Protective Mechanism Of Dl-3-n-butylphthalide On Cognitive Function,Oxidative Stress And Neuroinflammation In Vascular Dementia

With the aggravation of the aging process of the global population,the incidence of vascular dementia（VD）increases year by year,accounting for about 15%of all dementia cases,and is the most common dementia only second to Alzheimer’s disease（AD）.The treatment of VD has been listed as one of the key scientific research projects by World Health Organization（WHO）in the 21st century.However,there is still no ideal treatment for VD,so it is of great clinical significance and social value to explore the pathogenesis and preventive drugs of VD.In China,the prevalence of stroke is increasing year by year,and it is the first fatal disease in China,and stroke often leads to cognitive impairment.Post-stroke cognitive impairment（PSCI）refers to the cognitive decline after stroke,which is an important subtype of Vascular cognitive impairment（VCI）.The quality of life and life span of patients are affected seriously.However,PSCI is a preventable and treatable disease,so early identification and early treatment can reduce the occurrence of PSCI to a certain extent.Currently,clinically recognized humoral markers of PSCI is still lack.Neutrophilic/lymphocyte ratio（NLR）and platelet/lymphocyte ratio（PLR）are new blood inflammatory indexes.To some extent,the increase of the ratio represents the aggravation of oxidative stress and inflammatory response.Previous studies have confirmed that NLR and PLR are associated with the severity and prognosis of a variety of cognition-related diseases,but there are few reports on the studies of NLR and PLR in PSCI.Nuclear factor erythroid-2 related factor 2（Nrf2）is an important regulator of cell homeostasis.In recent years,the Nrf2 pathway has been considered as a key factor in ameliorating oxidative stress damage in cerebral ischemia and dementia.The effects of Nrf2 on antioxidant stress,inflammation elimination and autophagy regulation have been confirmed in many studies.Microglia are the main immune cells in the central nervous system（CNS）and play a central role in neuroinflammatory response.Studies have confirmed that microglia-mediated inflammation after ischemia-reperfusion injury（IRI）is involved in the occurrence and development of VD.The toxicity of microglia is mediated by the excessive production of many harmful substances,including tumor necrosis factor-alpha（TNF-α）,interleukin-6（IL-6）and interleukin-1β（IL-1β）.Toll-like receptors（TLRs）are a family of pattern recognition receptors that have been shown to be involved in neuroinflammation and dementia.Toll-like receptor 4（TLR4）is an important switch in microglia activation and function regulation.Activation of TLR4 induces a cascade of signals via the myeloid differentiation factor 88（MyD88）dependent pathway.Promote the phosphorylation and transfer of nuclear factor-Kappa B（NF-κB）into the nucleus,and finally promote the production of large quantities of neurotoxic pro-inflammatory cytokines（PICs）,such as TNF-α,IL-6 and IL-1β,etc.,cause inflammatory response and neuron damage,resulting in cognitive impairment.Dl-3-n-butylphthalide（NBP）,a natural drug extracted from celery seed,was approved by the State Food and Drug Administration for the treatment of ischemic stroke in 2002.Studies have confirmed that NBP can improve cognitive impairment in VD animal models by inhibiting inflammation,endoplasmic reticulum stress,oxidative stress and neuronal apoptosis,and promoting myelin formation and hemodynamics.Meanwhile,NBP can promote Nrf2 expression in various animal models.However,the specific mechanism of Nrf2 activation in VD treatment remains unclear.Based on the background,84 patients with first-onset cerebral infarction were enrolled in this study.According to the Mini-Mental State Examination（MMSE）score,they were divided into the cognitive impairment group and the non-cognitive impairment group to explore the relationship between NLR,PLR and PSCI.This study further explored the therapeutic mechanism of NBP in VD mice induced by repeated cerebral ischemia-reperfusion（RCIR）.The relationship between Nrf2 and microglia proliferation and TLR4/MyD88/NF-k B pathway was elucidated in order to provide new ideas for clinical diagnosis and treatment of VD.Part One Correlation between