| Object:Through observing the effects of Chinese herbal compound Guanxinkang（Gxk）and its decomposed formulas on PERK-eIF2αsignaling pathway in LDLR-/-atherosclerotic model mice,to explore the regulation mechanism of Guanxinkang and its decomposed formulas on hepatic cholesterol homeostasis and aortic cholesterol deposition.The dissection of Gxk was studied to deepen the understanding of TCM theory of AS treatment.Methods:60 LDLR-/-mice were induced by high-fat diet for 12 weeks,10 C57BL/6J mice with genetic background were set as normal group.The hyperlipidemia model mice were randomly divided into six group,and then be intervened by corresponding drug based on the groups.Each group was sacrificed after having been intervened for 12 weeks.Serum blood lipids were detected by biochemical analyzer;liver and aorta pathological changes were observed by HE staining;the expression levels of protein and gene of PERK,eIF2α,SREBP-2,ABCA1,ATF4 and Bcl-2 were determined by real-time fluorescence quantitative PCR and Western-blot.Results:1.Blood lipid resultsCompared with the model group,the expression levels of serum TG and TC in the Gxk group were significantly decreased,and the expression of HDL-C was significantly increased（p<0.05）,while the expression of LDL-C was not statistically different（p>0.05）.The expressions of serum TG,TC and LDL-C in the simvastatin group were significantly decreased,and the expression of HDL-C was significantly increased（p<0.05）.The expression of serum TC and LDL-C in the CF1 group was significantly decreased（p<0.05）,but there was no significant difference in the expression of TG and HDL-C（p>0.05）.The serum TG expression of the CF2 group was significantly increased,HDL-C expression was significantly increased（p<0.05）,while the expression of TC and LDL-C was not statistically different（p>0.05）.The expressions of serum TG and TC in the CF3 group were significantly decreased（p<0.05）,and the expressions of HDL-C and LDL-C were not statistically different（p>0.05）.2.HE staining resultsCompared with the model group,the liver tissue structure of the Gxk group was lightly destroyed,and some of the lipid droplets were visible in the visual field.The range was small and the hepatic cords were arranged in an orderly manner.The liver tissue damage of CF1 group of mice was lighter,and it was close to the Guanxinkang group.The CF2and CF3 group are severely damaged,second only to the model group.In the Gxk and simvastatin groups,the aorta was less damaged,no obvious plaque formation,and the vascular structure was complete.There were different degrees of plaque formation in the blood vessels of the three disassembled groups.Among them,the CF3 group were the most severe,and the plaque in CF1 group was smaller.3.Real-time PCR resultsThe results of mouse liver PCR showed that compared with the model group,the expression of PERK,eIF2αand SREBP-2 in the liver of Guanxinkang group was significantly decreased（p<0.05）.The expression of PERK and eIF2αin liver of simvastatin group was significantly decreased（p<0.05）,but not on SREBP-2（p>0.05）.The expressions of PERK,eIF2αand SREBP-2 in liver of three decomposed groups were significantly decreased（p<0.05）.Although the expression of ABCA1 gene in the liver of each group increased,there was no statistical difference（p>0.05）.The results of mouse aortic PCR showed that compared with the model group,the expression of ATF4 gene in the aorta of the Guanxinkang group was significantly decreased,and the expression of Bcl-2 gene was significantly increased（p<0.05）,while the expression of PERK and eIF2αgenes was down-regulated,but no statistical difference（p>0.05）.The expression of ATF4 gene in the aorta of the simvastatin group was significantly decreased,and the expression of Bcl-2 gene was significantly increased（p<0.05）,but had no significant effect on the expression of PERK and eIF2α（p>0.05）.The ATF4 gene in the aorta of the CF1 group was significantly decreased（p<0.05）,but had no significant effect on the expression of PERK,eIF2αand Bcl-2 genes（p>0.05）.The expression of ATF4 gene in the aorta of the CF2 group of mice was significantly decreased,and the expression of Bcl-2 gene was significantly increased（p<0.05）,but had no significant effect on the expression of PERK and eIF2α（p>0.05）.The expression of ATF4 gene in the aorta of the CF3 group was significantly decreased（p<0.05）,but had no significant effect on the gene expression of PERK,eIF2αand Bcl-2（p>0.05）.4.Western Blot resultsThe results of western blot analysis showed that the expressions of p-PERK,p-eIF2αand SREBP-2 proteins in the liver of Guanxinkang group were significantly down-regulated compared with the model group（p<0.05）.The expression of p-PERK and p-eIF2αprotein in the liver of simvastatin group was significantly decreased（p<0.05）,but the expression of SREBP-2 protein was not significantly（p>0.05）.The expression of p-PERK and p-eIF2αprotein in the liver of CF1 group was significantly decreased（p<0.05）,but the expression of SREBP-2 protein was not significantly affected（p>0.05）.There was no down-regulation of p-PERK,p-eIF2αand SREBP-2 proteins in the CF2 group（p>0.05）.The expressions of p-PERK,p-eIF2αand SREBP-2 in the CF3 group were significantly down-regulated（p<0.05）.Compared with the model group,the expression of ABCA1 in Guanxinkang group was significantly increased（p<0.05）,but there were no significant difference in the other groups（p>0.05）.The results of western blot analysis showed that the expression of p-PERK,p-eIF2αand ATF4 protein in the aorta of the Guanxinkang group was significantly decreased,and the expression of Bcl-2 protein was significantly increased（p<0.05）.The expression of ATF4protein in the aorta of the simvastatin group was significantly down-regulated,the expression of Bcl-2 protein was significantly up-regulated（p<0.05）,and the down-regulation of phosphorylation of PERK and eIF2αprotein was not significant（p>0.05）.The phosphorylation level of AIF2αprotein and the expression of ATF4 protein in the aorta of the CF1 group were significantly decreased（p<0.05）,but the phosphorylation level of PERK and Bcl-2 were not significant（p>0.05）.The ATF4 protein expression in the aorta of the two groups and the three groups were significantly down-regulated（p<0.05）,but not on PERK,eIF2αprotein phosphorylation and Bcl-2 protein（p>0.05）.Conclusion:1.Using LDLR-/-mice as a model,blood lipid changes and plaque formation were evident in mice.2.Gxk can effectively regulate blood lipids,and can maintain liver cholesterol homeostasis and relieve ectopic deposition of aortic cholesterol,thereby alleviating AS.3.Gxk’s regulation of blood lipids and"stabilization"of liver cholesterol may be related to the reduction of cholesterol synthesis,uptake and promotion of cholesterol transport and metabolism by activating ABCA1 and inhibiting PERK-eIF2α-SREBP-2 signaling pathway.Moreover,the Yiqi Huoxue drugs in Gxk（Astragalus,Motherwort,Salvia miltiorrhiza）have more inhibitory effects on the PERK-eIF2α-SREBP-2 signaling pathway.4.Gxk reduces the role of cholesterol in aortic ectopic deposition,possibly by inhibiting endoplasmic reticulum stress PERK-eIF2α-ATF4 signaling pathway,thereby increasing the expression of anti-apoptotic factor Bcl-2 and reducing apoptosis.Reduces cholesterol deposition in the aorta,thereby relieving AS.Moreover,the Yiqi Huatan drugs Gxk（Astragalus,Melon,Quercus,Pinellia）have more important role in inhibiting the PERK-eIF2α-ATF4 signaling pathway. |
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