DL-3-n-butylphthalide Alleviates Vascular Cognition Impairment By Regulating Endoplasmic Reticulum Stress And Shh/Ptch1 Signal Pathway In Rats

Vascular cognitive impairment?VCI?is caused by a variety of cerebrovascular risks factors,cerebral infartion and non-infartional cerebro-vascular diseases.VCI is a syndrome of containing different impairment of cognition,from mild cognitive impairment to vascular dementia.VCI include non-dementia cognitive dysfunction and vascular dementia?VD?.With the elderly population increasing and aging speeding up,the morbidity,morbidity,disability and mortality incidences of cerebrovascular disease has increased year by year.The ratio of VCI progressed to VD has risen year by year.In Europe VD has becomed the second largest type of dementia only to Alzheimer's disease?AD?.But in Asia,VD has becomed the largest type of dementia.Except limb dysfunction caused by cerebrovascular disease,it also causes cognitive dysfunction and mood disorders.VD is a chronic and progressive procedure.Because of the high incidence of disability and the decline of cognitive impairment,VD has seriously affected the daily life,the professional and social abilities of patients,which has brought heavy care and economic burden to families and society.VD can be prevented and cured consider of the role of vascular factors.It is urgent to treat VCI early and prevent VCI from developing into VD.It is important to take early positive and effective prevention and treatment to reduce the burden of patient's themselves,their family and the society.Vascular factors are the fundamental factor of VD.Increasing evidence has suggested that its progressive procedure is associated with diverse pathologies,such as chronic cerebral hypoperfusion,ischemia/reperfusion injury,oxidative stress,inflammatory reactions and so on.Several studies have characterized endoplasmic stress?ERS?as an important pathophy-siological status contributing to VD.The endoplasmic reticulum is an important organelle in the cell,which participates in the proper folding and secretion of proteins.ERS is a process that occurs when unfolded/misfolded proteins accumulate in the endoplasmic reticulum?ER?after a homeostasis disturbance.It can be triggered by specific stress conditions such as hyperglycemia,hypoxia,nutrient deprivation,calcium depletion,chemical toxicants,genetic mutations and so on.The major goal of ERS is to maintain cellular homeostasis within the ER by activating the unfolded protein response?UPR?.On acute stage,ERS is an adaptive reaction of the cell.It plays a beneficial role in the pathological process of disease.However,persistent ERS can exceed the recognition ability of the cell,and lead to apoptosis,which will accelerate the development of the disease.UPR are mediated through three pathways:1)PERK-eIF2??protein kinase R like endoplasmic reticulum kinase--eukaryotic translation initiation factor 2subunit??pathway;2)IRE1-XBP1-XBP1s?inositol-requiring enzyme1-X-box-binding protein-1-spliced XBP1?pathway;3)ATF6?activating transcription factor 6?pathway.UPR plays an important role in the development of VD.ERS can improve learning and memory ability of VD rats by inhibiting hippocampal neuronal apoptosis.However,the specific mechanisms for ERS are not known.Synapse play an important role in learning and memory[19].Synaptophysin?SYN?is a specific presynaptic marker for neurons.Postsynaptic density protein 95?PSD-95?is a postsynaptic protein of synapse.Growth associated protein 43?GAP43?is an indicator of neurite elongation and synapse formation.All of the proteins are essential for synaptic neurotransmission.They were observed as an extension of behavioral improvement to evaluate the effective of NBP in this study and termed as plasticity-related markers.Sonic hedgehog?Shh?,as a secreted multifunctional morphogen,plays important roles during neural development.It binds to Patched1?Ptch1?and leads to the downstream activation of the Smoothened co-receptor?Smo?and Gli transcription factor1?Gli1?.which can regulate cell growth,different-iation and survival in a variety of cells,including neurons.Shh/Ptch1pathway affects neural circuit formation at several stages on neurite extension,axon pathfinding and synapse formation.Shh/Ptch1/Smo/Gli activation