Exploration The Regulatory Effect Of AMPK Signal Pathway On Steatosis Of Primary Hepatocytes In Laying Hens Using PCR Array Technique

In order to study the regulatory effect of AMPK signal pathway on steatosis of primary hepatocytes in laying hens,Hyland brown laying hens aged from 34 to 40 weeks were selected to establish in vitro culture model of layer hepatocytes by in situ two-step perfusion.The hepatocytes were treated with oleic acid / palmitic acid(2:1)high fat culture medium of 1m M to construct steatosis model.The cells were divided into two groups: CK group(0m M oleic acid / palmitic acid)and FFA group(1m M oleic acid / palmitic acid).Then the cell proliferation activity of primary hepatocytes in laying hens was detected under ICelligence system,lipid accumulation was observed by oil red O staining and glycogen content was observed by glycogen staining,lipid related indexes(TC,TG),glucose content in supernatant(GLU),redox related indexes(SOD,T-AOC,MDA);were measured by biochemical detection kit,and apoptosis was observed by acridine orange / ethidium bromide staining.The level of reactive oxygen species and the change of mitochondrial membrane potential were measured by flow cytometry,and the m RNA expression of 84 genes in AMPK signal pathway was determined by PCR array.In addition,verification experiments were carried out on key genes and proteins(P-AMPK,ACC,P-ACC,AKT,Caspase-3,INSR).The results are as follows:1.Compared with CK group,FFA group showed a significant decrease in lipid accumulation and glycogen content,and ICelligence system assay showed that the cell proliferation ability of FFA group was lower than that of CK group,but there was no significant difference in cell activity between the two groups(P>0.05).2.Compared with CK group,the content of TG,TC and MDA in FFA group increased significantly or very significantly(P<0.05 or P<0.01),while the content of SOD and T-AOC decreased significantly(P<0.01),and the content of GLU in cell supernatant decreased significantly(P<0.05).3.Compared with CK group,the apoptosis rate and the level of reactive oxygen species and the mitochondrial membrane potential in FFA group increased significantly(P<0.01).4.Compared with CK group,the expression of m RNA of FASN,ACACB,ADRA2 A,ADRA2C,HMGCS1,PFKFB3,PFKFB4,m TOR,TXNIP,FOXO3,TSC1,TSC2 and TP53 in AMPK signal pathway of FFA group was significantly upregulated(P<0.05 or P<0.01),while the expression of m RNA of CRTC1,CPT2,CPT1 A,INSR,AKT1,PPARGC1 B and CAMKK2 was significantly down-regulated(P<0.05 or P<0.01).5.Compared with CK group,the expression of P-AMPK and P-ACC protein in FFA group was significantly higher than that in FFA group(P <0.01).The expression of Caspase-3 protein increased significantly(P <0.05);while the expression of AKT and INSR protein showed a decreasing trend.(P > 0.05).Conclusion: The expression of FASN,ACACB and ADRA2 A genes in AMPK signaling pathway was significantly up-regulated,while the expression of CRTC1,CPT2 and CPT1 A genes was significantly down-regulated under the influence of steatosis in primary hepatocytes of laying hens,resulting in metabolic disorder of lipids and carbohydrates,oxidative damage and changes in cell cycle.