| Background:Plethoric lipidosis in liver can interdict the autophagic flux of Kupffer cells,leading to the activation of inflammasomes and consequently exacerbating the inflammatory reaction caused by lipid deposition in the liver.Forkhead box protein O3 a is one of the important transcription factors that connects inflammatory reaction and energy metabolism.However,under the condition of high fat diet,the mechanism of Foxo3 a regulating the KCs autophagy and inhibiting inflammasomes’ activation remains unknown.Therefore,our experiment will focus on the Foxo3a’s variation and the way of adjusting the KCs autophagy flow and suppressing the activation of inflammasomes in KCs of nonalcoholic fatty liver model.Methods:1.Different concentrations of palmitic acid(PA 0,0.16,0.32,0.64 mM)and lipopolysaccharide(LPS 100 ng/ml)jointly stimulated KCs for 12 h in vitro:(1)Western blotting assay tested:(1)Autophagy related proteins,e.g Beclin 1 and LC3.(2)Nucleotide oligomerization domain(NOD)-like receptor family pyrin domain containing 3,e.g NLRP3,caspase 1 and apoptosis associated speck-like protein(ASC).(3)The expression of some upstream signal molecules binding to Foxo3 a such as adenosine monophosphate ativated protein kinase(AMPK),phosphatidyl inositol 3-kinase(PI3K)and Akt.(2)Enzyme-linked immuno sorbent assay(ELISA)tested the content of interleukin-1β(IL-1β)and IL-18.(3)We observed reactive oxygen species through dichlorofluorescein diacetate(DCFH-DA)fluorescence probe and fluorescence microscope.2.(1)We used Foxo3 a overexpression plasmid(Foxo3a-OE)and Foxo3a-short hairpin RNA plasmid(Foxo3a-shRNA)to transfect KCs respectively for 48 h,then used PA and LPS to stimulate KCs for 12h:(1)WB detected the expressional changes of autophagy related proteins and NLRP3 inflammasomes related proteins.(2)ELISA detected the content of IL-1β and IL-18 in cellular supernatant.(3)We observed the distribution of KCs autophagosomes through the transmission electron microscope.(2)KCs were infected by mRFP-EGFP-LC3 deficient adenovirus for 48 h.We utilized Foxo3 a agonist Iturin A to pretreat KCs for 1h firstly,then used PA and LPS to stimulate KCs for 12 h,finally we observed the distribution of LC3 marked autophagosomes through fluorescence microscope.3.(1)48 h after Foxo3a-OE transfected KCs,we utilized PA and LPS to stimulate KCs for 12 h,then utilized real-time quantitative polymerase chain reaction(RT-PCR)to detect expressional status of target molecule’s mRNA downstream transcripted by Foxo3 a.(2)We utilized Bim-OE and Bim-shRNA to transfect KCs in vitro for 48 h.Then we firstly utilized Foxo3 a inhibitor SC97 and agonist Iturin A to pretreat KCs for 1h,then utilized PA and LPS to stimulate KCs for 12 h.(1)WB detected the expressional change of autophagy related proteins and NLRP3 inflammasomes related proteins.(2)ELISA detected the content of cellular supernatant IL-1β and IL-18.4.24 mice(clean class)were randomly divided into 3 groups: coarse food diet group(CFD),high fat diet group(HFD)and high fat diet combining with Iturin A intraperitoneal injection group(Iturin A),and each group had 8 mice.After feeding the mice for 16 weeks,we took out their livers and peripheral blood respectively:(1)We detected liver’s steatosis condition through HE staining.(2)We detected mice’s liver function and blood lipid level through automatic biochemical analyzer.(3)We observed the distribution of autophagosomes in KCs through transmission electron microscope.(4)We separated liver’s KCs and extracted RNA and protein:(1)WB detected autophagy related proteins and NLRP3 inflammasomes related proteins.(2)RT-PCR detected mRNA’s level of IL-1β and IL-18.Results:1.The comparison between stimulating KCs in vitro with LPS combined with PA and stimulating KCs with LPS individually:(1)The expression of autophagy related protein Beclin 1 decreased in proportion while the expression of LC3Ⅱ/Ⅰ increased.(2)The expression level of NLRP3 inflammasomes related molecules like NLRP3,ASC,caspase 1 p10,IL-1β and IL-18 increased.(3)The expression level of Foxo3 a decreased,whereas that of p-Foxo3 a,p-Akt and p-PI3 K increased.The expression of p-AMPK remained unchanged.(4)The production of ROS increased evidently.2.(1)When KCs was over-expressed Foxo3 a,it could facilitate KCs autophagic flux evidently,and inhibit the activation of NLRP3 inflammasomes induced by PA combined with LPS.(2)When KCs was silence expressed Foxo3 a,it could further inhibit KCs autophagic flux,and further facilitate the activation of NLRP3 inflammasomes induced by PA combined with LPS.3.(1)When KCs was over-expressed Foxo3 a,in the PA combined with LPS group,only Bim’s mRNA expressional level was obviously lower than the Foxo3 a overexpressional group.(2)(1)In the condition of silence expressed Bim,even though KCs was given Foxo3 a agonist,KCs autophagic flux was still interdicted and the activation of NLRP3 inflammasoms was still facilitated in PA combined with LPS group.(2)In the condition of over-expressed Bim,even though KCs was given Foxo3 a inhibitor,KCs autophagy was still proceeding consequently and the activation of NLRP3 inflammasoms was still inhibited in PA combined with LPS group.4.Comparing with HFD group mice,in the Iturin A group mice:(1)Hepatic tissue steatosis and inflammatory reaction were milder.(2)Hepatic function and blood lipid level were lower.(3)There were more autophagosomes in hepatic tissue KCs.(4)The expressional level of Foxo3 a and Bim in KCs were obviously higher.Autophagy was activated and the activity of NLRP3 inflammasomes induced by high fat diet was lower.Conclusions:1.Free fatty acid interdicted autophagic flux of KCs and facilitated the activation of NLRP3 inflammasomes through affecting the phosphorylation of Foxo3 a.2.Foxo3 a inhibited the inflammasomes induced by free fatty acid through renewing KCs autophagic flux.3.Foxo3 a promoted autophagy of KCs and inhibited the NLRP3 inflammasomes induced by free fatty acid through facilitating transcriptional activity of Bim.4.Foxo3 a relieved liver steatosis and inflammatory reaction of the mice caused by lipidosis through facilitating transcriptional activity of Bim. |
