| BackgroundCerebrovascular diseases,with atherosclerosis(AS) being the main patho-change,do harm to human beings badly.The pathogenesy of AS is very complicated.Nowadays, the theory of "response to injury" and chronic inflammation have been widely accepted.The immunologic mechanism plays an important role in the formation and development of atherosclerosis.Those cells and moleculars,which participate in immunoreaction,can influence and interact with each other,and promote atherosclerosis.The role of oxidized low density lipoprotein(oxLDL) is being paid more and more attention.OxLDL is formed when LDL is oxidized,which is related with oxidative stress.OxLDL takes part in atherosclerosis through its cytotoxic effect, chemotaxis and its effect on fibrinolysis and coagulation.Furthermore,oxLDL is antigenic,resulting in generation of autoantibodies(oxLDL-Ab).OxLDL and oxLDL-Ab both are involved in atherosclerosis and play important roles.Many previous studies have investigated the roles of oxLDL and oxLDL-Ab.However,the results are inconsistent and to some extent contradictory.The discrepancy among these studies indicates the complexity of immunologic mechanism in AS.Atherosclerosis is a multi-stage,chronic and slowly progressive disease.It includes vascular endothelial cell damage,adhension of monocytes and lymphocytes,invasion into endothelium and transformation to foam cells,formatin of fatty streak,migration and hyperplasia of smooth muscle cells and formation of fibrous plaques,which gradually evolved into plaques,and the plaque instability or rupture leading to acute clinical events.AS is usually found in major arteries.Carotid is the main path of blood-supply to brain,and many atherosclerosis occurred there.The stability of atherosclerosis is very important as the rupture of plaques may induce stroke,which has high fatality and disability rates.The surgical operation on unstable plaques provides a new way of secondary prevention.Thus,it is very important to study the stablity of plaques.In the present study,we designed an epidemiologic study on patients with stable lesions and studied the association of blood lipids,oxLDL, oxLDL-Ab and other risk factors with stable carotid atherosclerosis.It can provide a basis for the prevention and cure of AS and is very meaningful.OxLDL takes part in atherosclerosis through various mechanisms,with inducing the vascular endothelial cell apoptosis being one of the early effects.Apoptosis is also called programmed cell death or cell suicide,which plays a crucial role in developing and maintaining health.It is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes.It occurs after sufficient cellular damage or some certain stimulus,and controlled by specific genes.In this process,a programmed sequence of events leads to the elimination of cells without releasing harmful substances.The end result of apoptosis is cell death without inflammation of the surrounding tissue.Apoptosis,especially apoptosis of the vascular endothelial cells,plays a crucial role in the occurrence and development of atherosclerosis.It affects the normal function of endotheliocyte, promotes the thrombopoiesis,and participates in vascular remodeling,and influences the morphous and structure of arteries.Garlic and garlic oil extracted have extensive pharmacologic actions,such as anti-pathogenic microorganism,anticancer,immunity enhancement,antioxygen, anti-aging,liver protection,prevention and cure of diabetes.Above all,they have outstanding preventive and therapeutical effects on diseases of cardiovascular and cerebrovascular system by multiple mechanisms,with protection of endothelial cells involved.Selenium(Se),the essential trace element,can also protect cardiovascular system.The level of Se has a significant inverse correlation with the incidence of cardiovascular system diseases.Se also has other biological effects,such as antioxygen and detoxication.It is a hot issue that whether garlic and selenium could synergize each other or not.However,the natural PH of garlic is from 5.3 to 5.8, while that of sodium selenite is about 11.0.Thus,the alltride would be destoryed when mixing them directly.Furthermore,the selenium in sodium selenite is inorganic, and it is difficult to determine the absorption and transfer ratios.Microcapsule can unite garlic oil and selenium,but it is impossible to study its effects by experiments. Selenium-enriched garlic provides an opportunity to unite garlic oil and selenium,and to evaluate their effects.It was reported that selenium-enriched garlic can protect cardiovascular system. However,the specific mechanism has not been elucidated,especially the effect on cell apoptosis.Therefore,we extracted oil from selenium-enriched garlic cultivated in Cangshan,and determined the contents of alltride and selenium.Then oxLDL was applied to ECV304 to induce apoptosis,which was intercepted by selenium-enriched garlic oil.By doing this,the effect of selenium-enriched garlic oil on endothelial cell damage induced by oxLDL was studied and the potential mechanisms