NLR,PLR and post-stroke cognitive impairment in patients with first-episode cerebral infarctionObjective:To investigate the relationship between neutrophil/lymphocyte ratio（NLR）,platelet/lymphocyte ratio（PLR）and post-stroke cognitive impairment in patients with first-stroke cerebral infarction.Methods:84 patients with first-onset cerebral infarction hospitalizedin the Department of Neurology of Hebei General Hospital from March 2020 to December 2021 were enrolled in this study.General informationon admission was collected,including gender,age,height,weight and laboratory examination results.Laboratory results included neutrophil count,lymphocyte count,platelet count,uric acid（UA）,homocysteine（Hcy）,triglyceride（TG）,total cholesterol（TC）,high density lipoprotein（HDL）,low density lipoprotein（LDL）,apolipoprotein A1（Apo A1）and apolipoprotein B（Apo B）.NLR and PLR were calculated,NLR=neutrophils/lymphocytes,and PLR=platelets/lymphocytes.At 3 months follow-up,MMSE scale was used to score,the subjects were divided into post-stroke cognitive impairment（PSCI）and post-stroke no cognitive impairment（PSNCI）.Independent sample T test or?2 test was used to compare the clinical data of the two groups.Pearson correlation analysis was used to analyze the relationship between PLR,NLR and cognitive dysfunction in patients with cerebral infarction.Logistic regression analysis was used to determine the possible influencing factors of PSCI in patients with initial cerebral infarction.Results:A total of 84 patients with first-episode cerebral infarction were included in this study,including 50 males（59.52%）and 34 females（40.48%）.PSCI 40 cases（47.62%）.Compared with the PSNCI group,there were no significant differences in age,gender,BMI,hypertension,diabetes,coronary heart disease,smoking history,drinking history,fasting blood glucose,TC,TG,HDL,LDL,UA,Apo A1 and Apo B（P>0.05）;There were statistically significant differences between Hcy,NLR and PLR groups（P<0.05）.Pearson correlation analysis showed that the severity of PSCI was positively correlated with PLR,NLR and Hcy（P<0.05）.Multivariate Logistic regression analysis showed that increased PLR level was an independent risk factor for PSCI（P<0.05）.Conclusions:1.The levels of NLR,PLR and Hcy were positively correlated with the degree of cognitive impairment in PSCI patients.PLR is an independent risk factor for PSCI.2.Inflammatory response is involved in the pathological process of PSCI,and PLR may be an early serum inflammatory marker of PSCI.Part Two Protective effects of Dl-3-n-butylphthalide on cognitive function,hippocampal histopathology and ultrastruc-ture in mice with vascular dementiaObjective:Repeated cerebral ischemia-reperfusion（RCIR）method was used to establish a VD mouse model.The mice were given 80mg/Kg or120mg/Kg NBP by gavage for 4 weeks.Using Morris Water maze（MWM）,Hematoxylin-Eosin staining（HE）and transmission electron microscopy（TEM）to observe the cognitive function,the histological and pathological changes and ultrastructural changes in the hippocampus of mice in each group.Methods:Identified wild-type（WT）male mice（background:CD1/ICR）aged 8-12w weeks and weighing 25-35g were randomly divided into Sham group,RCIR group,NBP₈₀group and NBP₁₂₀group.The Sham group and RCIR group were given the same volume of experimental corn oil once a day for 4 weeks.Morris water maze was conducted on the 23rd day after RCIR surgery.The escape latency,swimming speed and time spent in the target quadrant（%）of mice in each group were recorded to evaluate the spatial learning and memory ability.After the Morris water maze,4 mice were randomly selected and fixed by 4%paraformaldehyde perfusion.The brain tissues were conventionally embedded in paraffin,sectioned,stained with HE,and the histopathological changes of hippocampal CA1 region were observed under a microscope.In addition,3 or 4 mice from each group were randomly selected and fixed with electron microscope perfusion fixator to prepare electron microscope tissue samples in the hippocampal CA1 region,and the ultrastructural changes of neurons in the hippocampal CA1 region were observed by transmission electron microscopy.Results:1.In the stage of place navigation test phase,the escape latency of mice in each group was gradually shortened.On days 2-5,the escape latency in the RCIR group was