Study On The Mechanism Of Baicalin On Cognitive Function,White Matter Damage,and Neuroinflammation In Vascular Dementia

As life expectancies and the aging population across the world continue to rise,dementia has emerged as one of health challenge for humanity.According to the World Health Organization,dementia affects nearly 50 million people worldwide,and by 2050,this number will triple,placing a huge burden on global public health.Vascular dementia(VD)is a cognitive disorder caused by reduced cerebral blood flow and is widely considered to be the second most common type of dementia.However,the pathogenesis of VD is complex and not yet fully understood,and there is still a lack of effective therapeutic drugs.Therefore,actively exploring the pathogenesis of VD and finding effective therapeutic drugs for VD is a hot issue in medical research.Cerebral small vessel disease(CSVD),a primary vascular contributor to cognitive impairment in the elderly,contributes to 45% of all cases of dementia.As a result of its insidious onset and unknown etiology and pathogenesis,the prevention and treatment of CSVD have not yet made a breakthrough.Recent studies have revealed that systemic inflammation plays a crucial role in the pathogenesis of CSVD,the systemic immune-inflammation index(SII)has been proposed as a relatively new systemic inflammatory biomarker that more comprehensively reflects the systemic immune response and inflammatory status.In addition,several studies have found an association between elevated SII levels and the development of different imaging features of CSVD,and a correlation between elevated SII levels and cognitive impairment in a variety of different pathological states.However,no studies have yet examined the relationship between SII,CSVD,and cognitive impairment simultaneously.Chronic cerebral hypoperfusion(CCH)is considered as one of the main pathological mechanisms of CSVD.Its induced cerebral ischemia and hypoxia can cause demyelinating changes in white matter,which is a key factor in the degenerative process leading to cognitive decline and dementia.Secondary neuroinflammatory response to CCH accelerates the loss and limits the regeneration of oligodendrocytes,leading to progressive demyelination and insufficient remyelination in the white matter.Thus,promoting remyelination and inhibiting neuroinflammation may be an effective therapeutic strategy.Studies have shown that the Wnt signaling pathway plays an important role in both myelin formation and neuroinflammatory processes.GSK-3β is a critical kinase in the Wnt signaling pathway that negatively regulates the entry of β-catenin into the nucleus,which modulates the expression of Wnt target genes.In addition,during GSK3β inhibition,the increasing β-catenin levels appear to reduce the nuclear translocation of the pro-inflammatory transcription factor nuclear factor-kappa B(NF-κB).However,little is known about the role of Wnt/β-catenin signaling and NF-κB signaling in CCH-associated neuroinflammation and demyelination.Baicalin(BAI)is a flavonoid compound that exhibits multiple pharmacological functions,including anti-inflammatory,anti-tumor,anti-apoptotic,and anti-oxidant.BAI has low toxicity and is well tolerated when administered orally in both humans and rodents.Due to its ability to cross the blood-brain barrier into the cerebrospinal fluid,BAI exhibits strong cognitive protective effects in various neurological injury models.However,it remains to be explored whether BAI suppresses inflammation and promotes remyelination during CCH.On the basis of the above,to explore the associations of the SII with total CSVD burden and cognitive impairment,201 consecutive patients with complete clinical and laboratory data were included in this study.In addition,we further established a model of VD induced by CCH in rats by occluding the two common carotid arteries bilaterally to investigate whether postoperative application of BAI could alleviate CCH-induced cognitive impairment.And whether this neuroprotective effect was achieved by inhibiting neuroinflammation and promoting remyelination.Furthermore,we examined whether BAI’s protective effects on CCH are associated with the activation of Wnt/β-catenin and inhibition of NF-κB signaling.Part one SII is associated with cerebral small vessel disease burden and cognitive impairmentObjective: To explore the associations of the SII with total CSVD burden and cognitive impairment.Methods: We enrolled 201 consecutive patients in the retrospective study with complete clinical and laboratory data.Patient