Based On PCR Array Technology To Explore The Mechanism Of Huayu Qutan Fang Regulating Autophagy And Interfering With Liver Lipid Deposition

Objective:To establish a hyperlipidemia rat model and use autophagy as an entry point to explore the effect and mechanism of Huayu Qutan Recipe on liver lipid damage in hyperlipidemia rats.Materials and Methods:Thirty SD rats were randomly divided into the control group,the hyperlipidemia group and the Huayu Qutan group,with 10 rats in each group.The control group was fed with the normal diet,and the hyperlipidemia group and the Huayu Qutan group were given the high-fat diet,free to drink water,and molded for 60 days.Then,the Huatan Qutan group was given the Huayu Qutan Recipe 10 ml/(kg·d),once/day,for 30 days.The control group and the hyperlipidemia group were given an equal volume of phosphate buffered saline(PBS)at the same time every day.After 90 days fed,the biochemical analyzer detected serum cholesterol,triglyceride,low density lipoprotein cholesterol,and high density lipoprotein cholesterol.HE staining was used to observe the morphological changes of liver tissue.Oil red O staining was used to observe liver lipid deposition.The liver tissue was isolated and the expression of m TOR signaling pathway-related gene was detected by PCR array.RT-PCR was used to verify the expression of autophagy-related protein in rat liver tissue by Western blot.Results:1.Effect of Huayu Qutan Recipe on Blood Lipid Level in Rats with Hyperlipidemia.The results showed that compared with the control group,the serum levels of TC and LDL-C in the hyperlipidemia group increased significantly,and the level of HDL-C decreased significantly(P<0.01).The level of TG increased,but it was not statistically significant.Compared with the hyperlipidemia group,the serum levels of TC and LDL-C in the Huayu Qutan group were significantly decreased(P<0.01),and the HDL-C level was significantly increased(P<0.05).The level of TG decreased,but it was not statistically significant.2.Effect of Huayu Qutan Recipe on Morphological Changes and Lipid Damage of Liver Tissue in Rats with Hyperlipidemia HE staining showed that the hepatic cell line of the control group was normal;the morphology of hepatocytes was normal;the nucleus was round,large and centered.There was no lipid accumulation in the cytoplasm and no inflammatory cells infiltrated in the lobule.In the hyperlipidemia group,the liver fatty degeneration was obvious;the hepatocytes were swollen and round;the cytoplasm was loose and there was a large fat droplet.Some cells showed that the nucleus was squeezed into the cell membrane,and round fat vacuoles and inflammatory cells of different sizes were observed in the cytoplasm.In the Huayu Qutan group,the degree of hepatic steatosis was reduced;the number of lipid vacuoles was reduced and the volume of hepatocytes was decreased.Oil red O staining showed that the liver cells of the hyperlipidemia group showed oil red coloration and increased lipid droplets,which was diffuse;the number of lipid droplets in the liver cells of the Huayu Qutan group was significantly reduced.3.Effect of Huayu Qutan Recipe on expression of m TOR signaling pathway-related genes in liver of hyperlipidemic rats.There were 7 genes that changed simultaneously between the Huayu Qutan group and the hyperlipidemia group and the hyperlipidemia group and the control group.According to the fold change value greater than 2 for the up-regulated gene,less than 0.5 for the down-regulated gene,the differential genes are Akt1,Bcl2,Igf1,Mapk14,Mapk8,m TOR,Becn1.The results showed that compared with the control group,the expression levels of Aktl,Bcl2,Igf1,Mapk14 and Mapk8 genes in the hyperlipidemia group increased,and the expression levels of m TOR and Becn1 genes decreased.Compared with the hyperlipidemia group,the expression levels of Aktl,Bcl2,Igf1,Mapk14 and Mapk8 genes in Huayu Qutan group were down-regulated,and the expression levels of m TOR and Becn1 genes were up-regulated.4.RT-PCR method was used to verify differential gene expression in liver of each group.The above experimental verified for autophagy and m TOR related key genes,including Akt,Bcl2,m TOR,Beclin1(Becn1).The results showed that compared with the blank control group,Akt and Bcl2 m RNA levels were significantly increased in the hyperlipidemia group,and m TOR and Beclin1 m RNA levels were significantly down-regulated(P<0.01,P<0.05).Compared with the hyperlipidemia group,the Akt and Bcl2 m RNA levels in the Huayu Qutan Recipe group were significantly down-regulated,and the m TOR and Beclin1 m RNA levels were significantly up-regulated(P<0.01,P<0.05).5.Effect of Huayu Qutan Recipe on the expression of key proteins in autophagy induced by AMPK signaling pathway in liver of hyperlipidemia rats.Western blot was used to detect the expression levels of LC3A/B,p-m TOR,m TOR,p-AMPK,AMPK and ULK1 in each group.The results showed that compared with the blank control group,the expression levels of LC3A/B,p-AMPK and ULK1 in the hyperlipidemia group were significantly decreased(P<0.05,P<0.01),and the expression level of p-m TOR protein was significantly increased(P<0.01).Compared with the high-fat group,the expression levels of LC3A/B,p-AMPK and ULK1 in the Huayu Qutan Recipe group were significantly increased(P<0.05),and the expression level of p-m TOR protein was significantly decreased(P<0.01).Conclusion:1.Huayu Qutan Recipe can correct dyslipidemia in hyperlipidemia rats and improve liver lipid damage in hyperlipidemia rats.2.Huayu Qutan Recipe may improve liver lipid damage in rats with hyperlipidemia by regulating autophagy-related genes and regulating AMPK signaling pathway and affecting autophagy.