Neuroprotective Effect And Mechanisms Of HLY78 On Early Brain Injury After Subarachnoid Hemorrhage

Subarachnoid hemorrhage(SAH)is a common cerebrovascular disease with an incidence of about 5-10% of stroke.It has the epidemiological characteristics of high mortality and high morbidity.Early brain injury(EBI)is a group of pathophysiological changes that occur within 72 hours of SAH,characterized by decreased cerebral blood flow,brain edema,apoptosis,inflammatory,etc.EBI is the leading cause of acute death and long-term neurological dysfunction in SAH patients.Thus,inhibition of neuronal apoptosis and blood-brain barrier(BBB)destruction at the acute stage can improve neurological dysfunction following SAH.HLY78 is a small molecule phenanthridine alkaloid compound,which is transformed from lycorine.In vitro,HLY78 targets the DIX domain of Axin and promotes the association of Axin with low-density lipoprotein receptor-related protein 6(LRP6),which subsequently phosphorylate LRP6 and activate the downstream Wnt signaling pathway.Wnt pathway has been proved to be involved in the regulation of neuronal apoptosis in stroke and plays an important role in the formation and maintenance of the blood-brain barrier.However,to date,there is no study on HLY78 in SAH animal model.In the present study,we explored the neuroprotective effect of HLY78 on SAH and the underlying molecular mechanism by behavioral and histological methods.It provides a new research direction for reducing neuron apoptosis and blood-brain barrier damage caused by SAH.Part ? Effects of HLY78 on neurological function aftersubarachnoid hemorrhageObjectiveTo investigate the short-and long-term therapeutic effects of HLY78 on neurological deficits after SAH in rats and the safety of HLY78.MethodSprague-Dawley rats weighing 280-310 gram were used to induce SAH rat models,which were divided into sham,SAH + vehicle,and SAH + HLY78 groups.In order to explore the optimal dose of HLY78,the HLY78 treatment groups was divided into three subgroups according to the concentration of HLY78: SAH + HLY78(0.2 mg/kg),SAH + HLY78(0.6 mg/kg),SAH + HLY78(1.8 mg/kg).Based on the results of neurological function scores at 24 h after SAH induction,the optimal dosage of HLY78 was determined for the following research.The effects of HLY78 on the motor,cognitive and neurodegenerative changes after SAH were analyzed by the tests of foot-fault,Rotarod,Morris water maze,and Fluoro-Jade C staining.Finally,normal saline and HLY78(0.6 mg/kg)were given to the healthy rats to establish the Naive + vehicle(normal saline)and Naive + HLY78 groups.The toxicity of HLY78 in vivo was evaluated by HE staining and measuring the weight change of rats.Results1.HLY78 treatment can significantly improve the short-term neurological deficit after SAH in rats.Compared with sham group,the neurological scores at 24 hours of SAH + vehicle group were significantly decreased(p < 0.01).HLY78(0.6 mg/kg and 1.8mg/kg)significantly improved the neurological function score at 24 hours after SAH(p < 0.05),while HLY78(0.2 mg/kg)had no influence on the neurological deficits(p > 0.05).2.HLY78 significantly improved long-term neurological deficits after SAH.In the foot-fault test on 7 and 14 days,HLY78 significantly alleviated the foot-fault times of the bilateral forelimbs compared with SAH + vehicle group.In the Rotarod test,compared with SAH + vehicle group,the falling latency in both 5 rpm and 10 rpm trials was significantly prolonged in the SAH + HLY78 group(p < 0.05).In the Morris water maze test,HLY78 treatment significantly reduced the time and the swimming distance before rats found the platform(p < 0.05).And in the probe quadrant trial,the rats treated with HLY78 spent a longer duration in the probe quadrant than SAH + vehicle group(p < 0.05).In addition,Fluoro-Jade C staining showed that HLY78 significantly reduced the number of degenerated neurons(p < 0.05).3.The safety of HLY78 in vivo.There was no significant difference in the body weight between the Na?ve + NS and Na?ve + HLY78 groups at 1,7,14,and 21 days.HE staining showed no necrosis in normal tissues of liver and kidney.ConclusionIn this study,we found that HLY78 can significantly improve short-term and long-term neurological deficits after SAH,and the therapeutic dose applied in this study has no significant toxic effect on the body.Part ? Effect of HLY78 on neuronal apoptosis aftersubarachnoid hemorrhage and its potential mechanismObjective1.To explore the expression of p-LRP6 and LRP6 in the ipsilateral brain tissues after SAH.2.To investigate the effects of HLY78 on SAH-induced neuronal apoptosis.3.Using specific LRP6 small interfering RNA(siRNA)to explore the mechanism of the anti-apoptotic effect of HLY78.Method1.The rat SAH model was established by an intravascular puncture at the bifurcation of the left middle cerebral artery.Western blot and immunofluorescence were used to detect the endogenous p-LRP6 and LRP6 expression in the left cerebral cortex after SAH.2.HLY78 was administered intranasally at 1 h after the SAH induction.Western blot,transferase-mediated deoxy uridine triphosphate-biotin nick end labeling(TUNEL),and immunofluorescence were used to detect neuronal apoptosis at 24 h after SAH.3.LRP6 siRNA was injected intraventricularly at 24 h before the SAH model was established.The neurological function scores were evaluated again after siRNA administration.The expression of p-LRP6,LRP6,GSK3?,?