A Meta-analysis Of Homocysteine And In Vitro Experiment Of Its Effect On BV2 Cells In Alzheimer’s Disease

Part 1.Blood and CSF homocysteine levels in Alzheimer’s disease: a metaanalysis and meta-regression of case-control studiesObjective:Homocysteine(Hcy)is a sulfur-containing non-essential amino acid and produced as an intermediate product in the synthesis of methionine and cysteine.It has been widely reported that the elevated level of blood Hcy is an independent risk factor for Alzheimer’s disease(AD).However,there is much more controversy about the hot topic on the levels of Hcy in AD.This study will pool the data of published studies regarding the levels of Hcy in blood and cerebrospinal fluid(CSF)in AD,and investigate whether and what degree the Hcy levels in blood and CSF were elevated in AD patients compared with healthy controls to explore the factors related to the elevated Hcy levels in AD.Methods:1.A systematic literature search was carried out to collect data from case-control studies regarding Hcy and AD in the Pub Med and Embase databases up to February 5,2021.2.Screening of relevant studies.Note Express reference management software was used to screen relevant literature and to further remove duplicates.After the preliminary screening of the title and abstract,and the further screening by reading of the full-text papers,all studies meeting the inclusion criteria were identified and the Hcy concentrations and other related data were extracted.The NewcastleOttawa Quality Assessment Scale(NOS)was used to assess the quality of the included studies.3.For each included study,the ratio of AD to control in mean Hcy concentration(Ro M)and its 95% CI was calculated,then all values were combined using a random-effect model to derive a pooled result of Ro M and its 95% CI.4.In the meta-regression analysis,the standard average difference(SMD)was calculated and used as a single variable for analyzing blood follicle acid,vitamin B12 and MMSE score.The ratio of the mean concentration(log Ro M)as the response variable Y,with corresponding values of the folic acid,vitamin B12 and MMSE score as the self-variable X,the combination factor regression model.Results:1.The initial search in both the databases yielded 218 hits,added 14 from handsearching,with a total of 232 publications.After removing duplicates,we screened titles and abstracts,and excluded 93 studies.After carefully reading and reviewing full-text articles,36 studies that did not meet the inclusion criteria were excluded.At last,we identified a total of 35 studies,containing 2172 AD patients and 2289 healthy controls.2.Among all the studies included,there were 4 studies reported the Hcy levels in CSF and 33 in blood(24 in plasma and 9 in serum,2 in both CSF and plasma),11 reported MMSE scores,19 reported the levels of vitamin B12 and 19 reported the levels of blood folate.3.In the estimation of the degree of the elevated level of blood Hcy by use of Ro M,the pooled results showed that the Ro M of AD/controls in the blood Hcy level was 1.32(95% CI,1.25–1.40,p<0.001),with large heterogeneity across studies(I2=81.4%,p<0.001),indicating that AD patients have an over 30% increased blood Hcy level.4.The subgroup analysis showed that the Hcy level significantly differed between AD and controls in plasma(Ro M,1.31;95% CI,1.22–1.40,p<0.001;I2=84.5%,p<0.001)and in serum(Ro M,1.37;95% CI,1.28–1.47,p<0.001;I2=49.6%,p<0.05),The subgroup result remained significant and the value of effect size varies slightly.While in the serum analysis heterogeneity decreased obviously,and the change is small in the plasma analysis.This subgroup analysis partly explains the source of heterogeneity.5.To assess whether the CSF Hcy level significantly differed between patients with AD and healthy controls,4 studies consisting of 150 patients and 149 controls,were included.The pooled result showed that the pooled Ro M of AD to controls in the CSF Hcy level was 1.12(95% CI,0.90–1.39,p=0.293),indicating that the CSF Hcy level did not alter in AD patients compared with the healthy controls,and with considerable heterogeneity among studies(I2=69.4%,p = 0.02).6.To further explore sources of heterogeneity,a stepwise univariate meta-regression was performed to examine the effects of several key study population characteristics on the effect size.The results of meta-regression analyses revealed that the blood folate level was inversely associated with the blood Hcy level(95% CI,-0.9163 to-0.1810,p=0.006).No statistic significant associations were observed among MMSE