The Mechanism Of COX-2 Inhibitor Celecoxib Up-regulating Leukotrienes In Atherosclerosis And The Intervention Of Zileuton

Our previous studies have found that celecoxib,a cyclooxygenase-2?COX-2?inhibitor,can aggravate atherosclerosis?AS?lesions,and the mechanism may be related to the up-regulation of the level of the leukotrienes?LTs?.In this study,by using atherosclerotic animal model of apolipoprotein E deficient(ApoE^-/-)mice fed with high-fat diet,the mechanism of COX-2 inhibitor celecoxib up-regulating leukotrienes was investigated from the two inflammatory metabolic pathway,COX-2 and 5-lipoxygenase?5-LO?pathway,in addition,the 5-LO inhibitor zileuton was used to intervene to effects of celecoxib on atherosclerosis.The aim of this experiment is to further study the role of LTs up-regulation in celecoxib-exacerbated atherosclerosis,it will provide a new theory and a new idea for the rational use of COX-2 inhibitor,in addition,for the prevention and prediction of the AS-related adverse cardiovascular reaction caused by COX-2 inhibitor.Part ?: Mechanisms of celecoxib up-regulating leukotrienes in atherosclerosisObjective:To investigate the mechanism of the COX-2 selective inhibitor celecoxib up-regulating LTs in atherosclerosis from the COX-2 and 5-LO inflammatory metabolic pathways in high-fat diet fed ApoE^-/-mice.Methods:Twelve wild-type C57BL/6J male mice were used as normal control group,and twenty-four 6-week-old SPF ApoE^-/-male mice were randomly divided into two groups,the AS model group and the celecoxib group,12 in each group.After two weeks of adaptive feeding,a high-fat diet?normal chow + 0.15% cholesterol + 21% fat?was used in the AS model group and the celecoxib group,normal chow was used in the normal control group.18 weeks later,the protein expressions of COX-2,5-LO,and leukotriene C₄ synthetase?LTC₄S?,the key enzymes involved the synthesis of LTs,in aortic root sections were measured by immunohistochemistry method.The protein and gene expression levels of COX-1,COX-2,5-LO,and LTC₄S were detected by Western Blot and q RT-PCR respectively in aortic homogenates.Results:The results of immunohistochemistry showed that COX-2 was seldom expressed in the normal control group,and COX-2 positiveproducts were mainly located in nucleus and cytoplasm.In AS group and celecoxib group,COX-2 was highly expressed compared with normal control group?P<0.01?,and the positive expression of COX-2 in celecoxib group was significantly lower than that in AS model group?P<0.01?.5-LO was slightly expressed in the normal control group and mainly located in the nuclear membrane.In AS group and celecoxib group,5-LO was highly expressed compared with normal control group?P<0.01?.In celecoxib group,the 5-LO positive expression was significantly higher than that in AS model group?P<0.01?.LTC₄S was less expressed in normal control group.In AS group and celecoxib group,LTC₄S was highly expressed compared with normal control group?P<0.01?,and LTC₄S expression was mainly located in cytoplasm.The LTC₄S positive expression in celecoxib group was significantly higher than that in AS group?P<0.01?.Western Blot results showed that there were no significant differences of COX-1 protein expression among normal control group,AS model group and celecoxib group.The protein expression of COX-2,5-LO,and LTC₄S in the AS model group was significantly higher than that in the normal control group?P<0.01?.The expression of COX-2 protein in the celecoxib group was significantly lower than that in the AS model group?P<0.01?,while the expression of 5-LO and LTC₄S protein was significantly higher than that in the AS model group?P<0.01?.The PCR results showed that there was no difference in the expression of COX-1 gene between AS model group and celecoxib group.Comparing with the AS model group,the expression of COX-2 protein was significantly decreased?P<0.01?,while the expression of 5-LO and LTC₄S genes were significantly increased?P<0.01?.Conclusions:80 mg/kg/d celecoxib intragastric administration for 18 weeks increased the protein and m RNA expression of 5-LO and LTC 4S,while reduced COX-2 protein and gene expression in the aortas of ApoE^-/-mice,indicating that celecoxib may up-regulate aortic LTs levels through the 5-LO shunt mechanism.Part ? The Intervention of Celecoxib in Atherosclerosis by zileutonObjective:To investigate whether the 5-LO inhibitor zileuton can intervene the aggravating