| Objective:The aim of this study is to explore the correlation among the cognition of the elderly,the level of VD and inflammation,based on the cross-sectional survey of the population and laboratory tests.And to analyze the anti-inflammatory mechanism of VD on the AD model in vitro which was induced by Aβ1-42 in PC12 cells.The aim is to provide a scientific basis for the anti-inflammatory about the prevention and treatment of cognitive impairment.Methods:Part 1: A case-control study on the cognition,the level of VD and inflammation in the elderly1.Evaluation of cognition Stratified cluster random sampling was used to selected elderly people aged 65 and older in dense communities including Yingze District,Xinghualing District,Jiancaoping District,Wanbolin District,Xiaodian District and Jinyuan District in Taiyuan to conduct a questionnaire survey and select the MCI group based on the Montreal Cognitive Assessment(Mo CA)scores.The control group was selected in the community during the same period which was matched in age,gender,and education level.2.Sample size The sample size was determined according to the 1:1 ratio case-control study sample size formula.According to the 2010-2013 China National Nutrition and Health Survey,the VD deficiency rate of the elderly was 39.15%,and α=0.05,β=0.10,OR=2.The calculation result was N=176 pairs.Taking into account the possibility of missing data,the sample size was determined to be 180 pairs,that is,360 persons.3.Blood sample collection and pre-test processing The blood samples of the elderly was collected fast under investigation in the morning,and put them into 5 ml EDTA anti-coagulation blood collection tubes,then separated plasma and red blood cells,and store them in a refrigerator at-80°C.4.Detection of plasma 25(OH)D3 level,inflammatory factors IL-1β and IL-18 levels Enzyme-linked immunosorbent assay was used to detect plasma 25(OH)D3,the levels of plasma inflammatory factors IL-1β and IL-18.The current internationally recommended classification standards for the degree of VD deficiency are: 25(OH)D3<10 ng/m L(< 25nmol/L)for severe deficiency,<20 ng/m L(< 50nmol/L)for lack,21~29ng/ m L(52~72nmol/L)is insufficient,≥ 30 ng/m L(≥ 75nmol/L)is sufficient.Part 2: Correlation between VD and inflammatory factors in elderly MCI group1.Subject180 participants who were included in the case group were selected as the research objects based on the previous case-control study.According to the lack of VD(25(OH)D3level <20 ng/m L),the investigators were divided into sufficient VD group and VD deficiency group.2.Blood sample collection and pre-test processing The blood samples of the elderly was collected fast under investigation in the morning,and put them into 5 ml EDTA anti-coagulation blood collection tubes,then separated plasma and red blood cells,and store them in a refrigerator at-80°C.Part 3: Study on the protective effect of 1,25(OH)2D3 on Aβ1-42 induced pyrolysis in PC12 cells The cell injury model was induced by 20 μM Aβ1-42.The experiment was established as a control group,model group(20 μM Aβ1-42),1,25(OH)2D3 low-dose group(1 n M1,25(OH)2D3 + 20 μM)Aβ1-42),1,25(OH)2D3 medium dose group(10 n M 1,25(OH)2D3 +20 μM Aβ1-42)and 1,25(OH)2D3 high dose group(100 n M 1,25(OH)2D3 + 20 μM Aβ1-42).CCK-8 was detected the survival rate of PC12 cells,AO/EB double fluorescence staining was detected cell membrane permeability,biochemical was detected LDH release rate,ELISA was detected the content of IL-1β and IL-18,cellular immunofluorescence,Western Blot were detected the expression of related proteins in the pyrolysis pathway.Part 4: Study on the mechanism of Aβ1-42 inducing pyrolysis of PC12 cells The si RNA transfection technology was applied to inhibit the expression of Caspase-1 protein.The experiment was set up as a control group,model group(20 μM Aβ1-42),Caspase-1-si RNA group(50 n M Caspase-1-si RNA + 20 μM