| BackgroundGlobally,as the increased patients and the rapidly elevated prevalence,diabetes has been one of the most seriously and emergency public health problems in this century.Type 2 diabetes mellitus(T2DM)is commonly associated with abnormal serum lipid level and the percentage of dyslipidemia is 67.1% in it.At present,intraperitoneal injection of STZ is the most commonly used chemical method to create diabetic mice model.A study suggested that multiple intraperitoneal injection of STZ could successfully create model of T2 DM.On the contrary,high dose of STZ injection has been widely used to establish type 1 diabetes mellitus.While,in our experiments,we tried to establish T2 DM with hyperlipermia by a high-fat diet with a large dose of STZ and evaluated this mice model.It was reported that G protein coupled receptors have a closely relationship with diabetes and cardiovascular disease.Adrenaline receptor 3(beta 3 receptor,?3-AR),a membrane receptor,belongs to superfamily member of G protein coupled receptor.No w,there is a divergence in the role of the ?3-AR in myocardium.On the one hand,studies indicated that ?3-AR played a protective function in pressure overload hypertrophy and heart failure,evidenced by the obviously increased ?3-AR expression in the atrium and ventricle of the rat suffered from heart failure.On the other hand,studies revealed that the contentiously elevated expression of ?3-AR could lead to a progressive deterioration of cardiac function,via producing the excessive negative inotropic effects.It was worth noting that one study revealed that the myocardial apoptosis is obviously increased with the application of ?3-AR agonists.According to this phenomenon,it is reasonable to speculate that ?3-AR may be aggravated heart injury via increasing myocardial apoptosis.The disruption of mitochondrial dynamics has a close relationship with cardiovascular diseases,and the aggravated ROS production plays a nonegligible role in it.At the same time,ROS are thought to be associated with diabetic myocardium injury.Further,ROS also can lead to myocardial apoptosis by a direct injury to cell membranes,DNA and protein,when the produced ROS levels are more than the body's antioxidant capacities.Moreover,previous studies revealed that the disorders of mitochondrial dynamics resulted in mitochondrial dysfunction.And the fusion of mitochondrial membrane was mainly mediated by OPA1.Most importantly,studies showed that the expression of OPA1,in mice models,is significantly decreased in mitochondria of diabetic heart,combina tion with abnormal mitochondrial fusion and morphology.Hence,in our research,with the establishment of the model of T2 DM with dyslipidemia,we investigated the mechanism of ?3-AR/OPA1 in the myocardium injury induced by a high glucose and high fat.Methods:1.The SPF C57BL/6J male mice which have a healthy genetic background were divided into two groups: the control group(fed normal diet);the experimental group(fed a high fat which includes 60% fat heat(D12492))and combine with intraperitoneal in jection of 100 mg/kg STZ after 3 weeks a high fat diet.The index from the two mice groups were measured,including the weight,fasting blood-glucose and glucose tolerance test.The serum level of TG,TC,LDL-c,FFA were detected and the contents of serum insulin in mice was measured by ELISA kit.The serum BNP and cardiac ultrasound was detected.The oil red O staining of liver tissue was also detected.The expression of OPA1 was measured by q-PCR and western blot.2.The experimental groups were randomly divided into three groups,again: the control group(saline solution),the agonist group(BRL37344),and the inhibitor groups(SR52390A).The expressions of ?3-AR and OPA1 in cardiac tissue were both measured by q-PCR and western blot.The myocardial fibrosis was detected by Masson staining.Also,immunofluorscence staining was used to detect the OPA1 expression.3.Mouse myocardial cell lines(MCM)were divided into three groups: the high glucose + 0?mol/L palmitic acid sodium group;high glucose + 200?mol/L palmitic acid sodium group;high glucose + 400?mol/L palmitic acid sodium group,and were intervened in 8h and 16 h,respectively.The changes of myocardial apoptosis were measured by the flow cytometry.The expressions of ?3-AR and OPA1 were both measured by q-PCR and western blot.The knock-down of OPA1 myocardial cell model was created by the si RNA transfeciton technology,and the effective of siRNA transfecion was measured by q-PCR and western blot.The changes of ROS,apoptosis,mitochondrial membra ne potential,and the ATP were measured in each group.The changes of ROS and apoptosis after NAC intervention were measured.Results:1.The mice weight was increased with a high fat diet(D12492)which includes 60% fat heat,and lipid droplets were found by oil red O staining in liver tissue.Compared with the mice fed with a normal diet,the fasting blood-glucose,the serum levels of insulin,TG,TC,LDL-c,and FFA were significantly elevated and combination with an impaired glucose tolerance after intraperitoneal injection of STZ.In mice model of T2 DM with dyslipidemia,the serum levels of BNP were increased,cardiac diastolic function was impaired,and the expression of OPA1 were decreased both in mRNA and protein level.2.?3-AR agonists increased the expression of ?3-AR and conversely inhibited the OPA1 m RNA expression;On the contrary,the OPA1 expression was increased with the inhibition of ?3-AR.Consistent with the results of q-PCR,the OPA1 expression was measured by immunofluorescence staining.It was found that the fibrosis of myocardial tissue can be aggravated by ?3-AR agonists and attenuated by the ?3-AR antagonists.3.The myocardial apoptosis was increased with the elevated concentration of palmitic acid sodium and intervention time measured by the flow cytometry.The mRNA and protein expression of OPA1 was decreased and combined with increased ROS production after intervention with a high fat.The elevated levels of ROS,increased myocardial apoptosis,decreased mitochondrial membrane potential and ATP were found after OPA1 siRNA transfecion.With the application of NAC,the levels of ROS and myocardial apoptosis were obviously decreased.Conclusions:In this study,we established the mice model of T2 DM with dyslipidemia via a high-fat diet with a large dose of STZ.OPA1 attenuate myocardial apoptosis via inhibiting of oxidative stress in the condition of high glucose and high fat.The expression of ?3-AR is increased in myocardium tissue of mice suffered from T2 DM with dyslipidemia,which lead to the fibrosis of myocardium and ROS production via the inhibiting of OPA1.This study preliminarily expounded that,T2 DM with dyslipidemia can induce myocardial injury via the ?3-AR /OPA1 pathway,and this finding can provide intervention targets for clinical therapey. |
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