| BackgroundStroke is the main disease that causes death and disability in the world.Every year,5 million people die of stroke,almost 10%of the total deaths.China is one of the countries with the highest incidence rate of stroke worldwide.At present,the situation of stroke prevention and control is very serious in China.There are 12.42 million people over 40 years old in China who have had stroke.70%of the surviving patients have different degrees of disability,which causes serious economic burden to the society and families,and is an important influencing factor of returning to poverty due to illness.In recent years,with the increase of incidence rate of cerebrovascular disease and the coming of aging population,people begin to pay more and more attention to cerebral small vessel disease(CSVD).CSVD accounts for 25%-30%of all the causes of ischemic stroke.CSVD can not only cause acute symptoms,such as lacunar infarction,cerebral hemorrhage,but also can cause dementia,gait abnormalities,urinary retention and mood disorders and other occult symptoms.Professor Philip B.Gorelick’s research published in stroke in 2011 showed that 20%to 40%of Alzheimer’s disease was related to CSVD.However,the true pathogenesis of CSVD is not clear,which hinders the accurate diagnosis and effective treatment of CSVD.At present,the treatment of CSVD is still lack of specific treatment measures,most of which are still in the research stage.The current treatment targets include lipid-lowering,antiplatelet,anti-inflammatory,blood-brain barrier regulation,prostacyclin c-AMP system,etc.According to whether CSVD is the acute stage of stroke,it can be divided into acute stage treatment and non acute stage treatment.Since there is no guideline for the treatment of acute stroke in CSVD,the treatment of acute stroke in CSVD is the same as that of large vascular stroke,including hyperacute thrombolysis and antithrombotic treatment in acute phase.The non acute treatment of CSVD is mainly antithrombotic and prevention and control of vascular risk factors.According to the early pathological study published by C.Miller Fisher in Neurology,lipid hyaline degeneration and cellulosic necrosis of small artery wall in CSVD patients are caused by long-term hypertension,and a number of clinical studies also show the inseparable relationship between CSVD and hypertension.For example,CNSR(China National Stroke Registry)research suggests that the history of hypertension is significantly related to the recurrence of arteriolar occlusive stroke,but the current clinical research has not confirmed the causal relationship between CSVD and hypertension.The results of SPS3(The Secondary Prevention of Small Subcortical Strokes)indicated that the strong antihypertensive effect was safe and had the potential to reduce the trend of stroke recurrence(HR 0.81,95%Cl 0.64-1.03,P=0.08),but it was not beneficial to improve the cognitive function after lacunar infarction;the study of PRESERVER suggested that the strong antihypertensive effect would not reduce the cerebral blood flow of patients with severe white matter disease.Cerebral small vessel disease is a whole brain disease.The study of its mechanism includes several aspects from the age,vascular risk factors and other pathogenic causes to the clinical symptoms,from the structural and functional abnormalities of blood vessels to the damage of brain tissue,resulting in the abnormal connection of brain network,and finally leading to different clinical phenotypes.At present,there have been many researches focusing on clinical problems to explore the role of endothelial damage,blood-brain barrier damage,inflammation and stress in CSVD,so as to provide new therapeutic targets for the treatment of CSVD.Using single gene genetic CSVD as a natural model,the intervention target research was carried out to provide scientific basis for the study of complex CSVD intervention targets caused by multiple exposure factors.The etiology and pathogenesis of CSVD are not clear,the treatment and prevention are limited,so it is urgent to find new therapeutic targets.In the past,as a fat soluble vitamin,vitamin D played an important role in brain function and development,besides being related to cardiovascular disease,autoimmunity,inflammation and cancer,and was closely related to the occurrence and development of nervous system diseases through various mechanisms such as inflammation,immunity and neuroprotection.The lack of vitamin D may lead to corresponding neurological