was discussed.ObjectivesIn the present study,subjects who had thickened endarterium or plaques but no clinical events were recruited.A case-only study and a case-control study were designed to investigate the association of blood lipids,oxLDL,oxLDL-Ab and other risk factors with carotid atherosclerosis.The relationship between oxLDL and oxLDL-Ab was also studied.It was expected to elucidate the immunologic mechanism in atherosclerosis,and to provide scientific basis for prevention and cure of atherosclerosis.OxLDL was applied to ECV304 to induce cell apoptosis,and then selenium-enriched garlic oil extracted was applied.The cell survival rate,cell proliferation,apoptosis rate,changes of apoptotic associated proteins,as well as their mRNA levels were detected in order to study the damage of vascular endothelial cells induced by oxLDL, the effect and potential mechanism of selenium-enriched garlic oil on the damage.It was expected to make sure whether and how much selenium-enriched garlic oil could be protective,and to provide scientific basis for further studies of its effects.Methodsâ… .Observational study of the association of oxLDL and atherosclerosis in clinic1.Subjects(1) Case-only studyWe screened all the subjects over 40 years old who admitted to the teaching hospital of Shandong University of traditional Chinese medicine for ultrasonographic scanning of the carotid artery between February 2005 and December 2005.We excluded those who had severe hepatic,renal or thyroid disorders,or malignant tumor.We also excluded those who refused to participate in this study after explanation.Furthermore, the patients had to show increased IMT(≥1.0mm) and/or at least a measurable plaque in the carotid artery,and had no clinical events after 2 years.No one received vitamin B6,B12,folic acid or antioxidant in the last 2 weeks.(2) Case-control studyCases were the same as those in the case only study.Controls were those examined in the corresponding period but had no atherosclerosis lesions.Informed consents were obtained from all the subjects participating in the study.2.Carotid Ultrasonography ExaminationThe carotid ultrasonography examination was performed by a special messenger with ESAOTE ultrasonoscope.The intima-media thickness(IMT) of the common carotid artery was measured.A plaque was defined as IMT≥1.3mm or a distinct area with an IMT 50%thicker than the neighboring sites.The volumes of all plaques were calculated.3.MeasurementsInformation on the characteristics of the subjects,such as name,sex,age,occupation, average monthly incomings,education background,address,connecting method, smoking and drinking habits,physical exercise habits and family history were obtained by a unified questionnaire.Body weight,height and blood pressure were measured.4.Biochemical analysisVenous blood samples were taken in the morning after an overnight fast.Plasma and serum were separated respectively according to different demands and FPG(fasting plasma glucose) was determined within 2 hours by glucose oxidase method.Low density lipoprotein(LDL),high density lipoprotein(HDL),lipoprotein(a)(Lp(a)), total cholesterol(TC) and triglyceride(TG) levels were determined by enzymatic techniques.Angiotensinâ…¡(Angâ…¡) was determined by radioimmunoassay. Lipoprotein a(Lpa) was determined by emuls-strengthened projection turbidity method.Antibodies against Helicobacter pylori(Hp),Chlamydia pneumoniae(Cp) and cytomegalovirus(CMV) were assayed by ELISA.Circulating oxLDL and oxLDL-Ab were also measured by ELISA.5.Statistical analysisAll statistics were analyzed by SPSS 11.5 and SAS 9.0.A stepwise multiple regression analysis and logistic regression analysis were used to study the influencing factors for circulating oxLDL and oxLDL-Ab concentrations.A general linear regression analysis was used to study the influencing factors for IMT and plaque volumes.An unconditional logistic regression analysis and discriminant analysis were performed in the multivariate analysis in the case-control study.â…¡.In vitro study of intercepting the endothelial cell damage induced by oxLDL Garlic oil was prepared from general and selenium-enriched garlic with two methods, extraction with ethanol and olive oil,and water vapor distillation and extraction with olive oil.The homogenate of garlic was also prepared.The contents of alltride were determined by HPLC,and those of selenium were determined by hydride generation atomic fluorescence spectrometry.LDL was oxidative modified into oxLDL by incubation with copper ion in vitro,and estimated by AGE prestained with Sudan black and TBARS.Garlic oil prepared by water vapor distillation and extraction with olive oil was applied in the experiments.ECV304 were cultured in RPMI-1640 supplemented with 10%(v/v) heat-inactivated new-born calf serum and 100U/ml penicillin and streptomycin.Furthermore,the cell line was grown in a humidified incubator at 37℃and 5%CO2.ECV304 lied in logarithm growth period were digested with 0.25%trypsin,and adjusted to a density of 2×104 cells/ml with RPMI-1640.The survival rate