The Role Of PINK1-mediated Mitophagy Dysfunction In The Pathogenesis Of NASH

Objective: Nonalcoholic fatty liver disease(NAFLD)has been recognized as a major public health problem worldwide.The spectrum of NAFLD includes nonalcoholic simple fatty liver,nonalcoholic steatohepatitis(NASH)with or without fibrosis,cirrhosiss.Though the NASH is a key link in the process of NAFLD,the pathogenesis of disease progression from NAFL to NASH has not been fully understood.Recent studies have confirmed that mitochondrial dysfunction could induce the activation of NLRP3 inflammasome,aggravated the inflammatory response.PINK1/Parkin mediated mitophagy is one of the classical pathways to remove damaged mitochondria.The aim of this study is to investigate the effect of mitophagy on NLRP3-dependent inflammatory response during NASH disease progression by detecting PINK1/Parkin-mediated mitophagy.Methods:To induce NASH,the mice were constructed using the methionine-choline deficient diet(MCD),and the control groups were fed with Methionine-Choline sufficient diet(MCS).The levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),total superoxide dismutase(T-SOD),methane dicarboxylic aldehyde(MDA)and hydrogen peroxide(H2O2)were measured,the liver pathology was observed by hematoxylin-eosin(HE)staining,Masson staining and oil red O staining.The expression of PINK1,Parkin,NLRP3,caspase 1,ASC,IL1β and IL18 m RNA were detected in liver tissues by quantitative real-time PCR(RT-q PCR),the expressions of LC3,P62,PINK1,Parkin and NLRP3Ⅱ in liver tissues were analyzed by Western blot.Results:1 The NASH model in mice was built successfully.The mice in the MCS 6 4-week group and the MCS 8-week group were neat and shiny,the weight was increased,the activity and the mental were normal.However,the MCD 4-week group mice were hair messy,dull,lost weight,decreased activity,poor spirit,ate less.The MCD 8-week group mice were hair messy,dull,lost weight,reduced activity,apathetic,and food intake decreased significantly.The body weight in model groups were lower than that in 0-week group and contral groups(P <0.01),and the liver index in MCD 8-week group was higher than that in 0-week group,MCD 4-week group,and MCS 8-week group(P <0.01).2 The levels of serum ALT and AST in model groups were obviously higher than those in 0-week group and contral groups(P <0.05),and the levels of ALT and AST in MCD 8-week group were higher than those in MCD 4-week group.3 The liver of 0-week group and MCS 4-week group were dark red and shiny,soft,sharp edges,and liver density were normal;MCD 4-week group liver were yellow,volume increased,visible interstitial filling of light yellow fat spots,blunt edges;and MCS 8-week group liver were pale red,shiny,occasionally visible light yellow fat spots in the tissue;whereas the liver of MCD 8-week group were yellow,volume increased,visible a large amount of fat spots,density decreased,blunt edges.4 The histopathological staining in liver tissueHE staining showed that: 0-week group and MCS 4-week group: the structure of hepatic lobules were clear,the hepatocytes were radially arranged around the central vein,the structure of hepatocytes were intact,there was no degeneration,necrosis and inflammatory cell infiltration;MCD 4-week group: a mass of liver cells filled with fat vacuoles,a part of the liver structure were disordered,with occasional inflammatory cell infiltration;MCS 8-week group were occasionally scattered steatosis,without bubble-like changes and inflammatory cell infiltration;MCD 8-week group: the liver cells filled with a large number of fat vacuoles in different sizes,the structure of liver cells were disordered with balloon-like changes,there were a large number of inflammatory cell infiltration,mainly mononuclear cells and lymphocytes.Masson staining showed that: 0-week group and MCS 4-week group: the structure of liver lobules were complete,and the collagen fibers were only stained in the portal area;MCD 4-week group: hepatic lobulars’ structure survived,the fiber confined to the portal area,a small amount of collagen fibers could be seen in the perisinusoidal space;MCS 8-week group: the structure of liver lobules were complete,the collagen fibers were stained in the portal area and perisinusoidal space;MCD 8-week group: liver lobulars’ structure were disordered,the collagen fibers existed in the middle of hepatic lobules,showing significant necrosis of the liver cells and fibrous tissue hyperplasia,hyperplastic collagen fibers distributed in perisinusoidal space.Oil red O staining showed that: 0-week group and MCS 4-week group: there were no red lipid droplets in liver tissue;MCD 4-week group: a large number of red lipid droplets were found in the liver tissue,liver lobular endemic lipid droplet cells/total cell number of about 40% to 65%;MCS 8-week group: there were small amount of red lipid droplets in liver tissue,liver lobular endemic lipid droplet cells/total cell number of about 3% to 10%;MCD 8-week group: there were a large number of red fat droplets in liver tissue,part of them blent into pieces,liver lobules containing lipid droplets/total cell number of about 65% to 90%.Liver histopathological NAS scoring: the score of liver tissue in model groups were higher than that in 0-week group and contral groups(P <0.05),and the total score was more than 5 points,which was in accordance with NASH histological diagnosis.5 The levels of serum MDA and H2O2 in model groups were obviously higher than those in 0-week group and contral groups(P <0.05),while the level of serum SOD in model groups were lower than that in 0-week group and contral groups(P <0.05).6 The expression of NLRP3,caspase1,ASC,IL1β and IL18 m RNA in model groups were significantly higher than those in 0-week group and contral groups(P <0.05),while the expression of Parkin m RNA was lower.The expression of PINK1 m RNA in MCD 8-week group was higher than that in 0-week group,MCD 4-week group and MCS 8-week group(P <0.05).7 The expression of PINK1,Parkin in model groups were significantly lower than those in 0-week group and contral groups(P <0.05),while the expression of NLRP3 was higher.The expression of LC3Ⅱin MCD 4-week group was increased than that in 0-week group and MCD 4-week group,but in MCD 8-week group was decreased.The expression of P62 in MCD 8-week group was significantly higher than that in 0-week group,MCD 4-week group and MCS 8-week group(P <0.01).8 The expressions of PINK1 in NASH mouse were negatively correlated with the levels of NLRP3.Conclusions:1 PINK1/Parkin-mediated mitochondrial autophagy is involved in the pathological process of NASH.2 The dysfunction of mitophagy may up-regulate ROS and activate NLRP3 inflammasome,in this way to promote the progress of NASH disease.