Study Of The Mechanism Of 20-hydroxyeicosatetraenoic Acid Induce Myocardial Cell Apoptosis By Delta PKC In Diabetic Cardiomyopathy

20-hydroxyeicosatetraenoic acid?20-HETE?can be generated by the arachidonic acid hydroxylase catalyzed cytochrome CYP450?-peanuts,play an important role in arterial blood pressure,renal function and vascular tone regulating aspects.In recent years,the role and mechanism of 20-HETE in blood vessels is more extensive,but there is few report about the effect of 20-HETE on the heart and related mechanisms.Diabetic cardiomyopathy is one of the major cardiac complications in patients with diabetes mellitus.In this study,we aim to determine whether diabetic cardiomyopathy can lead to an increase in endogenous 20-HETE abnormalities,and the abnormal increase in 20-HETE is a major factor in the cause of diabetes related cardiac disease.In this experiment,the rats were intraperitoneal injected by urine streptomycin for 4 weeks as the animal model of diabetes,which was induced to diabetic myocardial disease of 20-HETE related research.In order to confirm whether the diabetic cardiomyopathy caused by 20-HETE,firstly,we examined myocardial mechanics parameters in diabetic rats after 20-HETE perfusion.We found that compared with normal rats,the heart weight,body weight and myocardial mechanics index were of myocardial cells in diabetic rats was decreased;and the treatment of diabetic cardiomyocytes with 20-HETE decreased these myocardial mechanics parameters significantly.This inhibitory effect of 20-HETE could be blocked by HET0016?20-HETE inhibitor?and rottlerin?delta PKC synthesis inhibitor?respectively.These results suggest that 20-HETE is involved in the myocardial injury induced by PKC.In order to determine whether myocardial injury induced by 20-HETE is involved in apoptosis process,Hoechst and TUNEL fluorescence methods were used to detect the apoptosis of myocardial cells.We found that compared with normal rats,the apoptosis rate of diabetic cardiomyocytes was increased;and the treatment of diabetic cardiomyocytes with 20-HETE increased the apoptosis rate significantly.This stimulatory effect of 20-HETE could be blocked by HET0016 and rottlerin respectively.These results demonstrate that 20-HETE is involved in the myocardial injury induced by PKC through apoptosis pathway.Some studies have indicated that the increase of reactive oxygen species in ROS cells can lead to apoptosis,so we hope to determine whether the myocardial injury induced by diabetes mellitus is caused by ROS.We found that compared with normal rats,ROS content of myocardial cells in diabetic rats was significantly increased;and the treatment of diabetic rats cardiomyocytes with 20-HETE increased the ROS content significantly.This stimulatory effect of 20-HETE could be blocked by HET0016 and rottlerin respectively.These results indicate that the myocardial injury induced by diabetes mellitus is caused by ROS,and 20-HETE can stimulate ROS in cardiac cells by PKC pathway.As the heart of NADPH oxidase is an important potential source of ROS,we first consider the diabetes induced myocardial cell ROS activity enhancement may be related to the activation of NADPH oxidase.Therefore,in order to confirm the content of ROS in diabetes induced myocardial cells increase is caused by stimulation of NADPH oxidase activity,we used NADPH oxidase chemiluminescence method for quantitative detection of its activity in myocardial cells,results showed that compared with normal rats,the activity of NADPH oxidase of myocardial cells in diabetic rats was increased;and the treatment of diabetic rats cardiomyocytes with 20-HETE increased the activity of NADPH oxidase significantly.This stimulatory effect of 20-HETE could be blocked by HET0016 and rottlerin respectively.These results indicate that 20-HETE can stimulate the activity of NADPH oxidase in cardiac muscle cells by PKC pathway,which leads to the increase of ROS content,and ultimately leads to the increase of myocardial cell apoptosis.The part above is the first molecular mechanism of myocardial cell apoptosis.In addition,caspase-3 is key apoptotic molecules.We used spectrophotometric method was established for the determination of myocardial cell apoptosis and caspase-3 protein activity,and the data obtained from the statistical analysis.The results showed that compared with normal rats,the activity of Caspase-3 of myocardial cells in diabetic rats was increased;and the treatment of diabetic rats cardiomyocytes with 20-HETE increased Caspase-3 activity significantly.This stimulatory effect of 20-HETE could be blocked by HET0016 and rottlerin respectively.These results indicate that in the heart of diabetic rats,20-HETE can stimulate the apoptosis protein Caspase-3 activity in myocardial cells by PKC pathway,which leads to an increase in the apoptosis rate of cardiomyocytes,which is the second molecular mechanism of myocardial cell apoptosis.Research has shown that,Ca²⁺ is closely related to the level of function and myocardial cells,so in order to detect diabetes induced apoptosis of cardiac function and myocardial cell injury whether it is achieved through the cardiac muscle cell induced by internal calcium overload,the experimental detection of diabetic rat myocardial cell calcium transient changes,the results found that compared to ordinary rat heart,the amplitude of Ca²⁺ of the heart in diabetic rats were increased and prolonged;and diabetic rat hearts treated by 20-HETE,the Ca²⁺ amplitude and duration were significantly higher than in diabetic rats;however,when the heart of diabetic rats treated by HET0016 and rottlerin respectively,the stimulatory effect of Ca²⁺ by 20-HETE could be blocked.These results indicate that in the heart of diabetic rats,20-HETE can increase the amplitude of Ca²⁺ and the time course of cardiac muscle cells by PKC pathway,which leads to the increase of apoptosis rate,and the part above is the third molecular mechanism of myocardial cell apoptosis.It has been reported that the Bcl-2 family members,which are activated by apoptosis,can regulate the release of apoptosis related proteins by acting on mitochondria,which determines the fate of cells.Among the members of the Bcl-2 family,Bax and Bcl-2 were well studied.The destruction of mitochondrial membrane potential can increase the expression of Bcl-2 and decrease the expression of Bax.Our Western blot results showed that compared with normal rats,the expression of apoptosis gene Bax was increased,apoptosis suppressor gene Bcl-2 was decreased in diabetic rats;and after the treatment of 20-HETE in diabetic rats,Bax was increased,whereas Bcl-2 was decreased more significantly.This stimulatory effect of 20-HETE could be blocked by HET0016 and rottlerin respectively.The part above is the last molecular mechanism of myocardial cell apoptosis.In conclusion,our results demonstrate that 20-HETE could induce apoptosis in diabetes cardiomyocytes,an effect that is likely mediated through multiple intrinsic apoptotic pathways involving ROS and Ca²⁺ pathway.