has been found in cortical astrocytes who appear to have numerous roles in maintaining normal brain function,including roles regulating synapse formation and even synaptic plasticity.The pathway can improve cognitive function.In the development of VD,many signal pathways have been reported involved.The activation of the Shh/Ptch1pathway has not been reported in the development of VD.Meanwhile,there is interaction between Shh/Ptch1 pathway and ERS.The loss of Sonic hedgehog can lead to ileal secretory cell modifications indicative of endoplasmic reticulum stress.In addition ERS induces hepatocyte production of Shh ligands which,in turn,provide paracrine pro-fibrogenic signals to neighboring cells.All of the above suggest Shh/Ptch1 activation is essential for cells regeneration to keep cells survival from injuries.However,the effect of Shh/Ptch1 pathway in a rat model of VD is not well understood.In addition,little is known about the effects of ERS together with Shh/Ptch1pathway.What is the relationship between them.NBP was first extracted from the seeds of Chinese celery.NBP has been approved to be effective not only in protecting against ischemic cerebral injury,but also in ameliorating vascular cognitive impairment in dementia patients and AD models.NBP treatment can also protect hippocampal neurons,reduce apoptosis and astrocyte reactivity and improve spatial memory deficits in CCH rats.NBP exerts its effects on cognitive deficits by preventing ischemic neuropathologic alterations,increasing acetylcholine synthesis,inhibiting oxidative damage,and so on.However,whether the protective effects of NBP in VD are related to ERS or/and Shh/Ptch1 pathway are needed to been explored.The relationship between NBP and Shh/Ptch1 pathway has not be researched before.In our study,we established VD rat model by ligation of bilateral common common carotid artery?BCCAO?.Treatment with 30mg/kg,60mg/kg and 120mg/kg NBP were administrated to observe:1)whether NBP had protective effects on the model;2)whether the protective effect of NBP is related to the expression of ERS-related proteins;3)whether the Shh/Ptch1 pathway was involved in the development of VD;4)whether the protective effect of NBP is related to the activation of the Shh/Ptch1 pathway;5)The possible interaction between ERS and Shh/Ptch1 pathway in the development of VD and nd whether the two mechanisms interacted with each other.Part One NBP with different doses improves cognitive function and pathological damage in VD ratsObjective:VD rat model was established by BCCAO method,and to further evaluate the protective effects of different doses?30mg/kg,60mg/kg and 120mg/kg?of NBP on cognitive function and pathological damage tested by Morris water maze and HE staining.Methods:Thirty male rats weighted 250-300g were randomly distributed into five groups:sham?sham-operated+corn oil?group,model?BCCAO+corn oil?group,NBP30?NBP 30mg/kg treated model group?group,NBP60?NBP60 mg/kg treated model group?group and NBP120?NBP 120 mg/kg treated model group?group.Each group was investigated at 4 weeks after operation.The NBP?dissolved with corn oil?or corn oil were administered via gavage everyday 24h after BCCAO.During the experiment,the animals'weights were measured weekly.After 28 days of continuous administration,all the survival mice were evaluated learning and memory abilities by Morris water maze test for six consecutive days.1)Place navigation test:each mouse received six trails per day for five consecutive days.The time to find the submerged platform?escape latency?was recorded in each trial as the learning score.2)Spatial probe test:The next day after the place navigation test,the mice were tested on a spatial probe trial in which the platform was removed,and the mice were placed in the opposite quadrant and allowed to swim freely for 60 s.The time percentage of rats spent in the target quadrant where the platform had been located and the count crossed the platform was recorded as memory score.After behavioural evaluation,pathological changes and normal neuron count of hippocampal CA1 region were examed by y HE staining.Results:In the Morris water maze,the spatial learning ability of the five groups differed at different time points.On the first day,no significant difference in