| Purpose:In this study,we investigated the possible molecular biological mechanism of action of Xiang Sha Liu Jun Zi Tang in correcting dyslipidemia through AMPK/Insig-1/SREBP2 signaling pathway using spleen-deficient hyperlipidemic rats and human hepatocellular carcinoma Hep G2 cells as the entry point for cholesterol synthesis.Material and method:Part Ⅰ: Effect of Xiang Sha Liu Jun Zi Tang on cholesterol synthesis via AMPK/Insig-1/SREBP2 pathway in rats with spleen deficiency hyperlipidemia and the mechanism1.60 SPF-grade SD rats were randomly divided into 4 groups,namely,the blank control group,the high-fat group,the spleen-deficiency hyperlipidemia group and the Xiang Sha Liu Jun Zi Tang group(11.34 g-kg-1-d-1).After 15 days,except for the blank control group,the other three groups were fed high-fat chow for 14 weeks with free intake and unlimited water intake to replicate the hyperlipidemia model.Gavage was administered at the 10 th week,and the conversion factor of body surface area between human and animals was used.The Xiang Sha Liu Jun Zi Tang group was given Xiang Sha Liu Jun Zi Tang 11.34 g-kg-1-d-1 by gavage,and the remaining three groups were given an equal volume of saline.4 weeks later,the aortic blood and liver of rats were taken for the experiment.2.The serum total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LLDL-C)were measured by automatic biochemical analyzer.Low density lipoprotein cholesterol(LDL-C)content.3.HE staining technique was used to observe the morphological and pathological changes of hepatocytes,and oil red O method was used to observe the liver lipid deposition.4.Realtime RT-PCR was used to detect the levels of ubiquitin protein ligase(gp78),insulin-inducible gene 1(Insig-1),sterol regulatory element binding protein 2(SREBP2),3-hydroxy-3-methylmicrocoenzyme coenzyme A reductase(HMGCR)mRNA.western blot was used to detect the levels of phosphorylated adenylate-activated protein kinase(p-AMPK),AMPK,gp78,Insig-1,SREBP2,HMGCR protein levels.Part Ⅱ: Effect of Xiang Sha Liu Jun Zi Tang on free fatty acid-induced cholesterol synthesis in human hepatocellular carcinoma Hep G2 cells via AMPK/Insig-1/SREBP2 pathway and the mechanism1.CCK-8 assay to screen the optimal administration concentration of Xiang Sha Liu Jun Zi Tang-containing serum.2.Hep G2 cells were treated with 1mM of free fatty acid mixture.Oil red O staining was performed to observe the lipid deposition in the cells of blank control group(Control group),free fatty acid intervention group(FFA group),blank serum intervention group(KB group),Xiang Sha Liu Jun Zi Tang drug-containing serum intervention group(XS group)and AMPK enzyme inhibitor Dorsomorphin intervention group(Dor group);TC,TG and FFA contents in each group were detected.3.Realtime RT-PCR was used to detect the levels of gp78,Insig-1,SREBP2 and HMGCR mRNA in Control,FFA,XS and Dor groups.western blot was used to detect the levels of p-AMPK,AMPK,gp78,SREBP2 and HMGCR in Control,FFA,XS and Dor groups,Insig-1,SREBP2,HMGCR protein levels.Results:Part Ⅰ1.Effects of Xiang Sha Liu Jun Zi Tang on blood lipid levels in rats with spleen deficiency hyperlipidemiaCompared with the blank control group,serum TC,TG and LDL-C levels were significantly increased and HDL-C levels were significantly decreased in the high-fat and spleen-deficiency hyperlipidemic rats(P<0.05,P<0.01);compared with the high-fat group,serum LDL-C levels were increased and HDL-C levels were decreased in the spleen-deficiency hyperlipidemic rats(P<0.05,P<0.01);compared with the spleen-deficiency hyperlipidemic group,serum TC,TG and LDL-C levels were significantly decreased and HDL-C levels were significantly increased in the Xiang Sha Liu Jun Zi Tang group(P<0.05,P<0.01).Compared with the spleen deficiency and high fat group,TC,TG and LDL-C were significantly lower and HDL-C was significantly higher in the Xiang Sha Liu Jun Zi Tang group(P<0.01).2.Effects of Xiang Sha Liu Jun Zi Tang on liver histopathology of rats with spleen deficiency hyperlipidemiaHE staining showed that the nuclei of the liver in the blank control group were rounded in the middle,the sinusoids were normal,and the cell cords were clearly visible.The foaminess of liver tissue was significantly reduced,the swelling of hepatocytes was improved,the nuclei tended to be normalized,and neatly arranged hepatocytes were visible.The oil red O method observed that the hepatocytes in the blank control group rats were regularly arranged,without orange-red lipid droplets,with blue nuclei and normal morphological structure of liver sinusoids;compared with the blank control group,the hepatocytes in the high-fat group were significantly enlarged,with diffuse orange-red lipid droplets visible in the visual field and nuclei of different sizes and significantly increased lipid accumulation;compared with the high-fat group,the liver tissue