Influence Of Nec-1 On The Necroptosis Pathway Of Rat Glomerular Endothelial Cells Cultured Under High Glucose Condition

Objective: To investigated whether Nec-1,a specific inhibitor of the necroptosis pathway,can alleviate necroptosis and inflammatory reaction of rat glomerular endothelial cells(RGECs)under the stimulate of high glucose.its mechanism is also studied in vitro.Methods: Rat glomerular cells were cultured in vitro and detected the expression of Anti-hemophilic Factor VIII for identification to be RGECs.RGECs were divided into four groups: control group,mannitol group,high glucose group,high glucose+100?mol/L Nec-1 group.Immunocytochemistry and immunofluorescence were used to detect the quantification and positioning of the protein expression of RIP1,RIP3 and IL-6 in each group.The m RNA expression of RIP1,RIP1 and IL-6 were detected by real-time PCR.Western blotting(WB)were used to detect the protein expression of RIP1,RIP3 and IL-6.Results: 1.Cell identification: The cytoplasm of rat glomerular cells were expressed Anti-hemophilic Factor VIII,which is identified as rat glomerular endothelial cells(RGECs).2.Immunocytochemistry: RIP1,RIP3 and IL-6 were mainly expressed in RGECs cytoplasm,and their expression of high glucose group were significantly increased as compared with those of control group(P<0.01)and mannitol group(P<0.01),however,the expression of the RIP1,RIP3 and IL-6 in high glucose+Nec-1 group were significantly decreased as compared with those of high glucose group(P<0.01)significantly,while the rest of the groups were no obvious difference(P>0.05).3.Immunofluorescence: target protein were mainly expressed in RGECs cytoplasm,and there fluorescence intensity of high glucose group were significantly brighter than those of control group(P<0.01)and mannitol group(P<0.01),compared with high glucose group,the fluorescence of the RIP1,RIP3 and IL-6 in high glucose+100?mol/L Nec-1 group were significantly weakened,while the rest of the groups were no obvious difference.4.RT-PCR:m RNA relative expression of RIP1,RIP3 and IL-6 of high glucose group were obviously increased as compared with those of control group(P<0.01)and mannitol group(P<0.01),however,the expression of the RIP1,RIP3 and IL-6 in high glucose+100?mol/L Nec-1 group were significantly decreased as compared with those of high glucose group(P<0.01)significantly,while the rest of the groups were no obvious difference(P>0.05).5.WB: The protein quantity of RIP1,RIP3 and IL-6 in high glucose group were significantly increased as compared with those of control group(P<0.01)and mannitol group(P<0.01),however,the expression of the RIP1,RIP3 and IL-6 in high glucose+100?mol/L Nec-1 group were significantly decreased as compared with those of high glucose group(P<0.01)significantly,while the rest of the groups were no obvious difference(P>0.05).Conclusion: Under high glucose stimulation,the necroptosis of RGECs shows a significant increasing,which lead to more inflammatory reaction and injury.After intervention with Nec-1,a specific blocker of necroptosis,the expression of the RIP1,RIP3 and IL-6 have been downregulated,which is indicate that Nec-1 may reduce the inflammatory response of RGECs by inhibiting the necroptosis pathway.