Protective Effect Of The Total Flavonoids Of Rhododendron On Hypoxic-injury In Cerebral Vascular Endothelial Cells Via Promoting The H₂S Production To Inhibit The RhoA-ROCK Pathway

Background:Ischemic brain disease is one of the leading causes of human death worldwide,and its incidence increases with age.Cerebral ischemia/reperfusion（I/R）injury refers to the injury caused by the restoration of blood supply to the brain after a period of ischemia.At present,we have not fully understood the specific mechanism of hypoxic ischemic brain disease,and the development of drugs for its treatment has not made great progress.Studies have shown that the Rho A-ROCK pathway is involved in the pathological process of ischemic brain injury.Ischemic brain injury can induce Rho A activation,up-regulation of ROCK₁and ROCK₂expression and activity,leading to neuronal cell death.Rats with cerebral ischemic injury have reduced hippocampal nerve damage,and studies have confirmed that endothelial-derived H₂S can inhibit the Rho A-ROCK signaling pathway against ischemic brain injury.Total flavones of rhododendra（TFR）is an effective part of flavonoids extracted from rhododendron,which has the effect of protecting cardiovascular and cerebrovascular.Previous studies have found that TFR has a significant anti-ischemic brain injury effect,and initially showed that hypoxic injury can lead to the reduction of CSE protein expression and H₂S generation in cerebral vascular endothelial cells.Whether there is protective effect of vascular endothelial cells and its relationship with H₂S generation.Similarly,the study of the effect of TFR on the Rho A-ROCK signaling pathway in the cerebral vascular endothelium has not been reported.Therefore,this study aimed to investigate whether TFR has protective effect on hypoxic injury of cerebral vascular endothelial cells in rats,and to explore its relationship with H₂S generation and Rho A-ROCK signaling pathway.Provide new treatment ideas for ischemic brain diseases.Purpose:1.To study whether TFR has protective effect on cerebral vascular endothelial cell injury in H/R rats.2.To study the relationship between the protective effect of TFR on cerebral vascular endothelial cells of H/R rats and H₂S and Rho A-ROCK signaling pathway.Method:The primary rat cerebral vascular endothelial cells were cultured to construct a hypoxia-reoxygenation model（H/R）.Grouped as Control,Model,ACh（1μmol/L）,TFR（90mg/L,270mg/L,810mg/L）,ACh（1μmol/L）+PPG（100μmol/L）,TFR（810mg/L）+PPG（100μmol/L）,ACh（1μmol/L）+ASP（100μmol/L）,TFR（810mg/L）+ASP（100μmol/L）,CCG-1423（1μmol/L）,TFR（810mg/L）+CCG-123（1μmol/L）group.After 8 h of hypoxia and 6 h of reoxygenation,cell viability was detected by CCK-8kit,LDH activity,MDA and H₂S content were detected by kit,CSE,3-MST,Rho A,ROCK₁,ROCK₂protein expressions were detected by Western blot,and Rho A,ROCK₁,ROCK₂activity was detected by ELISA,Results:1.Compared with the control group,the cell viability of the model group was significantly decreased,and the levels of MDA and LDH were significantly increased（P<0.01）.The TFR dose groups（90,270,810 mg/L）significantly attenuated the cell viability caused by H/R injury.decreased（P<0.01 or P<0.05）,and attenuated the increase in MDA content and LDH activity（P<0.05）.In addition,administration of ACh（1μmol/L）had a similar effect to TFR.The results showed that TFR could inhibit H/R injury-induced intracellular enzyme leakage and lipid peroxidation in rat cerebral vascular endothelial cells,and had a protective effect on it.Compared with the control group,the H₂S content of vascular endothelial cells in the model group was significantly decreased after H/R injury,and after adding TFR,the H₂S content in endothelial cells was significantly increased in the 270 mg/L and 810 mg/L TFR groups,respectively（P<0.05）.Compared with the control group,the expressions of CSE and 3-MST proteins in the cerebral vascular endothelial cells of the H/R model group were significantly decreased（P<0.05）.In the administration group,270 mg/L and 810 mg/L TFR treatments significantly increased the expression of CSE protein（P<0.05）.For protein3-MST,810 mg/L TFR group could increase the expression of 3-MST（P<0.05）.Compared with the control group,H/R injury resulted in a significant increase in the protein expressions and activities of ROCK₁,ROCK₂and Rho A in the model group（P<0.01 or P<0.05）.In contrast,TFR（270 and 810 mg/L）could significantly reduce the expression and activity of ROCK₁,ROCK₂and Rho A proteins（P<0.05）,and ACh（1μmol/L）had similar effects to the TFR group（P<0.01）or P<0.05)2.After pretreatment with H₂S synthase CSE inhibitor（PPG 100μmol/L）for 30 min,compared with TFR（810 mg/L）group,the activity of cerebral vascular endothelial cells and LDH activity in H/R rats in TFR+PPG group were significantly reduced.significantly increased（P<0.01）,and the H₂S content of rat cerebral vascular endothelial cells was significantly decreased（P<0.05）.After PPG pretreatment,the effect of ACh（1μmol/L）was similar to that of TFR group（P<0.01 or P<0.05）.The results showed that the effect of TFR against H/R injury in rat cerebral vascular endothelial cells was related to CSE,and TFR could promote the production of H₂S from CSE in cerebral vascular endothelial cells.After PPG pretreatment,compared with TFR（810 mg/L）,the effect of TFR+PPG group on inhibiting H/R-induced increase of ROCK₁,ROCK₂and Rho A protein expression and activity in cerebral vascular endothelial cells of rats was significantly weakened（P<0.05,P<0.01）.These results suggest that the effect of TFR on inhibiting the expression and activity of ROCK₁,ROCK₂and Rho A proteins in H/R rat cerebral vascular endothelial cells may be related to CSE.3.After pretreatment with H₂S synthase 3-MST inhibitor aspartic acid（ASP 100μmol/L）for 30 min,compared with the TFR group,the viability of cerebral vascular endothelial cells in the TFR+ASP group decreased slightly,but there was no statistical significance.The activity of LDH in the cells increased slightly,and the content of H₂S in the cells did not decrease significantly.Compared with the TFR group,there was no significant difference in the expression and activity of Rho A protein in the TFR+ASP group.The above results indicated that the effect of TFR and ACh against H/R cerebral vascular endothelial cell injury and the generation of H₂S in 3-MST were not significantly related.4.After pretreatment with Rho A inhibitor CCG-1423（1μmol/L）for 30 min,compared with the TFR group,the cell viability of the TFR+CCG-1423 group decreased,and the LDH content in the cell supernatant increased（P<0.05）.After adding the inhibitor,the protective effect of TFR on H/R injury of rat cerebral vascular endothelial cells was weakened.Compared with the TFR group,the Rho A activity of the cells in the TFR+CCG-1423 group was increased（P<0.05）,indicating that the inhibitory effect of TFR on Rho A was weakened after adding the inhibitor.The above results further indicate that TFR protects cerebral vascular endothelial cells from damage caused by H/R by inhibiting the Rho A-ROCK signaling pathway.Conclusion:1.TFR has a protective effect on cerebral vascular endothelial cell injury in H/R rats2.TFR can promote H₂S synthase CSE to generate H₂S,and inhibit the Rho A-ROCK signaling pathway via H₂S in H/R rat cerebral vascular endothelial cells.