disorders and affect the prognosis of patients.Vitamin D supplementation can also prevent and treat vascular damage by reducing blood pressure,inhibiting renin-angiotension-aldosterone system,and inhibiting atherosclerosis.At present,many studies have shown that vitamin D deficiency is closely related to the clinical severity and functional prognosis of ischemic stroke,but there are few reports about vitamin D deficiency and cerebral small vessel disease.Vitamin D was discovered and named in 1992.Its main source is intestinal absorption and skin synthesis.Serum 25-hydroxy vitamin D has a long half-life and is relatively stable in the blood circulation.Its concentration is considered to be the most reliable indicator to determine the overall vitamin D status,so it can be used to determine whether there is vitamin D deficiency in patients.The role of vitamin D is mediated by vitamin D receptor(VDR),which is a ligand activated transcription factor that controls gene expression or gene function.VDR gene is located on human chromosome 12q12-ql4,with 14 exons.It is reported that there are about 470 polymorphic sites in VDR gene,most of which are FokI.TaqI,BsmI and Apal.A series of gene research results have confirmed the relationship between VDR gene variation and diabetes,tumor,multiple sclerosis,Parkinson’s disease and other diseases.The exact relationship between 25-hydroxy vitamin D and cerebral small vessel disease is only reported in the research report of Korean and Indian scholars.So far,only one study in China has reported the relationship between vitamin D deficiency and the total MRI burden of patients with cerebral small vessel disease in Fujian.Therefore,our research population comes from two relatively large first-class ternary general hospital in Shandong and Beijing in northern China.The purpose of this study is to study the possible relationship between the level of 25-hydroxy vitamin D and the risk of cerebral small vessel disease,so as to clarify its possible correlation,and further explore the role of hypertension in it,so as to provide a new target for the prevention and treatment of cerebral small vessel disease.However,vitamin D plays a role under the guidance of vitamin D receptor.Only one Indian study reported that VDR gene polymorphism is related to the pathogenesis of vascular dementia caused by cerebral small vessel disease through literature search.There is no research report on VDR gene polymorphism and cerebral small vessel disease in other populations in the world including China.The determination of genetic susceptibility factors may increase our understanding of the etiology and pathology of cerebral small vessel disease,and help to determine new treatment objectives,provide experimental and theoretical basis for further elucidating the pathogenesis of cerebral small vessel disease,so as to reduce the serious social burden of cerebral small vessel disease.Part One Correlative study between serum 25-hydroxy vitamin D level and cerebral small vessel diseasePurposeThe main purpose of this study is to explore the correlation between 25-hydroxy vitamin D and cerebral small vessel disease.MethodsFrom January 2017 to December 2017,106 patients with cerebral small vessel disease and 115 normal controls were enrolled in this study.All subjects had a detailed physical examination of the nervous system.NIHSS scale and Rankin scale were used to assess the severity of stroke and the degree of disability after stroke.The vitamin status was determined by measuring 25-hydroxy vitamin D.Subjects were divided into three subgroups:vitamin D deficiency(≤12ng/ml),insufficiency(12-20ng/ml)and sufficiency(≥20ng/ml).Baseline survey including blood pressure,height and weight was carried out for all patients at admission or outpatient.The baseline survey was conducted by trained and qualified clinical doctors and investigators.The main contents of the baseline survey include:demographic information(general information);clinical characteristics and symptoms;physical signs;laboratory tests(red blood cells,white blood cells,platelets,fibrinogen,fasting blood glucose,glycohemoglobin,hemoglobin,glycoalbumin,uric acid,homocysteine,total cholesterol,low-density lipoprotein,high-density lipoprotein,triglyceride,C reactive protein,albumin);related basic disease history(hypertension,diabetes,hyperlipidemia,heart history and peripheral vascular disease);family history of stroke;lifestyle(smoking and drinking).We use chi square test to analyze the correlation