| Ischemic stroke(IS)is a common clinical disease in which cerebral ischemia and hypoxia are caused by the reduction or interruption of blood flow caused by cerebral vascular blockage,which leads to brain tissue damage.Ischemic stroke is often accompanied by vascular recanalization and blood flow reperfusion,which further aggravates cellular metabolic disorders and structural damage,leading to severe neurological function defects,namely cerebral ischemia reperfusion injury(CIRI).Oxidative stress response plays a key role in cerebral ischemia reperfusion injury.The sudden increase of reactive oxygen species can cause neuronal apoptosis,brain edema and blood brain barrier damage.During ischemia,reactive oxygen species accumulate continuously in cells,reducing the effect of antioxidants.Oxidative stress may lead to autophagy and aggravate brain injury after blood supply to ischemic tissue is restored.Therefore,reducing oxidative stress,inhibiting autophagy,finding possible therapeutic targets and developing effective neuroprotective drugs are the key to ameliorate cerebral ischemia-reperfusion injury.Several studies have shown a link between silent information regulator 1(SIRT1)and autophagy,which regulates inflammatory response,oxidative stress,apoptosis and autophagy.Forkhead box protein O1(FoxO1),an important downstream molecule of SIRT1,serves as an important deacetylating substrate and is a key factor regulating autophagy.Betulinic acid(BA)is a natural triterpenoid compound with pentacyclic lupane structure,which is mainly extracted from birch bark.Studies have shown that betulinic acid pretreatment can significantly reduce the expression of 3-nitrotyrosine(one of the active nitrogen products involved in oxidative stress injury)in the brain tissues of cerebral ischemia-reperfusion mice,suggesting that betulinic acid can protect cerebral ischemia-reperfusion injury through anti-oxidative stress.Therefore,it is speculated whether betulinic acid can improve cerebral ischemia reperfusion injury by regulating autophagy,and whether the regulation pathway is SIRT1/FoxO1 signaling pathway.This study to establish a cerebral ischemia reperfusion(I/R)rat model and OGD/R cell model.The protective effect of betulinic acid on cerebral ischemia reperfusion injury was discussed from autophagy level,and the possible regulation mechanism through SIRT1/FoxO1 signaling pathway was also discussed.1.Betulinic acid improves cerebral ischemia reperfusion injuryObjective: To explore the protective effect of betulinic acid on cerebral ischemia reperfusion injury.Methods: Thirty male SD rats were randomly divided into Sham group(Sham group,n=10),cerebral ischemia reperfusion group(I/R group,n=10),betulinic acid+cerebral ischemia reperfusion group(BA+I/R group,n=10).Cerebral ischemia-reperfusion(I/R)model was established in I/R group by MCAO method with middle cerebral artery occlusion.The rats were ischemic for 1 h and reperfusion for 24 h.Cerebral ischemia-reperfusion model was established in BA+I/R group after continuous intragastric administration of betulinic acid(50 mg/kg)for 7 days.The neurological injury of rats was observed by Longa 5-point neurological function score.TTC staining was used to observe the cerebral infarction volume of rats.He staining was used to observe the histopathological damage.The release of reactive oxygen ROS,an indicator of oxidative stress,was detected by immunofluorescence method.The secretion of MDA,MPO and SOD related to oxidative stress was detected by ELISA.The apoptosis of cerebral cortex nerve cells was observed by TUNEL method.Results: The neurological function score of the I/R group was significantly increased,with severe cerebral infarction,severe pathological injury,excessive release of oxidative stress products and neuronal cell apoptosis,which were significantly different from that of the Sham group(P< 0.05).The results showed that the nerve function of I/R group was impaired.In BA+I/R group,the neurological function score decreased,the volume of cerebral infarction decreased,the number of intact neurons increased,the degeneration necrosis was improved,the oxidative stress level was reduced,and apoptosis was inhibited.Conclusion: Betulinic acid can alleviate cerebral ischemia-reperfusion injury,inhibit oxidative stress,decrease neuronal apoptosis and improve neurological dysfunction in rats.2.Betulinic acid alleviates brain injury by inhibiting abnormal autophagy during cerebral ischemia reperfusionObjective: To explore the effect of betulinic acid on the level of autophagy in cerebral ischemia reperfusion injury.Methods: Thirty male SD rats were randomly divided into Sham group(Sham group,n=10),cerebral ischemia reperfusion group(I/R group,n=10),betulinic acid+cerebral ischemia reperfusion group(BA+I/R group,n=10).The presence of autophagosomes was observed by transmission electron microscopy.The expression of autophagy related proteins LC3-II/I,Beclin1 and p62 in brain tissues was observed by immunofluorescence and Western blot.In vitro,oxygen and glucose deprivation/repoxygenation(OGD/R)model was established,oxygen and glucose deprivation for2 h,repoxygenation for 24 h.BA and BA combined with rapamycin,an autophagy agonist,were administered