Mechanism Of Aflatoxin B1-induced Renal Cell Pyroptosis Through Inhibition Of Mitophagy

Pyroptosis is a form of inflammatory cell death,hyperpyrosis can lead to high levels of cell death,tissue damage,and organ failure.Aflatoxin B1(AFB1)is carcinogenic,mutagenic and teratogenic.The kidney is the main organ of AFB1 toxicity.Studies have confirmed that AFB1 can accumulate in the kidney and cause renal damage,including renal tissue disorders and dysfunction.However,there has been no study on AFB1’s effect on renal cell pyroptosis.Therefore,the aim of this thesis was to investigate the effects of AFB1 on renal cell pyroptosis and to determine its specific mechanisms.The main studies and results are as follows.(1)To investigate the effect of AFB1 on scorch death of NRK-52 E cells and mouse kidney cells.(1)In order to study the toxic effect of AFB1 on NRK-52 E cells,we used MTT test to screen out the toxic dose of AFB1 of 1.25-40 μM.(2)In order to study the effect of AFB1 on renal function in mice,we found that compared with the Control group,the contents of urea nitrogen(BUN),creatinine(CRE)and uric acid(UA)in the renal tissues of mice in the AFB1 group were significantly increased.A vacuolar lesion of the renal tubules occurs and the space between the renal sacs is widened.(3)In order to study the effect of AFB1 on scorch death of renal cells,western blot detection showed that,AFB1 significantly increased the expressions of NLRP3,GSDMD-N,caspase-1,interleukin 1β(IL-1β),interleukin 18(IL-18)and lactate dehydrogenase(LDH)in mouse kidney cells and NRK-52 E cells.These results indicated that AFB1 could induce pyrosis of NRK-52 E cells and mouse renal cells.(2)To investigate the effect and mechanism of AFB1 on lysosome membrane permeability(LMP)and cathepsin B(CTSB).(1)To study the effect of AFB1 on lysosome function,we first detected the expressions of lysosome associated membrane protein 1(LAMP1)and CTSB in mice and NRK-52 E cells.It was found that AFB1 down-regulated the expression level of LAMP1 protein and up-regulated the expression level of CTSB protein.It indicated that AFB1 caused damage to the lysosome of NRK-52 E cells.(2)In order to investigate the effect of AFB1 on LMP,we detected the immunofluorescence staining of NRK-52 E cells’ LMP-related indexes Acridine orange(AO),Lysotracker Red(LTR)and CTSB,and found that AFB1 could induce LMP.(3)In order to study the influence of CTSB on NLRP3,we detected the colocalization of CTSB and NLRP3 by immunofluorescence staining,and found that AFB1 could improve the co-localization of CTSB and NLRP3.These results indicated that AFB1 promoted the release of CTSB into the cytoplasm and activated NLRP3 by inducing LMP.(3)To investigate the effect and mechanism of AFB1 on mitochondrial damage and mitophagy.(1)In order to investigate the effect of AFB1 on mitochondrial autophagy,we found that AFB1 down-regulates the expressions of PINK1 and Parkin proteins,while up-regulates the expressions of LC3 and P62 proteins.The addition of mitochondrial autophagy activator CCCP significantly increased the expressions of PINK1,Parkin and LC3,while decreased the expression of P62.LC3 and Mito-Tracker Red colocalization further demonstrated that AFB1 could inhibit mitochondrial autophagy.(2)In order to study the effect of AFB1 on reactive oxygen species(ROS),we detected mitochondrial membrane potential(MMP)and found that AFB1 reduced MMP,indicating that AFB1 could induce mitochondrial damage.Next,we found that AFB1 treatment significantly increased intracellular ROS and intracmitochondrial ROS(mt ROS).After the addition of CCCP,ROS levels decreased,indicating that AFB1 induced ROS levels to increase by inhibiting mitochondrial autophagy.(3)In order to study the effect of ROS on LMP,we added ROS inhibitor NAC and found that NAC could reverse the results of AO and LTR,indicating that the increase of ROS could promote the occurrence of LMP.These results indicate that AFB1 can induce LMP by inhibiting mitophagy,leading to the rise of intracellular ROS.In conclusion,this study illustrates the molecular mechanism of AFB1 triggering renal cell pyroptosis by inhibiting mitophagy,promoting ROS rise,inducing LMP and thus releasing CTSB to activate NLRP3 at both in vivo and in vitro levels.