The Effect And Mechanism Study Of Sapidolide A Protecting Against Acute Liver Injury By Regulating NLRP3 Inflammasomes In Macrophaes

Objective:Acetaminophen(APAP)is widely used as an over-the-counter analgesic and antipyretic,which overdose cause acute liver damage,even death.At present,only N-Acetyl-L-cysteine(NAC)can alleviate liver parenchymal cell damage based on the APAP metabolic mechanism,but its narrow therapeutic time window greatly limits its clinical application.In recent years,activated macrophages secrete pro-inflammatory cytokines and chemokines,and further recruit immune cells to mediate inflammation and then exacerbate APAP-induced liver injury(AILI),which suggested that macrophages had broad prospects in alleviating the inflammation phase of AILI.Therefore,it is urgent to find anti-inflammatory drugs for the inflammation phase of AILI to expand the existing treatment methods.Sapidolide A(SA),extracted from the Baccaurea ramiflora Lour,is a new type of sesquiterpene lactone compound.It has shown that some sesquiterpenoids had anti-inflammatory activities,which inhibitted the production of pro-inflammatory factors in macrophages.However,there is no studies about the effect of SA on AILI and the molecular mechanism of SA exerting its anti-inflammatory function.Our study will use different models and experimental methods,firstly to explore whether SA has protective effect on the AILI mice,and explore whether SA affect the inflammation process of AILI by reducing the secretion of inflammatory factors or the recruitment of inflammatory cells.Then,we will explore the molecular mechanism about SA regulating macrophages to alleviate AILI.Our research will provide potential therapeutic targets and theoretical basis for the discovery of natural compounds to protect AILI targetting on macrophages.Methods:AILI model was established on wild-type C57BL/6 male mice,and SA was administrated to investigate its therapeutic effect on AILI.(1)The degree of liver damage was investigated by detecting two biochemical indicators of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST).(2)Hematoxylin&eosin(H&E)staining was used to detect liver histopathology of AILI mice.(3)Td T mediated d UTP Nick End Labeling(TUNEL)staining was used to detect hepatocyte apoptosis in liver tissues.(4)Western Blotting was used to detect the expression level of phosphorylated c-Jun N-terminal kinase(p-JNK),high mobility group protein B1(HMGB1),caspase-1 and cleaved-caspase-1 protein.(5)q RT-PCR was used to detect the m RNA level of Il-6,Tnf-α,Ifn-γ,monocyte markers(Cd68),Nlrp3,Asc and Il-1βin liver.(6)Immunohistochemical staining was used to detect the expression level of macrophage marker(CD68)in liver tissue.(7)Flow cytometry was used to detect the number of monocytes(CD11b~+F4/80~+)in liver.(8)Enzyme linked immunosorbent assay(ELISA)was used to detect the secretion levels of inflammatory factors Il-1βand IL-18 in serum.The inflammasome activation(pyrolysis)model,M1/M2 type polarization model and conditioned medium model were established on mouse bone marrow-derived macrophages(BMDM)to investigate the mechanism of SA alleviating the inflammation of AILI.(1)Sulforhodamine B(SRB)staining was used to detect the survival rate of BMDM and L02 cells.(2)q RT-PCR was used to detect the m RNA levels of Nlrp3,Il-1βand Il-18 in BMDM.(3)Western Blotting was used to detect the levels of caspase-1,IL-1β,cleaved-caspase-1 and cleaved-IL-1βin cells lysates and supernatants.(4)Immunofluorescence staining was used to detect the formation of apoptosis-associated speck-like protein containing CARD(Apoptosis-associated speck-like protein containing a CARD,ASC)speck.Results1.Effect study of SA protecting inflammation of acetaminophen-induced liver injury(AILI)(1)The test results of serum level of ALT and AST showed that SA effectively inhibitted the increase of ALT and AST in AILI mice.(2)The H&E staining results showed that SA significantly improved the structure of injury liver tissue and reduced inflammatory cell infiltration in AILI mice.(3)The TUNEL staining of liver tissue showed that the TUNEL positive area was significantly reduced after SA treatment in AILI mice,which greatly improved cells apoptosis.(4)Western Blotting results show that SA significantly down-regulated the expression level of p-JNK and HMGB1 in the liver in AILI mice,which effectively reduced liver tissue damage and inhibited inflammation.(5)The test results of serum level of ALT and AST showed that SA did not affect the up-regulation of ALT and AST caused by APAP administration after 6 hours.