Role Of Mitochondrial Autophagy Regulating NLRP3 Inflammasome In BDE-47 Induced Liver Injury In Mouse

Objective: 2,2 ’,4,4’-tetrabromodiphenyl ether(BDE-47)is one of the most common brominated flame retardants.It can enter the human body through respiratory tract,digestive tract,skin and other ways to produce a variety of toxicity.As the most important detoxification and metabolism organ in the human body,the liver is the main target of BDE-47 injury.More and more evidence shows that BDE-47 has a variety of hepatotoxic effects,including oxidative stress,DNA damage,inflammation,apoptosis,steatosis and fibrosis.Inflammatory bodies,as an important part of the natural immune system,play an important role in autoimmune diseases,infectious diseases and metabolic diseases.Among them,NLRP3 inflammatory bodies play an important role in the immune system and inflammatory diseases.The activated NLRP3 inflammatory bodies have strong proinflammatory activity and can cause the expression of a variety of inflammatory mediators.The activation mechanisms of NLRP3 inflammatory bodies include many kinds,among which mitochondrial damage and its release of mitochondrial signals are the most common.Mitochondria are the main sites for aerobic respiration of cells.When it is damaged,it will start mitochondrial autophagy to clear the damaged mitochondria,regulate the quantity and quality of mitochondria,and enhance the cell’s self-defense ability.Some studies have shown that mitochondrial autophagy can participate in the regulation of the activation of NLRP3 inflammatory bodies.However,it is unclear whether mitochondrial autophagy can regulate the activation of NLRP3 inflammatory bodies in BDE-47 induced liver injury in mice.Therefore,this experiment established a mouse model of BDE-47 exposure and intervention of autophagy activator(rapamycin)to study the role of mitochondrial autophagy in the liver injury of mice exposed to BDE-47,in order to explore the relationship between NLRP3 inflammatory bodies and mitochondrial autophagy in the liver injury of BDE-47.To provide scientific basis for prevention and control of BDE-47 induced liver injury.Methods: 1.Forty adult ICR male mice(6-8 weeks old)were selected as experimental objects and randomly divided into 5 groups.BDE-47 was dissolved in corn oil and poisoned by gavage every morning.The exposure dose group was divided into 5groups:control group(corn oil);12.5 mg/kg/day exposure group;25 mg/kg/day exposure group;50mg/kg/day exposure group;100mg/kg/day exposure group,continuous exposure for 4 weeks.ALT and AST liver function indexes in serum of mice were detected with the kit;HE staining was used to detect the changes of liver tissue structure in mice;The damage of mitochondria in mice was observed by transmission electron microscope;The changes of membrane potential and reactive oxygen species were detected by flow cytometry;The expression of NLRP3 in mouse liver was detected by immunofluorescence assay;Detection of NLRP3,Caspase-1 and IL-1β in mouse liver by Western Blot.The protein expression levels of IL-18,TNF-a,PINK1,Parkin,LC3,P62.2.Thirty two adult male ICR mice(6-8 weeks old)were selected as experimental objects and randomly divided into four groups for the intervention experiment of rapamycin(RAPA),which were divided into: control group(corn oil+normal saline);50mg/kg/day BDE-47 group;5 mg/kg/day RAPA group;5mg/kg/day RAPA+50mg/kg/day BDE-47 group.The mice were intraperitoneally injected with normal saline or RAPA one hour before intragastric exposure every morning for 4 weeks.ALT and AST liver function indexes in serum of mice were detected with the kit;HE staining was used to detect the changes of liver tissue structure in mice;The changes of membrane potential in mice were detected by flow cytometry;Detection of NLRP3,Caspase-1 and IL-1β in mouse liver by Western Blot.The protein expression levels of IL-18,TNF-α,PINK1 and Parkin.Results: 1.BDE-47 exposure induced changes in liver function of mice: With the increase of BDE-47 exposure dose,the content of ALT and AST in serum of mice increased significantly or extremely significantly(P<0.05).2.BDE-47 exposure caused pathological changes in the liver tissue of mice: HE staining showed that the gap of liver cells in mice exposed to BDE-47 was significantly enlarged,loosely arranged,changed in shape,and there was inflammatory cell infiltration.3.Effect of BDE-47 exposure on liver mitochondria of mice: Transmission electron microscopy showed that mitochondria of liver cells exposed to BDE-47 were swollen and mitochondrial membrane was broken.The liver mitochondrial membrane potential and intracellular ROS decreased with the increase of exposure dose,and there were significant differences between the 50mg/kg/day and 100 mg/kg/day groups and the control group(P<0.01).4.Effect of BDE-47 exposure on liver mitochondrial autophagy in mice: The expression levels of Parkin,PINK1,LC3II/LC3 I related proteins in liver mitochondria autophagy decreased with the increase of BDE-47 exposure dose,which was significantly different from the control group(P<0.05).5.Effect of BDE-47 exposure on activation of NLRP3 inflammatory bodies and downstream inflammatory factors in mouse liver: protein results showed that Caspase-1,NLRP3,IL-1β,IL-18,TNF-α The protein content showed an upward trend with the increase of exposure dose,and the expression levels of Caspase-1 and IL-18 protein in 50 mg/kg/day and 100 mg/kg/day groups were significantly higher than those in the control group(P<0.05);NLRP3 and TNF-α The expression of protein in 25,50,100 mg/kg/day group was significantly higher than that in the control group(P<0.05);IL-1β The expression of protein in 12.5,25,50 and 100 mg/kg/day groups was significantly different(P<0.05).The immunofluorescence results also showed that the expression of NLRP3 inflammatory body protein increased,and the fluorescence intensity increased with the increase of exposure.6.Autophagy activator increased the level of mitochondrial autophagy related protein in mice liver: protein results showed that compared with BDE-47 group,PINK1 and Parkin protein content in BDE-47+rapamycin group increased significantly(P<0.05).7.Activate mitochondrial autophagy to alleviate the effect of BDE-47 exposure on liver function of mice:Compared with BDE-47 group,the contents of ALT and AST enzymes in the liver of BDE-47+rapamycin group mice decreased significantly(P<0.05).8.Activating mitochondrial autophagy alleviates the pathological changes of mice liver caused by BDE-47 exposure: HE staining results showed that compared with BDE-47 group,after adding rapamycin,the swelling of liver cells was reduced,the morphology was restored,and there was no obvious inflammatory cell infiltration.9.Activating mitochondrial autophagy alleviates the decrease of liver mitochondrial membrane potential in mice exposed to BDE-47: Flow cytometry results showed that compared with BDE-47 group,the decrease of liver mitochondrial membrane potential caused by BDE-47 was reversed after adding rapamycin(P<0.01).10.Activating the expression of mitochondrial autophagy reduces the expression of NLRP3 and its downstream inflammatory factors in the liver of mice exposed to BDE-47: Compared with BDE-47 group,the proteins of Caspase-1,NLRP3,IL-18,IL-1β,TNF-α in mice liver of BDE-47+rapamycin group were significantly decreased,and the decreasing trend was statistically different(P<0.05 or P<0.01).Conclusion: Activation of mitochondrial autophagy can inhibit the activation of inflammatory bodies regulating NLRP3,thereby alleviating the liver damage caused by BDE-47 in mice.