The Effect Of Selenium In Diabetes Aggravate D Ischemic Reperfusion Injury Through Stabilizing Mitochondrial Fission/fusion Imbalances

Objective:The purpose of this study is to clarify the reventive and therapeutic role of selenium and selenoproteins in hyperglycemia aggravate cerebral ischemia injury,and whether this effect is related to stabilize mitochondrial pathway,especi-ally the stability of mitochondrial division/fusion.Methods: Part 1:In the logarithmic growth phase,HT22 cells were divided into control group,the lactic acid group(LA),hyperglycemia+hypoxia group(HG+H),lactic acid + sodium selenite groups(LA +Se)and hyperglycemia+hypoxia+sodium selenite group(HG+H+Se);Sel H HT22 cell and V-HT22 cells were divided into control group,the lactic acid group(LA),and hyperglycemia+hypoxia group(HG+ H).Given corresponding treatment to cell group,cell activity,morphology and cell apoptosis were detected.Part 2: Experimental animals were randomly divided into normal blood sugar group(NH),diabetes group(GH),diabetes+ sodium selenite group(GH+Se),diabetes + insulin group(GH+insulin)and diabetes+ sodium selenite+insulin group(GH+Se+ insulin).Each group have 10 SD rats and respectively with blank control group(control)and sham operation group(sham),I/R24 h and I/R72 h.Type I diabetes model was induced by streptozocin(ST Z,60mg/kg).After diabetes success 4 weeks sodium selenite(0.4mg/kg.d)and Protamine Zinc insulin(2U/d)were given lasting 4 weeks.The focal cerebral ischemia reperfusion model was induced by the middlecerebral artery ischemia(MCAO).Iba1,GFAP,Mitochondrial fission/fusin markers and autophagy marker was detected by HE staining,Nissl's staining,electron microscopy,immunohistochemical and western blotting.Part 3: In the logarithmic growth phase,HT22 cells were divided into control group,the lactic acid group(LA),hyperglycemia + hypoxia group(HG + H),lactic acid + sodium selenite group(LA+ Se)and hyperglycemia+hypoxia+sodium selenite group(HG+H+Se);Sel H-HT22 cell and V-HT22 cells were divided into control group,the lactic acid group(LA),and hyperglycemia + hypoxia group(HG+H).Given corresponding treatment to cell group,mitochondrial membrane potential,mitochondrial fission/fusin markers,autophagy marker was detected.Results: Part 1:(1)Cell viability:the cell viability of HT22 cells treated with 5m M LA or 50 m M HG+H was decreased with the increased of time,and cell viability signifi-cantly increased in plus 100 n M sodium selenite groups.After incubation with LA and HG+H,Sel H-HT22 and V-HT22 cells viability were decline gradually as time prolonged.But the degree of decreased in Sel H-HT22 group was less than V-HT22 group.(2)Light microscopic fndings : LA and HG+H exposure resulted in HT22 cell shrinkage and nuclear condensation,suggesting cell death.Concurrent treatment with sodium selenite ameliorated the changes.LA and HG+H exposure resulted in Sel H-HT22 and V-HT22 cells shrinkage,nuclear condensation,even separated from bottle wall.And the changes of Sel H-HT22 cell was few than V-HT22 cells.(3)Results of Annexin V/PI double dyeing: LA and HG+H exposure resulted the late apoptosis and secondary necrosis cells was significantly increased in HT22 cell.Concurrent treatment with sodium selenite significantly reduced thelate apoptosis and secondary necrosis.LA and HG+H exposure resulted in the late apoptosis and secondary necrosis of Sel H-HT22 and V-HT22 cells significantly increased.And the late apoptosis and secondary necrosis percent of Sel H-HT22 cells were less than V-HT22 cells.(4)Expression of Endo G: LA and HG+H exposure resulted in Endo G which is transported into the nucleus of HT22 cells significantly increased.And the transported Endo G was decreased in c oncurrent treatment with sodium selenite groups.LA and HG+H exposure induced transported Endo G increased in Sel H-HT22 and V-HT22 cells.And transported Endo G in Sel H-HT22 cells were less than V-HT22 cells.