The Research On Mechanism Of Inflammasome Activation In Macrophages Induced By Cisplatin Injuring Renal Tubule Cells

BackgroundCisplatin is widely used as anticancer drug,It's most important side effect is acute kidney injury(AKI).25?35%of patients with a single cisplatin treatment would developed AKI.The renal toxicity of cisplatin reduces the quality of life of cancer patients,so that patients have to reduce the dose of cisplatin or even stop the treatment.At present,the study on the pathogenesis of AKI induced by cisplatin shows that the aseptic inflammatory reaction in renal tissue caused by cisplatin is the main mechanism of AKI induced by cisplatin.Cisplatin in renal tubular epithelial cell accumulation induced renal tubular epithelial cell apoptosis and necrosis,the macrophages that exist in renal tubules could trend to damage of renal tubular epithelial cells when tubular epithelial cell injured,releasing pro-inflammatory cytokines and chemokines that caused further damage of renal tubular epithelial cells.However,there is a lack of in-depth study on the mechanism of activation and the relationship between AKI and the renal tubular epithelial cell damage.Cisplatin induced renal tubular epithelial cell necrosis could released damage associated molecular patterns(DAMP),DAMP could activate the innate immune system,mononuclear macrophages plays an important role in the innate immune system.There are DAMP type recognition receptor(PRR)distributed in cell membrane and intracellular.Nod like receptors(NLR)is found in mononuclear macrophage,NLR.conversion molecular protein ASC and cysteine protease caspase could component NLRP3 inflammasome,DAMP act on mononuclear macrophage NLR could activate caspase-1 in NLRP3 inflammasome,produce IL-1??IL-18.Animal experiments have confirmed that knockdown of caspase gene and blockade of IL-18 can mitigated AKI,suggesting that during the progress of cisplatin induced AKI,mononuclear macrophage NLRP3 inflammasome activation may be an important mechanism.In the study of AKI induced by ischemia and reperfusion,blocking the effect of DAMP can reduce the damage of renal tubular epithelial cells.In coculture of renal tubular epithelial cells and monocytes suggesting that renal tubular epithelial cells could release cytokines to regulate the activation of monocytes.Chemotherapy drugs induced tumor cells could release DAMP,activate monocytes and macrophages to attack tumor cells.These studies suggest that the renal tubular epithelial cells may release DAMP to activate macrophages.Based on the above background,our study based on the construction of cisplatin induced acute renal tubular epithelial cell injury model,use the damaged renal tubular epithelial cell supernatant on mononuclear macrophage,observe the mononuclear macrophage NLRP3 inflammasomes is able to be activated or not,and explore the mechanism of NLRP3 inflammasome activation in this system.1.Damage of renal tubular epithelial cells by cisplatinObjective:To establish a model of acute renal tubular epithelial cell injury induced by cisplatin,and to detect the changes of cell proliferation,apoptosis,necrosis,ROS level,and the level of ATP in NRK-52 cells with cisplatin induced injury.Methods:NRK-52E cells were treated with different concentrations of cisplatin.Cell proliferation,apoptosis and necrosis were detected by cell counting,MTT and caspase3,7 kit.Flow cytometry and immunofluorescence staining were used to detect the levels of ROS and mitochondrial ROS in the cells.The levels of ATP were detected by ATP kit and immunofluorescence staining.Results:Cisplatin could inhibit the proliferation,increase the intracellular ROS level,and promote the intracellular ATP release to the extracellular after treated with cisplatin.SummaryCisplatin acts on renal tubular epithelial cells could decrease the proliferation of cells,increase the ROS level and promote the release of ATP.2.Cisplatin damaged renal tubular epithelial cells activated the NLRP3 inflammasome in macrophagesObjective:Use the supernatant of cisplatin injured renal tubular epithelial as conditioned media,treated on macrophage cells(THP-1).Use PCR,Western blot,ELISA to detect the activation of NLRP3 inflammasome in macrophages.Treating different inhibitors on injured THP-1 cells,so as to clarify the damage induced by conditioned media on macrophage NLRP3 inflammasome activation.Methods:Treating THP-1 cells with conditioned media,detecting the leveal of IL-1beta by PCR,Western blot and ELISA.Use NF-?B inhibitor Bayll-7082,pan caspase inhibitor z-VAD-fmk,Caspase-1 inhibitor Z-YVAD-FMK,antioxidant ebselen,NAC,ATP sensitive K channel inhibitor glibenclamide,KCl,P2X receptor inhibitor PPADs act in injured macrophages,detecting the level of IL-1 beta.Results:Conditionanl media act in macrophages could activate the NLRP3 inflammasome.It's activation dependents on the activation of NF-?B,caspase-1 activation.The decreased intracellular ROS level could inhibit the activation of NLRP3 inflammasome.SummaryCisplatin injured renal tubular epithelial cells could release DAMP to activate NLRP3 inflammasome in macrophages.3.Cisplatin damaged renal tubular epithelial cells could release ATP and activated the NLRP3 inflammasome in macrophagesObjective:To investigate the effect of ATP on the activation of NLRP3 in THP-1 cells.Method:Use ATP scavengers apyrase clear the ATP among the conditioned media,use of antioxidant NAC reduced the ATP level among conditioned media,then detect the level of IL-lbeta in macrophages.Treated P2 receptor inhibitor Suramin,P2X receptor inhibitor PPADs and P2X7 receptor inhibitor A-438079 in THP-1 cells,and detect the changes of the levels of IL-1beta.Results:The decreasion of ATP could inhibit the activation of NLRP3 in THP-1 cells,and the use of ATP receptor antagonist could inhibit the activation of NLRP3.SummaryCisplatin injured renal tubular epithelial cells could release ATP as DAMPs that activated NLRP3 inflammasome in macrophages.4.Cisplatin damaged renal tubular epithelial cells could restrain autophagy and activated the NLRP3 inflammasome in macrophagesObjective:To investigate the autophagy condition when conditioned media act on THP-1 cells.And investigate the relationship between autophagy and the activation of NLRP3 inflammasome.Methods:Detect the autophagy level when conditioned media act on THP-1 cells.After adding mitochondrial autophagy enhanced agent andrographolide,inflammasome inhibitor z-VAD-fmk,Z-YVAD-FMK in macrophages,detecting the activation of NLRP3 inflammasome and the level of ROS.Results:Detecting after conditioned meida act on THP-1 cells,the change of intracellular LC3 expression level.After blocking the NLRP3 inflammasome activation,the LC3 expression level change in macrophage cells.application of immunofluorescence method to detect the mitochondrial autophagy changes.Using mitochondrial autophagy enhanced agent Andrographolide to regulate macrophage mitochondrial autophagy,after that detecting macrophage mitochondrial ROS and the change of NLRP3 inflammasome activation.SummaryCisplatin induced injured enal tubular epithelial cells release something decrease the level of autophagy in macrophages,the activation of NLRP3 in macrophages was related with down-regulation of autophagy.Statistical methodAll values are presented as mean ± SEM.The significance of differences among mean values was determined by One-way ANOVA.Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS20.0.The accepted level of significance was P<0.05.ConclusionCisplatin induced damage of renal tubular epithelial cells,could activate the NLRP3 inflammasome through the release of ATP.upregulation of macrophage mitochondrial autophagy can inhibit the damage of renal tubular epithelial cells on macrophage NLRP3 inflammasome activation.