The Mechanism Of Oxidative Stress In Rat Aortic Injury Induced By Doxorubicin

Objective:1.To investigate the effects and mechanisms of doxorubicin（DOX）on rat aortic vascular smooth muscle cells;2.To investigate the mechanism of oxidative stress in rat aortic injury induced by DOX.Methods:1.Part I:the rat aortic vascular smooth muscle cells were divided into four groups,saline control group（control group）;doxorubicin 1/3 IC₅₀group（0.33μM DOX group）;doxorubicin IC₅₀group（1μM DOX group）and doxorubicin 4/3 IC₅₀group（1.33μM DOX group）.After 24 hours of administration,collected the cells and detected the changes of NO,ROS,CAT and ATP content in the rat aortic vascular smooth muscle cells;Western Blot was used to detect the expression levels of total PKC,PKCα,PKCβ1,PKCβ2,p22^phox,NOX2,eNOS,iNOS,TXNIP and PKCδin the rat aortic vascular smooth muscle cells.2.PartⅡ:8-week-old SD male rats with a weight requirement of（300±15）g are randomly divided into 7 groups after measuring blood pressure,normal saline control group（n=16,ip normal saline 1.25 mL/kg,once every three days,6 times）;doxorubicin group（n=16,ip DOX 2.5 mg/kg,once every three days,6 times）;DOX+apocynin（APO）group（n=16,ip DOX 2.5 mg/kg,once every three days,6times;ig APO 50 mg/kg,2 times per 3 days,12 times）;APO group（n=16,ig APO 50mg/kg,2 times per 3 days,12 times）;solvent control group（n=16,ig 10%propylene glycol+2%anhydrous ethanol+88%normal saline,10 mL/kg,2 times per 3 days,12 times）;DOX+acetylcysteine（NAC）group（n=16,ip DOX 2.5 mg/kg,once every three days,6 times;ig NAC 100 mg/kg,2 times per 3 days,12 times）;NAC group（n=16,ig NAC 100 mg/kg,2 times per 3 days,12 times）.No intragastric administration on the same day as DOX group.Weigh the rats before administration,adjust the dose according to weight.The tail arterial systolic blood pressure,diastolic blood pressure,mean blood pressure and heart rate of rats in each group were measured after the last administration.The rats were anesthetized with 10%chloral hydrate（0.3 mL/100g,ip）,and blood was collected from the abdominal aorta,The contents of reactive oxygen species（ROS）,angiotensin II（Ang-Ⅱ）,nitric oxide（NO）,malondialdehyde（MDA）,superoxide dismutase（SOD）,catalase（CAT）,glucocorticoid,norepinephrine（NA）and ATP in serum were detected;The vascular reactivity of rat aorta to KCl,PE and ACh were measured using a constant temperature perfusion system;HE staining,Masson staining and transmission electron microscope were used to observe the morphological changes of rat aorta tissues;Western Blot was used to analyze the protein expression levels of total PKC,AT₁,PKCα,NOX2,p22^phox,iNOS,eNOS and PKCδin rat aorta tissues of each group.Results:1.DOX decreased the survival rate of rat aortic vascular smooth muscle cells in a concentration and time-dependent manner,increasing intracellular NO and ROS content and decreasing the antioxidant enzyme CAT activity,oxidative stress damaged mitochondria,resulting in a decrease in ATP level.2.The expression of PKCα,PKCβ1,PKCβ2,p22^phox,eNOS,iNOS and TXNIP were increased in rat aortic vascular smooth muscle cells after treated by DOX.3.The expression of NOX2 in rat aorta vascular smooth muscle cells did not change significantly,but the expression level of total PKC was down-regulated,and different dose of DOX had different effects on the expression level of PKCδ.4.Chronic administration of DOX decreased diastolic blood pressure,but had no effect on systolic blood pressure,mean blood pressure and heart rate of rats.5.Chronic administration of DOX increased the contractile response to KCl and PE and decreased the endothelium-dependent vasodilation response to ACh.NADPH oxidase inhibitor APO and reactive oxygen species inhibitor NAC could improve the vascular response dysfunction induced by DOX administration.6.Chronic administration of DOX increased the content of Ang-Ⅱ、ROS、NO and MDA in the rat serum and decreased SOD and CAT enzyme activity,leading to oxidative stress in rats.The intervention of NADPH oxidase inhibitor APO and reactive oxygen inhibitor NAC can reduce the content of Ang-Ⅱ、ROS、NO and MDA to some extent,and recover the enzyme activity of SOD and CAT,partialy.7.Chronic administration of DOX decreased the content of glucocorticoid in rat serum and had no effect on NA content,while the intervention of NADPH oxidase inhibitor APO and reactive oxygen inhibitor NAC reduced the glucocorticoid content in rat serum further,and had no effect on NA content.8.Chronic administration of DOX increased the content of ATP in rat serum,while the intervention of NADPH oxidase inhibitor APO and reactive oxygen inhibitor NAC reduced the content of ATP,to some extent.9.Chronic administration of DOX resulted in aortic endothelial cell shedding,endometrial folds,endothelial injury,smooth muscle cell proliferation,media thickening,fibrosis;moderate and severe swelling of mitochondria,blurring of adventitia and disappearance of the crest.The treatment of APO and NAC improved the aortic endothelial injury caused by DOX administration,alleviated the degree of fibrosis and mitochondrial injury.10.Chronic administration of DOX could upregulate the expression of total PKC,AT₁,PKCα,NOX2,p22^phoxand iNOS in rat aorta tissue,and downregulate the expression of PKCδ,but there was no significant change in the expression of eNOS.To some extent,the intervention of APO can reverse the increase of protein expression such as total PKC、AT₁、PKCα、NOX2 and iNOS induced by DOX,but the intervention of APO had no effect on the increase of p22^phoxand the decrease of PKCδinduced by DOX;To some extent,the intervention of NAC can reverse the increase of protein expression such as AT₁、PKCα、NOX2,P22^phoxand iNOS as well as the decrease of PKCδinduced by DOX,but the intervention of NAC had no effect on the increase of total PKC induced by DOX.Conclusion:1.DOX can reduce the survival rate of rat aortic vascular smooth muscle cells in a concentration and time-dependent manner.The increase of NO content in rat aortic vascular smooth muscle cells may be related to the up-regulated expression of eNOS and iNOS proteins.The increased production of ROS may be related to the increased expression of p22^phox,PKCα,PKCβ1 and PKCβ2.These factors and the increased expression of TXNIP may mediate the decrease of antioxidant capacity of cells and participate in the occurrence of oxidative stress induced by DOX.2.Chronic administration of DOX led to oxidative stress injury in rat aorta,which were characterized by proliferation of smooth muscle cells,thickening of media,fibrosis,moderate and severe swelling of mitochondria,the disappearance of the crest;the contractile reactivity of aorta to KCl and PE increased;endothelial cells fall off,caused endothelial dysfunction and decreased endothelial-dependent diastolic reactivity of the aorta to ACh.It may induce aortic injury by up-regulating Ang-Ⅱand AT₁of the renin-angiotensin system,and then through the increased expression of PKC and PKCαto mediate the activation of NOX2,induce ROS production and lead to oxidative stress.NADPH oxidase inhibitor APO and ROS inhibitor NAC can improve these changes caused by DOX,which may be related to the inhibition of ROS production.