significantly longer than that in Sham group（P<0.01）.NBP significantly shortened the escape latency in VD mice at day 3-5（P<0.01）.We also found that the escape latency of VD mice treated with 120mg/kg NBP was similar to that of Sham mice,without significant statistical difference.On day 4 and 5,the latency of escape in NBP₁₂₀group was significantly shorter than that in NBP₈₀group（P<0.01）.There was no difference in swimming speed among the four groups,suggesting that the cognitive difference of mice in each group was not affected by limb motor function.In the spatial probe test phase,compared with the Sham group,the time of the RCIR group to spent in the target quadrant was significantly shorter（P<0.001）.With NBP treatment,the space exploration ability of VD mice was improved,and the time spent in the target quadrant was significantly longer than that of RCIR group（P<0.05）.2.In the Sham group,the pyramidal neurons in hippocampal CA1 aera were tightly ranked in order.The neurons were clear in shape and moderate in size with normal microstructure.In contrast,in the RCIR group,neuronal shrinkage and loss,and pyknosis of the cytoplasm were observed.NBP could reverse the presence of injured neurons in the hippocampal CA1 region.3.The nucleus of hippocampal neurons were large,round,the nuclear membrane structure was complete,and the chromatin was uniformly distributed in fine granular form;while in the RCIR group,the hippocampal neurons were irregular in shape,the chromatin agglomerated,nuclear membrane and organelles dissolved or disappeared.After NBP treatment,neuron damage in the hippocampus was reduced.Conclusions:1.In this study,The Mouse model of VD was prepared by RCIR method.MWM test showed that the model was stable and reliable.2.NBP could improve the cognitive function of VD mice,reduce the pathological damage and improve the ultrastructure of hippocampus in VD mice,and has a neuroprotective effect.Part Three Effects of Dl-3-n-butylphthalide on apoptosis and autophagy in vascular dementia miceObjective:To investigate the effect of NBP on neuronal apoptosis and autophagy.TUNEL staining was used for quantitative analysis of apoptotic cells and Western blot（WB）was used to detect B-cell lymphoma-2（Bcl-2）,Bcl-2 associated X protein（Bax）,microtubule-associated protein 1 light chain3（LC3）and p62,to further clarify the regulation of NBP on apoptosis and autophagy of hippocampal neurons in VD mice.Methods:The expression of TUNEL cells in the hippocampal CA1region was observed under an optical microscope,and the degree of hippocampal injury was evaluated by apoptotic index.In addition,6 mice were randomly selected from each group.After anesthesia,the hippocampus was decapitated,and the expression levels of apoptosis-related proteins Bcl-2and Bax,as well as autophagy related proteins LC3 and p62 were detected by Western blot.Results:1.TUNEL staining showed that there were more TUNEL-positive cells in hippocampal CA1 region of RCIR group（apoptosis index:P<0.001）,NBP significantly reduced the number of apoptotic cells（P<0.001）,and NBP₁₂₀group was superior to NBP₈₀group in improving apoptosis index（P<0.05）.2.The expression of Bax and Bcl-2 was detected by Western blot.Low levels of Bax and high levels of Bcl-2 were found in the Sham group.Moreover,increased levels of Bax and reduced levels of Bcl-2 were found in the RCIR group than in the Sham group（P<0.001）.NBP was able to reverse these levels.3.Compared with the Sham group,LC3Ⅱ/Ⅰlevel increased and P62level decreased in the RCIR group（P<0.05）,moreover,LC3Ⅱ/Ⅰand p62 levels was further increased/decreased after NBP treatment（P<0.05）.Meanwhile,compared with NBP₈₀group,the promotion of NBP₁₂₀group were more significant（P<0.05）.Conclusions:1.The expression level of Bcl-2 was decreased in the hippocampus of VD mice,while the expression level of Bax was increased,and the bcl-2/Bax ratio was significantly decreased,suggesting that the apoptosis of hippocampal neurons in VD mice was overactivated.NBP has anti-apoptotic effect and can reverse these changes.2.LC3Ⅱ/Ⅰexpression increased and P62 level decreased in hippocampus of VD mice,suggesting active autophagy.After NBP application,LC3Ⅱ/Ⅰexpression was further increased,p62 level was further decreased,and autophagy was further activated.These results suggest that NBP can play a neuroprotective