Aβ1-42),NC-si RNA group(50 n M NC-si RNA + 20 μM Aβ1-42),1,25(OH)2D3 dose group(100 n M1,25(OH)2D3 + 20 μM Aβ1-42),Caspase-1-si RNA+1,25(OH)2D3 dose group(50 n M Caspase-1-si RNA + 100 n M 1,25(OH)2D3 + 20 μM Aβ1-42).Cellular immunofluorescence,Western Blot were detected the expression of related proteins in the pyrolysis pathway,AO/EB double fluorescence staining was detected cell membrane permeability,biochemical was detected LDH release rate,ELISA was detected the content of IL-1β and IL-18.Results:Part 1: A case-control study on the cognition,the level of VD and inflammation in the elderly A total of 360 elderly people in the community were included in this case-control study,with an average age of(73.06 ± 5.53)years old,including 181 women(50.3%)and179 men(49.7%).According to the Mo CA score,the elderly were divided into normal group(n=180)and cognitive impairment group(n=180).After comparing the Mo CA total scores and various scores of the two groups of elderly people,it was found that the MCI group’s scores in the Mo CA total score,visual space,naming,attention,language,abstraction,recall and orientation were all lower than normal group,the difference was statistically significant(P < 0.001).After comparing the demographic characteristics,lifestyle and health status of the two groups of elderly people,it was found that there were no statistically significant in gender(P = 0.916),age(P = 0.342),and education level(P = 0.070)between the normal group and the MCI group.In terms of other demographic characteristics,lifestyle and health status,there was no statistical significance between the two groups(P > 0.05).The average 25(OH)D3 concentration of 360 elderly people surveyed in this case-control study was 14.43(11.58,18.17)ng/m L,of which 16 subjects(4.4%)were severely deficient in 25(OH)D3,271 subjects(75.3%)were deficiency in 25(OH)D3,the adequate in 34 subjects(9.4%),the deficiency and severe deficiency accounted for 79.7%.The25(OH)D3 levels of the normal group and the cognitive impairment group were 14.78(11.81,20.46)ng/m L and 14.16(11.31,17.38)ng/m L respectively.After comparing the overall levels of the two groups,it was found that the MCI group was significantly lower than the normal group(P = 0.031).The median IL-1β level of the inflammatory factor was 6.66 pg/m L(5.96 pg/m L,7.41 pg/m L),of which the median IL-1β level in the normal group was 6.12 pg/m L(5.58 pg/m L,6.68 pg/m L),the median IL-1β level in MCI group was 7.20 pg/m L(6.63 pg/m L,7.95 pg/m L),compared with the normal group,the level of plasma inflammatory factor IL-1β in the elderly in MCI group was significantly increased(P < 0.001).The median IL-18 level of the overall inflammatory factor was229.88 pg/m L(206.63 pg/m L,259.42 pg/m L),and the median IL-18 level of the normal group was 214.51 pg/m L(186.61 pg/m L,247.63 pg/m L)./m L),the median IL-18 level in MCI group was 246.92 pg/m L(222.5 pg/m L,277.11 pg/m L).Compared with the normal group,the plasma inflammatory factor level of IL-18 was significantly higher(P <0.001).Spearman correlation was applied to analyze the total cognitive Mo CA scores of the elderly and the levels of various domains and 25(OH)D3.It was found that the plasma25(OH)D3 levels of the elderly were positively correlated with the Mo CA scores,the correlation coefficient r = 0.257,P < 0.001.The correlation results with the scores of each domains of cognition showed that in the abstract and recall part,cognition was positively correlated with 25(OH)D3 level,the correlation coefficient was r = 0.121,P =0.022;r = 0.131,P = 0.013.The plasma IL-1β level of the elderly was negatively correlated with the Mo CA score,the correlation coefficient r =-0.440,P < 0.001,and the IL-18 level was negatively correlated with the Mo CA score,the correlation coefficient r=-0.434,P < 0.001.The correlation results with the scores of various cognitive domains show that