demographics,general clinical information and blood chemistries were collected and cognitive function was assessed using the MMSE score.Based on Mini-Mental State Examination(MMSE)scores and years of education,the patients were divided into cognitive impairment group and no cognitive impairment group.The SII was calculated as platelet count × neutrophil count / lymphocyte count.The total CSVD burden was assessed based on magnetic resonance imaging,which was considered severe when the score exceeded 2.We performed 2-tailed Student’s t test,Mann-Whitney U test,χ2 test,Logistic regression models,Spearman’s correlation,and mediation analysis to evaluate the associations of SII with CSVD burden and cognitive impairment.Results:1.A total of 201 patients were enrolled in the current analysis.The cognitive impairment group included 68 patients(33.83%),and the no cognitive impairment group included 133 patients(66.17%).Compared with patients in the no cognitive impairment group,patients with cognitive impairment were older,less educated,had a higher proportion of combined hypertension,lower HDL-C levels,and higher t Hcy levels(all P<0.01),and patients in the cognitive impairment group had higher levels of SII and CSVD total load scores(all P<0.001).2.After adjustment for confounding factors in the multivariate binary logistic regression model,SII(OR=1.003;95%CI:1.002-1.004;P<0.001)or severe CSVD burden(OR=2.571;95%CI:1.212-5.456;P=0.014)was significantly associated with the risk of cognitive impairment.Moreover,SII was significantly related to the severity of the CSVD burden(OR=1.002;95%CI:1.001-1.003;P=0.005).3.Based on SII tertiles,the eligible patients were divided into 3 groups(Tertile 1: <414.06,Tertile 2:414.06-571.37,Tertile 3: >571.37).Patients in both the Tertile2 and Tertile3 groups had an elevated risk of cognitive impairment compared to the Tertile1 group,with a linear trend(P<0.001),and the Tertile3 group was significantly associated with total severe CSVD burden(P<0.001).4.Spearman’s correlation analyses revealed that SII levels were negatively associated with MMSE scores(rs=-0.391,P<0.001),and positively associated with the total CSVD burden score(rs=0.361,P<0.001).5.Mediation analysis demonstrated that the presence of severe CSVD burden partly mediated the effect of higher SII levels on cognitive impairment.Conclusions:1.The increased level of SII is an independent risk factor for cognitive impairment.As SII levels increase,the risk of cognitive impairment increases.2.The increased level of SII is an independent risk factor for severe CSVD burden.As the SII level increases,the severity of the total CSVD burden increases.3.The presence of severe CSVD burden partly mediated the effect of higher SII levels on cognitive impairment.Part two BAI improved cognitive impairment and hippocampal pathological damage in VD rats induced by CCHObjective: Using CCH leading to VD as an entry point for the study,we investigated the effects of BAI on cognitive function and hippocampal histopathology in CCH rats.Methods: Male Sprague-Dawley(SD)rats of SPF grade were assigned to four groups: Sham group,CCH+vehicle group,CCH+BAI-50 group and CCH+BAI-100 group.The model of VD induced by CCH was established by bilateral common carotid artery occlusion(BCCAO).The CCH+BAI-50 group and CCH+BAI-100 group were given 50 mg/kg and 100 mg/kg BAI solution by gavage once daily for 28 days,respectively.On the day following the end of the administration,spatial learning and memory behaviors were evaluated using the MWM test,including a 5-day place navigation test and a1-day spatial probe test.Three rats were chosen from each group shortly after the MWM test.These rats were transcardially perfused for brain extraction,and paraffin embedding and hippocampal parts were sliced.The morphology of hippocampal CA1 area neurons was examined by Nissl staining.Results:1.During place navigation testing,the escape latency was significantly decreased during training,and statistically significant differences were observed for the main effects of the day(F=50.484,P<0.001)and group(F=12.394,P<0.001).However,there was no significant interaction between the two(F=0.639,P>0.05).From days 1-5,CCH+vehicle group rats displayed significantly longer escape latencies than the Sham rats(day 1-2: P<0.05;day3: P<0.001;day 4: P<0.01;day 5: P<0.001).After treatment with 50mg/kg BAI,the escape latencies in rats were reduced compared with the CCH+vehicle group(day 3-4: P<0.05;day 5: P>0.05).The escape latencies were shortened more significantly in rats after treatment with 100mg/kg