| Stroke is of great concern because of its high incidence, mutilation and mortality rate.60%-80% of stroke is cerebral infarction of which atherosclerosis is the major cause. Thus, thorough understanding of atherosclerosis mechanism can hopefully reduce the incidence of cerebral infarction.During the last decade, tremendous researches have been carried out in elucidating the mechanism of atherosclerosis formation, and in spite of encouraging discoveries of its relation with inflammation and hyperlipidemia and etc, the mechanism still remains unclear.In addition to the endothelium, blood vessel walls are composed of many constituents such as matrix basement, and smooth muscle cells. All these components form a coherent tissue that requires, for proper function, a constant exchange of information via electrical and chemical signals. A key pathway that allows such communication is gap junction (GJ). GJ channels, composed of six connexins to form a channel in the cell membrane, allow the direct exchange of ions small metabolites, and other secondary messengers molecules between cell in contact. Recently, it was found that gap junction was closely related to athereosclerosis, but the mechanism is unclear.On the other hand, a great deal of studies have been focused on the relationship between the Single-nucleotide polymorphism of Connexin 37 (Cx37) and athereosclerosis, coronary heart disease(CHD), Myocardial infarction(MI). It is found that Cx37 C1019T is a Single-gene marker of athereosclerosis, and it is related with CHD and MI. Similar to CHD and MI, cerebral infarction has the common pathologic manifestation of athereosclerosis. However, the relationship between the Single-nucleotide polymorphism of Connexin 37 (Cx37) and athereosclerosis cerebral infarction is unclear.Thus, the influence of oxidized low density lipoprotein (ox-LDL), high-density lipoprotein (HDL) and octanol on the expression of connexin of cultured human umbilical vein endothelial cell were observed and its possible mechanism was studied. Moreover, the relationship between the SNP of Connexin 37 (Cx37) and athereosclerosis cerebral infarction was also studied.Objiective:Our aim was to observe the influence of oxidized low density lipoprotein(ox-LDL) on the expression of connexin and the apotosis of cultured Human umbilical vein endothelial cell (HUVE-12), as well as to investigate the influence and the mechanism of HDL and octanol on the expression of connexin and apotosis of cultured endothelial cells induced by ox-LDL.Methods:HUVE-12 cells were divided into seven groups and were exposed to different conditions. The groups included:①control group: normal DMEM culture medium;②-④ox-LDL group:HUVE-12 were incubated with 25μg/ml ox-LDL,50μg/ml ox-LDL and 100μg/ml ox-LDL for 24 hours;⑤HDL group:HUVE-12 was incubated with 100μg/ml HDL for 24 hours;⑥HDL and ox-LDL group:HUVE-12 were coincubated with 100μg/ml HDL and 50μg/ml ox-LDL for 24 hours;⑦octanol and ox-LDL group:HUVE-12 was coincubated with 1mm octanol and 50μg/ml ox-LDL for 24 hours. MTT method was carried out to evaluate the proliferation of HUVE-12 while viability of HUVE-12 was detected by Trypan blue staining, cell apotosis was examined through flow-cytometry. The expressions of Cx37,40,43, Bcl-2, Bax and Caspase-3 mRNA were examined by real time RT-PCR, also the proteins expression was examined by immunocy-tochemistry and western blot.Result:1. Expressions of proteins and mRNA of Cx37 and Cx 40 were decreasing when HUVE-12 incubated with 25μg/ml,50μg/ml and 100μg/ml ox-LDL for 24h. By contrast, expressions of proteins and mRNA of Cx 43 were significantly elevated comparing with the control group (P<0.05). The results showed that after HUVE-12 were exposed to various concentration of ox-LDL for 24h, cell proliferation was downregulated and its viability decreased significantly in a concentration-dependent manner while cell apotosis was increased (P<0.05). Expressions of proteins and mRNA of Bcl-2 were decreased, while expressions of proteins and mRNA of Bax and caspase-3 were increasing. The specific values for Bcl-2/Bax in groups with different ox-LDL concentrations were significantly elevated (P<0.05).2. Comparing with the 50μg/ml ox-LDL group, expressions of proteins and mRNA of Cx37 and Cx 40 in HDL group were upregulated, when the mRNA expression of Cx43 was downregulated (P＜0.05). Cell proliferation and viability in HDL group