-catenin,Cleaved Caspase 3,Bax,and Bcl-2 were detected by Western blot.Result1.Western blot results showed that the endogenous expression of p-LRP6 was significantly increased at 6 to 72 h and reached the peak at 24 h after SAH(p < 0.01).Immunofluorescence results showed that p-LRP6 was expressed on both neurons and astrocytes.2.Western blot results showed that compared with the SAH + vehicle group,the expression of Bcl-2 in the HLY78 treated group was significantly increased,while the cleaved caspase 3 and Bax was decreased(p < 0.01).TUNEL staining showed that the number of TUNEL positive neurons in SAH + vehicle group was significantly higher than that in sham group(p < 0.01).And HLY78 significantly decreased the number of TUNEL positive neurons(p < 0.01).3.The results of mechanism research showed when compared with SAH + HLY78 + scrambled siRNA group,the neurological scores of rats injected with LRP6 siRNA were significantly decreased(p < 0.05).Western blot analysis showed that compared with the SAH + vehicle group,the expression levels of p-LRP6,p-GSK-3?,?-catenin,and Bcl-2 in the HLY78 treatment group were significantly increased(p < 0.05),while p-?-catenin,Bax,Cleaved Caspase 3 expression levels were decreased(p < 0.01).After the LRP6 was silenced by the specific siRNA,the expression of p-LRP6,p-GSK-3?,?-catenin,and Bcl-2 decreased,while the expression levels of p-?-catenin,Bax,and cleaved casepase 3 increased(p < 0.05).ConclusionThe study found that HLY78 can inhibit neuronal apoptosis caused by SAH,and the LRP6-mediated Wnt/?-catenin signaling pathway is involved in the regulation of HLY78's anti-apoptotic effect.This study provides new ideas for the treatment of neuronal apoptosis induced by SAH.Part ? Effect of HLY78 on blood-brain barrier aftersubarachnoid hemorrhage and its potential mechanismObjective1.To study the effect of HLY78 treatment on blood-brain barrier disruption caused by SAH.2.Specific LRP6 siRNA was used to study the protective effect and potential mechanism of HLY78 on blood-brain barrier after SAH.Method1.The SAH model was established in rats,and the expression levels of endogenous p-LRP6 and LRP6 in the left brain tissue were detected by Western blot at 0,6,12,24 and 72 hours after SAH.Immunofluorescence was used to detect whether p-LRP6 was expressed on endothelial cells(ECs).2.HLY78 was administered intranasally 1 hour after the establishment of the SAH model.The brain water content of rats was quantitatively analyzed by dry and wet weight methods at 24 hours after SAH.Evans blue solution(EB)was injected intraperitoneally to quantitatively analyze Evans blue exudation and evaluate the integrity of the blood-brain barrier.3.LRP6 siRNA was micro pumped through the right ventricle 24 hours before the SAH model was established.The neurological scores after siRNA administration were evaluated.Western blot technique was utilized to detect the expression levels of p-LRP6,LRP6,?-catenin,Occludin,ZO-1,and Claudin-5.Result1.Results of Western blot showed endogenous p-LRP6 was significantly up-regulated within 6 to 72 hours after SAH(p < 0.01),and reach the highest peak at 24 hours.Immunofluorescence showed that p-LRP6 was expressed on vascular endothelial cells in the ipsilateral cerebral cortex.Compared with the sham group,p-LRP6 expression was significantly increased on vascular endothelial cells in the SAH group.2.The results of brain tissue water content and Evans blue extravasation tests showed that compared with the SAH + vehicle group,the brain water content in the HLY78 treated group was significantly decreased(p < 0.01).Moreover,less Evans blue extravasation was also observed in the HLY78 treated group(p < 0.01).3.Rats modified Garcia and beam balance scores after injection of lateral ventricle LRP6 siRNA were significantly lower than SAH + HLY78 + scramble siRNA(p < 0.01).Western blot results showed that compared with the SAH + vehicle group,the expression of p-LRP6,?-catenin,Occludin,ZO-1,and Claudin-5 was up-regulated in the HLY78 treatment group(p <0.01).After LRP6 siRNA silenced LRP6 expression,p-LRP6,?-catenin,Occludin,ZO-1,and Claudin-5 expressions significantly decreased(p <0.01).ConclusionThis study showed that HLY78 could reduce the blood-brain barrier damage caused by SAH and improve neurological deficits by activating the Wnt/?-catenin signaling pathway.This study provides a new target for treating SAH-induced blood-brain barrier disruption.