Acetic Acid Controls Inflammatory Disease By Suppressing NLRP3 Inflammasome

BackgroundInflammasome refers to a class of large molecular platforms in activated immune cells.IL-1β,which is the downstream of inflammasomes,plays a central role in inflammatory response and inflammatory disease.In mammals,almost all of the IL-1βis producted by mononuclear phagocyte system,NLRP3 inflammasome activation in macrophage has been proved to closely related to many disease including insulin resistance,coronary atherosclerosis,inflammatory bowel disease.NLRP3 inflammasome is the most widely studied category among all inflammasome in mammalian at present,and it emerged as a potential target for therapy.Acetate,as a member of shrot chain fatty acid,is one of the important metabolites.Large amounts of acetic acid can be produced by gut microbiota,the histone deacetylation of cells can also produce acetic acid.The widespread existence of acetic acid in the body suggests that acetate possess various biological activities.It is reported that acetic acid can improve the prognosis of heart failure patients to prevent myocardial remodeling,but the role of acetic acid in inflammatory disease is not clear.This study was designed to explore the protective effect of acetate on NLRP3 inflammasome-denpend inflammatory disease and look into the underlying mechanisms.Methods1.To establish a disease model of LPS-induced endotoxemia and observe the therapeutic effect of acetic acid.Intraperitoneal injected with LPS is utilized to induce endotoxemia: 6~8 weeks male C57BL/6J mice are randomly divided into 3 groups: Control group: mice were intraperitoneal injected with PBS solution.Endotoxemia group: mice were intraperitoneal injected with LPS,followed by intraperitoneal injection with PBS solution 30 minute later.Acetate-treatment group: mice were intraperitoneal injected with LPS,followed by intraperitoneal injection with acetate-PBS solution 30 minute later.6 hours later,blood were harvested from heart with Sevoflurane anesthesia,and the concentration of IL-1β,IL-6 and TNF-αin serum was determined.2.To establish a disease model of MSU-induced and alum-induced peritonitis and observe the therapeutic effect of acetic acid.Intraperitoneal injected with MSU/ alum is utilized to induce peritonitis: 6~8 weeks male C57BL/6J mice are randomly divided into 3 groups,Control group: mice were intraperitoneal injected with PBS solution.peritonitis group: mice were intraperitoneal injected with MSU/alum solution 10mg/kg and 2mg/kg respectly,followed by intraperitoneal injection with PBS solution 30 minute later.Acetate treatment group: mice were intraperitoneal injected with MSU/alum solution 10mg/kg and 2mg/kg respectly,followed by intraperitoneal injection with acetate-PBS solution 30 minute later.6 hours later,sacrificed mices,peritoneal lavage was performed with PBS solution.Use flow cytometry to detect the number of cells in the lavage fluid,and concentration of IL-1β in the lavage fluid was determined.3.To establish the model of inflammasome activation in vitro and observe the effect of acetic acid on it.For cell culture,cell form marrow cavity of tibia and femur was harvested,stimulate the cells with macrophage colony stimulating factor(M-CSF)and Penicillin-Streptomycin Solution,replace the medium four days later to get bone marrow-derived macrophage(BMDM).Intraperitoneal injected with 5% thioglycolate salt by 2ml and performe peritoneal lavage with PBS solution to get peritoneal macrophage(PM).Stimulate the BMDM or PM with LPS(200ng/ml)for 3 hours.After that,treat the cell with acetate at 20/25/30 mM or isometric PBS solution for 30 minutes,and then,challenge with adenosine triphosphate(5mM)or nigericin(20μM)for 30 minutes,collect the surpernate and determine the concentration of IL-1β.4.Stimulate the BMDM or PM with LPS(200ng/ml)for 3 hours.After that,cells were treated with,in turn,3-MA/Bafilomycin A1,the inhibitors of autophagy,at proper concentration for 30 minutes,acetate at 20/25/30 mM or isometric PBS solution for 30 minutes,adenosine triphosphate(5mM)or nigericin(20μM)for 30 minute,collect the surpernate and determine the concentration of IL-1β.5.Stimulate the BMDM or PM with LPS(200ng/ml)for 3 hours.After that,cells were treated with,in turn,acetate at 20/25/30 mM or isometric PBS solution for 30 minutes,adenosine triphosphate(5mM)or nigericin(20μM)for 30 minute,wash the cell 3 times with cold PBS solution and scrap it within cell lysis buffer.Determine the total concentration of protein.The protein levels of indicated molecμles which reflect autophagy and inflammasome was examined by western blot.6.Stimulate the BMDM or PM with LPS(200ng/ml)for 3 hours.After that,cells were treated with,in turn,KH7,an inhibitors of adenylyl cyclases,at 5μM or H89,an inhibitor of PKA,at 5μM for 30 minutes,acetate at 20/25/30 mM or isometric PBS solution for 30 minutes,adenosine triphosphate(5mM)or nigericin(20μM)for 30 minute,collect the surpernate and determine the concentration of IL-1β.7.Stimulate the BMDM or PM with LPS(200ng/ml)for 3 hours.After that,cells were treated with,in turn,acetate at 20/25/30 mM or isometric PBS solution for 30 minutes,adenosine triphosphate(5mM)or nigericin(20μM)for 30 minutes,wash the cell 3 times with cold PBS solution and scrap it within NP40 lysis buffer.Preprecipitation the cell lysate with normal IgG and precipitation targart protein with corresponding antibody overnight.The ubiquitination levels of NLRP3 inflammasome was determined by western blot.Results1.In LPS-induced endotoxemia,the concentration of proinflammatory cytokine IL-1β,IL-6 and TNF-αwas elevated in response to LPS injection,whereas the concentration in acetate-treatment group was lower compare to endotoxemia group.2.In MSU-induced and alum-induced peritonitis,the concentration of proinflammatory cytokine IL-1βin lavage fluid elevated compared with control group whereas the concentration in acetate-treatment group was lower compare to peritonitis group,as well as the total cell counts.3.LPS-primed BMDM or PM,products IL-1βand release it to supernate in response to ATP or nigericin,acetate-treatment down-regulate the concentration of IL-1βin cell culture medium.4.The inhibitor of autophagy,3-MA and Bafilomycin A1,reverse the inhibitory effect of acetate on IL-1β,in the meantime,either 3-MA or Bafilomycin A1 can not induce IL-1β release when use alone.5.Acetate-treatment change the expression level of proteins involving in autophagy,promote the degeneration of NLRP3 inflammasome but has no effect on ASC6.The inhibitory effect of acetate on IL-1βwas abrogated by inhibit the activity of adenylyl cyclase and PKA.Consistently,the ubiquitination level of NLRP3 inflammsome was elevated after acetate treatment.ConclusionAcetate downregulated the production of IL-1β by inhibiting NLRP3 inflammasome activation,and alleviated LPS-induced endotoxemia and MSU-induced and alum-induced peritonitis,acetate promoted NLRP3 inflammasome degeneration via Gpr43-sAC-PKA pathway,which elevated the ubiquitination level of NLRP3 inflammasome.