The Effect Of DHA On Oxidative Stress In Heart Of Type2Diabetic Rats

Objective: Diabetes has become a threat to human health and life. Nearly95%of diabetic patients are type2diabetes （T2DM）. T2DM causes systemiccomplications and cardiac complication is the main reason for the highmortality rate. Oxidative stress is one of the most important mechanisms thatleeds to cardiac complication of diabetes.Oxidative stress results from the too much active substance in the tissuecells-including reactive oxygen species （ROS） and reactive nitrogen species（RNS）. Active substances increase antioxidant substances scavengingcapability, which result in oxidation of biological macromolecules （includinglipids, carbohydrates, proteins and DNA）.In diabetes, due to sustainedlong-term stimulution of hyperglycemia, the active substances in theorganization increase and antioxidant system function declines. Oxidation andantioxidant imbalance causes oxidative stress.Docosahexaenoic acid is one of the n-3polyunsaturated fatty acids,which has antioxidation and can prevent cardiac complication. Can DHAimprove the oxidative stress in heart of T2DM rats? It has not been reported.For above purposes, in this experiment oxidative stress, antioxidantenzyme activity in heart of type2diabetic rats fed diet with DHA andwithout DHA were assayed to provide experimental evidence for using DHAfor prevention of type2diabetes cardiac complications.Methods:1AnimalsThe type2diabetes rat model made by Hou LG et al. of our researchgroup was used as subjects. The rats were divided into three groups: controlgroup, diabetes mellitus group and diabetes mellitus administered DHA group.Cardiac tissues were removed for the detection of ROS content, T-AOC activity （T-AOC）, the contents of advanced glycation end-products （AGEs）,Protein carbonizations （PCG）, malondialdehyde （MDA）, Hydroxynonenal（4-HNE）,4-hydroxy-2-hexenal （4-HHE） and activities of antioxidativeenzymes.2Determination of the T2DM rat myocardial oxidative stress level.2.1Detection of the Total Antioxidant （T-AOC） Capacity in cardiac tissues.Tissue’s antioxidant can restore Fe3+to Fe2+, and the latter can form stableComplex with Phenanthroline. Antioxidant capacity can be measured bycolorimetry. Result is expressed in antioxidant per mg protein sample （U/mgprot）.2.2Detection of the content of reactive oxygen species （ROS） in cardiactissues.We stained myocardial sections of different test groups by fluorescentprobe-dihydroethidium （DHE）. According to fluorescence microscope’s redfluorescence, we can judge quantity of ROS content in different groups.2.3Detection of the content of advanced glycation end-products （AGEs）incardiac tissues.The result could be detected by Enzyme-linked Immunosorbent Assayand expressed in AGEs content per mg protein sample （nmol/μg prot）.2.4Detection of the content of protein carboxide groups （PCG） in cardiactissues.The result could be detected by2，4-dinitrophenylhydrazine colorimetryand expressed in protein carboxide per mg protein sample（nmol/mg prot）.2.5Detection of the content of malondialdehyde （MDA） in cardiac tissues.Thiobarbituric acid （TBA） colorimetry method was used to determine thecontent of MDA in the tissues, which was expressed in protein MDA per mgprotein sample （nmol/mg prot）.3Determination of the T2DM rat cardiac antioxidant activity.3.1Detection of the activity of T-SOD in cardiac tissues.We detected the inhibition rate of SOD to superoxide anion radical by themethod of xanthine oxidase to calculate the activity of SOD. The results are expressed by a per milligram of sample protein activity unit （U/mg prot）.3.2Detection of the activity of CuZn-SOD in cardiac tissues.We detected the inhibition rate of SOD to superoxide anion radical by themethod of xanthine oxidase, then calculate the activity of SOD, subsequentlyafter Mn-SOD activity was inhibited to determine the activity of CuZn-SODin cardiac tissues. The activity of CuZn-SOD was expressed as （U/mg prot）.3.3Detection of the GSH-Px’s activity.Firstly we need to detect the H₂O₂’s consumption in unit time by DTNBcolorimetry and then calculate GSH-Px’s activity. The result could beexpressed in enzyme activity per mg protein sample （U/mg prot）.3.4Detection of the CAT’s activity.Firstly we need to detect the H2O2’s consumption in unit time byammonium molybdate colorimetry and then calculate CAT’s activity. Theresult could be expressed in enzyme activity per mg protein sample （U/mgprot）.4The change of T2DM rat myocardium n-3and n-6fatty acid metabolites.4.1Detection of the content of4-hydroxynonenal （4-HNE） in cardiac tissues.We stained the tissue slice’s4-HHE antibody of normal control, DMgroup and DHA group by Immunohistochemistry; then scanned pictures byfluorescence microscope to detect the integrated optical density. The resultcould be expressed mean optical density of each group.4.2Detection of the content of4-hydroxy-2-hexenal （4-HHE） in cardiactissues.We stained the tissue slice’s4-HHE antibody of normal control, DMgroup and DHA group by Immunohistochemistry; then scanned pictures picby fluorescence microscope to detect the integrated optical density. The resultcould be expressed mean optical density of each group.Results:1DHA improves T2DM rat myocardium Oxidative stress’s level.1.1The levels of T-AOC （U/mgprot）.The levels of T-AOC in Con, DM and DHA groups were1.48±0.11, 1.16±0.15and1.54±0.39respectively. The content of T-AOC in myocardiumof DM group was lower than that in Con group （P<0.05）；The content ofT-AOC in myocardium of DHA group was higher than that in DM group（P<0.05）. There were no significant differences between DHA and Con groups.