there were negative correlations in each cognitive domains.Multivariate logistic regression was used to analyze the risk factors of cognitive impairment.The results found that diabetes(OR = 1.987,95%CI: 1.054~3.744)was positively correlated with the risk of cognitive impairment,which was a risk factor for cognitive impairment;income level(OR = 0.823,95%CI: 0.684~0.990),BMI(OR =0.687,95%CI: 0.513~0.918)and VD(OR = 0.366,95%CI: 0.249~0.538)were negatively correlated with the risk of cognitive impairment,which were protective factors for cognitive impairment.In the multivariate logistic regression model,it was found that after adjusting for variables such as age,gender,education level,income level,BMI,hypertension,diabetes,hyperlipidemia,smoking,drinking,exercise,etc.,IL-1β was found to be in Q2,Q3,and Q4 were 4.221,7.848,and 12.619 times of Q1(Q2: OR3 =4.221,95% CI3: 2.031 ~ 8.773;Q3: OR3 =7.848,95% CI3: 3.740 ~ 16.465;Q4: OR3 = 12.619,95% CI3: 5.889 ~ 27.040),IL-18 in Q4 is 3.373 times that of Q1(Q4: OR3 = 3.373,95% CI3: 1.736 ~ 6.553),which were risk factors for cognitive impairment.Part 2: Correlation between VD and inflammatory factors in elderly MCI group180 participants who were included in the case group were selected as the research objects based on the previous case-control study.According to the deficiency of VD(25(OH)D3 level <20 ng/m L),the investigators were divided into VD sufficient group and VD deficiency group.After comparing the demographic characteristics,lifestyle and health status of the two groups of elderly people,there were no difference between the two groups(P > 0.05).The median IL-1β level of the 180 elderly with cognitive impairment selected in this study was 6.39 pg/m L(5.77 pg/m L,7.36 pg/m L),of which the median IL-1β level in VD sufficient group was 5.58 pg/m L(5.05 pg/m L,6.12 pg/m L),and the median IL-1β level in the VD deficient group was 6.61 pg/m L(5.85 pg/m L,7.56 pg/m L),compared with the VD sufficient group,the level of plasma inflammatory factor IL-1β in the elderly in the VD deficient group was significantly increased,and the difference was statistically significant(P < 0.016).The median IL-18 level of the overall inflammatory factor was222.56 pg/m L(187.57 pg/m L,255.62 pg/m L),and the median IL-18 level of the VD sufficient group was 185.98 pg/m L(172.64 pg/m L,208.49)pg/m L),the median IL-18 level in the VD deficient group was 228.66 pg/m L(201.83,257.93 pg/m L).Compared with the VD sufficient group,the plasma inflammatory factor IL-18 level in the elderly in the VD deficient group was significant increased,the difference was statistically significant(P < 0.036).Spearman correlation was used to analyze the plasma 25(OH)D3 level and the inflammatory factors IL-1β and IL-18 levels in the elderly with cognitive impairment,and it was found that the plasma 25(OH)D3 level and the IL-1β level were negatively correlated,r =-0.168,P < 0.025;plasma 25(OH)D3 level was negatively correlated with IL-18 level,r =-0.257,P = 0.000.In the multivariate logistic regression model,after adjusting for variables such as age,gender,education level,income level,BMI,hypertension,diabetes,hyperlipidemia,smoking,drinking,exercise,etc.,it was found that those subjects with VD deficiency,which has high levels of inflammatory factors,had a risk of 4.066 times then those with sufficient VD(Q4: OR3 = 4.066,95% CI3: 1.654~ 9.995).There was no correlation between VD deficiency and inflammatory factor IL-1β.Part 3: Study on the protective effect of 1,25(OH)2D3 on Aβ1-42 induced pyrolysis in PC12 cells According to the results of the CCK-8 experiment,20 μM Aβ1-42 and 1,10,100 n M1,25(OH)2D3 intervention doses were selected for subsequent experiments.Compared with the control group,the cell survival rate of the model group was significantly reduced(P < 0.01).Compared with the model group,the cell survival rate of 1,10,100 n M1,25(OH)2D3 