| Objective Based on the establishment of hepatocyte L02 injury model stimulated by Nano-Co,the toxic damage effect of Nano-co stimulation was observed and the activation of NLRP3 inflammasome in hepatocytes was studied.To investigate the mechanism of NLRP3 inflammasome activation induced by Nano-Co through mtROS.This will enrich and improve the understanding of the potential health hazards of nanomaterials,and provide a theoretical basis for the study of the toxicological mechanism of Nano-Co and the potential therapeutic prospects for NLRP3.Methods 1.The uptake of Nano-Co by hepatocyte L02 was observed by transmission electron microscope(TEM).2.L02 cells were exposed to different concentrations of Nano-Co to establish a hepatocyte injury model.3.The cytotoxic effect of Nano-Co stimulation was detected by CCK-8 kit and LDH kit.The expressions of NLRP3,ASC and Caspase-1 p20 were detected by Western blot after Nano-Co exposure.The levels of IL-1β and IL-18 released were detected by enzyme-linked immunosorbent assay(ELISA).4.The effect of regulating NLRP3 expression on the expression of caspase1 p20,IL-1β and IL-18 induced by Nano-Co: RNA interference was used to regulate the expression of NLRP3,Western blot was used to detect the expression of Caspase-1 p20,and ELISA kit was used to detect cellular IL-1β and IL-18 expression levels.5.The levels of ROS and mtROS in cells stimulated by Nano-Co were detected by ROS,mitochondrial ROS(mtROS)kit.6.To verify the regulation of mtROS on the activation of NLRP3 inflammasome induced by Nano-Co: total ROS scavenger NAC and mtROS specific scavenger MitoTEMPO(TEMPO)were used to pretreat cells respectively,and then stimulated cells with Nano-Co.The expression of Caspase-1 p20 and other molecules was detected by Western blot.The expression levels of IL-1β and IL-18 in the cell supernatant were determined by ELISA.7.The effect of regulating the expression of NF-κB on the expression of Caspase-1 p20,IL-1β and IL-18: the cells were pretreated with NF-κB inhibitor and then exposed to Nano-Co,and the expression of Caspase-1 p20 was detected by Western blot assay.The expressions of IL-1 β and IL-18 were detected by ELISA kit.Results 1.The uptake and endocytosis of Nano-Co in hepatocytes and the ultrastructural changes of hepatocytes were observed under transmission electron microscope(TEM).2.Compared with the control group,the cell viability was decreased and the release of LDH was increased in different concentrations of Nano-Co stimulation group.The results showed that Nano-Co had cytotoxic effect on L02 cells in a time-and concentration-dependent manner.3.After L02 cells were exposed to different concentrations of Nano-Co for 24 hours,western blot showed that the protein expression of NLRP3 increased with the increase of Nano-Co concentration,but there was no significant difference in the expressions of pro-caspase-1(Pro-Casp1),pro-IL-1 β and ASC(P>0.05).Nano-Co induced the expressions of Casp1(20-kDa)and mature IL-1 β(17-kDa)protein in L02 cells,and 7.5 μg/mL Nano-Co stimulated the cells.The activation proteins Casp1(20-kDa)and IL-1 β(17-kDa)of inflammatory corpuscles in NLRP3 were significantly increased(P<0.05).The results of ELISA showed that the expressions of IL-18 and IL-1β in 7.5 μg/mL group and 10 μ g/mL Nano-Co stimulation group were significantly increased(P<0.05).After transfection of siRNA NLRP3 successfully inhibited the expression of NLRP3,the results showed that the release of LDH in L02 cells exposed to Nano-Co was decreased,and the expressions of Casp1(20-kDa)and IL-1 β(17-kDa)were effectively inhibited(P<0.01).4.After L02 cells were treated with different concentrations of Nano-Co,the results showed that ROS and mtROS increased with the increase of Nano-Co concentration.After pretreated with NAC(ROS inhibitor)and TEMPO(mtROS inhibitor)respectively,western blot results showed that TEMPO group and NAC group significantly inhibited the expressions of NLRP3,Casp1(20-kDa)and IL-1β(17-kDa)related molecule(P< 0.01).5.Hepatocytes were pretreated with BAY11-7082(NF-κB inhibitor)and then exposed to Nano-Co.The results of western blot showed that the expression of NF-κB in hepatocytes increased in a concentration-dependent manner in different concentration stimulation groups,and the expression of p65 was significantly inhibited in NAC inhibition group(P<0.01).However,the expression of Casp p20 was significantly inhibited in NF-κB inhibition group(P<0.05),the ELISA results showed that the expression levels of IL-1β and IL-18 did not change significantly.Conclusion 1.Nano-Co induces oxidative stress in cells,causing an increase in ROS and mtROS production,further promoting the activation of NLRP3 inflammasome in hepatocyte L02.2.Oxidative stress injury and NLRP3 may be therapeutic targets for liver injury induced by Nano-Co,which provide a basis for the prevention and treatment of Nano-Co cytotoxicity. |
