MCAO）was used to establish the model of cerebral ischemia-reperfusion injury in mice.1.1 Experimental grouping:wild type CSE mice（WT）and knockout CSE mice（KO）were randomly divided into 9 groups,with 10 rats in each group:Sham WT group,MCAO WT group,TFR40mg/kg WT group,TFR80mg/kg WT group,TFR120mg/kg WT group,Na HS4.8mg/kg WT group,Sham KO group,MCAO KO group,TFR120mg/kg KO group.1.2 Experimental grouping:wild type ROCK₂ mice（WT）and heterozygous knockout ROCK₂ mice（HK）were randomly divided into 8 groups,with 10 mice in each group:Sham WT group,Sham+KD025 WT group,MCAO WT group,MCAO+KD025 WT group,TFR120mg/kg WT group,Sham HK group,MCAO HK group,TFR120mg/kg HK group.1.3 The middle artery occlusion method was used to established the model of cerebral ischemia-reperfusion（I/R）injury in mice,after 2 hours of ischemia,the reperfusion was restarted for 24 hours.1.4 Laser speckle imaging（LSI）was observed to detect the changes of cerebral blood flow in mice before ischemia,20 minutes after ischemia and 20 minutes after reperfusion.1.5 According to the neurological function score of Zea Longa,assess the neurological function of mice.1.6 The cerebral infarction volume of mice was detected by TTC staining.1.7 The activities of lactate dehydrogenase（LDH）and nerve specific enolase（NSE）in serum were measured by using the kits.1.8 The content of H₂S in cerebral vessels was detected by methylene blue method.1.9 The activities of Rho A,ROCK₁ and ROCK₂ were detected by ELISA kits.2.0 Western blot was used to determine the expression of Rho A,ROCK₁,and ROCK₂proteins in the hippocampus.2.Primary cultured rat hippocampal neurons cells and rat cerebral vascular endothelial cells.After cells were identified by using immunofluorescence,the two cells were co-cultured by Transwell method.2.1 Experimental grouping:NC+EC^WT was divided into:Control,H/R,H/R+TFR（810mg/L）,H/R+Na HS（200μmol/L）,NC+EC^KO was divided into:Control,H/R,H/R+TFR（810mg/L）,H/R+Na HS（200μmol/L）。2.2 Except the control group,other groups were exposed to hypoxia incubator（94%N₂-5%CO₂-1%O₂）for 10 hours and then reoxygenated for 14 hours.2.3 The cell viability of rat hippocampal neurons were measured by CCK-8 kits.2.4 The cell apoptosis of rat hippocampal neurons were detected by flow cytometry and cell nuclear staining.2.5 The activities of lactate dehydrogenase（LDH）and nerve specific enolase（NSE）in rat hippocampal neurons were detected by using kits.2.6 The content of H₂S in rat hippocampal neurons was measured by methylene blue method.2.7 The activities of Rho A and ROCK₂ in rat hippocampal neurons were detected by using ELISA kit.2.8 The expression of Rho A and ROCK₂ proteins in hippocampal neurons of rats were detected by western blot.Results:1.The results of cerebral blood flow showed that:before operation,the cerebral blood flow of the left and right sides of the mice was approximately the same;during ischemia,the cerebral blood flow in the right side of mice decreased significantly,while the left side did not;after reperfusion,the cerebral blood flow of the left and right sides of the mice recovered approximately the same.Compared with the two Sham groups,the right cerebral blood flow of mice in the two MCAO groups decreased significantly due to brain I/R injury;Compared with the MCAO WT group,pretreatment with 40,80 and 120 mg/kg TFR and 4.8 mg/kg Na HS significantly inhibited the decrease of cerebral blood flow in the right side of mice.However,compared with MCAO KO group,pretreatment with 120 mg/kg TFR had no significant effect on cerebral blood flow in mice.2.In CSE WT mice,compared with Sham group,MCAO group mice cerebral I/R injury induced neurological deficit and cerebral infarction.40,80 and 120 mg/kg TFR and 4.8mg/kg Na HS reduced the neurological score and cerebral infarction volumn of model group mice,the high dose TFR and Na HS had the most significant effect.In CSE KO mice,the treatment of 120 mg/kg TFR had no significant effect on the neurological deficit and cerebral infarction volumn caused by cerebral I/R injury in mice.3.Compared with the mice in Sham WT or Sham KO groups,MCAO WT or MCAO KO groups mice cerebral I/R injury increased LDH and NSE levels in serum.In CSE WT mice,40,80 and 120 mg/kg TFR significantly inhibited the increase of LDH and NSE levels in serum,and 4.8 mg/kg Na HS also remarkable inhibited the increase of LDH and NSE levels;In CSE KO mice,the high dose of 120 mg/kg TFR had no significant effect on the serum LDH and NSE levels by mice cerebral I/R injury.4.The decrease of H₂S content caused by