| BackgroundThe incidence and the mortality of acute renal injury(AKI)is high,is high.There is still a lack of effective treatment for AKI.Mitochondria are the main energy source of cell metabolism,and also an important organelle to regulate cell death.If cell damage or hypoxia,it can be activated by the release of Caspase system or ROS to start different types of cell death.The renal tubular epithelial cell mitochondria dysfunction activates a variety of cell death pathway,which is caused by various pathogenic factors,my be an important mechanism for the occurrence and development of AKI.Recent studies show that the pyroptosis of renal tubular epithelial cells in AKI is the main way of cell death,but its mechanism and signaling pathway are not clear at present.Our previous study showed that the Nlrp3(ACHT,LRR and PYD domains-containing protein 3)plays an important role in the pathogenesis of AKI,and it plays a key role in the pyroptosis of renal tubular epithelial cells,but its upstream regulatory pathway and its mechanism are not clear.The current studies have shown that mitochondrial dysfunction plays a central role in Nlrp3 activation,and the dynamin-related protein 1 is the key molecular switch of mitochondrial fission,which is involved in the occurrence and development of mitochondrial dysfunction.The latest research shows that the Nlrp3 can be activated by RNA virus throughing Drp1.But the interaction and regulation mechanism of Drp1,mitochondrial dysfunction and Nlrp3 are still unknown.Based on the above background,we have proposed the hypothesis that Drp1 regulates the pyroptosis of renal tubular epithelial cells in AKI through the regulation of mitochondrial /Nlrp3 axis.Therefore this project intends to first verify that kidney epithelial cells in AKI existing pyroptosis.To study the interaction and regulation mechanism of Drp1,mitochondria and inflammatory molecules,We intervene the axis of Drp1,mitochondrial /Nlrp3.It may explain the mechanism of the pyroptosis of renal tubular epithelial cells in acute renal failure AKI,and provide a theoretical basis for the next step in the search for new therapeutic strategies for the treatment of AKI.ObjectiveIn this study,the use of hypoxia / re-oxygenation(H/R)treatment of human renal tubular epithelial cells(HK-2 cells)was proposed to imitate the ischemia reperfusion injury of human renal tubular epithelial cells.The expression levels of inflammatory factors IL-1 and caspase-1 and the pyroptosis of renal tubular epithelial cells were detected to investigate the effect of ischemia/reperfusion(I/R)on the inflammatory response and pyroptosis of human renal tubular epithelial cells.The expression of Nlrp3,ROS,and Drp1 in HK-2 cells were detected to the possible mechanism of activation of inflammatory factor IL-1b and pyroptosis in I/R HK-2 cells.Blocking Drp1 activity by using Mdivi-1,the expression of Drp1,Nlrp3,IL-1b,ROS,LDH and the pyroptosis in I/R cells were detected to explore the role of Drp1 in inflammation and pyroptosis and its possible mechanisms.Finally explore the key role of Drp1 in the Molecular mechanism of activation of mitochondrial dysfunction in /Nlrp3 inflammatory pathway and the pyroptosis in HK-2.Method1.Amplification of si Nlrp3 adenovirus in vitro,calculate the virus titer and MOI value,has been prepared for follow-up tests.2.Human renal tubular epithelial cells were cultured,and Ischemia reperfusion model of human renal tubular epithelial cells were imitated by hypoxia/re-oxygenation(H/R)treatment.3.The situation of pyroptosis in each group by flow cytometry.4.AU5800 Beckman automatic biochemical analyzer was used to detect the level of lactate dehydrogenase in cell supernatant of each group.5.Laser scanning confocal microscope was used to observe the expression level of reactive oxygen species in each group.6.The expression of Nlrp3,caspase-1,Drp1 and IL-1b protein in the cells of each group was detected by blot Western method.Results1.The pyroptosis exists in human renal tubular epithelial cells induced by hypoxia and re oxygenation.