(5)Results of mitochondrial membrane potential : Orange red/green fluorescence ratio decreased significantly(P < 0.01)in HT22 cells exposured to LA and HG+H.And there were less decreased in c oncurrent treatment with sodium selenite groups(P < 0.05).LA and HG+H exposure resulted in Orange red/green fluorescence ratio of Sel H-HT22 and V-HT22 cells decreased significantly.And the ratio is more lower in V-HT22 cells(P < 0.05).Part 2:(1)Blood glucose:blood glucose of diabetes rats were higher than NH group,and postoperative blood glucose significantly higher than the other time points(P<0.01).Blood glucose of GH+Se group was higher than GH+insulin and GH+Se+insulin(P<0.01).But there was no difference between GH+insulin and GH+Se+insulin.(2)Rate of weight change:Expect NH group,rate of diabetes rats were all decling significantly as compared with NH group(P < 0.01).Concurrent treatment with sodium selenite and/or insulin made rate increased in GH+Se,GH+insulin and GH+Se+insulin groups.Rate of GH group was higher than GH+insulin andGH+Se+insulin group(P < 0.01).But there was no difference between GH and GH+Se group.(3)Brain function scores:There were no nerve injury symptoms in sham group.After operation the brain function scores of GH group were higher than NH group,and also higher than GH+insulin and GH+Se+insulin group.there was no difference between GH and GH+Se group.(4)Infarct volume and infarction area :After ischemia for 30 min and reperfusion for 24 h and 72 h brain slices were staining by TTC.At 24 h of I/R,infarct size of diabetes rats were significantly larger than the NH group.At 24 h of I/R,the rest of group staining were completely red except GH group.After ischemia for 20 min and reperfusion for 24 h brain slices were staining by TTC,and the results shown no significant infarction in sham group.Infarct volume of diabetes rats were significantly larger than the NH group.Infarct volume of GH group were significantly larger than GH+Se,GH+insulin and GH+Se+insulin group.And infarct volume of GH+Se+insulin group was significantly smaller than GH+Se group.(5)The effect on rat peripheral blood serum selenium protein : only GH+Se+insulin group rats serum selenium protein increased obviously as compared with the NH,GH+Se and GH+insulin group(P < 0.05).(6)Light and electron microscopic findings:(1)HE staining: Mild cerebral edema,capillary hyperplasia,few degeneration and nucleus pycnosis was observed in diabetes.At 24 h of I/R,cerebral edema was observed in NH group,while worse cerebral edema and neuronal pycnosis were appeared in diabetic group.And damage of GH+ Se+insulin group was the lightest.At 72 h of I/R,even worse cerebral edema was showed in GH group,and there were more neuronal pycnosis,a large number of blood capillary and inflammatory cellswere observed.There were ameliorated in GH+Se,GH+insulin and GH+Se+insu-lin group.(2)Nissl's staining:In NH group cell body and protuberant were clear and nuclei were pale,nissl bodies were abundant and deep dyeing,and background are slightly pale blue;the dyeing of cytoplasm in diabetes rats were light than NH group.At 24 h of I/R,nissl bodies were reduced in NH group,and even worse in GH group.Concurrent treatment with sodium selenite and/or insulin ameliorated above changes.(3)Electron microscopy: After ischemia for 30 min and reperfusion for 24 h,the brain tissue were used to observe ultrastructure.Injury of glial cells were worse than nerve cells.And there were also some injured changes at uninjured side.(7)The change of microglia in penumbra:There were a small amount of stationary phase microglia,which had small cell body and thin branches in control group,and there had no difference between groups.After ischemia reperfusion,Iba1 positive cells were quickly activated and migrated to the damaged area.At 24 h of I/R,the number of Iba1 positive cells were same as control.But the shape became to irregular,cell body became to bigger,and branches became to think.The number of GH+Se and GH+Se+insulin group is much more than others.At 72 h of I/R,the number of Iba1 positive cells were significantly increased as compared to control and I/R 24 h,and shape of it became to ameboid.Compared to NH group the number of Iba1 positive cells were significant increased in the rest 4 groups.The number of GH+Se+insulin and GH+ insulin group were more than GH group.