Role Of LOX In Hemodynamically Induced Intracranial Aneurysm Formation

Aneurysmal subarachnoid hemorrhage,as one of the cerebral vascular diseases,accounts for about 85%of all spontaneous subarachnoid hemorrhage,seriously threatening human health.According to the WTO,the incidence of the whole world is about 9.1/100,000,the annual incidence in Beijing area is 2/100,000,is slightly lower than the world.however,considering the early insufficient understanding of the disease in our country,there are many patients died before they arrived at the hospital.The actual incidence of our country will be greater than the estimated.Aneurysmal subarachnoid hemorrhage is very dangerous and has a high fatality rate.However,under normal circumstances,it is difficult to predict the growth and rupture of intracranial aneurysms,causing great distress for the discovery and treatment.In recent years,the high wall shear stress was found to be related to the growth and rupture of intracranial aneurysms by using numerical simulation of hydrodynamic computational techniques in combination with morphological indicators and hemodynamic parameters.Hemodynamic changes are only the initial factors of aneurysm growth and rupture,and how it further evolves into"pathological changes"through"mechanical changes"remains a difficulty in this field.Lysyl oxidase is an important catalytic enzyme in the activation and expression of collagen fibers and elastic fibers in vivo.Its abnormal expression is closely related to the pathological changes of the destructive remodeling of vascular wall(degradation of the extracellular matrix of vascular wall).Therefore,we hypothesized that the expression of LOX in local endothelial cells was significantly altered after the hemodynamic changes in the cerebral vessels,and that it was possible to cause the occurrence of aneurysms through a series of responses.The main purposes of this study were as follows:1.To explore the effect of changes in shear force on the expression of endothelial LOX;2.Animal experiments were conducted to study the relationship between LOX and hemodynamically induced intracranial aneurysm formation.Methods involved in the present study,the original generation of endothelial cell culture,and the extraction of adherent cells of total RNA extraction,bioinformatics analysis technology,quantitative PCR,endothelial cells hemodynamic model establishment,hemodynamics of rats model,adeno-associated virus sieve,package and drop test,cell apoptosis detection,flow cytometry and Western blotting,medical statistics.Part Ⅰ The effect of shear force changes on endothelial cell LOX expression at the cellular levelObjectives:To explore the effects of shear force changes on LOX expression in rat endothelial cells at the cellular level.Methods:Primary,intracranial artery endothelial cells of SD rat were cultured and divided into three gourps,static group(0 dyn/cm~2),low shear force group(12 dyn/cm~2),and normal shear force group(24 dyn/cm~2)and high shear force group(36 dyn/cm~2).They were placed into the parallel plate flow chamber to provide stable shear force stimulation through a smart peristaltic pump.After 24 hours of stimulation,the total RNA of the adherent cells was extracted and passed.Realtime-PCR was used to determine the expression of mRNA,LOX protein was collected,the expression of LOX protein was detected by Western blotting,and apoptosis was confirmed by detecting the expression level of Cleaved-Caspase 3 protein.Results:1.The expression of LOX was statistically different between the groups.With reference to the quiescent group,the expression of LOX in the low shear and high shear groups increased(p<0.05),and the expression of LOX in the normal shear group decreased(p<0.05).The normal shear force and the high shear force and low shear force group were further compared,and the two were statistically different(p<0.05).Compared the high shear force group with the low shear force group showed the statistically significant difference between them(p<0.05).It can be seen that under normal shear force,the expression of LOX is the lowest,while under high shear and low shear,the expression of LOX is correspondingly increased,but under high shear,the expression of LOX is increased.The most obvious is high.2.The LOX gene was expressed in a small amount in quiescent cells,and the expression was decreased after stimulation with normal shear force.The difference between the two groups would be statistically significant(p<0.05).The expression of RNA was increased with high shear force and low shear force,and there was a statistically significant difference(p<0.05).The expression of RNA was highest under high shear(p<0.05).The expression level of Cleaved-Caspase 3 protein was increased in the low shear force and high shear force group when compared with the static group(p<0.05),but there was no significant difference between the normal shear force group and the normal shear force group.