| BackgroundThe change of angiopoietin-1(Ang-1)/angiopoietin-2(Ang-2)ratio is an important mechanism in the cerebral ischemia-reperfusion induced by the blood-brain barrier injury in the cerebral vascular parricides,microvascular endothelial cells and astrocytes.Docosahexaenoic acid(DHA)is a highly unsaturated fatty acid necessary for the human body and is important for maintaining the normal structure and physiological function of the cell membrane.DHA can reduce the cerebral ischemia-reperfusion injury in rats,maintain blood-brain barrier integrity and reduce blood-brain barrier permeability;further study found that DHA can up-regulate Ang-1 expression,reduce Ang-2 secretion,Protecte the deprivation of rat brain microvascular endothelial cells.Src-inhibited protein kinase C substrate(SSeCKS)is a substrate for protein kinase C,a cytoskeletal cleavage factor that plays an important role in maintaining cell attachment,cell morphology,and regulating cell permeability.Up-regulate the expression of Ang-1 in astrocytes and improve the blood-brain barrier function.In this study,we prepared an evaluation model of oxidative glucose deprivation/reoxygenation and reperfusion injury in human cerebral vascular cells(HBVPs)to evaluate this worthy of study whether the molecular mechanism of regulation of Ang expression is related to cerebrovascular pericytes,endothelial cells and astrocytes SSeCKS.ObjiectveTo investigate the molecular mechanism of doeosahexecnoic acid(DHA)in oxidative glucose deprivation/reoxygenation and reperfusion injury in human brain vascular pericytes(HBVPs):the relationship with Src suppressed C kinase substrates(SSeCKS)expression.Methods1.HBVPs in good growth state were randomly divided into 10 groups(n=60):Control group(Group C);Oxygen glucose deprivation for 6h/Reoxygenation and recovery of sugar for 2h(Group Q6R2);Oxygen glucose deprivation for 6h/Reoxygenation and recovery of sugar for 6h(Group Q6R6);Oxygen glucose deprivation for 6h/Reoxygenation and recovery of sugar for 12h(Group Q6R12);Oxygen glucose deprivation for 12h/Reoxygenation and recovery of sugar for 2h(Group Q12R2);Oxygen glucose deprivation for 12h/Reoxygenation and recovery of sugar for 6h(Group Q12R6);Oxygen glucose deprivation for 12h/Reoxygenation and recovery of sugar for 12h(Group Q12R12);Oxygen glucose deprivation for 24h/Reoxygenation and recovery of sugar for 2h(Group Q24R2);Oxygen glucose deprivation for 24h/Reoxygenation and recovery of sugar for 6h(Group Q24R6);Oxygen glucose deprivation for 24h/Reoxygenation and recovery of sugar for 12h(Group Q24R12).C group was normal culture,the rest of the group according to the time of the oxygen glucose deprivation and reoxygenation and recovery time to do the appropriate treatment.which the glucose-deprived environment was cultured in a three-gas incubator containing 94%N2:5%CO2:1%O2 as a sugar-free serum-free medium and the environment of reoxygenation was 5%CO2 incubator with serum containing medium.The cell morphology,adherence and shedding were observed by inverted microscope.The cell viability was detected by CCK-8.Apoptosis was detected by Annexin-V/PI double staining and flow cytometry.2.HBVPs in good growth state were randomly divided into 8 groups(n=48):Normal control group(group C),Oxygen glucose deprivation/Reoxygenation group(group Q);High dose DHA pretreatment group(group H);Low-dose DHA pretreatment group(group L);Normal control + SSeCKS gene silencing group(group S);Oxygen glucose deprivation/Reoxygenation glycoside + SSeCKS gene silencing group(group QS);High dose DHA pretreatment SSeCKS gene silencing group(group HS);Low dose DHA pretreatment SSeCKS gene silencing group(group LS).Group C was cultured according to normal cell culture conditions,Group Q in sugar-free serum-free medium in three-gas incubator for 24 hours than the replacement of sugar-free serum-free medium for sugar-containing serum medium culture incubator for 6h.DHA pretreatment groups were added to the DHA medium with the final concentration of 40?M and 10?M before the glucose deprivation treatment for 1 hour,and then the subsequent treatment was carried out.Each SSeCKS gene silencing group was subjected to siRNA transfection according to the instructions of SSeCKS siRNA(h)(Santa,USA)and supporting transfection reagent(Santa,USA),and tested the silencing efficiency.Analysis the SSeCKS gene silencing effect by western blot;LDH ELISA kit was used to detect the cytotoxicity of each group.CCK-8 kit was used to detected the cell viability.and apoptosis was detected by flow cytometry.Annexin-V/PI double staining was used to detect the cell apoptosis after the reoxygenation for 2 h,6 h,12 h and 24 h.3.HBVPs in good growth state were randomly divided into 8 groups(n=48):Normal control group(group C),Oxygen glucose deprivation/Reoxygenation group(Q group);High dose DHA pretreatment group(H group);Low-dose DHA pretreatment group(L group);Normal control + SSeCKS gene silencing group(S group);Oxygen glucose deprivation/Reoxygenation glycoside + SSeCKS gene silencing group(QS group);High dose DHA pretreatment SSeCKS gene silencing group(HS group);Low dose DHA pretreatment SSeCKS gene silencing group(LS group).Group C was cultured according to normal cell culture conditions,Group Q in sugar-free serum-free medium in three-gas incubator for 24 hours than the replacement of sugar-free serum-free medium for sugar-containing serum medium culture incubator for 6h.DHA pretreatment groups were added to the DHA medium with the final concentration of 40?M and 10?M before the glucose deprivation treatment for 1 hour,and then the subsequent treatment was carried out.Each SSeCKS gene silencing group was subjected to siRNA transfection according to the instructions of SSeCKS siRNA(h)(Santa,USA)and supporting transfection reagent(Santa,USA),and tested the silencing efficiency.The levels of Ang-1,Ang-2 and VEGF in the supernatant of the culture supernatant by ELISA kits and the levels of Ang-1,Ang-2 and VEGF protein expression by Western Blot at 2h,6h,12h and 24h after reoxygenation.Results1.The cells in the control group showed good growth in the inverted microscope