respectively.The cells were divided into Control group,OGD/R group,BA+OGD/R group and BA+OGD/R +RAPA group.Cell viability was detected by CCK8 assay.Western blot was also used to detect the expression of autophagy related proteins LC3-II/I,Beclin1 and p62 at the cell level.The expression of reactive oxygen species(ROS)was detected by immunofluorescence.The contents of MDA,MPO and SOD were detected by ELISA.Apoptosis was detected by flow cytometry.Results: Tem results showed that the number of autophagosomes in the brain tissue of rats in I/R group increased.Compared with I/R group,the number of autophagosomes in BA+I/R group was decreased.Immunofluorescence and Western blot results showed that compared with Sham group,the expression of autophagy related proteins LC3-II/I and Beclin1 in I/R group was increased,and the expression of p62 was decreased(P< 0.05).The expression of Beclin1 and LC3-II/I was significantly decreased and the expression of p62 was increased in BA+I/R group(P<0.05).The results showed that betulinic acid could inhibit abnormal autophagy induced by cerebral ischemia reperfusion injury in rats.Further in vitro experiments,CCK8 detection results showed that the cell viability was higher when the concentration of betulinic acid was 10 μM for 24 h.In addition,compared with the Control group,the expression of autophagy related proteins LC3-II/I and Beclin1 increased in OGD/R group,the expression of p62 decreased,the release of ROS increased,the content of MDA and MPO significantly increased,the content of SOD significantly decreased,and the apoptosis rate increased(P< 0.05).Compared with OGD/R group,the expression of autophagy related proteins Beclin1 and LC3-II/I in BA+OGD/R group was significantly decreased,the expression of p62 was increased,the release of ROS was decreased,the content of MDA and MPO was significantly decreased,the content of SOD was significantly increased,and the apoptosis rate was decreased(P< 0.05).RAPA,an autophagy agonist,can block this effect.These results indicated that betulic acid could regulate the expression of autophagy related proteins at the cellular level,reduce oxidative stress and prevent cell apoptosis by inhibiting autophagy,and this effect was inhibited by autophagy agonists.Conclusion: Betulinic acid can regulate the expression of autophagy related proteins,and alleviates brain injury by inhibiting abnormal autophagy during cerebral ischemia reperfusion.3.Betulinic acid improves cerebral ischemia reperfusion injury by inhibiting autophagy through SIRT1/FoxO1 pathwayObjective: To explore the regulatory mechanism of betulinic acid inhibiting autophagy through SIRT1/FoxO1 pathway to improve cerebral ischemia reperfusion injuryMethods: Thirty male SD rats were randomly divided into Sham group(Sham group,n=10),cerebral ischemia reperfusion group(I/R group,n=10),betulinic acid+cerebral ischemia reperfusion group(BA+I/R group,n=10).The expressions of SIRT1/FoxO1 pathway related proteins SIRT1 and Ac-Foxo1 were observed and detected by immunofluorescence and Western blot.In vitro,oxygen and glucose deprivation/repoxygenation(OGD/R)model was established,oxygen and glucose deprivation for 2 h,reoxygenation for 24 h.BA and BA combined with SIRT1 inhibitor Ex527 were administered respectively.The cells were divided into Control group,OGD/R group,BA+OGD/R group and BA+OGD/R+Ex527 group.Western blot was used to detect the expression of SIRT1/FoxO1 pathway related proteins SIRT1 and Ac-Foxo1.The release of ROS was detected by immunofluorescence method.The expression of autophagy related proteins LC3-II/I,Beclin1 and p62 were detected by immunofluorescence and Western blot.Results: The results of immunofluorescence and Western blot showed that compared with Sham group,the expression of SIRT1 was decreased and the expression of Ac-FoxO1 was increased in I/R group(P< 0.05).Compared with I/R group,the expression of SIRT1 was increased and the expression of Ac-FoxO1 was decreased in BA+I/R group(P< 0.05).These results suggest that betulinic acid can activate SIRT1/FoxO1 pathway and alleviate cerebral ischemia-reperfusion injury.Further in vitro experiments,SIRT1 expression was observed to decrease and Ac-FoxO1 expression was observed to increase in OGD/R group.Compared with OGD/R group,SIRT1 expression was increased and Ac-FoxO1 expression was decreased in BA+OGD/R group.Intervention with SIRT1 inhibitor Ex527 can block this effect.These results suggest that betulinic acid can also activate SIRT1/FoxO1 pathway at the cellular level.In addition,ROS release was increased in OGD/R group,the expression of autophagy related proteins LC3-II/I and Beclin1 was increased,and the expression of p62 was decreased(P< 0.05).Compared with OGD/R group,ROS release decreased,autophagy related proteins LC3-II/I and Beclin1 expression decreased,and p62 expression increased in BA+OGD/R group.Intervention with SIRT1 inhibitor Ex527 could block this effect.These results suggest that betulinic acid can inhibit autophagy through SIRT1/FoxO1 pathway,reduce oxidative stress,and improve cerebral ischemia reperfusion injury.Conclusion: Betulinic acid may inhibit autophagy and ameliorate cerebral ischemia reperfusion injury through SIRT1/FoxO1 signaling pathway. |
