The Function And Regulatory Mechanism Of NLRP12 In Alcohol-induced Acute Liver Injury

Background: Alcohol-induced acute liver injury(ALI)is characterized by abnormal disorder of liver structure and function,often accompanied by excessive inflammatory response,which is sometimes called alcoholic hepatitis(AH).In recent years,a large number of studies have shown that the activation of macrophage in the liver induced by ethanol and the secretion of inflammatory cytokines are indispensable factors leading to the occurrence of alcoholic liver injury.Specifically,alcohol is the most important cause which could trigger the immune receptor activation and activate liver macrophage through multiple inflammatory signaling pathways,which involve the transcription and synthesis of a large number of inflammatory cytokines especially proinflammatory cytokines.And the production of proinflammatory cytokines will accelerate the development of alcoholic liver disease further.During the process of inflammatory response,NOD-like receptors,a family of immune receptors,have always been one of the research hotspots,and several members of NOD-like receptors play crucial role in the occurrence,progression,regulation and other aspects of inflammatory response.NLRP12,a member of the NOD-like receptor family,has been widely reported to regulate inflammation in a variety of tumor and inflammation-related diseases,and its anti-inflammatory effect in most diseases also provides ideas for the introduction of the reason for selected topic.Therefore,this study focused on the regulatory effect of NLRP12 on the regulatory effect of NLRP12 on inflammation,as well as the possible mechanism of this regulatory effect and the effect on the apoptosis on hepatocyte and AML-12 cell line,so as to provide new research ideas and therapeutic targets for the treatment of alcoholic liver injury.Objective:(1)To explore the regulatory effect of NLRP12 on liver injury,steatosis,the level of inflammation cytokines originated from liver macrophage in alcohol-induced acute liver injury and the possible mechanism;(2)To explore the possible effect of NLRP12 in the regulation of inflammatory response on the apoptosis of hepatocyte and AML-12 cell line.Methods: In vivo,C57BL/6J mice were randomly divided into 6 groups: control liquid-diet group(CD-fed),ethyl alcohol liquid-diet group(EtOH-fed),control liquid-diet+empty vector adenovirus group(CD-fed+Ad-EGFP),ethyl alcohol liquid-diet+empty vector control virus group(EtOH-fed+Ad-EGFP),control liquid-diet+NLRP12-overexpression group(CD-fed+Ad-NLRP12-EGFP)and control liquid-diet+NLRP12-overexpression group(EtOH+Ad-NLRP12-EGFP).The total modeling period included 16 days,consisting of the adaptation of feeding(TP4030C diet)for 5 days.From the sixth day,EtOH-fed diet groups were provided with 5% TP4030 A diet,and CD-fed groups were supplied with TP4030 C diet.At 7 am on the 16 th day,mice offered with TP4030 A were given 20 percent ethanol by gavage,while mice supplied with TP4030 C diet were given equal saline,and lavage dose is calculated on the weight of mice(5g/kg).All mice were put to death after the gavage for 9 hours.The blood was taken for the detection of liver injury indexes such as ALT,AST and the content of inflammatory cytokines such as TNF-α,IL-6 and IL-1β.The liver tissue was used for detection of m RNA and protein expression of NLRP12 and inflammation-related cytokines.Additionally,the liver tissue is used for double fluorescent dye,oil red staining,HE staining to verify the building of the mouse model and the change of liver steatosis and injury after the overexpression of NLRP12.In vitro,50 m M EtOH was used to activate RAW 264.7 cells.Firstly,the level of inflammation cytokines in the supernatant induced by alcohol was used to determine whether the cells could be activated,and the extracted protein and m RNA were analyzed for the expression of NLRP12.Secondly,after the use of interfering RNA(NLRP12-si RNA)and the overexpression of NLRP12 by adenovirus in vitro,the protein expression and m RNA level of NLRP12,inflammatotion cytokines and NF-κB-related protein were analyzed by Western blot and qRT-PCR.In addition,after the overexpression and silence of NLRP12 in RAW264.7 cell line,the supernatant was used to cultured AML-12 cells for 24 h respectively,and the apoptosis ratio of AML-12 cells and apoptotic protein indexes were detected,so as to explore whether the inflammatory regulation effect of NLRP12 altered the apoptosis of AML-12 cells.Result: In vivo,obvious liver steatosis and injury were shown according to serological,pathological and inflammatory cytokines expression in the EtOH-fed group,indicating that the model was successfully established.Compared with the CD-fed group,the m RNA and protein expression of NLRP12 were abnormally up-regulated in the EtOH-fed group,and the result of immunohistochemical showed that NLRP12 was highly expressed in the EtOH-fed group,and the result of immunofluorescence double-staining indicated that NLRP12 was co-localized with CD68(macrophage marker).After NLRP12 was overexpressed in mice injected with adenovirus through caudal vein,the results and analysis of pathological biopsy and serum liver injury indexes showed that the degree of hepatic steatosis and injury was reduced,and the expression of proinflammatory cytokines were also significantly decreased.In vitro,compared with the control group,the level of NLRP12 and inflammation cytokines were elevated in RAW264.7 treated with 50 m M alcohol.After the overexpression of NLRP12,the level of proinflammatory cytokines decreased significantly,while after the down-regulation of NLRP12,the level of several inflammation cytokines showed an upward trend.Furthermore,after NLRP12 was overexpressed in RAW 264.7 cells,the apoptosis ratio of AML-12 cells was dramatically decreased,while the apoptosis rate of AML-12 cells was increased after the silence of NLRP12 in RAW26.7 cells.Conclusion: NLRP12 could inhibit the activation of macrophage and the expression of inflammation cytokines through the negative regulation of NF-κB signaling pathway and the negative regulatory role for inflammation of NLRP12 reduced the apoptosis of hepatocyte and AML-12 cells.