(8)The change of astrocytes in penumbra: There were a small amount of astrocytes in control group.The astrocytes in NH group had small cell body and thin branches.The cell body became bigger and branches thinker in diabe tes rats groups.And the number of GFAP positive cells in GH group much more than NHgroup(P<0.05).After ischemia reperfusion,the number of GFAP positive cells increased,the cell body became bigger and branches coarsened.At 24 h of I/R,the number of GFAP positive cells in each group were increased than control.At 72 h of I/R,the number of GFAP positive cells in each group were significantly increased as compared to I/R24 h,and the number of GH group obviously higher than other groups.(9)Expression of mitochondrial fission/fusin markers:(1)Mitochondrial fission markers Fis1 and Drp1: Expression of Fis1 and Drp1 increased significantly in GH and GH+Se groups comparing to NH group.After ischemia reperfusion 24 h,expression of Fis1 and Drp1 increased significantly in each group comparing to its control except GH group.And futher increased at 72 h of I/R,which contain GH group.At 24 h of I/R,GH and GH+Se+insulin group increased more than other groups.And expression of Fis1 and Drp1 in GH group were higher than GH+Se,GH+ insulin and GH+Se+insulin group.At 72 h of I/R,expression of Fis1 and Drp1 in diabetes rats were obviously incresed,and GH gr o u p w e r e h i gh e r t h a n G H + S e,G H + i ns u l i n a n d G H + S e + i n s u li n gr o u p.(2)Mitochondrial fusin markers Opa1 and Mfn-2:There were no differences between control group.After ischemia reperfusion 24 h,expression of Opa1 and Mfn-2 increased significantly in each group comparing to its control.And it declined at 72 h of I/R,except GH group.At 24 h of I/R,bes ides GH+Se+insulin and GH+Se group,expression of Opa1 and Mfn-2 of other 3 groups were lower than NH group.At 72 h of I/R,expression of Opa1 and Mfn-2 increased significantly in GH group comparing to other groups.(10)Autophagy marker LC3 I/II: Ratio of LC3 II/ LC3 I in diabetes rats were significantly increased.After ischemia reperfusion 24 h,ratio of LC3 II/ LC3 I increased significantly in each group comparing to its own control,in which GHgroup was the highest one.And futher decreased after ischemia reperfusion 72 h.Part 3(1)Mitochondrial electron microscopy:There were abundant endoplasmic reticulum and mitochondria,mostly elongated,in the control cells.Mitochondria in LA and HG+H-treated cells became swelling.And distended rough endoplasmic reticulum,abundant presence of lysosomes and autophagosomes were observed.Selenite concurrent treatment and Sel H ameliorated LA and HG+H caused alterations in the mitochondrion,endoplasmic reticulum and lysosome.(2)Expression of mitochondrial fission/fusin markers:(1)Mitochondrial fission markers Fis1,Drp1 and p-Drp1: LA and HG+H exposure induced higher expression of Fis1,Drp1 and p-Drp1.Selenite concurrent treatment and Sel H ameliorated this increase.(2)Mitochondrial fusin markers Opa1 and Mfn-2: LA and HG+H exposure induced higher expression of Opa1 and Mfn-2.Futher increased were observed at selenite concurrent treatment and Sel H groups.(3)Autophagy marker LC3 I/ LC3II: Ratio of LC3 II/ LC3 I in LA and HG+H treatment groups were significantly increased.Selenite concurrent treatment and Sel H ameliorated this increase.Conclusion: 1.Sodium selenite and Sel H can significantly alleviate hyperglycemia aggravated neuronal damage.2.Sodium selenite and Sel H possible alleviate hyperglycemia aggravated cerebral ischemia reperfusion injury through stabilize mitochondrial division/fusion imbalances and reduce autophagy.