Effect Of CD40/CD40L On The Onset And Development Of Cerebral Infarction And Related Mechanism

Background:Cerebral infarction (CI), also known as ischemic cerebrovascular disease, is mainly due to cerebral artery atherosclerosis (AS) and thrombosis. All these pathogenesis lead to cerebral artery stenosis even occlusion. In clinical practice, it may cause focal acute cerebral insufficiency. CD40/CD40L is a pair of complementary transmembrane glycoprotein belonging to the tumor necrosis factor receptor (TNFR) and tumor necrosis factor (TNF) superfamily respectively. CD40/CD40L genes that are involved in immune regulation, inflammation, hypercoagulable and other pathophysiological states can activate the inflammatory response, lead to endothelial dysfunction, and activate the platelets. CD40/CD40L system plays an important role in the development of AS and AS plaque rupture. Many studies have confirmed that CI is a polygenic disease, which is influenced by many genetic and environmental factors. Many AS related genes and their polymorphism are associated with the susceptibility to CI. In recent years, more and more research found that human CD40/CD40L genes have multiple single nucleotide sequence polymorphisms (SNPs), and these functional SNPs can change the amount and activity of CD40and CD40L, cause the corresponding changes of the downstream biological effects, and then influence the development of a range of autoimmune diseases and inflammatory diseases, including CI.Objective:To determine the association between CD40rsl883832(C/T) polymorphisms and the risk of CI in Han population and its possible molecular mechanism.Methods:Blood samples were collected from CI patients and age-and sex-matched healthy control groups. Peripheral blood DNA was extracted by Phenolic-chloroform method. CD40rsl883832(C/T) polymorphism was genotyped by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Real-time PCR was performed to determine CD40mRNA expression from the peripheral blood mononuclear cells (PBMCs) of CI patients and age-and sex-matched healthy controls. χ2test was used to analyze the difference in CD40rs1883832(C/T) genotype distribution between the cases and the controls. Association between CD40genetic polymorphisms and CI risk was analyzed by unconditional logistic regression. ANOVA was used to analyze difference in sCD40L concentration in the plasma and PBMCs CD40mRNA expression between cases and controls.Results:1. The frequencies of CD40rs1883832polymorphism CC, CT and TT genotypes in the healthy control population were35.4%,48.4%and16.2%, and those of rs1883832polymorphism CC, CT and TT genotypes in the CI patients were28.1%,53.5%and18.4%. The distribution of rs1883832genotypes in the CI case group and the control group had significant difference (P=0.028, P<0.05). The carrier rate of CI patients with T allele was significantly higher than that of the healthy control group (P=0.009, P<0.05). Stratified analysis with gender, age, and CI etiology showed that:1) The frequency of male CI patients with CC genotype was significantly lower than that in the healthy control group, with significant difference (P=0.013, P<0.05);2) The frequency of CI patients older than50with CC genotype was significantly lower than that in the healthy control group, with significant difference (P0.006, P<0.01);3) The frequency of lacunar infarction patients with CC genotype was significantly lower than that in the healthy control group (P=0.028, P<0.05).2. The plasma sCD40L concentration in people with CD40rs1883832polymorphism TT and CT genotypes was higher than in those with CC genotype.3. The PBMCs CD40mRNA expression in people with CD40rs1883832polymorphism TT genotypes was higher than in those with CC or CT genotype.Conclusion:1. CD40rs1883832polymorphism is associated with cerebral infarction susceptibility in Han population, and T allele increases the risk.2. T allele carrier of CD40rs1883832polymorphism is a risk allele carrier. Background:Cerebral infarction, also known as ischemic cerebral vascular disease, is mainly due to cerebral artery atherosclerosis and thrombosis, which caused luminal narrowing or occlusion, and focal acute cerebral blood insufficiency. The main clinical manifestation of CI was neurologic dysfunction caused by ischemia, hypoxia, and necrosis of the brain tissue. Vascular endothelial cells were an important composition of blood vessels. Many vasoactive factors were synthesized and released by EC, so EC plays an important role in the cerebrovascular autoregulation of cardiovascular disease. Modern research has shown that the initial factor for many cardiovascular diseases was vascular endothelial damage. CD40was expressed in endothelial cells. The downstream signaling pathways