BAI(day 3-4: P<0.01;day 5: P<0.001).2.The results of the spatial probe test showed that the rats in the CCH+vehicle group stayed in the target quadrant for a shorter period of time(P<0.001)and crossed the platform less frequently(P<0.001)than the rats in the Sham group.Compared with the CCH+vehicle group,the rats in CCH+BAI-100 group spent more time in the target quadrant(P<0.01)and crossed the platform more frequently(P<0.05).3.Nissl staining revealed a normal neuronal structure of the hippocampal CA1 pyramidal cells in rats in the Sham group,with moderately sized neurons that were tightly arranged.Conversely,tissue damage was observed in the CCH+vehicle group,as indicated by the presence of pyramidal cell remnants,loosely arranged neurons,deterioration of the neuronal structure,and neuronal shrinkage.The administration of BAI partly reversed the morphological changes and increased neuronal survival.Conclusions:1.The rat model of VD induced by CCH was successfully established by BCCAO.This model is stable and reliable and can be used to explore the pathogenesis of VD and find effective therapeutic drugs.2.BAI mitigated hippocampal neuronal damage,which in turn improved cognitive impairment caused by CCH.Part three Effects of BAI on white matter injury and remyelination in rats with vascular dementiaObjective: White matter injury induced by CCH is closely related to the development of VD.This part of the study further investigated the protective effect of BAI on white matter injury in CCH-induced VD rats and further observed whether BAI could promote remyelination.Methods: The animals were grouped and administered as in part two.To label proliferating cells,5’-bromo-2’-deoxyuridine(Brd U,50mg/kg)was administered intraperitoneally to three rats in each group once per day for 14 successive days beginning at 24 h following operation.Shortly after the MWM test 28 days postoperatively,three rats injected with Brd U were transcardially perfused for brain extraction,and paraffin embedding and the corpus callosum parts were sliced.Luxol Fast Blue(LFB)staining was used to assess white matter integrity in each group,and immunofluorescence staining was used to observe the number of Olig2+ and Olig2+/Brd U+ cells in the corpus callosum of each group.The remaining rats were subjected to fresh corpus callosum tissue sampling,and the expression of MBP and Olig2 proteins in each group was detected by western blot.Results:1.The results of LFB staining showed that myelin density in the corpus callosum of rats in the CCH+vehicle group was significantly lower than that in the Sham group(P<0.01),and MBP protein expression was also significantly decreased(P<0.01).However,BAI(100mg/kg)treatment significantly increased myelin density(P<0.01)and MBP protein expression(P<0.05).Although the intensity of LFB staining and MBP protein levels were higher in the CCH+BAI-100 group than in the CCH+BAI-50 group,there was no statistical difference(P>0.05).2.Immunofluorescence results showed that the number of Olig2+ cells in the corpus callosum of rats in the CCH+vehicle group was significantly less than that in the Sham group(P<0.01),and Olig2 protein expression was significantly decreased(P<0.01).Treatment with BAI(50mg/kg and100mg/kg)significantly increased the number of Olig2+ cells(P<0.05 and P<0.01).Olig2 protein expression level was also significantly increased by BAI(100 mg/kg)treatment(P<0.05).3.While the CCH+vehicle group had more Olig2+/Brd U+ cells than the Sham group,no significant differences were observed between the two groups(P>0.05).This endogenous response was amplified induced by BAI treatment(100 mg/kg)(P<0.01).Conclusions:1.CCH rats exhibited learning and memory deficits,accompanied by reduced myelin density in the corpus callosum and downregulated MBP protein expression levels,suggesting that CCH can successfully mimic white matter damage in VD.BAI treatment could reduce myelin loss and thus improve cognitive function.2.BAI promoted Olig2 protein expression in the corpus callosum and oligodendrocyte regeneration,which in turn promoted myelin formation and exerts neuroprotective effects.Part four Effect of BAI on white matter neuroinflammation in rats with vascular dementiaObjective: To observe the effect of BAI on the microglia phenotype in the corpus callosum of CCH-induced VD rats and whether it can inhibit neuroinflammation to exert neuroprotective effects.Methods: The animals were grouped and administered as in part two.Paraffin sections prepared in part three were applied,and the number of Iba1+cells in the corpus callosum