| PART 11,25(OH)2D3 INHIBITS HIGH GLUCOSE INDUCED-OXIDATIVE STRESS IN INS-1 CELLSObjective:to confirm the effect of high glucose on the oxidative stress and the Nrf2 system in rat islet β-cells;to find the effects of different concentrations of 1,25(OH)2D3 on high glucose induced-oxidative stress and Nrf2 system;to expore the optimum concentration of 1,25(OH)2D3 at which can effectively inhibit the oxidative stress induced by high glucose.Methords:the INS-1 cells were clutured in different concentrations of glucose and divided into 5 groups(5.5,11.1,15,25,35mmol/L),the ROS was detected by DCFH-DA,and the index of oxidative stress(4-HNE),total Nrf2 and nuclear Nrf2 expression was detected by Western blot.Then the cells were combined or mono-treated with different glucose(NG:11.1 mmol/L,HG:25mmol/L)and 1,25(OH)2D3(10-9,10-8,10-7,10-6mol/L)concentrations.ROS was detected by DCFH-DA,4-HNE,total Nrf2 and nuclear Nrf2 expression was detected by Western blot.Results:High glucose can increase ROS and 4-HNE,which can reflect oxidative stress,in INS-1 cells in a concentration-dependent way.Besides high glucose can also increase the expression of Nrf2 both in total cells and nucleus.1,25(OH)2D3 can inhibit high glucose-induced oxidative stress in INS-1 cells in a dose-dependent way.1,25(OH)2D3 can also increase the Nrf2 expression in total cell and nucleus in INS-1 cells treated with high glucose.Conclusion:high glucose could conduct oxidative stress in isletβ-cells,which can be inhibited by high 1,25(OH)2D3 in a dose dependent manner.Besides,1,25(OH)2D3 can active Nrf2 system in a dose-dependent way.PART 2 THE EFFECTS AND MECHAINSMS OF 1,25(OH)2D3 ALONE OR IN COMBINE WITH VDR SILENCING BY LENTIVIRUS TRANSFECTION ON HIGH GLUCOSE-INDUCED OXIDATIVE DAMAGE IN INS-1 CELLSObjective:to construct the stable transfective VDR silencing islet-β-cells;to confirm whether the anti-oxidative damage effects of 1,25(OH)2D3 is through the VD/VDR classic pathway,and associated with Nrf2 system activation;to explore the relationship between the GSK-3β/Fyn pathway and the effect on the activation of Nrf2 system by 1,25(OH)2D3.Methods:transfected INS-1 cells with Lenti-shVDR and selected stable-transfected INS-1 cells with puromycin.The mRNA and protein expression of VDR were detected by qRT-PCR and Western bolt.The INS-1 cells were treated with normal glucose,high glucose alone or in combine with 1,25(OH)2D3,superoxide anion was detected by using DHE Fluorescent Probe,indicators associated with oxidative stress,apoptosis,Nrf2 system,and GSK-3β/Fyn were detected by Western blot.mRNA expressions of HO-1,NQO1 were detected by qRT-PCR.Then we treated the INS-1 cells with high glucose,1,25(OH)2D3 alone or in combine with Nrf2 siRNA,and Western blot were used to detect the expression of indicators about oxidative stress,apoptosis and Nrf2 system.Finally,INS-1 cells were treated with NG,HG and 1,25(OH)2D3 with or without Licl,an inhibitor of GSK-3β,and indicators of GSK-3β/Fyn and Nrf2 system were dectected by Western blot.Resultes:the MOI of 50 could efficiently infected the INS-1 cells and silenced the VDR expression over 70%detected by Western blot or qRT-PCR.1,25(OH)2D3 can significantly inhibit HG induced-oxidative in INS-1 cells and increase the expression of nuclear Nrf2 and its target genes;the effects of 1,25(OH)2D3 were inhibited by VDR silencing.Nrf2 siRNA treatment could inhibit the protective effects of 1,25(OH)2D3 in INS-1 cells cultured with HG.Besides,Licl,the inhibitor of GSK-3β,could inhibit the activity of GSK-3β/Fyn pathway and upregluted the activity of Nrf2 in INS-1 cells cultured with HG.1,25(OH)2D3 treatment had a mimicked effects with the inhibitor of GSK-3β in INS-1 cells treated with high glucose.Conclusion:Lenti-shVDR could efficiently infected the INS-1 cells and silenced the VDR expression over 70%.In vitro,1,25(OH)2D3 can inhibit HG induced oxidative damage in islet β-cells throught the VD/VDR classic pathway,and might be associated with modulating the activity of Nrf2 system through GSK-3β/Fyn pathway.PART 3 THE EFFECTS AND MECHANISMS OF 1,25(OH)2D3 ALONE OR IN COMBINE WITH VDR SILENCING BY LENTIVIRUS TRANSFECTION ON ANTI-OXIDATIVE DAMAGE IN TYPE 1 DIABETIC RATSObjective:to explore the effects of 1,25(OH)2D3 with or without VDR silencing on oxidative damage in pancreas of diabetic rats and its potential mechanisms.Methods:sixty SD-rats were randomly assigned into following 5 gourps:normal glucose group,diabetic group,diabetic+1,25(OH)2D3 treatment group,diabetic+1,25(OH)2D3+shVDR treatment group,diabetic+shVDR treatment group,each group contained 12 rats.The diabetic rat model was constructed by single interperitoneal injection with STZ.8-OHDG and Nrf2 in pancreas of each group was detedcted by immunohistochemistry.Western blot was used to detect of the expression of VDR,indicators of oxidative stress,apoptosis,Nrf2 system,and GSK-3β/Fyn pathway.qRT-PCR was used to detect the mRNA expression of VDR,HO-1 and NQO1.Results:We successfully established rat models of each group.1,25(OH)2D3 has a tend to decrease the high glucose induced by DM,but there is no significant difference compared with DM group.1,25(OH)2D3 can significantly improve the level of HbA1C which can be increased by DM.Compared with NG group,the level of oxidative stress and apoptosis was increased in the DM,DM+VD+shVDR,DM+shVDR group,while it can be improved in DM+VD group.Besides,1,25(OH)2D3 can significantly increase the activity of Nrf2 system and decrease the GSK-3β/Fyn pathway compared with DM group,but had no effect on VDR silencing rats.Conclusion:once interperitoneal injection of STZ could successfully establish the diabetic rat model;Lenti-shVDR could effeciently tranfected the diabetic rats and could silence the VDR stably.1,25(OH)2D3 could improve the the anti-oxidative stress and apoptosis in pancreas of DM rats through VD/VDR classic pathway.This effect may be associated with the interaction effect of GSK-3β/Fyn pathway and Nrf2 system. |
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