were enhanced significantly while cell apotosis was ameliorated (P＜0.05). Expressions of proteins and mRNA of Bcl-2 were upregulated, while the expressions of proteins and mRNA of bax and caspase-3 were downregulated. The specific values for Bcl-2/Bax was elevated (P<0.05). Expressions of proteins and mRNA of Cx37 and Cx 40 in HDL+ox LDL group were upregulated, when expressions of protein and mRNA of Cx43 were downregulated (P<0.05). Cell proliferation and viability in HDL+ ox-LDL group were enhanced significantly while cell apotosis was decreased (P＜0.05). Expressions of proteins and mRNA of Bcl-2 were upregulated, while expressions of proteins and mRNA of bax were downregulated, and mRNA expression of caspase-3 was upregulated. The specific values for Bcl-2/Bax was elevated (P＜0.05).3. Comparing with the 50μg/ml ox-LDL group, expressions of proteins and mRNA of Cx37 and Cx 40 in octanol+ox-LDL group were upregulated, when expressions of proteins and mRNA of Cx43 were downregualted (P＜0.05). What's more, Cell proliferation and viability in octanol+ox-LDL group were enhanced significantly while cell apotosis was decreased (P<0.05). Expressions of proteins and mRNA of Bcl-2 were increased, while expressions of proteins and mRNA of bax and caspase-3 were decreased. The specific values for Bcl-2/Bax was elevated significantly (P＜0.05).Conclusion:1. ox-LDL could induce the remodeling of connexin in HUVE-12 in virto, and the effect was in a concentration-dependent manner. It was suggested that ox-LDL could lead to AS by the remodeling of connexin. HUVE-12 cell proliferation and viability were significantly downregulated in a concentration-dependent manner by ox-LDL while cell apotosis was enhanced. The effect of ox-LDL on cell apotosis of HUVE-12 could be related to mitochondrial apoptosis pathway which could be a mechanism of AS causing by ox-LDL.2. HDL could prevent the remodeling of connexin in HUVE-12 and decrease cell apotosis of HUVE-12 induced by ox-LDL. It indicated that HDL could play a role in anti-AS by this pathway.3. Similarly with the effect of HDL, octanol could prevent the remodeling of connexin and decrease cell apotosis in HUVE-12 induced by ox-LDL. The effect of octanol on cell apotosis caused by ox-LDL could also be related to mitochondrial apoptosis pathway. ox-LDL could affect connexins and gap junction in endothelial cells and it could result in the chain of mitochondrial apoptosis pathway which lead to cell apotosis.Objective:Our aim was to investigate the relationship between connexin 37 gene polymorphisms and atherothrombotic cerebral infarction.Methods:A case-control study was carried out in which connexin 37 C1019T and I1297D gene polymorphisms in 245 paitents with atherothrombotic cerebral infarction (ACI group) and 145healthy subjects (control group) were analyzed with polymerase chain reaction (PCR)-restriction fragment length polymorphism(RFLP).Result:1. The results showed that there were Cx37 C1019T and I1297D single-nucleotide polymorphism (SNP) in han population of Hunan province. In control group Cx37 C1019T CC, CT and TT genotype frequencies were 63.3%,29.0% and 9.7%, while C and T allele frequencies were 77.9% and 22.1%. Cx37 I1297D II, ID and DD genotype frequencies were 47.7%,42.6% and 9.7%, while I and D allele frequencies were 69.0% and 31.0%.2. In ACI group Cx37 C1019T CC, CT and TT genotype frequencies were 70.6%,25.7% and 3.7%,, while C and T allele frequencies were 83.5% and 16.5%. But no differences were detected of genotypes and allele frequencies among patients with ACI group and control group (x 2=3.765, P=0.152 and x 2=3.692, P=0.055). Cx37 I1297DⅡ, ID and ID genotype frequencies were 45.3%,42.9% and 11.8%, while I and D allele frequencies were 66.7%and 33.3%. But no differences were detected of those genotypes and allele frequencies among patients with ACI group and control group (x 2=0.526,P=0.769 and x 2=0.458,P=0.499).Conclusion1. There were Cx37 C1019T and I1297D single-nucleotide polymorphism (SNP) in han population of Hunan Province. The Cx37 C1019T C allele and Cx37 I1297D I allele were dominant respectively in both groups.2.Cx37gene C1019T和I1297D polymorphism were not associated with an increased risk of CI in Han population of Hunan Province, China, and might not be considered as an independent predictor of atherothrombotic cerebral infarction. |
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