(p>0.05).When the high shear force group was used as a reference,the difference was statistically significant(p<0.05).The normal shear force and the high shear force and low shear force group were further compared,and the two were statistically different(p<0.05).At the same time,it can be found that the expression of Cleaved-Caspase3 protein is most obvious under the action of high shear force.Conclusion:Parallel plate flow chambers can better simulate hemodynamics stimulation of endothelial cells in vitro.Under the action of normal shear force,the expression of LOX was the lowest in endothelial cells.Under the action of high shear force and low shear force,the expression of LOX changed significantly,but under the action of high shear force,LOX expression increased and the Cleaved-Caspase 3 protein expression was also pronounced.PartⅡ Animal experiments study the relationship between LOX and hemodynamically induced intracranial aneurysm formationObjectives:1.To verify the results of cell experiments using animal experiments;2.To construct an adeno-associated virus that interferes with LOX expression,specifically to interfere with the expression of LOX;3.To study the relationship between LOX and hemodynamically induced intracranial aneurysm formation.relationship.Methods:1.Sixty-six SD rats were randomly divided into control group(n=18),sham operation group(n=24),modeling group(n=24).Modeling group established blood flow by microsurgery technique.The model was the ligation of the left external carotid artery at the beginning of the SD rat,the left pterygopalatine artery,and the right common carotid artery end;the sham operation group only performed vascular exploration,no vascular ligation;the control group did not do any treatment;After 3 months,the rat intracranial blood supply arterial resin specimens were prepared,immunohistochemical detection of brain vascular LOX expression,Realtime-PCR technique was used to determine LOX gene expression;2.Exogenous effective RNAi vector was screened and adeno-associated virus was performed.Packaging,specific interference with LOX gene expression;3,72 SD rats were selected again,randomly divided into modeling group(n=24),interference group(n=24)and negative control group(n=24).The hemodynamic model was established.The model group was given the model alone.The intervention group was given adeno-associated virus in the tail vein,the negative control group was given the negative control adeno-associated virus in the tail vein,and the rats were sacrificed 3 months later to prepare the intracranial blood supply.The expression of LOX gene in brain blood vessels was detected,and the expression of LOX gene was determined by Realtime-PCR technique.Results:1.Compared with the sham operation group and the control group,the modeling group showed a significant increase in the tumor rate.The difference was statistically significant(p<0.05).The immunohistochemistry suggested that the LOX expression of the traffic complex increased,and the LOX gene was determined by Realtime-PCR.Increased expression(p<0.05);2.Construction of adeno-associated virus with good expression,mean titer of 5.05E+12;3.After adeno-associated virus-specific interference with LOX expression,the rate of intracranial aneurysm formation was called model group.Compared with the negative control group,the difference was statistically significant(p<0.05).Immunohistochemistry suggested that the LOX expression of the traffic complex was lower than that of the model group and the negative control group.Realtime-PCR method was used to determine the expression of LOX-related mRNA compared with the model group.The negative control group was reduced(p<0.05).Conclusion:The overall trend of animal experiment results is consistent with cell experiments.High shear stimulation can lead to an increase in the expression of LOX in the intracranial arteries,and at the same time lead to aneurysmal changes in the intracranial blood supply,and tumor-like changes can be effectively inhibited after specifically inhibiting the expression of LOX.Summary:High shear stimulation can lead to a significant increase in the expression of LOX,which has been verified in both cell and animal experiments.In vitro cell experiments can lead to increased apoptosis,and animal experiments can lead to increased tumorigenic rate of intracranial aneurysms;specificity Inhibition of LOX expression can reduce the tumor formation rate and inhibit the development of aneurysms.This study preliminarily clarified the relationship between LOX and hemodynamic-induced intracranial aneurysm formation,and the specific mechanism can be studied later,which is expected to improve the prevention and treatment of aneurysmal subarachnoid hemorrhage in China.