(6)The H&E staining showed that SA did not improve the damage structure of liver lobules caused by APAP administration after 6 hours.(7)The SRB staining showed that SA did not affect the decrease of L02 cells survival rate caused by APAP.(8)Western Blotting results showed that SA does not affect the up-regulation of p-JNK and cleaved-PARP expression levels caused by APAP.The above results indicated that SA improved the later stage of AILI without affecting the early stage of liver parenchymal damage.(9)The results of q RT-PCR showed that SA significantly inhibitted the up-regulated m RNA levels of Il-6,Tnf-αand Ifn-γin AILI mice.(10)The results of q RT-PCR showed that SA significantly inhibitted the up-regulated m RNA levels of macrophage markers Cd68 and F4/80 in AILI mice.(11)The CD68 immunohistochemical staining showed that SA significantly reduced the CD68-positive area in AILI mice.(12)The results of flow cytometry showed that SA significantly reduced the number of macrophages(CD11b~+F4/80~+cells)in AILI mice.The above results indicated that SA significantly inhibitted the inflammation in the later stage of AILI.2.SA alleviates the inflammation by inhibiting the activation of inflammasomes in macrophage(1)The results of SRB staining showed that SA significantly increased the survival rate of BMDM in the pyroptosis model;and its protective effect on different degrees of pyroptosis caused by ATP administration for 30,60,90,and 120 minutes.(3)The results of q RT-PCR showed that SA did not increase the m RNA levels of Il-6 and Inos in BMDM,and did not inhibit the up-regulated m RNA levels of Il-6 and Inos caused by LPS,which indicated that SA had no effect on M1 polarization of BMDM.(4)The results of q RT-PCR showed that SA did not increase the m RNA levels of Fizz1 and Arg1 in BMDM,and did not promote the up-regulated m RNA levels of Fizz1 and Arg1 caused by IL-4,which indicated that SA had no effect on M2 polarization of BMDM.The above results indicated that SA significantly inhibitted the pyroptosis of BMDMs without affecting its polarization.(5)The results of SRB staining showed that SA significantly reduced the decreasing survival rate of L02 cells in the macrophage pyroptosis conditioned medium model.(6)Western Blotting results showed that SA significantly inhibitted the up-regulated level of p-JNK and cleaved-PARP in L02 cells in the macrophage pyroptosis conditioned medium model.(7)The results of q RT-PCR showed that SA inhibitted the up-regulated m RNA of Nlrp3,Il-1βand Il-18 caused by LPS in BMDM.(8)Western Blotting results showed that SA inhibitted the up-regulated levels of NF-κB and IL-1β caused by LPS in BMDM.(9)ASC immunofluorescence staining results showed that SA significantly inhibitted the formation of ASC speck in BMDM with activated-inflammasomes.(10)Western Blotting results showed that SA significantly down-regulated the expression of cleaved-caspase-1 and cleaved-IL-1βin supernatants in BMDM with activated-inflammasomes.(11)SA significantly inhibited the decline of BMDM survival rate after APAP-conditioned medium administration was observed.(12)Western Blotting results showed that SA inhibitted the up-regulation of cleaved-caspase-1 expression caused by APAP conditioned medium in BMDM supernatants.The above results indicated that SA could against pyroptosis of BMDM by inhibiting the activation of NLRP3 inflammasomes.(13)The results of q RT-PCR showed that SA down-regulated the m RNA levels of Nlrp3,Asc and Il-1βin AILI mice.(14)Western Blotting results showed that SA down-regulated the expression of cleaved-caspase-1 in AILI mice.(15)ELISA results showed that SA significantly inhibited the secretion of IL-1βand IL-18 in the serum of AILI mice.The above results indicated that SA inhibitted the secretion of IL-1βand IL-18 by inhibiting the activation of NLRP3 inflammasomes in macrophages,and in turn,result in reducing inflammation to finally alleviate AILI.Conclusion:Our study found that by inhibiting the inflammation in the later stage of AILI,SA could significantly alleviate AILI in vivo,whereas SA showed no effect in the early stage.Further studies found that SA could inhibit the inflammation by inhibiting the activation of NLRP3 inflammasomes in macrophages and reduced the damage of liver cells in the later stage of AILI.Our study not only revealed the therapeutic effect of SA on AILI,but also provided a theoretical basis for the design and discovery of new natural drugs.