of cells was determined by trypan blue exclusion,and the viability of cells was determined by MTT.The concentrations of garlic oil and Se with the inhibition rates lower than 10%were calculated and used in the study.The cells were divided into 8 groups,control group,oxLDL group,selenium-enriched garlic oil group,oxLDL and selenium-enriched garlic oil group,general garlic oil group, oxLDL and general garlic oil group,selenium group,oxLDL and selenium group.The final concentration of general garlic oil was 5μl/L,selenium-enriched garlic oil being 2.5μl/L,selenium being 0.02μg/ml,and oxLDL being 22μg/ml.Cells were treated with garlic oil for 24h followed by oxLDL in the groups with both oxLDL and garlic oil.The viability of cells was determined by MTT,the apoptosis rate was determined by flow cytometry stained with Annexin V-FITC/PI.The morphology of cells was assessed by Giemsa's staining,the expressions of Bc1-2,Bax and caspase-3 were analyzed using western blot,and the mRNA levels of Bc1-2,Bax and caspase-3 were detected by reverse transcription polymerase chain reaction(RT-PCR).Resultsâ… .Observational study of the association of oxLDL and atherosclerosis in clinic1.Case-only study(1) Correlation between circulating oxLDL and oxLDL-AbSpearman's rank correlation test showed that there was no linear correlation between circulating oxLDL and oxLDL-Ab.(2) Relationship between circulating oxLDL and atherosclerosisSpearman's rank correlation test showed that circulating oxLDL was weakly correlated with IMT(rs=0.195,P=0.084) and not correlated with plaque volumes (rs=0.093,P=0.410).However,the multivariate analysis showed that oxLDL was correlated with neither IMT nor plaque volumes. (3) Relationship between circulating oxLDL-Ab and atherosclerosis Spearman's rank correlation test showed that circulating oxLDL-Ab was correlated with neither IMT(rs=0.005,P=0.962) nor plaque volumes(rs=0.043,P=0.703).The multivariate analysis showed that oxLDL-Ab was correlated with neither IMT nor plaque volumes.(4) Risk factors for carotid atherosclerosisIn the general linear regression models,only age and hypertension were independent factors associating with IMT and plaque volumes.2.Case-control study(1) Relationship between circulating oxLDL and atherosclerosisThe case-control study showed that oxLDL was a risk factor for atherosclerosis.(2) Relationship between circulating oxLDL-Ab and atherosclerosisThe case-control study showed that oxLDL-Ab was a protecive factor for atherosclerosis.(3) Risk factors for carotid atherosclerosisIn the case-control study,age,BMI,TC,TG and oxLDL were independent risk factors for carotid atherosclerosis after confounding factors were controlled,with the contribution of TG being the greatest(OR=7.399,95%CI:1.498-36.554);while oxLDL-Ab played a protective role(OR=0.793,95%CI:0.714-0.880).(4) Prediction of carotid atherosclerosisStepwise discriminate analysis showed that both oxLDL and oxLDL-Ab were predictable for carotid atherosclerosis.â…¡.In vitro study of intercepting the endothelial cell damage induced by oxLDL1.The contents of alltrideThe contents of alltride in different components of general and selenium-enriched garlic were determined.The results showed that the alltride was extracted successfully by olive,and the garlic oil extracted had high alltride content.2.The contents of seleniumThe contents of selenium in different components of general and selenium-enriched garlic were determined.The results showed that the content of selenium in selenium-enriched garlic was 2609 times as high as that in general garlic.Most of the selenium in selenium-enriched garlic was water-soluble.When the garlic oil was prepared by water vapor distillation and olive oil,the content of selenium in selenium-enriched garlic oil was 142 times as high as that in general garlic oil. Selenium mostly existed in water-solution,with the proportion being 71.42%.When the garlic oil was extracted with ethanol and olive oil,the content of selenium in selenium-enriched garlic oil was 181 times as high as that in general garlic oil. Selenium mostly existed in the emulsion phase after extraction,with the proportion being 48.76%.3.The effects of general garlic oil,selenium-enriched garlic oil and selenium on cell growthECV304 lied in logarithm growth period were cultured with general or selenium-enriched garlic oil(concentrations being 2.5-40μl/L) or selenium (concentrations being 0.04-0.64μg/ml).Cells were collected after 24 hours,48 hours and 72 hours respectively.Count 200 cells and the results showed that the cell survival rates were higher than 90%,indicating that general garlic oil or selenium-enriched garlic oil or selenium at these concentrations had no obvious side effects on cells.4.The effects of general garlic oil,selenium-enriched garlic oil,selenium and oxLDL on cell viabilityECV304 lied in logarithm growth period were cultured with general or selenium-enriched garlic oil(concentrations being 5-320μl/L) or selenium (concentrations being 0.04-2.56μg/ml) or oxLDL(concentrations being 10-160μg/ml) for 