effect of celecoxib on atherosclerosis in ApoE^-/-mice fed by high-fat diet.Methods:Twelve wild-type C57BL/6J male mice were used as normal controls,and thirty six-week-old SPF ApoE^-/-male mice were randomly divided into 3 groups,AS model group,celecoxib group and celecoxib+zileuton group,12 in each group.After two weeks adaptive feeding,the normal control group mice were fed with normal diet,and mice of AS model group,celecoxib group and celecoxib+zileuton group were fed with high-fat diet?normal chow + 0.15% cholesterol + 21% fat?.18 weeks later,B-type ultrasound scan was performed to observe the formation of the atherosclerotic lesion in aorta.The plaques area ratio?%?in aortic arch and the blood vessel diameter of abdominal aorta were measured by image pro plus 6.0 software.The photographs of the entire aortas were taken under a stereomicroscope and then the entire aortas were stained with oil red O to observe the formation of atherosclerotic lesions and to measure the positive area ratio?%?by image pro plus 6.0 software.In addition,the aortic root sections were stained withhematoxylin-eosin?HE?,and then the plaque area ratio?%?and intima-media thickness?IMT?were measured by image pro plus 6.0 software.Results:The results from B-type ultrasound scan,the photographs of the entire aortas taken under a stereomicroscope,oil red O stain of the entire aorta and HE staining of aorta roots all showed that the obvious atherosclerotic lesions have formed in the aortas of AS model group.The range and extent of aortic therosclerotic lesions in the celecoxib group were significantly more serious than those in the AS group,however,the range and extent of aortic therosclerotic lesions in the celecoxib +zileuton group were significantly less alleviative than those in the celecoxib group.In the B-type ultrasound scan measurement results,comparing with the normal control group,the aortic arch plaque area ratio?%?of the AS model group was significantly increased?P<0.01?.Comparing with the AS group,the aortic arch plaque area ratio?%?was increased in the celecoxib group?P<0.01?.Compared with the celecoxib group,the aortic arch plaque area ratio?%?in the celecoxib +zileuton group was decreased?P<0.01?,but had no significant difference compared with the AS group?P>0.05?.The blood vessel diameter of abdominal aorta in the AS model group was significantly lower than that in the normal control group?P<0.01?.The lumen of the abdominal aorta was extremely narrow in the celecoxib group,and the blood vessel diameter of abdominal aorta was significantly reduced compared with the AS model group?P<0.01?.The blood vessel diameter of the abdominal aorta in the celecoxib+zileuton group was significantly enlarged than that in the celecoxib group?P<0.01?.In the oil red O stain of the entire aorta measurement results,the entire aorta plaques area ratio?%?of the AS group was significantly increased comparing with the normal control group?P<0.01?,indicating successful modeling.Compared with the AS group,the plaques area ratio?%?of the enitre aorta in the celecoxib group was significantly increased?P<0.01?.The plaques area ratio?%?of the enitre aorta in the celecoxib + zileuton group was significantly lower than that of celecoxib group?P <0.01?,but had no significant differences compared with the AS model group?P >0.05?.In the HE staining of aorta root section measurement results,the plaque area ratio?%?in the AS model group was significantly increased comparing with the normal control group?P<0.01?.Compared with the AS model group,the plaque area ratio?%?of celecoxib group was significantly increased?P<0.05?.The plaque area ratio?%?in the celecoxib+zileuton group was significantly smaller than that of the celecoxib group?P<0.05?,but has no significant differences comparedwith the AS model group?P<0.05?.IMT statistics showed that,the IMT of the AS model group increased significantly comparing with the normal control group?P<0.01?.Compared with the AS model group,the IMT of the celecoxib group increased significantly?P<0.01?.Compared with celecoxib group,the IMT value of celecoxib+Zileuton group was significantly decreased?P<0.01?,but had no significant differences compared with the AS model group?P >0.05?.Conclusion:80 mg/kg/d celecoxib intragastric administration for 18 weeks aggravates AS lesions in Apo E-/-mice,5-LO inhibitor zileuton can reduce the aggravating effect of celecoxib on AS.