Study Of EGCG On The Regulation Of Lipid Metabolism In Canine Hepatocytes

Epigallocatechin-3-gallate(EGCG)is the main component of biological activity in tea and tea polyphenols,which function as antioxidant,cancer prevention,anti-inflammatory and bacteria,free radical scavenging,hypolipidemic,blood pressure lowering and blood sugar lowering,prevention of cardiovascular and cerebrovascular diseases and other biological activities.Hyperlipidemia is a common nutrient metabolic disease with abnormally elevated blood lipids in pet dogs,which poses a great threat to the health of pet dogs.In this experiment,canine hepatocytes were cultured in vitro and different concentrations of EGCG were added to the canine hepatocytes to study their effects on the related protein and gene expression in the signal transduction pathway of AMPK.It was initially stated that EGCG reduced the lipogenesis of canine hepatocytes.In this experiment,healthy canine were selected and primary canine hepatocytes were isolated and cultured by two-steps perfusion method.Different concentrations of EGCG were added.After a period of time,the optical concentration and reaction time of EGCG were determined.After primary canine hepatocytes were cultured 48 hours,different concentrations of EGCG primary liquor and AMPK? inhibitors BML-275(0,0.01,0.1 and 1.0 ?M EGCG,10 ?M BML-275,0.1 ?M EGCG+10 ?M BML-275)were co-cultured with hepatocytes.Morphological changes of canine hepatocytes were observed using inverted microscope and projection electron microscope.The gene expression levels of PPAR?,SREBP-1c,ACO,CPTI,FAS,ApoE,ApoA1 and ACC? were detected real-time fluorescence quantitative PCR(qRT-PCR).The protein expression levels of AMPK?,p-AMPK?,PPAR?,SREBP-1c,ACO,CPTI,FAS,ApoE,ApoA1,ACC? and p-ACC? were detected by western blotting(WB).Nucleoplasm distribution of PPAR? and SREBP-1c were conducted by Cell immunofluorescence.The effect of EGCG on the expression of PPAR? and SREBP-1c in the nucleus was detected by electrophoretic mobility shift assay(EMSA).The activity of AMPK?,ACC? and CPTI and the content of AMP and ATP were determined by enzyme-linked immuno sorbent assay(ELISA).The contents of TG and VLDL were detected by automatic biochemical analyzer.The results show:1.Changes in cell morphology,cell viability and cell membrane integrity: After 4hours,hepatocytes adhered to the wall and the edges of the cells were spherical orpolygonal.After 48 hours of culture,the cells adhered firmly and grew.Pseudopodia,polygonal,abundant cytoplasmic contents,closely packed cells,clear intercellular spaces,good cell growth state were reflected in microscopic view.LDH leakage rate between 0-48 hours gradually decreased and the lowest level is at 48 hours,which indicate that the integrity of the cytomembrane and the growth situation of hepatocytes were best.48 hours was selected as the optimal time for cells to start adding stimulatory factors.2.The best concentration and reaction time of EGCG are 0.1 ?M,30 min.After adding the same concentration of 0.1 ?M EGCG,the expression of p-AMPK protein at the 10 min and the 30 min point was significantly higher than that in the control group(P <0.01),and at the 30 min was the highest phosphorylation level of AMPK?.3.Expression levels of genes mRNA in AMPK? signaling pathway: Compared with the control group,the mRNA expression of PPAR?,ACO and CPTI increased with the increase of EGCG concentrations(P <0.01),and significantly decreased in the BML-275 inhibitor group(P <0.05 or P <0.01).Compared with 0.1 ?M EGCG group,the mRNA expression of PPAR?,ACO and CPTI in EGCG and BML-275 group significantly reduced(P <0.01).Compared with the control group,the mRNA expression levels of SREBP-1c,FAS,ApoE,ApoA1 and ACC? decreased with the increase of EGCG concentration(P <0.01),and the BML-275 inhibitor group significantly increased(P <0.05 or P <0.01).Compared with 0.1 ?M EGCG,the mRNA expression levels of SREBP-1c,FAS,ApoE,ApoA1 and ACC? significantly increased in the EGCG and BML-275 group(P <0.01).4.The expression levels of related protein in AMPK? signaling pathway:Compared with the control group,the expression levels of p-ACC?,p-AMPK?,PPAR?,ACO and CPTI increased with the increase of EGCG concentrations(P <0.05 or P <0.01).Compared with the 0.1 ?M EGCG group,the expression levels of p-ACC?,p-AMPK?,PPAR?,ACO and CPTI BML-275 in inhibitor group was significantly lower(P <0.05 or P <0.01),and significantly decreased in the 0.1 ?M EGCG and BML-275 group(P <0.05 or P <0.01).Compared with the control group,the protein expression of SREBP-1c,FAS,ApoE,ApoA1 and ACC? decreased with the increase of EGCG concentrations(P <0.05 or P <0.01),and increased significantly in the BML-275 inhibitor group(P <0.05 or P <0.01).Compared with0.1 ?M EGCG group,the protein expression of SREBP-1c,FAS,ApoE,ApoA1 and ACC? significantly increased in the EGCG and BML-275 group(P <0.05 or P<0.01).5.Immunofluorescence results of PPAR? and SREBP-1c were observed by laser confocal microscopy: Compared with the control group,nuclear expression level of PPAR? in the 0.1 ?M EGCG group was significantly increased,and the nuclear expression level was also significantly higher in BML-275 inhibitor and EGCG and BML-275 groups than that in the control group.Compared with the control group,the nuclear expression level of SREBP-1c in 0.1 ?M EGCG group was significantly reduced.The nuclear expression level of SREBP-1c in BML-275 inhibitor group and EGCG and BML-275 group were significantly increased.6.EMSA results showed that the transcriptional activity of PPAR? increased with the increase of EGCG concentrations,and significantly decreased in the BML-275 inhibitor group(P <0.05 or P <0.01).Compared with the control group,the transcriptional activity of SREBP-1c decreased with the increase of EGCG concentrations(P <0.05 or P <0.01),and increased significantly in the BML-275 inhibitor group(P <0.01).Compared with the 0.1 ?M EGCG group,the transcriptional activity of PPAR? significantly decreased in the EGCG and BML-275groups(P <0.05).7.Compared with the control group,enzyme activities of AMPK? and CPTI increased with the increase of EGCG concentrations(P <0.01),decreased significantly in BML-275 inhibitor group(P <0.01).Compared with 0.1 ?M EGCG,enzyme activities of AMPK? and CPTI significantly decreased in EGCG and BML-275 group(P <0.01).Enzyme activity of ACC? decreased with the increase of EGCG concentration(P <0.01)and significantly increased in the BML-275 inhibitor group was(P <0.01).Compared with the 0.1 ?M EGCG group,enzyme activity of ACC? in the EGCG and BML-275 group significantly increased(P <0.05).8.Compared with the control group,the content of TG and VLDL decreased with the increase of EGCG concentration(P <0.05 or P <0.01).In summary,EGCG activate the AMPK signaling pathway in canine hepatocytes,accelerate the lipid oxidation,reduce the synthesis of fatty acids and the content of TG and VLDL in canine hepatocytes,thereby reduce the accumulation of hepatic lipids.These results are expected to lay a theoretical and experimental basis for the development of lipid-lowering products for canine via clarifying the lipid metabolism regulation mechanism of EGCG on canine hepatocytes.