escape latency was observed among the five groups.According to the analysis using the SNK test,the escape latency of Model group was significantly longer than the Sham group and NBP-treated groups beginning on day 2?P<0.05?.Compared with the Sham group,the escape latency of the Model group was significantly longer on days 3-5?P<0.05?.From the second day,rats in the NBP30 and NBP60 groups showed shorter mean latencies than the Model group.Meanwhile,the NBP120 group exhibited significantly shorter escape latency than the Model group.Although,there was no significance among the NBP treated groups.But there was no significance between the NBP120 group and Sham group on the day 5.In the probe trial,the rats in Model group spent less time in the target quadrant?P<0.05?than rats in the Sham group.Improvements were observed significantly in the NBP-treated groups?P<0.05?.However,differences were not observed between rats treated with different doses of NBP.On day 28 after BCCAO,the pyramidal neurons in the CA1 region of the hippocampus in the Sham group were tightly ranked in order.The neurons were clear and moderate in size with normal microstructure.In the Model group,obvious pathological changes were exhibited with loosely arranged neurons,neuronal shrinkage,loss and light color staining.Administration of NBP,especially 120 mg per day,reversed the morphologic changes.Conclusion:The VD rat model prepared by BCCAO showed significant loss of spatial learning and memory in Morris water maze test at 4 weeks after operation,accompanied by less normal neuron.Different doses of NBP treatment significantly improved the impaired spatial learning and memory function and increased the normal neuron count.The protective effect was significant especially in the NBP₁₂₀ group.Part Two NBP with different doses protect synaptic plasticity related proteins in hippocampus of rats with vascular dementiaObjective:After VD rat model was prepared,the changes of SYN,GAP43 and PSD95 in hippocampus of rats were observed at 4 weeks,and to explore whether different doses?30mg/kg,60mg/kg and 120mg/kg?of NBP would up-regulate the expression of SYN,GAP 43 and PSD95.Methods:Grouping and model preparation were carried out in accor-dance with Part I.Western blot and Real Time-PCR assay were used for evaluating the expression of SYN,GAP43 and PSD95 after BCCAO among Sham group,Model group and different doses of NBP-treated groups.Results:We performed western blots and RT-PCR to determine whether SYN,GAP43 and PSD95 was involved in the neuroprotective effects of NBP on the rats of VD.At both protein and mRNA levels,the expressions of SYN,GAP43 and PSD95 were reduced in the Model group compared to Sham group?P<0.01?.Treatment with 30mg/kg,60mg/kg and 120mg/kg NBP significantly up-regulated the expressions of the three markers than Model group,especially the NBP60 and NBP120 groups?P<0.01?.At the protein level,the expression of SYN and PSD95 exhibited the most significance in the NBP120 group.The expression of PSD95 was significantly difference among NBP treated groups?P<0.01?.As we can see there were no significantly differences between the NBP120 group and Sham group.While At the mRNA level,NBP120 group significantly up-regulated the expression of SYN and GAP43 than NBP60 and NBP30 groups?P<0.05?.Whereas there was no dose-response pattern between NBP-treated groups at both levels.Conclusion:Different doses of NBP had protective effects in improving the cognitive function of VD rats by up-regulating the expression of SYN,GAP 43 and PSD 95 at both protein and gene levels.Part Three NBP with different doses regulate the expressions of mar-kers related to ERS in hippocampus of rats with vascular dementiaObjective:After VD rat model was prepared,the expression of PERK,elF2?,IRE1,XBP1,XBP1s,ATF6,GRP78,CHOP and caspase-12 in hippocampus of rats were observed at 4 weeks and to determine whether endoplasmic reticulum stress play an important role in the progressive of VD.Meanwhile,to observe the effects of different doses of NBP on the expression of PERK,elF2?,IRE1,XBP1,XBP1s,ATF6,GRP78,CHOP and caspase-12 in hippocampus and to explore whether different doses?30mg/kg,60mg/kg and 120mg/kg?of NBP have a protective effects by regulate the expressions of markers related to ERS