of classification variables,and t test to test the correlation between continuous variables.Logistic regression analysis was used to test the correlation between 25-hydroxy vitamin D and cerebral small vessel disease.The odds ratio and 95%confidence interval were calculated by logistic regression analysis.Results1.Comparison of clinical data between cerebral small vessel disease group and control groupA total of 221 subjects were enrolled,106 of them were CSVD patients and 115 were control group.Among 106 CSVD patients,80(75.5%)were men and the mean age was 61.6±13.2 years.The scores of NIHSS and Rankin scale in CSVD group were 3(1-5)and 1(0-2),respectively.Compared with controls,patients with CSVD were associated with men,stroke history,smoking history,hyperlipidemia,higher systolic pressure,higher diastolic pressure and LDL(P<0.05).2.25-hydroxy vitamin D and cerebral small vessel diseaseThe levels of 25(OH)D were classified as sufficient in 11 patients(10.4%)vs 25 controls(21.7%),insufficient in 19 patients(17.9%%)vs 43 controls(37.4%),and deficient in 76 patients(71.7%)vs 47 controls(40.9%).The mean serum 25(OH)D level was much lower in CSVD patients(10.22+5.6 ng/ml)compared with controls(14.6±7.5 ng/ml)(P=0.001).Logistic regression analysis revealed the level of 25(OH)D as an independent predictor of CSVD(OR 0.772,95%CI 0.691-0.862,P-0.001)after adjustment for covariate gender,stroke history,smoking history,hyperlipidemia,systolic blood pressure,diastolic blood pressure and LDL.Compared with the sufficient group of 25(OH)D,we also found the ORs of CSVD in the deficient and insufficient group were 5.609(95%CI 2.006-15.683,P=0.001)and 1.077(95%CI 0.338-3.428,P=0.9)after adjusting for potential confounders,respectively.3.Correlation analysis of 25-hydroxy vitamin D level and hypertension interaction with cerebral small vessel diseaseWe also found there was a significant interaction between vitamin D status and hypertension on the presence of cerebral small vessel disease(Pinteraction=0.001).Compared with sufficient group,in hypertensives with vitamin D deficient and insufficient group,the OR increased to 9.738(95%CI 2.398-39.540,P=0,001)and 1.108(95%CI 0.232-5.280,P=0.898),respectively.However,we did not find such interaction between vitamin D status and hypertension in non-hypertensive patients.Conclusion1.There is a significant correlation between the risk of cerebral small vessel disease and 25-hydroxy vitamin D deficiency.2.The interaction between hypertension and vitamin D deficiency increases the possibility of cerebral small vessel disease.3.Further study on the relationship between 25-hydroxy vitamin D and cerebral small vessel disease has certain clinical value.Part Two Correlative study between vitamin D receptor gene polymorphism and cerebral small vessel diseasePurposeThe purpose of this study was to explore the relationship between the four polymorphisms of vitamin D receptor(VDR)gene Fokl,TaqI,ApaI and BsmI and the genetic susceptibility of cerebral small vessel disease.MethodsAll the subjects were the same as the first part of the study population,all of them were Han nationality.PCR-RFLP was used to detect the gene in the case group and the control group.Firstly,the blood genomic DNA was amplified by PCR reaction in advance,and the corresponding primers were designed by reference.The PCR products were digested by enzyme,and the genotypic frequency of SNP locus was counted by imaging with ultraviolet transmission instrument.Finally,10%of the PCR products were randomly selected for sequencing,genotypes were detected and compared with the above RFLP results.Spss22.0 was used to analyze the genotype data.Chi square test and logistic regression were used to test the genotypes and allele frequency differences of SNP loci,analyze the dominant genetic mode,recessive genetic mode and superimposed genetic mode,and predict the gene disease risk association.Results1.The four loci of FokI,TaqI,Apal and BsmI of VDR gene were tested by chi square,all P values were greater than 0.05,which were consistent with Hardy Weinberg equilibrium,indicating that they were representative of the population.2.Through the analysis of the genotype distribution of FokI,TaqI,ApaI and BsmI of VDR gene,we found that,(1)in the overall sample,FokI has a significant correlation with the incidence of cerebral small vessel disease,and there is a significant difference in the genotype distribution between the case group and the control group(P=0.004).After sex