LOX-1 Down-Regulation Relieves Liver Damage Of The Rat With Diabetes By The Means Of AMPK/FoxO1 Signal

Objective: The siRNA was used to knockdown LOX-1 in liver of db/db mice to observe the effect of downregulation of LOX-1 on AMPK/ Fox O1 signaling pathway and FNDC5 in liver tissue of db/db mice,and to explore the effect and possible mechanism of LOX-1 on liver injury of diabetic mice.Methods: Thirty SPF grade male db/db mice aged 7-8 weeks were divided into db/db LOX-1 knockdown group(AAV-shLOX-1),db/db empty vector group(AAV-sh CON)and db/db blank control group(db/db)according to random number table method,with 10 mice in each group.Eight homologous wild-type male db/m mice of the same week age were normal control group(NC).Construct adeno-associated virus 9 LOX-1 knockout vector(AAV9-LOX-1).Mice in AAV-shLOX-1 group and AAV-sh CON group were transfected with AAV9 through tail vein,mice in db/db group were injected with equal volume of normal saline through tail vein,and mice in NC group were not treated.Fasting blood glucose(FBG)and body weight of mice were measured at the same time every week.After 14 weeks,the mice were sacrificed and serum and liver were collected.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of mouse liver.The transfection of AAV9-LOX-1 in mouse liver was observed under fluorescence microscope.The m RNA expression levels of LOX-1,AMPK,Fox O1,IL-6 and FNDC5 in mouse liver were detected by QRT-PCR.The protein expression levels of LOX-1,AMPK,Fox O1,IL-6,FNDC5,p-AMPK and p-Fox O1 in mouse liver were detected by Western blotting.The contents of ALT,TG and FNDC5 in serum of mice were determined by enzyme-linked immunosorbent assay(ELISA).Results:1.Compared with the NC group,db/db mice were generally in a poor state,showing dull response,lethargy and little movement,dry hair color,obvious symptoms of polydipsia,polydipsia and polyuria,and obesity.Subcutaneous fat and abdominal fat increased significantly,and the liver was milky yellow,enlarged,and round with blunt edges.The body weight,liver index and FBG of db/db mice were significantly increased(P<0.05).2.HE staining showed that compared with NC group,the structure of liver lobules in AAV-shLOX-1 group,AAV-sh CON group and db/db group were disorganized,and a large number of vacuole-like lipid droplets appeared in hepatocytes,showing obvious steatodegeneration.After LOX-1 knockdown,the number of lipid droplets in hepatocytes of AAV-shLOX-1 group was significantly reduced,and steatosis was significantly alleviated.3.Fluorescence microscopy showed that the expression of green fluorescent protein(GFP)could be observed in the liver of mice in AAV-shLOX-1 group and AAV-sh CON group,but no GFP was observed in the liver of mice in db/db group and NC group.4.Compared with NC group,the m RNA and protein expression levels of LOX-1 in liver of db/db mice were increased(P<0.05).After transfection with AAV9,the expression levels of LOX-1 m RNA and protein in liver of AAV-shLOX-1 group were decreased(P<0.05).Compared with AAV-sh CON group and db/db group,LOX-1 m RNA was decreased by 40.1%and 44.4%.LOX-1 protein decreased by 16.0% and 18.7%(P<0.05).5.Compared with NC group,the m RNA and protein expression levels of AMPK and FNDC5 in liver of AAV-shLOX-1,AAV-sh CON and db/db groups were decreased(P<0.05),while the m RNA and protein expression levels of Fox O1 and IL-6 were increased(P<0.05).The expression levels of P-AMPK and P-Fox O1 protein were decreased(P <0.05),and the changes of above indexes could be reversed after LOX-1 knockdown(P<0.05).6.Compared with NC group,the serum ALT and TG of db/db mice were increased and the serum FNDC5 was decreased(P<0.05).LOX-1 knockdown could decrease serum ALT and TG levels and increase FNDC5 levels in AAV-shLOX-1 group(P<0.05).7.Pearson correlation analysis of FNDC5 in liver and serum of mice in each group showed that FNDC5 in liver was negatively correlated with LOX-1,IL-6,TG,ALT,liver index,FPG and body weight,with statistical significance(P<0.05).Serum FNDC5 was negatively correlated with LOX-1,IL-6,TG,ALT,liver index,FBG and body weight,with statistical significance(P<0.05).Conclusions: In diabetic condition,the downregulation of LOX-1 can reduce the steatosis and inflammation in the liver of db/db mice,improve the blood lipids and liver function of db/db mice,and thus alleviate the progression of liver injury in diabetic mice.The mechanism may be related to the interference in the signal transduction of AMPK/Fox O1 signaling pathway by regulating the protein expression of related signal molecules.