MCAO injury is inhibited significantly in CSE WT mice pretreated with 80,120 mg/kg TFR and 4.8 mg/kg Na HS.However,in CSE KO mice,120mg/kg TFR had no significant effect on the content of H₂S of cerebral vessels;in addition,the content of H₂S in the cerebral vessels of mice in Sham KO group was significantly lower than that in Sham WT group,and the knockout of CSE obviously reduced the generation of H₂S.5.Compared with Sham group,cerebral I/R njury increased in Rho A,ROCK₁ and ROCK₂ activities and protein expression in the hippocampus of CSE WT mice.120 mg/kg TFR and 4.8 mg/kg Na HS significantly inhibited the increase in Rho A,ROCK₁ and ROCK₂activities and protein expression.40 and 80 mg/kg TFR also inhibited the increase in Rho A,ROCK₁ and ROCK₂ activities and protein expression.However,120 mg/kg TFR did not significantly effect in Rho A,ROCK₁ and ROCK₂ activities and protein expression in hippocampus of CSE KO mice.6.Compared with the Sham group of ROCK₂ WT mice and ROCK₂ HK mice,two MCAO groups mice cerebral I/R injury induced significant decrease of cerebral blood flow,neurological deficit,cerebral infarction and increase of serum LDH and NSE activities.However,the ROCK₂ inhibitor KD025 100mg/kg and the ROCK₂ heterozygote KO could significantly attenuate these changes caused by MCAO.Compared with the MCAO WT group or the MCAO HK group,120 mg/kg TFR significantly reduced the decrease of cerebral blood flow,neurological deficit,cerebral infarction and the increase of serum LDH and NSE activity caused by brain I/R injury,but the weakening effect of TFR120 HK group was significantly weaker than that of TFR120 WT group.7.After the co-culture of hippocampal neurons cells and cerebral vascular endothelial cells,compared with the two control groups,the cell viability of the two model groups decreased significantly.In NC+EC^WT co-culture,TFR（810 mg/L）and Na HS（200μmol/L）significantly increased cell viability after H/R.In NC+EC^KOco-culture,Na HS（200μmol/L）significantly inhibited the decrease of cell viability,but TFR（810 mg/L）had no significant effect on the decrease of cell viability after H/R.8.In the co-culture of NC+EC^WT and NC+EC^KO,compared with the control group,the model group caused cell membrane damage by H/R injury,increased LDH and NSE levels,and Na HS（200μmol/L）inhibited the increase of LDH and NSE levels.In NC+EC^WT co-culture,pretreatment with TFR（810 mg/L）significantly inhibited the increase of LDH and NSE levels after H/R injury,but in NC+EC^KO co-culture,compared with H/R model group,TFR（810 mg/L）did not significantly decrease the LDH and NSE levels.9.In NC+EC^WT co-culture,compared with the control group,the model group caused neuron cells apoptosis,and the fluorescence intensity of apoptotic cells was significantly enhanced.Compared with H/R model group,TFR（810 mg/L）and Na HS（200μmol/L）can significantly reduce the cells fluorescence intensity,and inhibit the cells apoptosis.However,in NC+EC^KO co-culture,TFR（810 mg/L）did not inhibit H/R injury-induced cell apoptosis,while Na HS（200μmol/L）inhibited neuronal apoptosis significantly.10.In NC+EC^WT co-culture and NC+EC^KO co-culture,compared with the control group,the model group induced a significant decrease in the cell H₂S content.Na HS（200μmol/L）inhibited the decrease of H₂S content caused by H/R injury.In NC+EC^WT co-culture,compared with the H/R model group,the pretreatment of TFR（810 mg/L）significantly inhibited the reduction of H₂S content.However,in NC+EC^KO co-culture,TFR（810 mg/L）had no effect above,and had no obvious difference compared with H/R model group.11.In NC+EC^WT co-culture,H/R model group significantly increased the activities of Rho A and ROCK₂ and protein expression.Compared with H/R model group,TFR（810 mg/L）and Na HS（200μmol/L）significantly inhibited the increase of Rho A and ROCK₂activities and protein expression by H/R injury.However,in NC+EC^KO co-culture,compared with H/R model group,Na HS（200μmol/L）significantly inhibited the increase of Rho A and ROCK₂activities and protein expression,but TFR（810 mg/L）pretreatment had no significant effect on Rho A and ROCK₂ activities and protein expression.Conclusion:1.TFR protects against cerebral I/R injury by promoting the production of H₂S by CSE in cerebral vascular endothelial cells.2.Down-regulation of Rho A-ROCK pathway plays a protective role in brain I/R injury.3.The protective effect of H₂S-mediated TFR produced by CSE in vascular endothelium on hypoxic-ischemic brain injury is related to the inhibition of Rho A-ROCK pathway in nerve cells.