of rats in each group was observed using immunofluorescence staining.Western blot was used to observe the expression of Iba1,i NOS and Arg-1 protein in the corpus callosum of rats in each group.The production and release of tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β)and interleukin-10(IL-10)were measured by enzyme-linked immunosorbent assay(ELISA)in the corpus callosum of rats in each group.Results:1.Immunofluorescence results showed that the number of Iba1+ cells was increased in the CCH+vehicle group of rats compared with the Sham group(P<0.001).However,BAI(50mg/kg and 100mg/kg)treatment significantly reduced the number of Iba1+ cells(P<0.01 and P<0.001,respectively).Consistent with the immunofluorescence staining results,western blot results further confirmed the increased expression of Iba1 in the CCH+vehicle group rats(P<0.001).After BAI treatment(100mg/kg),Iba1 protein expression was significantly downregulated(P<0.01).2.Western blot results showed that the expression of i NOS protein was significantly increased in the corpus callosum of CCH+vehicle rats compared with Sham group(P<0.01),while Arg-1 protein expression was not significantly changed(P>0.05).INOS protein expression was significantly down-regulated by BAI(100mg/kg)treatment(P<0.05),and Arg-1 protein expression was significantly up-regulated(P<0.001).In addition,Arg-1protein expression in the corpus callosum was higher in the CCH+BAI-100 group compared with the CCH+BAI-50 group(P<0.05).3.ELISA results showed that TNF-α and IL-1β levels were significantly higher in the CCH+vehicle group than in the Sham group(both P<0.001).BAI treatment significantly inhibited the production and release of TNF-α and IL-1β(both P<0.05,CCH+BAI-50 vs.CCH+vehicle;P<0.001 and P<0.01,respectively,CCH+BAI-100 vs.CCH+vehicle)and promoted the production of the anti-inflammatory factor IL-10(P<0.001,CCH+BAI-100 vs.CCH+vehicle).In addition,IL-10 levels were higher in the CCH+BAI-100 group compared with the CCH+BAI-50 group(P<0.05).Conclusions:1.BAI inhibited microglia polarization to the pro-inflammatory phenotype in CCH rats,inhibited the production and release of pro-inflammatory factors,and attenuated white matter injury.2.BAI could modify microglia polarization to the anti-inflammatory phenotype in CCH rats,promoting the production and release of anti-inflammatory factors.The favorable effects of BAI on remyelination and cognitive recovery could be partially attributed to a favorable microenvironment for oligodendrocyte regenerationPart five Effects of BAI on white matter Wnt/β-catenin and NF-κB signaling pathways in rats with vascular dementiaObjective: To observe the regulatory effects of BAI on Wnt/β-catenin and NF-κB signaling pathways in the corpus callosum of CCH-induced VD rats.Methods: The animals were grouped and administered as in part two.Western blot was used to observe the protein expression levels of GSK3β,p-GSK3β,β-catenin and NF-κB in the corpus callosum in each group of rats.Results:1.Western blot results showed that GSK3β protein expression in the corpus callosum of the CCH+vehicle group was not significantly different from that of the Sham group(P>0.05),and although the expression of p-GSK3β was slightly higher than that of the Sham group,this difference did not reach statistical significance(P>0.05).After BAI treatment(50mg/kg and100mg/kg),the p-GSK3β protein expression level was significantly increased(P<0.01 and P<0.001,respectively)and the p-GSK3β/GSK3β ratio was significantly up-regulated(P<0.05 and P<0.01,respectively).In addition,both total and nuclear β-catenin protein expressions were slightly elevated in the CCH+vehicle group compared with the Sham group,but the differences did not reach statistical significance(both P>0.05).100mg/kg BAI treatment significantly increased total β-catenin protein expression(P<0.05),while50mg/kg and 100mg/kg BAI administration significantly increased the expression of nuclear β-catenin protein(P<0.01 and P<0.001,respectively).In addition,the nuclear β-catenin protein expression level was higher in the CCH+BAI-100 group compared with the CCH+BAI-50 group(P<0.05).2.The protein of nuclear NF-κB was increased in the corpus callosum of rats following CCH compared with the Sham group(P<0.001),while BAI treatment(50mg/kg and 100mg/kg)ameliorated the enhancement(P<0.05 and P<0.001,respectively).Conclusions: BAI ameliorated cognitive impairment in CCH-induced VD rats through its pro-remyelination and anti-inflammatory capacities,possibly by activating the Wnt/β-catenin and suppressing the NF-κB signaling.