The Effect Of STING Signal Pathway On Liver Injury And Hepatic Glucose And Lipid Metabolism And Its Mechanism Study

BackgroundObesity has become a serious public health problem in the world and its incidence has increased year by year.Nonalcoholic fatty liver disease(NAFLD)is a common result of obesity and metabolic syndrome.Hepatocyte injury and metabolic disorders are hallmarks of NAFLD,yet the pathogenesis of NAFLD is complex including immune-related reactions,oxidative stress,lipid peroxidation damage,abnormal fat metabolism and hormone secretion,environmental and genetic factors,etc.These mechanisms are not well understood,and effective measures for preventing and treating NAFLD are lacking.In recent years,more and more research has proved that abnormal immune activation plays an important role in the pathogenesis of NAFLD.Stimulator of interferon genes(STING)is an endoplasmic reticulum protein,as an important signal molecule in immune and inflammatory reactions.It is considered to be an important molecule in the host immune response during pathogen infection.STING is able to identify the DNA released into the host cytoplasm by microbes,and recruits TBK1(Tank-binding kinase 1)and a transcription factor IRF3(interferon regulatory factor)to form a compound,then translocates to the nucleus.In this process,IRF3 is phosphorylated by TBK1,forming a dimer and entering the nucleus.IRF3 can combine with the promoter of the target gene(IFN a,IFN b,NF-?B,ICAM-1,etc.),then promote the expression of inflammatory cytokines and lead to inflammatory response.Meanwhile,phosphorylated IRF3 can be associated with mitochondrial Bcl-2 related X protein(Bax)and activate the mitochondrial apoptotic pathway that depends on caspase 3 and 9 and causes mitochondria-associated apoptosis.It is worth noting that STING,in addition to identifying exogenous DNA in the cytosol,can cause certain autoimmune diseases by identifying its own DNA.STING and IRF3 have recently been shown to play an important role in early alcoholic liver disease and liver fibrosis with the study of STING.There are many similarities in the pathological processes of NAFLD and alcoholic liver disease.However,little is known about the role of STING signal pathway in the development of NAFLD.ObjectsWe aimed to explore that the effect of STING signal pathway on hepatocyte inflammation,apoptosis,and glucose and lipid metabolism.Methods1.Mouse model:C57BL/6 mice were randomly divided into two groups.Some were fed normal chow diet(NCD),whereas others were fed high-fat diet(HFD).After 12 weeks,some livers were snap-frozen in liquid nitrogen at-80? for RT Q-PCR and western blot analysis.Other parts of the liver pieces fixed in 4%paraformaldehyde were embedded in paraffin.The slides were used for morphological observation and immunohistochemical(IHC)staining to detect the expression of STING.2.Cell model:We used a free fatty acids(FFA)-induced hepatocyte steatosis with L-02 human liver cells which were processed by oil red O staining to assess lipid content for subsequent experiments.3.Cell transfection:To down-regulated the expression of STING and IRF3,L-02 cells were transfected with small interfering RNA(siRNA)using the Lipofectamine 2000 Transfection Reagent for 6h.4.western blot:Liver tissues and L-02 cells were homogenized in RIPA buffer.Protein samples were separated with 10%SDS-polyacrylamide gells,and then proteins were transferred to nitrocellulose paper.The membranes were blocked and after incubation with appropriate primary antibodies overnight at 4 C.After multiple washes,secondary antibodies were incubated for 1 h at room temperature and proteins were visualized using enhanced chemiluminescence.5.Quantitative real-time PCR(qRT-PCR):Total RNA from liver tissues and L-02 cells was extracted with TRIzol reagent and then reverse-transcribed.qRT-PCR was conducted with the SYBR Green PCR kit to detect the expression of STING.6.Immunohistochemical(IHC):Paraffin sections were dewaxed,washed and blocked with protein-blocking solution and then incubated with primary and secondary antibodies,and DAB as the chromogen before being examined under an optical microscope.7.Statistical Analysis:Data were analyzed using Student's t-test or one-way ANOVA,and p<0.05 was taken to indicate a statistically significant difference.Results1.The STING signal pathway was activated in livers of obese mice and in FFA-induced L-O2 cells.(1)IHC staining showed that the levels of STING were up-regulated in livers of HFD-fed mice.(2)qRT-PCR and western blot also revealed that the expression of STING and its downstream factor IRF3 were up-regulated in livers of HFD-fed mice and in FFA-induced L-02 cells.2.The STING signal pathway was involved hepatic inflammation and apoptosis in NAFLD.(1)Hoechst 33342 staining showed that knocking-down either STING or IRF3 led to a significant reduction of hepatic apoptosis.(2)Western blot revealed that the levels of the nuclear factor ?B(NF-?B)signaling pathway,inflammatory cytokines(TNF-alpha?IL-1? and IL-6)and apoptotic signaling(Bax/Bcl-2?Cleaved/total caspase-3 and Cleaved-PARP/PARP)were significantly down-regulated with knocking-down either STING or IRF3.3.The STING signal pathway was involved hepatic glucose and lipid metabolic disorders in NAFLD.(1)Glycogen PAS staining and Oil red O staining showed that knocking-down either STING or IRF3 led to increase PAS positive staining and decrease Oil red O positive staining.(2)Western blot revealed that the levels of insulin signaling(p-AKT/t-AKT)and glycolysis enzymes(GCK,PFK,and PK)increased.The levels of expression of GSK3? and hepatic gluconeogenesis enzymes(G-6-Pase,PEPCK,and PC)were lower.Meanwhile,SREBP-1c decreased,while PPARa levels were higher with knocking-down either STING or IRF3.In summary,STING/IRF3 knockdown enhanced glycogen storage and alleviated lipid accumulation,which were associated with increased hepatic enzymes in glycolysis and lipid catabolism,and attenuated hepatic enzymes in gluconeogenesis and lipid synthesis.ConclusionsOur study is the first time to suggest the expression of STING up-regulated in livers of obese mice and in FFA-induced L-02 cells.Meanwhile,our study also suggests that the STING signal pathway promotes hepatocyte injury and dysfunction by inducing inflammation and apoptosis and by disturbing glucose and lipid metabolism.This pathway may be a novel therapeutic target for preventing NAFLD development and progression.