intervention group was significantly improved(P < 0.05 or P < 0.01).The results of AO/EB staining was showed that compared with the control group,the cell death rate was significantly increased(P < 0.01).Compared with the model group,the cell death rate of the 1 n M 1,25(OH)2D3 intervention group was reduced,and the cell death rate of the 10 n M 1,25(OH)2D3 and 100 n M 1,25(OH)2D3 intervention groups was reduced more(P < 0.05 or P < 0.01).The cell death rate of the 100 n M 1,25(OH)2D3intervention group was significantly lower than that of the 1 n M 1,25(OH)2D3 and 10 n M1,25(OH)2D3 intervention groups(P < 0.01,P < 0.05).Compared with the control group,the LDH release and IL-1β and IL-18 secretion in the model group were significantly increased(P < 0.01).Compared with the model group,1 n M,10 n M and 100 n M1,25(OH)2D3 pretreatment the cell LDH release and IL-1β and IL-18 secretion in the intervention group were significantly reduced(P < 0.01),and the 100 n M 1,25(OH)2D3intervention group cell LDH release rate and the IL-1β and IL-18 secretion levels was improved significantly compared with 1 n M and 10 n M(P < 0.01,P < 0.05).By semi-quantitative analysis of the fluorescence intensity of ASC and Caspase-1protein,it was found that compared with the control group,the model fluorescence intensity was higher(P < 0.01);compared with the model group cells,there was no significant difference in the fluorescence intensity of ASC protein in 1 n M 1,25(OH)2D3treated intervention group(P > 0.05),but the fluorescence intensity of Caspase-1 protein was reduced(P < 0.05).The cells in the intervention group pretreated with 10 n M and100 n M 1,25(OH)2D3 were significantly reduced both(P < 0.01);the decrease in cells in the 100 n M 1,25(OH)2D3 intervention group was more significant than that in the 1 n M1,25(OH)2D3 intervention group(P < 0.01).Western Blot results were showed that compared with the control group,the protein expression levels of NLRP1,ASC,Pro-Caspase-1,Caspase-1 and GSDMD-N in the model group were significantly decreased(P < 0.01).Compared with the model group,there was no significant difference in NLRP1 protein expression in the intervention group pretreated with 1 n M 1,25(OH)2D3(P > 0.05),but the expression level in the 10 n M and100 n M 1,25(OH)2D3 groups was significantly reduced(P < 0.01);the expression of Pro-Caspase-1 protein was no significantly difference in 1 n M and 10 n M 1,25(OH)2D3groups(P > 0.05),the expression was significantly reduced in 100 n M 1,25(OH)2D3group(P < 0.01);the expression of ASC,Caspase-1 and GSDMD-N in the 3 intervention groups were significantly reduced(P < 0.01);compared with 1 n M and 10 n M 1,25(OH)2D3 intervention group,the expression of NLRP1,ASC,Pro-Caspase-1,Caspase-1 and GSDMD-N were more significant(P < 0.05 or P < 0.01).Part 4: Study on the mechanism of Aβ1-42 inducing pyrolysis of PC12 cells Through semi-quantitative analysis of the fluorescence intensity of ASC and Caspase-1 protein,it was found that compared with the control group,the fluorescence intensity of Aβ1-42 was higher(P < 0.01);compared with the Aβ1-42 group,there was no significant difference in the fluorescence intensity of ASC protein in the Caspase-1-si RNA group(P > 0.05).The fluorescence intensity of ASC and Caspase-1protein in the 1,25(OH)2D3 intervention group and Caspase-1-si RNA + 1,25(OH)2D3intervention group were significantly reduced(P < 0.01);the decrease in the 100 n M1,25(OH)2D3 intervention group was more significant than that in the 1 n M 1,25(OH)2D3intervention group(P < 0.01).Compared with the Caspase-1-si RNA group,the NC-si RNA group had stronger fluorescence intensity of the two proteins(P < 0.01),and there was no statistical difference compared with the Aβ1-42 group(P > 0.05).Western Blot results were showed that compared with the control group,the expressions of NLRP1,ASC,Pro-Caspase-1,Caspase-1 and GSDMD-N in Aβ1-42 