| Objective: To explore whether Nec-1 can reduce the release of inflammatory mediators and damage in renal tissue of diabetic nephropathy rats through inhibiting the signaling pathway of necroptosis.Methods: In the experiment,adult male SD rats were divided into diabetic nephropathy group(DN group)and normal control group(NC).After successful modeling,the DN group was divided into the DN group,the DN+vehicle(DV),the DN+ Nec-1(Nec-1);then the NC group was divided into the NC group,the NC+Nec-1 group(NN).Four observation points were observed at 4,8,12 and 16 weeks in each group,with 6 rats at each time point.The DN group was established by a one-time intraperitoneal injection of streptozotocin(STZ)at 50mg/kg.The Ne group and NN group were given Nec-1 gavage at a single dose of 3.5mg/kg starting from the first day after the successful modeling,and every other day.For DV group,Nec-1 solubilizer DMSO was given by gavage as 10% DMSO-PBS starting from the first day after successful modeling,and was given once every other day.Blood and urine samples were taken before the execution to detect serum creatinine,blood glucose,serum urea and 24-hour urine protein.Renal pathological paraffin section for general pathology;The expression of receptor interaction protein 1(RIP1),receptor interaction protein 3(RIP3)and interleukin-6(IL-6)protein and m RNA were determined by immunohistochemistry,western blot,and Real-Time PCR.And impact of Nec-1 on the above indicators of rat with diabetic nephropathy could be assessed.Results: 1.The Blood and urine biochemistry tests: 1)blood glucose and 24-hour urine protein:(1)Blood glucose and urine protein at different time points in DN and DV groups were significantly higher than those in NC and NN groups(P < 0.01),while blood glucose and urine protein at different time points in the Nec-1 group were significantly lower than those in the DN and DV groups(P < 0.01).(2)There was no significant difference in blood glucose an urine protein between the comparison of NC and NN groups,DN and DV groups at different time points(P > 0.05).(3)Blood glucose,urine protein in DN and DV groups increased with the time increasing(P < 0.05).2)Serum creatinine and urea:(1)Serum creatinine and urea in DN and DV groups were significantly increased at different time points than in NC and NN groups(P < 0.01),while serum creatinine and urea in Nec-1 group at different time points were significantly reduced compared with DN and DV groups(P < 0.01).(2)Serum creatinine and urea were not significant difference between the comparison of NC and NN groups,DN and DV groups at different time points(P>0.05).(3)There was no significant difference in serum creatinine and urea over time between DN and DV groups.2.The renal pathology: In the DN and DV groups,we see a variety of glomerulus enlargement,the growth of the membranes,the increase of the cells,the expansion of the cells,the expansion of the renal tubules,the granules and the cavitation of the bubbles.While the above renal pathological changes in Nec-1 group were alleviated at different degrees compared with those in DV and DN groups.The morphology of glomeruli and tubules was basically normal in NC and NN groups.3.Immunohistochemistry:(1)In DN and DV groups,it was found that the amount of RIP1,RIP3 and IL-6 in rats,kidney tissue were significantly higher than those in NC and NN groups at different time points(P < 0.01),while the amounts of RIP1,RIP3 and IL-6 in Nec-1 group at different time points were significantly lower than those in DN and DV groups(P < 0.01).(2)The amounts of RIP1,RIP3 and IL-6 at different time points were not significant difference between the comparison of NC and NN groups,DN and DV groups(P > 0.05).4.Western-blot:(1)In DN and DV groups,it was found that the amount of RIP1,RIP3 and IL-6 in rats’ kidney tissue were significantly higher than those in NC and NN groups at different time points(P < 0.01),while the amount of RIP1,RIP3 and IL-6 protein at different time points in the Nec-1 group were lower than those in the DN and DV groups(P < 0.05).(2)The amounts of RIP1,RIP3 and IL-6 at different time points were not significant difference between the comparison of NC and NN groups,DN and DV groups(P > 0.05).5.Real-Time PCR:(1)In DN and DV groups,it was found that the relative amount of RIP1,RIP3 and IL-6m RNA in rats,kidney tissue were significantly higher than those in NC and NN groups at different time points(P < 0.01),while the relative amount of RIP1,RIP3 and IL-6m RNA in Nec-1 group at different time points were significantly lower than those in DN and DV groups(P < 0.01).(2)The amounts of RIP1,RIP3 and IL-6m RNA at different time points were not significant difference between the comparison of NC and NN groups,DN and DV groups(P > 0.05).Conclusion: Activation of necroptosis pathway may be involved in the release of inflammatory mediators and injury in renal tissue of diabetic nephropathy rats.The expression of RIP1,RIP3 and IL-6 in renal tissues of diabetic nephropathy rats were decreased after the intervention with Nec-1.Nec-1 may reduce the release of inflammatory mediators and damage in renal tissue of diabetic nephropathy by inhibiting the necroptosis pathway. |
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