in the spleen deficiency high-fat group was obviously edematous,and the lipid droplet content The treatment with Xiang Sha Liu Jun Zi Tang can significantly reduce the orange-red lipid droplets in hepatocytes and improve the structure of liver sinusoids and nuclei,and significantly improve the lipid deposition in liver tissue.3.Effects of Xiang Sha Liu Jun Zi Tang on mRNA expression of liver gp78,Insig-1,SREBP2 and HMGCR genesCompared with the blank control group,the liver Insig-1 mRNA expression was decreased(P<0.05)and gp78,SREBP2,HMGCR mRNA expression was increased(P<0.01)in rats in the high-fat group and the spleen-deficiency high-fat group;compared with the spleen-deficiency high-fat group,the Insig-1 mRNA expression was increased(P<0.01)in rats in the Xiang Sha Liu Jun Zi Tang group,and gp78,SREBP2,HMGCR mRNA expression was decreased(P < 0.01);there was no statistical significance between the high-fat group and the spleen-deficient high-fat group.4.Effects of Xiang Sha Liu Jun Zi Tang on liver p-AMPK,AMPK,gp78,Insig-1,SREBP2,HMGCR protein expressionCompared with the blank control group,the liver p-AMPK/AMPK and Insig-1 protein expressions were decreased in the high-fat group and the spleen-deficient high-fat group(P <0.01);gp78,SREBP2 and HMGCR protein expressions were increased(P < 0.01,P < 0.05);compared with the high-fat group,the spleen-deficient high-fat group p-AMPK/AMPK and Insig-1 protein expressions were decreased and SREBP2 protein expression increased in the spleen-deficient high-fat group(P < 0.01,P < 0.05);compared with the spleen-deficient high-fat group,p-AMPK/AMPK and Insig-1 protein expression increased in the Xiang Sha Liu Jun Zi Tang group rats(P < 0.01,P < 0.05);gp78,SREBP2 and HMGCR protein expression decreased(P < 0.01,P < 0.05).Part Ⅱ1.CCK8 method for screening the optimal administration concentration of Xiang Sha Liu Jun Zi Tang-containing serumThe effect of Xiang Sha Liu Jun Zi Tang-containing serum on cell viability after 24 h on Hep G2 cells was dose-dependent,and 10% Xiang Sha Liu Jun Zi Tang-containing serum had less effect on cell viability.2.The oil red O staining and TC,TG and FFA contents of the cells in Control,FFA,KB,XS and Dor groupsCompared with the Control group,the number of orange-red lipid droplets in the cytoplasm of cells in the FFA and KB groups was significantly increased,and the contents of TC,TG and FFA in the cells were higher(P < 0.01);between the FFA and KB groups,there was no significant difference in the field of view,and the contents of TC,TG and FFA were not statistically significant.Compared with the FFA group,the number of lipid droplets in the XS group was smaller and the contents of TC,TG and FFA were lower(P < 0.01);compared with the XS group,the number of orange-red lipid droplets in the cytoplasm of cells in the Dor group was significantly higher and the contents of TC,TG and FFA in the cells were higher(P < 0.05 or P < 0.01).3.Effects of mRNA expression of gp78,Insig-1,SREBP2 and HMGCR genes in cells of Control group,FFA group,XS group and Dor groupCompared with the Control group,the expression levels of gp78,SREBP2,and HMGCR mRNA were increased in the FFA group(P < 0.01,P < 0.05),and the expression levels of Insig-1 mRNA were all decreased(P < 0.01).Compared with the FFA group,the mRNA expression levels of gp78,SREBP2,and HMGCR were decreased(P < 0.01,P < 0.05)and Insig-1 mRNA expression levels were increased(P < 0.01)in the XS group.After the addition of AMPK enzyme inhibitor Dorsomorphin,the expression levels of gp78,SREBP2,HMGCR mRNA were increased(P < 0.01,P < 0.05)and Insig-1 mRNA expression levels were decreased(P < 0.05)in the Dor group compared with the XS group.4.Effects of Xiang Sha Liu Jun Zi Tang-containing serum on the expression of p-AMPK,AMPK,gp78,Insig-1,SREBP2,HMGCR proteins in cellsCompared with the Control group,gp78,SREBP2,HMGCR protein expression was increased in the FFA group(P < 0.01)and p-AMPK/AMPK,Insig-1 protein expression was decreased in the FFA group(P < 0.01).Compared to the FFA group,gp78,SREBP2,HMGCR protein expression was decreased(P < 0.01,P < 0.05)and p-AMPK/AMPK,Insig-1 protein expression was increased(P < 0.01)in the XS group.After adding the AMPK enzyme inhibitor Dorsomorphin,gp78,SREBP2,HMGCR protein expression was increased(P < 0.01,P < 0.05)and p-AMPK/AMPK,Insig-1 protein expression was decreased(P < 0.01)in the Dor group compared to the XS group.Conclusion:1.Xiang Sha Liu Jun Zi Tang can effectively correct the dyslipidemia and lipid deposition in spleen-deficient hyperlipidemic rats and free fatty acid-induced human hepatocellular carcinoma Hep G2 cells.2.Xiang Sha Liu Jun Zi Tang interferes with cholesterol synthesis and corrects dyslipidemia by regulating the expression of related genes and proteins in the AMPK/Insig-1/SREBP2 pathway. |
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