| Background Cardiovascular diseases are the most important causes of morbidity and mortality in diabetic patients,who have a two to fourfold increase in the risk of coronary artery disease.BK channels,expressed on vascular smooth muscle cells(SMCs),act as breaks for the increase in vascular tone that occurs after membrane depolarization and elevation of cytosolic calcium.Recent studies have found that docosahexaenoic acid(DHA)can protect cardiovascular system,such as anti-arrhythmias,reducing blood lipids and blood pressure,prevention of myocardial infarction.However,the underlining mechanisms remain virtually unknown.It may be related to the activation of BK channels on coronary SMCs.Objective To investigate the effects and mechanisms of DHA on diabetic coronary function.Methods(1)Streptozotoc in-induced diabetic rat models were established by injection intraperitoneally,and DHA-treated rat models were established by intragastric administration of 0.1 g DHA per day for 8 weeks.(2)The effects of different concentrations of DHA on coronary artery function in normal groups and diabetic groups,with and without pre-incubation of BK channel inhibitor IBTX,and the effects of BK channel inhibitor IBTX or BK channel activitor NS 1619 on coronary arteries function of DHA-treated rat models were detected by vascular teision measurement.(3)Coronary SMCs were isolated by enzyme digestion.(4)The effects of DHA on coronary SMCs BK channels were recorded by patch clamp in single channel configuration.(5)The mRNA and protein expressions of BK channels in DHA-treated rat models were determined by qRT-PCR technique and the western blotting technique,respectively.Results(1)Streptozotocin-induced diabetic rat models were established successfully.The weight and blood glucose were(190.75±4.32)vs(248.89±18.41)g(P<0.05,n=60)and(7.29±0.31)vs(30.21±1.35)mmol/L(P<0.05,n=60)before and after diabetes,respectively,and the successful rate was 91.6%.(2)When coronary arteries were exposed to 0.1 ?mol/L,1 ?mol/L,3?mol/L,10 ?mol/L,30 ?mol/L and 100 ?mol/L DHA,the percentages of coronary vasoconstriction were(3.01±1.65%),(4.22±2.45%),(4.44±2.94%),(20.89±3.34%),(50.27±6.37%)and(68.54±8.63%)(P<0.05,n=13)in normal rats,while the percentages were(1.21±1.17%),(1.44±2.93%),(4.38±3.02%),(10.66±3.86%),(15.28±4.62)and(36.82±5.83%)(P<0.05,n=13)in diabetic rats.(3)When coronary arteries were exposed to 0.1?mol/L,1 ?mol/L,3 ?mol/L,10?mol/L,30 ?mol/L and 100 ?mol/L DHA with pre-incubation of IBTX,the percentages of coronary vasoconstriction were(2.03±1.82%),(2.52±1.14%),(3.23±1.56%),(3.18±2.34%),(3.39±1.59%)and(9.58±2.63%)(P<0.05,n=5)in normal rats,while the percentages were(0.98±1.72%),(0.77±1.69%),(1.24±1.57%),(1.17±1.52%),(1.68±2.62)and(1.57±1.34%)in diabetic rats(P>0.05,n=5).(4)When coronary arteries exposed to 100 nmol/L IBTX and 60 mmol/L KCl,the percentages of coronary vasoconstriction between IBTX and KCl(IBTX/KCl%)were(79.24±4.36%)in control groups,(87.25±2.13%)in DHA-treated control groups,(29.81±4.08%)in diabetic control groups,and(70.73±5.27%)in DHA-treated diabetic groups(P<0.05,n=12).(5)When exposed to 30 ?mol/L NS1619,the percentages of coronary vasodilation(NS1619/KCl%)were(46.89±7.80%)in control groups,(48.59±4.00%)in DHA-treated control groups,(15.28±2.21%)in diabetic control groups,and(44.04±3.11%)in DHA-treated diabetic groups(P<0.05,n=8).(6)At test potential 60 mV,1 ?mol/L calcium,the open probabilities(NPo)of BK channels were(0.091±0.009),(0.090±0.0102),(0.093±0.092),(0.098±0.0091),(0.099±0.013),(0.101±0.025),(0.3712±0.0456),(0.751±0.029)and(0.866±0.046)(P<0.05,n=5)with perfusion of 0 ?mol/L,0.01 ?mol/L,0.03 ?mol/L,0.1 ?mol/L,0.3 ?mol/L,1?mol/L,3?mol/L,5 ?mol/L and 10 ?mol/L DHA respectively in normal groups.In diabetic groups,the NP0 of BK channels were(0.0161±0.011),(0.015±0.010),(0.017±0.013),(0.016±0.0017),(0.019±0.0112),(0.018±0.020),(0.153±0.021),(0.3127±0.019)and(0.450±0.025)(P<0.05,n=5)at 0 ?mol/L,0.01?mol/L,0.03?mol/L,0.1 ?mol/L,0.3?mol/L,1 ?mol/L,3 ?mol/L,5 ?mol/L and 10?mol/L DHA respectively.DHA activated BK channels in a dose-dependent manner and the NPo of BK channels in diabetic groups were significantly decreased under the same concentrations of DHA(P<0.05).(7)The NP0 of BK channels at test potential 20 mV,40 mV,60 mV,80 mV and 100 mV were(0.004±0.001),(0.023±0.009),(0.254±0.055),(0.667±0.042)and(1.109±0.034)(P<0.05,n=10)in control groups;the NP0 were(0.006±0.001),(0.039±0.013),(0.302±0.002),(0.779±0.050)and(1.295±0.051)(P<0.05,n=10)in DHA-treated control groups;the NP0 were(0.000±0.001),(0.001±0.002),(0.014±0.004),(0.072±0.027)and(0.270±0.021)(P<0.05,n=10)in diabetic control groups;the NP0 were(0.006±0.000),(0.023±0.006),(0.150±0.050),(0.428±0.015)and(0.896±0.042)(P<0.05,n=10)in DHA-treated diabetic groups.(8)The mRNA levels of BK channel a subunit(BK-?)were unchanged in DHA-treated non-diabetic and diabetic groups,compared to those of DHA-untreated rats.The BK channel ?1 subunit(BK-?1)mRNA expressions were(1.25±0.15)in control groups,(1.28±0.12)in DHA-treated control groups,(0.77±0.13)in diabetic control groups,and(0.78±0.10)in DHA-treated diabetic groups(P<0.05,n=8).(9)The protein levels of BK-a were unchanged in DHA-treated non-diabetic and diabetic groups,compared to those of DHA-untreated untreated rats.The BK-?1 protein expressions were(1.22±0.04)in control groups,(1.18±0.04)in DHA-treated control groups,(0.20±0.03)in diabetic control groups,and(1.14±0.07)in DHA-treated diabetic groups(r<0.05,n=8).Conclusions(1)Reduced NP0 of BK channel and decreased BK channel mRNA and protein expressions in diabetic rats may contribute to diabetic coronary vasculopathy.(2)On the setting of DHA,the NPo of BK channels in normal and diabetic coronary SMCs were both augmented,while the NPo of diabetic rats were lower than those of normal rats.Treatment with DHA increased diabetic coronary BK channels NPO which was reduced in diabetic rats.(3)DHA can relax coronary in a concentration-dependent manner both in normal and diabetic rats,but the percentages of coronary vasodilation were significantly lower in diabetic rats than those in normal rats.Effects of DHA on coronary arteries function in both groups were abolished after pre-treatment with IBTX.BK channels sensitive vasoreactivity are impaired in diabetic groups and can be restored by treatment with DHA.(4)DHA can protect diabetic coronary by increasing BK-?1 subunit expression and activating BK channels. |
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