role in VD mice by activating autophagy.3.It is suggested that NBP could inhibit apoptosis and regulate autophagy in a dose-dependent manner to a certain extent.Part Four Effects of Dl-3-n-butylphthalide on oxidative stressand Nrf2 pathway in hippocampus of vascular dementia miceObjective:Biochemical methods were used to detect superoxide dismutase（SOD）,malondialdehyde（MDA）and 8-isoprostanes F2α（8-iso PGF2α）in mice of different groups.Western blot and quantitative real-time polymerase chain reaction（q RT-PCR）were used to detect neuclear-Nrf2and NADPH:quinine oxidoreductase-1（NQO1）and Heme Oxygenase-1（HO-1）protein and gene levels.Methods:8 mice were randomly selected from each group.After anesthesia,the hippocampus was cut off and the hippocampus homogenate was prepared.SOD activity and MDA content in the hippocampus of mice in each group were determined according to the kit instructions.In addition,10mice were randomly selected from each group,and the level of 8-iso PGF2αin mice hippocampus was detected by ELISA.Six mice in each group were randomly selected.After anesthesia,the hippocampus was decaptured,and the expression levels of Nrf2,HO-1 and NQO1 in the hippocampus of mice in each group were detected by Western blot and q RT-PCR.Results:1.Compared to the Sham group,we found that the SOD activity was significantly decreased（P<0.001）,the MDA content and the level of 8-iso PGF2αwere markedly increased in the RCIR group（P<0.001）.NBP treatment was effective in stimulating the activities of SOD and inhibiting the production of MDA and 8-iso PGF2α（P<0.05）.For SOD activity and 8-iso PGF2α,there was a significant difference between the NBP₈₀group and the NBP₁₂₀group（P<0.05）.2.Western blot and q RT-PCR analysis showed that the levels of neuclear-Nrf2,HO-1 and NQO1 in RCIR group were higher than those in the Sham group（P<0.05）,and the expression levels in NBP group were further increased（P<0.05）.The increase in NBP₁₂₀group was more significant（P<0.05）.We further studied the expression changes of some other downstream effectgenes of Nrf2.Compared with the RCIR group,m RNA expression levels of GCLM,GSTM1,PRDX1 and TXNRD1 were significantly increased after NBP treatment after RCIR injury（P<0.05）.The expressions of GCLM,GSTM1,PRDX1 and TXNRD were more significantly increased in NBP₁₂₀group（P<0.05）.Conclusions:1.RCIR injury can induce the occurrence of oxidative stress in the hippocampus,and NBP can reduce the level of oxidative stress in the hippocampus of VD mice.2.NBP can activate Nrf2/HO-1 pathway and increase the expression of neuclear-Nrf2,HO-1 and NQO1 in hippocampus after RCIR injury.Part Five Effects of Dl-3-n-butylphthalide on neuroinflamma-tion in hippocampus of vascular dementia miceObjective:To further determine whether NBP plays a neuroprotective role through anti-inflammatory in VD mice.The levels of TNF-α,IL-6 and IL-1βin the hippocampus of mice in each group were determined by enzyme-Linked immunosorbent assay（ELISA）.Ionized calcium binding Adapter molecule（Iba-1）proliferation and protein levels of TLR4/MyD88/NF-κB signaling pathway were detected by immunofluorescence label and Western blot.Methods:Four mice in each group were randomly selected and given5-bromodeoxyuridine（Brd U）intraperitoneal injection to mark the prolifera-tion of microglia cells.After death,the brain tissues were embedded in paraffin and sected for Iba-1⁺/Brd U⁺immunofluorescence double labeling.In addition,10 mice from each group were randomly selected,and the levels of TNF-α,IL-6 and IL-1βin the hippocampus of mice were detected by ELISA.The protein levels of TLR4/MyD88/NF-κB pathway and Iba-1 were detected by Western blot.Results:1.Compared with Sham group,TNF-α,IL-6 and IL-1βlevels in RCIR group were significantly increased（P<0.001）,and excessive elevation of PICs was significantly inhibited in NBP group(especially NBP₁₂₀group)after 4weeks of NBP treatment（P<0.05）.The expression of PICs in NBP₈₀group and NBP₁₂₀group was statistically different（P<0.05）.2.RCIR injury promoted TLR4/MyD88/NF-κB signaling（P<0.001）,while NBP could reverse the promotion（P<0.05）.In addition,the inhibition of NBP for TLR4/MyD88/NF-κB pathway was more obvious in the NBP₁₂₀group（P<0.05）.3.Compared to the Sham group,the RCIR group showed an increased number of Iba-1⁺/Brd U⁺cells（P<0.001）.NBP reduced microglial