（P>0.05）. It indicated that DHA enhanced the T-AOC in myocardium ofT2DM rat.1.2The content of ROS （IOD）.The levels of ROS in Con, DM and DHA groups were3.97±0.23，13.38±1.42，5.23±0.74respectively，The content of ROS in DM group was higherthan that in Con group （P<0.05）. The content of ROS in DHA group waslower than DM group （P<0.05）. Compared with Con, the level of ROS inmyocardium of DHA group made no difference （P>0.05）. It indicated thatDHA droped the ROS in myocardium of T2DM rat.1.3The content of AGEs （nmol/μg prot）.The content of AGEs in Con, DM and DHA groups were0.11±0.009,0.14±0.008and0.12±0.007respectively. The content of AGEs inmyocardium of DM group was higher than that in Con group （P<0.05）；Thecontent of AGEs in myocardium of DHA group was lower than DM group（P<0.05）.Compared with Con, the level of AGEs in myocardium of DHAgroup made no difference（P>0.05）. It indicated that DHA droped the AGEs inmyocardium of T2DM rat.1.4The content of PCG （nmol/mg prot）.The levels of Protein carbonylation in Con, DM and DHA groups were3.79±0.34,5.22±0.67and3.80±0.39respectively. The content of Proteincarbonylation in myocardium of DM group was higher than that in Con group（P<0.05）；The content of Protein carbonylation in myocardium of DHA groupwas lower than DM group （P<0.05）. There was no statistical differencesbetween Con and diet group （P>0.05）. It indicated that DHA droped the PCGin myocardium of T2DM rat.1.5The content of MDA （nmol/mg prot）.The levels of MDA in Con, DM and DHA groups were1.18±0.05, 1.26±0.09and1.54±0.18respectively. The content of MDA in myocardium ofDM group was higher than that in Con group （P<0.05）. The content of MDAin DHA group was higher than Con （P<0.05）. It indicated that DHA increasedthe MDA in myocardium of T2D Mrat.2The activity of T2DM myocardium Antioxidant enzyme.2.1The activity of T-SOD （U/mg prot）.The levels of T-SOD in Con, DM and DHA groups were147.74±27.31,142.98±23.54and138.69±24.40respectively. The content of T-SOD inmyocardium of DM group was lower than that in Con group, however, theresult had no significant differences. There were no significant differencesbetween DHA and DM groups. It indicated that DHA had no significant effecton the T-SOD in myocardium of T2DMrat.2.2The activity of CuZn-SOD （U/mg prot）.The levels of CuZn-SOD in Con, DM and DHA groups were92.81±10.09,94.81±19.63and96.61±15.70respectively. There were no significantdifferences among them. It indicated that DHA had no significant effect on theCuZn-SOD in myocardium of T2DMrat.2.3The activity of GSH-Px （U/mg prot）.The levels of GSH-Px in Con, DM and DHA groups were171.73±19.83,139.23±11.60and134.39±22.13respectively. Activity of GSH-Px inmyocardium had no significant differences between Con and DM group. Theresult was same between DM and DHA group.（P>0.05）. It indicated thatDHA had no significant effect on the activity of GSH-Px in myocardium ofT2DMrat.2.4The activity of CAT （U/mg prot）.The levels of CAT in Con, DM and DHA groups were10.50±1.30,17.94±3.93and16.74±1.91respectively. The content of CAT inmyocardium of DM group was higher than that in Con group.（P<0.05）；Thecontent of CAT in myocardium of DHA trended to be lower than DM groupthough there were no significant differences between+DHA and DM group. Itindicated that DHA had no significant effect on the activity of CAT in myocardium of T2DMrat.3Change of Fatty acid metabolites of n-3and n-6in myocardial tissue.3.1The content of4-HNE （IOD）.The levels of4-HNE in Con, DM and DHA groups were7.90±0.54,17.65±1.27and5.93±0.53respectively. The content of4-HNE in myocardiumof DM group was higher than that in Con group.（P<0.05）；The content of4-HNE in myocardium of DHA group was lower than that in Congroup.（P<0.05）. The content of4-HNE in myocardium of DM group washigher than that in4-HNE group.（P<0.01）. It indicated that DHA droped theproduction of4-HNE in myocardium of T2DMrat.3.2The content of4-HHE （IOD）.The levels of4-HNE in Con, DM and DHA groups were1.06±0.13,4.58±0.44and16.50±0.93respectively. The content of4-HNE in myocardiumof DM group was higher than that in Con group.（P<0.05）；The content of4-HNE in myocardium of DHA group was lower than that in DMgroup.（P<0.01）. It indicated that DHA’s oxidation was enhanced andgenerated a lot of metabolite-4HNE.In conclusion, the diet with DHA effectively inhibits the T2DM ratcardiac oxidative stress and DHA oxidized itself may play a role among themechanism.Conclusion:1DHA can improve the oxidative stress in heart of type2diabetic rat.2DHA can be used as a non-enzyme antioxidant to protect diabetic heart.