Effects Of Genistein On Fatty Liver Hemorrhagic Syndrome And Its Mechanism

Fatty liver hemorrhagic syndrome（FLHS）is a metabolic disease mostly observed in laying hens,which is characterized by fatty degeneration and hemorrhage in the liver.FLHS usually causes a drop-in egg production rate,causing serious losses to the poultry industry.The pathogenesis of FLHS in laying hens is still unclear,and its pathogenesis is similar to human metabolic associated fatty liver disease.The current research showed that NLRP3-GSDMD mediated pyrolysis was closely related to the occurrence of the disease.Genistein is a phytoestrogen with a variety of biological activities and plays a protect role in FLHS of laying hens,however,its mechanism is still unclear.After genistein enters the body,it mainly exerts its biological activity by binding to estrogen receptor（ER）.ERαis the main ER expressed in the liver,plays an important role in regulating lipid metabolism,insulin resistance and inflammation.Our previous studies found that ERαwas a transcriptional regulator of NLRP3,which could regulate the expression of NLRP3 and the activation of NLRP3 inflammasome.Therefore,we hypothesis that genistein could regulate NLRP3-GSDMD mediated pyroptosis by binding to ERα,thereby alleviate FLHS of laying hens.The main contents and results of this topic are as follows:1.The effects of genistein on FLHS induced by high-energy and low-protein diet in laying hensTo study the regulation role of genistein on FLHS of laying hens,we first established FLHS model by using high-energy and low-protein（HELP）diet and adding different concentrations of genistein to the diet.The results showed that genistein inhibited lipid deposition in the liver,and improved insulin resistance.We further tested the expression of lipid metabolism-related genes and found that genistein inhibited fatty acid uptake genes（LPL and cd36）and synthesis genes（SCD-1,ACC,SREBP-1 and FAS）expression（P<0.01）,and promoted fatty acidβ-oxidation genes（AMPKα,CPT-1 and PPARα）expression（P<0.01）.Western blot results showed that genistein up-regulated the phosphorylation level of AMPKαand the expression of CPT-1（P<0.01）,down-regulated the expression of ACC,SREBP-1 and FAS（P<0.01）,indicating that genistein inhibited lipid synthesis and promoted fatty acidβ-oxidation to reduce lipid deposition.Moreover,we found that genistein inhibited the gene and protein expressions of NLRP3 inflammasome components（NLRP3,ASC and Caspase-1）and GSDMD,and decreased the expression of IL-1βand IL-18（P<0.01）,indicated that genistein could inhibit liver inflammation.Further,we detected the m RNA and protein expressions of ERαand ERβfound that genistein up-regulated the m RNA and protein levels of ERα（P<0.01）,and no significant difference was found in ERβ（P>0.05）.Collectively,genistein could block fat deposition of FLHS,inhibit NLRP3-GSDMD mediated pyroptosis,and activate ERα.2.The effects of ERαon NLRP3-GSDMD mediated pyroptosis induced by HFD/FFATo clarify the correlation between ERαand high fat diet（HFD）-induced inflammation,we first constructed ERαknockout mice and ERαknockdown cell lines and used HFD/FFA to establish in vivo and in vitro fatty liver model.The results showed that ERαdeletion aggravated the lipid deposition of hepatocytes induced by HFD/FFA,resulting in more serious liver injury.Liver injury is often accompanied by inflammation,consistent with our results that ERαdeletion increased both the m RNA transcription levels and protein expressions of NLRP3 inflammasome components（NLRP3,ASC and Caspase-1）and GSDMD（P<0.01）,leading to increased release of IL-1βand IL-18（P<0.01）.In addition,we found that ERαknockdown increased the release of LDH（P<0.01）,resulting in an increase in the number of PI positive cells,suggesting that deletion of ERαaggravated the NLRP3-GSDMD mediated pyroptosis.In contrast,selective ERαagonist inhibited the expression of NLRP3,ASC and Caspase-1 induced by FFA（P<0.01）,as well as inhibited the oligomerization of ASC and formation of ASC specks.At the same time,selective ERαagonist down-regulated the expression of GSDMD,resulting in the decrease of IL-1β,IL-18and LDH released into extracellular（P<0.05）,and decreased the number of PI positive cells.These above results suggested that