regeneration（P<0.05）.While the levels of Iba-1 protein in the RCIR group were higher the Sham group（P<0.001）,lower levels were observed in the NBP group（P<0.05）.Conclusions:1.Microglia were activated and proliferated significantly in VD mouse model.NBP could significantly inhibit the proliferation of microglia and reduce the level of Iba-1.2.The increased expression of PICs in VD mice suggested that hippocampal injury is accompanied by neuroinflammation.NBP can reduce the production of TNF-α,IL-6 and IL-1βin the hippocampus of VD mice,and reduce the occurrence of central inflammatory cascade.3.The TLR4/MyD88/NF-κB pathway was activated in VD mice,and NBP inhibited the overactivation of TLR4/MyD88/NF-κB pathway.Part Six Dl-3-n-butylphthalide improves cognitive function ofvascular dementia mice via Nrf2 pathwayObjective:Nrf2 knockout(Nrf2^-/-)mice were used to further explore whether the antioxidant,anti-apoptotic and anti-inflammatory effects of NBP on VD mice were mediated by Nrf2 pathway.Methods:Grouping and administration of WT mice were the same as in Part two.Male mice with Nrf2^-/-genotype（background:CD1/ICR）,aged 8-12weeks and weighing 25-35g,were randomly divided into Nrf2^-/-+RCIR group and Nrf2^-/-+NBP₁₂₀group.On the 2nd day after surgery,NBP₁₂₀group was given NBP intragastric administration at a dose of 120mg/kg,and RCIR group was given equal volume of experimental corn oil intragastric administration once a day for 28 days.Brd U was administered as part five,with 4 mice in each group.Morris water maze,HE staining,TEM,TUNEL staining,immunofluorescence double label,ELISA and Western blot were used to detect WT and Nrf2^-/-mice.Results:1.Western blot anlysis showed that neuclear-Nrf2,HO-1 and NQO1protein levels were consistently low in Nrf2^-/-mice.Compared with WT+RCIR group,Nrf2^-/-+RCIR group significantly reduced the expression of neuclear-Nrf2,HO-1 and NQO1 proteins（P<0.05）.Meanwhile,depletion of Nrf2 weakened the up-regulation of Nrf2,HO-1 and NQO1 by NBP.In WT mice,NBP significantly improved MDA content induced by RCIR.Compared with WT+RCIR group,MDA content in Nrf2^-/-+RCIR group was further increased（P<0.001）.In Nrf2^-/-mice,the anti-oxidative stress effect of NBP was weakened,and there were no significant differences in SOD activity,MDA and 8-iso PGF2αcontent between Nrf2^-/-+NBP₁₂₀group and Nrf2^-/-+RCIR group（P>0.05）.Compared with WT+RCIR group,MDA content in Nrf2^-/-+RCIR group was significantly higher（P<0.001）.MWM found that in WT mice,NBP significantly shortened the escape latency and increased the time spent in the target quadrant of VD mice.However,NBP did not reverse these learning and memory disorders in Nrf2^-/-mice.HE staining and ultrastructural analysis of hippocampus showed that NBP significantly reduced the necrosis and death of neuron cells in WT+RCIR mice.Compared with WT mice,neuronal degeneration and necrosis of Nrf2^-/-mice were further aggravated after RCIR injury,and neuronal injury of Nrf2^-/-+VD mice was not significantly improved after NBP treatment.3.TUNEL staining showed that in WT mice,compared with RCIR group,the number of apoptotic cells after NBP treatment was significantly reduced（apoptosis index:P<0.01）,which was consistent with the results of the second part of the study.The number of apoptotic cells in Nrf2^-/-+RCIR mice was not significantly decreased after NBP treatment（P>0.05）.4.In WT mice,NBP significantly inhibited the regeneration of hippocampal microglia and decreased the level of Iba-1 protein in VD mice,but the inhibition of NBP on Nrf2^-/-+RCIR mice was blocked（P>0.05）.In WT mice,TNF-α,IL-6 and IL-1βlevels in hippocampal tissue of VD mice after NBP treatment were significantly decreased;in Nrf2^-/-mice,the levels of TNF-α,IL-6 and IL-1βin the hippocampus of Nrf2^-/-+NBP₁₂₀group were not significantly decreased（P>0.05）.In WT mice,RCIR injury promotes TLR4/MyD88/NF-κB signaling,and NBP inhibits overactivation of this pathway.However,in Nrf2^-/-mice,the inhibition of NBP disappeared completely（P>0.05）.Conclusions:1.The antioxidant,anti-inflammatory and anti-apoptotic neuroprotective effects of NBP on VD mice were all realized via Nrf2 signaling pathway.2.This study confirmed for the first time that NBP regulates the TLR4/MyD88/NF-κB pathway through Nrf2 in RCIR-induced VD mouse model to play a neuroprotective role.