of CD40including nuclear factor-kappa B were activated after the activation of CD40, with subsequent up-regulation of proinflammatory and proatherogenic genes, thereby affecting the endothelial and circulatory function. Curcumin was a yellow acidic phenolic compound extracted from the plant curcuma longa, which was the main pharmacological active ingredient of turmeric. Curcumin inhibited NF-κB, caused the down-regulation of NF-κB downstream gene expression, and played an important role in anti-oxidation, anti-inflammation, and anti-proliferation.Objective:To explore the change of CD40expression in vascular endothelial injury induced by Ang II, and to determine whether the protective effect of curcumin on endothelial cells was mediated by the inhibition of CD40/CD40L signaling pathway.Methods:The Ang Ⅱ model experiment included a normal control group, an Ang Ⅱ(10-5M) injury group,3curcumin(10-6M,3×10-6M,10-5M) groups, a telmisartan (10-5M) group and a Hcy (10-3M) injury group,3curcumin (10-6M,3×10-6M,10-5M) groups, a rosuvastatin (10-5M) group. Apoptosis was detected by Hoechst33258staining;CD40 mRNA expression in the human umbilical vein endothelial cells (HUVEC-C) was detected by real-time PCR; and CD40protein expression in HUVEC-C cells was detected by Western Blot.Results:1. In the endothelial cells, CD40mRNA expression in the Ang Ⅱ group was higher than that in the control group, with significant difference (P<0.05). Curcumin inhibited the upregulation of CD40mRNA induced by Ang Ⅱ, and high concentration of curcumin significantly inhibited the upregulation of CD40mRNA expression induced by Ang Ⅱ (P<0.05);2. Ang Ⅱ significantly upregulated CD40protein expression compared with control group, with significant difference (P<0.05). Pretreatment with high concentration of curcumin significantly inhibited the upregulation of CD40protein expression induced by Ang Ⅱ compared with Ang Ⅱ group (P<0.05);3. Hey significantly upregulated CD40mRNA expression compared with control group, with significant difference (P<0.05). Pretreatment with curcumin inhibited the upregulation of CD40mRNA expression, and high concentration of curcumin significantly inhibited the upregulation of CD40protein expression induced by Ang Ⅱ (P<0.05);4. Hey significantly upregulated CD40protein expression compared with control group, with significant difference (P<0.05). Pretreatment with high concentration of curcumin significantly inhibited the upregulation of CD40protein expression induced by Hey (P<0.05).Conclusion:In HUVEC-C curcumin can down-regulate CD40mRNA and protein expression induced by Ang Ⅱ or Hey, and protect HUVEC-C from endothelial injury induced by Ang Ⅱ or Hey. Background:CD40/CD40L signal pathway is an important auxiliary stimulate of T cell activation. Besides its key role in regulating T cell immune response, it is also associated with B cell activation, proliferation, differentiation and the production of antibodies. CD40/CD40L system mediated inflammatory immune response in the peripheral blood mononuclear cells (PBMCs), so it may be involved in the occurrence and development of AS. CD40rsl883832may affect the incidence and development of cardiovascular disease by altering the levels of CD40/CD40L system. The primary culture of PBMCs according to genotypes would be an ideal cell model to examine the cell response to damage and the protection of drugs.Objective:Primary cultured PBMCs were divided into2groups according to the genotype of CD40rsl883832. We detected the CD40expression in each group to determine the association between CD40rsl883832(C/T) polymorphisms and different responses to Hey and curcumm.Methods:Blood samples (30ml) from each volunteer were collected to extract peripheral blood DNA and to isolate the PBMCs. CD40rs1883832(C/T) polymorphism was genotyped by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Primary cultured PBMCs were divided into a normal control group, an Hey injury group, a curcumin group and a rosuvastatin group. Real-time PCR was performed to determine CD40mRNA expression. CD40protein expression level in the PBMCs was detected by Western Blot.Results:1. Incubation of PBMCs with10-3M Hey for24h led to about3fold increase of CD40mRNA expression in PBMCs with T allele, which was significantly higher than in PBMCs without T allele (about2folds;2. The protein level of CD40was upregulated by10-3M Hey in the2groups, and PBMCs with T allele showed a significantly higher degree of CD40protein level;3. The increased level of CD40was partially inhibited by curcumin and statin. PBMCs with T allele were more sensitive to curcumin and rosuvastatin.Conclusion:PBMCs with T allele are more damaging after Hcy administration, and PBMCs with T allele were more sensitive to curcumin.