| Objective: SD rat diabetic model was constructed by intraperitoneal injection with streptozotocin,To explore the expression and meaning of c-FLIP of the necroptosis signaling pathways markers in the kidney tissues in diabetic nephropathy of rats,and c-FLIP mediated necroptosis pathways in the pathogenesis of diabetic nephropathy.Methods: Adult Sprague-Dalwley(SD)male rats were divided into the diabetic and control groups(DN and NC).The DN group was constructed by intraperitoneal injection with streptozotocin(55mg/kg).The 6 rats of the two groups were sacrificed at week 2、4、8、12 and 16 respectively,meanwhile blood glucose,body weight,kidney weight and24-hours urinary protein monitored at the same time points respectively.Paraffin sections of renal tissue of rats at the same time points were stained by hematoxylin-eosin(HE)and Periodic Acid-Schiff(PAS).The expression of c-FLIP(cellular FLICE-inhibitory protein)protein,receptor interaction protein 1(RIP1),receptor interaction protein 3(RIP3)and mRNA were detected by immunohistochemistry,western blot,and Real-Time-PCR.Analysis.Their relationship was analyzed.Results: 1.Blood and urine:1)blood glucose:(1)The blood glucose in different time points in Group DN showed obviously higher expression levels than that of Group NC(P<0.01).2)24-hour urinary protein:(1)The 24-hour urinary protein in different time points in Group DN showed obviously higher expression levels than that of Group NC(P<0.01).(2)The24-hour urinary protein were gradually increased,and have siginificanted.(P<0.01).3)The serum creatinine and serum urea:(1)The serum creatinine and serum urea in different time points in Group DN showed obviously higher expression levels than that of Group NC(P<0.01).(2)The serum creatinine and serum urea both had no significant difference in different time points in Group DN(P>0.05).2.Renal pathology:(1)In different points in DN group the kidneys of rats were white and sweeling.Glomerulus,mesangial matrix andnumber of cells were increased with dilatation of partial tubular andBowman’s capsule,tubular epithelial cell granules and vacuolar degeneration in different time points inGroup DN.(2)The kidney weight index in different time points in Group DN showed obviously higher expression levels than that of Group NC(P<0.01).(3)The kidney weight index were gradually increased,and had siginificanted.(P < 0.05).3.Immunohistochemistry:(1)c-FLIP was mainly staining in glomerular and renal tubular cells cytoplasm.The staining of c-FLIP in differenttime points in Group DN showed obviously lower than those of Group NC(P<0.01).The staining of c-FLIP have respectively compared each other in DN group,no significant difference were found between 2 weeks and 4 weeks,the staining of c-FLIP at8,12 and 4 weeks were obviously lower at 2 and 4weeks(P < 0.01).Also there were no significant different at 8 and 12 weeks,the staining of c-FLIP at16 weeks were obviously lower at 8 and 12 weeks(P<0.01).(2)RIP1 and RIP3 were mainly staining in glomerular cell cytoplasm.The staining of RIP1,RIP3 in different time points in Group DN were significantly higher than that of Group NC(P<0.01).However,their expression at 8,12 and4 weeks were obviously higher than those at 2 and 4weeks(P < 0.01).There were no significant different between 8 and 12 weeks,their expression at16 weeks were obviously higher than those at 8 and 12 weeks(P<0.01).(3)In the DN group,The expression levels of c-FLIP were strong negatively correlation with RIP1 and RIP3 each other(r=-0.933,P <0.01;r=-0.954,P<0.01);and urinary protein,the kidney weight index,the serum creatinine and urea nitrogen are also showed a negative correlation(r=-0.945,P < 0.01;r=-0.922,P <0.01;r=-0.543,P < 0.01;r=-0.494,P < 0.01).4.Western Blot:(1)The expression of c-FLIP potein in different time points in Group DN showed obviously lower than those of Group NC(P<0.01);The expression of c-FLIP potein have respectively compared each other in DN group,no significant difference were found between 2 weeks and 4 weeks,the expression of c-FLIP potein at 8,12 and 16 weeks was obviously lower than those at 2 and4weeks(P < 0.01).Also there were no significant different at 8 and 12 weeks,the expression of c-FLIP potein at 16 weeks was obviously lower than those at 8 and 12weeks(P < 0.01).(2)The expression of RIP and RIP3 poteins in different time points inGroup DN were significantly higher than those of Group NC(P<0.01).The expression of RIP1 and RIP3 poteins have respectively compared each other in DN group,no significant difference were found between 2 weeks and 4 weeks,the expression of RIP1 and RIP3 poteins at 2 and 4 weeks were obviously lower than those at 8,12 and 16 weeks(P <0.01).Also there were no significant different at 8 and 12 weeks,the expression of RIP1 and RIP3 poteins at 8 and 12 weeks were obviously lower than those at 16 weeks(P<0.01).(3)In the DN group,The expression levels of c-FLIP were strong negatively correlation with RIP1 and RIP3 each other(r=-0.985,P < 0.01;r=-0.986,P < 0.01);and urinary protein,the kidney weight index,the serum creatinine and urea nitrogen are also showed a negative correlation(r=-0.930,P < 0.01;r=-0.907,P < 0.01;r=-0.575,P < 0.01;r=-0.558,P < 0.01).5.RT-PCR:(1)The expression of c-FLIP mRNA in differenttime points in Group DN showed obviously lower than those of Group NC(P<0.01).The expression of c-FLIPmRNA have respectively compared each other in DN group,no significant difference were found between 2 weeks and 4 weeks,the expression of c-FLIPmRNA at 8,12 and 16 weeks were obviously lower than those at 2 and 4 weeks(P < 0.01).Also there were no significant different at 8 and 12 weeks,the expression of c-FLIP potein at 16 weeks was obviously lower than those at 8 and 12 weeks(P < 0.01).(2)The expression of RIP1 mRNA and RIP3 mRNA in different time points in Group DN were significantly higher than those of Group NC(P<0.01).The expression of RIP1 mRNA and RIP3 mRNA have respectively compared each other in DN group,no significant difference were found between 2 weeks and 4 weeks,the expression of RIP1mRNA、RIP3mRNA at 2 and 4 weeks were obviously higher than those at 8,12 and 16 weeks(P<0.01).Also there were no significant different at8 and 12 weeks,the expression of RIP1 mRNA 、 RIP3 mRNA at 8 and 12 weeks were obviously higher than those at 16 weeks(P < 0.01).(3)In the DN group,The expression levels of c-FLIPmRNA were strong negatively correlation with RIP1 and RIP3 each other(r=-0.850,p < 0.01;r=-0.869,p < 0.01);and urinary protein,the kidney weight index,the serum creatinine and urea nitrogen are also showed a negative correlation(r=-0.877,P<0.01;r=-0.895,P<0.01;r=-0.643,P<0.01;r=-0.499,P<0.01).Conclusion: 1 、 In the kidney tissues of diabetic Rats,The expression of c-FLIP are obviously lower than those of the normal control group,Which may contribute to the tissue injury of DN rats.2 、 Also,The Activation of necroptosis pathway may contribute to the tissue injury of DN rats.3 、 The expression levels of c-FLIP were strong negatively correlation with RIP1 and RIP3 each other.The lower levels of c-FLIP expression can activate necroptosis pathway,which may promote the renal injury of diabetes mellitus. |
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