Lycopene Alleviates Hepatic Ischemia Reperfusion Injury By Enhancing Kupffer Cells Autophagy And Inhibiting NLRP3 Inflammasome Activation

Background and Aims:Partial hepatectomy(PH)and liver transplantation(LT)are the most effective treatments for the patients with hepatic malignant tumor and end-stage of liver diseases.Hepatic ischemia reperfusion injury(HIRI)is a major cause for acute postoperative live dysfunction and liver failure.However,the prevention and the treatment of HIRI are still limited.Hepatic Kupffer cells(KCs)play a major role in the pathophysiology of HIRI.KCs targeted intervention is an effective strategy for prevention and treatment of HIRI.Lycopene(Lycopene)is a natural carotenoid,which exists in a variety of fruits and vegetables.It is known to have antioxidant and anti-inflammatory properties.Rat HIRI animal model has confirmed that lycopene can effectively reduce HIRI by reducing hepatocellular injury and hepatic inflammation.However,the effect of lycopene on KCs in the pathophysiology of HIRI and its mechanism have not been elucidated.The purpose of this study is to use mouse HIRI model to clarify the effect of lycopene on KCs activation in the pathophysiology of HIRI and its specific mechanisms.Methods:C57BL/6 Mice were administered with either olive oil(10ml/kg body weight)as control or lycopene(20mg/kg body weight)by gavage for 2 weeks,then we established HIRI model to investigate the effects of lycopene on liver injury,inflammatory response,inflammatory cells aggregation and KCs autophagy in mice.Then by using 3-Methyladenine(3-MA)to inhibit autophagy activity to reverse verify that lycopene affects the liver injury of HIRI model by affecting KCs autophagy.Finally,we constructed a mouse model of HIRI treated with Nrf2-si RNA and HO-1-si RNA,through tail vein injection to explore the molecular mechanism of lycopene affecting autophagy of KCs.The specific experiments are as follows: ELISA was used to detect the levels of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in mice.H & E staining was used to detect the degree of liver cell injury.Myeloperoxidase(MPO)was used to detect the level of MPO in liver tissue.TUNEL was used to detect the degree of liver cell apoptosis.Western Blot was used to detect the expression of Bcl-2,Bcl-xl,P62,LC3 B,NLRP3,C-caspase-1,Pro-caspase-1,ASC,IL-1β,Pro-IL-1β,Nu-Nrf2,Cyto-Nrf2,HO-1 in liver tissue or KCs.Immunohistochemistry was used to detect the aggregation of F4/80+macrophages and Ly6G+ neutrophils in liver tissue.ELISA was used to detect the expression of TNF-α,IL-1β,IL-18 in liver tissue.The expression of TNF-α,IL-1 β,IL-6,IL-10 and NLRP3 in liver tissue was detected by q PCR,the expression level of LC3 B and NLRP3 in liver tissue was detected by immunofluorescence,and the number of autophagosomes in KCs was detected by transmission electron microscope.Results:In this study,we observed that the level of aspartate aminotransferases(AST),alanine aminotransferase(ALT),and the percentages of hepatocellular apoptosis in mice pretreated with lycopene were significantly lower than control mice.Lycopene inhibited F4/80+ macrophage and Ly6G+ neutrophil accumulation,which further decreased the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin 6(IL-6).Interestingly,lycopene induced increased autophagy in KCs,which was evidenced by elevated autophagosomes and the increased protein level of LC3 B.In these KCs,lycopene-induced upregulation of autophagy inhibited NOD-like receptor family pyrin domain-containing 3 protein(NLRP3)inflammasome activation,which was demonstrated by the reduced m RNA and protein levels of NLRP3,cleaved caspase-1,an apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),and IL-1β.Furthermore,3-methyladenine,an autophagy inhibitor,abolished lycopene’s inhibitory effect on the NLRP3 inflammasome in KCs,which led to increased hepatic IR injury.Further molecular mechanism studies showed that the protein levels of nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase 1(HO-1)were elevated in KCs isolated from IR-stressed mice pretreated with lycopene.Nrf2-si RNA or HO-1-si RNA could block the autophagy activation enhanced by lycopene in KCs,resulting in the activation of the NLRP3 inflammasome and aggravated hepatic IR injury.Nrf2-si RNA or HO-1-si RNA could block the autophagy activation enhanced by lycopene in KCs,resulting in the activation of the NLRP3 inflammasome and aggravated hepatic IR injury.Conclusion:In summary,we found that lycopene could effectively reduce hepatocellular injury,hepetocyte apoptosis and inflammatory response in mice HIRI model.Lycopene suppressing NLRP3 inflammasome activation through enhancing Nrf2/HO-1-mediated autophagy in KCs.The results of this study are expected to provide a scientific basis for the clinical application of lycopene and provide new strategies for the prevention and treatment of HIRI.