stratification,the difference of FokI genotype distribution was only observed in women(P=0.001);(2)For TaqI,there was no significant difference in the statistical value of its genotype in the overall sample(P=0.901),even after we stratified according to sex,the genotype distribution was not statistically significant,P=0.683 in men,P=0.725 in women,and there was no correlation between it and the incidence of cerebral small vessel disease;(3)For ApaI,there was no statistical significance in the distribution of genotypes in the overall sample(P=0.933),in men(P=0.973),in women(P=0.972),even if we stratified by sex,it was not statistically significant.Therefore,there is no significant correlation between Apal and cerebral small vessel disease;(4)Similarly,for BsmI,the statistical value of its genotype in the overall sample was not statistically significant(P=0.320),P=0.448in men,P=0.528 in women,and there is no statistical difference,which has no correlation with the incidence of cerebral small vessel disease.3.By analyzing the distribution of FokI,TaqI,Apal and BsmI alleles of VDR gene,there was a significant difference in the distribution of FokI alleles of VDR gene between the case group and the control group(P=0.001).Carrying the FokI minimum allele f could significantly increase the risk of cerebral small vessel disease(OR=2.156,95%CI=1.362-3.414,P=0.001).After sex stratification,there was no significant difference in the frequency distribution of the median gene in men(P=0.212),while in women,carrying the FokI minimum allele f increased the risk of cerebral small vessel disease(OR=4.529,95%CI 2.136-9.606,P<0.001).The distribution of TaqI,ApaI,Bsml allele frequency between the case group and the control group was(0.932,0.757,0.789),P value was greater than 0.05,indicating no statistical significance.Then we stratified according to gender,P=0.443 in TaqI men,P=0.407 in TaqI women,P=0.869 in ApaI men,P=0.812 in ApaI women,P=0.737 in BsmI men and P=0.260 in Bsml women,all of which showed no statistical difference,indicating that TaqI,Apal and BsmI were not related to the incidence of cerebral small vessel disease.4.After removing age,smoking history,hyperlipidemia,hypertension,diabetes and other confounding factors,the results of multivariate logistic regression analysis showed that in the dominant model of the overall sample,FokI locus was still significantly related to the risk of cerebral small vessel disease(OR=2.042,95%CI 1.073-3.886,P=0.03).FokI locus is also associated with the risk of cerebral small vessel disease in the dominant model of female cerebral small vessel disease(OR=10.323,95%CI 2.54-41.956,P=0.001),but the correlation was not found in the dominant model of male cerebral small vessel disease.In the superposition model of the population sample,FokI was still significantly associated with the risk of cerebral small vessel disease(OR=1.784,95%CI 1.045-3.045,P=0.034).The FokI locus was also associated with the risk of cerebral small vessel disease in the female model(OR=3.826,95%CI 1.469-9.964,P=0.006);The correlation was not found in the male model.In the total sample,male and female recessive model,no significant correlation between FokI locus and the risk of cerebral small vessel disease was found.TaqI,Apal and BsmI were not found to be related to the risk of cerebral small vessel disease in the dominant model,superposition model and recessive model of the total sample,male and female samples.5.In the cerebral small vessel disease group,the mutation homozygous type FF at FokI site significantly reduced the value of 25-hydroxy vitamin D,compared with the wild type FF,the p value of t-test is less than 0.05,which is statistically significant.The p value of 25-hydroxy vitamin D in the heterozygous type FF was greater than 0.05 after t-test compared with the wild type FF,which was not statistically significant,but the correlation was not found in the control group.There was no significant difference in 25-hydroxy vitamin D between the other three genotypes of the case group and the control group.Conclusion1.In the Han population of northern China,the minimum allele of FokI can significantly increase the risk of cerebral small vessel diseases,and this correlation is only observed in women.2.The minimum allele of FokI can reduce the level of 25-hydroxy vitamin D.3.There is no correlation between TaqI,ApaI,BsmI and cerebral small vessel disease in the Han population of northern China. |
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