Breviscapine On Expression Of ICAM-1 And ICAM-1 MRNA In Cerebrovascular Endothelial Cells In Smoking Rats

Breviscapine on expression of ICAM-1 and ICAM-1 mRNA in cerebrovascular endothelial cells in smoking ratsIntroductionCigarette smoking is one of the important preventable factors of proceeding and development of heart and cerebral disease. Some studies indicate that cigarette smoking can increase the expression of ICAM-1 and there is positive correlation between the increase of ICAM-1 and smoking quantity. ICAM-1 is mostly expressed by vascular endothelial cells. After stroke it mediates adherence of neutrophilic granulocyte and vascular endothelial cells and emigration of neutrophilic granulocyte, educe various effects and induce ischemic damage.Nuclear factor-kB (NF-kB) is an important cellular signal transduction molecule which can regulate immunization and inflammation gene expression and it can regulate the expression of ICAM-1. Protein kinase C (PKC) is one kind of upstream kinase of NF-kB, generally participates cellular signal transduction, cellular differentiation, gene activation and so on. PKC can active NF-kB through participates phosphorylation of inhibitor of kB (IkB) and decolles IkB and NF-kB. PKC inhibitor can block this pathway.In this study we use Breviscapine injection as PKC inhibitor. Through investigate the effect of PKC inhibitor on expression of ICAM-1 which is induced by smoking, we study the intervention effect and best time window of therapy of PKC inhibitor on cerebral infarct under smoking.Materials and Methods1,Experimental materials (1)Experimental animals: 66 healthy male Wistar rats, aged eight weeks, weights are 280～320g, offered by the animal department of CMU.(2)Main reagents: ICAM-1 (CD54) immunohistochemistry and in situ hybrization reagents are offered by Wuhan Boster, Breviscapine injection is offered by Yunnan pharmaceuticals company, DECP is offered by sigma.(3)Main instruments: optical microscope, paraffin wax cut slices, image analysis system.2,Experimental methods(1)The groups of experimental animals: 66 Wistar rats were randomly divided into non-infarct group (n=18) and infarct group (n=48). Rats in non-infarct group were no subject MCAO and were randomly divided into non-smoking group (n=6), smoking group (n=6) and smoking+PKC inhibitor group (n=6). 4 hours after giving PKC inhibitor rats in non-infarct group were sacrificed. Rats in infarct group were subject MCAO and were randomly divided into smoking group (n=24) and smoking+PKC inhibitor group (n=24).(2)Preparation of smoking rats models: Referencing Chow smoke perfuse model, using kind of cigarette, rats were passive smoke four times each day, each time five, until 12w.(3)Preparation of MCAO models: Reference Zea longa to prepare left middle cerebral artery occlusion models. The criterion of success models is that the rats flexed right forelimb when raised by the tail, Homer sign in the homonymy, circled toward the right side or fell down to the right side and there is no subarachnoid hemorrhage.(4)Immunohistochemistry to examine the expression of ICAM-1 and in situ hybrization to examine the expression of ICAM-1 mRNA: After perfuse the brain were removed and then were embedded in paraffin. Coronal sections of 5μm were cut. Immunohistochemistry and in situ hybrization were performed to examine the expression of ICAM-1 and ICAM-1 mRNA.(5)Statistical treatment: Taken two sections of each animal, data were acquired from vascular endothelial cells in 5 different cortical fields, measure the average grey scale of their kytoplasm, calculate the average grey scale of vascular endothelial kytoplasm in each animal cerebral cortex. The results were analyzed using t-test or ANOVA and processed by SPSS 13.0. A value P＜0.05 was considered statistically significant.ResultsThere was very little expression of ICAM-1 and ICAM-1 mRNA in cerebral cortical vascular endothelial kytoplasm of rats in non-smoking group. There was most expression of ICAM-1 and ICAM-1 mRNA in cerebral cortical vascular endothelial kytoplasm of rats in smoking group. The expression of ICAM-1 and ICAM-1 mRNA was less in smoking+PKC inhibitor group.The analysis of variance by repeated measures of kytoplasm grey scale at various time points in infarct group indicates that the earlier PKC inhibitor is given, the more effect of block the increase of ICAM-1 and ICAM-1 mRNA expression.ConclusionsPKC inhibitor can decrease the expression of ICAM-1 and ICAM-1 mRNA in cerebral cortical vascular endothelial cells of smoking rats. PKC inhibitor can prevent occurrence of cerebral infarction in smoking rats which have no cerebral infarction. It can protect brain of smoking rats which have cerebral infarction through block the expression of ICAM-1 and ICAM-1 mRNA, and medicine at earlier period can get better effect.