24 to 72 hours.Cell viability was detected by MTT and the results showed that the inhibition effects of general garlic oil,selenium-enriched garlic oil,selenium and oxLDL on cell viability were enhanced with time or cencentrations increasing. General garlic oil at a concentration of 5μl/L,selenium-enriched garlic oil at 2.5μl/L or selenium at 0.02μg/ml almost had no inhibition effect on normocellular viability,with the inhibition rates being 3.46%,4.86%and 4.54%,respectively.The differences with that of control group were not significant(P＞0.05).OxLDL at a concentration of 22μg/ml inhibited the cell viability greatly,with the inhibition rates being 24.60%,and had significant difference with that of control group(P＜0.05).After general garlic oil,selenium-enriched garlic oil or selenium was applied,the inhibition of cell viability induced by oxLDL was significantly depressed,with the inhibition rates reduced to 7.23%,3.70%and 5.68%,respectively.The differences with that of oxLDL group were significant(P＜0.05).5.The effects of selenium-enriched garlic oil and oxLDL on cell morphology The cell morphology was observed under immersion objective aider Giemsa's staining. The cells in control group showed big and round nucleus.Their structures were integrity and shapes were in order.The morphology of the cells treated with oxLDL was irregular and apoptosis was induced.When cells were treated with selenium-enriched garlic oil in advance,the morphology was almost normal,and cell apoptosis was observed occasionally.6.The effects of general garlic oil,selenium-enriched garlic oil,selenium and oxLDL on cell apoptosisThe cell apoptosis was detected by Annexin V-FITC/PI staining.The results showed that the apoptosis rate in oxLDL group was significantly elevated(P＜0.05).The apoptosis rates in general garlic oil group,selenium-enriched garlic oil group or selenium group were not significantly different from that in control group(P＞0.05). However,both garlic oil and selenium could inhibit the apoptosis induced by oxLDL, and the apoptosis rates were significantly different from that in oxLDL group (P＜0.05).7.The effects of selenium-enriched garlic oil and oxLDL on expression of Bax,Bc1-2 and caspase-3The results of western blot showed that the expressions of Bax and caspase-3 in oxLDL group were significantly high,while that of Bc1-2 was low compared with that in control group.When cells were treated with selenium-enriched garlic oil in advance, the expressions of Bax and caspase-3 were significantly decreased,while that of Bc1-2 was increased,and they were all similar with that in control group. 8.The effects of selenium-enriched garlic oil and oxLDL on mRNA of Bax,Bc1-2 and caspase-3The results of RT-PCR showed that oxLDL decreased the mRNA level of Bc1-2 significantly(16.1%,P＜0.05),and increased the mRNA levels of Bax(17.5%,P＜0.05) and caspase-3(61.0%,P＜0.05).The ratio of Bc1-2 to Bax was decreased meanwhile.When cells were treated with selenium-enriched garlic oil in advance,the mRNA level of Bc1-2 increased by 11.1%,and was significantly different from that in oxLDL group(P＜0.05).The mRNA level of Bax decreased by 2.9%,and was no longer significantly different from that in control group(P＞0.05),although the difference with that in oxLDL group was not significant(P＞0.05).The mRNA level of caspase-3 decreased by 25.7%,and was significantly different from that in oxLDL group(P＜0.05),although it was still significantly higher than that in control group(P＜0.05).The ratio of Bc1-2 to Bax was increased and similar with that in control group.Conclusions1.There was no linear relationship between circulating oxLDL and oxLDL-Ab in patients with stable atherosclerotic lesions.2.The circulating oxLDL levels in carotid atherosclerotic patients were significantly higher than those in controls,but had no association with the degree of pathological changes in patients with stable atherosclerotic lesions.3.The circulating oxLDL-Ab levels in carotid atherosclerotic patients were significantly lower than those in controls,but had no association with the degree of pathological changes in patients with stable atherosclerotic lesions.4.Both oxLDL and oxLDL-Ab can predict whether a person has a high risk of developing atherosclerosis,and can be used to identify those needing ultrasonography examination.5.OxLDL had obvious cytotoxicity,and could induce the vascular endothelial cell apoptosis.This may be related with the changes of expressions of Bc1-2,Bax and caspase-3 proteins from the mRNA levels.This was one of the early effects of oxLDL on atherosclerosis.6.Selenium-enriched garlic oil at a relatively low range of concentrations could inhibit the cytotoxicity of oxLDL.It intercepted the vascular endothelial cell apoptosis induced by oxLDL through reversing the expressions of Bc1-2,Bax and caspase-3 proteins from the mRNA levels.By this action,selenium-enriched garlic oil could protect the vascular endothelial cells and inhibit the occurrence of atherosclerosis.
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