The Relationship Between Hyperhomocysteinemia And Ischemic Cerebrovascular Disease And Its Inflammatory Mechanism

Background The global burden of disease research shows that stroke has become the first cause of death in China in 2010.Its mortality and disability rate are high,which seriously affects the life quality of patients and imposes a heavy burden on patients,families and society.There are currently 13 million stroke patients in China,and about 75% of them are ischemic stroke.The most common ischemic stroke is cerebral small vessel disease(CSVD),including lacunar infarction(LI)and ischemic white matter lesion(WML).With the widespread use of technologies such as computed tomography(CT)and nuclear magnetic resonance(MR)imaging,a large number of CSVDs have been discovered.In recent years,great progress has been made in the etiology,pathogenesis,clinical and imaging studies of CSVD.Epidemiological surveys show that Asians have a higher burden of CSVD and that the risk of CSVD increases with age.To reduce the serious family and social burden of CSVD,it has become an important target for clinical prevention and treatment.Hyperhomocystinemia(HHcy)refers to an increase in the content of free and protein-bound homocysteine ??and mixed sulfide in plasma or serum,caused by methionine metabolic disorders.Homocysteine(Hcy is an important risk factor for the onset of cardiovascular disease,but the relationship between HHcy and brain large arteriosclerosis is more controversial.Domestic reports of HHcy are associated with increased intracranial and extracranial arteriosclerosis stenosis and increased stenosis.It is reported in foreign countries that HHcy only has an important effect on cerebral small vessel ischemia,but not on the main artery.We found in the clinic that HHcy is closely related to brain and small arterial lesions.Studies have shown that HHcy can cause atherosclerosis through inflammation,accelerate the process of atherosclerosis,and increase the risk of ischemic stroke.Hypersensitive C-reactive protein(hs-CRP)is a commonly used inflammatory marker in clinical practice and plays an important role in the inflammatory process of atherosclerosis.It is a highly sensitive marker for judging the risk and prognosis of atherosclerotic cerebral infarction.hs-CRP can damage endothelial cells by activating inflammatory reaction and triggering oxidative stress.At the same time,endothelial cell dysfunction is an early indicator and initial mechanism of atherosclerosis and vascular disease,and endothelial cell function damage induces vascular sclerosis and abnormal proliferation of smooth muscle cells.The exact mechanism by which HHcy causes vascular endothelial cell damage has not been elucidated.Studies have shown that HHcy increases inflammation of the aortic adventitia,which leads to endothelial cell dysfunction through cysteinyl aspartate specific proteinase(Caspase-1)-dependent inflammation.However,HHcy causes an increase in inflammatory response,impairs vascular endothelial cell functions,and the specific mechanism and process of ischemic CSVD is still unclear,it is worthy of further study to guide the development of appropriate intervention strategies in the clinic.Objective1.To study the effect of HHcy on atherosclerosis(carotid plaque,intima-media thickness(IMT),cervical/intracranial stenosis);2.To study the effect of HHcy on ischemic CSVD(LI and WML);3.To study the hs-CRP level in patients with ischemic CSVD,IMT thickening and carotid instability plaque.4.To study the effect of HHcy induced mouse carotid proliferating cell nuclear antigen(PCNA)expression including serum inflammatory factors(interleukin1?,IL-1?;tumor necrosis factor-?,TNF-?;and interleukin-6,IL-6),and release of caspase-1 in mouse cerebrovascular vessels.Then,explore the effect of HHcy on inflammatory response and release of pro-inflammatory factors,and further study the relationship between HHcy-induced receptor family pyrin domain-containing 3(NLRP3)activation and vascular endothelial cell dysfunction.Methods A total of 153 inpatients in our department of neurology between 2017.1-2018.02 and1357 middle-aged and in-patients in our hospital were selected for study.The relationship between Hcy and age,gender and menopause was compared.Hcy in the small vessel occlusion(LI)group was compared with the inpatient/non-stroke hospitalized patients;the ischemic WML score was scored according to the Fazekas classification,and according to the