(1)the normal control group: the lactate dehydrogenase LDH in the cell culture supernatant was 36.90±1.89 IU/L,and the flow cytometry showed that the rate of pyroptosis was only 0.115%(P<0.05)in the normal control group.(2)Hypoxia / reoxygenation oxygen(H / R)group: compared with the normal control group,after hypoxia/re-oxygenation 24 h,the lactate dehydrogenase of cell supernatant increased up to 58.80±6.58 IU / L,the difference is statistically significant(P < 0.05);and The pyroptosis rate of human renal tubular epithelial cells increased significantly to 5.27%,the difference is statistically significant(P < 0.05);indicating that hypoxia/re-oxygenation can significantly increase the pyroptosis of human renal tubular epithelial2.Hypoxia/re-oxygenation increased the expression of Nlrp3,IL-1b,caspase-1 in human renal tubular epithelial cells,the si Nlrp3 Can reduce the expression of Nlrp3,,IL-1b,and caspase-1,and decrease the rate of pyroptosis of human renal tubular epithelial cells.(1)In normal control group,the levels of Nlrp3,caspase-1 and IL-1b.(2)Hypoxia / re oxygenation(H/R)group: compared with the normal control group,the Nlrp3 and caspase-1 activation of human renal tubular epithelial cells were increased,and the level of inflammatory factor IL-1(P<0.05)was significantly increased after hypoxia / re oxygenation(24h);(3)Hypoxia/re-oxygenation+Nlrp3 silencing adenovirus(H/R+si Nlrp3)group :Compared with hypoxia / re oxygenation(H/R)group,After hypoxia reoxygenation 24 h,the pyroptosis of human renal tubular epithelial cell death increased(1.78%)was significantly lower than that of hypoxia / reoxygenation group(5.27%)(P < 0.05),the activation of Nlrp3,caspase-1 and IL-1b in the renal tubular epithelial cells significantly reduced.show that dead si Nlrp3 effectively alleviate the pyroptosis in the hypoxia reoxygenation induced human renal tubular epithelial cells.and Nlrp3 also involved in the apoptosis.3.The expression of Drp1 and ROS in human renal tubular epithelial cells was up-regulated by hypoxia/re-oxygenation.Mdivi-1 could inhibit the activation of Drp1,reduce the expression of ROS,and decrease the level of Nlrp3,caspase-1 and IL-1b,and reduce the pyroptosis of the cells.(1)In normal control group,the levels of Drp1 and ROS were lower in human renal tubular epithelial cells.(2)hypoxia / re oxygenation(H/R)group: compared with the normal group,the expression of 24 h in human renal tubular epithelial cells was significantly increased(P<0.05),and the expression of ROS in the cells was also significantly increased after hypoxia/re-oxygenation.(3)Hypoxia/re-oxygenation+Mdivi-1(H/R+Mdivi-1)group:Compared with hypoxia/re-oxygenation(H/R)group,Mdivi-1 could not significantly reduce the expression of Drp1,but the expression of ROS in cells was significantly decreased,the level of pyroptosis(1.8%)was significantly lower than that of hypoxia/re-oxygenation group(5.27%)(P < 0.05),and the activation of Nlrp3,caspase-1 and IL-1 significantly decreased(P < 0.05),It was suggested that inhibition of Drp1 activation could effectively reduce the level of ROS and decrease the expression of Nlrp3,caspase-1 and IL-1 in human renal tubular epithelial cells.Drp1 was involved in the pyroptosis of human renal tubular epithelial cells induced by hypoxia/re-oxygenation.Conclusion1.the pyroptosis induced by hypoxia /re-oxygenation is the important ways of the death of renal tubular epithelial cells.2.Hypoxia/re-oxygenation induced mitochondrial dysfunction,may through upregulation of Drp1,and then induced inflammation of the Nlrp3 of expression,which could cause the inflammation reaction and the pyroptosis in the HK-2 cells.This may be an important mechanism of acute ischemic renal injury. |
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