Role Of Mitophagy-mediated NLRP3 Inflammasome Activation In Cadmium-induced Chicken Liver Damage

Cadmium,a toxic heavy metal and widespread exists in environment,which is harmful to human and animal health.Liver is the main target organ of cadmium toxicity.However,the pathogenesis of cadmium-induced chicken liver injury is still unclear.In this study,the toxic mechanism of cadmium-induced chicken liver injury was explored in vivo and in vitro.In vivo test,200 male Hyland white chicken（1-day-old）were used as exprimental animals,and randomly divided into 4 groups（50 bird/group）.Fed the chicken with 0 mg/kg（Control group）,35 mg/kg（L-Cd group）,70 mg/kg（M-Cd group）and 140 mg/kg（H-Cd group）cadmium-treatment diet,respectively.The chicken were continuously administrated for 90 d.Liver microstructure and ultrastructure were observed,the level of Cd accumulation in liver,the contents of mineral elements,serum liver function index,hepatic detoxification metabolism function,inflammatory response related factors,NLRP3 inflammasome activation,mitochondria damage and mitophagy level were measured.The part of in vitro test used avian leghorn male hepatoma cell line（LMH cells）as the research object,add 0,0.5,1 or 2μM Cd Cl₂ to the culture system for 24 h.The cell viability,cell morphology,cellular inflammatory response related factors,NLRP3 inflammasome activation,mitochondria damage and mitophagy level were detected.Then,the mitophagy inducer CCCP was added to the culture system for intervention of cadmium exposure test.cell viability,NLRP3 inflammasome activation,and mitophagy level were detected.The results were shown as follows:（1）The results of in vivo test were as follows:1)The structure and function of PINK1 and Parkin were predicted by bioinformatic tools.The rabbit anti-chicken Parkin and PINK1 polyclonal antibodies with strong specificity and high immunocompatibility were obtained.The detection technology platform for mitocphagy of chicken hepatocytes was established.2)Cadmium exposure resulted in a slow increase of body weight,the reduction of crow size,tibia length and liver weight,the disordered arrangement of hepatocytes,the increase of Kupffer cells,caryolysis and fragmentation of nuclear,swelling of mitochondria,disappearance of mitochondrial crista.Cadmium exposure caused abnormal liver function related index（AST,ALT,GGT,IBIL,TP）,enhanced hepatic oxidation products（MDA and H₂O₂）,reduced hepatic total antioxidant capacity（T-AOC）and antioxidant enzymes（CAT,POD,T-SOD,Mn-SOD,Cu Zn-SOD）activities,These results indicated that cadmium exposure could change hepatic structure and function,cause hepatic oxidative stress,which leads to significant hepatotoxicity.3)Cadmium exposure increased cadmium accumulation and the content of Be,Ba,Na,V,Cr,Fe,Co;decreased the contents of Ca,Ag,Cu,Zn,Mn,Se;inhibited xenobiotic nuclear receptor（AHR,CAR,PXR）response and disrupted CYP450s（total CYP450、AH、NCR、APND、ERND）.35 mg/kg cadmium activated Nrf2-mediated PhaseⅡdetoxifying enzyme.140 mg/kg cadmium inhibited signaling pathway.Cadmium exposure decreased P-GP and MRP1 transcriptional level.These results indicated that cadmium exposure could inhibit nuclear receptor response,disrupt CYP450s homeostasis,inhibit PhaseⅡ,Ⅲdetoxification metabolism reaction,reduced cadmium excretion,lead to cadmium accumulation and element homeostasis disorder in liver,which aggravate the hepatotoxicity of cadmium.4)Cadmium exposure caused inflammatory cell infiltration in liver and fibrosis in portal area,increased pro-inflammatory factors（TNF-α,IL-1β,IL-18）contents of serum and liver,decreased anti-inflammatory factor（IL-10）content of serum and liver.Besides,cadmium exposure promoted the activation of NLRP3 inflammasome and the expression of its downstream factors（Caspase1,IL-1β,IL-18）.These results indicated that cadmium exposure could cause liver inflammation by activating NLRP3 inflammasome to result in liver damage.5)Cadmium exposure disrupted mitochondrial structure,reduced mitochondrial membrane potential and mt DNA copy number,decreased the expression levels of mitochondrial biogenesis and mitochondrial fusion related regulatory factors（SIRT1,PGC-1α,NRF1,TAFM,Mfn1,Mfn2）,increased the expression levels of mitochondrial fission and mitophagy related regulatory factors（MFF、Fis1、PINK1、Parkin、LC3、ATG3、ATG5、ATG9、Beclin-1）.These results indicated that cadmium exposure could destroy mitochondrial structure,interfere with mitochondrial biogenesis and mitochondrial dynamics,which induced liver mitophagy.（2）The results of in vitro test were as follows:1)Cadmium exposure inhibited LMH cell viability,promoted NLRP3 inflammasome activation and inflammatory factors release,induced cell inflammatory injury.Meanwhile,cadmium exposure damaged mitochondrial structure and function,and caused mitophagy.However,with increasing cadmium dosage,mitophagy activity was decreased.These results indicated that mitophagy and NLRP3 inflammasome activation played important roles in cadmium-induced LMH cell inflammatory injury.2)After CCCP intervented mitophagy reaction,inhibits LMH cells cadmium-induced NLRP3inflammasome activation,inflammatory factors release and cell viability decrease induced by cadmium.These results indicated that mitophagy could relieve cadmium-induced hepatotoxicity via regulatting NLRP3 inflammasome activation.Taken together,cadmium exposure could disturb hepatic element homeostasis,inhibit hepatic detoxification metabolism function,cause hepatic oxidative stress and activate NLRP3inflammasome,leading to hepatocytes inflammatory injury.Meanwhile,cadmium exposure caused hepatocytes mitochondrial damage and mitophagy.Increasing the mitophagy level could inhibit NLRP3 inflammasome activation in hepatocytes,alleviating cadmium-induced hepatocytes inflammatory injury.This study confirmed that mitophagy,by removing damaged mitochondria,inhibited NLRP3 inflammasome activation to relieve cadmium-induced liver inflammatory damage.