in hippocampus of rats with vascular dementia.Methods:Grouping and model preparation were carried out in accordance with Part I.Western blot and Real Time-PCR assay were used for evaluating the expression of PERK,elF2?,IRE1,XBP1,XBP1s,ATF6,GRP78,CHOP and caspase-12 after BCCAO among Sham group,Model group and different doses of NBP-treated groups.Results:We detected the protein expression of PERK,elF2?,IRE1,XBP1,XBP1s,ATF6,GRP78,CHOP and caspase-12.Except for GRP78treated 30mg/kg NBP,the results showed that the expressions of other proteins treated with NBP significantly decreased compared to Model group?P<0.05?.There were no significant differences in the expression of XPB1among NBP-treated groups.NBP₁₂₀ group significantly deregulated the expression of all the proteins except for XPB1 and elF2?(equals in NBP₁₂₀and NBP₆₀ groups)?P<0.05?.There was no dose-response pattern between NBP-treated groups.Data are shown in Fig.4A.Except for proteins,we also detected the mRNA expression of PERK,elF2?,IRE1,XBP1,XBP1s,ATF6,GRP78,CHOP and caspase-12.Interestingly,the mRNA levels of CHOP and elF2?were significantly increased in NBP₃₀ group compared to Model group?P<0.01?and there was no significantly differences between NBP₆₀ group and Model group.Besides CHOP and elF2?,all the other markers were significantly decreased in NBP₁₂₀group compared to Model group?P<0.01?.As to PERK and IRE1,there was no significantly differences between NBP₃₀ group and Model group.Similarly,the NBP₁₂₀ group exhibited the most significantly differences among treated groups except for caspase-12(equals in NBP₁₂₀ and NBP₆₀ groups)?P<0.01?.Conclusion:The markers of edoplasmic reticulum stress in hippocampal tissue were confirmely activated in the VD model.The expression of all the markers were up-regulated in the model group.After given 30 mg/kg,60mg/kg and 120 mg/kg of NBP,the expression of PERK,elF2?,IRE1,XBP1,XBP1s,ATF6,GRP78,CHOP and caspase-12 were down-regulated.NBP played neuroprotective effects by inhibiting apoptosis of neurons and promoting cell survival to lowering by down-regulating the markers related with ERS,especially in NBP120 group.Part Four NBP with different doses regulate the expressions of markers related to Shh/Ptch1 pathway in hippocampus of rats with vascular dementiaObjective:After VD rat model was prepared,the changes of Shh,Ptch1,Smo and Gli1 in hippocampus of rats were observed at 4 weeks,and to explore whether different doses?30mg/kg,60mg/kg and 120mg/kg?of NBP would regulate the expression of Shh,Ptch1,Smo and Gli1.Methods:Grouping and model preparation were carried out in accordance with Part I.Western blot and Real Time-PCR assay were used for evaluating the expression of Shh,Ptch1,Smo and Gli1.After BCCAO among Sham group,Model group and different doses of NBP-treated groups.Results:We performed Western blots and RT-PCR to detect the expression of Shh,Ptch1,Smo and Gli1.At both protein and mRNA levels,the expression of Shh,Ptch1,Smo and Gli1 were observed decreased in the hippocampus of the rats in the Model group compared to those in the Sham group?P<0.01?.At the protein level,all treated groups were significantly up-regulated the expressions of the four markers than Model group?P<0.01?.Especially the NBP120 group exhibited the most significantly increased?P<0.05?.While at mRNA level,the treatment of NBP60 and NBP120groups had nearly the same effect on the expression of Shh and Gli1.Although all treated groups had protective effects on the expression of Ptch1and Smo,there were no differences between three treated groups.Conclusion:The expression of Shh/Ptch1 pathway in hippocampal neurons of VD rats decreased after BCCAO,which indicated that the pathway was involved in the development of VD.After NBP was given,the pathway is activated variously to promote the survival of hippocampal neurons by promote synaptic formation and regulate its plasticity.The activation of the pathway plays a neuroprotective role and can improve cognitive function of VD.The neuroprotective effects of the Shh/Ptch1pathway in VD may play alone,may play together with ERS.Or we can say the pathway was a survival way while ERS was a death way.The balance of the two determines the fate of cells,life of death.The specific mechanisms need further studies.