severity of WML,To study the effects of HHcy on large atherosclerosis,the Hcy and WML were severely compared in different groups.Comparison between non-stenosis in the cervical and intracranial stenosis group,plaque-free groups and carotid unstable plaque group,and normal IMT group and the IMT ?1 group were carried out.To study the effect of hs-CRP on arteriosclerosis and WML,hs-CRP comparison was executed between luminal and non-stroke control groups,unstable plaques and plaque-free groups between WML groups(Fazekas0-1and 4-6)with different severity.Then,for the mechanism study,HHcy mouse model was prepared by intragastric administration of 1.5% methionine in C57BL/6J male mice.After 8 weeks of model preparation,serum inflammatory factors IL-1?,TNF-? and IL-6 were detected by enzyme-linked immunosorbent assay.The level of PCNA was detected in the carotid artery by immunohistochemistry.The expression of caspase-1 in cerebrovascular was detected by immunofluorescence.Expression of Caspase-1 and Endothelin-1(ET-1)was detected by immunoblotting.At the same time,human microvascular endothelial cell-1(HMEC-1)was stimulated by HHcy for 24 hours.The contents of IL-1? and Caspase-1 in the supernatant of the cells were determined by immunoblotting.Then,the levels of intracellular ET-1,vascular cell adhesion molecule-1(VCAM-1),NLRP3,NF-?B,pro-Caspase-1,and Caspase-1 were detected after treating HMEC-1 cells with Hcy.Results1.Hcy of the cervical/intracranial artery stenosis group was significantly higher than that in the non-stenosis group(P < 0.05),and that in the IMT(>1)group was significantly higher than that in the normal IMT group(P < 0.05).2.Hcy of WML group with Fazekas 2-3 score was significantly higher than that of 0-1 score(P < 0.05);Hcy of WML with Fazekas 4-6 score was significantly higher than that of 2-3score(P < 0.05);Hcy of LI group was significantly higher than that of physical examination group(p < 0.05);Hcy of LI group was significantly higher than that of hospitalized non-stroke group(P < 0.05);3.The hs-CRP of LI group was significantly higher than that of non-stroke group(P < 0.05);the hs-CRP of Fazekas 4-6 WML was significantly higher than that of Fazekas 0-1 WML(P <0.05);the hs-CRP of unstable plaque group was significantly higher than that of non-plaque group(p < 0.05).4.The levels of IL-1?,TNF-? and IL-6 in the serum of HHcy model mice were increased,with IL-1? the most obvious increased.Caspase-1 expression was increased in HHcy model mice.The expression of Caspase-1 in the carotid artery of the model group was increased.HHcy increased the content of IL-1? and Caspase-1 in the supernatant of HMEC-1 cells.Meanwhile,HHcy decreased the activity of HMEC-1 cells and increased the release of LDH.ET-1 and VCAM-1 expression increased cytoplasmic NLRP3,Caspase-1 levels,and increased nuclear NF-?B p65 protein expression.Conclusion HHcy can cause or aggravate the occurrence/development of IMT thickening,carotid instability atherosclerotic plaque,and cervical/intracranial artery stenosis by increasing the inflammatory response,eventually leading to the occurrence and development of ischemic CSVD.We should pay attention to HHcy prevention and treatment.To reduce the occurrence and development of ischemic CSVD(IL,WML),inflammatory response might be highlighted as one of the targets of interventional ischemic CSVD.HHcy causes vascular endothelial damage in ischemic CSVD.The mechanism deserves further study.A further in-depth study found that HHcy increases the expression of pro-IL-1?,pro-Caspase-1 by activating NF-?B signaling pathway,followed by increasing the expression of NLRP3,activates NLRP3 inflammasome and activation and release of Caspase-1 and IL-1?.Synergistic effects of pro-inflammatory factors IL-1?,IL-6,TNF-? promote the development and progression of vascular endothelial cell inflammation,leading to vascular endothelial cell injury(with decreased endothelial cell viability and increased LDH release).Furthermore,the number of PCNA positive cells were increased,indicating that vascular smooth muscle cells were proliferated excessively,and vascular remodeled(IMT thickening,plaque formation,arterial stenosis),which causing or aggravating arteriosclerosis,eventually producing ischemic cerebral small vessel disease such as lacunar infarction and white matter lesion.Interventions targeting NLRP3 inflammasome activation to inhibit inflammation might help to prevent or treat ischemic CSVD caused by HHcy.