and vitamin B12(95% CI,-0.3709 to 0.2831,p=0.084;95% CI,-0.8756 to 0.5044,p=0.578).7.For evaluation of the study quality,the NOS scores were high with values ranging from 5 to 9.We used the Begg’s and Egger’s tests to assess publication bias.In the comparison of blood Hcy levels between patients with AD and controls,there was no evidence of publication bias(Begg’s test: p=0.15,Egger’s test: p=0.07),which was also confirmed by the symmetry of the funnel plot.In the comparison of CSF Hcy level between patients with AD and controls,we also did not find potential publication bias(Begg’s test: p=0.73,Egger’s test: p=0.91,as well as the symmetry of the funnel plot).The sensitivity analysis demonstrated that the summarized estimate was not substantially altered by removal of a single study.Conclusions:1.Regardless of dementia severity,the blood Hcy level is significantly higher in AD patients than in control subjects,and there is an approximate one-third increase in blood Hcy in AD patients.2.The elevated Hcy level in AD patients was robustly associated with the decreased level of blood folate in AD,but neither with that of blood vitamin B12 nor with the degree of dementia,indicating that the higher the Hcy concentration in the blood,the lower the levels of folate acid in blood.Alzheimer’s patients can reduce the concentration of homocysteine by supplementing with folate instead of vitamin B12,thereby improving the AD process.3.The level of Hcy in CSF did not alter significantly in AD compared with healthy controls.Part 2 Experimental studies of inflammatory factors induced by homocysteine in cultivated BV2 cells and their expressions in AD model miceObjective:1.To observe the effect of exogenous homocysteine on the expression levels of IL-1β in in vitro-cultivated BV2 cells.2.To explore the mechanisms of Hcy’s effect on inflammatory cytokines.3.To investigate the changes of serum pro-inflammatory cytokines in transgenic AD mice,for the purpose of providing preliminary data for exploring the association of homocysteine with inflammatory factors in AD.Methods:1.The Western Blot technique was used to detect the IL-1β protein expression level in BV2 cells after the homocysteine treatment.2.The ELISA was used to detect the IL-1β protein expression level in cultured microglia.3.The Western Blot technique was also used to detect the expression of TREM-1.4.The luminex liquid-phase chip microarray was used to detect the levels of the serum IL-1β and other cytokines such as TNF-α,IL-2,IL-4,IL-6,IL-16,IL-10,INF-γ molecules in mice.Results:1.The Western Blot results showed that IL-1β protein expression levels were significantly increased after the Hcy treatment.2.ELISA results showed that IL-1β protein expression levels were significantly increased when treating BV2 cells at 36 h using 100 μM homocysteine compared to the control group.3.The Western Blot results showed that the concentrations of homocysteine were 100 μM,200 μM and 300 μM compared with the non-used lipid polysaccharide pretreatment group,the expression of NLRP3 inflammasome increased.Compared to non-homocysteine treatment groups,no statistical differences were observed in NLRP3 inflammasome expression.4.The Western Blot results showed that homocysteine inhibited the expression of TREM-1 on BV2 cells,and the higher the concentration of homocysteine,the greater the inhibitory effect.5.Comparison of 3×Tg AD transgenic mice,2×Tg AD tri-transgenic mice and wild mice serum IL-1β,TNF-α,IL-2,IL-4,IL-6,IL-16,IL-10,INF-γ protein expression levels by the luminex liquid phase suspension array.Using Prism statistical software statistics showed the serum IL-1β protein expression level in 3×Tg AD mice was significantly higher than the wild mice(591.18±118.68,p<0.05),while there was no statistically significant of TNF-α,IL-2,IL-4,IL-6,IL-16,IL-10,INF-γ protein expression levels in the transgenic mice.Conclusion:1.Hcy promotes increased IL-1β secretion in in vitro-cultivated BV2 cells,indicating that Hcy may lead to the increased excretion of inflammatory cytokines in light of in vitro evidence.2.Hcy has ability to activate the LPS-triggered NLRP3 inflammasome.3.TREM-1 molecules associated with NLRP3 inflammation may not be involved in the activation of the Hcy-induced NLRP3 inflammasome.4.As a common AD model,with a significant increase in blood IL-1β level,the 3×Tg AD model simulated inflammatory factor abnormalities in AD patients.However,whether the increase in the IL-1β level is directly related to the rise in Hcy needs further experimental validation.