Jiedu Huoxue Formula Regulates PI3K/Akt/mTOR Signaling Pathway Intervention The Mechanism Of Atherosclerosis Action In APOE^-/- Mice

AS is a common chronic inflammatory disease in clinical practice today,and acute vascular embolic events triggered by rupture of vulnerable plaques are its main cause of morbidity and mortality.The inflammatory response not only exacerbates the AS process,but also deteriorates the atheromatous plaque towards rupture.Auto Phagy is the main catabolic program of cells and is an important way to maintain the stability of the body’s internal environment.Macrophages are the key inflammatory cells in the development of AS,and their inflammatory response and degree of autophagy affect the stability of plaques.Our team has found that induction of autophagy can suppress vascular inflammatory response and stabilize vulnerable plaques.In this study,we further investigated the pathway regulation mechanism of APOE^-/-AS mice on serum lipid and inflammatory factor levels,aortic pathology and autophagy level,starting from macrophage autophagy and inflammatory response,to provide theoretical support for the scientific prevention and treatment of AS vulnerable plaques with the Jiedu Huoxue Formula.Methods:Six C57BL/6J mice and 36 APOE^-/-mice were selected and fed in groups.The model group,herbal treatment group and rapamycin group were fed with high-fat diet to replicate the AS model,and were instilled with saline,detoxification and blood activation formula and rapamycin at week8,and dissected and sampled at week 11.The levels of serum TC,TG,HDL,LDL and inflammatory factors MCP-1 and IL-6 were measured by enzyme marker,the aortic roots were stained with HE,the formation and number of autophagosomes in aortic tissues were observed by transmission electron microscopy,and the expression levels of Beclin-1,LC3 protein,PI3K,Akt and mTOR pathway protein in aortic roots were measured by Western Blot.Results:1.Comparison of body mass change of mice in each group:The body mass of mice was measured once every two weeks,and it was found that the body mass of mice in each group increased compared with the beginning at the seventh week,and the growth of mice fed with high-fat diet was obvious.The mice in the blank group and the control group fed with normal chow were in good mental condition,with shiny fur and flexible activities;the mice in the groups fed with high-fat chow were in slightly poorer mental condition and were more obese.At the eighth week,we started to administer the drug and continued to feed the mice with the same diet for four weeks,and found that the mass of the mice in the middle and high dose groups and the rapamycin group decreased significantly compared with the model group（P<0.05）.2.Serum lipid results of mice in each group:TC,TG,HDL and LDL levels were higher in mice in each group fed with high-fat chow than in the blank group and the control group fed with normal chow.Compared with the model group:TG was lower in the low-dose group of Chinese medicine than the model group（P<0.05）;TC,TG and LDL were lower in the middle and high-dose groups and rapamycin group（P<0.05）;HDL levels were higher in each group than the model group（P<0.05）.3.The expression of serum MCP-1 and IL-6 in mice of each group:compared with the control group:the levels of MCP-1 and IL-6 in the model group were increased（P<0.05）,and the levels of MCP-1 in the blank group were decreased（P<0.05）.Compared with the model group:MCP-1 levels were decreased in the low-dose group of Chinese medicine（P<0.05）,and MCP-1and IL-6 levels were significantly decreased in the middle and high-dose groups and rapamycin group（P<0.05,P<0.01）.4.HE staining of the aortic root showed that the aortic vessel walls of mice in the blank group were structurally intact,and no atheromatous plaques were seen.In the control group,the inner wall of blood vessels was uneven and some of the walls were thickened.In the model group,the inner wall of blood vessels was uneven and smooth,and atheromatous plaques could be seen,with inflammatory cells infiltrating in the plaques.In the Chinese medicine group and rapamycin group,the intimal wall was not smooth,and a small amount of atheromatous plaque could be seen,and the degree of inflammatory cell infiltration and vessel wall thickness were reduced compared with the model group.5.Transmission electron microscopy autophagosome observation results:autophagosomes were dominated by bilayer membrane structure.A small number of autophagosomes and intact mitochondria were observed in the aortic tissues of mice in the blank and control groups;the number of autophagosomes and mitochondria in the model group was much smaller,and the mitochondrial structure was incomplete.The mitochondrial structure was still intact in the treatment group,and autophagosomes and autophagolysosomes were seen in the medium-dose and rapamycin groups of TCM,and the number of autophagosomes and autophagolysosomes increased in the low-dose and high-dose groups.6.Western Blot method was used to determine the expression of Beclin-1and LC3 proteins and PI3K,Akt and mTOR pathway proteins in the aortic root:（1）Comparison of Beclin-1 and LC3 protein expression levels among groups:compared with the control group,Beclin-1 protein expression decreased in the model group（P<0.05）,and there was no significant difference in protein expression in the blank group（P>0.05）.Compared with the model group,Beclin-1,LC3II protein expression and LC3II/Ⅰratio increased significantly in the Chinese medicine group and rapamycin group（P<0.05,P<0.01）.（2）Comparison of PI3K,Akt,mTOR protein expression levels in each group:compared with the control group,PI3K,Akt,mTOR protein expression increased in the model group（P<0.05,P<0.01）,and there was no significant difference in protein expression in the blank group（P>0.05）.Compared with the model group,mTOR protein expression decreased in the low dose group of Chinese medicine（P<0.05）,and PI3K,Akt,and mTOR protein expression significant decline in the middle and high dose groups of Chinese medicine and rapamycin group（P<0.05,P<0.01）.Conclusion:1.Detoxifying and revitalizing formula effectively reduces body mass and serum lipid level in AS mice.2.Detoxifying and revitalizing blood formula reduces the expression levels of MCP-1 and IL-6 in serum of AS mice,decreases the inflammatory response in plaque and reduces the vulnerability of plaque.3.Detoxifying and invigorating blood can inhibit atheroma plaque formation and reduce inflammatory cell infiltration in AS mice.4.Detoxifying and invigorating blood can increase the number of autophagosomes in AS mice and induce macrophage autophagy to suppress intraplaque inflammatory response.5.Detoxifying and revitalizing formula significantly upregulated Beclin-1 and LC3II protein expression and decreased PI3K,Akt and mTOR protein expression,suggesting that its mechanism of inhibiting inflammatory response and stabilizing vulnerable plaques may be related to direct inhibition of mTOR signaling molecules and indirect inhibition of their upstream PI3K/Akt pathway factor activation.