and the nucleus was clearly visible,and the number of viable cells decreased with the prolongation of hypoxia reoxygenation time.We can see a small number of cells were round floating in the culture dish by microscopic observation after Hypoxia for 24h and reoxygenation for 2h,and round floating cells increased significantly after reoxygenation for 6h,After 9h to 12h reoxygenation,more than 50%floating cells were observed under microscope.The survival rate of the normal cells in the control group was 100%,and the survival rate was only 50%when hypoxia for 24h and reoxygenation for 6h,and the rate was 30%when hypoxia for 24h and reoxygenation for 24h.The apoptotic rate of HBVPs cells was closely related to the time of glucose deprivation/reoxygenation and recovery.After hypoxia and reoxygenation,the cell apoptosis rate was significantly increased.Compared with the control group,the apoptotic rate of the rats was significantly higher than that of the control group(P<0.05).2.According to the recommended conditions,the content of SSeCKS protein decreased significantly at 24 h after transfection for 12 h,and reached the peak at 24 h after transfection,and the inhibitory rate was best after 50 h after transfection.Compared with group C,the leakage of LDH in Q group was significantly increased(P<0.05),and in group L was lower than that in Q group(P<0.05),and group H was significantly higher than that in group Q(P<0.05).There was no significant difference in LDH leakage between group S and group C(P>0.05),and group C decreased compared with group QS(P<0.05),the difference is not obvious between group LS,HS and QS.there were not significantly different between group L and group LS,group H and group HS(P>0.05).With the prolongation of hypoxia/reoxygenation time,the leakage of LDH gradually increased,and the increase correspondingly at each time(P<0.05);Compared with group C,the survival rate of group C was stable at about 50%,and the survival rate of group L and group H was significantly higher than that of group L(P<0.05),and group H was higher than that of group L.(P<0.05).,and the survival rate of group S was not significantly decreased(P>0.05).there was no significant difference between the group QS,the group LS and the group HS too(P<0.05).Compared with group C,there was no significant increase in group S in apoptosis(P>0.05),and the increase in group QS,group LS and group HS significantly(P<0.01).while no significant difference between the three groups compared with each other(P>0.05).but the group HS was higher than that group H(P<0.01)and group LS was higher than group L(P<0.01)too,With the prolongation of reoxygenation and recovery time,the apoptotic rate increased gradually,and the difference was significant(P<0.05);At the same time point,the contents of SSeCKS protein in the other groups were significantly lower than those in group C(P<0.05).Compared with group Q,L and H increased significantly(P<0.05).Compared with group S,there were not significantly between group QS,LS and HS(P>0.05).There were significantly different between group L and group LS,group H and group HS too(P<0.05).The content of SSeCKS protein decreased gradually in the same group(P<0.05).3.Compared with group C,the secretion and protein content of Ang-1 in the other groups were significantly lower than those in group C at different time points(P<0.01).Compared with group Q,the secretion and protein content of group L and group D increased significantly(P<0.05),and the high dose DHA group increased higher than the low dose group(P<0.05).Compared with group QS,there was no significant difference in Ang-1 secretion and protein content between group LS and HS(P>0.05).Compared with group L,the content of group LS decreased(P<0.05)and the group HS was also lower than group H(P<0.05).Compared with the two groups,the secretion and protein content of the corresponding groups gradually increased(P<0.05);Compared with group C,the secretion and expression of Ang-2 in group Q were significantly increased(P<0.05),and there was no change in group S(P>0.05).Compared with group Q,group L and group H decreased(P<0.05),and group H decreased more significantly(P<0.05).Compared with group S,group QS,LS and HS increased significantly(P<0.01).Compared with group L,group LS secretion and protein content increased significantly;Compared with group C,the secretion and protein content of VEGF in group Q were significantly increased(P<0.05),and there was no change in group S(P>0.05)compared with group C(P<0.05).Compared with group Q,group L and group H decreased(P<0.05),and group H decreased more significantly(P<0.05),Compared with group S,group QS,LS and HS increased significantly(P<0.01),Compared with group L,in group LS secretion and protein content increased significantly,Compared with group H,group HS also showed a significant increase(P<0.05),group QS,LS and HS were not significantly different(P>0.05).There was no significant difference between different time points(P>0.05).Conclusion1.The cells were significantly damaged and the cell viability was stable at about 50%after 24 hours of hypoxia and reoxygenation for 6h,.The apoptotic rate was decreased and the results were stable.2.HBVPs were pretreated with 40?M and 10?M DHA pretreated with oxygen glucose deprivation/reoxygenation.The results showed that HBVPs could significantly improve the cell status,improve the survival rate and reduce the apoptosis,and the protective effect of DHA was more obvious.3.The expression of SSeCKS was down-regulated in HBVPs with glucose deprivation/reoxygenation,and it was up-regulated by DHA pretreatment.And the expression of Ang-1 was down-regulated,Ang-2 expression was up-regulated and Ang-1/Ang-2 ratio decreased after further SSeCKS gene silencing,while the protective effect of DHA on glucose deprivation/reoxygenation and recovery of HBVPs disappeared,It suggesting that DHA can increased the ratio of Ang-1/Ang-2 and reduced the damage of HBVPs by sugar and glucose deprivation/reoxygenation,and the mechanism was related to the up-regulation of SSeCKS expression. |
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