The Effects And Mechanisms Of Ablation Of JIP3 On Liver Injury Induced By NAFLD Mice

BackgroundThe prevalence of obesity-related metabolic diseases,including non-alcoholic fatty liver disease?NAFLD?,diabetes and coronary heart disease,is increasing year by year,and current treatment measures are mainly aimed at the consequences of the disease rather than the causes of metabolic disorders.Therefore,exploring the pathogenesis of obesity-related diseases has important guiding significance for the clinical prevention and treatment of obesity and related metabolic diseases.NAFLD refers to hepatic fat metabolism disorder caused by alcohol and other factors that determine liver damage.Its main features are hepatic steatosis and lipid metabolism disorders.The initiating factors of NAFLD are closely related to obesity and insulin resistance.Fat accumulation in the liver can easily cause oxidative stress and lipid peroxidation,which induces hepatocyte injury,inflammation and fibrosis,and promotes the progression of NAFLD.JNK interacting protein 3?JIP3?,a member of the JIP family,was initially identified as a scaffold protein for the c-jun amino-terminal kinase?JNK?signaling pathway.JIP3 has enhanced ability to form heterodimers.Multifaceted JIP3 scaffold proteins are associated with TLR.TLR recognizes pathogen-specific molecular motifs and induces responses involving NF-?B and MAPK?including ERK1/2 and JNK?,and participates in cell proliferation,apoptosis,neuronal development,transmitter transport and other physiological activities.Currently,studies on JIP3 mainly focus on the central nervous system,and it is still unclear whether it can affect liver injury and lipid metabolism in NAFLD by regulating the NF-?B and MAPK signaling pathway.ObjectiveThe purpose of this study was to investigate the effect and molecular mechanism of JIP3 on liver injury in NAFLD mice by using JIP3 knockout mice(JIP3^-/-mice)as the research object and establishing a NAFLD model through a high-fat diet.MethodsMale mice(wild type and JIP3^-/-type)aged 6-8 weeks and weighing 18-20g were randomly divided into 4 groups?n=12/group?after 1 week of adaptive feeding:?1?chow diet feeding wild type control group?WT/Con?;?2?chow diet feeding JIP3^-/-group(JIP3^-/-/Con);?3?high fat diet feeding wild type group?WT/HFD?;?4?high fat diet feeding JIP3^-/-group(JIP3^-/-/HFD).The total feeding time was 16 weeks,during which the body weight and blood glucose of the mice were measured weekly.At 12weeks,4 rats in each group were randomly selected for blood lipid determination and HE staining to evaluate whether the NAFLD model was established successfully.IPGTT and IPITT were performed after a 12h fast at the weekend of 16.The next day,mice were placed under anesthesia and body fat was assessed by dual energy X-ray absorptiometry,and the liver tissue was stored in liquid nitrogen.Cultured AML-12 cells were transfected with JIP3siRNA and divided into four groups:?1?normal control group?con?;?2?high-fructose group?HFr?;?3?high-fructose JIP3silence group?siJIP3?;?4?siRNA control group?siNC?.The venous blood was taken for biochemical and hormone detection;the expression of p-NF-?B and p-JNK in liver tissue or cells was detected by Western Blot;IL-1?TNF-?,IL-6,IL-18 and COX2 were detected by quantitative real-time PCR;DCF analysis of ROS production and quantitative measurement of H₂O₂,MDA,XO,SOD and TAC;mouse liver tissue formaldehyde fixation,HE staining to observe morphological changes;immunohistochemistry and immunofluorescence detection of expression and localization of p-NF-?B and p-JNK protein.Results1.JIP3 knockout attenuates metabolic disorders and liver damage in mice on high-fat dietCompared with the normal diet group,the body weight and body fat content of the HFD group were significantly increased,and the fasting glucose and insulin levels were significantly increased.The results of IPGTT and IPITT indicated that the glucose tolerance of the mice was impaired and the insulin resistance was obvious.After JIP3 gene knockout,the body weight and fat content of mice were decreased,and fasting glucose and insulin sensitivity were improved.Liver steatosis,serum ALT,AST and blood lipid levels were significantly increased in HFD group,TC and TG levels were significantly increased in liver tissue,while liver injury and steatosis index were improved in JIP3 knockout mice.2.JIP3 knockout improves oxidative stress and inflammatory response in mice induced by high fat dietCompared with the normal diet group,the expressions of inflammatory cytokines?IL-1?,TNF-?,IL-6,IL-18 and COX2?in serum and liver tissues of mice in the HFD group were up-regulated,the expression levels of oxidative stress injury indicators?ROS,H₂O₂,O₂^-,MDA,XO,iNOS?were increased,and the levels of antioxidant cytokines?SOD,TAC?were decreased.After JIP3 knockout,the level of inflammatory cytokines in mice decreased,the level of antioxidant stress factors increased,and the indicators of oxidative stress injury were down-regulated.3.JIP3 knockout affects the molecular mechanism of lipid metabolism,oxidative stress and inflammatory responseJIP3 knockout down-regulated the expression of lipid-related genes,including SREBP1c,FAS,SCD1,ADFP and ChREBP?.Western blot showed that the expressions of TXNIP,NLRP3,ASC and Caspase-1 in oxidative stress related genes induced by HFD were up-regulated,while the expressions of these genes were decreased in JIP3 knockout mice.Compared with WT/Con mice,the expressions of antioxidant factors HO-1 and Nrf-2 in liver tissues of JIP3^-/-/HFD mice were significantly up-regulated,and the transcriptional activity of TLR4/MyD88/NF-?B signaling pathway was decreased.4.JIP3 down-regulation improves lipid genesis,oxidative stress and inflammation in high fructose-induced AML-12cellsLipidogenesis-related genes?SREBP1c,FAS,SCD1,FDAP and ChREBP-??were highly expressed in HFr and down-regulated in the JIP3 knockout group.The expression of TXNIP,NLRP3,ASC and Caspase-1 was increased in HFr-incubated cells and was significantly reduced in JIP3 knockdown cells.JIP3 silenced cells cultured in HFr significantly down-regulated ROS,H₂O₂,O₂^-,MDA,XO,iNOS,and,on the contrary,SOD and TAC levels were up-regulated.JIP3 silencing significantly reduced HFr-induced expression of TLR4/MyD88/p-NF-?B and p-JNK.ConclusionJIP3 gene knockout can improve liver injury in NAFLD mice,and reduce oxidative stress and inflammatory response,which may be related to the inhibition of TLR4/MyD88/NF-?B and JNK signaling pathways.