Hepatocytes Derived Increased SAA1 Promotes Intrahepatic Platelet Aggregation And Aggravates Liver Inflammation In NAFLD

Objective:Non-alcoholic fatty liver disease(NAFLD)is the pathological manifestation of metabolic syndrome in liver.The pathology of NAFLD may evolve from the initial simple steatosis to non-alcoholic steatohepatitis,liver fibrosis and even liver cancer.This pathological process includes both the lipotoxic damage caused by excessive lipid deposition to hepatocytes and the inflammatory damage caused by the aggregation and activation of various immune cells.Recently,more and more studies have found that platelets play an important role in liver disease and homeostasis.In particular,anti-platelet therapy can reduce intrahepatic platelet aggregation and improve the inflammation of fatty liver.A previous study has confirmed that SAA is a gene closely related to high-fat diet induced obesity,and SAA1 can promote liver insulin resistance.Obese people are often accompanied by insulin resistance,dyslipidemia and NAFLD.Here,based on the identification of highly expressed SAA1 and large platelet aggregation in fatty liver tissue,we will elucidate the effect of SAA1-mediated TLR2 signaling on platelet aggregation and activation,and explore whether steatosis-induced SAA1 exacerbates liver inflammation by promoting platelet aggregation in NAFLD.Methods:1.NAFLD mouse model was established by feeding high fat diet for 16 weeks.The histopathological changes of the liver were observed by HE staining,and the expression of SAA1 in the liver tissue of normal mice and NAFLD mice was detected by Western blot and Real Time-PCR.2.Oil red O staining was used to observe lipid droplet deposition in normal cultured and Oleic acid-induced L-02 cells.The expression of SAA1 in normal cultured L-02hepatocytes and ole acid-induced steatotic L-02 cells was detected by Western blot and Real-time PCR.3.CD42b expression was detected in normal liver tissue and NAFLD liver tissue by immunohistochemical staining to determine the aggregation of platelets in liver tissue.4.The platelets were preincubated by SAA1(10μg/ml),then stimulated by ADP(2.5μM or 5μM),Thrombin(0.04 U/ml or 0.08U/ml)or Collagen(1μg/ml or 2μg/ml)respectively to determine the platelet aggregation by the aggregator,Western blot was used to detect the expression of p-AKT473,AKT,p-GSK-3β,GSK-3β,p-P38,P38,p-SYK and SYK.5.Platelets were stimulated by SAA1 or Tirofiban,and then incubated on pre-coated collagen slides,while unstimulated platelets were incubated on BSA-coated and collagen coated slides respectively.Forty-five minutes later,platelet adhesion was detected by TRITC-phalloidin staining.6.L-02 cells were transfected with LV-EGFP and LV-sh RNA-SAA1.After oleic acid induction,the conditioned medium was collected to incubate platelets,then these platelets were stimulated with Thrombin for determining the aggregation rate and assessing the expression of P-selectin by flow cytometry,the expression of p-AKT473,AKT,p-GSK-3β,GSK-3β,p-P38,P38,p-SYK and SYK were detected by Western blot.7.Platelets were incubated by SAA1(10μg/ml)only or SAA1(10μg/ml)in combination with TLR2 Block AB(4μg/ml),and then stimulated by ADP(2.5μM),Thrombin(0.04 U/ml)or Collagen(1μg/ml)to determine platelet aggregation by aggregators(A)or to detect the expression of p-AKT473,AKT,p-GSK-3β,GSK-3β,p-P38,P38,p-SYK and SYK by Western blot.8.Liver tissues were collected from normal mice,HFD mice injected with Lv-sh RNA-NC or Lv-sh RNA-SAA1 respectively,and the expression of CD42b,CD3,F4/80 and LY6G was detected by immunohistochemistry C.Flow cytometry was used to analyze the ratio of CD4~+cell and CD8~+cells in liver nonparenchymal cells from normal mice and HFD mice injected with Lv-sh RNA-NC or Lv-sh RNA-SAA1.Results:1.After 16 weeks of high-fat diet,lipid deposition was observed in the liver tissues of NAFLD mice,and the expression of SAA1 was increased.2.Obvious vacuolar lipid droplets were deposited in hepatocytes after oleic acid induction,and the expression of SAA1 was increased after in steatotic hepatocytes.3.A small amount of scattered distribution of platelets in liver sinus was observed in normal liver tissues,and,a large number of platelets were obviously gathered in liver sinusoids of NAFLD mice.4.SAA1 could enhance platelet aggregation when stimulating with low concentration of platelet agonists(ADP,Thrombin or Collagen).when stimulated with high doses of platelet agonists(ADP or Collagen),although SAA1 also showed an enhanced platelet aggregation effect,there was no statistical difference.5.There were only a small amount of platelets adhering on the BSA-coated cell slides,and the untreated platelets showed normal adhesion and expansion on the collagen-coated cell slides.The increased number and volume of SAA1 incubated platelets adhering on the collagen-coated cell slides were also be observed.Tirofiban incubation leaded to less platelets adhering to the collagen-coated cell slides and the volume of platelets is also smaller.6.Thrombin stimulated platelets expressed more p-AKT473,p-GSK-3β,p-p38,and p-SYK than the unstimulated platelets,and a further increase in these molecules were induced when stimulated with SAA1 in combination with thrombin.7.The CM from Lv-sh RNA-SAA1 transfected L-02 cells induced by oleic acid did not effectively enhance platelet aggregation.Similarly,p-selectin and platelet activation-related molecules(p-AKT473,p-GSK-3β,p-p38 and p-SYK)expression were significantly reduced.8.The aggregation rate of platelets incubated with SAA1 was significantly increased,and the aggregation degree of platelets was significantly reduced when stimulated with SAA1 in combination with TLR2 signal blocking antibody(TLR2 Block AB).The expression of platelet activation-related molecules(p-AKT473,p-GSK-3β,p-p38and p-SYK)decreased when TLR2 signaling was inhibited.9.The inhibition of SAA1 expression in vivo resulted into the decrease of intrahepatic platelet aggregation and the infiltration of inflammatory cells(CD3~+cells,F4/80~+cells and LY6G~+cells)in liver tissue of NAFLD mice.In addition,the proportion of CD8~+cells in non-parenchymal cells in liver was also significantly reduced.Conclusions:Our findings identify that high-fat diet-induced hepatic highly expressed SAA1aggravates fatty liver inflammation by promoting intrahepatic platelet aggregation via TLR2 signaling,these data also suggest that SAA1 may serve as a potential target for ameliorating NAFLD.