group were significantly decreased(P < 0.05 or P < 0.01).Compared with Aβ1-42 cells,the expressions of NLRP1 and ASC protein in the 1,25(OH)2D3 and Caspase-1-si RNA +1,25(OH)2D3 intervention groups were significantly reduced(P < 0.01);the expression of Pro-Caspase-1 protein in Caspase-1-si RNA and Caspase-1-si RNA + 1,25(OH)2D3intervention group was significantly reduced(P < 0.01);the expressions of Caspase-1and GSDMD-N proteins in Caspase-1-si RNA,1,25(OH)2D3 and Caspase-1-si RNA +1,25(OH)2D3 intervention groups were significantly decreased(P < 0.01).Compared with the Caspase-1-si RNA intervention group,the expressions of NLRP1 and ASC protein in the 1,25(OH)2D3 and Caspase-1-si RNA + 1,25(OH)2D3 intervention groups were significantly lower(P < 0.01);Pro-Caspase-1,Caspase-1 and GSDMD-N proteins in the Caspase-1-si RNA intervention group were significantly reduced compared to the NC-si RNA intervention group(P < 0.01);the expression of GSDMD-N protein in the Caspase-1-si RNA + 1,25(OH)2D3 intervention group was lower than that in the Caspase-1-si RNA intervention group(P < 0.05).The results of AO/EB staining was showed that compared with the control group,the cell death rate in the Aβ1-42 group was significantly increased(P < 0.01).Compared with the Aβ1-42 group,the red fluorescence was decreased,and the cell death rate was decreased in the Caspase-1-si RNA group,1,25(OH)2D3 and the Caspase-1-si RNA +1,25(OH)2D3 group(P < 0.01).Compared with the Caspase-1-si RNA group,the NC-si RNA group had stronger red fluorescence(P < 0.01),and there was no statistical difference compared with the Aβ1-42 group(P > 0.05).Compared with the control group,the release of LDH and the secretion of IL-1β and IL-18 in the Aβ1-42 group were significantly increased(P < 0.01).Compared with Aβ1-42 group,the release of LDH and the secretion of IL-1β and IL-18 were significantly reduced in the Caspase-1-si RNA group,1,25(OH)2D3 group and Caspase-1-si RNA + 1,25(OH)2D3 group(P < 0.01).The NC-si RNA group was statistically significant compared with the Caspase-1-si RNA group(P < 0.01),and there was no statistical difference compared with the Aβ1-42 group.The secretion level of IL-1β in Caspase-1-si RNA +1,25(OH)2D3 group was lower than that in Caspase-1-si RNA group(P < 0.05).Conclusion:1.The Vitamin D deficiency was quite common in 360 elderly people investigated by this research in 6 communities in Taiyuan.The plasma level of 25(OH)D3 in MCI patients was significantly lower than normal elderly people;there was a positive correlation between cognition and plasma level of 25(OH)D3.The levels of plasma IL-1βand IL-18 in elderly patients with MCI were significantly higher than those in normal elderly patients;there was a negative correlation between cognition and plasma IL-1β and IL-18.Diabetes was found to be a risk factor for cognitive impairment.Higher incomes and the levels of VD were protective factors for cognition.Normally obese people have better cognition than thin people.Normal and slightly overweight people have better cognition than thin people.And higher levels of IL-1β and IL-18 were risk factors for MCI.2.The levels of plasma inflammatory factors IL-1β and IL-18 in the elderly with cognitive impairment in the VD deficiency group were higher than those in VD sufficient group;there was a negative correlation between the plasma 25(OH)D3 and IL-1β and IL-18 in the elderly with cognitive impairment,and VD deficiency was the risk factor in MCI with higher level of IL-18.3.Pyrolysis could be induced by Aβ1-42,and 1,25(OH)2D3 could play a neuroprotective effect by inhibiting the pyrolysis in PC12 cells induced by Aβ1-42.4.Inhibiting the expression of Caspase-1 by si RNA,the expression of the key pore-forming protein of GSDMD-N-pyrolysis could be inhibited,thereby reducing cell membrane damage and the maturation and release of LDH,IL-1β,and IL-18. |
PDF Full Text Request