| Background : Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia,which caused by abnormal insulin secretion or dysfunction.Diabeticchronic complications are the leading causes of morbidity and mortality in patients.The most common cause of death among people with diabetes is cardiovascular disease,such as myocardial infarction and heart rhythm.Large conductance calcium activated potassium channels(BK channels)are the most widely distributed potassium channels in the smooth muscle cells of coronary arteries and play an important role in the regulation of resting membrane potential and vascular tone.n-3 PUFAs are essential human fatty acids,mainly including eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA).n-3 PUFAs has been shown to prevent of myocardial infarction and reduce other adverse cardiovascular events through anti-oxidative stress,anti-inflammatory,lipid-lowering and vasodilatation.In recent years,n-3 PUFAs is known to activate BK channels and have protective effects on the cardiovascular system.However,the underlying mechanisms through which DHA activates BK channels remain unclear,may be related to the effect of CYP450 oxidase。Methods(1)Type I diabetic rat models were established by intraperitoneal injection of streptozotocin.Daily fish oil was intragastrically administered and SKF525 A was intraperitoneal injected.(2)Rat coronary smooth muscle cells were isolated by three-step enzymatic digestion.(3)BK channel currents were recorded bywhole-cell patch clamp.The effects of different concentrations of DHA or EPA on the BK channel currents in rat coronary artery smooth muscle cells were determined before and after SKF525 A incubation.BK currents in rat coronary artery smooth muscle were recorded in each group.(4)The effect of different concentrations of DHA/EPA on coronary artery diastolic function in the absence of incubation and incubation of BK channel inhibitor(IBTX)was detected by microvascular tone assay;BK channel inhibitor IBTX and agonist NS1619 were tested.The coronary artery constraction induced by BK channel inhibitor IBTX and relaxation induced by BK channel agonist NS1619 were detected in each group.(5)Western Blotting and qRT-PCR were used to detect the expression of BK channel subunit protein and gene in rat coronary artery smooth muscle cells.(6)Intracellular calcium ion imaging technique was used to detect the change of intracellular calcium concentration in the eight groups of coronary arterial smooth muscle cells.Results(1)The rat model of type I diabetes mellitus was successfully established.The body weights of the rats before and after the modeling were(202.1.75±3.52)g and(294.32±21.52)g(P<0.05,n=40);the blood glucose levels were(7.28±0.49)and(31.23±0.85)mmol/L(P<0.05,n=40),the success rate of the model was 92.4 %.(2)Perfusion of 0.01 μmol/L,0.03 μmol/L,0.1 μmol/L,0.3 μmol/L,1 μmol/L,3 μmol/L,and 10 μmol/L DHA or EPA perfusion can increase the currents of BK channels and the increasements were concentration dependended.After incubated with SKF525 A,the activation of BK channels by DHA or EPA was significantly inhibited.(3)After pre-contracted with ET-1 of coronary artery in normal group and diabetic group,0.1 μmol/L,1 μmol/L,3 μmol/L,10 μmol/L,30 μmol/L and 100 μmol/L DHA or EPA caused concentration-dependent vasodilatation.And DHA/EPA induced-dilation in diabetic group was weaker than the normal group.After incubation of IBTX,the diastolic effect of DHA or EPA on rat coronary arteries was significantly inhibited.(4)There were no significant differences in BK-α subunits and BK-β1 gene and protein expression groups in normal group,normal fish oil fed group,SKF525 A normal group,fish oil fed and SKF525 A normal group rats.(P>0.05,n=6).There was no significant difference in gene and protein expression of BK-α subunit between diabetic group,fish oil fed diabetic group,SKF525 A diabetic group,and fish oil fed with SKF525 A diabetic group(P>0.05,n=6).The relative expressions of BK-β1 subunit gene were 0.91±0.06,1.38±0.17,0.92±0.10 and 1.17±0.06(P <0.05,n=6),respectively and the relative expressions of β1 subunit protein were 0.99±0.06,1.89±0.17,0.92±0.05 and 1.43±0.18(P<0.05,n=6),respectively,in diabetic group,fish oil fed diabetic group,SKF525 A diabetic group,and fish oil fed with SKF525 A diabetic group.(5)There was no significant difference in BK currents of coronary artery smooth muscle cells between normal group,fish oil fed normal group,normal SKF525 A group,and fish fed with SKF525 A normal group.Compared with the diabetic group,there was no significant difference in BK currents in the SKF525 A diabetic group.The BK current in the fish fed and SKF525 A diabetic group was slightly higher than both the diabetic group and the SKF525 A diabetic group but lower than the fish oil fed diabetic group(P<0.05,n=6).(6)KCl and IBTX induced vasoconstriction were detected in coronary arteries of all the eight groups.The IBTX/KCl% values in the blood vessels have no significantly difference between the normal four groups(P>0.05,n=5-6).The fish oil fed and SKF525 A diabetic group had a higher IBTX/KCl% value than the diabetic group and the SKF525 A diabetic group,which was lower than the fish oil fed diabetic group(P<0.05,n=5-6).As for the proportion of BK channel activator NS1619 in pre-contraction with KCl,the ratio of fish oil fed and SKF525 A diabetic group was higher than that of diabetic group and SKF525 A diabetic group,and lower than that of fish oil fed group(P<0.05,n=5-6).(7)There was no significant difference in ratio of intracellular calcium ion fluorescence signal(Ratio)among the normal four groups(P>0.05,n=5).In the four diabetes groups,there was no difference between the diabetic group and the SKF525 A diabetic group(P>0.05,n=5-6).The intracellular calcium concentration in the fish oil fed and SKF525 A diabetic group was significantly lower than that in the diabetic group and higher than the fish oil fed group(P<0.05,n=5-6).Conclusions(1)Both EPA and DHA,the main components of n-3 PUFAs,can respectively activate the BK channels in coronary artery smooth muscle cells of diabetic rats,and plays a protective role from coronary arteryrelaxation.(2)The BK channels of coronary artery smooth muscle cells were impaired in diabetic rats,and n-3 PUFAs can increase the expression of BK-β1,the BK channels currents,and decrease the intracellular calcium ion concentration,improve the coronary artery function through the CYP450 epoxygenase pathway or direct action.That may provide a new strategy for preventing and retarding the progress of coronary heart disease in patients with diabetes mellitus. |
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