ERαcould inhibit NLRP3-GSDMD mediated pyroptosis induced by HFD/FFA.3.The mechanism of ERαrelieving liver lipid deposition and inflammation via inhibiting NLRP3-GSDMD mediated pyroptosisTo further clarify the mechanism of ERαalleviated liver inflammation by inhibiting NLRP3-GSDMD mediated pyroptosis,this experiment established fatty liver model in ERαknockout mice and inhibited NLRP3-GSDMD mediated pyroptosis by adding NLRP3specific inhibitor MCC950.The results showed that MCC950 significantly inhibited fat deposition,insulin resistance and inflammation in ERα^-/-mice induced by HFD,manifested by the down-expressed genes related to fatty acid intake and synthesis（P<0.01）,promoted expressions of fatty acidβoxidation genes（P<0.01）.Meanwhile,MCC950 inhibited levels of TG and TC（P<0.05）,inhibited the AUC of GTT and ITT（P<0.01）,and reduced release of IL-1βand IL-18（P<0.01）.Similar results have also been found in ERαknockdown cell lines added MCC950,as the protein expression of ASC,caspase-1,IL-1β,IL-18 and GSDMD decreased,as well the IL-1β,IL-18 and LDH release（P<0.01）and PI positive cells number.The above experimental results confirmed that the alleviated effect of ERαon fatty liver is related to the inhibition of NLRP3-GSDMD mediated pyrolysis.4.Study on the relationship between ERαand GSDMDThe above results proved that the improve role of ERαin fatty liver is connected with GSDMD,as the absence of ERαled to a significant increase of the expression of GSDMD,and the specific activation of ERαcould significantly inhibit the expression of GSDMD.However,the regulatory mechanism between ERαand GSDMD is still unclear.To further explore whether ERαcan inhibit pyroptosis by directly interacting with GSDMD,Co-IP and GST pull-down assays were performed.Results of Co-IP and GST pull-down proved the direct interaction between ERαand GSDMD,and laser confocal technology confirmed colocalization of ERαand GSDMD.GSDMD knockdown cells and control cells were pretreated with ERαinhibitor and added FFA,GSDMD knockdown reduced the number of red lipid droplets,and TG level（P<0.01）.Knockdown of GSDMD inhibited expressions of NLRP3,ASC and Caspase-1,ultimately led to decrease of IL-1β,IL-18,and LDH release（P<0.01）,and PI-positive cells.The above results indicated that ERαcould directly interact with GSDMD and inhibit pyroptosis induced by FFA.5.The mechanism of genistein relieves liver injury induced by HFD via activating ERαTo explore whether genistein could inhibit NLRP3-GSDMD mediated pyroptosis via targeting ERαto alleviate liver injury,WT and ERα^-/-mice were feeding with HFD and adding genistein.We found that genistein could inhibit lipid deposition,down-regulate the expression of NLRP3 inflammasome components（NLRP3,ASC and Caspase-1）,and inhibit the expression of GSDMD,as well significantly decrease the release of IL-1βand IL-18（P<0.01）,indicating that genistein had a significant inhibitory effect on HFD-induced liver injury of WT mice.However,the study found that the genistein had no longer obvious inhibitory effect on steatosis,insulin resistance and inflammation in HFD-induced ERα^-/-mice any more.It was confirmed by the no change of accumulation of lipid droplets in the liver,as well the level of fatty acid intake genes,fatty acid synthesis genes and fatty acidβ-oxidation genes（P>0.05）.Meanwhile,the expression of NLRP3,ASC,Caspase-1 and GSDMD were not changed significantly compared with ERα^-/-mice without genistein treatment（P>0.05）.All the results indicated that genistein inhibited NLRP3-GSDMD mediated pyroptosis via targeting ERαto alleviate liver injury induced by HFD.In summary,the expression of NLRP3 and GSDMD were significantly increased during the occurrence of FLHS in laying hens induced by HELP diet,and genistein inhibited the expression NLRP3 and GSDMD by activating ERα.Genistein played improve role on the fatty degeneration and inflammation of laying hens induced by HELP diet.It is the first time to confirm that ERαcould directly interact with GSDMD and inhibit the occurrence of pyroptosis.Genistein could inhibite NLRP3-GSDMD mediated pyroptosis by activating ERαto ameliorate FLHS of laying hens.This study enriched the pathogenesis of FLHS and will provided a new perspective for the control and prevention of FLHS.